Spermatogonial Apoptosis Research Articles

Disrupted sperm mirna expression profiles revealed a fingerprint of impaired spermatogenesis in oligozoospermia males

MicroRNAs (miRNAs) are short, single stranded noncoding RNA molecules that function as post-transcriptional regulators inhibiting mRNA translation. Sperm microRNAs have been recognized as playing a key role in testicular function. However, there are limited studies investigating the importance of miRNAs in male factor infertility. The aim of this study was to correlate specific sperm miRNA expression in association with male factor infertility diagnoses to further the understanding of miRNAs in impaired spermatogenesis. Research study. Sperm samples were donated with patient consent: oligozoospermia (<10 million/ml; n=7; mean paternal age = 37.7 years), 0% normal morphology (Kruger strict criteria; n=6; mean paternal age = 41.7 years), and normozoospermic controls (n=7; mean paternal age = 38.1 years). miRNA was isolated from individual sperm samples (n=19) to determine the expression profiles of 10 specific miRNAs using qPCR with Taqman® miRNA assays (Thermo Scientific). Statistical analysis was performed with REST© software (Qiagen), significance at P<0.05. There were no significant differences observed between the groups for paternal age. None of the 10 specific miRNAs displayed significant differences in expression levels for the 0% normal morphology group compared to normozoospermic controls or oligozoospermia males. Conversely, oligozoospermia males displayed altered sperm miRNA expression profiles compared to normozoospermic controls, specifically, 3 miRNAs (122, 449 and 15b) exhibited decreased expression (P<0.05), while 2 miRNAs (141 and 200a) showed increased expression (P<0.05). Target gene analysis of these altered miRNAs identified several important spermatogenesis genes including, among others BCL2 (a regulator of spermatogonial apoptosis) and NOTCH1 (differentiation and survival of germ cells). Interestingly, both upregulated miRNAs, miR-141 (6.02 fold; P<0.05) and 200a (3.05 fold; P<0.05), are known suppressors of sperm associated antigen 9 (SPAG9), which is a critical gene located in the acrosomal compartment that has been implicated in capacitation and the sperm-oocyte interaction. Significant suppression of SPAG9 expression could represent one molecular mechanism contributing to compromised spermatogenesis in oligozoospermic males. MiRNAs function post-transcriptionally to regulate downstream target gene expression typically by inhibiting or repressing mRNA translation. Oligozoospermia displayed significant disruptions to specific sperm miRNA expression profiles revealing a fingerprint of impaired spermatogenesis resulting in low sperm concentration. In contrast, it would appear that for these specific miRNAs there was no association with abnormal sperm morphology. Determining the underlying mechanisms associated with male factor infertility will assist in improving clinical diagnosis and patient management during infertility treatment.

Effects of cadmium exposure on Iberian ribbed newt (&lt;i&gt;Pleurodeles waltl&lt;/i&gt;) testes

To characterize the histomorphologic effects of cadmium on adult newt testes, male Iberian ribbed newts (6 months post-hatching) were intraperitoneally exposed to a single dose of 50 mg/kg of cadmium, with histologic analysis of the testes at 24, 48, 72, and 96 h. Beginning 24 h after cadmium exposure, apoptosis of spermatogonia and spermatocytes was observed, and congestion was observed in the interstitial vessels of the testes. Throughout the experimental period, the rates of pyknotic cells and TUNEL and cleaved caspase-3 positivity were significantly higher in the spermatogonia and spermatocytes of cadmium-treated newts compared with control newts. There were no significant differences between cadmium-treated and control newts in phospho-histone H3 positivity in the spermatogonia and spermatocytes. These results suggest that spermatogonia and spermatocytes in adult Iberian ribbed newts are highly sensitive to cadmium. This is the first report of the histomorphologic characteristics of cadmium-induced testicular dysfunction in newts.

