Loss of connexin 43 in Sertoli cells provokes postnatal spermatogonial arrest,reduced germ cell numbers and impaired spermatogenesis

For the reason that adult Sertoli cell specific connexin 43 knockout (SCCx43KO) mice show arrested spermatogenesis at spermatogonial level or Sertoli cell only tubules and significantly reduced germ cell (GC) numbers, the aims of the present study were (1) to characterize the remaining GC population and (2) to elucidate possible mechanisms of their fading. Apoptosis was analyzed in both, KO and wild type (WT) male littermates during postnatal development and in adulthood using TUNEL. Although GC numbers were significantly reduced in KO at 2 and 8 days postpartum (dpp) when compared to WT, no differences were found concerning apoptotic incidence between genotypes. From 10 dpp, the substantial GC deficiency became more obvious. However, significantly higher apoptotic GC numbers were seen in WT during this period, possibly related to the first wave of spermatogenesis, a known phenomenon in normal pubertal testes associated with increased apoptosis. Characterization of residual spermatogonia in postnatal to adult KO and WT mice was performed by immunohistochemical reaction against VASA (marker of GCs in general), Lin28 and Fox01 (markers for undifferentiated spermatogonia) and Stra8 (marker for differentiating spermatogonia and early spermatocytes). During puberty, the GC component in SCCx43KO mice consisted likely of undifferentiated spermatogonia, few differentiating spermatogonia and very few early spermatocytes, which seemed to be rapidly cleared by apoptosis. In adult KOs, spermatogenesis was arrested at the level of undifferentiated spermatogonia. Overall, our data indicate that Cx43 gap junctions in SCs influence male GC development and differentiation rather than their survival.     

Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis

The transition from mitosis to meiosis is unique to germ cells. In murine embryonic ovaries and juvenile testes, retinoic acid (RA) induces meiosis via the stimulated by retinoic acid gene 8 (Stra8), but its molecular pathway requires elucidation. We present genetic evidence in vivo and in vitro that neuregulins (NRGs) are essential for the proliferation of spermatogonia and the initiation of meiosis. Tamoxifen (TAM) was injected into 14-day post-partum (dpp) Sertoli cell-specific conditional Nrg1(Ser-/-) mutant mice. TAM induced testis degeneration, suppressed BrdU incorporation into spermatogonia and pre-leptotene primary spermatocytes, and decreased and increased the number of STRA8-positive and TUNEL-positive cells, respectively. In testicular organ cultures from 5-6 dpp wild-type mice and cultures of their re-aggregated spermatogonia and Sertoli cells, FSH, RA [all-trans-retinoic acid (ATRA), AM580, 9-cis-RA] and NRG1 promoted spermatogonial proliferation and meiotic initiation. However, TAM treatment of testicular organ cultures from the Nrg1(Ser-/-) mutants suppressed spermatogonial proliferation and meiotic initiation that was promoted by FSH or AM580. In re-aggregated cultures of purified spermatogonia, NRG1, NRG3, ATRA and 9-cis-RA promoted their proliferation and meiotic initiation, but neither AM580 nor FSH did. In addition, FSH, RAs and NRG1 promoted Nrg1 and Nrg3 mRNA expression in Sertoli cells. These results indicate that in juvenile testes RA and FSH induced meiosis indirectly through Sertoli cells when NRG1 and NRG3 were upregulated, as NRG1 amplified itself and NRG3. The amplified NRG1 and NRG3 directly induced meiosis in spermatogonia. In addition, ATRA and 9-cis-RA activated spermatogonia directly and promoted their proliferation and eventually meiotic initiation.     

TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence.These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.     

Developmental regulation of the bcl-2 family during spermatogenesis: insights into the sterility of bcl-w-/- male mice

Expression of bcl-w, a close relative of bcl-2 is essential for male fertility in mice. Although the initial wave of spermatogenesis in bcl-w -/- mice proceeds normally until 3-4 weeks of age, adults fail to produce sperm. To clarify why bcl-w is essential for adult but not juvenile spermatogenesis, we investigated the expression pattern of eight bcl-2 family members. We found that both the level and pattern of expression varied in different cell types during juvenile and adult spermatogenesis. Anti-apoptotic genes bcl-w, bcl-2 and bcl-xL were all expressed in spermatogonia during juvenile spermatogenesis, but only bcl-w was detected in spermatogonia of adult mice. A similar shift was evident in Sertoli cells. This developmental regulation may co-ordinate physiological germ cell apoptosis in wild type mice and account for the time of onset for pathological germ cell apoptosis in bcl-w -/- animals.     

