Concomitant loss of proapoptotic BH3-only Bcl-2 antagonists Bik and Bim arrests spermatogenesis

The BH3-only proteins of the Bcl-2 family initiate apoptosis through the activation of Bax-like relatives. Loss of individual BH3-only proteins can lead either to no phenotype, as in mice lacking Bik, or to marked cell excess, as in the hematopoietic compartment of animals lacking Bim. To investigate whether functional redundancy with Bim might obscure a significant role for Bik, we generated mice lacking both genes. The hematopoietic compartments of bik-/-bim-/- and bim-/- mice were indistinguishable. However, although testes develop normally in mice lacking either Bik or Bim, adult bik-/-bim-/- males were infertile, with reduced testicular cellularity and no spermatozoa. The testis of young bik-/-bim-/- males, like those lacking Bax, exhibited increased numbers of spermatogonia and spermatocytes, although loss of Bik plus Bim blocked spermatogenesis somewhat later than Bax deficiency. The initial excess of early germ cells suggests that spermatogenesis fails because supporting Sertoli cells are overwhelmed. Thus, Bik and Bim share, upstream of Bax, the role of eliminating supernumerary germ cells during the first wave of spermatogenesis, a process vital for normal testicular development.     

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.     

Deciphering the pathways of germ cell apoptosis in the testis

A growing body of evidence demonstrates that germ cell death both spontaneous (during normal spermatogenesis) and that induced by suppression of hormonal support or increased scrotal temperature occurs via apoptosis. The mechanisms by which these proapoptotic stimuli activate germ cell apoptosis are not well understood. In order to provide some insight, here we report the key molecular components of the effector pathways leading to caspase activation and increased germ cells apoptosis triggered by mildly increased scrotal temperature. Short-term exposure (43 °C for 15 min) of the testis to mild heat results, within 6 h, in stage- and cell-specific activation of germ cell apoptosis in rats. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum (ER) into paranuclear areas, cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP. Furthermore, Bax is co-localized with ER in the susceptible germ cells as assessed by combined two-photon and confocal microscopy and Western blot analyses of fractionated testicular lysates. In additional studies, using gld and lpr^cg mice, which harbor loss-of-function mutations in Fas-ligand (FasL) and Fas, respectively, we demonstrated that heat-induced germ cell apoptosis is not blocked, thus providing further evidence that the Fas signaling system is dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.     

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.     

An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.     

Effect of the W mutation, for white belly spot, on testicular germ cell differentiation in mice.

The effect of the mutation for white belly spot controlled by the dominant gene W on spermatogenesis in mice was examined by experimental cryptorchidism and its surgical reversal. The course of spermatogenesis from spermatogonia to spermatid was normal in intact testes of W/+ mice. In cryptorchid testes, there was no difference in the number and activity of Type A spermatogonia between the testes of W/+ and +/+ mice, in mitotic and labelling indices. Although surgical reversal of the cryptorchid testis resulted in regenerative differentiation of germ cells in both genotypes, the recovery of cell differentiation in the W/+ testis was slower than in the +/+ testis. There were fewer germ cells, such as intermediate-Type B spermatogonia or more advanced ones, in W/+ testes. On Day 17 after surgical reversal, cell associations in W/+ testes were abnormal and the numbers of intermediate-Type B spermatogonia, spermatocytes and spermatids were approximately 70, 50 and 15%, respectively, of those in +/+ testes. These results indicate that the W gene affects spermatogenic cell differentiation in adult mice.     

The inflammatory mediators TNFα and nitric oxide arrest spermatogonia GC-1 cell cycle

During an inflammatory process of the testis, the network of somatic, immune, and germ cell interactions is altered leading to organ dysfunction. In testicular biopsies of infertile men, spermatogenesis impairment is associated with reduced spermatogonia proliferation, increased number of immune cells, and content of pro-inflammatory cytokines. TNFα-TNFR and nitric oxide (NO)-NO synthase systems are up-regulated in models of testicular damage and in human testis with maturation arrest. The purpose of this study was to test the hypothesis that TNFα-TNFR system and NO alter the function of spermatogonia in the inflamed testis. We studied the effect of TNFα and NO on GC-1 spermatogonia cell cycle progression and death by flow cytometry. GC-1 cells expressed TNFR1 and TNFR2 (immunofluorescence). TNFα (10 and 50 ng/ml) and DETA-Nonoate (0.5 and 2 mM), a NO releaser, increased the percentage of cells in S-phase of the cell cycle and reduced the percentage in G1, inducing also cell apoptosis. TNFα effect was not mediated by oxidative stress unlike NO, since the presence of N-acetyl-l-cysteine (2.5 and 5.0 mM) prevented NO induced cell cycle arrest and death. GC-1 spermatogonia overpass NO induced cell cycle arrest but no TNFα, since after removal of NO, spermatogonia progressed through the cell cycle. We propose TNFα and NO might contribute to impairment of spermatogenesis by preventing adequate functioning of the spermatogonia population. Our results showed that TNFα and NO impaired spermatogonia cell cycle, inducing GC-1 arrest in the S phase.     

