Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis.
ABSTRACT The population of early A spermatogonia includes stem cells that possess spermatogonial stem cell properties. Recent reports suggest that these cells have the ability to regain pluripotent properties. Here, we show that expression of the pluripotency marker undifferentiated embryonic cell transcription factor 1 (UTF1) is restricted to distinct germ cells within the testis. In embryonic and neonatal testes, all gonocytes were found to strongly express UTF1. During further testicular development, expression of UTF1 was restricted to a subset of A spermatogonia and with the increase in age the number of cells expressing UTF1 decreased even more. Ultimately, in the adult rat testis, only a small subset of the A spermatogonia expressed UTF1. Remarkably, even in testes of vitamin A-deficient rats, in which the early A spermatogonia (A(s), A(pr), and A(al)) are the only type of spermatogonia, only a subset of the spermatogonia expressed UTF1. In the adult rat testis, expression of UTF1 is restricted to a subpopulation of the ZBTB16 (PLZF)-positive early A spermatogonia. Furthermore, the observed distribution pattern of UTF1-expressing cells over the different stages of the cycle of the seminiferous epithelium suggests that the expression of UTF1 is restricted to those A(s), A(pr), and short chains of A(al) spermatogonia that are in the undifferentiated state and therefore maintain the ability to differentiate into A1 spermatogonia in the next round of the epithelial cycle or possibly even in other directions when they are taken out of their testicular niche.
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ABSTRACT: Whole mounted segments of seminiferous tubules from rat testes have been used to investigate the morphology and proliferative activity of the undifferentiated type A spermatogonial population. This has led to the formulation of a new model for spermatogonial stem cell renewal. Three groups of undifferentiated A spermatogonia were classified according to their topographical arrangements as isolated, paired, and aligned spermatogonia. It was proposed that the isolated (as well as a few paired) spermatogonia, which were always present throughout the seminiferous epithelium, are the functional stem cells and should therefore be designated as As. Through sporadic divisions, the As spermatogonia both maintain their own numbers and give rise to pairs of cells which are destined to eventually differentiate. The latter undergo several synchronous divisions in succession, thereby forming increasingly longer chains of aligned spermatogonia. The proliferation of these chains, primarily in stages I–V, leads to a gradual expansion in the size of the undifferentiated type A population. When the population attains its maximal size in stage V, mitotic activity among the aligned cells ceases, and all of these cells morphologically transform without further division into typical A1 spermatogonia. Subsequently, the cohort of A1 cells synchronously divides in stage IX to begin the long process of spermatogonial maturation. The isolated (and a few paired) cells, which do not undergo this transformation and remain quiescent during the stage IX peak of mitosis, form a residual stock of stem cells, that, during the course of another cycle, rebuild the population of aligned A spermatogonia. In this way, a continual supply of type A1 spermatogonia which will cyclically differentiate is insured.The Anatomical Record 02/1971; 169(3):533 - 557.
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ABSTRACT: Transforming growth factor beta (TGFbeta) inhibits proliferation and promotes the migration of primordial germ cells (PGCs) towards explants of gonadal ridges in vitro. However, its effects in vivo are still unclear. Here, we analyzed the behavior of PGCs in embryos lacking TGFbeta signaling via the type I receptor ALK5. TGFbeta in vivo was neither a chemoattractant for PGCs, nor did it affect their proliferation during migration towards the gonadal ridges up to embryonic day (E)10. Unexpectedly, the absence of TGFbeta signaling in fact resulted in significant facilitation of PGC migration out of the hindgut, due to the reduced deposition of collagen type I surrounding the gut of Alk5-deficient mutant embryos. Migratory PGCs adhere strongly to collagen; therefore, reduced collagen type I along the gut may result in reduced adhesion, facilitating migration into the dorsal mesenterium and gonadal ridges. Our results provide new evidence for the role of TGFbeta signaling in migration of PGCs in vivo distinct from that described previously.Developmental Biology 09/2005; 284(1):194-203. · 3.87 Impact Factor