Testicular germ cells apoptosis following exposure to chronic stress in rats

ObjectiveTo evaluate the effects of chronic stress on testosterone hormone level and germ cells apoptosis in testes and the inhibitory role of glucocorticoid receptor antagonist, RU486. MethodsAdult male Sprague–Dawley rats were randomly assigned to four groups (n = 6): control; stress; RU486; stress/RU486. Animals in RU486 and stress/RU486 subjected to subcutaneous injections of 2.5 mg RU486/kg 1 h before stress session. Rats were submitted to chronic restraint stress (1 h daily for 12 consecutive days) whereas control animals were not subjected to stress. Serum testosterone assay was performed and the occurrence of DNA fragmentation in the testis sections was examined using TUNEL staining. ResultsChronic restraint stress significantly induced a decrease in serum testosterone level with increase in apoptosis in spermatogonia. Systemic administration of RU486 significantly restored serum testosterone levels and attenuates stress-induced apoptosis in spermatogonia. ConclusionThe restraint stress-induced change in serum testosterone levels and seminiferous tubules apoptosis closely associated with the glucocorticoid receptor.

Alteration of protein prenylation promotes spermatogonial differentiation and exhausts spermatogonial stem cells in newborn mice.

Spermatogenesis in adulthood depends on the successful neonatal establishment of the spermatogonial stem cell (SSC) pool and gradual differentiation during puberty. The stage-dependent changes in protein prenylation in the seminiferous epithelium might be important during the first round of spermatogenesis before sexual maturation, but the mechanisms are unclear. We have previous found that altered prenylation in Sertoli cells induced spermatogonial apoptosis in the neonatal testis, resulting in adult infertility. Now we further explored the role of protein prenylation in germ cells, using a conditional deletion of geranylgeranyl diphosphate synthase (Ggpps) in embryonic stage and postmeiotic stage respectively. We observed infertility of Ggpps−/− Ddx4-Cre mice that displayed a Sertoli-cell-only syndrome phenotype, which resulted from abnormal spermatogonial differentiation and SSC depletion during the prepubertal stage. Analysis of morphological characteristics and cell-specific markers revealed that spermatogonial differentiation was enhanced from as early as the 7th postnatal day in the first round of spermatogenesis. Studies of the molecular mechanisms indicated that Ggpps deletion enhanced Rheb farnesylation, which subsequently activated mTORC1 and facilitated spermatogonial differentiation. In conclusion, the prenylation balance in germ cells is crucial for spermatogonial differentiation fate decision during the prepubertal stage, and the disruption of this process results in primary infertility.

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis.

SummaryCell cycle control during spermatogenesis is a highly complex process owing to the control of the mitotic expansion of the spermatogonial cell population and following meiosis, induction of DNA breaks during meiosis and the high levels of physiological germ‐cell apoptosis. We set out to study how E2F1, a key controller of cell cycle, apoptosis, and DNA damage responses, functions in the developing and adult testis. We first analyzed the expression pattern of E2f1 during post‐natal testis development using RNA in situ hybridization, which showed a differential expression pattern of E2f1 in the adult and juvenile mouse testes. To study the function of E2f1, we took advantage of the E2F1−/− mouse line, which was back‐crossed to C57Bl/6J genetic background. E2f1 loss led to a severe progressive testicular atrophy beginning at the age of 20 days. Spermatogonial apoptosis during the first wave of spermatogenesis was decreased. However, already in the first wave of spermatogenesis an extensive apoptosis of spermatocytes was observed. In the adult E2F1−/− testes, the atrophy due to loss of spermatocytes was further exacerbated by loss of spermatogonial stem cells. Surprisingly, only subtle changes in global gene expression array profiling were observed in E2F1−/− testis at PND20. To dissect the changes in each testicular cell type, an additional comparative analysis of the array data was performed making use of previously published data on transcriptomes of the individual testicular cell types. Taken together, our data indicate that E2F1 has a differential role during first wave of spermatogenesis and in the adult testis, which emphasizes the complex nature of cell cycle control in the developing testis.

