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Spermatogenesis in humans is comprised of a series of highly complicated cellular events, necessary to support the production of an upward of 200 million sperm daily from puberty through the entire adulthood of a healthy man. Recent advances in the field using the techniques of cell and molecular biology, genetics, and biochemistry have unraveled many of the mysteries in spermatogenesis. In this Chapter, we highlight some recent advances in the field regarding the biology of human spermatogenesis. We also summarize and discuss recent advances regarding the regulation of spermatogenesis in humans. Due to rapid advances in our understanding of spermatogenesis and the large number of published reports in the literature in the last 2–3 decades, we focus on rapidly developing areas to stimulate the interest of our readers, in particular in areas that offer advances for the treatment of infertility in men.
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3
Human Spermatogenesis and Its
Regulation
Haiqi Chen PhD, Dolores Mruk PhD, Xiang Xiao PhD
and C. Yan Cheng PhD
Contents
Introduction................................................................ 49
Structure and Composition of the Seminiferous
Epithelium.................................................................. 50
Sertoli Cells and the BloodTestis Barrier (BTB).... 52
CellCell Interactions in the Testis ........................... 53
Spermatogonia and Self-renewal ............................... 54
Spermatocytes and Meiosis ....................................... 56
Spermatids and Spermiogenesis ................................ 58
Cycle of the Seminiferous Epithelium ...................... 58
Factors that Regulate Spermatogenesis ..................... 59
Hormonal Regulation................................................. 59
Small Non-coding RNAs (sncRNAs)........................ 62
Obesity ....................................................................... 63
Summary .................................................................... 64
References .................................................................. 64
Introduction
In human spermatogenesis, undifferentiated
spermatogonia derived from spermatogonial
stem cells give rise to diploid spermatocytes
which undergo meiosis I/II to produce haploid
spermatids [13]. Spermatids, in turn, undergo
spermiogenesis to form functional spermatozoa
[35]. After puberty at *12 years of age, a
healthy man produces upwards of 200 million
sperm daily from his 20 s through 50 s [6,7],
which declines gradually with a reduction of
*50% by his 80 s [8,9]. Spermatogenesis is
composed of a series of cellular events which
includes (i) self-renewal of spermatogonial stem
cells and spermatogonia via mitosis, (ii) trans-
formation and differentiation of spermatocytes,
(iii) generation of haploid spermatids via meiosis
I/II, and (iv) nal morphological maturation of
spermatids to become spermatozoa via spermio-
genesis. These processes thus involve multiple
cellular events in the testis and are under com-
plex controls by regulatory hormonal and sig-
naling axes. The most important of these
regulatory pathways is the hypothalamicpitu-
itarytesticular axis involving both FSH and LH,
and testosterone and estradiol-17ßas key regu-
lators [1014]. Since the subject of hormonal
regulation of spermatogenesis in men has been
extensively reviewed [1015], we focus our
discussion herein on other topics in which
advances have been made in recent years.
H. Chen D. Mruk C.Y. Cheng (&)
Center for Biomedical Research Population Council,
1230 York Ave., New York, NY 10065, USA
e-mail: y-cheng@popcbr.rockefeller.edu
H. Chen
e-mail: hqchen1990@gmail.com
D. Mruk
e-mail: mruk@popcbr.rockefeller.edu
X. Xiao
Department of Reproductive Physiology,
Zhejiang Academy of Medical Sciences, Hangzhou,
Zhejiang, China
e-mail: xxiao@zjams.cn
©Springer International Publishing AG 2017
S.J. Winters and I.T. Huhtaniemi (eds.), Male Hypogonadism,
Contemporary Endocrinology, DOI 10.1007/978-3-319-53298-1_3
49
sjwint01@louisville.edu
A better understanding of human spermatogen-
esis is necessary since about 15% of married
couples are infertile, and half of these cases are
attributed to male factors, indicating that about
7% of men in the general population are infertile
[16]. In this context, it is of interest to note that
male reproductive health can be considerably
altered by the environment and life style factors
[1721]. Semen quality is believed to be
declining [2225], illustrating the global trend of
impaired male reproductive health. While 618%
of non-obstructive azoospermia or severe oligo-
zoospermia cases can be attributed to Y chro-
mosome microdeletions [26], as many as 25
40% of infertile men have no identiable cause
for hypospermatogenesis [27,28]. In fact, infer-
tility is an emerging global public health issue
right after cancer and cardiovascular diseases.
Interestingly, experiments using mouse genetic
models, including
knockout/knockin/gene-trapped, transgenic, and
chemical-induced point mutant mice, have iden-
tied more than 400 genes pertinent to sper-
matogenesis [28,29], and the use of proteomics,
epigenomics, and genomics have identied
>2300 genes pertinent to spermatogenesis and/or
testis function. However, how this information
applies to humans remains largely unknown [30].
Future directions in omicsresearch pertinent to
human spermatogenesis have been recently
reviewed [30,31]; herein, we focus on the local
regulation of spermatogenesis and highlight areas
of research which can serve as helpful examples
to guide future investigations.
Structure and Composition
of the Seminiferous Epithelium
In adult humans, as in rodents, the functional unit
that produces sperm via spermatogenesis is the
seminiferous tubule. Spermatogenesis is sup-
ported by testosterone produced by Leydig cells
in the interstitium, and by estradiol-17ßproduced
by Sertoli and germ cells [1015], although there
is evidence that human Leydig cells also produce
estradiol-17ß[32]. In men, each testis has *400
600 seminiferous tubules, the length of all the
tubules combined per pair of testes is *400
meters, and each tubule has a diameter of
*200 µm[3,33]. Spermatogenesis occurs as
shown in the cross section of a tubule (Fig. 3.1),
consisting of Sertoli cells and germ cells at dif-
ferent stages of development. These germ cells
include undifferentiated spermatogonia, dark type
A spermatogonia, pale type A spermatogonia,
type B spermatogonia; preleptotene, leptotene,
zygotene and pachytene primary spermatocytes,
and secondary spermatocytes; as well as Sa, Sb,
Sc, Sd1, and Sd2 spermatids until Sd2 transform
into spermatozoa [34]. Sertoli and germ cells near
the base of the seminiferous epithelium are in
close contact with the basement membrane,
which is a modied form of extracellular matrix
and is composed of mostly collagen (type IV) and
laminins, heparin sulfate proteoglycan, and
nidogen/entactin [35,36]. Behind the basement
membrane is the type 1 collagen layer, to be
followed by the myoid cell layer, lymph, and then
lymphatic endothelium. Collectively, they con-
stitute the tunica propria [35,36] (Fig. 3.1).
During spermatogenesis, germ cells, in particular
post-meiotic spermatids, rely almost solely on
Sertoli cells for structural, nutritional, and para-
crine support since haploid spermatids are
metabolically quiescent cells with relatively little
cytosol to support their cellular functions [37,
38]. Thus, the Sertoli cell is known as the
motheror nursecell, with each Sertoli cell
supporting *3050 developing germ cells based
on morphometric analysis in the rat testis [39].
In humans, Sertoli cells undergo mitotic pro-
liferation in two separate phases. The rst phase
takes place shortly after birth during the
neonatal-infantile period when the Sertoli cell
population increases via mitotic proliferation
[40]. During puberty, Sertoli cells again rapidly
divide mitotically [40], giving rise to
*500 10
6
Sertoli cells per testis (each adult
human testis weighs *1819 gm). The Sertoli
cell number declines to *300 10
6
by
5085 years of age, associated with a reduction
in daily sperm output [8]. Each testis has
*4.5 10
6
Sertoli cells in mice [41] and 30
40 10
6
Sertoli cells in rats [4244] compared
to 500 10
6
in men [8], and a daily sperm
50 H. Chen et al.
sjwint01@louisville.edu
production of 8x [41], 70x [45], and 200x10
6
[46], respectively. The number of Sertoli cells is
determined by FSH, thyroid hormones, growth
hormone, and several paracrine growth factors
[40]. Outside the tubules in the interstitial space
lay the Leydig cells, broblasts, and some
macrophages and microvessels (Fig. 3.1). Leydig
cells produce testosterone (T) under the inuence
of luteinizing hormone (LH) since LH receptors
are limited to Leydig cells in human and rodent
testes. Interestingly, the T level in the interstitial
uid and seminiferous tubule uid is at least
Type B spermatogonium
Preleptotptene
spermatocyte
Pachytene
spermatocyte
Elongated
spermatid
Sertoli cell
nucleus
Adluminal (Apical) compartment
Basal
compartment
BTB
BTB
Sertoli cell
nucleus
F-actin bundle
Apical ES protein
complex
Desmosomal protein
Gap junction protein
Endoplasmic
reticulum
Tight junction
protein complex
Basement membrane
Basal ES
protein complex
Type 1 collagen
layer
Blood
vessel
Blood
vessel
Myoid cell layer
Leydig
cell
Leydig
cell
Elongating
spermatid
Lymph
Type A spermatogonium
Lymphatic
endothelium
Sertoli cell
Sertoli cell
Tunica
propria
Intermediate filaments
Fig. 3.1 Structure and cellular composition of the sem-
iniferous epithelium in the human testis. The seminiferous
epithelium, as seen in this schematic drawing of a cross
section of a seminiferous tubule, is composed of Sertoli
and germ cells. The BTB is constituted by actin-based
tight junctions, basal ectoplasmic specialization (ES), and
gap junctions, as well as the intermediate lament-based
desmosome. As one of the tightest bloodtissue barriers in
the human body, the BTB divides the seminiferous
epithelium into the adluminal (apical) and basal compart-
ments. Spermatogonia (both type A, B and undifferenti-
ated) and preleptotene spermatocytes reside in the basal
compartment, whereas the other primary and secondary
spermatocytes and post-meiotic spermatids are found in
the apical compartment. The most notable junction in the
mammalian testis including humans is the ES, which is
typied by the presence of actin microlament bundles
sandwiched in-between cisternae of endoplasmic reticu-
lum and the apposing Sertoli cellcell plasma membranes.
The ES at the BTB is called basal ES while the ES at the
Sertolispermatid interface is designated apical ES. Since
developing germ cells (e.g., preleptotene spermatocytes)
and developing spermatids are being transported across
the seminiferous epithelium, Sertoli cellSertoli cell and
Sertoligerm cell junctions are continuously remodeling
throughout the 6 stages of the epithelial cycle of
spermatogenesis in the human testis
3 Human Spermatogenesis and Its Regulation 51
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70- and 50-fold higher than the T level in the
systemic circulation in the rat [47]. In humans,
the intratesticular T (e.g., interstitial uid) level is
also 100-fold higher than the systemic circulation
[4850], illustrating a considerably higher T
level is maintained in the testis to support
spermatogenesis.
Sertoli Cells and the BloodTestis
Barrier (BTB)
The seminiferous epithelium of the mammalian
testis is further divided into two functional
compartments: (1) the basal, and (2) the adlu-
minal (apical) compartments (Fig. 3.1), due to
the presence of the Sertoli cell bloodtestis bar-
rier (BTB) located near the basement membrane.
The BTB is constituted by coexisting actin-based
basal ectoplasmic specialization (basal ES) and
tight junction (TJ), as well as coexisting basal ES
and gap junction, and intermediate
lament-based desmosome [51,52]. As such,
undifferentiated spermatogonia, both type A and
B spermatogonia, and preleptotene spermato-
cytes differentiated from type B spermatogonia
reside in the basal compartment. More advanced
primary spermatocytes, secondary spermato-
cytes, and all haploid spermatids including
spermatozoa reside in the adluminal compart-
ment (Fig. 3.1).
Briey, the BTB provides a unique microen-
vironment in the adluminal compartment for
meiosis I/II and post-meiotic spermatid devel-
opment so that these events are segregated from
the systemic circulation, behind an immunolog-
ical barrier. Studies have shown, however, that
the BTB contributes minor signicance to the
immune privilege of the testis since germ cells
residing in the basal compartment are equally
immunogenic, containing multiple testis-specic
antigens [53,54]. Instead, the testis immune
privilege is maintained mostly by Sertoli cells
through their secretory immunosuppressive bio-
molecules: cytokines, bioactive lipids and pep-
tides, and androgens from Leydig cells [55].
The BTB in humans is established at puberty by
*1213 years of age when Sertoli cells cease to
divide and become fully differentiated, con-
comitant with the onset of meiosis I/II [40]. It is
of interest to note that human Sertoli cells, sim-
ilar to Sertoli cells in rodents, can be
de-differentiated, becoming mitotically active
when they are exposed to fetal bovine serum
[40], such as cultured in media containing 5
10% fetal bovine serum (v/v) [56]. Under such
conditions, Sertoli cells can be maintained
through multiple passes in vitro without detect-
able changes in their functional and physiologi-
cal properties [57].
BTB function in rodents is dependent on
testosterone [5861]. However, treatment of nor-
mal men with testosterone plus levonorgestrel
(LNG), which is capable of suppressing sper-
matogenesis and causing azoospermia or
oligospermia with suppression of intratesticular
androgen levels, did not affect BTB function since
the distribution of claudin-3 remained relatively
unaltered [62]. However, in a more recent study in
which healthy men were treated with T plus LNG
together with the GnRH antagonist acyline or the
5a-reductase inhibitor dutasteride (in order to
provide added suppression of spermatogenesis),
there was considerable down-regulation of
claudin-11, connexin-43, and vinculin at the BTB
compared to men treated wth T + LNG alone [63].
Collectively, these ndings suggest that human
BTB is regulated in a manner similar to rodents.
Some species differences exist, however, such as
the extent of androgen-dependency, since it
appears that a considerable reduction in the
intratesticular T level is necessary to suppress the
BTB integrity in humans.
Due to the limited access to normal human
testes for functional analysis, it is difcult to
compare the regulation of the human BTB
integrity with that of rodents. With advances in
cell and tissue culture techniques, however,
human Sertoli cells, using media containing fetal
bovine serum, have been cultured and main-
tained successfully for several weeks in vitro [56,
57,6466], with a functional tight junction-
permeability barrier [56,64]. Using this
approach, the organization of F-actin that sup-
ports BTB integrity has been investigated at the
human Sertoli cell BTB. It is now known that
52 H. Chen et al.
sjwint01@louisville.edu
human Sertoli cell F-actin organization is main-
tained, similar to that of rodents, by actin binding
proteins, including Arp2/3 (actin related protein
2/3) complex and Eps8 (epidermal growth factor
receptor pathway substrate 8) that confers bran-
ched actin network and bundled actin microla-
ments, respectively [57]. Furthermore, exposure
of human Sertoli cells to environmental toxi-
cants, such as CdCl
2
, or bisphenol A (BPA),
rapidly perturbs F-actin organization in these
cells. Changes in the distribution of cell adhesion
proteins at the BTB, including the TJ-protein
ZO-1, and the basal ES proteins N-cadherin and
ß-catenin, from the cell surface to the cell cytosol
[57], likely reect degradation via an
endosome-dependent degradation pathway.
These changes disrupt integrity and function of
the BTB. It is expected that much information
will be obtained using this in vitro system to gain
insight into the biology of the human BTB.