The testicular cycle of Gambusia affinis holbrooki (Teleostei: Poeciliidae)

Fifteen male mosquito fish ( Gambusia affinis holbrooki ) were collected in 1989 on the 15th of each month to perform a quantitative histologic study of the annual testicular cycle including a calculation of the gonadosomatic index, testicular volume, and the total volume per testis occupied by each germ cell type. The cycle comprises two periods: spermatogenesis and quiescence. The spermatogenic period begins in April with the development of primary spermatogonia into secondary spermatogonia, spermatocytes and round spermatids. In May, the first spermatogenic wave is completed and the testicular volume begins to increase up to June when the maximum testicular volume and gonadosomatic index are reached. Germ cell proliferation with successive spermatogenetic waves continues until August. In September germ cell proliferation ceases and neither secondary spermatogonia nor spermatocytes are observed. However, spermiogenesis continues until October. In November, spermiogenesis has stopped and the testis enters the quiescent period up to April. During this period only primary spermatogonia and spermatozoa are present in the testis. In addition, a few spermatids whose spermiogenesis was arrested in November are observed. Testicular release of spermatozoa is continuous during the entire spermatogenesis period. The spermatozoa formed at the end of this period (September-October) remain in the testis during the quiescent period and are released at the beginning of the next spermatogenesis period in April. Developed Leydig cells appear all year long in the testicular interstitium, mainly around both efferent ducts and the testicular tubule sections showing S₄ spermatids.     

Recombinant human insulin-like growth factor I stimulates all stages of 11-ketotestosterone-induced spermatogenesis in the Japanese eel, Anguilla japonica, in vitro.

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-ketotestosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type B spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.     

An overview of cell renewal in the testis throughout the reproductive cycle of a seasonal breeding teleost, the gilthead seabream (Sparus aurata L)

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the testicular regression process that occurs in both seasonal testicular involution and sex change. Insofar as fish reproduction is concerned, little is known about cell renewal and elimination during the reproductive cycle of seasonal breeding teleosts with asynchronous spermatogenesis. We have previously described how acidophilic granulocytes infiltrate the testis during postspawning where, surprisingly, they produce interleukin-1beta, a known growth factor for mammalian spermatogonia, rather than being directly involved in the elimination of degenerative germ cells. In this study, we are able to discriminate between spermatogonia stem cells and primary spermatogonia according to their nuclear and cytoplasmic diameters and location in the germinal epithelium, finding that these two cell types, together with Sertoli cells, proliferate throughout the reproductive cycle with a rate that depends on the reproductive stage. Thus, during spermatogenesis the spermatogonia stem cells, the Sertoli cells, and the developing germ cells (primary spermatogonia, A and B spermatogonia, and spermatocytes) in the germinal compartment, and cells with fibroblast-shaped nuclei in the interstitial tissue proliferate. However, during spawning, the testis shows few proliferating cells. During postspawning, the resumption of proliferation, the occurrence of apoptotic spermatogonia, and the phagocytosis of nonshed spermatozoa by Sertoli cells lead to a reorganization of both the germinal compartment and the interstitial tissue. Finally, the proliferation of spermatogonia increases during resting when, unexpectedly, both oogonia and oocytes also proliferate. This proliferative pattern was correlated with the gonadosomatic index, testicular morphology, and testicular and gonad areas, suggesting that complex mechanisms operate in the regulation of gonocyte proliferation in hermaphrodite fish.     

Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.)

Spermatogenesis in male Atlantic halibut (Hippoglossus hippoglossus L.) was investigated by sampling blood plasma and testicular tissue from 15-39-month-old fish. The experiment covered a period in which all fish reached puberty and completed sexual maturation at least once. The germinal compartment in Atlantic halibut testis appears to be organized in branching lobules of the unrestricted spermatogonial type, because spermatocysts with spermatogonia were found throughout the testis. Spermatogenesis was characterized histologically, and staged according to the most advanced type of germ cell present: spermatogonia (Stage I), spermatogonia and spermatocytes (Stage II), spermatogonia, spermatocytes and spermatids (Stage III), spermatogonia, spermatocytes, spermatids and spermatozoa (Stage IV), and regressing testis (Stage V). Three phases could be distinguished: first, an initial phase with low levels of circulating testosterone (T; quantified by RIA) and 11-ketotestosterone (11-KT; quantified by ELISA), spermatogonial proliferation, and subsequently the initiation of meiosis marked by the formation of spermatocytes (Stage I and II). Secondly, a phase with increasing T and 11-KT levels and with haploid germ cells including spermatozoa present in the testis (Stage III and IV). Thirdly, a phase with low T and 11-KT levels and a regressing testis with Sertoli cells displaying signs of phagocytotic activity (Stage V). Circulating levels of 11-KT were at least four-fold higher than those of T during all stages of spermatogenesis. Increasing plasma levels of T and 11-KT were associated with increasing testicular mass throughout the reproductive cycle. The absolute level of, or the relation between, testis growth and circulating androgens were not significantly different in first time spawners compared to fish that underwent their second spawning season. These results provide reference levels for Atlantic halibut spermatogenesis.     