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ABSTRACT: Adult germline stem cells are capable of self-renewal, tissue regeneration and production of large numbers of differentiated progeny. We show here that the classical mouse mutant luxoid affects adult germline stem cell self-renewal. Young homozygous luxoid mutant mice produce limited numbers of normal spermatozoa and then progressively lose their germ line after birth. Transplantation studies showed that germ cells from mutant mice did not colonize recipient testes, suggesting that the defect is intrinsic to the stem cells. We determined that the luxoid mutant contains a nonsense mutation in the gene encoding Plzf, a transcriptional repressor that regulates the epigenetic state of undifferentiated cells, and showed that Plzf is coexpressed with Oct4 in undifferentiated spermatogonia. This is the first gene shown to be required in germ cells for stem cell self-renewal in mammals.Nature Genetics 07/2004; 36(6):647-52. · 35.21 Impact Factor
Expression of the pluripotency marker UTF1 is restricted to a
subpopulation of early A spermatogonia in rat testis
Maaike P A van Bragt1, Hermien L Roepers-Gajadien1, Cindy M Korver2, Jan Bogerd1,
Akihiko Okuda3, Bart J L Eggen4, Dirk G de Rooij1,2and Ans M M van Pelt2
1Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, 3584 CH Utrecht,
The Netherlands,2Center for Reproductive Medicine, Academic Medical Center, 1105 AZ Amsterdam,
The Netherlands,3Division of Developmental Biology, Research Center for Genomic Medicine,
Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan and
4Department of Developmental Genetics, Groningen Biomolecular Sciences and Biotechnology Institute,
University of Groningen, 9751 NN Haren, The Netherlands
Correspondence should be addressed to A M M van Pelt at Center for Reproductive Medicine, Academic Medical Center, Room
F2-131-2, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; Email: email@example.com
The population of early A spermatogonia includes stem cells that possess spermatogonial stem cell properties. Recent reports suggest that
these cells have the ability to regain pluripotent properties. Here, we show that expression of the pluripotency marker undifferentiated
Aal) are the only type of spermatogonia, only a subset of the spermatogonia expressed UTF1. In the adult rat testis, expression of UTF1 is
As, Apr, and short chains of Aalspermatogonia that are in the undifferentiated state and therefore maintain the ability todifferentiate intoA1
spermatogonia in the next round of the epithelial cycle or possiblyeven in other directions when theyare taken out of their testicular niche.
Reproduction (2008) 136 33–40
Spermatogenesis is a continuous process starting with
spermatogonial stem cells (de Rooij & Russell 2000,
unipotent stem cells only being able to form cells of the
However, recent reports suggest that these cells have a
greater potential. Testes of both neonatal and 6-week-old
mice were reported to contain pluripotent cells or cells
abletoobtainthese propertiesinvitro (Kanatsu-Shinohara
et al. 2004, Guan et al. 2006, Seandel et al. 2007).
Previously, it was described that primordial germ cells
(PGCs), of both human and mouse origin, could give rise
to pluripotent stem cells (Matsui et al. 1992, Shamblott
et al. 1998). Finally, PGCs, gonocytes, and spermatogo-
nial stem cells express several genes believed to be
essential for pluripotency, e.g., Pou5f1 (previously
octamer-binding transcription factor 3/4, Oct3/4) and
Sry box 2 (Sox2) (Pesce et al. 1998, Shi et al. 2006).
Another pluripotency-associated gene is Utf1 (Okuda
et al. 1998). This gene was found to be expressed in
embryonic stem (ES) cells, embryonic carcinoma (EC)
Chuva de Sousa Lopes et al. 2005). In adult mice, Utf1
mRNA was only detected in the ovary and testis (Okuda
was found to be localized to the nucleus and subnuclear
fractionation, and mobilityassays revealed that UTF1 was
a chromatin-associated protein with histone-like proper-
ties (van den Boom et al. 2007). Differentiation of EC cells
is accompanied by a rapid reduction in UTF1 levels,
Nishimoto et al. 1999, van den Boom et al. 2007).
important mechanism by which POU5F1 maintains the
stem cell state of ES cells (Nishimoto et al. 2005).
Experiments indicated that UTF1 plays a role in the
proliferation rate and the teratoma-forming capacity of ES
cells (Nishimoto et al. 2005). However, recently it was
q 2008 Society for Reproduction and Fertility
ISSN 1470–1626 (paper) 1741–7899 (online)
Online version via www.reproduction-online.org
of a specific epigenetic profilethatallows lineage-specific
differentiation of ES and EC cells while it is not involved in
stem cell renewal (van den Boom et al. 2007).
