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Sexual maturation of the Mongolian gerbil
(Meriones unguiculatus): a histological,
hormonal and spermatic evaluation
Maria Etelvina Pinto-Fochi
,Ana Carolina Negrin
˜o Roberto Taboga
and Rejane Maira Go
Department of Biology, Institute of Biosciences, Letters and Exact Sciences, Sa
˜o Paulo State
University – IBILCE/UNESP, Rua Cristo
˜o Colombo, 2265, CEP 15054-000, Sa
˜o Paulo, Brazil.
Department of Structural and Functional Biology, Institute of Biology, University of Campinas –
UNICAMP, Box 6109, CEP 13083-970, Campinas, Sa
˜o Paulo, Brazil.
Department of Morphology, Institute of Biosciences, Sa
˜o Paulo State University – IB/UNESP,
Box 510, 18618-000, Botucatu, Sa
˜o Paulo, Brazil.
Corresponding author. Email: email@example.com
Abstract. This study determined the phases of sexual development of the male Mongolian gerbil (Meriones
unguiculatus) based on an integrative analysis of testicular morphology, hormonal data and sperm parameters. Male
gerbils were analysed at 1, 7, 14, 21, 28, 35, 42, 50, 60, 70, 90, 100 and 120 days of age. Body, testicular and epididymal
weights increased up to Day 70, 60 and 90, respectively. The impuberal phase, characterised by the presence of gonocytes,
extended until Day 14. The prepubertal period lasted until Day 42, when puberty was achieved and a drastic increase in
serum testosterone levels, mature adult Leydig cells and elongated spermatids was observed. Gerbils at 60 days of age
showed a remarkable number of spermatozoa in the testis, epididymidis caput/corpus and cauda, and at Day 70 the
maximum daily sperm production was reached. However, the gerbil may be considered sexually mature only from Day 90
onward, when sperm reserves become stable. The total transit time of spermatozoa along the epididymis of sexually
mature gerbils was 11 days, with 1 day in the caput/corpus and 10 days in the cauda. These data cover a lacuna regarding
the reproductive parameters of this rodent and provide foundations for its use in testicular toxicology studies.
Additional keywords: daily sperm production, epididymal sperm transit time, puberty, sperm motility.
Received 24 February 2014, accepted 9 October 2014, published online 3 December 2014
The Mongolian gerbil (Meriones unguiculatus), also known as
the Mongolian squirrel, is a murine rodent of the Gerbillinae
subfamily found in arid regions of China and Mongolia. The
gerbil was introduced as a laboratory rodent in the 1960s
(Schwentker 1963;Rich 1968) and, in recent decades, it has
assumed an important role in biological and biomedical
experiments alongside other classic species such as the rat
(Rattus norvegicus), the mouse (Mus musculus) and the hamster
(Mesocricetus auratus). This rodent has been widely used as an
experimental model in different areas of scientific research
such as immunology (Jeffers et al. 1984;Nawa et al. 1994;
Wiedemann et al. 2009), cell culture (Moritomo et al. 1991) and
neurophysiology (Moller et al. 1979;Cao et al. 2005).
In the last decade there has been an emphasis concerning
gerbil reproductive biology. Several descriptive or experimental
studies have focussed on the prostate (dos Santos et al. 2003;
Corradi et al. 2004;Santos et al. 2006;Go´es et al. 2007;
Rochel et al. 2007;Taboga et al. 2009;Fochi et al. 2013) or
epididymis (Domeniconi et al. 2006,2007), as well as the sex-
steroid milieu (Blottner et al. 2000;Siegford et al. 2003;Juana
et al. 2010). A general description of the postnatal development
of the testes in the gerbil was made by Ninomiya and Nakamura
(1987).Segatelli et al. (2000,2002) showed that the seminifer-
ous epithelium cycle of this small rodent includes 12 stages;
the relative frequency of each stage and the duration of
whole spermatogenesis (47.5 days) were estimated using
H-thymidine injection and autoradiography (Segatelli et al.