BARD1 expression during spermatogenesis is associated with apoptosis and hormonally regulated

The BRCA1-binding RING-finger domain protein BARD1 may act conjointly with BRCA1 in DNA repair and in ubiquitination, but it may also induce apoptosis in a BRCA1-independent manner. In this study, we have investigated BARD1 expression during spermatogenesis. In contrast with BRCA1, which is expressed only in meiotic spermatocytes and early round spermatids, BARD1 is expressed during all stages of spermatogenesis. However, while spermatogonia expressed full-length BARD1 mRNA, later stages of spermatocyte precursors express predominantly a novel, shorter splice form BARD1beta. BARD1beta lacks the BRCA1-interacting RING finger but maintains its proapoptotic activity. Consistently, BRCA1 can counteract the proapoptotic activity of full-length BARD1 but not of BARD1beta. Several lines of evidence suggest that BARD1 is involved in proapoptotic signaling in testis: i) both BARD1 isoforms are mostly found in cells that stain positive for TUNEL, Bax, and activated caspase 3; ii) BARD1beta, capable of inducing apoptosis even in the presence of BRCA1, is specifically expressed in BRCA1-positive later stages of spermatogenesis; iii) antiapoptotic hormonal stimulation leads to BARD1 downregulation; and iv) BARD1 expression is associated with human pathologies causing sterility due to increased germ cell death. Our data suggest that full-length BARD1 might be involved in apoptotic control in spermatogonia and primary spermatocytes, while a switch to the BRCA1-independent BARD1beta might be necessary to induce apoptosis in BRCA1-expressing meiotic spermatocytes and early round spermatids.     

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.     

Apoptose et spermatogenèse

Onset of spermatogenesis is associated with a wave of apoptosis, which limits its efficacy during the first cycles in most mammals. After the first cycles, the actual efficacy of spermatogenesis always remains below the theoretical yield. Among the germinal cells, spermatogonia are the main targets of physiological apoptosis. This physiological apoptosis partly depends on the relationships between germ cells and Sertoli cells. The impact of the Sertoli cell/germ cell number ratio on the efficacy of spermatogenesis is well accepted, the concept of density-dependent regulation in the seminiferous tubule was proposed in the early eighties. Since the steps of spermatogenesis require a continuous progression of the cell cycle rather than an arrest, germ cells might therefore be more sensitive to apoptosis. This may also lead to severe disturbances between proliferation and cell death. The first experiments designed to elucidate the mechanisms of germ cell apoptosis were based on hormonal deprivation or cryptorchidism. However, the link between hormonal or cellular action and cell survival remained to be established. Analysis of signal transduction pathways involved in germ cell apoptosis and their regulation were the next steps. The involvement of bcl-2 family genes has been confirmed, although the expression of some of its members remains more controversial. Data derived from overexpression of some genes (Bcl-2, Bcl-xl) or resulting from gene inactivation (Bax) at the testicular level have highlighted the role of these genes in the control of germ cell apoptosis and have also provided some evidence for the strict requirement for density-dependent regulation of spermatogenesis. More recently, variations in the pattern of expression of these genes or proteins helped to explain some of the discrepancies in the literature. The place of the Fas/Fas ligand system during the first cycle of spermatogenesis remains a matter of debate, with controversies concerning the precise site of expression of this oncogene and its receptor. Conversely, its role in the testis after chemotoxic or radiotoxic treatments is well established. However, the normal fertility of animals with a spontaneous inactivation of Fas or Fas L genes does not support a physiological role of these factors during spermatogenesis. While factors involved in TNF/TNF R1 (Tumor Necrosis Factor) are under study, some data have been reported concerning the role of TRAIL (TNFalpha Related Apoptosis Inducing Ligand) and its active or decoy receptors in the testis. Among the oncogenes which may modulate the apoptotic process, Kit/Stem Cell Factor is particularly interesting, as Kit is expressed in some germ cells and Leydig cells, whereas SCF is expressed by Sertoli cells. Its impact during gonadal development and in the survival and proliferation of differentiated spermatogonia has been clearly established. Using a transgenic mice model, in which the Kit gene was inactivated by the insertion of a nls-lacZ sequence in its first exon, we showed that one single copy of the gene was unable to sustain physiological spermatogenesis and fertility in male mice. Our results also suggest that the Kit gene might be expressed at different steps of spermatogenesis, with different signal transduction pathways and biological actions. Finally, analysis of the signal transduction pathways involved in testicular apoptosis and their mechanisms of control is one of the key steps to a better understanding of both impairment of spermatogenesis and the pathogenesis of certain germ cell tumours.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

Overexpression of Bcl-W in the testis disrupts spermatogenesis: revelation of a role of BCL-W in male germ cell cycle control

To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken beta-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5'-bromo-2'deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.