Suppression of Stra8 expression in the mouse gonad by WIN 18,446

Bis-(dichloroacetyl)-diamines (BDADs) are compounds that inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully investigated. It has been proposed that BDADs may function via inhibition of testicular retinoic acid (RA) biosynthesis. We employed an organ culture technique and the expression of a marker for RA activity, Stra8 (stimulated by retinoic acid gene 8), to investigate if the BDAD WIN 18,446 inhibited the biosynthesis of RA from retinol (ROL) in neonatal and adult murine testis and in the embryonic murine gonad. After culturing either whole testes or germ cells isolated from mice at 2 days postpartum (dpp) with WIN 18,446 or with WIN 18,446 plus ROL, Stra8 expression was suppressed, demonstrating that WIN 18,446 inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-responsive LacZ reporter gene to demonstrate limited RA induction of LacZ expression in 2-dpp testes cultured with WIN 18,446 plus ROL. The expression of Stra8 was downregulated in adult mouse testis tubules cultured with WIN 18,446 when compared to tubules cultured with the vehicle control. WIN 18,446 also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 h. These murine results provide critical insights regarding how the BDADs can inhibit spermatogenesis by blocking the ability of vitamin A to drive germ cell development. In addition, these techniques will be useful for screening novel inhibitors of RA biosynthesis as potential male contraceptives.     

Spermatogonial depletion in adult Pin1-deficient mice

Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1-/- mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1-/- testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1-/- testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.     

Multiple effects of retinoids on the development of Sertoli, germ, and Leydig cells of fetal and neonatal rat testis in culture

We investigated the effect of retinoids on the development of Sertoli, germ, and Leydig cells using 3-day culture of testes from fetuses 14.5 and 18.5 days post-conception (dpc) and from neonates 3 days postpartum (dpp). Addition of 10(-6) M and 3.10(-8) M retinoic acid (RA) caused a dose-dependent disruption of the seminiferous cords in 14.5-day-old fetal testes, without any change in the 5-bromo-2'-deoxyuridine (BrdU) labeling index of the Sertoli cells. RA caused no disorganization of older testes, but it did cause hyperplasia of the Sertoli cells in 3-dpp testes. Fragmentation of the Sertoli cell DNA was not detected in control or RA-treated testes at any age studied. The cAMP produced in response to FSH was significantly decreased in RA-treated testes for all studied ages. Both 10(-6) M and 3.10(-8) M RA dramatically reduced the number of gonocytes per 14.5-dpc testis. This resulted from a high increase in apoptosis, which greatly exceeded the slight increase of mitosis. RA caused no change in the number of gonocytes in testes explanted on 18.5 dpc (the quiescent period), whereas it increased this number in testes explanted on 3 dpp (i.e., when gonocyte mitosis and apoptosis resume). Lastly, RA and retinol (RE) reduced both basal and acute LH-stimulated testosterone secretion by 14.5-dpc testis explants, without change in the number of 3beta-hydroxysteroid dehydrogenase-positive cells per testis. Retinoids had no effect on basal or LH-stimulated testosterone production by older testes. In conclusion, RE and RA are potential regulators of the development of the testis and act mainly negatively during fetal life and positively during the neonatal period on the parameters we have studied.     

Implication of nanos2-3'UTR in the expression and function of nanos2

Translational control of gene expression is an important component of the regulation of cellular differentiation and development. To elucidate the function of the 3'untranslated region (UTR) of the nanos2 gene in mice, we compared the phenotypes of lacZ knock-in mice with or without a native nanos2 3'UTR and found that this region of the nanos2 gene has a potential role during translational regulation in germ cells. The nanos2-3'UTR functions to repress the translation of mRNA in oocytes, but enhances the production of protein in the male gonads. To further understand the significance of the nanos2 3'UTR in vivo, we generated the mouse line nanos2pA/pA, which lacks this region endogenously. In nanos2(-/pA) mice, the number of germ cell-depleted seminiferous tubules was increased when compared with that of nanos2pA/pA mice, indicating a dose-dependent defect in spermatogenesis. These results suggest that the level of nanos2 protein is critical for normal spermatogenesis, and that this pathway may be regulated through the nanos2-3'UTR. We found that the defects in nanos2pA/pA and nanos2(-/pA) mice were caused by apoptosis of gonocytes in the embryonic gonads and gonocyte/spermatogonia in neonatal testes. In addition, it was noted that the nanos2 expression was restricted to a particular subset of spermatogonia after birth, which indicates that nanos2 plays a role in the maintenance and differentiation of gonocytes/spermatogonia in neonatal testes.     

White-spotting mutations affect the regenerative differentiation of testicular germ cells: demonstration by experimental cryptorchidism and its surgical reversal.