2004). Regarding the immature testis, in previous reports we
have described the neonatal differentiation of gonocytes (Pinto
et al. 2010a) and characterised Leydig cell populations
(Pinto et al. 2010b). Thus, although the gerbil can be considered
an excellent model for the study of issues related to the male
reproductive system, a serious lacuna still persists concerning
Reproduction, Fertility and Development, 2016, 28, 815–823
Journal compilation !CSIRO 2016 www.publish.csiro.au/journals/rfd
the phases of testis development before adult age and sperm
parameters, mainly sperm reserves and epididymal transit
time. A detailed description of Mongolian gerbil sexual devel-
opment supported by testicular histology, steroidogenesis and
sperm production and reserve data are crucial to comprehen-
sion of its reproductive function and application in toxicologi-
cal and developmental studies. Therefore, this study assessed
the stages of sexual development of the Mongolian gerbil by
means of an integrated evaluation of the morphological and
physiological changes in the testis as well as some sperm
Materials and methods
Mongolian gerbils were kept in the Animal Breeding Center of
˜o Paulo State University (UNESP), Institute of Biosciences,
Humanities and Exact Sciences (IBILCE; Sa
˜o Paulo, Brazil)
under controlled temperature (23–258C), humidity (40–60%)
and luminosity (12 h light : 12h dark cycle). All animals were
given free access to water and rodent feed (Labina; Purina,
Paulı´nia, Brazil) ad libitum. Experimental procedures were
performed in accordance with the National Council for Control
of Animal Experimentation (CONCEA) and approved by the
Ethical Committee for Animal Research of the Bioscience
Institute/UNESP (Protocol CEEA no. 31/07). Male gerbils were
used at the following ages: 1, 7, 14, 21, 28, 35, 42, 50, 60, 70, 90,
100 and 120 days of age. The offspring were obtained by mating
female gerbils (90 days of age) in oestrus with male gerbils of the
same age, in a ratio of 1 : 1. The births were evaluated daily in the
morning and the birth date was considered to be Day 0. Only one
male pup from every litter was used for each age, i.e. n¼5 for
ages 1–50 days and n¼15 for the remaining ages. Gerbils were
weighed, anesthetised with ketamine (800 mL kg
) and xyla-
zine (200 mL kg
) and killed by CO
after death the animals were decapitated for collection of blood
and the testes and epididymis were removed and weighed.
Histological analyses of the testes were performed on five ani-
mals per age. The left testes were fixed in Bouin’s fluid for 6 h,
washed for several days in 70% ethanol and processed for
embedding in Paraplast (Merck, Darmstadt, Germany). Paraffin
sections were stained with haematoxylin–eosin (HE) and used
for immunocytochemistry. The right testes were fixed in 2.5%
glutaraldehyde, 1% tannic acid, 3.5% sucrose and 5 mM calcium
chloride in 0.1 M cacodylate buffer, pH 7.4, for 2 h at 48C. After
1 h in this solution, the testes were cut into smaller fragments and
fixed for 1 h more in the same solution. Testicular fragments
were post-fixed in 1% osmium tetroxide in cacodylate buffer
for 2 h and embedded in araldite 502 (Electron Microscopy
Sciences, Hatfield, PA, USA). One micron-thick sections were
stained with a solution of 1% toluidine blue and 1% borax in
water for light microscopic analyses. The analyses were per-
formed using an Olympus BX60 photomicroscope (Olympus,
Hamburg, Germany) and the images were digitalised using the
software Image-Pro Plus 6.0 for Windows (Media Cybernetics,
Bethesda, MD, USA).
Stages of testicular development
Histological analyses of the testes were performed in paraffin
sections stained with HE to determine the different stages
of postnatal testicular development: impuberal, prepubertal,
pubertal and adult, according to Courot et al. (1970). These
phases were determined based on the analysis of characteristics
of the seminiferous cords/tubules regarding the presence of
gonocytes, spermatogonia, primary spermatocytes, elongated
spermatids and spermatozoa, as well as the lumen formation
process. The gonocytes and germ cells at different stages of
differentiation were identified based on the descriptions of Pinto
et al. (2010a)and Segatelli et al. (2002), respectively.
The presence of mature adult Leydig cells (ALC) was
also examined using combined analysis of thick sections and
immunocytochemistry for the enzyme 17b-hydroxysteroid
dehydrogenase (17b-HSD), according to previously published
descriptions (Chamindrani Mendis-Handagama and Ariyaratne
2001). To detect 17b-HSD immunoreactivity, the sections were
deparaffinised and rehydrated, then antigen retrieval was per-
formed in citrate buffer, pH 6.0, at 978C for 45 min. Blocking of
endogenous peroxidases was obtained by covering the slides
with 3% H
in methanol for 20 min. The tissue sections were
treated with Background Sniper solution (Biocare Medical,
Concord, CA, USA) for 15 min to block non-specific protein
linkage. Sequentially, sections were incubated overnight at
48C with primary rabbit IgG anti-human 17b-HSD antibody
(sc-32872; Santa Cruz Biotechnology, Santa Cruz, CA, USA)
diluted 1 : 100 in 3% bovine serum albumin (BSA). The sections
were then incubated with a biotinylated anti-rabbit antibody
followed by the avidin–biotin complex ABC kit (Santa Cruz
Biotechnology) for 45 min at 378C. The immunoreaction was
revealed with diaminobenzidine (DAB) for 3 min and the sec-
tions were counterstained with haematoxylin. The washes were
accomplished with 0.05% Tween 20 in phosphate-buffered
saline (PBS), pH 7.6. The negative control was obtained by
omission of the primary antibody.