Persistent DNA Damage in Spermatogonial Stem Cells After Fractionated Low-Dose Irradiation of Testicular Tissue

Testicular spermatogenesis is extremely sensitive to radiation-induced damage, and even low scattered doses to testis from radiation therapy may pose reproductive risks with potential treatment-related infertility. Radiation-induced DNA double-strand breaks (DSBs) represent the greatest threat to the genomic integrity of spermatogonial stem cells (SSCs), which are essential to maintain spermatogenesis and prevent reproduction failure. During daily low-dose radiation with 100mGy or 10mGy, radiation-induced DSBs were monitored in mouse testis by quantifying 53 binding protein 1 (53BP-1) foci in SSCs within their stem cell niche. The accumulation of DSBs was correlated with proliferation, differentiation, and apoptosis of testicular germ cell populations. Even very low doses of ionizing radiation arrested spermatogenesis, primarily by inducing apoptosis in spermatogonia. Eventual recovery of spermatogenesis depended on the survival of SSCs and their functional ability to proliferate and differentiate to provide adequate numbers of differentiating spermatogonia. Importantly, apoptosis-resistant SSCs resulted in increased 53BP-1 foci levels during, and even several months after, fractionated low-dose radiation, suggesting that surviving SSCs have accumulated an increased load of DNA damage. SSCs revealed elevated levels of DSBs for weeks after radiation, and if these DSBs persist through differentiation to spermatozoa, this may have severe consequences for the genomic integrity of the fertilizing sperm.

Male Infertility: Role of Cellular, Biochemical and Molecular Events

Infertility is a unique category of human functional disability, where the fertility disturbances of one partner may only become evident through the other partner’s problem, while optimal reproductive function in one partner may compensate for impaired function in the other. It is estimated that approximately 15% of married couples are infertile. Male factor infertility contributes to about 50% manifested as quantitative abnormality (azoospermia, cryptozoospermia and oligozoospermia), or as qualitative abnormality (asthenospermia, teratozoospermia and necrospermia) or both. Investigation of apoptosis in spermatogonia, spermatocytes and spermatids in the testis has been done extensively and many apoptotic factors have been identified. Testicular apoptosis has been reported in human specimens, but its correlation with serum gonadotropins and testosterone levels is not clear. It is assumed that somatic cells can die in the apoptotic, the autophagic, or the necrotic way but the mechanisms involved in the sperm death are obscure and the biological significance of apoptosis in ejaculated sperm is yet to be elucidated. Thus the goal of this article is to identify and discuss common themes in mitochondrial function and the apoptotic pathway related to mammalian reproduction. Apart from this finding the correlation of apoptosis with serum gonadotropins and testosterone levels will also help in correlating the processes taking place in spermatozoa responsible for male infertility.

Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection

Mumps commonly affects children 5-9 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cell-only syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients' fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-κB signaling pathways were constitutively activated in GGPPS(-/-) Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS(-/-) Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility.

Unilateral Cryptorchidism in Mice Mutant forPtgds

The pathophysiology of cryptorchidism, abnormal testicular descent, remains poorly understood. In this study, we show that both heterozygous and homozygous mice deficient for lipocalin-type prostaglandin D(2) (PGD(2) ) synthase (Ptgds) presented unilateral cryptorchidism affecting the second phase of testicular descent in 16% and 24% of cases, respectively. The adult cryptorchid testes show an increase in spermatogonia apoptosis along with a global decrease in the tubule size parameters, whereas the gubernaculum of newborn mutants present some histological abnormalities. Disruption of the inguinoscrotal phase did not present impairment of the androgen pathway but rather a decrease in Rxfp2 mRNA expression in the gubernaculum. These observations led us to investigate the role of the PGD(2) signaling pathway in human testicular migration through PTGDS sequencing of DNA from 29 children with cryptorchidism. However, none of the investigated cases presented mutations in the PTGDS gene. Nevertheless, our results identify the PTGDS enzyme as a novel component in the cryptorchidism puzzle.

