Asymmetric Distribution of UCH‐L1 in Spermatogonia Is Associated With Maintenance and Differentiation of Spermatogonial Stem Cells

Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy‐terminal hydrolase 1 (UCH‐L1) is expressed in type A (A_s, A_pr, and A_al) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH‐L1 was associated with self‐renewal versus differentiation divisions of SSCs as defined by co‐localization of UCH‐L1^high and PLZF, a known determinant of undifferentiated SSCs, versus co‐localization of UCH‐L1^low/? with proteins expressed during SSC differentiation (DAZL, DDX4, c‐KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH‐L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH‐L1 and PLZF in situ in seminiferous tubules. Expression level of UCH‐L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH‐L1‐positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self‐renewal and differentiation in mammalian spermatogenesis. J. Cell. Physiol. 220: 460–468, 2009. © 2009 Wiley‐Liss, Inc.     

The new function of two ubiquitin C-terminal hydrolase isozymes as reciprocal modulators of germ cell apoptosis.

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. The two ubiquitin C-terminal hydrolase (UCH) enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. These two UCH isozymes have 52% amino acid identity and share significant structural similarity. A new function of these two closely related UCH enzymes during spermatogenesis which is associated with germ cell apoptosis has been analyzed. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. During spermatogenesis, apoptosis controls germ cell numbers and eliminates defective germ cells to facilitate testicular homeostasis. In this paper, I review the distinct function of the two UCH isozymes in the testis of gad and Uchl3 knockout mice, which are strongly but reciprocally expressed during spermatogenesis. In addition, the importance of UCHL1-dependent apoptosis for normal spermatogenesis and sperm quality control is discussed.     

Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. Ubiquitin C-terminal hydrolase (UCH) L1 is thought to associate with monoubiquitin to control ubiquitin levels. Previously, we found that UCHL1-deficient testes of gad mice have reduced ubiquitin levels and are resistant to cryptorchid stress-related injury. Here, we analyzed the function of UCHL1 during the first round of spermatogenesis and during sperm maturation, both of which are known to require ubiquitin-mediated proteolysis. Testicular germ cells in the immature testes of gad mice were resistant to the early apoptotic wave that occurs during the first round of spermatogenesis. TUNEL staining and cell quantitation demonstrated decreased germ cell apoptosis and increased numbers of premeiotic germ cells in gad mice between Postnatal Days 7 and 14. Expression of the apoptotic proteins TRP53, Bax, and caspase-3 was also significantly lower in the immature testes of gad mice. In adult gad mice, cauda epididymidis weight, sperm number in the epididymis, and sperm motility were reduced. Moreover, the number of defective spermatozoa was significantly increased; however, complete infertility was not detected. These data indicate that UCHL1 is required for normal spermatogenesis and sperm quality control and demonstrate the importance of UCHL1-dependent apoptosis in spermatogonial cell and sperm maturation.     

Ubiquitin C-terminal hydrolase-L1 (Uch-L1) correlates with gonadal transformation in the rice field eel

The ubiquitin-proteasome pathway is crucial for a variety of biological processes, including spermatogenesis. Ubiquitin C-terminal hydrolase-L1 (Uch-L1) is thought to associate with monoubiquitin to control ubiquitin levels. Here, we report the identification of Uch-L1 cDNA from the testis of the rice field eel, a natural sex reversal vertebrate, by using cDNA microarray analysis. Uch-L1 encodes a protein of 220 amino acids that shows high homology to Uch-L1 of vertebrates, especially fish species. Both mRNA and protein are mainly expressed in testis, ovotestis and ovary, as well as in the brain. Immunohistochemistry analysis revealed differential expression of Uch-L1 in three kinds of gonads. In the ovary, expression of Uch-L1 was observed mainly in the developing ovary and slightly in the mature ovary. In ovotestis during the intersex stage, Uch-L1 was expressed in the male gonad epithelium and degraded ovary. In testis, expression was observed in developing germ cells, including spermatogonia and spermatocytes. Furthermore, Uch-L1 was upregulated during gonadal transformation, especially from the beginning of the intersex stage onwards. Native-PAGE showed that Uch-L1 underwent dimerization and oligomerization in gonads, and that the relative level of dimerization/oligomerization decreased during gonadal transformation. Simultaneously, ubiquitin polypeptide expression was upregulated during this process. These results suggest that Uch-L1, via the ubiquitin-proteasome system, may play an important role not only in gametogenesis, but also in the gonadal transformation process in the rice field eel.     