Serum hormone levels
After blood sample collection, the serum was separated by
centrifugation (1200gat 48C) for 20 min and stored at "208C for
subsequent hormone assays. The serum testosterone and oes-
tradiol levels were determined with automatic equipment
(VITROS ECi-Johnson and Johnson Ultra-Sensitive Quimio-
luminescent analysis) using specific reagents supplied by
Johnson and Johnson (Langhorne, PA, USA). Eight animals
were used from each group and the test was performed in trip-
licate. The sensitivity of the method was 1–1500 pg mL
testosterone and 0.1–3814 ng mL
Ten animals at Days 60, 70, 90, 100 and 120 were used for sperm
count analyses. The right testis and epididymis were removed,
weighed and immediately frozen for use. The testis sperm
number, daily sperm production (DSP), sperm number and
transit time in the epididymis were estimated. Homogenisation-
resistant testicular spermatids and spermatozoa in the caput/
corpus and cauda epididymidis were also estimated as described
816 Reproduction, Fertility and Development M. E. Pinto-Fochi et al.
previously (Robb et al. 1978;Fernandes et al. 2007), with
adaptations as described below.
The testis was decapsulated, weighed and homogenised in
4 mL Saline-Triton-Merthiolate solution (STM solution; 0.9%
NaCl containing 0.05% Triton X-100 and 0.01% Merthiolate),
followed by sonication for 30 s. The samples were diluted
10 times and an aliquot was transferred to a Neubauer chamber
(Laboroptik Ltd, Lancing, UK). Homogenisation-resistant sper-
matids were counted in quadruplicate for each animal to esti-
mate the total number of spermatids per testis. The variation
between the quadruplicates was less than 20%. To calculate
DSP, the total number of homogenisation-resistant spermatids
was divided by a time divisor. The time divisor is the number of
days of the seminiferous cycle in which the homogenisation-
resistant spermatids are present in the seminiferous epithelium
(Amann 1970,2008). Based on previous publications (Segatelli
et al. 2000) showing that gerbil spermatids complete their
morphological differentiation and the nucleus completes its
condensation and takes on its definitive shape, together with
our observations of spermatids in homogenised testicular paren-
chyma, we concluded that the nuclei resistant to homogenisation
in testicular homogenates were those in Step 13, 14 and 15
spermatids, found in Stages I to VI of the cycle of the seminifer-
ous epithelium in the gerbil (Segatelli et al. 2004). The duration
of these stages is 5.81 days (Segatelli et al. 2004), thus, the time
divisor of 5.81 was used to estimate DSP in gerbil. Then, the
DSP per gram of testis was calculated in order to determine the
The gerbil epididymis exhibits a particular shape: the caput
and cauda regions are voluminous, composed of a coiled
epididymidal duct, whereas the corpus is very slender and
contains the uncoiled epididymidal duct (Domeniconi et al.
2007). For sperm counts, this organ was separated into two
segments: one containing the caput/corpus and the other the
cauda. Each segment was weighed and homogenised in an
amount of STM solution according to its weight (1 mL of
STM for each 200 mg of caput/corpus and 1 mL of STM for
each 100 mg of cauda) followed by sonication for 30 s. The
samples were diluted 20 times and an aliquot was transferred to a
Neubauer chamber. Spermatozoa were counted in quadruplicate
per animal. The sperm transit time along the epididymis was
determined by dividing the number of spermatozoa in each
portion by the DSP. The values are expressed as 10
Immediately after euthanasia, the cauda of the left epididymis
was collected. Spermatozoa were obtained with the aid of a
needle by means of rinsing with 1.0 mL of modified human tubal
fluid (HTF) medium (Irvine Scientific, Santa Ana, CA, USA) at
348C. A Makler counting chamber (Sefi-Medical, Haifa, Israel)
warmed to 348C was loaded with a small aliquot of sperm
solution (10 mL). Sperm motility was assessed by visual esti-
mation (100 spermatozoa per animal, in duplicate) under a
phase-contrast microscope (Olympus BX60) at 200#magnifi-
cation. Spermatozoa were classified as immotile, motile without
progression or motile with progressive movement.