CellCell Interactions in the Testis
Due to the presence of the BTB, germ cell
development, in particular post-meiotic spermatid
development, relies almost exclusively on the
structural, nutritional, and paracrine support of
Sertoli cells. This is mediated via cell junctions at
the Sertoligerm cell interface, involving multiple
genes and their proteins [37,38,67,68]
(Fig. 3.2). Even though undifferentiated sper-
matogonia [e.g., spermatogonial stem cells
(SSCs)], type A and type B spermatogonia, and
preleptotene spermatocytes reside outside the
BTB, they also rely on the Sertoli cell for func-
tional and structural support through the expres-
sion of unique genes (Fig. 3.2). This is
particularly true for SSCs which are located at the
stem cell nichesusually at the base of the
seminiferous epithelium wherein three seminif-
erous tubules meet, adjacent to the microvessels
in the interstitium. In short, SSCs rely on Sertoli
cells for structural and functional support, besides
biomolecules from the microvessels [69,70].
Sertoli cells also modulate spermatogenesis
through cross talk with Leydig cells. Sertoli cells
stimulate Leydig cell differentiation but inhibit
Leydig cell steroidogenesis [7173]. One of the
paracrine factors produced by Sertoli cells that
modulates Leydig cell function has been partially
characterized [74]. Studies using genetic models
Fig. 3.2 Cellcell interaction within the testis and its
functional relationship to the hypothalamicpituitary
testicular axis. The classic hormonal regulatory axis that
exerts its regulatory effects on the testis through the
release of gonadotropin-releasing hormone (GnRH) from
the hypothalamus, which in turn regulates the secretion of
luteinizing hormone (LH) and follicle-stimulating hor-
mone (FSH) from the pituitary gland. Testosterone pro-
duced by interstitial Leydig cells provides the feedback
loop to modulate the production of GnRH from the
hypothalamus and thereby LH and FSH from the pituitary
gland. In humans and primates in contrast to rodents,
testosterone has no direct feedback effect on the pituitary
[240]. Inhibin produced by Sertoli cells provides selective
feedback to the production of FSH. There is cross talk
among germ cells (including spermatogonial stem cells)
and Sertoli cells in the seminiferous epithelium, as well as
Leydig cells and peritubular myoid cells in the intersti-
tium. Studies have shown an array of growth factors,
genes and hormones are involved. Abbreviations: CSF1,
colony stimulating factor 1; BMP4, bone morphogenetic
protein 4; GDF9, growth differentiation factor-9; GnRH,
gonadotropin-releasing hormone; P-Mod-S, peritubular
myoid cell modulates Sertoli cell factor; TGF-b1, trans-
forming growth factor-1; Wt1, Wilmstumor 1
3 Human Spermatogenesis and Its Regulation 53
sjwint01@louisville.edu
have shown that Sertoli cells maintain Leydig
cell number (both fetal and adult Leydig cells)
and support Leydig cell development [75,76], as
well as determine Leydig cell differentiation
status and cell fate through the Wt1 gene [77]. On
the other hand, Sertoli cells also interact with
germ cells to support spermatogenesis through
multiple paracrine factors [37,78,79] (Fig. 3.2).
There is also functional cross talk between
Sertoli cells and the tunica propria, in particular
peritubular myoid cells (Fig. 3.2). For instance,
Sertoli cells work in concert with peritubular
myoid cells in the production and deposition of
extracellular matrix (ECM) components [80]to
produce and maintain the basement membrane,
which is a modied form of ECM [35,36].
Studies have also suggested that peritubular
myoid cells modulate Sertoli cell function
wherein myoid cells produce a paracrine factor
known as P-Mod-S that regulates Sertoli cell
function [81]. However, the identity of this factor
remains to be claried. Another report has shown
that while peritubular myoid cells do not secrete
clusterin or a
2
-macroglobulin (a wide-spectrum
protease inhibitor [82]), co-cultures of myoid
cells with Sertoli cell promote Sertoli cell a
2
-
macroglubulin and clusterin secretion [83]. It
must be noted, however, that unlike Sertoli cells,
which can be maintained in culture in serum-free
medium (e.g., F12/DMEM with several growth
factors [84]) for up to *2-wk, peritubular myoid
cell cultures require the presence of serum pro-
teins, such as *510% fetal calf serum for their
survival [83].
The most conclusive study thus far to illus-
trate the importance of peritubular myoid cells in
spermatogenesis is the generation of a peritubular
myoid cell-specic androgen receptor
(AR) knockout (PTM-ARKO) mouse model
[85]. PTM-ARKO male mice have reduced
seminiferous tubule uid production and are
azoospermic and infertile, with reduced expres-
sion of certain androgen-dependent Sertoli cell
genes [85]. These ndings illustrate that ARs in
Sertoli cells fail to assume the function of ARs in
myoid cells to sustain spermatogenesis. Further-
more, these ndings demonstrate that Sertoli cell
androgen-dependent gene expression is
modulated by peritubular myoid cells, unequiv-
ocally demonstrating that Sertoli and peritubular
myoid cells are functionally connected. Interest-
ingly, studies using Amh-Cre to induce expres-
sion of the Diphtheria toxin receptor in Sertoli
cells to cause controlled, cell-specic, and acute
ablation of the Sertoli cell population in adult
mice have shown that Sertoli cells also control
peritubular myoid cell fate, including its differ-
entiation status and cell population [76]. Using
this approach, Sertoli cells were shown to mod-
ulate testicular vascular network development,
including modulating circulating testosterone
levels in adult mice [86]. It should be noted that
much of the information stated above and
depicted in Fig. 3.2 is derived from studies in
rodents. Clearly, further studies are needed to
understand the cellcell interactions between
Sertoli cells, Leydig cells, and peritubular myoid
cells in the human testis.
Spermatogonia and Self-renewal
Spermatogonia are the progenitor cells of all
germ cells and are designated type A and type B
[87,88]. In humans, type A spermatogonia are
further categorized into progenitor dark type A
spermatogonia (A
d
) and pale type A spermato-
gonia (A
p
) which are capable of undergoing
mitotic proliferation for self-renewal [89]
(Fig. 3.3). A
p
are the predominant spermatogonia
that survive radio- and chemotherapy while A
d
are largely eliminated during such treatment,
illustrating that A
p
are the potential spermato-
gonial stem cells [90]. At present, it is generally
accepted that A
d
constitute a pool of reserve
spermatogonia which remain quiescent but can
give rise to A
p
when needed and are considered
to be the truespermatogonial stem cells in
humans [4,91]. The molecular basis for the
regulation of human spermatogonia remains lar-
gely unknown. cKIT, a transmembrane protein
tyrosine kinase receptor (also known as stem cell
factor receptor or CD117), and its ligand cKIT
ligand (also known as stem cell factor (SCF)) are
known to be involved in the differentiation of
spermatogonia in humans, rodents, and primates
54 H. Chen et al.
sjwint01@louisville.edu
3 Human Spermatogenesis and Its Regulation 55
sjwint01@louisville.edu
[91]. cKIT is expressed in spermatogonia, round
spermatids, and spermatozoa as well as in Leydig
cells whereas its ligand or SCF is primarily
expressed in Sertoli cells in humans [92]. How-
ever, cKIT is not expressed in spermatocytes or
elongating spermatids [93]. Expression of cKIT
in type A spermatogonia varies across the semi-
niferous tubules, being highest in proliferating
A
p
spermatogonia and type B spermatogonia
[9395], illustrating the involvement of
cKIT/SCF in spermatogonial self-renewal and
differentiation. The reduced staining of cKIT in
type A spermatogonia has been shown in men
with subfertility, and its reduced expression is
associated with an increase in apoptosis in type A
spermatogonia [96].
GFRA1, a6-INTEGRIN, PLZF, GPR125,
SALL4, and THY1 are undifferentiated sper-
matogonia markers in mouse, monkey, and man,
whereas UTF1 (undifferentiated embryonic cell
transcription factor 1) and FGFR3 (broblast
growth factor receptor 3, also known as CD333)
are molecular markers specic for human type A
spermatogonia [91]. The molecular basis for the
transition of undifferentiated spermatogonia to
type A spermatogonia has been established in
rodents and involves retinoic acid (RA), the
active metabolite of vitamin A, which is also
involved in the initiation of meiosis [97]. Indeed,
treatment of mice with the pan-retinoic acid
receptor antagonist, BMS-189453 that blocks the
action of RA in the mouse testis, leads to
reversible infertility due to meiotic arrest [98,
99]. The notion that RA is involved in
spermatogenesis in humans is based on ndings
that bisdichloroacetyldiamine WIN 18,446, an
inhibitor of retinoic acid synthesis, inhibits
ALDH1A2 and induces reversible infertility in
mice, rats, monkeys, and men [100103]. More
study is needed to dene the role of retinoic acid
in human spermatogonial differentiation.
Spermatogonial differentiation is a
gonadotropin-dependent event in primates [104]
since FSH treatment increases the number of A
p
and type B spermatogonia in monkeys [105,
106]. Emerging evidence suggests that FSH
plays a role in spermatogonial differentiation in
men [107]. However, in normal adult men,
gonadotropins (FSH and LH) serve as sper-
matogonial survival factors by regulating the
intrinsic apoptotic pathway to promote germ cell
survival but have no effect on germ cell prolif-
eration [108].
Spermatocytes and Meiosis
Spermatocytes, namely leptotene, zygotene,
pachytene, and diplotene spermatocytes are
found in the human seminiferous epithelium
[109] following the initiation of meiosis in
humans at puberty [110] (Figs. 3.3 and 3.4).
Diplotene spermatocytes undergo meiosis I to
form secondary spermatocytes (with haploid
number of chromosomes but 2n content of
DNA), which rapidly progress to meiosis II so
that a single secondary spermatocyte produces
two haploid round Sa spermatids. In this context,
bFig. 3.3 Comparison of germ cell development in the
human versus the rat testis. While the development of
germ cells through spermatogenesis is similar between the
two species, there are some notable differences such as
during spermatogonial self-renewal and differentiation. In
the rat, Asingle spermatogonia (A
s
) divide and form
Apaired spermatogonia (A
pr
), which further divide to
form Aaligned spermatogonia (A
al
). The differentiating
A
al
undergo 5 cell divisions (A
1-4
and In) to form type B
spermatogonia. In humans, both Adark spermatogonia
(A
d
) and Apale spermatogonia (A
p
) are capable of
undergoing self-renewal through mitosis, and A
p
also
give rise to type B spermatogonia, the latter of which are
then differentiate to form preleptotene spermatocytes. In
the human testis, preleptotene spermatocytes formed in
stage III (concomitant with spermiation that takes place at
late stage II) of the epithelial cycle are transported across
the BTB to enter the adluminal compartment to prepare
for meiosis I/II which takes place at stage VI of the
epithelial cycle, and Sa spermatids, the equivalent of
round spermatids in the rat, are found in stage I. In the rat,
a single diploid A
s
can give rise to 4096 haploid sperm;
whereas in the human, only 16 sperm are derived from
one type A spermatogonium. The number of different
types of germ cells that can be derived from a single
committed type A spermatogonium is indicated. This
gure was prepared based on the information of earlier
reports [1,51,127] and in the model of rat spermatoge-
nesis it is noted that A
pr
and A
al
spermatogonia have been
shown to be able to revert to A
s
[241]
56 H. Chen et al.
sjwint01@louisville.edu
it is of interest to note that, unlike other somatic
cells, neither spermatocytes nor spermatids
metabolize glucose; instead, they rely on lactate
supplied by Sertoli cells as their energy source
[111,112]. In brief, glucose is taken up by Ser-
toli cells via the specic glucose transporter
GLUT1 and is processed into lactate glycolyti-
cally [113] by testis-specic lactate dehydroge-
nase C4 (LDHC4). Lactate is transported out of
Sertoli cells by a monocarboxylate transporter
MCT1 and taken up by meiotic and post-meiotic
germ cells via their specic monocarboxylate
transporter, MCT2 [111]. Studies have shown
that energy metabolism of Sertoli and germ cells
is regulated by FSH, steroids, insulin and para-
crine factors, and their disruption leads to male
infertility [112,114]. It is also noted that defects
in meiosis, such as meiotic maturation arrest
Fig. 3.4 A schematic drawing illustrating the six stages
of the epithelial cycle (I-VI) in the human testis, and the
associated germ cell types in the seminiferous epithelium
in each of these stages. It is noted that spermiation takes
place in late stage II, and preleptotene spermatocytes arise
in stage III, which are being transported across the BTB in
stage III. Meiosis takes place at stage VI of the cycle, and
the entire epithelial cycle takes 16 days to complete. The
duration of each stage, from I to VI, is also annotated.
This drawing was prepared and modied from earlier
reports [4,34,242], and recent advances in the staging of
human spermatogenesis have recently been discussed
[243]
3 Human Spermatogenesis and Its Regulation 57
sjwint01@louisville.edu
(or early maturation arrest) is found in *10% of
men with non-obstructive azoospermia (NOA),
with the histopathological features of reduced
tubule diameter, reduce germ cell number, and
degenerating spermatocytes [115].
Spermatids and Spermiogenesis
The process during which spermatids undergo
extensive morphological transformation through
round Sa spermatids, Sb1, Sb2, Sc, Sd1, and Sd2,
and eventually to spermatozoa is known as
spermiogenesis [116] (Figs. 3.3 and 3.4). During
this process, no further cellular divisions occur,
newly formed round spermatids from secondary
spermatocytes are characterized by a small
spherical nucleus with the usual array of cyto-
plasmic organelles, including the Golgi appara-
tus, mitochondria, and centrioles [116]. Each
spermatid undergoes considerable morphological
changes that begin with a large granule called the
acrosomic granule which is generated by several
small proacrosomic granules in the area of the
Golgi apparatus that grows over the nucleus. As
spermiogenesis continues, the acrosome trans-
forms further, and nuclear chromatin condensa-
tion begins [117]. At the nal steps, the
spermatid nucleus completes its chromatin con-
densation, and the majority of the cytoplasm
eventually detaches from the spermatid to form
the residual body, which is engulfed by the
Sertoli cell and processed into a phagosome. The
phagosome is then transported to the base of the
Sertoli cell for lysosomal degradation [118]. In
humans, spermiogenesis can be disrupted, lead-
ing to: (i) late maturation arrest, manifested by an
arrest in development in early spermatids with
dark round nuclei, and (ii) hypospermatogenesis,
with an arrest in further development of con-
densed oval spermatids [115]. Mature sperma-
tozoa, once formed, are released into the tubule
lumen via the nal cellular process of sper-
matogenesis at spermiation [5,119]. While the
molecular mechanism underlying spermiation
remains relatively unexplored, studies in rodents
have shown that spermiation is tightly regulated
by the spatiotemporal expression of signaling
molecules such as p-FAK-Tyr
407
, as well as actin
binding/regulatory proteins (e.g., the Arp2/3
complex, Eps8), involving degeneration of api-
cal ES and generation of laminin fragments [5,
119]. However, this process is poorly understood
in humans except that it is likely dependent on
FSH and testosterone. So far, human male
infertility has not been attributed to defects of
spermiation alone [3].