In order to learn more about a possible role of UTF1 in
spermatogenesis, we now have studied the localization
of UTF1 in the testis at different developmental stages
during pre- and postnatal life. During testicular develop-
ment, the expression of UTF1 was restricted to the
gonocytes and A spermatogonia. Cell counts revealed a
decrease in the number of UTF1-positive cells during
testicular development and expression in the adult rat
testis was found to be restricted to a subpopulation of
early A spermatogonia.
Utf1 mRNA is expressed in testis of neonatal,
pubertal, and adult rats
To determine Utf1 mRNA expression in the rat testis of
different ages, we first obtained the rat Utf1 sequence.
Adult rat total testis RNA was subjected to RT-PCR using
primers derived from the human and mouse Utf1 cDNA
The resulting rat Utf1 cDNA sequence (GenBank acc.
no. EU176857) encodes a predicted protein of 338
amino acids and comparison between rat and mouse or
human UTF1 showed an overall amino acid identity of
93 and 65% and similarity of 96 and 73% respectively
(Fig. 1). Previously, based on the human and mouse
UTF1 protein sequences, two conserved domains, CD1
and CD2, were identified (Fukushima et al. 1998).
Analysis of the corresponding domains in rat UTF1
revealed identities of 96 and 87% for CD1 and 100 and
86% for CD2 compared with mouse and human UTF1
respectively (Fig. 1).
To determine Utf1 expression during rat testicular
development, RT-PCRs were performed on total testis
RNA of rats of various ages (Fig. 2). Utf1 mRNA was
detected in neonatal, pubertal, and adult rat testes and in
the testes of vitamin A-deficient (VAD) rats (Fig. 2).
Gonocytes and spermatogonia express UTF1
To determine which cell types in the testis express UTF1,
we performed immunohistochemistry at different
developmental stages during pre- and postnatal life. At
16 days post coitum (dpc), a strong staining for UTF1
was present in the nuclei of all gonocytes (Fig. 3A).
Figure 1 Amino acid sequence of rat UTF1 protein
compared with the mouse and the human UTF1
protein sequences. Identities are marked by the
gray boxes. The two conserved domains are
underlined (Fukushima et al. 1998).
M P A van Bragt and others
Reproduction (2008) 136 33–40www.reproduction-online.org
In gonocytes undergoing mitosis, the condensed
chromosomes were stained for UTF1 (Fig. 3A). At 18
dpc and 4 days post partum (dpp), the expression was
also strong and localized to the nuclei of all gonocytes
(Fig. 3B and C). At 9 dpp, when gonocytes have migrated
to the basal membrane and have become spermatogo-
nia, UTF1 expression was detected in the nuclei of
almost all A spermatogonia (Fig. 3D). At 13 dpp, staining
was present in A spermatogonia, but not in B
spermatogonia or preleptotene spermatocytes (Fig. 3E).
At later stages during development and in adult rat testes,
when spermatogenesis was complete, UTF1 expression
was restricted to A spermatogonia (Fig. 3F–J). However,
in testes of older rats, A spermatogonia that did not
express detectable UTF1 levels were also observed. In
VAD animals, seminiferous tubules only contain early A
spermatogonia (As, Apr, and Aal), Sertoli cells and
sporadically preleptotene spermatocytes (Mitranond
et al. 1979, Van Pelt & De Rooij 1990). Immunohis-
tochemistry showed that in the VAD rat testis, UTF1 is
expressed in spermatogonia (Fig. 3K). Interestingly, also
in VAD testes, spermatogonia were observed that did not
show expression of UTF1. Immunolocalization experi-
ments were repeated with three other antibodies
directed against UTF1, all confirming the localization
of UTF1 to the nuclei of gonocytes and A spermatogonia.
In accordance with the immunolocalization of UTF1 to
the spermatogonia in VAD rat testes, RT-PCR results
confirmed the expression of Utf1 in spermatogonia
isolated from these testes (Fig. 4). We furthermore
found that the early A spermatogonia marker Zbtb16
(previously promyelocytic leukemia zinc finger, PLZF)
and the activators of Utf1 transcription Pou5f1 and Sox2
were also expressed by the early A spermatogonia
isolated from the VAD rat testes (Fig. 4).
The number of UTF1-positive cells decreases with age
The expression pattern of UTF1 during testicular develop-
ment indicated that the number of UTF1-positive cells in
the testis decreased with age. To quantify this finding, the
Figure 2 RT-PCR for Utf1 on total testis of rat of several ages and
VAD total testis. Numbers express age in days post partum. C, cDNA;
K, RT control.