Pum 1 sequesters apoptosis during spermatogenesis

RNA binding proteins have long been known to play a role in spermatogenesis. The laboratory of Norman Hecht was one of the leaders in this field, having characterized three RNA binding proteins that regulate mRNAs during sperm differentiation. Polypyrimidine tract-binding protein 2 (PTB2) has two functions—stabilizing mRNA transcripts in the cytoplasm and being involved in splicing.1 Using in vivo cross-linking of protein to RNA, several mRNA and small RNA targets were identified in the testis.2 Translin is another protein that both stabilizes mRNAs from a specific transcription factor, and is associated with small RNAs.3 Perhaps the most interesting is the DNA/RNA binding protein MSY2. At low concentrations, MSY2 activates transcription, but at higher concentrations, the protein stabilizes the mRNAs that it had previously activated.4 This plays a particularly useful role in spermiogenesis as it stabilizes and stores the transcripts of several genes that are required long after transcription has stopped. MYSY2 has also recently been shown to bind specifically to small RNAs 25–33 nt in length, suggesting a broader role for this protein than is currently known.5 Recently, Chen et al.6 have added another protein to the known group of RNA-binding proteins that are involved in spermatogenesis. These authors had been interested in a Drosophila protein, Pum, which post-transcriptionally regulates several genes in the germline. In their current work, they performed a detailed study of the mouse homologue PUM1. They first showed that it is highly expressed in the testis, though other tissues had significant levels of PUM1. In the testis, immunocytochemical localization indicated that Pum 1 is expressed in the cytoplasm of spermatocytes and spermatids. Pum 1 knockout males have reduced sperm counts and reduced litter sizes, but they remain fertile. The testes of Pum1−/− mice show much higher levels of apoptosis in spermatogonia, and reduced levels of spermatogenesis. The authors then used a genome wide RNP-Chip assay to identify RNAs that were specifically associated with PUM1 in the testis. They identified 3687 transcripts that represented 1527 genes. These genes were then analyzed by MetaCore to identify 11 biochemical pathways that were enriched in the 1527 genes. One of these was a pathway that regulates p53 that included nine of the genes identified as binding to PUM1. They then confirmed that all nine proteins were increased by Western blots. To confirm that PUM1 has a regulatory effect on the p53 pathway, they crossed Pum1−/− mice with a line that has a mutant p53 gene and showed that the apoptosis in the testis was reduced. The authors conclude that spermatogonia are normally removed by apoptosis during spermatogenesis, but that this process must be regulated; otherwise, too many of the spermatogonia will be lost. PUM1 is a strong candidate for at least some of this regulation, and its mode of action is through mRNA binding. However, in contrast to MSY2, for example, PUM1 seems to inhibit translation of the mRNAs permanently, rather than stabilizing the mRNA for translation at its appropriate time. As demonstrated by the results from both groups, the issue of how RNA-binding proteins regulate spermatogenesis is far from understood. It is likely that MSY2 and PTB2 play a role in regulating small RNAs, but understanding how they do this will have to evolve as the still new field of micro RNA continues to explode. For PUM1, the immediate question is how does its interaction with the other 8 pathways and 1518 genes contribute to spermatogenesis? It would also be interesting to know whether the Pum1−/− mice crossed with the mutant p53 mice had restored fertility. If not, this might be an important model for understanding other roles PUM1 has in male fertility. Another question is whether PUM1, like MSY2 and PTB2, also binds to non-coding small RNAs and plays a role in regulating these important cell modifiers. This work highlights, once again, the importance of RNA-binding proteins during spermiogenesis. It is likely that we have still only scratched the surface on this important molecular process.

Benzo[a]pyrene Reduces Testosterone Production in Rat Leydig Cells via a Direct Disturbance of Testicular Steroidogenic Machinery

Background: Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is a ubiquitous environmental pollutant that is currently suspected of being an endocrine disruptor. The testis is an important target for PAHs, yet insufficient attention has been paid to their effects on steroidogenesis in Leydig cells.Objective: We hypothesized that long-term exposure to low concentrations of B[a]P might disrupt testosterone production in Leydig cells via an alteration of steroidogenic proteins.Results: Oral exposure to B[a]P reduced serum and intratesticular fluid testosterone levels in rats. However, we did not observe serious testicular atrophy or azoospermia, although spermatogonial apoptosis was significantly increased. Compared with control cells, Leydig cells primed with B[a]P in vivo produced less testosterone in response to human chorionic gonadotropin (hCG) or dibutyl cyclic adenosine monophosphate in vitro. Of note, the reduction of testosterone levels was accompanied by decreased expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD), as well as increased levels of cytochrome P450 side chain cleavage (P450scc), in Leydig cells. The up-regulation of P450scc expression after exposure to B[a]P appears to be associated with a compensatory mechanism for producing the maximum amount of pregnenolone with the minimum amount of transported cholesterol by StAR; the down-regulation of 3β-HSD may occur because B[a]P can negatively target 3β-HSD, which is required for testosterone production.Conclusions: B[a]P exposure can decrease epididymal sperm quality, possibly by disturbing testosterone levels, and StAR may be a major steroidogenic protein that is targeted by B[a]P or other PAHs.