Parametric data were initially analysed by analysis of variance
(one-way ANOVA) and subsequently by Tukey’s test. For non-
parametric data, the Kruskal–Wallis test followed by Dunn’s
test was used. Both tests are for multiple comparisons with
significance levels of 5% (P#0.05). All statistical evaluations
were performed using the software Statistica 7.0 (StatSoft, Inc.,
Tulsa, OK, USA).
Body weight of gerbils increased until Day 70 and remained
stable until Day 120, with the most notable increase occurring
between Days 35 and 42 (23.3 g $2.4 to 39.5 g $4.6; Fig. 1a).
On the other hand, testicular weight increased until Day 60 and
remained unchanged after this age. This increase was more
outstanding from Day 35 to 60, thus testicular weight increased
100 Body weight (g)
Body weight (g)
Testis weight (mg)
1 7 14 21 28 35 42
Days after birth
Days after birth
Epididymal weight (mg)
60 70 90 100
Fig. 1. (a) Body (g), testis (mg) and (b) epididymal (mg) weight of gerbils.
Values represent the mean $s.e.m. a, b, c, d, e, f, g ¼statistically significant
difference between groups with different letters.
Sexual maturation of Mongolian gerbil Reproduction, Fertility and Development 817
,75% between Days 35 and 42 and doubled between Days 50
and 60 (Fig. 1a). The epididymal weight increased between
Days 60 and 90 and stabilised thereafter. This increase in the
epididymis was mainly due to the increased weight of the cauda,
since the caput/corpus weight did not change (Fig. 1b).
Stages of testicular development
Histological analysis of the testis demonstrated that the
impuberal stage, characterised by the predominant presence of
gonocytes and the absence of a lumen in the seminiferous cords,
extended until Day 14 (Fig. 2a). Until Day 7 the majority of
gonocytes were localised in the central region of the seminif-
erous cords, but at Day 14 90% of total gonocytes were observed
at the base of the seminiferous cords and from Day 21 onward
gonocytes were not visible. The prepubertal period occurred
between Days 14 and 42. At Day 21, leptotene spermatocytes
were observed (Fig. 2b), demonstrating that meiosis had already
started. In the subsequent week (Day 28), it was possible to
visualise spermatocytes in the zygotene stage and tubules with a
lumen (Fig. 2c). The expansion of the lumen and pachytene
spermatocytes were observed at Day 35 (Fig. 2d). At Day 42 the
tubular diameter increased remarkably and elongated sperma-
tids were already found (Fig. 2e), as well as mature ALC
(Fig. 3a,b), indicating the onset of puberty. Mature ALC
exhibited a flattened polyhedral shape and cytoplasm without
lipid droplets (Fig. 3a) and positivity for the enzyme 17b-HSD
(Fig. 3b). The presence of free spermatozoa in the testis of
gerbils at Day 60 indicates full spermatogenesis (Fig. 2f).
Serum hormone levels
Serum testosterone levels did not change in gerbils between Days
14 and 35 and exhibited a marked increase from Day 35 to 50
(46.3 $9.9 vs 660.85 $20.5) and an additional increase from
Day 70 to 90, stabilising thereafter (Fig.4). Serum oestrogen levels
showed no significant variation from Day 21 to 35, increased by
,27% at Day 42 and remained stable thereafter (Fig. 4).
Fig. 2. Histological paraffin sections of gerbil testis at different stages of postnatal development stained with HE,
showing the seminiferous tubules (inset) or detail of the seminiferous epithelium. (a) 14, (b) 21, (c) 28, (d) 35,
(e) 42, ( f) 60 days of age. G, gonocyte; L, spermatocyte in leptotene; Z, spermatocyte in zygotene; P, spermatocyte
in pachytene; rs, round spermatid; es, elongated spermatid; * free spermatozoon. Bar ¼20 mm; inset bar ¼50 mm.
818 Reproduction, Fertility and Development M. E. Pinto-Fochi et al.
The sperm parameters of gerbils at Days 60 to 120 are presented
in Table 1. The counts revealed that both the testis and epidid-
ymis of gerbils at Day 60 already had a reasonable number of
spermatozoa. Sperm number in the testis and DSP increased in
animals until Day 70 and then remained stable after this age.