The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.

We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.     

Crlz-1 Is Prominently Expressed in Spermatogonia and Sertoli Cells during Early Testis Development and in Spermatids during Late Spermatogenesis

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.     

Proteomic analysis of brain proteins in the gracile axonal dystrophy (gad) mouse, a syndrome that emanates from dysfunctional ubiquitin carboxyl-terminal hydrolase L-1, reveals oxidation of key proteins

Ubiquitin carboxyl-terminal hydrolase L-1 (UCH L-1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L-1 as one specific target of protein oxidation in Alzheimer's disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L-1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L-1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor alpha/ATP synthase and neurofilament-L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.     

γ—Aminobutyric acid transporter （GAT1） overexpression in mouse affects the testicular morphology

γ-Aminobutyric acid and GABAergic receptors were previously reported to be distributed in reproductive systems besides CNS and predicted to participate in the modulation of testicular function.γ-Aminobutyric acid transporter was implicated to be involved in this process.However,the potential role of γ-aminobutyric transporter in testis has not been explored.In this study,we investigated the existence of mouse γ-aminobutyric acid transporter subtype I (mGAT1) in testis.Wild-type and transgenic mice,which overexpressing mGAT1 in a variety of tissues,especially in testis,were primarily studied to approach the profile of mGAT1 in testis.Mice with overexpressed mGAT1 develop normally but with reduced mass and size of testis as compared with wild-type.Testicular morphology of transgenic mice exhibited overt abnormalities including focal damage of the spermatogenic epithelium accompanied by capillaries proliferation and increased diameter of seminiferous tubules lumen.Reduced number of spermatids was also found in some seminiferous tubules.Our results clearly demonstrate the presence of GAT1 in mouse testis and imply that GAT1 is possibly involved in testicular function.     

Potentiation of ATP-induced currents due to the activation of P2X receptors by ubiquitin carboxy-terminal hydrolase L1

Mammalian neuronal cells abundantly express a de-ubiquitinating isozyme, ubiquitin carboxy-terminal hydrolase L1 (UCH L1). Loss of UCH L1 function causes dying-back type of axonal degeneration. However, the function of UCH L1 in neuronal cells remains elusive. Here we show that overexpression of UCH L1 potentiated ATP-induced currents due to the activation of P2X receptors that are widely distributed in the brain and involved in various biological activities including neurosecretion. ATP-induced inward currents were measured in mock-, wild-type or mutant (C90S)-UCH L1-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in both wild-type and C90S UCH L1-transfected cells, suggesting that hydrolase activity was not involved but increased level of mono-ubiquitin might play an important role. The increased currents were dependent on cAMP-dependent protein kinase (PKA) and Ca2+ and calmodulin-dependent protein kinase (CaMKII) but not protein kinase C. In addition, ATP-induced currents were likely to be modified via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32) that is regulated by PKA and phosphatases. Our finding shows the first evidence that there is a relationship between UCH L1 and neurotransmitter receptor, suggesting that UCH L1 may play an important role in synaptic activity.     