Cycle of the Seminiferous Epithelium
In human as well as rodent testes, the distinctive
cellular associations in the seminiferous epithe-
lium along the tubule can be dened into different
stages which appear cyclically throughout sper-
matogenesis, known as the epithelial cycle of
spermatogenesis [88,120,121]. In the testis of
rats, mice, and humans, an epithelial cycle is
composed of I-XIV, I-XII, and I-VI stages,
respectively [4,122,123]. It is of interest to note
that spermiation and meiosis I/II take place at stage
VIII and XIV, VIII and XII and II and VI, in the rat,
mouse, and human testis, respectively. It takes
about 16 days to complete an epithelial cycle in
the human [124] versus 8.6 days in the mouse
[125] and 12.8 days in the rat [126]. This is the
time it takes for a given spot in a seminiferous
tubule at a specic stage of the epithelial cycle,
such as at stage II, when observed under the
stereomicroscope, to undergo cyclic staging and
become stage II again. However, the duration of
spermatogenesis, i.e., from type A spermatogonia
to spermatozoa, is *68 days (4.2 cycles) in
humans versus *35 days (4 cycles) and
*58 days (4.5 cycles) in mice and rats as noted in
Fig. 3.4 for humans (for reviews, see [1,127,
128]). This is the time it takes for a single human
diploid Ap spermatogonium (progenitor germ cell,
wherein Ad spermatogonium is the regenerative
reserve stem cell) to develop into multiple haploid
spermatozoa which requires *4.2 cycles. In this
context, it is noted that the staging of the epithelial
cycle is largely dened according to changes in the
Golgi region of developing spermatids, namely the
acrosome, when it is visualized by the periodic
acid Schiffs reaction (PAS). In mouse, rat, and
58 H. Chen et al.
sjwint01@louisville.edu
human, spermatids can be divided into 16, 19, and
6 steps, respectively, throughout spermiogenesis
before they develop and transform into sperma-
tozoa. The classication of human spermatids into
6 steps of Sa, Sb1, Sb2, Sc, Sd1, and Sd2 was
based on the use of osmium-dichromate as xative
for the human testis, which was then stained with
osmium and examined by transmission electron
microscopy [117].
Factors that Regulate
Spermatogenesis
Numerous factors are known to affect human
spermatogenesis. Below, we highlight regulators
that are well established or are rapidly develop-
ing in the eld.
Hormonal Regulation
Hormonal regulation of spermatogenesis in
humans is a complex biological event involving
both testosterone and estradiol-17ß[10,13,14,
129,130] and is tightly regulated by the
hypothalamicpituitarytesticular axis by
follicle-stimulating hormone (FSH) and luteiniz-
ing hormone (LH) that exert their effects on Sertoli
and Leydig cells, respectively. Besides these hor-
mones, inhibins, activins, follistatin, and other
paracrine factors are also involved. Since the
hormonal regulation of spermatogenesis in
humans has been eminently reviewed recently [11,
13,131136], interested readers are encouraged to
seek further information from these earlier
reviews. Below is a summary of recent ndings
pertinent to the hormonal regulation of human
spermatogenesis.
FSH
FSH is an important regulator of spermatogenesis
through effects on Sertoli cells where FSH
receptors are expressed. FSH activates at least
ve signaling pathways in Sertoli cells:
cAMP-PKA, MAPK, PI3 K-AKT, intracellular
Ca, and phospholipase A2 [137139]. Gene
expression proles indicate that FSH regulates a
panoply of Sertoli cell genes. FSH promotes
Sertoli cell proliferation before puberty, while
after puberty, FSH activates Sertoli cell to sup-
port germ cell development. Despite the impor-
tant role of FSH in spermatogenesis, this
hormone, unlike testosterone (see below), is not
considered essential to, but rather ne-tunes
spermatogenesis. For instance, deletion of the
FSH-ßsubunit in mice led to infertility in
FSH-decient females due to a block in follicu-
logenesis, but FSH-decient males were fertile
even though the testes of these mice were
reduced in size [140,141]. Furthermore, men
with inactivating mutations of the FSH-bgene or
the FSH receptor have impaired spermatogenesis
but generally remain fertilean observation
explained in part by the nding that the FSH
receptor possesses low-level constitutive activity
in the absence of FSH [129,142144] (see
Chap. 6). On the other hand, it is of interest to
note that men with congenital complete hypog-
onadotropic hypogonadism (CHH) are usually
treated with hCG (human chorionic gonado-
trophin) together with FSH to induce testis
development, spermatogenesis, and fertility (see
Chap. 20). Also, men with idiopathic oligoas-
thenoteratozoospermia treated with recombinant
FSH for 3 months were found to have consid-
erable improvement in seminal parameters vs.
untreated controls [145,146].
LH
The primary function of LH is to stimulate
the production of testosterone by Leydig cells
[147]. LHCG-receptor (luteinizing hormone/
choriogonadotropin receptor, also called
LHCG-R or LHR) expression begins in fetal
Leydig cells [148]. Human males with inacti-
vating mutations of the LHCG-R develop
ambiguous genitalia and are testosterone-
decient indicating that hCG/LH signaling is
essential for testosterone production in the fetus
and adult [149151]. LH stimulates testosterone
production by stimulating the expression of key
steroidogenic enzyme genes as well as tran-
scription factors that are required for testosterone
biosynthesis to support spermatogenesis and
other male reproductive function [152]. Ligand
3 Human Spermatogenesis and Its Regulation 59
sjwint01@louisville.edu
binding to the LHCG-R stimulates adenylate
cyclase, increases cAMP production and phos-
phorylates target proteins through protein kinases
A and C, activates ERK1 and 2, and increases
calcium signaling [148]. Interestingly, accumu-
lating evidence supports the notion that
steroidogenesis in human fetal testes is highly
sensitive to environmental toxicants or elected
lifestyle (e.g., cigarette smoking) which are dis-
ruptive to LH-mediated testosterone production
by Leydig cells [17,153].
Intratesticular Testosterone
(ITT) Microenvironment
Testicular aspirations of normal men have shown
that T is the predominant intratesticular sex steroid
[48,49,154]. ITT concentration in men averages
609 ±50 ng/ml, which is much higher than the
average serum T level of 3.7 ±0.3 ng/ml [48
50]. This gradient between the testis and serum is
similar to ndings in rodents, wherein ITT in rat
testes is *100-fold higher than the serum T level
[47,155]. Collectively, these ndings support the
notion that a high ITT level is necessary to main-
tain spermatogenesis. For instance, the high level
of T in the testis is known to support spermatid
adhesion, spermiogenesis, and BTB function in
rodent testes [5,51,156]. In this context, it is of
interest to note that the ITT concentration at
*2000 nM vesurs *12 nM of T in serum in
normal men is considerably higher than that of
SHBG/ABP (sex hormone binding globulin/
androgen binding protein, *50 nM; note:
SHBG/ABP reduces androgen bioavailability),
suggesting that most ITT is bioavailable [154].
Studies by Jarow et al. found that *70% of the
total ITT is bioactive based on a novel androgen
bioactivity assay [154]. Interestingly, neither ITT
concentrations nor intratesticular bioactive
androgen levels were strongly correlated with
serum T concentration (correlation coefcients,
r= 0.38) nor serum bioactive androgen level
(r= 0.46). Yet SHBG/ABP levels in the testis and
serum in humans were strongly correlated [48,
154]. These data thus fail to explain the disparity
between intratesticular and serum bioactive
androgen levels [48,154,157]. In contrast, Roth
et al. found that T levels in testicular aspirations
(i.e., to obtain ITT) and serum of fertile men in
samples obtained simultaneously were strongly
and positively correlated (r= 0.67) [158]. The
different results in the two studies may be due to
different assay approaches. In this context, it is of
interest to note that a recent report has shown that
high ITT is not necessary to support spermatoge-
nesis in humans [159], and proposed that the ITT
level may be high only because T is synthesized
locally by Leydig cells in the interstitial space of
the testis. Thus, the physiological signicance of
the ITT level to support spermatogenesis requires
additional further studies.
Importantly, the earlier report of Roth et al.
also demonstrated considerable variations in ITT
levels in fertile men which correlated very
strongly with serum LH levels (r = 0.87). It was
hypothesized that LH secretory pulsatility might
cause pulsatility in ITT concentrations [158]. In
fact, ITT pulsatility was suggested by an earlier
study that quantied T and estradiol concentra-
tions over 4 h in cannulated gonadal veins of
men with varicocele [32]. While the importance
of ITT pulsatility to spermatogenesis remains to
be established, it may inuence the cyclic nature
of spermatogenesis during the epithelial cycle.
For instance, the requirements of the seminifer-
ous epithelium to support androgen-dependent
cellular events at late stage II during spermiation
are quite different from stages VI when meiosis
takes place. As such, it is likely that the ITT level
required at stage II is different from stage VI and
other stages to support spermatogenesis.
Nonetheless, the ITT concentration necessary to
support spermatogenesis in humans has not been
established by quantifying T levels in intrates-
ticular uid obtained during micro-TESE (mi-
crodissection testicular sperm extraction) [160].
Furthermore, studies in rodents have shown that
T, besides playing a crucial role in maintaining
germ cell adhesion in particular developing
spermatids, modulates endocytic vesicle-mediated
protein trafcking [161]. Due to the high level of
cellular activities in the epithelium to maintain
the daily sperm production rate, many cellular
proteins, in particular those found at the Sertoli
Sertoli or Sertoligerm cell interface, are being
re-cycled [162]. Studies have shown that the
60 H. Chen et al.
sjwint01@louisville.edu
relative ratio of ITT/cytokines within the
micro-environment of the seminiferous epithe-
lium may be crucial to govern these protein
recycling events during the epithelial cycle [163].
These studies, while recently performed in
rodents, can now be extended to humans by
using the human Sertoli cell in vitro system [57].
Androgen Receptor (AR)
T exerts its effects through AR signaling [156,
164166]. In humans, AR expression is restricted
to Sertoli cells [167,168]. Studies using murine
models have shown that specic deletion of AR
in Sertoli cells (SC-specic AR KO) leads to
meiotic arrest and early spermatogenic matura-
tion arrest [169,170], and also terminal differ-
entiation of haploid spermatids [171].
Collectively, these ndings illustrate the signi-
cance of AR-mediated action in spermatogenesis,
in particular murine meiosis and spermiogenesis.
In men, the AR expression level visualized by
immunohistochemistry in Sertoli cells was higher
in those with NOA (non-obstructive azoosper-
mia) vs. OA (obstructive azoospermia), and a
signicant positive correlation was observed
between FSH levels and Sertoli cell AR expres-
sion in OA patients [167]. However, efforts to
relate AR function or distribution to infertility in
humans have been unsuccessful. For instance, no
correlation was found between AR expression in
Sertoli cells with serum T levels, or serum LH,
and FSH levels in men with NOA [167]. Also,
exogenous FSH was shown to trigger an increase
in AR expression in the human testis [167].
However, hCG therapy had no apparent effect,
thereby underscoring the signicance of
FSH-dependent Sertoli cell AR expression [167].
Exon 1 of the AR contains a CAG trinu-
cleotide repeat that encodes a polyglutamine tract
in the N-terminus of the AR. This region is
required for AR interaction with transcriptional
co-regulators, and variation in CAG repeat
length, even within normal alleles, inuences AR
function in that shorter repeats are associated
with a more transcriptionally active receptor.
Furthermore, an expanded polyglutamine
tract confers toxic properties responsible for
neuronal and non-neuronal degeneration in the
neurological disorder spinal and bulbar muscular
atrophy in which affected men have small testes
and gynecomastia (SBMA) [172]. Attempts to
correlate the severity of spermatogenic dysfunc-
tion with CAG-encoded polyglutamine length
polymorphism in the AR gene have, however,
yielded inconsistent and conicting results [173
176]. Interestingly, an insertion mutation near the
beginning of the CAG repeat in exon 1 of the AR
gene was found in an azoospermic man [177].
Treatment of an infertile man who had a point
mutation of p.Val686Ala in the AR
ligand-binding domain with prolonged high-dose
testosterone therapy was found to produce
marked improvement in sperm count and semen
quality [178]. Combined with intracytoplasmic
microinjection, testosterone treatment resulted in
fertility. A point mutation in the transactivation
domain (TAD) of the AR gene was found in an
infertile man with gynecomastia with a high FSH
level, small testes, and Sertoli cell-only syn-
drome [179]. However, a fertile man with
gynecomastia was found to have a p.Pro69Ser
mutation within the AR ligand-binding domain
but to display no considerable defects in semen
quality [180]. Taking these data collectively, it is
anticipated that more AR mutation-associated
male infertility cases will be identied. As more
data are available, a systemic analysis is war-
ranted to relate mutation(s) of the AR gene with
idiopathic male infertility. At this point, based on
the available data, it is likely that FSH-dependent
AR expression is crucial for spermatogenesis.
However, more accurate functional assays are
necessary to pin-point the importance of AR in
male infertility.
Estrogen
Testes are known to produce a considerable
amount of estradiol-17ßvia aromatase, which is
essential to maintain spermatogenesis [10,12,
181]. In humans, the role of estrogen in sper-
matogenesis remains a subject of debate. Emerg-
ing evidence, however, supports the notion that
estrogen is involved in testis development, uid
resorption in the rete testis, maintenance of sper-
matogenesis, and in the maturation of spermatozoa
[10,181,182]. Most importantly, aromatization of
3 Human Spermatogenesis and Its Regulation 61
sjwint01@louisville.edu
T to estradiol-17ßin the hypothalamus provides an
important negative feedback signal to gonado-
tropin secretion [10]. Aromatase is expressed by
human Leydig cells, Sertoli cells, spermatocytes,
spermatids, and spermatozoa (its presence in
spermatogonia remains unknown) [181] while
estrogen is produced mostly by Leydig cells in the
adult human testis [10].
Estrogen exerts its effects via estrogen recep-
tors ERa(ESR1) and ERß(ESR2) [10,12].
Studies in rodents have shown that ERais
expressed by Leydig cells and peritubular myoid
cells; whereas ERßis found in some Leydig
cells, but mostly in Sertoli cells and germ cells
[183,184]. In short, ERais predominant in the
interstitium and ERßin the seminiferous epithe-
lium. Subsequent studies, however, have
demonstrated that ERais also expressed in Ser-
toli and germ cells in the rat testis [185]. In
human testes, the binding of estradiol-17ßto
human sperm was rst reported in 1981 [186]. In
1998, human sperm were shown to express both
the ERamRNA and protein [187]. Subsequent
studies have conrmed the presence of ERßas
well as ERain human sperm [12]. It is now
generally accepted that ERais expressed in
spermatogonia, pachytene spermatocytes, and
early round spermatids; whereas ERßis expres-
sed in pachytene spermatocytes, early round
spermatids, Sertoli cells, and Leydig cells in
human testes [188,189]. A genetic analysis study
in 300 infertile Indian men and 255 fertile nor-
mal subjects identied single nucleotide poly-
morphisms (SNPs) (found in 4 subjects) and
mutations (found in 8 subjects) in the ERßgene,
suggesting ERßgene mutations are a cause of
spermatogenesis failure in men. These infertile
men displayed normal reproductive tract and
serum hormone levels [190]. The few men
identied with aromatase deciency due to
autosomal recessive inheritance of mutations in
the CYP19A1 gene tend to have small testes and
an abnormal semen analysis. Additionally, hor-
mone levels in men with mutation in ERaand
decient in aromatase have considerable changes
in serum T, estradiol, LH, and/or FSH compared
to normal subjects (see Chap. 1). While the
number of patients is small, these ndings
illustrate the signicance of estrogen in human
spermatogenesis. Furthermore, genetic and
pharmacologically induced estrogen deciency
leads to reduced libido in men [191,192]. In
summary, estrogen is known to be involved in
the development of the testis, maintenance of
male reproductive tract, uid resorption in the
rete testis, and in particular sperm maturation in
humans [12,181,193,194].