The same pattern was observed for spermatogenic efficiency.
The sperm counts per gram of tissue stabilised at Day 100 for the
epididymidis caput/corpus and at Day 90 for the cauda. The
transit time from Day 90 onward was ,1 day in the epididymidis
caput/corpus and ,10 days in the cauda.
From Day 60 onward the percentage of spermatozoa that were
motile with progressive movement, motile without progression
or immotile stabilised at ,56%, ,22% and ,22%, respectively
There has been increasing interest in different aspects of the
reproductive organs of the Mongolian gerbil, particularly the
prostate (dos Santos et al. 2003;Corradi et al. 2004;Santos et al.
2006;Go´es et al. 2007;Rochel et al. 2007;Taboga et al. 2009;
Fochi et al. 2013) and epididymis (Domeniconi et al. 2006,
2007). However, the experimental and toxicological knowledge
on this rodent is still incipient, partly due to the lack of infor-
mation on its sexual maturation and rates of sperm production
and reserves. This paper fulfills this deficiency by presenting a
detailed overview of gerbil testicular development based on
morphological aspects, the synthesis of steroids, sperm counts,
epididymal transit time and sperm motility.
Ninomiya and Nakamura (1987) found that spermatogenesis
in the gerbil commences at ,2 weeks of age, when spermato-
gonial mitoses are first observed. The present data, as well as a
previous analysis of neonatal testis development (Pinto et al.
2010a), confirmed the findings of Ninomiya and Nakamura
(1987) concerning the onset of spermatogenesis. Considering
that the duration of spermatogenesis in the gerbil is 47.5 days
(Segatelli et al. 2004) and that, at Day 60 spermatozoa were
already observed in the epididymis, it can be concluded that, in
fact, spermatogenesis begins at around 14 days of age. Accord-
ing to Ninomiya and Nakamura (1987), the testes of animals at
7 weeks of age exhibited few seminiferous tubules with sperma-
tozoa, but at 10 weeks, the majority of tubules presented
spermatozoa. Furthermore, they also verified that spermatozoa
first appeared in the epididymis at 10 weeks with a remarkable
increase at 12 weeks. We first observed spermatozoa in the
epididymis earlier (,9 weeks or Day 60) than Ninomiya and
Nakamura (1987), which may be due to differences between the
laboratory lineages employed in these studies.
With respect to the levels of sex steroids, it is known that the
production of androgens in rodents in the fetal and neonatal
periods is due to the fetal Leydig cell population (O’Shaugh-
nessy et al. 2006); these cells are very common in the gerbil
testis up to Day 35 (Pinto et al. 2010b). Serum testosterone
Fig. 3. Thick section stained with toluidine blue and immunolocalisation of the enzyme 17b-
hydroxysteroid dehydrogenase (17b-HSD) in the interstitial tissue of gerbil at 42 days of age. The
immature adult Leydig cells (I) were recognised by the presence of lipid droplets (arrow) and the absence
of labelling for 17b-HSD. In mature adult Leydig cells (M), the opposite was noted, i.e. the lack of lipid
droplets and immunoreactivity for 17b-HSD. md, myoid cell; mc, macrophages; en, endothelial cells;
pe, pericytes; bv, blood vessels. Bar ¼20 mm.
1 7 14 21 28 35 42 50 60 70 90 100 120
Days after birth
Testosterone levels (pg mL !1)
en levels (n
Fig. 4. Serum levels of testosterone and oestrogen in gerbils at 14 to
120 days of age. Values represent the mean $s.e.m. a, b, c, d ¼statistically
significant differences between groups with different letters.
Sexual maturation of Mongolian gerbil Reproduction, Fertility and Development 819
levels were not different from Day 14 to 35. Even though newly
formed ALC were observed at Day 28 (Pinto et al. 2010b),
studies in the rat indicate a smaller secretory capacity compared
with mature ALC (Eckstein et al. 1987). The abrupt increase in
serum testosterone at Day 42 was coincident with the appear-
ance of mature ALC at this age.