Exposure to Retinoic Acid in the Neonatal but Not Adult Mouse Results in Synchronous Spermatogenesis1

Retinoic acid (RA) is required for germ cell differentiation, the regulation of which gives rise to a constant production of mature sperm. In testes from 3-day postpartum (dpp) RARE-hsplacZ mice, periodic regions positive for beta-galactosidase activity were observed along the length of the seminiferous tubules. Periodicity was abolished by treatment of neonates with exogenous RA at 2 dpp. To assess the consequences, 2-dpp mice were treated with RA, and the long- and short-term effects were assessed. Long-term effects of neonatal RA exposure included a delay in the appearance of advanced germ cells and the absence of a spermatogenic wave (synchronous spermatogenesis) in the adult. In contrast, RA exposure in vitamin A-sufficient adults did not result in synchronous spermatogenesis but rather induced apoptosis in a subset of spermatogonia. Shortly after (24 h) neonates were exposed, altered expression of known germ cell differentiation and the (Stra8, Kit, Sycp3, and Rec8) meiosis markers and an increase in the number of STRA8 and SYCP3 immunopositive cells were observed relative to those of vehicle controls. However, 48 and 72 h after exposure, a significant reduction in the number of STRA8 and SYCP3 immunopositive cells occurred. Immunohistochemical analysis of a marker for apoptosis demonstrated neonatal exposure resulted in increased germ cell apoptosis, as observed in the adult. Additionally, RA exposure resulted in increased Cyp26a1 expression of the RA-degrading enzyme. Thus, while RA treatment of neonatal and adult mice resulted in apoptosis of spermatogonia, synchronous spermatogenesis occurred only after neonatal RA exposure.

Reduced Expression of an RNA-binding Protein by Prolactin Leads to Translational Silencing of Programmed Cell Death Protein 4 and Apoptosis in Newt Spermatogonia

Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3'-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3'-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.

GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules

BackgroundApoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules.ResultsStable mRHBDD1 knockdown GC-1 cells were sensitive to apoptotic stimuli, PS341 and UV irradiation. In vitro, they survived and proliferated normally. However, they lost the ability to survive and differentiate in mouse seminiferous tubules.ConclusionOur findings suggest that mRHBDD1 may be associated with mammalian spermatogenesis.

Photoperiod-Modulated Testis Maturation in Atlantic Cod (Gadus morhua, L.)1

Precocious male puberty is a significant problem in Atlantic cod aquaculture. While photoperiod manipulation can inhibit testis growth, a detailed analysis of effects on spermatogenesis is missing. Starting July 1, 2004, prepubertal fish were exposed to different photoperiod regimens in indoor tanks for 17 mo. Testis histology, germ cell dynamics (proliferation and apoptosis), and plasma androgen levels were analyzed. In the natural light (NL) group, testis growth started in September 2004 and was completed in February 2005, when a 2-mo spawning period started. In the constant light (LL) group, none or very few spermatogenic cysts were recruited into spermatogenesis, and apoptotic germ cell loss was high. A change of photoperiod from NL to LL at winter solstice (December 21, 2004) resulted in premature (2 mo) completion of the reproductive cycle, while changing from LL to NL at winter solstice triggered faster than normal testis development. Plasma testosterone levels increased in the NL group from spermatogonial proliferation toward meiosis, while those of 11-ketotestosterone increased toward spermiogenesis and spermiation. Plasma androgen levels did not rise under LL conditions. Comparing fish with developing testes from all groups indicated that low androgen levels were associated with a high incidence of spermatogonial apoptosis; we also found that androgen receptor mRNA expression was most prominent in Sertoli cells in contact with growing spermatogonial clones. Our data show that an inhibitory photoperiod (LL) reduced or blocked differentiation of spermatogonia, increased apoptosis (particularly among proliferating spermatogonia), and was associated with reduced androgen levels, a situation possibly reflecting insufficient gonadotropic stimulation.