Small Non-coding RNAs (sncRNAs)
As noted above, spermatogenesis is a highly
complicated process that requires the intriguing
participation of multiple genes at the transcrip-
tional and post-transcriptional levels to produce
spermatozoa. Studies have shown that germ cells,
including spermatogonia, spermatocytes, and
post-meiotic spermatids all contain abundant
levels of non-coding RNAs, such as siRNA
(small interfering RNA, 2025 nucleotides),
miRNA (microRNA, 2124 nucleotides), and
piRNAs (Piwi-interacting RNA, 2631 nucleo-
tides) [195197]. The most important sncRNAs
that are involved in spermatogenesis are miRNAs
and piRNAs and the associated pathways based
on studies of genetic models in rodents [198
201]. These small RNAs are short single-stranded
non-coding nucleotides that are known to directly
disrupt target mRNAs through degradation,
causing translation repression to block protein
synthesis [20,202]. Since miRNA can bind to
more than one mRNA, a specic miRNA can
regulate the function of more than a single gene.
Small RNAs including miRNAs and piRNA are
mostly stored in the chromatoid body which is a
dense structure in the germ cell cytosol composed
of mainly RNAs and RNA-binding proteins and
are involved in the regulation of germ cell apop-
tosis, proliferation, and differentiation, as well as
spermatogonial stem cell self-renewal [200,203,
204]. Studies have shown an alteration in the
expression patterns of small RNAs in infertile
men [205207]. For instance, miR-141, miR-429,
and miR-7-1-3p were up-regulated in men with
idiopathic non-obstructive azoospermia (NOA)
[206] while miR-34c-5p, miR-122, miR-146b-5p,
62 H. Chen et al.
sjwint01@louisville.edu
miR-181a, miR-374b, miR-509-5p, and
miR-513a-5p were markedly down-regulated in
men with NOA. These miRNAs are possibly
involved in regulating germ cell apoptosis [207].
In this context, it is of interest to note that
miRNAs and siRNAs are processed by an
RNase III endonuclease Dicer, the deletion of
which in mice leads to infertility. Dicer is essen-
tial for haploid spermatid differentiation [208]
and for the assembly and maintenance of cell
junctions in the seminiferous epithelium during
spermatogenesis [209]. Specic deletion of Dicer
in Sertoli cells also leads to infertility with com-
plete absence of spermatozoa in seminiferous
tubules and progressive testicular degeneration
[210]. While the role of Dicer in human fertility
remains to be elucidated, it is obvious that small
regulatory RNAs represent a tempting target for
non-hormonal male contraception. Also, much
work is needed to better understand the role of
small RNAs in human spermatogenesis, and their
use as diagnostic markers to monitor infertility,
for example, men who are at risk because of
industrial exposure to toxicant-induced infertility.
Obesity
According to NIH guidelines (see http://www.
nhlbi.nih.gov/health/health-topics/topics/obe/
diagnosis), adults with a body mass index
(BMI) >30 (kg/m
2
) are obese. Based on the latest
statistics in 2014, the obese adult population in the
U.S. is at 27.7% (http://www.gallup.com/poll/
181271/obesity-rate-inches-2014.aspx), which is
double the worlds average of 13% (http://www.
who.int/mediacentre/factsheets/fs311/en/), illus-
trating an alarming trend given the health risks
associated with obesity. A lower sex hormone
binding globulin (SHBG) level is found in men
with obesity and/or diabetes, which in turn lowers
the total testosterone level (see Chap. 16 and
[211]). Emerging evidence suggests a negative
correlation between rising BMI and sperm count,
sperm concentration, and motile sperm, which
impedes male fertility [212214]. Interestingly,
while other studies suggest that obesity has no
major impact on fertility, semen quality,
gonadotropin levels, or other sperm parameters
despite reduced testosterone levels [215,216].
The reason(s) behind the different conclusions is
not immediately known. Nonetheless, studies
have demonstrated possible mechanism(s) by
which obesity might lead to reduced spermato-
genesis capacity and daily sperm output, as well
as reduced sperm count and sperm quality. One
proposed mechanism is hyperestrogenism in
which serum estrogens are considerably higher in
obese men due to an increase in peripheral con-
version of testosterone to estrogens by aromatase
in adipose tissue [217219], coupled with a
reduced testosterone production [214]. The
excessive estrogens in the systemic circulation
thus inhibit the release of LH and FSH from the
pituitary gland via the negative feedback on the
hypothalamus, thereby reducing T production,
intratesticular T levels, and the testosterone/
estrogen ratio. The reduced T and FSH levels
result in suppressed spermatogenesis. However,
the correlation between high levels of estradiol
and obesity is controversial and is further affected
by low SHBG [220], suggesting the need for
additional studies. Nonetheless, this mechanism
is supported by ndings that obese men have
considerably lower levels of inhibin B than
healthy men [221223]. Elevated levels of
cytokines are another potential cause of
hypospermatogenesis in obese men [224]. Other
mechanisms that might cause defects in human
spermatogenesis in obesity include reduced levels
of SHBG, insulin and leptin resistance, sleep
apnea, and adiponectin deciency [225229].
Additionally, factors involved in the pathogenesis
of obesity, such as high calorie diet, genetic, and
epigenetic disorders, might also play a role in
perturbing sperm production [230,231].
Besides the disruptive effects of obesity on
spermatogenesis, there is emerging evidence that
obesity also affects the molecular structure of
testicular germ cells and mature spermatozoa,
such as an impairment of acrosome reaction
[232], leading to altered growth in offspring,
increased susceptibility to disease in adults [233],
and erectile dysfunction [234]. Studies also
suggest that high-fat diets can affect the epige-
netic content of sperm or the endocrine content
3 Human Spermatogenesis and Its Regulation 63
sjwint01@louisville.edu
of seminal uid, which in turn affects early fetal
development [213].
Bariatric surgery [235], such as Roux-en-Y
gastric bypass surgery [236], has been reported to
increase T levels and improve sexual function in
obese men. Much of the increase in T, however,
results from an increase in the level of SHBG
[236]. Interestingly, there are case reports in
which bariatric surgery was followed by
impaired semen parameters, possibly by per-
turbing absorption of vitamins and trace elements
[237,238]. However, one study of six men
showed no disruptive changes of bariatric sur-
gery on semen parameters, coupled with an
increase in urinary total T levels [239].
Summary
Spermatogenesis is a series of cellular events that
take place in the seminiferous tubules of the testis.
In this review, we provide a brief overview of
human spermatogenesis, from spermatogonial
self-renewal via mitosis, meiosis, post-meiotic
spermatid development via spermiogenesis, to
the release of sperm at spermiation. We discuss the
role of Sertoli cell and especially the role of the
BTB in spermatogenesis. We also highlight some
specic areas of research that deserve future
attention. Due to the lack of human testis samples
for analysis, in particular those from normal sub-
jects, the study of human spermatogenesis lags far
behind studies in rodents. However, human Sertoli
cells obtained at biopsy can now be cultured
in vitro and maintained up to weeks and months
where they remain mitotically active. These cells
can be subcultured for creative experiments. We
also briey discuss some emerging elds of
research that focus on factors affecting human
spermatogenesis. These factors may deserve more
attention by investigators in future years.
Acknowledgements The authors wish to thank Drs.
Stephen Winters and Ilpo Huhtaniemi for their helpful
remarks, comments, insightful thoughts, and suggestions
during the preparation of this manuscript for this book.
Without this valuable help, this Chapter could not have
been written.This work was supported by grants from the
National Institutes of Health, R01 HD056034 (to C.Y.C.),
U54 HD029990 Project 5 (to C.Y.C.); National Natural
Science Foundation of China (NSFC) 31371176 (to X.
X.), China Qianjiang Talents Program QJD1502029 (to
X.X.) and Zhejiang Province Department of Science
Technology Funding 2016F10010 (to X.X.)
References
1. Ehmcke J, Schlatt S. A revised model for spermato-
gonial expansion in man: lessons from non-human
primates. Reproduction. 2006;132:67380.
2. Schlatt S, Ehmcke J. Regulation of spermatogene-
sis: an evolutionary biologists perspective. Semin
Cell Dev Biol. 2014;29:216.
3. Neto FT, Bach PV, Najari BB, Li PS, Goldstein M.
Spermatogenesis in humans and its affecting factors.
Semin Cell Dev Biol. 2016.
4. Amann RP. The cycle of the seminiferous epithe-
lium in humans: a need to revisit? J Androl.
2008;29:46987.
5. ODonnell L, Nicholls PK, OBryan MK, McLach-
lan RI, Stanton PG. Spermiation: the process of
sperm release. Spermatogenesis. 2011;1:1435.
6. Amann RP, Howards SS. Daily spermatozoal
production and epididymal spermatozoal reserves
of the human male. J Urol. 1980;124(2):2115.
7. Johnson L, Petty CS, Neaves WB. A comparative
study of daily sperm production and testicular
composition in humans and rats. Biol Reprod.
1980;22(5):123343.
8. Johnson L, Zane RS, Petty CS, Neaves WB.
Quantication of the human Sertoli cell population:
its distribution, relation to germ cell numbers, and
age-related decline. Biol Reprod. 1984;31:78595.
9. Zitzmann M. Effects of age on male fertility. Best
Pract Res Clin Endocrinol Metab. 2013;27:61728.
10. ODonnell L, Robertson KM, Jones ME, Simp-
son ER. Estrogen and spermatogenesis. Endocr
Rev. 2001;22:289318.
11. McLachlan R, ODonnell L, Meachem S, Stanton P,
de Kretser D, Pratis K, et al. Hormonal regulation of
spermatogenesis in primates and man: insights for
development of the male hormonal contraceptive.
J Androl. 2002;23:14962.
12. Carreau S, Hess RA. Oestrogens and spermatoge-
nesis. Philos Trans R Soc Lond B Biol Sci.
2010;365:151735.
13. Sharpe RM. Regulation of spermatogenesis. In: The
Physiology of Reproduction. Eds. Knobil, E., Neill,
J.D. New York, Raven Press. 1994. pp. 1363434.
14. ODonnell L, Meachem SJ, Stanton PG, McLach-
lan RI. Endocrine regulation of spermatogenesis. In:
Neill JD, editor. Physiology of Reproduction. 3rd
ed. Amsterdam: Elsevier; 2006. p. 101769.
15. OShaughnessy PJ. Hormonal control of germ cell
development and spermatogenesis. Semin Cell Dev
Biol. 2014;29:5565.
64 H. Chen et al.
sjwint01@louisville.edu
16. Kilchevsky A, Honig S. Male factor infertility in
2011: semen quality, sperm selection and
hematospermia. Nat Rev Urol. 2012;9:6870.
17. Sharpe RM. Environmental/lifestyle effects on
spermatogenesis. Phil Trans R Soc Lond B Biol
Sci. 2010;365:1697712.
18. Cheng CY, Wong EWP, Lie PPY, Li MWM, Su L,
Siu ER, et al. Environmental toxicants and male
reproductive function. Spermatogenesis. 2011;1:213.
19. Wei Y, Schatten H, Sun QY. Environmental
epigenetic inheritance through gametes and impli-
cations for human reproduction. Hum Reprod
Update. 2015;21(2):194208.
20. Campion S, Catlin N, Heger N, McConndell EV,
Pacheco SE, Saffarini C, et al. Male reprotoxicty
and endocrine disruption. EXS. 2012;101:31560.
21. Wan HT, Mruk DD, Wong CKC, Cheng CY.
Targeting testis-specic proteins to inhibit spermato-
genesis - lesson from endocrine disrupting chemi-
cals. Expert Opin Ther Targets. 2013;17:83955.
22. Geoffroy-Siraudin C, Loundou Ad, Romain F,
Achard V, Courbiere B, Perrard MH, et al. Decline
of semen quality among 10932 males consulting for
couple infertility over a 20-year period in Marseille.
France. Asian J Androl. 2012;14:58490.
23. Jorgensen N, Vierula M, Jacobsen R, Pukkala E,
Perheentupa A, Virtanen HE, et al. Recent adverse
trends in semen quality and testis cancer incidence
among Finnish men. Int J Androl. 2011;34:e3748.
24. Virtanen HE, Sadov S, Vierula M, Toppari J. Finland
is following the trend-sperm quality in Finnish men.
Asian J Androl. 2013;15(2):1624.
25. Handelsman DJ, Cooper TG. Falling sperm counts
and global estrogenic pollution: what have we
learned over 20 years? Asian J Androl. 2013;15
(2):15961.
26. Foresta C, Moro E, Ferlin A. Y chromosome
microdeletions and alterations of spermatogenesis.
Endocr Rev. 2010;22:22639.
27. Greenberg SH, Lipshultz LI, Wein AJ. Experience
with 425 subfertile male patients. J Urol.
1978;119:50710.
28. Krausz C, Escamilla AR, Chianese C. Genetics of
male infertility: from research to clinic. Reproduc-
tion. 2015;150(5):R15974.
29. Jamsai D, OBryan MK. Mouse models in male
fertility research. Asian J Androl. 2011;13:13951.
30. Carrell DT, Aston KI, Oliva R, Emery BR, De
Jonge CJ. The omicsof human male infertility:
integrating big data in a systems biology approach.
Cell Tissue Res. 2016;363(1):295312.
31. Com E, Melaine N, Chalmel F, Pineau C. Pro-
teomics and integrative genomics for unraveling the
mysteries of spermatogenesis: the strategies of a
team. J proteomics. 2014;107:12843.
32. Winters SJ, Troen P. Testosterone and estradiol are
co-secreted episodially by the human testis. J Clin
Invest. 1986;78:8703.
33. Muller J, Skakkebaek NE. Quantication of germ
cells and seminiferous tubules by stereological
examination of testicles of 50 boys who suffered
from sudden death. Int J Androl. 1983;6:14356.
34. Clermont Y. The cycle of the seminiferous epithe-
lium in man. Am J Anat. 1963;112:3551.
35. Dym M. Basement membrane regulation of Sertoli
cells. Endocr Rev. 1994;15:10215.
36. Siu MKY, Cheng CY. Dynamic cross-talk between
cells and the extracellular matrix in the testis.
BioEssays. 2004;26:97892.
37. Mruk DD, Cheng CY. Sertoli-Sertoli and
Sertoli-germ cell interactions and their signicance
in germ cell movement in the seminiferous epithe-
lium during spermatogenesis. Endocr Rev.
2004;25:747806.
38. Cheng CY, Mruk DD. Cell junction dynamics in the
testis: Sertoli-germ cell interactions and male contra-
ceptive development. Physiol Rev. 2002;82:82574.
39. Weber JE, Russell LD, Wong V, Peterson RN.
Three dimensional reconstruction of a rat stage V
Sertoli cell: II. Morphometry of Sertoli-Sertoli and
Sertoli-germ cell relationships. Am J Anat.
1983;167:16379.
40. Sharpe RM, McKinnell C, Kivlin C, Fisher JS.
Proliferation and functional maturationof Sertoli cells,
and their relevance to disorders of testis function in
adulthood. Reproduction. 2003;125:76984.
41. Auharek SA, Avelar GF, Lara NLM, Sharpe RM,
Franca LR. Sertoli cell numbers and spermatogenic
efciency are increased in inducible nitric oxide
synthase (iNOS) mutant-mice. Int J Androl.
2011;34:e6219.
42. Berndtson WE, Thompson TL. Changing relation-
ships between testis size, Sertoli cell number and
spermatogenesis in Sprague-Dawley rats. J Androl.
1990;11:42935.
43. Wing TY, Christensen AK. Morphometric studies
on rat seminiferous tubules. Am J Anat.
1982;165:1325.
44. Wang ZX, Wreford NG, de Kretser DM. Determi-
nation of Sertoli cell numbers in the developing rat
testis by sterological methods. Int J Androl.