The effect of white-spotting (W) mutations on differentiation of testicular germ cells was investigated by using experimental cryptorchidism and its surgical reversal. All mutant mice used in this study (Wv/+, Wsh/+, Wf/+ and Wf/Wf) showed normal fertility and well-ordered spermatogenesis, as in congenic +/+ mice. In the cryptorchid testis, which contains only type A spermatogonia as germ cells, the number and the proliferative activity of type A spermatogonia in mutant mice were comparable to +/+ mice. On the other hand, surgical reversal of the cryptorchid testis in mutants resulted in impaired regenerative differentiation of germ cells. Although complete recovery of spermatogenesis was observed in +/+ mice, testicular weight in Wsh/+, Wf/+ and Wf/Wf mice recovered to approximately 60-70% of intact levels, and some portions of seminiferous epithelium showed incomplete spermatogenesis. In Wv/+ mice, however, ability to recover the weight was completely lost, and only type A spermatogonia existed as germ cells in seminiferous tubules 3 mo after surgical reversal. These results suggest that W mutation affects the differentiation through type A spermatogonia to type B spermatogonia, indicating the functional significance of W (c-kit) in early spermatogenesis.     

Apoptosis,onset and maintenance of spermatogenesis: evidence for the involvement of Kit in Kit-haplodeficient mice

Kit/stem cell factor (SCF ) has been reported to be involved in survival and proliferation of male differentiating spermatogonial cells. This kinetics study was designed to assess the role of Kit/SCF during spermatogenesis in mice, and the extent of male programmed germ cell death was measured between 8 and 150 days of age. For this purpose, 129/Sv inbred mice in which one Kit allele was inactivated by an nlslacZ sequence insertion (Kit(W-lacZ/+)) were compared with 129/Sv inbred mice with wild-type alleles at the Kit locus. Four different approaches were used: 1) morphometric study to assess spermatogenesis, 2) flow cytometry to study testicular cell ploidy, 3) in situ end labeling to detect apoptosis, and 4) follow-up of reporter gene expression. Spermatogenesis was lower in Kit(W-lacZ/+) heterozygous mice both at the onset of spermatogenesis and during adulthood. Indeed, greater apoptosis occurred at the onset of spermatogenesis. This was followed in the adult by a smaller seminiferous tubule diameter and a lower ratio between type B spermatogonia and type A stem spermatogonia in Kit(W-lacZ/+) mice compared with Kit(+/+) mice. These differences are probably related to the Kit haplodeficiency, which was the only difference between the two genotypes. Germ cell counts and testicular cell ploidy revealed delayed meiosis in Kit(W-lacZ/+) mice. Reporter gene expression confirmed expression of the Kit gene at the spermatogonial stage and also revealed Kit expression during the late pachytene/diplotene transition. These results suggest involvement of Kit/SCF at different stages of spermatogenesis.     

Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes

Retinoids have pleiotropic effects on embryonic development and are essential for spermatogenesis in the adult, where they act via nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We used immunohistochemistry to examine the cellular localization of RARs and RXRs in the rat testis from Day 13.5 postconception (13.5 dpc) until Day 8 postpartum (8 dpp), and these findings were compared with those for immature and adult testes. RARalpha and RARbeta were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20. 5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RARgamma from 8 dpp. Immunoreactivity for RXRalpha was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRbeta was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXRgamma gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RARgamma and RXRgamma, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.     

Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats

Under normal condition in the most mammals, spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. The present study was designed to determine the effects of ghrelin treatment on in vivo quality and quantity expression of apoptosis and proliferation specific indices in rat testicular germ cells. Twenty eight adult normal rats were subdivided into equal control and treatment groups. Treatment group received 3 nmol of ghrelin as subcutaneous injection for 30 consecutive days or vehicle to the control animals. The rats from each group (n=7) were killed on days 10 and 30 and their testes were taken for immunocytochemical evaluation and caspase-3 assay. Immunohistochemical analysis indicated that the accumulations of Bax and PCNA peptides are generally more prominent in spermatocytes and spermatogonia of both groups. Likewise, the mean percentage of immunoreactive spermatocytes against Bax increased (P<0.01) in the ghrelin-treated group on day 10, while despite of 30% increment in the Bax level of spermatocytes in the treated rats on day 30, however, it was not statistically significant. During the experimental period, only a few spermatogonia represented Bax expression and the changes of Bax immunolabling cells were negligible upon ghrelin treatment. Likewise, there were immunostaining cells against Bcl-2 in each germ cell neither in the control nor in the treated animals. In fact, ghrelin balanced Bax/Bcl-2 ratio toward at increase of Bax level in the spermatocytes and therefore may stimulate apoptosis in these germ cells. In contrast, ghrelin administration significantly suppressed proliferation-associated peptide PCNA in the spermatocytes as well as spermatogonia (P<0.05). Whereas, caspase-3 activity did not show any marked alteration during the experiment in both groups (P>0.05). Upstream of Bax substance parallel to down-regulation of PCNA demonstrate that ghrelin may prevent massive accumulation of germ cells during normal spermatogenesis. These observations also indicate that ghrelin may be considered as a modulator of spermatogenesis in normal adult rats and could be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors.     

Long glucocorticoid-induced leucine zipper (L-GILZ) protein interacts with ras protein pathway and contributes to spermatogenesis control

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.     

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.