DSP is the number of spermatozoa produced per day by a
testis or the two testes of an individual (Amann 1970,2008). The
DSP is a quantitative indicator of success in spermatogenesis
and, when expressed per gram of testicular parenchyma
), it reflects the efficiency of sperm production, which
is quite useful for comparisons of experimental conditions. DSP
per testis and per gram of testis can be estimated by quantitative
testicular histology (Amann 1970;Franc¸a 1992) or by the
method used here based on homogenates of testicular paren-
chyma and counts of homogenisation-resistant spermatids
(Amann 1970;Robb et al. 1978). The second method is simplest
and probably the most accurate. This method requires a time
divisor to convert the number of counted cells per unit volume or
mass of testis to the number of sperm cells produced each day
(Amann 1970). As previously mentioned in the Methods
section, the time divisor refers to the number of seminiferous
cycle days in which the homogenisation-resistant spermatids are
present in the seminiferous epithelium (Amann 1970,2008).
In this study the nuclei resistant to homogenisation in testicular
homogenates were those in Step 13, 14 and 15 spermatids, found
in Stages I to VI of the cycle of the seminiferous epithelium in
the gerbil; the duration of these stages is 5.81 days and thus the
time divisor was 5.81 days. In the rat, homogenisation-resistant
spermatids are Step 17–19 spermatids and the time divisor
is 6.1 days (Clermont et al. 1959), while in the mouse, the
homogenisation-resistant spermatids are Step 14–16 spermatids
and the time divisor is 4.84 days (Oakberg 1956). Thus, the
procedures adapted here can be widely accepted for DSP
estimation and the time divisor of gerbil is similar to those used
in other laboratory rodents.
To our knowledge, this is the first report on sperm number
and DSP determination in Mongolian gerbil by the method of
counting homogenisation-resistant spermatids (Robb et al.
1978). Blottner et al. (2000) have determined the sperm number
per testis and per gram of testis of sexually mature gerbils by
another method (haemocytometer) and the numbers observed in
our study are consistent with the values reported by these
authors. It was observed that the DSP in the sexually mature
gerbil is 12 $0.6 (10
per testis per day) with a spermatogenic
efficiency of 26 $4.1 (10
of testis). Previous studies have
Table 2. Sperm motility
Sperm motility of Mongolian gerbils at 60 to 120 days of age. Values represent the mean $s.e.m.
Age Motile with progressive movement (%) Motile without progression (%) Immotile (%)
60 days 56.29 $4.3 22.00 $3.7 21.71 $2.8
70 days 57.43 $3.0 21.71 $3.6 20.86 $3.8
90 days 56.57 $4.9 20.71 $3.9 22.71 $4.2
100 days 55.57 $5.6 20.86 $4.5 23.57 $4.8
120 days 57.71 $4.6 20.14 $3.1 22.14 $4.4
Table 1. Sperm count parameters
Sperm parameters of Mongolian gerbils at 60 to 120 days of age. Values represent the mean $s.e.m.
Values within rows with different superscript letters
differ significantly (P#0.05)
Parameter Gerbil age
60 days 70 days 90 days 100 days 120 days
Spermatid number (10
per testis) 55.1 $3.9
Spermatid number (10
testis) 120.0 $11.6
per gerbil) 9.5 $0.7
testis) 20.6 $2.0
Caput/corpus epididymidal sperm number (10
Caput/corpus epididymidal sperm number (10
Cauda epididymidal sperm number (10
Cauda epididymidal sperm number (10
Sperm reserves (10
Epididymal sperm transit time (days)
Caput/corpus 0.7 $0.2
Cauda 4.8 $0.8
Total 5.5 $1.0
Data for one epididymis multiplied by 2.
820 Reproduction, Fertility and Development M. E. Pinto-Fochi et al.
demonstrated that the DSP per testis of gerbils is 18 $3 and
spermatogenic efficiency is 33 $5(Segatelli et al. 2004). The
difference between our data and those of Segatelli et al. (2004)
can be explained by the different methods used. Segatelli et al.
(2004) used the method of quantitative testicular histology
proposed by Franc¸a (1992), while we used the method of
homogenisation-resistant spermatids described by Robb et al.
(1978), as previously discussed. Generally, there is an inverse
relationship between the length of the spermatogenic cycle and
spermatogenic efficiency. Species in which the spermatogenic
cycle length is shorter show a higher spermatogenic efficiency,
while species that present a longer spermatogenic cycle have a
lower spermatogenic efficiency (Franc¸a et al. 2005). This is the
case for several South American rodents, in which the relation-
ship (spermatogenic cycle length/spermatogenic efficiency)
occurs, like the spiny rat (8.6 days/82 #10
et al. 2010), agouti (9.5 days/52 #10
), paca (11.5 days/
;Costa et al. 2010) and capybara (11.9 days/10 #10
Paula et al. 1999). The duration of the cycle in rats is 12.9 days
(Leblond and Clermont 1952) and their spermatogenic efficien-
cy is 24 #10
of testis (Robb et al. 1978), while in mice the
duration is 8.6 days (Oakberg 1956) and the spermatogenic
efficiency is 47 #10
of testis (Franc¸a et al. 2005).