Pathways involved in testicular germ cell apoptosis in immature rats after FSH suppression

FSH is a key regulator of testis function, required for the establishment of full complements of Sertoli and germ cells during postnatal testis development and for the maintenance of spermatogenesis in the adult. FSH plays an important role in germ cell survival rather than proliferation, in the window between 14 and 18 days of testicular development, which coincides with the cessation of Sertoli cell proliferation and the onset of germ cell meiosis during the first wave of spermatogenesis. This study aimed to identify the pathway(s) of apoptosis regulated by changes in FSH levels in 14 - to 18-day-old rats, using a model of in vivo FSH suppression by passive immunoneutralization with a rat anti-FSH antibody. Apoptotic pathways were identified by immunohistochemistry using pathway-specific proteins as markers of the intrinsic (activated caspase 9) and extrinsic (activated caspase 8) pathways, followed by quantification of cell numbers using stereological techniques. In addition, RT-PCR was used to assess the expression of pathway-specific genes. We previously reported a 2.5-fold increase in spermatogonial apoptosis in these samples after 4 days of FSH suppression, and now show that this increase correlates with a 9.8-fold (P<0.001) increase in the frequency of caspase 9-positive spermatogonia in the absence of caspase 8 immunoreactivity. By contrast, spermatocytes exhibited both increased caspase 9 (7.5-fold; P<0.001) and caspase 8 (5.7 fold; P<0.001) immunoreactivities after 4 days of FSH suppression. No significant change in the transcription levels of candidate genes required for either pathway was detected. This study demonstrates that, in the seminiferous tubules, FSH suppression induces spermatogonial apoptosis predominantly via the intrinsic pathway, while spermatocyte apoptosis occurs via both the intrinsic and extrinsic pathways.

Follicle-Stimulating Hormone Affects Spermatogonial Survival by Regulating the Intrinsic Apoptotic Pathway in Adult Rats1

Follicle-stimulating hormone plays a key role in spermatogonial development in adult rats via poorly understood mechanisms. We aimed to identify the role of this hormone in the regulation of germ cell apoptosis and proliferation in adult rats by suppression of FSH action following passive immunoneutralization with a rat FSH antibody for 4 and 7 days. Apoptosis and proliferation were identified by TUNEL and proliferating cell nuclear antigen labeling methods, respectively. Intrinsic and extrinsic apoptotic pathways were identified by immunohistochemistry, stereological techniques, and RT-PCR by assessing pathway-specific proteins and genes. Following FSH suppression for 4 and 7 days, we have previously reported a 30% decrease in spermatogonial number, with increased apoptosis in a stage-specific manner. The present study also shows stage-specific increases in apoptosis with no changes in proliferation. This increase in apoptosis was attributable to an increase in spermatogonial apoptosis via the intrinsic rather than extrinsic pathway, as shown by increased activated caspase 9-positive spermatogonia. The concomitant suppression of FSH and LH/testosterone showed that testosterone alone or together with FSH was more important in spermatocyte and spermatid survival by regulating both apoptotic pathways. A reduction in the level of the intrinsic pathway transcript Bcl2l2 (apoptosis suppressor gene) following FSH suppression for 4 days shows that FSH regulates some components of the intrinsic pathway. This study reveals that FSH predominantly acts as a survival factor for spermatogonia by regulating the intrinsic pathway while having no affect on germ cell proliferation in rats in vivo.

Hypogonadal Mouse, a Model to Study the Effects of the Endogenous Lack of Gonadotropins on Apoptosis1

Testicular apoptosis is involved in the regulation of germ cell numbers, allowing optimal sperm production. Apoptosis has been described to occur in response to the absence of hormonal stimulation of the testis. Here we investigate the effect of the physiological lack of gonadotropins from birth using the hypogonadal (homozygous for the mutant allele Gnrh1(hpg)) mouse as a model. We pursued a concerted strategy using microarray analysis and RT-PCR to assess transcript levels, TUNEL to quantify the incidence of apoptosis, and Western blotting to assess the respective contribution of the extrinsic and intrinsic apoptotic pathways. Our results indicate a large increase in apoptosis of both somatic and germ cell compartments in the hpg testis, affecting Sertoli cells as well as germ cells of all ages. We confirmed our observations of Sertoli cell apoptosis using anti-Mullerian inhibiting substance staining and staining for cleaved fodrin alpha. In the somatic compartment, apoptosis is primarily regulated via the membrane receptor (extrinsic) apoptotic pathway, while in the germ cell compartment, regulation occurs via both the mitochondrial (intrinsic) and membrane receptor (extrinsic) apoptotic pathways, the latter potentially in a stage-specific manner. This study is the first report of spermatogonial apoptosis in response to gonadotropin deficiency as well as the first report of Sertoli cell apoptosis in response to gonadotropin deficiency in the mouse.