1989;12:5864.
45. Johnson L, Petty CS, Neaves WB. A comparative
study of daily sperm production and testicular
composition in humans and rats. Biol Reprod.
1980;22:123343.
46. Amann RP, Howards SS. Daily spermatozoal
production and epididymal spermatozoal reserves
of the human male. J Urol. 1980;124:2115.
47. Turner TT, Jones CC, Howards SS, Ewing LL,
Zegeye B, Gunsalus GL. On the androgen microen-
vironment of maturing spermatozoa. Endocrinol-
ogy. 1984;115:192532.
48. Jarow JP, Chen H, Rosner W, Trentacoste S,
Zirkin BR. Assessment of the androgen environ-
ment within the human testis: Minimally invasive
method to obtain intratesticular uid. J Androl.
2001;22:6405.
49. Roth MY, Page ST, Lin K, Anawalt BD, Mat-
sumoto AM, Synder CN, et al. Dose-dependent
3 Human Spermatogenesis and Its Regulation 65
sjwint01@louisville.edu
increase in intratesticular testosterone by very
low-dose human chorionic gonadotropin in normal
men with experimental gonadotropin deciency.
J Clin Endocrinol Metab. 2010;95:380613.
50. Takahashi J, Higashi Y, Lanasa JA, Winters SJ,
Oshima H, Troen P. Studies of the human testis.
XVII. Gonadotropin regulation of intratesticular
testosterone and estradiol in infertile men. J Clin
Endocrinol Metab. 1982;55:107380.
51. Cheng CY, Mruk DD. The blood-testis barrier and
its implications for male contraception. Pharmacol
Rev. 2012;64:1664.
52. Pelletier RM. The blood-testis barrier: the junctional
permeability, the proteins and the lipids. Prog
Histochem Cytochem. 2011;46:49127.
53. Cheng YH, Wong EWP, Cheng CY. Cancer/testis
(CT) antigens, carcinogenesis and spermatogenesis.
Spermatogenesis. 2011;1:20920.
54. Yule TD, Montoya GD, Russell LD, Williams TM,
Tung KSK. Autoantigenic germ cells exist outside the
blood testis barrier. J Immunol. 1988;141:11617.
55. Meinhardt A, Hedger MP. Immunological, para-
crine and endocrine aspects of testicular immune
privilege. Mol Cell Endocrinol. 2011;335:608.
56. Chui K, Trivedi A, Cheng CY, Cherbavaz DB,
Dazin PF, Huynh ALT, et al. Characterization and
functionality of proliferative human Sertoli cells.
Cell Transplant. 2011;20:61935.
57. Xiao X, Mruk DD, Tang EI, Wong CKC, Lee WM,
John CM, et al. Environmental toxicants perturb
human Serotli cell adhesive function via changes in
F-actin organization medicated by actin regulatory
proteins. Hum Reprod. 2014;29:127991.
58. ODonnell L, Stanton P, Bartles J, Robertson D.
Sertoli cell ectoplasmic specializations in the sem-
iniferous epithelium of the testosterone-suppressed
adult rat. Biol Reprod. 2000;63:99108.
59. Meng J, Holdcraft RW, Shima JE, Griswold MD,
Braun RE. Androgens regulate the permeability of
the blood-testis barrier. Proc Natl Acad Sci USA.
2005;102:16696700.
60. Chung NPY, Cheng CY. Is cadmium
chloride-induced inter-Sertoli tight junction perme-
ability barrier disruption a suitable in vitro model to
study the events of junction disassembly during
spermatogenesis in the rat testis? Endocrinology.
2001;142:187888.
61. Xiao X, Mruk DD, Lee WM, Cheng CY. c-Yes
regulates cell adhesion at the blood-testis barrier
and the apical ectoplasmic specialization in the
seminiferous epithelium of rat testes. Int J Biochem
Cell Biol. 2011;43:65165.
62. Ilani N, Armanious N, Lue YH, Swerdloff RS,
Baravarian S, Adler A, et al. Integrity of the
blood-testis barrier in healthy men after suppression
of spermatogenesis with testosterone and levonor-
gestrel. Hum Reprod. 2012;27(12):340311.
63. McCabe MJ, Tarulli GA, Laven-Law G, Matthies-
son KL, Meachem SJ, McLachlan RI, et al. Gon-
adotropin suppression in men leads to a reduction in
claudin-11 at the Sertoli cell tight junction. Hum
Reprod. 2016;31(4):87586.
64. Robillard KR, Hoque MT, Bendayan R. Expression
of ATP-binding cassette membrane transporters in
rodent and human Sertoli cells: relevance to the
permeability of antiretroviral therapy at the
blood-testis barrier. J Pharmacol Exp Ther.
2012;340:96108.
65. Hoque MT, Kis O, De Rosa MF, Bendayan R.
Raltegravir permeability across blood-tissue barriers
and the potential role of drug efux transporters.
Antimicrob Agents Chemother. 2015;59(5):257282.
66. Jesus TT, Oliveira PF, Silva J, Barros A, Ferreira R,
Sousa M, et al. Mammalian target of rapamycin
controls glucose consumption and redox balance in
human Sertoli cells. Fertil Steril. 2016;105(3):
82533 e3.
67. Bardin CW, Gunsalus GL, Cheng CY. The cell
biology of the Sertoli cell. In: Desjardins C,
Ewing L, editors. Cell and molecular biology of
the testis. New York: Oxford University Press;
1993. p. 189219.
68. Cheng CY, Mruk DD. Biochemistry of Sertoli
cell/germ cell junctions, germ cell transport, and
spermiation in the seminiferous epithelium. In:
Griswold MD, editor. Sertoli Cell Biology. 2nd
ed. Amsterdam: Elsevier; pp; 2015. p. 33383.
69. de Rooij DG. The spermatogonial stem cell niche.
Microsc Res Tech. 2009;72:5805.
70. Phillips BT, Gassei K, Orwig KE. Spermatogonial
stem cell regulation and spermatogenesis. Philos
Trans R Soc Lond B Biol Sci. 2010;365:166378.
71. Zwain I, Morris PL, Cheng CY. Identication of an
inhibitory factor from a Sertoli clonal cell line
(TM4) that modulates adult rat Leydig cell steroido-
genesis. Mol Cell Endocrinol. 1991;80:11526.
72. Zwain IH, Cheng CY. Rat seminiferous tubular
culture medium contains a biological factor that
inhibits Leydig cell steroidogenesis: its purication
and mechanism of action. Mol Cell Endocrinol.
1994;104:21327.
73. Saez JM, Avallet O, Naville D, Perrard-Sapori MH,
Chatelain PG. Sertoli-Leydig cell communications.
Ann N Y Acad Sci. 1989;564:21031.
74. Tio S, Koppenaal D, Bardin C, Cheng C. Purica-
tion of gonadotropin surge inhibiting factor from
Sertoli cell-enriched culture medium. Biochem
Biophys Res Commun. 1994;199:122936.
75. Rebourcet D, OShaughnessy PJ, Monteiro A,
Milne L, Cruickshanks L, Jeffrey N, et al. Sertoli
cells maintain Leydig cell number and peritubular
myoid cell activity in the adult mouse testis.
PLoS ONE. 2014;9(8):e105687.
76. Rebourcet D, OShaughnessy PJ, Pitetti JL, Mon-
teiro A, OHara L, Milne L, et al. Sertoli cells
control peritubular myoid cell fate and support adult
Leydig cell development in the prepubertal testis.
Development. 2014;141(10):213949.
77. Wen Q, Zheng QS, Li XX, Hu ZY, Gao F,
Cheng CY, et al. Wt1 dictates the fate of fetal and
66 H. Chen et al.
sjwint01@louisville.edu
adult Leydig cells during development in the mouse
testis. Am J Physiol Endocrinol Metab. 2014;307
(12):E113143.
78. Jegou B. The Sertoli-germ cell communication
network in mammals. Int Rev Cytol. 1993;147:
2596.
79. Griswold M. Interactions between germ cells and
Sertoli cells in the testis. Biol Reprod. 1995;52:
2116.
80. Skinner M, Tung P, Fritz I. Cooperativity between
Sertoli cells and testicular peritubular cells in the
production and deposition of extracellular matrix
components. J Cell Biol. 1985;100:19417.
81. Skinner MK, Fetterolf PM, Anthony CT. Purica-
tion of a paracrine factor, P-Mod-S, produced by
testicular peritubular cells that modulates Sertoli
cell function. J Biol Chem. 1988;263:288490.
82. Cheng CY, Grima J, Stahler MS, Guglielmotti A,
Silvestrini B, Bardin CW. Sertoli cell synthesizes
and secretes a protease inhibitor, a
2
-macroglobulin.
Biochemistry. 1990;29:10638.
83. Zwain IH, Grima J, Stahler MS, Saso L, Cailleau J,
Verhoeven G, et al. Regulation of Sertoli cell a
2
-
macroglobulin and clusterin (SGP-2) secretion by
peritubular myoid cells. Biol Reprod. 1993;48:
1807.
84. Mruk DD, Cheng CY. An in vitro system to study
Sertoli cell blood-testis barrier dynamics. Methods
Mol Biol. 2011;763:23752.
85. Welsh M, Saunders PT, Atanassova N, Sharpe RM,
Smith LB. Androgen action via testicular peritubu-
lar myoid cells is essential for male fertility.
FASEB J. 2009;23:421830.
86. Rebourcet D, Wu J, Cruickshanks L, Smith SE,
Milne L, Fernando A, et al. Sertoli Cells Modulate
Testicular Vascular Network Development, Struc-
ture, and Function to Inuence Circulating Testos-
terone Concentrations in Adult Male Mice.
Endocrinology. 2016;157(6):247988.
87. Allen E. Studies on cell division in the albino rat.
J Morphol. 1918;31:13385.
88. Clermont Y. Kinetics of spermatogenesis in mam-
mals: seminiferous epithelium cycle and spermato-
gonial renewal. Physiol Rev. 1972;52(1):198236.
89. Clermont Y. Renewal of spermatogonia in man.
Am J Anat. 1966;118(2):50924.
90. Schulze C. Morphological characteristics of the
spermatogonial stem cells in man. Cell Tissue Res.
1979;198(2):1919.
91. Boitani C, Di Persio S, Esposito V, Vicini E.
Spermatogonial cells: mouse, monkey and man
comparison. Semin Cell Dev Biol. 2016.
92. Sandlow JI, Feng HL, Cohen MB, Sandra A.
Expression of c-KIT and its ligand, stem cell factor,
in normal and subfertile human testicular tissue.
J Androl. 1996;17(4):4038.
93. Unni SK, Modi DN, Pathak SG, Dhabalia JV,
Bhartiya D. Stage-specic localization and expres-
sion of c-kit in the adult human testis. J Histochem
Cytochem. 2009;57(9):8619.
94. Medrano JV, Marques-Mari AI, Aguilar CE,
Riboldi M, Garrido N, Martinez-Romero A, et al.
Comparative analysis of the germ cell markers
c-KIT, SSEA-1 and VASA in testicular biopsies
from secretory and obstructive azoospermias. Mol
Hum Reprod. 2010;16(11):8117.
95. Gassei K, Ehmcke J, Dhir R, Schlatt S. Magnetic
activated cell sorting allows isolation of spermato-
gonia from adult primate testes and reveals distinct
CFRa1-positive subpopulations in men. J Med
Primatol. 2010;39:8391.
96. Feng HL, Sandlow JI, Sparks AE, Sandra A,
Zheng LJ. Decreased expression of the c-kit recep-
tor is associated with increased apoptosis in
subfertile human testes. Fertil Steril. 1999;71
(1):859.
97. Hogarth CA, Griswold MD. The key role of vitamin
A in spermatogenesis. J Clin Invest. 2010;120:
95662.
98. Chung SS, Wang X, Roberts SS, Griffey SM,
Reczek PR, Wolgemuth DJ. Oral administration of
a retinoic acid receptor antagonist reversibly inhibits
spermatogenesis in mice. Endocrinology. 2011;152:
2492502.
99. Chung SS, Wang X, Wolgemuth DJ. Prolonged
Oral Administration of a Pan-Retinoic Acid Recep-
tor Antagonist Inhibits Spermatogenesis in Mice
With a Rapid Recovery and Changes in the
Expression of Inux and Efux Transporters.
Endocrinology. 2016;157(4):160112.
100. Amory JK, Muller CH, Shimshoni JA, Isoherra-
nen N, Paik J, Moreb JS, et al. Suppression of
spermatogenesis by bisdichloroacetyldiamines is
mediated by inhibition of testicular retinoic acid
biosynthesis. J Androl. 2011;32(1):1119.
101. Paik J, Haenisch M, Muller CH, Goldstein AS,
Arnold S, Isoherranen N, et al. Inhibition of retinoic
acid biosynthesis by the bisdichloroacetyldiamine
WIN 18,446 markedly suppresses spermatogenesis
and alters retinoid metabolism in mice. J Biol
Chem. 2014;289(21):1510417.
102. Heller CG, Moore DJ, Paulsen CA. Suppression of
spermatogenesis and chronic toxicity in men by a
new series of bis(dichloroacetyl) diamines. Toxicol
Appl Pharmacol. 1961;3:111.
103. Drobeck HP, Coulston F. Inhibition and recovery of
spermatogenesis in rats, monkeys, and dogs med-
icated with bis(dichloroacetyl) diamines. Exp Mol
Pathol. 1962;1:25174.
104. Plant TM. Undifferentiated primate spermatogonia
and their endocrine control. Trends Endocrinol
Metab. 2010;21:48895.
105. van Alphen MM, van de Kant HJ, de Rooij DG.
Follicle-stimulating hormone stimulates spermato-
genesis in the adult monkey. Endocrinology.
1988;123:144955.
106. Simorangkir DR, Ramaswarmy S, Marshall GR,
Pohl CR, Plant TM. A selective monotropic eleva-
tion of FSH but not that of LH, amplies the
proliferation and differentiation of spermatogonia in
3 Human Spermatogenesis and Its Regulation 67
sjwint01@louisville.edu
the adult rhesus monkey (Macaca mulatta). Hum
Reprod. 2009;24:158495.
107. Selice R, Ferlin A, Garolla A, Caretta N, Foresta C.
Effects of endogenous FSH on normal human
spermatogenesis in adults. Int J Androl. 2011;34:
e5117.
108. Ruwanpura SM, McLachlan RI, Matthiesson KL,
Meachem SJ. Gonadotrophins regulate germ cell
survival, not proliferation, in normal adult men.
Hum Reprod. 2008;23:40311.
109. Schulze W, Rehder U. Organization and morpho-
genesis of the human seminiferous epithelium. Cell
Tissue Res. 1984;237(3):395407.
110. Feng CW, Bowles J, Koopman P. Control of
mammalian germ cell entry into meiosis. Mol Cell
Endocrinol. 2014;382(1):48897.
111. Boussouar F, Benahmed M. Lactate and energy
metabolism in male germ cells. Trends Endocrinol
Metab. 2004;15:34550.
112. Dias TR, Alves MG, Silva BM, Oliveira BF. Sperm
glucose transport and metabolism in diabetic indi-
viduals. Mol Cell Endocrinol. 2014;396:3745.
113. Oliveira PF, Martins AD, Moreira AC, Cheng CY,
Alves MG. The Warburg effect revisited - lession
from the Sertoli cell. Med Res Rev. 2015;35:
12651.