Although the cycle of the gerbils has an intermediate length
between rats and mice, 10.6 days, their spermatogenic efficien-
cy (26 $4.1 #10
) resembles that of rat and differs from those
observed for South American rodents.
The data on sperm counts and transit time in the epididymis
of the gerbil presented here are novel. These data show that the
total number of spermatozoa observed in the caput/corpus of
the epididymis of adult gerbils was smaller when compared
with the rat (,10 vs ,140 #10
per organ; Robb et al. 1978).
However, if we consider the sperm number per gram of caput–
corpus, the difference is smaller (,166 vs ,250 #10
Robb et al. 1978). Previous studies reported that few spermato-
zoa were seen in this region of the epididymis (Domeniconi et al.
2007). Regarding the cauda epididymidis, the total number of
spermatozoa observed in the mature gerbil (,120 #10
organ) was somewhat lower than in the rat (,200 #10
Robb et al. 1978); however, contrary to what occurs in the
caput/corpus when we consider the sperm number per gram of
cauda, the sperm number was considerably higher in the gerbil
(,1130 vs ,460 #10
;Robb et al. 1978). This indicates
the sperm storage capacity in the cauda epididymidis of the
gerbil and indicates that it is almost three times higher than that
in the rat, when expressed per gram of tissue.
The sperm transit time along the epididymis was determined
by the ratio between sperm reserves and DSP, as suggested by
Robb et al. (1978). Thus, it was found that the transit time of
spermatozoa through the epididymis of sexually mature gerbil is
11 days. The sperm transit time along the epididymis for most
mammalian species varies between 9 and 11 days (Amann et al.
1976;Franc¸a et al. 2005). Among rodents there is wide variation
in this value; for example, for hamster it is 15 days and for mouse
it is 5.5 days (Amann et al. 1976;Franc¸a et al. 2005). The gerbil is
similar to most species in relation to total transit time (11 days),
which is close to the rat, for which this time varies between 8 and
10 days, depending on the lineage (Robb et al. 1978;Franc¸a et al.
2005). In general, the time required for sperm maturation within
the caput and corpus ranges from 2 to 5 days (Amann et al. 1993;
Franc¸a et al. 2005). However, an intriguing finding was the rapid
sperm transit time along the caput/corpus of the epididymis
(,1 day) and the longer one in the cauda (,10 days). This
may be explained by the particular structure ofcorpus segment in
this species that appeared as a slender and straight segment of the
epididymidal duct connecting caput and cauda epididymidis
(Domeniconi et al. 2007). Moreover, it was reported that the
functionality of corpus in this species might be lower compared
with the other two segments, since histoenzymatic reactions
showed that this region has lower reactivity to the enzymes
related to metabolism of this organ such as succinate dehydroge-
nase (SDH), ATPase, acid and alkaline phosphatases previously
assessed in the corpus epididymidis by Domeniconi et al. (2006).
Additionally, Domeniconi et al. (2007) reported the absence of
clear cells in the epithelium of this region. On the other hand, the
cauda epithelium was characterised by a large number of these
cells intercalated between the principal cells and an expressive
presence of dark–narrow cells (Domeniconi et al. 2007). It is
known that the clear and narrow cells are related to secretory
activities responsible for acidification of the luminal fluid (Hermo
and Robaire 2002). Intraluminal acidification maintains sperm
quiescence in the epididymidis duct, preventing premature acti-
vation of acrosomal enzymes (Verma 2001). Although the data
of Domeniconi et al. (2007) support the roleof the cauda segment
in sperm storage, as classically known for most mammals, the
high sperm number per gram and transit time in this segment,
added to the atypical corpus structure, may indicate that a portion
of sperm maturation in the gerbil occurs in the proximal cauda.
Sperm maturation in the epididymis consists of a wide
spectrum of physiological and biochemical alterations that
improve its capacity for fertilisation (Hermo and Robaire
2002). Indeed, the analysis of sperm motility is one of the most
important parameters used in the evaluation of sperm quality
(Bonde et al. 1998;Winkle et al. 2009;Fernandez et al. 2011).