Figure 3 UTF1 expression in rat testes of different
ages. (A) 16 dpc, (B) 18 dpc, (C) 4 dpp, (D) 9 dpp,
(E) 13 dpp, (F) 17 dpp, (G) 24 dpp, (H) 42 dpp,
(I) 56 dpp, (J) 70 dpp, (K) adult VAD rat, and
(L) negative control 70 dpp. Asterisk, mitotic
gonocytes; arrows, positive spermatogonia;
arrowheads, negative spermatogonia. Bars:
(A and I) 100 mm, (B–D and L) 40 mm, (E–H, J and
K) 50 mm. Figure shows the immunolocalization
of UTF1 performed with the AB3383 antibody.
UTF1 in the testis
www.reproduction-online.org Reproduction (2008) 136 33–40
numbers of cells expressing UTF1 and ZBTB16 were
determined. ZBTB16 is a transcriptional repressor known
to be required for the self-renewal of spermatogonial stem
cells and its expression is known to be restrictedtoearly A
numbers of UTF1- and ZBTB16-positive cells per tubule
cross-section were scored in adjacent sections of rat testis
of different ages (Fig. 5). While the numbers of ZBTB16-
positive cells increased during testicular development,
the number of UTF1-positive cells clearly decreased.
Indicating that in the adult rat, there is a clear reduction in
UTF1-positive A spermatogonia, whereas the number of
ZBTB16-positive A spermatogonia increased during testi-
UTF1 expression is restricted to a subpopulation of
early A spermatogonia
To study which type of A spermatogonia expressed UTF1,
we performed double immunofluorescent labeling for
spermatogonia also expressed UTF1. However, at 56 and
70 dpp, not all ZBTB16-positive spermatogonia also
expressed UTF1 (Fig. 6). Cells positive for UTF1 but
negative for ZBTB16 were not observed. These findings
indicate that in the adult rat testis, UTF1 expression was
restricted to a subpopulation of early A spermatogonia.
Pattern of the UTF1-positive cells during the
Determining which early A spermatogonia express UTF1
is extremely difficult as early A spermatogonia are
morphologically indistinguishable (de Rooij 1998).
However, the numbers of the different types of early A
spermatogonia follow a specific pattern during the
epithelial stages. Studying the numbers of UTF1-
expressing cells during the epithelial stage could thus
possibly reveal the identity of UTF1-expressing cells.
The numbers of UTF1-positive spermatogonia per
stages (Fig. 7). During epithelial stages VII–X, the highest
number of UTF1-positive spermatogonia was observed.
The number decreased slowly from stages X until II–III,
after which an increase in the numbers of UTF1-positive
VII. In comparison, the average number of ZBTB16-
positive spermatogonia per tubule cross-section remained
relatively constant with exception of the decrease
observed in stage XIV (Fig. 7). During all stages, the
number of ZBTB16-positive spermatogonia was higher
than the number of spermatogonia that expressed UTF1.
At stage VII, the biggest overlap between UTF1 and
ZBTB16 expression was observed.
The mechanisms whereby spermatogonial stem cells
self-renew, differentiate, and possibly maintain their
pluripotent potential are not yet fully known. In order to
unravel the underlying mechanisms, the identification of
proteins involved in one or more of these processes is
crucial. The pluripotency-associated protein UTF1 is a
prime candidate for being involved in the process of self-
renewal and/or differentiation of spermatogonial stem
cells and possibly in maintaining the pluripotent
potential of these cells.
The rat sequence of the Utf1 gene was found to be
highly identical to that of mouse, but considerably
different from the human sequence (Fukushima et al.
1998). The identities of the conserved domains are
higher compared with the overall identity and therefore
the rat sequence supports the existence of the two
conserved domains (Fukushima et al. 1998). Utf1 mRNA
expression was found in the testes of both developing as
well as the adult rats, indicating that Utf1 expression is
not restricted to a certain developmental stage.
In embryonic and postnatal (4 dpp) rat testis,
expression of UTF1 protein was found to be restricted
to the gonocytes. Following further testicular develop-
ment, expression of UTF1 was only observed in A
spermatogonia. However, the number of cells per tubule
cross-section that expressed UTF1 decreased with age,
and non-expressing type A spermatogonia were
observed in the adult rat testes, suggesting that not all
types of A spermatogonia expressed UTF1.