114. Oliveira BF, Alves MG, Rato L, Silva J, Sa R,
Barros A, et al. Inuence of 5a-dihydrotestosterone
and 17b-estradiol on human Sertoli cells metabo-
lism. Int J Androl. 2011;34:e61220.
115. McLachlan RI, Rajpert-De Meyts E, Hoei-Hansen
CE, de Kretser DM, Skakkebaek NE. Histological
evaluation of the human testisApproaches to
optimizing the clinical value of the assessment:
Mini Review. Hum Reprod. 2007;22:216.
116. Clermont Y, Leblond CP. Spermiogenesis of man,
monkey, ram and other mammals as shown by the
periodic acid-Schiff technique. Am J Anat. 1955;96
(2):22953.
117. de Kretser DM. Ultrastructural features of human
spermiogenesis. Z Zellforsch Mikrosk Anat.
1969;98(4):477505.
118. Clermont Y, Morales C, Hermo L. Endocytic
activities of Sertoli cells in the rat. Ann NY Acad
Sci. 1987;513:115.
119. Cheng CY, Mruk DD. Biochemistry of Sertoli
cell/germ cell junctions, germ cell transport, and
spermiation in the seminiferous epithelium. In:
Sertoli Cell Biology, 2nd Edition. Ed. Griswold,
M.D., Amsterdam, Elsevier; pp. 333383. doi: 10.
1016/B978-0-12-417047-6.00012.0. 2015.
120. Roosen-Runge EC. Kinetics of spermatogenesis in
mammals. Ann N Y Acad Sci. 1952;55(4):57484.
121. Leblond CP, Clermont Y. Denition of the stages of
the cycle of the seminiferous epithelium in the rat.
Ann N Y Acad Sci. 1952;55(4):54873.
122. Hess RA, de Franca LR. Spermatogenesis and cycle
of the seminiferous epithelium. Adv Exp Med Biol.
2008;636:115.
123. Xiao X, Mruk DD, Wong CK, Cheng CY. Germ
cell transport across the seminiferous epithelium
during spermatogenesis. Physiology (Bethesda).
2014;29(4):28698.
124. Heller CG, Clermont Y. Spermatogenesis in man:
an estimate of its duration. Science. 1963;140
(3563):1846.
125. Oakberg EF. Duration of spermatogenesis in the
mouse and timing of stages of the cycle of the
seminiferous epithelium. Am J Anat. 1956;99
(3):50716.
126. Clermont Y, Leblond CP, Messier B. Duration of
the cycle of the seminal epithelium of the rat. Arch
Anat Microsc Morphol Exp. 1959;48(Suppl):3755.
127. Ehmcke J, Wistuba J, Schlatt S. Spermatogonial
stem cells: questions, models and perspectives.
Human Reprod Update. 2006;12:27582.
128. de Kretser DM, Kerr JB. The cytology of the testis.
In: Knobil E, Neill JB, Ewing LL, Greenwald GS,
Markert CL, Pfaff DW, editors. The Physiology of
Reproduction, vol. 1. New York: Raven Press;
1988. p. 837932.
129. McLachlan RI, ODonnell L, Meachem SJ, Stan-
ton PG, De Kretser DM, Pratis K, et al. Identica-
tion of specic sites of hormonal regulation in
spermatogenesis in rats, monkeys, and man. Recent
Prog Horm Res. 2002;57:14979.
130. Handelsman DJ. Hormonal regulation of spermato-
genesis: insights from constructing genetic models.
Reprod Fertil Dev. 2011;23:50719.
131. Itman CM, Mendis SH, Barakat B, Loveland KL.
All in the family: TGF-b family action in testis
development. Reproduction. 2006;132:23346.
132. Foresta C, Selice R, Garolla A, Ferlin A.
Follicle-stimulating hormone treatment of male
infertility. Curr Opin Urol. 2008;18:6027.
133. De Kretser DM, Hedger MP, Loveland KL,
Phillips DJ. Inhibins, activins and follistatin in
reproduction. Hum Reprod Update. 2002;8:52941.
134. Winters SJ, Moore JP. Paracrine control of gonado-
trophs. Semin Reprod Med. 2007;25:37987.
135. Winters SJ, Moore JP. Intra-pituitary regulation of
gonadotrophs in male rodents and primates. Repro-
duction. 2004;128:1323.
136. McLachlan R, Wreford N, ODonnell L, de Kret-
ser D, Robertson D. The endocrine regulation of
spermatogenesis: independent roles for testosterone
and FSH. J Endocr. 1996;148:19.
137. Huhtaniemi I. A short evolutionary history of
FSH- = stimulated spermatogenesis. Hormones
(Athens). 2015;14:46878.
138. Stanton PG. Regulation of the blood-testis barrier.
Semin Cell Dev Biol (in press; PMID:27353840;
DOI:101016/jsemcdb201606018). 2016.
139. Griswold MD, Heckert L, Linder C. The molecular
biology of the FSH receptor. J Steroid Biochem
Mol Biol. 1995;53:2158.
140. Kumar TR, Wang Y, Lu N, Matzuk MM. Follicle
stimulating hormone is required for ovarian follicle
68 H. Chen et al.
sjwint01@louisville.edu
maturation but not male fertility. Nat Genet.
1997;15:2014.
141. Kumar TR. FSHbeta knockout mouse model: a
decade ago and into the future. Endocrine. 2009;36
(1):15.
142. Plant TM, Marshall GR. The functional signicance
of SH in spermatogenesis and the control of its
secretion in male primates. Endocr Rev.
2001;22:76486.
143. Walker WH, Cheng J. FSH and testosterone
signaling in Sertoli cells. Reproduction.
2005;130:1528.
144. OShaughnessy PJ, Johnston H, Willerton L,
Baker PJ. Failure of normal adult Leydig cell
development in androgen-receptor-decient mice.
J Cell Sci. 2002;115:34916.
145. Caroppo E, Niederberger C, Vizziello GM, DAmato
G. Recombinant human follicle-stimulating hormone
as a pretreatment for iodiopathic oligoasthenoterato-
zoospermic patients undergoing intracytoplasmic
sperm injection. Fertil Sertil. 2003;80:1398403.
146. Santi D, Granata AR, Simoni M. Follicle-
stimulating hormone treatement of male idiopathic
infertility improves pregancy rate: a meta-analysis.
Endocr Connect. 2015;4:4658.
147. Saez JM. Leydig cells: endocrine, paracrine, and
autocrine regulation. Endocr Rev. 1994;15:574626.
148. Ascoli M, Fanelli F, Segaloff DL. The lutropin/
choriogonadotropin receptor, a 2002 perspective.
Endocr Rev. 2002;23:14174.
149. Themmen APN, Huhtaniemi IT. Mutations of
gonadotropins and gonadotropin receptors: Eluci-
dating the physiology and pathophysiology of
pituitary-gonadal function. Endocr Rev. 2000;21:
55183.
150. Latronico AC, Segaloff DL. Naturally occurring
mutations of the luteinizing-hormone receptor:
Lessons learned about rerproductive physiology
and G protein-coupled receptors. Am J Human
Genet. 1999;65:94958.
151. Latronico AC, Arnhold IJ. Inactivating mutations of
the human luteinizing hormone receptor in both
sexes. Semin Reprod Med. 2012;30:3826.
152. Winters SJ. Endocrine evaluatiojn of testicular
function. Endocrinol Metab Clin North Am.
1994;23:70923.
153. Scott HM, Mason JI, Sharpe RM. Steroidogenesis
in the fetal testis and its susceptibility to disruption
by exogenous compounds. Endocr Rev. 2009;30
(7):883925.
154. Jarow JP, Wright WW, Brown TR, Yan X,
Zirkin BR. Bioactivity of androgens within the
testes and serum of normal men. J Androl. 2005;26
(3):3438.
155. Cheng CY, Gunsalus GL, Morris ID, Turner TT,
Bardin CW. The heterogeneity of rat androgen
binding protein (rABP) in the vascular compartment
differs from that in the testicular tubular lumen:
further evidence for bidirectional secretion of
rABP. J Androl. 1986;7:1759.
156. Wang RS, Yeh S, Tzeng CR, Chang C. Androgen
receptor roles in spermatogenesis and fertility:
lessons from testicular cell-specic androgen recep-
tor knockout mice. Endocr Rev. 2009;30:11932.
157. Nieschlag E, Wickings EJ, Mauss J. Endocrine
testicular function in vivo and in vitro in infertile
men. Acta Endocrinol. 1979;90:54451.
158. Roth MY, Lin K, Amory JK, Matsumoto AM,
Anawalt BD, Snyder CN, et al. Serum LH correlates
highly with intratesticular steroid levels in normal
men. J Androl. 2009;31:13845.
159. Oduwole OO, Vydra N, Wood NE, Samanta L,
Owen L, Keevil B, et al. Overlapping dose
responses of spermatogenic and extragonadal
testosterone actions jeopardize the principle of
hormonal male contraception.
FASEB J. 2014;28:256676.
160. Shinjo E, Shiraishi K, Matsuyama H. The effect of
human chorionic gonadotropin-based hormonal
therapy on intratesticular testosterone levels and
spermatogonial DNA synthesis in men with
non-obstructive azoospermia. Andrology. 2013;1
(6):92935.
161. Yan HHN, Mruk DD, Lee WM, Cheng CY.
Blood-testis barrier dynamics are regulated by
testosterone and cytokines via their differential
effects on the kinetics of protein endocytosis and
recycling in Sertoli cells. FASEB J. 2008;22:
194559.
162. Xiao X, Wong EWP, Lie PPY, Mruk DD,
Wong CKC, Cheng CY. Cytokines, polarity pro-
teins and endosomal protein trafcking and signal-
ingthe Sertoli cell blood-testis barrier in vitro as a
study model. Methods Enzymol. 2014;534:18194.
163. Li MWM, Mruk DD, Lee WM, Cheng CY.
Cytokines and junction restructuring events during
spermatogenesis in the testis: An emerging concept
of regulation. Cytokine Growth Factor Rev.
2009;20:32938.
164. Smith LB, Walker WH. The regulation of sper-
matogenesis by androgens. Semin Cell Dev Biol.
2014;30:213.
165. Pihlajamaa P, Sahu B, Janne OA. Determinants of
receptor- and tissue-specic actions in androgen
signaling. Endocr Rev. 2015;36(4):35784.
166. Walker WH. Non-classical actions of testosterone
and spermatogenesis. Philos Trans R Soc Lond B
Biol Sci. 2010;365:155769.
167. Kato Y, Shiraishi K, Matsuyama H. Expression of
testicular androgen receptor in non-obstructive
azoospermia and its change after hormonal therapy.
Andrology. 2014;2:73440.
168. Griswold M. The central role of Sertoli cells in
spermatogenesis. Semin Cell Dev Biol.
1998;9:4116.
169. De Gendt K, Swinnen J, Saunders P, Schoonjans L,
Dewerchin M, Devos A, et al. A Sertoli
cell-selective knockout of the androgen receptor
causes spermatogenic arrest in meiosis. Proc Natl
Acad Sci USA. 2004;101:132732.
3 Human Spermatogenesis and Its Regulation 69
sjwint01@louisville.edu
170. Chang C, Chen Y, Yeh S, Xu D, Wang R, Guillou F,
et al. Infertility with defective spermatogenesis and
hypotestosteronemia in male mice lacking the
androgen receptor in Sertoli cells. Proc Natl Acad
Sci USA. 2004;101:687681.
171. Holdcraft RW, Braun RE. Androgen receptor
function is required in Sertoli cells for the terminal
differentiation of haploid spermatids. Development.
2004;131:45967.
172. La Spada AR, Wilson EM, Lubahn DB, Hard-
ing AE, Fischbeck KH. Androgen receptor gene
mutations in X-linked spinal and bulbar muscular
atrophy. Nature. 1991;352:779.
173. Delli Muti N, Agarwal A, Buldreghini E, Gioia A,
Lenzi A, Boscaro M, et al. Have androgen receptor
gene CAG and GGC repeat polymorphisms an
effect on sperm motility in infertile men? Androlo-
gia. 2014;46(5):5649.
174. Fietz D, Geyer J, Kliesch S, Gromoll J,
Bergmann M. Evaluation of CAG repeat length of
androgen receptor expressing cells in human testes
showing different pictures of spermatogenic impair-
ment. Histochem Cell Biol. 2011;136:68997.
175. Giagulli VA, Carbone MD, De Pergola G, Guasta-
macchia E, Resta F, Licchelli B, et al. Could
androgen receptor gene CAG tract polymorphism
affect spermatogenesis in men with idiopathic
infertility? J Assit Reprod Genet. 2014;31:68997.
176. Casella R, Maduro MR, Misfud A, Lipshultz LI,
Yong EL, Lamb DJ. Androgen receptor gene
polyglutamine length is associated with testicular
histology in infertile patients. J Urol.
2003;169:2247.
177. Chen YH, Xu HY, Wang ZY, Zhu ZH, Li CD,
Wu ZG, et al. An insertion mutation in the androgen
receptor gene in a patient with azoospermia. Asian J
Androl. 2015;17(5):8578.
178. Tordjman KM, Yaron M, Berkovitz A, Botchan A,
Sultan C, Lumbroso S. Fertility after high-dose
testosterone and intracytoplasmic sperm injection in
a patient with androgen insensitivity syndrome with
a previously unreported androgen receptor muta-
tion. Andrologia. 2014;46:7036.
179. Xu HY, Li CD, Tang LL, Wang LL, Yu X, Gu XM,
et al. An infertile man with gynecomastia caused by
a novel mutation of the androgen receptor gene.
Asian J Androl. 2015;17(3):50910.
180. Petroli RJ, Hiort O, Struve D, Maciel-Guerra AT,
Guerra-Junior G, Palandi de Mello M, et al.
Preserved fertility in a patient with gyknecomastia
associated with the p.Pro695Ser mutation in the
androgen receptor. Sex Dev. 2014;8:3505.
181. Carreau S, Wolczynski S, Galeraud-Denis I. Aro-
matase, estrogens and human male reproduction. Phil
Trans R Soc Lond B Biol Sci. 2010;365:15719.
182. Hess RA. Estrogen in the adult male reproductive
tract: A review. Reprod Biol Endocrinol. 2003;1
(52):114.
183. Zhou Q, Nie R, Prins GS, Saunders PTK, Katzenel-
lenbogen BS, Hess RA. Localization of androgen
and estrogen receptors in adult male mouse repro-
ductive tract. J Androl. 2002;23:87081.
184. Kotula-Balak M, Gancarczyk M, Sadowska J,
Bilinska B. The expression of aromatase, estrogen
receptor a and estrogen receptor b in mouse Leydig
cells in vitro that derived from crytorchid males.
Eur J Histochem. 2005;49:5962.
185. Lucas TF, Siu ER, Esteves CA, Monteiro HP,
Oliverira CA, Porto CS, et al. 17b-estradiol induces
the translocation of the estrogen receptors ESR1 and
ESR2 to the cell membrane, MAPK3/1 phosphory-
lation and proliferation of cultured immature rat
Sertoli cells. Biol Reprod. 2008;78:10114.
186. Cheng CY, Boettcher B, Rose RJ, Kay DJ, Tin-
neberg HR. The binding of sex steroids to human
spermatozoa. An autoradiographic study. Int J
Androl. 1981;4:117.
187. Durkee TJ, Mueller M, Zinaman M. Identication
of estrogen receptor protein and messenger ribonu-
celic acid in human spermatozoa. Am J Obstet
Gynecol. 1998;178:128897.