Animals at Day 60 presented similar percentages of motility
compared with sexually mature animals, which is evidence that,
although the DSP and sperm reserves have not yet attained their
maximum, the animals of this age already show rates of sperm
motility typical of adulthood.
Various stages of testicular development precede testicular
maturity in mammals: impuberal, prepubertal, pubertal and
sexual maturity (Courot et al. 1970). Based on histological
and hormonal analysis and sperm parameters, the testicular
development phases of gerbil were established. It is known that
the spectrum of sperm parameters is very wide and assessing of
sperm function characteristics relevant to fertility would require
the application of modern technologies and techniques beyond
microscopy (Petrunkina et al. 2007;Petrunkina and Harrison
2011). However, this study evaluated only some sperm para-
meters such as sperm counts, epididymal transit time and sperm
motility. The gerbil’s testes from birth to 14 days of age show
seminiferous cords with a predominant presence of gonocytes
and the absence of a lumen, signs that characterise the impuberal
stage. In the prepubertal phase (Days 14 to 42) the disappearance
of gonocytes occurs along with proliferation of germ cells in the
seminiferous epithelium. Prepubertal animals exhibit testicular
Sexual maturation of Mongolian gerbil Reproduction, Fertility and Development 821
cords in the process of lumen formation, which does not occur
homogeneously and synchronously along the testis. Puberty is
defined as the age at which a male individual achieves repro-
ductive capacity for the first time (Robb et al. 1978). Animals at
Day 60 showed remarkable sperm production and reserves.
Although these analyses were not performed on animals youn-
ger than 60 days of age, the characteristics proposed by Courot
et al. (1970) that indicate the onset of puberty, such as tubular
diameter increases, the appearance of elongated spermatids and
mature ALC, in addition to a drastic increase in serum testoster-
one levels, were already observed in animals at Day 42.
Additionally, published data for the rat indicate that, at Day
50, a small amount of sperm production and reserves can be
observed (Robb et al. 1978). Thus, our data indicate that puberty
begins in the gerbil at around 42 days of age. Previously, the
onset of puberty in gerbils was determined by anatomical and
hormonal measures (Siegford et al. 2003). Our findings confirm
the observations of Siegford et al. (2003) that puberty in male
gerbil begins between Days 43 and 48.
It is known that the maximum reproductive capacity of a
male in terms of sperm production or epididymal sperm reserves
is not attained until the testes reach adult size (Amann 1970). In
the case of the gerbil, the adult testis weight was stabilised at
Day 60; however, the first DSP maximum occurred at Day 70
(11.5 $1.5 #10
) and the maximum sperm reserve at Day 90
255.gerbil). Thus, the gerbil may be considered
sexually mature only at 90 days of age. Therefore, male gerbils
at 60 days of age are able to reproduce; however, if sexually
mature males are required for physiological studies, only gerbils
of at least 90 days of age should be used. This evidence indicates
that the gerbil differs from the mouse, which becomes sexually
mature at ,45 days of age (Kilborn et al. 2002) and resembles
the rat, which reaches sexual maturity at around 100 days of age
(Robb et al. 1978).
The data presented here demonstrate a substantial refinement
in the stages of sexual development, in addition to establishing
sperm parameters (DSP, spermatogenic efficiency, sperm
reserve, sperm transit time along the epididymis and sperm
motility) for the Mongolian gerbil (Meriones unguiculatus).
Thus, we determined that the impuberal phase extends until Day
14, the same period when spermatogenesis begins. The prepu-
bertal period occurs between Days 14 and 42; puberty is attained
at ,42 days of age and sexual maturity occurs at Day 90.
Furthermore, we found that, in terms of laboratory rodents, the
gerbil has a similar reproductive profile with the rat, except for
higher a sperm number per gram and transit time in the cauda
epididymidis. This range of information provides new founda-
tions for future investigations involving the reproductive biology
of this rodent, which has become an important experimental
model in reproductive biology research.
This article is part of the thesis presented by M. E. P. F. to the Institute of
Biology, UNICAMP, in partial fulfilment of the requirement for a PhD degree.
The authors thank Mr. Luis Roberto Falleiros, Jr., as well as all other
researchers at the Microscopy and Microanalysis Laboratory (IBILCE/
UNESP).This work was supported by the following sponsors: Sa
˜o Paulo State
Research Foundation (FAPESP), Coordinating Body for Training University
(CAPES)and Brazilian National Research and Development Council (CNPq).
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