Even in the testes of VAD rats, in which the early A
spermatogonia are the only type of spermatogonia,
expression of UTF1 was restricted to the spermatogonia.
This finding was supported by RT-PCR which showed
expression of Utf1 and its upstream factors Pou5f1 and
Sox2 in A spermatogonia isolated from VAD rat testes.
Figure 4 RT-PCR for Utf1, Pou5f1, Sox2, and Zbtb16 (PLZF) on
spermatogonia isolated from VAD rat testis. C, cDNA; K, RT control.
Figure 5 NumberGS.E.M. of UTF1- and ZBTB16-positive cells per
tubule cross-section in the testes of rats of various ages. ND, not
determined. Asterisk indicates that a significant difference (P%0.05)
between the number of UTF1- and ZBTB16-positive cells.
M P A van Bragt and others
Reproduction (2008) 136 33–40www.reproduction-online.org
Surprisingly, however, in VAD rat testis spermatogonia
that did not express UTF1 were also observed, indicating
that UTF1 expression was restricted to a subpopulation
of the early A spermatogonia.
As the different types of A spermatogonia are
morphologically very difficult to distinguish, co-locali-
zation studies of UTF1 with ZBTB16 were performed in
order to reveal the identity of the UTF1-positive cells. In
mice, expression of ZBTB16 in the testis is known to be
restricted to early A spermatogonia (Buaas et al. 2004,
Costoya et al. 2004). To our knowledge, localization of
ZBTB16 in rat testes has not been described before. The
expression pattern of ZBTB16 we observed in both cross-
sections as well as in whole-mount seminiferous tubules
of rat testes was similar to the expression seen in mice
(data not shown). In addition, we determined the
number of ZBTB16-positive spermatogonia during the
cycle of the seminiferous epithelium, which to our
knowledge has not been shown before.
The co-localization studies of UTF1 with ZBTB16
showed an almost complete overlap in A spermatogonia
of 42 dpp old rats. However, in adult (56 and 70 days old)
rats only partial co-localization was observed; cells that
expressed UTF1 always expressed ZBTB16, but cells
expressing ZBTB16 did not always express UTF1. These
observations are in accordance with our cell counts of the
numbers of UTF1- and ZBTB16-positive cells per tubule
cross-section in the testes of rats of various ages (Fig. 5).
At 42 dpp, when in almost all cells UTF1 and ZBTB16 are
co-expressed, counting revealed equal numbers of UTF1-
three times more ZBTB16-expressing cells were counted
compared with the number of cells expressing UTF1,
thereby confirming the co-localization experiments that
not all ZBTB16-positive cells expressed UTF1.
Thus, expression of UTF1 in the adult rat testis is
restricted to a subpopulation of early A spermatogonia as
shown by the lower number of UTF1-positive cells
compared with the number of ZBTB16-positive cells, the
partial co-localization of UTF1 with ZBTB16, and the
observation that in the testis of VAD rats not all early A
spermatogonia expressed UTF1.
The important question then is which types of early A
spermatogonia in the adult rat testis expressed UTF1.
Unfortunately, our attempts to immunohistochemically
stain for UTF1 on whole mounts of seminiferous
tubules were not successful, not allowing us to identify
the UTF1-expressing spermatogonia. In order to shed
more light on this issue, we determined the number
of UTF1-positive spermatogonia during the cycle of the
seminiferous epithelium. A pattern was found in which
the number of UTF1-positive spermatogonia peaked in
stages VII through X, gradually decreasing thereafter
until stage III, and then increasing again. This pattern is
quite different from that of the total numbers of early A
spermatogonia throughout the epithelial cycle that
shows an increase from about stages X to III, almost
similar numbers from stages III until VII and sharply
decreasing thereafter as the Aal spermatogonia differ-
entiate into A1 spermatogonia (Huckins 1971). As Aal
spermatogonia form the largest group within the
population of early A spermatogonia, their pattern is
Figure 6 Co-localization of UTF1 and ZBTB16 in
56 dpp rat testes. (A and B) Partial co-localization of
UTF1 and ZBTB16 can be observed in the testis of
two different 56 dpp rats. (C) Negative control.
Figure 7 Average numberGS.E.M. of UTF1- and ZBTB16-positive
cells per tubule cross-section for the different epithelial stages in
10-week-old rat testes.
UTF1 in the testis
www.reproduction-online.orgReproduction (2008) 136 33–40