188. Fietz D, Bergmann M, Hartmann K. In situ
hybridization of estrogen receptors a and b and
GPER in the human testis. Methods Mol Biol.
2016;1366:189205.
189. Pelletier G, El-Alfy M. Immunocytochemical local-
ization of estrogen receptors a and b in the human
reproductive organs. J Clin Endocrinol Metab.
2000;85:483540.
190. Khattri A, Pandey RK, Gupta NJ, Chakravarty B,
Deenadayal M, Singh L, et al. Estrogen receptor
beta gene mutations in Indian infertile men. Mol
Hum Reprod. 2009;15(8):51320.
191. Carani C, Rochira V, Faustini-Fustini M, Balestri-
eri A, Granata AR. Roel of oestrogen in male sexual
behaviour: Insights from the natural model of
aromatase deciency. Clin Endocrinol. 1999;51:
51724.
192. Finkelstein JS, Yu EW, Burnett-Bowie SA. Gon-
adal steroids and body composition, strength, and
sexual function in men. N Engl J Med. 2013;369:
2457.
193. Sharpe RM. Hormones and testis development and
the possible adverse effects of environmental
chemicals. Toxicol Lett. 2001;120:22132.
194. Stahl PJ, Stember DS, Goldstein M. Contemporary
management of male infertility. Annu Rev Med.
2012;63:52540.
195. Song R, Hennig GW, Wu Q, Jose C, Zheng H,
Yan W. Male germ cells express abundant endoge-
nous siRNAs. Proc Natl Acad Sci USA. 2011;108:
13159131564.
196. Song R, Ro S, Michaels JD, Park C, McCarrey JR,
Yan W. Many X-linked microRNAs escape meiotic
sex chromosome inactivation. Nat Genet.
2009;41:48893.
197. Holt JE, Stanger SJ, Nixon B, McLaughlin EA.
Non-coding RNA in spermatogenesis and epididy-
mal maturation. Adv Exp Med Biol. 2016;886:95
120.
70 H. Chen et al.
sjwint01@louisville.edu
198. de Mateo S, Sassone-Corsi P. Regulation of sper-
matogenesis by small non-coding RNAs: role of the
germ granule. Semin Cell Dev Biol. 2014;29:84
92.
199. Inui M, Martello G, Piccolo S. MicroRNA control
of signal transduction. Nat Rev Mol Cell Biol.
2010;11:25263.
200. Eisenberg I, Kotaja N, Goldman-Wohl D, Imbar T.
microRNA in Human Reproduction. Adv Exp Med
Biol. 2015;888:35387.
201. Bartel DP. MicroRNAs: genomics, biogenesis,
mechanism, and function. Cell. 2004;116:28197.
202. Brodersen P, Voinnet O. Revisiting the principles of
microRNA target recognition and mode of action.
Nat Rev Mol Cell Biol. 2009;10:1418.
203. Meikar O, Da Ros M, Korhonen H, Kataja N.
Chromatoid body and small RNAs in male germ
cells. Reproduction. 2011;142:195209.
204. Yadav RP, Kotaja N. Small RNAs in spermatoge-
nesis. Mol Cell Endocrinol. 2014;382(1):498508.
205. Lian J, Zhang X, Tian H, Liang N, Wang Y,
Liang C, et al. Altered microRNA expression in
patients with non-obstructive azoospermia. Reprod
Biol Endocrinol. 2009;7:13.
206. Wu W, Qin Y, Li Z, Dong J, Dai J, Lu C, et al.
Genome-wide microRNA expression proling in
idiopathic non-obstructive azoospermia: signicant
up-regulation of miR-141, miR-429 and
miR-7-1-3p. Hum Reprod. 2013;28(7):182736.
207. Wang C, Yang C, Chen X, Yao B, Yang C, Zhu C,
et al. Altered prole of seminal plasma microRNAs
in the molecular diagnosis of male infertility. Clin
Chem. 2011;57(12):172231.
208. Korhonen HM, Meikar O, Yadav RP, Papaioan-
nou MD, Romero Y, Da Ros M, et al. Dicer is
required for haploid male germ cell differentiation
in mice. PLoS ONE. 2011;6(9):e24821.
209. Korhonen HM, Yadav RP, Da Ros M, Chalmel F,
Zimmermann C, Toppari J, et al. DICER regulates
the formation and maintenance of cell-cell Junctions
in the mouse seminiferous epithelium. Biol Reprod.
2015;93(6):139.
210. Papaioannou MD, Pitetti JL, Ro S, Park C, Aubry F,
Schaad O, et al. Sertoli cell Dicer is essential for
spermatogenesis in mice. Dev Biol. 2009;326
(1):2509.
211. Winters SJ. Monitoring testosterone levels in
testosterone-treated men. Curr Med Res Opin.
2016;32(2):2712.
212. Kiess W, Wagner IV, Kratzsch J, Korner A. Male
obesity. Endocrinol Metab Clin North Am.
2015;44:76172.
213. Chambers TJG, Anderson RA. The impact of
obesity on male fertility. Hormones. 2015;14:5638.
214. Stokes VJ, Anderson RA, George JT. How does
obesity affect fertility in menand what are the
treatment options? Clin Endocrinol. 2015;82
(5):6338.
215. Teerds KJ, de Rooij DG, Keijer J. Functional
relationship between obesity and male reproduction:
from humans to animal models. Hum Reprod
Update. 2011;17(5):66783.
216. Ramlau-Hansen CH, Hansen M, Jensen CR,
Olsen JC, Bonde JP, Thulstrup AM. Semen quality
and reproductive hormones according to birth-
weight and body mass index in childhood and adult
lief: two decades of follow-up. Fertil Sertil.
2010;94:6108.
217. de Boer H, Verschoor L, Ruinemans-Koerts J,
Jansen M. Letrozole normalizes serum testosterone
in severely obese men with hypogonadotropic
hypogonadism. Diabetes Obesity Metabol.
2005;7:2115.
218. Schneider G, Kirschner MA, Berkowitz R,
Ertel NH. Increased estrogen production in obese
men. J Clin Endocrinol Metab. 1979;48:6338.
219. Ghosh S, Ashcraft K, Jahid MJ, April C, Gha-
jar CM, Ruan J, et al. Regulation of adipose
oestrogen output by mechanical stress. Nat Com-
mun. 2013;4:1821.
220. Dhindsa S, Batra M, Kuhadiya N, Dan-
dona P. Oestradiol concentrations are not elevated
in obesity-associated hypogonadotrophic hypogo-
nadism. Clin Endocrinol. 2014;80(3):464.
221. Burger HG. Physiological principles of endocrine
replacement: estrogen. Horm Res. 2001;56(Suppl
1):825.
222. Gutorova NV, Kleshchyov MA, Tipisova EV,
Osadchuk LV. Effects of overweight and obesity
on the spermogram values and levels of reproduc-
tive hormones in the male population of the
European north of Russia. Bull Exp Biol Med.
2014;157:958.
223. Giagulli VA, Kaufman JM, Vermeulen A. Patho-
genesis of the decreased androgen levels in obese
men. J Clin Endocrinol Metab. 1994;79:9971000.
224. Veldhuis J, Yang R, Roelfsema F, Taka-
hashi P. Proinammatory Cytokine Infusion Atten-
uates LHs Feedforward on Testosterone Secretion:
Modulation by Age. J Clin Endocrinol Metab.
2016;101(2):53949.
225. Mah PM, Wittert GA. Obesity and testicular
function. Mol Cell Endocrinol. 2010;316:1806.
226. Nielsen TL, Hagen C, Wraae K, Brixen K,
Petersen PH, Haug E, et al. Visceral and subcuta-
neous adipose tissue assessed by magnetic reso-
nance imaging in relation to circulating androgens,
sex hormone-binding globulin, and luteinizing hor-
mone in young men. The Journal of clinical
endocrinology and metabolism. 2007;92(7):2696
705.
227. Landry D, Cloutier F, Martin LJ. Implications of
leptin in neuroendocrine regulation of male repro-
duction. Reprod Biol Endocrinol. 2013;13:114.
228. Luboshitzky R, Lavie L, Shen-Orr Z, Herer O.
Altered luteinizing hormone and testosterone secre-
tion in middle-aged obese men with obstructive
sleep apnea. Obes Res. 2005;13:7806.
229. Ec Tsai. Matsumoto AM, Fujimoto WY,
Boyko EJ. Association of bioavailable, free, and
3 Human Spermatogenesis and Its Regulation 71
sjwint01@louisville.edu
total testosterone with insulin resistance: Inuence
of sex hormone-bindign globulin and body fat.
Diabetes Care. 2004;27:8618.
230. Hammoud AO, Gibson M, Peterson CM, Hamil-
ton BD, Carrell DT. Obesity and male reproductive
potential. J Androl. 2006;27:61926.
231. Rato L, Alves MG, Cavaco JE, Oliveeira PF.
High-energy diets: a threat for male fertility?
Obesity Rev. 2014;15:9961007.
232. Samavat J, Natali I, DeglInnocenti S, limberti E,
Cantini G, Di Franco A, et al. Acrosome reaction is
impaired in spermatozoa of obese men: a prelimi-
nary study. Fertil Steril. 2014;102:127481.
233. Palmer NO, Bakos HW, Fullston T, Lane M. Impact
of obesity on male fertility, sperm function and
molecular composition. Spermatogenesis. 2012;2
(4):25363.
234. Feldman HA, Johannes CB, Derby CA, Klein-
man KP, Mohr BA, Araujo AB, et al. Erectile
dysfunction and coronary risk factors: prospective
results from the Massachusetts male aging study.
Prev Med. 2000;30:32838.
235. Lucchese M, Maggi M. Hypogonadism as a new
comorbidity in male patients selection for bariatric
surgery: towards an extended concept of metabolic
surgery? Obes Surg. 2013;23:20189.
236. Hammoud A, Gibson M, Hunt SC, Adams TD,
Carrell DT, Kolotkin RL, et al. Effect of Roux-en-Y
gastric bypass surgery on the sex steroids and
quality of life in obese men. J Clin Endocrinol
Metab. 2009;94(4):132932.
237. di Frega AS, Dale B, Di Matteo L, Wilding M.
Secondary male factor infertility after Roux-en-Y
gastric bypass for morbid obesity: case report.
Human Reprod. 2005;20:9978.
238. Lazaros L, Hatzi E, Markoula S, Takenaka A,
sokitis N, Zikopoulos K, et al. Dramatic reduction
in sperm parameters following bariatric surgery:
report of two cases. Andrologia. 2012;44:42832.
239. Legro RS, Kunselman AR, Meadows JW, Kes-
ner JS, Krieg EFJ, Rogers AM, et al. Time-related
increase in urinary testosterone levels and stable
semen analysis parameters after bariatric surgery in
men. Reprod Biomed Online. 2015;30:1506.
240. Kawakami S, Winters SJ. Regulation of leutinizing
hormone secretion and subunit messenger ribonu-
cleic acid expression by gonadal steroids in peri-
fused pituitary cells from male monkeys and rats.
Endocrinology. 1999;140:358793.
241. Nakagawa T, Sharma M, Nabeshima Y, Braun RE,
Yoshida S. Functional hierarchy and reversibility
within the murine spermatogenic stem cell com-
partment. Science. 2010;328(5974):627.
242. Heller CG, Clermont Y. Kinetics of the germinal
epithelium in man. Recent Prog Horm Res.
1964;20:54575.
243. Muciaccia B, Boitani C, Berloco BP, Nudo F,
Spadetta G, Stefanini M, et al. Novel stage classi-
cation of human spermatogenesis based on acro-
some development. Biol Reprod. 2013;89(3):60.
72 H. Chen et al.
sjwint01@louisville.edu
... Type B spermatogonia further differentiate to spermatocytes (leptotene, zygotene, pachytene, and diplotene), which are present upon puberty in humans. Diplotene spermatocytes undergo meiosis I to form secondary haploid spermatocytes and further undergo morphological transformations from round spermatids to spermatozoa (Chen et al., 2017). ...
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Increasing rates of infertility associated with declining sperm counts and quality, as well as increasing rates of testicular cancer are contemporary issues in the United States and abroad. These conditions are part of the Testicular Dysgenesis Syndrome, which includes a variety of male reproductive disorders hypothesized to share a common origin based on disrupted testicular development during fetal and neonatal stages of life. Male reproductive development is a highly regulated and complex process that relies on an intricate coordination between germ, Leydig, and Sertoli cells as well as other supporting cell types, to ensure proper spermatogenesis, testicular immune privilege, and endocrine function. The eicosanoid system has been reported to be involved in the regulation of fetal and neonatal germ cell development as well as overall testicular homeostasis. Moreover, non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics with abilities to block eicosanoid synthesis by targeting either or both isoforms of cyclooxygenase enzymes, have been found to adversely affect male reproductive development. This review will explore the current body of knowledge on the involvement of the eicosanoid system in male reproductive development, as well as discuss adverse effects of NSAIDs and analgesic drugs administered perinatally, focusing on toxicities reported in the testis and on major testicular cell types. Rodent and epidemiological studies will be corroborated by findings in invertebrate models for a comprehensive report of the state of the field, and to add to our understanding of the potential long-term effects of NSAID and analgesic drug administration in infants.
... However, scRNA-seq fails to profile developing germ cells in the native context of a seminiferous tubule, the spatially confined functional unit of spermatogenesis, due to cell dissociation. The difficulty of studying spermatogenesis using scRNA-seq is further compounded by somatic cell types co-existing with the germ cells in the testis (Chen et al., 2017;Griswold, 2018;Smith and Walker, 2014). Failure of scRNA-seq to capture the spatial interaction between the germ cell lineage and the somatic cell lineage impedes a comprehensive understanding of spermatogenesis. ...
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Single-cell RNA sequencing has revealed extensive molecular diversity in gene programs governing mammalian spermatogenesis but fails to delineate their dynamics in the native context of seminiferous tubules, the spatially confined functional units of spermatogenesis. Here, we use Slide-seq, a spatial transcriptomics technology, to generate an atlas that captures the spatial gene expression patterns at near-single-cell resolution in the mouse and human testis. Using Slide-seq data, we devise a computational framework that accurately localizes testicular cell types in individual seminiferous tubules. Unbiased analysis systematically identifies spatially patterned genes and gene programs. Combining Slide-seq with targeted in situ RNA sequencing, we demonstrate significant differences in the cellular compositions of spermatogonial microenvironment between mouse and human testes. Finally, a comparison of the spatial atlas generated from the wild-type and diabetic mouse testis reveals a disruption in the spatial cellular organization of seminiferous tubules as a potential mechanism of diabetes-induced male infertility.
... Testosterone has an important role in spermatogenesis because it stimulates protein synthesis in all types of spermatogenic cells, leading to sperm development. So, the decrease in testosterone secretion causes impairment of protein synthesis in germ cells with subsequent degenerative changes of germ cells [26]. Figure 9: Box blot graph of (a) testicular tissue SCF level (relative expression) and (b) serum testosterone level (ng/ml) among different study groups. ...