Ubiquitin editing enzyme UCH L1 and microtubule dynamics: Implication in mitosis

Microtubules are essential components of the cytoskeleton and are involved in many aspects of cell responses including cell division, migration, and intracellular signal transduction. Among other factors, post-translational modifications play a significant role in the regulation of microtubule dynamics. Here, we demonstrate that the ubiquitin-editing enzyme UCH L1, abundant expression of which is normally restricted to brain tissue, is also a part of the microtubule network in a variety of transformed cells. Moreover, during mitosis, endogenous UCH L1 is expressed and tightly associated with the mitotic spindle through all stages of M phase, suggesting that UCH L1 is involved in regulation of microtubule dynamics. Indeed, addition of recombinant UCH L1 to the reaction of tubulin polymerization in vitro had an inhibitory effect on microtubule formation. Unexpectedly, Western blot analysis of tubulin fractions after polymerization revealed the presence of a specific ~50 kDa band of UCH L1 (not the normal ~25 kDa) in association with microtubules, but not with free tubulin. In addition, we show that along with 25 kDa UCH L1, endogenous high molecular weight UCH L1 complexes exist in cells, and that levels of 50 kDa UCH L1 complexes are increasing in cells during mitosis. Finally, we provide evidence that ubiquitination is involved in tubulin polymerization: the presence of ubiquitin during polymerization in vitro by itself inhibited microtubule formation and enhanced the inhibitory effect of added UCH L1. The inhibitory effects of UCH L1 correlate with an increase in ubiquitination of microtubule components. Since besides being a deubiquitinating enzyme, UCH L1 as a dimer has also been shown to exhibit ubiquitin ligase activity, we discuss the possibility that the ~50 kDa UCH L1 observed is a dimer which prevents microtubule formation through ubiquitination of tubulins and/or microtubule-associated proteins.     

Rete testis and the adjacent seminiferous tubules during postembryonic development in mice

Testicular compartment that includes rete testis and the adjacent transitional zone (TZ) of seminiferous tubules has been examined only by light and electron microscopy until now. However, recent data suggest that adult Sertoli cells (SCs) located in this compartment are capable to commence active proliferation both in vitro and in vivo, and hence, are not completely differentiated. The present study is first to investigate mouse rete testis and TZ during the postembryonic development and is intended to determine new protein markers for cells of this compartment, the state of their differentiation, and also their proliferative activity. It was demonstrated that rete testis cells were stained for SC marker Wt1 transiently, until day 25 of postembryonic development, then the staining disappeared. Another SC marker Dmrt1 that involved in the process of SC differentiation was not expressed in the rete testis cells during the postnatal development and in the adult state. One more feature that distinguished rete testis cells from SCs was lower proliferative activity of rete testis cells in 2–6 days old mice. SCs from TZ expressed Wt1 at all ages examined. However, at earlier ages, they were heterogeneous on Dmrt1 expression, and only by day 25, Dmrt1 expression was completely disappeared from TZ SCs. It is interesting that on day 18 when SCs in seminiferous tubules complete differentiation and exit from cell cycle proliferation of TZ SCs was at significantly higher level. It is also showed that in 3D culture, Wt1+ cells isolated from rete testis and TZ of 60 days old GFP male mice were capable to form seminiferous tubules de novo in cooperation with testicular cells from 6 days old mice.     

Spermatogenesis in white-lipped peccaries (Tayassu pecari)

We describe here morphological and functional analyses of the spermatogenic process in sexually mature white-lipped peccaries. Ten sexually mature male animals, weighing approximately 39 kg were studied. Characteristics investigated included the gonadosomatic index (GSI), relative frequency of stages of the cycle of seminiferous epithelium (CSE), cell populations present in the seminiferous epithelium in stage 1 of CSE, intrinsic rate of spermatogenesis, Sertoli cell index, height of seminiferous epithelium and diameter of seminiferous tubules, volumetric proportion of components of the testicular parenchyma and length of seminiferous tubules per testis and per gram of testis. The GSI was 0.19%, relative frequencies of pre-meiotic, meiotic and post-meiotic phases were, respectively 43.6%, 13.8% and 42.6%, general rate of spermatogenesis was 25.8, each Sertoli cell supported an average 18.4 germinative cells, height of seminiferous epithelium and diameter of seminiferous tubules were, respectively, 78.4 microm and 225.6 microm, testicular parenchyma was composed by 75.8% seminiferous tubules and 24.2% intertubular tissue, and length of seminiferous tubules per gram of testis was 15.8m. These results show that, except for overall rate of spermatogenesis, the spermatogenic process in white-lipped peccaries is very similar to that of collared peccaries, and that Sertoli cells have a greater capacity to support germinative cells than most domestic mammals.     

Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory

The neuronal ubiquitin/proteasomal pathway has been implicated in the pathogenesis of Alzheimer's disease (AD). We now show that a component of the pathway, ubiquitin C-terminal hydrolase L1 (Uch-L1), is required for normal synaptic and cognitive function. Transduction of Uch-L1 protein fused to the transduction domain of HIV-transactivator protein (TAT) restores normal enzymatic activity and synaptic function both in hippocampal slices treated with oligomeric Abeta and in the APP/PS1 mouse model of AD. Moreover, intraperitoneal injections with the fusion protein improve the retention of contextual learning in APP/PS1 mice over time. The beneficial effect of the Uch-L1 fusion protein is associated with restoration of normal levels of the PKA-regulatory subunit IIalpha, PKA activity, and CREB phosphorylation.     

UCH‐L1 promotes invasion of breast cancer cells through activating Akt signaling pathway

As a de‐ubiquitin enzyme, ubiquitin C‐terminal hydrolase (UCH)‐L1 has been shown to be overexpressed in several human cancers. However, the function of UCH‐L1 in invasion of breast cancers is still unclear. Here we report that the expression of UCH‐L1 is significantly higher in cancer cells with higher invasive ability. While ectopic UCH‐L1 expression failed to alter cell proliferation in MCF‐7 cells, it caused a significant upregulation of cellular invasion. Furthermore, siRNA mediated knockdown of UCH‐L1 led to suppression of invasion in UCH‐L1 overexpressing MCF‐7 cells. In order to identify molecular mechanisms underlying these observations, a novel in vitro proximity‐dependent biotin identification method was developed by fusing UCH‐L1 protein with a bacterial biotin ligase (Escherichia coli BirA R118G, BioID). Streptavidin magnetic beads pulldown assay revealed that UCH‐L1 can interact with Akt in MCF‐7 cells. Pulldown assay with His tagged recombinant UCH‐L1 protein and cell lysate from MCF‐7 cells further demonstrated that UCH‐L1 preferentially binds to Akt2 for Akt activation. Finally, we demonstrated that overexpression of UCH‐L1 led to activation of Akt as evidenced by upregulation of phosphorylated Akt. Thus, these findings demonstrated that UCH‐L1 promotes invasion of breast cancer cells and might serve as a potential therapeutic target for treatment of human patients with breast cancers.     

Mice that express enzymatically inactive cathepsin L exhibit abnormal spermatogenesis

The finding of large, stage-specific changes in secretion of procathepsin L by rat Sertoli cells has led to the hypothesis that this proenzyme promotes the survival, replication, or differentiation of spermatogenic cells. Experiments described herein used a mouse model to test this hypothesis. To prove that mice are appropriate for this purpose, we first demonstrate that mature mouse Sertoli cells express cathepsin L mRNA in the same stage-specific manner as rat Sertoli cells and they also secrete procathepsin L. To test whether catalytically active cathepsin L is required for normal spermatogenesis, we examined the testes of 110- to 120-day-old furless mice, which express catalytically inactive cathepsin L. Morphologic examination of testes of furless mice revealed both normal and atrophic seminiferous tubules. Enumeration of atrophic tubules in furless and control mice demonstrates that lack of functional cathepsin L results in a 12-fold increase in seminiferous tubule atrophy. To determine whether lack of functional cathepsin L affects the production of male germ cells in apparently normal, nonatrophic tubules, we compared numbers in control and furless mice of preleptotene spermatocytes, pachytene spermatocytes, and round spermatids per Sertoli cell. Results demonstrate that the lack of functional cathepsin L causes a 16% reduction in formation of preleptotene spermatocytes and a 25% reduction in differentiation of these cells into pachytene spermatocyte. These results suggest that procathepsin L either directly or indirectly has two distinct functions in the testis. This proenzyme prevents atrophy of seminiferous tubules and promotes the formation of preleptotene spermatocytes and the differentiation of these meiotic cells into pachytene spermatocytes.     