Doxorubicin Affects Testicular Lipids with Long-Chain (C18-C22) and Very Long-Chain (C24-C32) Polyunsaturated Fatty Acids

Doxorubicin disrupts spermatogenesis by causing apoptosis of spermatogonia and primary spermatocytes. The aim of this study was to examine the effect of this agent on adult rat testicular lipids and their fatty acids. A single dose (7.5 mg/kg) and a multidose regime (3 mg/kg once a week for 4 weeks) were evaluated. Both treatments resulted in the gradual loss of spermatogenic cells and determined a marked reduction in testicular size and weight 9 weeks after their start. Germ cell loss was accompanied by a decrease in phospholipids, including glycerophospholipids and sphingomyelin. Concomitantly, glycerophospholipids lost selectively their major polyunsaturated fatty acid (PUFA), 22:5n-6, and sphingomyelin lost its major very long-chain PUFA (VLCPUFA), 28:4n-6 and 30:5n-6. The molecular species from which the lost polyenes originated were thus a trait of germ cells. A transient peak of 16:0-ceramide was observed 48 h after the single dose. In both doxorubicin regimes, sphingomyelin and ceramide with reduced amounts of VLCPUFA after about 4 weeks and with no VLCPUFA after 9 weeks resulted. By contrast, triglycerides and especially cholesterol esters (CE) tended to accumulate in the testes undergoing germ cell death, probably in the surviving Sertoli cells, their fatty acid patterns suggesting that initially, these lipids retained part of the PUFA coming from, or no longer used for, the synthesis of germ cell glycerophospholipids. As the latter decreased, CE accumulated massively 9 weeks after starting doxorubicin treatment, 20:4n-6 becoming their major PUFA. Part of these CEs may derive from surviving steroidogenic cells.

Apoptosis and Molecular Pathways in the Seminiferous Epithelium of Aged and Photoinhibited Syrian Hamsters (Mesocricetus auratus)

Aging and short photoperiod exposure induce germ cell apoptosis in the Syrian hamster; however, the specific germ cells affected and the molecular pathways triggered have not been elucidated. We analyzed germ cell apoptosis and the expression of the Fas/Fas-L system, Bcl-2 family, and p53 in aged and photoinhibited hamsters and compared with those young maintained in natural photoperiod. Aging increased apoptosis in spermatogonia and spermatocytes, but in photoinhibited hamster testes only an increase in apoptotic spermatocytes was observed. Apoptosis was higher in aged hamsters in stages I-IV, V-VI and VII-VIII. Aging increased apoptosis of spermatogonia in stages I-IV and V-VI. Apoptotic pachytene spermatocytes were significantly higher in stages I-IV, V-VI, and VII-VIII in aging. Apoptotic preleptotene and pachytene spermatocytes were higher in aging, but no differences were observed in leptotene-zygotene. Fas-L was expressed by Sertoli cells, of young, aged, and photoinhibited hamsters. Bcl-x(L) was strongly expressed in germ cells on young hamsters and slightly in aging and after short photoperiod exposure. Spermatocytes of photoinhibited hamsters were intensively stained with Fas, Bax, Bcl-xs/L, and p53. In conclusion, aging increases apoptosis in spermatogonia and spermatocytes, depending on the stage of the seminiferous epithelium cycle, whereas after a short photoperiod exposure only an increase in apoptotic spermatocytes is observed. The results suggest that Fas, Bcl-x(L), Bax, and p53 participate in germ cell apoptosis induction after short photoperiod exposure, whereas only Bcl-x(L) is involved in aging.