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Methotrexate (MTX) is a folic acid antagonist, widely used as a chemotherapeutic and immunosuppressive drug, but it is toxic to reproductive systems. In recent years, the era of stem cell applications becomes a promising point as a possible therapeutic agent in male infertility. This study is aimed at evaluating the therapeutic effects of stem cells at histological, molecular, biochemical, and functional levels in a methotrexate-induced testicular damage model. Material and Methods. Thirty rats were divided randomly into three groups (ten rats each): group 1 (control): animals received an intraperitoneal injection of 2 ml phosphate-buffered saline per week for 4 weeks, group 2 (MTX-treated group): animals were intraperitoneally injected with methotrexate (8 mg/kg) once weekly for 4 weeks, and group 3 (ADMSC-treated group): methotrexate-treated animals received a single dose of 1×106 stem cells/rat at the 5th week. At the 8th week, blood samples were collected for hormonal analysis; then, animals were sacrificed. The testes were dissected; the right testis was stained with hematoxylin and eosin. Random sections were taken from group 3 and examined with a fluorescent microscope. The left testis was divided into two specimens: the first was used for an electron microscope and the second was homogenized for molecular and biochemical assessments. Results. Group 2 showed significant histological changes, decreased free testosterone level, decrease in stem cell factor expression, and dysfunction of the oxidation state. The results revealed significant improvement of these parameters. Conclusion. Transplantation of adipose tissue-derived stem cells (ADMSCs) can improve the testicular damage histologically and functionally in a rat model.
... The generation of sperm is a complex process dependent upon the relationships between multiple cell types within the testis. [1] Pinpointing the causes of infertility therefore requires an understanding of the cellular niche where sperm are generated. Advances in single cell sequencing of testicular tissue have begun to shed light on the transcriptomic signatures of the cell types within the niche, [2][3][4][5][6][7][8] however the development of in vitro tools to study their interactions and functionalities remains elusive. ...
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Infertility is thought to be caused by genetic mutations and dysfunction in the cellular niche where spermatogenesis takes place. An understanding of the specialized cellular processes which drive spermatogenesis is needed to develop treatments; however, the development of in vitro systems to study these cells has been hindered by our reliance on rarely available human testicular tissues for research. Human induced pluripotent stem cells (hiPSCs) can be used to derive human testicular-like cells, and thus provide an avenue for the development of in vitro testicular model systems. Therefore, this study set out to engineer a human testicular tissue model using hiPSCs for the first time. We demonstrate the ability of hiPSC-derived testicular cells to self-organize and mature into testicular-like tissues using organoid culture. Moreover, we show that hiPSC-derived testicular organoids promote testicular somatic cell maturation and spermatogenesis up to the post-meiotic spermatid stage. These hiPSC-derived testicular organoids have the potential to replace rarely available primary testicular tissues to further infertility research in an in vitro setting.
... However, scRNA-seq fails to profile developing germ cells in the native context of a seminiferous tubule -the spatially-confined functional unit of spermatogenesis -due to cell dissociation. The difficulty of studying spermatogenesis using scRNA-seq is further compounded by somatic cell types co-existing with the germ cells in the testis (Chen et al., 2017;Griswold, 2018;Smith and Walker, 2014). Failure of scRNA-seq to capture the spatial interaction between the germ cell lineage and the somatic cell lineage impedes a comprehensive understanding of spermatogenesis. ...
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Single-cell RNA sequencing has revealed extensive molecular diversity in gene programs governing mammalian spermatogenesis but fails to delineate their dynamics in the native context of seminiferous tubules — the spatially-confined functional units of spermatogenesis. Here, we use Slide-seq, a novel spatial transcriptomics technology, to generate a comprehensive spatial atlas that captures the spatial gene expression patterns at near single-cell resolution in the mouse and human testis. By using Slide-seq data, we devise a computational framework that accurately localizes testicular cell types in individual seminiferous tubules. Unbiased spatial transcriptome analysis systematically identifies spatially patterned genes and gene programs, nominating genes with previously underappreciated but important functions in spermatogenesis. Using the human testicular spatial atlas, we identify two spatially segregated spermatogonial populations composed of stem cells at distinct transcriptional states. Finally, a comparison of the spatial atlas generated from the wild type and diabetic mouse testis reveals a disruption in the spatial cellular organization in diabetic seminiferous tubules.
... In the seminiferous tubules, SSCs through the process of spermatogenesis give rise to large numbers of undifferentiated spermatogonia via mitosis [73]. Spermatogonia differentiate into diploid spermatocytes to generate haploid spermatids in meiosis I/II [74]. During spermiogenesis, the spermatids undergo to form functional spermatozoa (also known as sperm cells) [75] (Fig. 1). ...
Infertility is defined as not being able to become pregnant or to conceive a child after one year or longer of regular unprotected intercourse. Male infertility refers to a male's inability to cause pregnancy that can result from deficiencies in semen quality, sperm concentration, or abnormal sperm function. Till now, there are few effective methods for the treatment of a couple with male infertility. In the past few years, stem cell-based therapy as a promising strategy has emerged for the treatment of male infertility. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into any type of cell. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are two pluripotent populations that can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. Both undifferentiated hiPSCs and hESCs are powerful candidates for the treatment of male infertility. Generation of male germ cells from hPSCs can provide new mechanistic insights into the regulation of spermatogenesis and have a great opportunity to families with infertility. Therefore, a robust, reproducible, and low-cost culture method that supports hPSCs differentiation into male germ cells is necessary. However, very few studies have focused on the derivation of sperm-like cells from hiPSCs and the details of hPSCs differentiation into male germ cells have not been fully investigated. Therefore, in this review, we focus on the in vitro differentiation potential of hiPSCs into male germ cells.
... During the 6 stages of spermatogenesis, the junctions between Sertoli cells and reproductive cells are in a constant remodeling process to allow transportation through the SE. It is in the ad-luminal compartment that meiosis I and II, the formation up to the spermatozoa stage and spermiation take place [10]. ...
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The multiplication and development of germ cells in the seminiferous tubules of the testicles occur through a complex series of cellular events that are controlled by multiple signals. It is composed of 6 stages in humans. Spermatogonial stem cells are self-renewed via mitosis, meiosis and contribute to the formation of haploid spermatids from diploid spermatocytes. Through the process of spermiogenesis, spermatids undergo maturation and are transformed into functional spermatozoa which are released at spermiation after the breakage of intercellular bridges attaching the spermatids to Sertoli cells. Spermatogenesis is a continuous process requiring the contribution of numerous cell and regulatory factors. Its understanding is essential in order to advance research for treatment of male infertility. The different stages of spermatogenesis along with the main roles of Sertoli cell and BTB will be reviewed. Some emerging fields in research regarding the new classification was briefly examined for a better understanding of the complexity of the process.
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Background Infertile men with non-obstructive azoospermia (NOA) have impaired spermatogenesis. Dilated and un-dilated atrophic seminiferous tubules are often present in the testes of these patients, with the highest likelihood of active spermatogenesis in the dilated tubules. Little is known about the un-dilated tubules, which in NOA patients constitute the majority. To advance therapeutic strategies for men with NOA who fail surgical sperm retrieval we aimed to characterize the spermatogonial stem cell microenvironment in atrophic un-dilated tubules.Methods Testis biopsies approximately 3x3x3 mm3 were obtained from un-dilated areas from 34 patients. They were classified as hypospermatogenesis (HS) (n=5), maturation arrest (MA) (n=14), and Sertoli cell only (SCO) (n= 15). Testis samples from five fertile men were included as controls. Biopsies were used for histological analysis, RT-PCR analysis and immunofluorescence of germ and Sertoli cell markers.ResultsAnti-Müllerian hormone mRNA and protein expression was increased in un-dilated tubules in all three NOA subtypes, compared to the control, showing an immature state of Sertoli cells (p<0.05). The GDNF mRNA expression was significantly increased in MA (P=0.0003). The BMP4 mRNA expression showed a significant increase in HS, MA, and SCO (P=0.02, P=0.0005, P=0.02, respectively). The thickness of the tubule wall was increased 2.2-fold in the SCO-NOA compared to the control (p<0.05). In germ cells, we found the DEAD-box helicase 4 (DDX4) and melanoma-associated antigen A4 (MAGE-A4) mRNA and protein expression reduced in NOA (MAGE-A: 46% decrease in HS, 53% decrease in MA, absent in SCO). In HS-NOA, the number of androgen receptor positive Sertoli cells was reduced 30% with a similar pattern in mRNA expression. The γH2AX expression was increased in SCO as compared to HS and MA. However, none of these differences reached statistical significance probably due to low number of samples.Conclusions Sertoli cells were shown to be immature in un-dilated tubules of three NOA subtypes. The increased DNA damage in Sertoli cells and thicker tubule wall in SCO suggested a different mechanism for the absence of spermatogenesis from SCO to HS and MA. These results expand insight into the differences in un-dilated tubules from the different types of NOA patients.
Chapter
Genetic factors, including hereditary and somatic mutations in regulatory sequences, can regulate gene expression. Epigenetic mechanisms also regulate gene expression through modifications in chromatin structure that do not involve changes in the DNA sequence. Multiple lines of evidence in animal, in vitro model, as well as in humans, have linked epigenetic modifications with human health with a particular impact being noted in the systems (male and female) of reproduction. Epigenetic reprogramming does not occur only in intrauterine life but also in germ cell development and early embryogenesis, affecting developmental cues for gene expression during the lifetime. The transmission of epigenetic marks that might play a role in reproduction occurs through DNA methylation, histone modification, microRNAs (miRNAs), and chromatin remodeling. The regulation of gene expression through these mechanisms is a key mechanism for transcriptome dysregulation in human reproductive disorders. This chapter provides a comprehensive summary of the role of epigenetics in reproductive diseases.
Chapter
Spermatogenesis is comprised of a series of cellular events that lead to the generation of haploid sperm. These events include self-renewal of spermatogonial stem cells (SSC), proliferation of spermatogonia by mitosis, differentiation of spermatogonia and spermatocytes, generation of haploid spermatids via meiosis I/II, and spermiogenesis. Spermiogenesis consists of a series of morphological events in which spermatids are being transported across the apical compartment of the seminiferous epithelium while maturing into spermatozoa, which include condensation of the genetic materials, biogenesis of acrosome, packaging of the mitocondria into the mid-piece, and elongation of the sperm tail. However, the biology of spermiation remains poorly understood. In this review, we provide in-depth analysis based on the use of bioinformatics tools and an animal model that mimics spermiation through treatment of adult rats with adjudin, a non-hormonal male contraceptive known to induce extensive germ cell exfoliation across the seminiferous epithelium, but nost notably elongating/elongated spermatids. These analyses have shed insightful information regaridng the biology of spermiation.
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The testicular vasculature forms a complex network, providing oxygenation, micronutrients and waste clearance from the testis. The vasculature is also instrumental to testis function as it is both the route by which gonadotropins are delivered to the testis, and by which testosterone is transported away to target organs. Whether Sertoli cells play a role in regulating the testicular vasculature in postnatal life has never been unequivocally demonstrated. In this study we utilized models of acute Sertoli cell ablation and acute germ cell ablation, to address whether Sertoli cells actively influence vascular structure and function in the adult testis. Our findings suggest that Sertoli cells play a key role in supporting the structure of the testicular vasculature. Ablating Sertoli cells (and germ cells), or germ cells alone results in a similar reduction in testis size, yet only the specific loss of Sertoli cells leads to a reduction in total intratesticular vascular volume, number of vascular branches, and numbers of small micro-vessels; loss of germ cells alone has no effect on the testicular vasculature. These perturbations to the testicular vasculature leads to a reduction in fluid exchange between the vasculature and testicular interstitium, which reduces gonadotropin-stimulated circulating testosterone concentrations, indicative of reduced Leydig cell stimulation and/or reduced secretion of testosterone into the vasculature. These findings describe a new paradigm by which the transport of hormones and other factors into and out of the testis may be influenced by Sertoli cells, and highlights these cells as potential targets for enhancing this endocrine relationship.
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Based on enumeration of maturation-phase spermatids in testicular homogenates from adult men and rats, daily sperm production per gram of testicular parenchyma (DSP/g) was almost seven times greater in rats (21.1 ± 0.9 × 10⁶ vs 3.1 ± 0.5 × 10⁶ ; X̄ ± SEM, n = 10). The relative inefficiency of the human testis was uniformly expressed in cranial, equatorial and caudal regions. Furthermore, DSP/g was highly correlated between paired testes from individual men (r = +0.97, n = 10), and differences between paired testes were not significantly different from zero (P>0.4). Histometric analysis of glutaraldehyde-perfused testes revealed differences (P<0.05) in relative testicular compositions between humans and rats (n = 3) including higher proportions of parenchyma, seminiferous tubules, seminiferous epithelium and germinal cells in the rat testis. Humans exceeded rats only in the proportions of testis occupied by nongerminal components such as tunic, interstitium, tubule boundary tissue and Sertoli cells. Histometric determination of the proportion of the testicular parenchyma occupied by these nuclei and consideration of average volume of round spermatid nuclei yielded DSP/g estimates of 17.4 ± 1.8 × 10⁶ in rats (not significantly different from the result by the homogenization method) and 8.5 ± 1.3 × 10⁶ in humans (almost three times higher than the value obtained by the homogenization method). Although the disparity between values for human DSP/g obtained by the two methods cannot yet be explained, several lines of evidence suggest that the time divisor used for human material processed by the homogenization method is too long, thus yielding an underestimation of daily sperm production. While its absolute accuracy is questionable, the homogenization method remains a rapid and precise technique for estimating DSP/g in humans. Histometric analysis and histometric estimation of DSP/g support the concept that the human is less efficient in sperm production than the rat, but these methods suggest that the rat is only about twice as efficient per gram of testicular parenchyma as the human.
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The purpose of this review is to describe the endocrine and local testicular factors that contribute to the regulation of the blood-testis barrier (BTB), using information gained from in vivo and in vitro models of BTB formation during/after puberty, and from the maintenance of BTB function during adulthood. In vivo the BTB, in part comprised of tight junctions between adjacent somatic Sertoli cells, compartmentalizes meiotic spermatocytes and post-meiotic spermatids away from the vasculature, and therefore prevents autoantibody production by the immune system against these immunogenic germ cells. This adluminal compartment also features a unique biochemical milieu required for the completion of germ cell development. During the normal process of spermatogenesis, earlier germ cells continually cross into the adluminal compartment, but the regulatory mechanisms and changes in junctional proteins that allow this translocation step without causing a ‘leak’ remain poorly understood. Recent data describing the roles of FSH and androgen on the regulation of Sertoli cell tight junctions and tight junction proteins will be discussed, followed by an examination of the role of paracrine factors, including members of the TGFβ superfamily (TGFβ3, activin A) and retinoid signalling, as potential mediators of junction assembly and disassembly during the translocation process.
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Spermatogenesis is an extraordinary complex process. The differentiation of spermatogonia into spermatozoa requires the participation of several cell types, hormones, paracrine factors, genes and epigenetic regulators. Recent researches in animals and humans have furthered our understanding of the male gamete differentiation, and led to clinical tools for the better management of male infertility. There is still much to be learned about this intricate process. In this review, the critical steps of human spermatogenesis are discussed together with its main affecting factors.
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In all mammals, spermatogonia are defined as constituting the mitotic compartment of spermatogenesis including stem, undifferentiated and differentiating cell types, possessing distinct morphological and molecular characteristics. Even though the real nature of the spermatogonial stem cell and its regulation is still debated the general consensus holds that in steady-state spermatogenesis the stem cell compartment needs to balance differentiation versus self-renewal. This review highlights current understanding of spermatogonial biology, the kinetics of amplification and the signals directing spermatogonial differentiation in mammals. The focus will be on relevant similarities and differences between rodents and non human and human primates.
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We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of doses as low as 1.0 mg/kg/day with dosing periods of 4, 8, and 16 weeks. 100% sterility was observed in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from two of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at two levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.