Phagocytosis plays an important role in clearing dead cells caused by mono(2-ethylhexyl) phthalate administration

The role of phagocytosis in eliminating apoptotic spermatogenic cells caused by mono(2-ethylhexyl) phthalate (MEHP) was studied. Twenty-one-day-old C57Bl/6N male mice were given a single dose of 800 mg/kg MEHP in corn oil by oral gavage and sacrificed at 1, 3, 5, 7 and 9 days after initial exposure. At the same time, the role of phagocytosis in MEHP related apoptosis was examined using microinjection of annexin V into the seminiferous tubules of living mice. Results showed that mice treated with MEHP had a lower rate of testis weight gain (lower regression line) and a significant TUNEL-positive spermatogenic cell number compared to control. However, this incident was reversible, and the number of TUNEL-positive cells returned to normal after 9 days. Mice microinjected with annexin V and later treated with MEHP showed a large amount of TUNEL-positive cells compared to mice treated with MEHP only. This clearly proves that phagocytosis plays an efficient and highly important role in eliminating dead cells in the injured testis of mice treated with MEHP.     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

Taenia crassiceps: A secretion-substance of low molecular weight leads to disruption and apoptosis of seminiferous epithelium cells in male mice

The present research was performed to isolate and study the effects of a low molecular weight (<1300 Da) parasite-associated substance, obtained from peritoneal fluids of female mice infected with Taenia crassiceps cysticerci, on seminiferous epithelium cells of male mice testis. The results showed an intense disruption of Sertoli cells and germ cells within the seminiferous tubules of experimental mice, along with the destruction of their gap junction (GJ). Significant generalized apoptosis of germ cells within seminiferous tubules was determined by TUNEL staining (P = 0.0159). In addition, a significant number of infiltrating macrophages were found in the luminal space of these seminiferous tubules (P < 0.0001). Finally, electron microscopy studies revealed structural and morphological abnormalities in the somatic cells (Sertoli and Leydig cells) and in the germ cells, primarily in the round and elongate spermatids.     

Leydig cell-specific expression of DAX1 improves fertility of the Dax1-deficient mouse

Dax1 is an orphan nuclear receptor expressed in both Leydig and Sertoli cells of the testis. Mutation of DAX1 in humans causes adrenal failure and hypogonadotropic hypogonadism. Targeted mutagenesis of Dax1 in mice reveals a primary gonadal defect characterized by overexpression of aromatase and cellular obstruction of the seminiferous tubules and efferent ductules, leading to germ cell death and infertility. Transgenic expression of DAX1 under the control of the müllerian-inhibiting substance promoter, which is selectively expressed in Sertoli cells, improves fertility but does not fully correct the histological abnormalities in the testes of Dax1 knockout (Dax1KO) mice. We therefore hypothesized that Dax1 may also play a crucial role in other somatic cells of the testis, namely the Leydig cells. A 2.1-kilobase fragment of the murine LH receptor 5'-promoter (LHR-DAX1) was used to generate transgenic mice that selectively express DAX1 in Leydig cells. Expression of the LHR-DAX1 transgene caused no observable phenotype in wild-type mice but improved fertility when expressed in Dax1KO males (rescue [RS]). Although testicular size was not increased in LHR-DAX1 RS animals, aromatase expression was restored to normal levels, and sperm production was increased. Testicular pathology was only slightly improved in RS mice compared to Dax1KO animals. Taken together with the result of previous studies of DAX1 expression in Sertoli cells, we conclude that the testis phenotype of Dax1KO mice reflects the combined effects of Dax1 deficiency in both Sertoli and Leydig cells.