An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.     

Role of cytochrome C in apoptosis: increased sensitivity to tumor necrosis factor alpha is associated with respiratory defects but not with lack of cytochrome C release

Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-alpha-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways.     

Germ cell differentiation and proliferation in the developing testis of the South American plains viscacha, Lagostomus maximus (Mammalia, Rodentia)

Cell proliferation and cell death are essential processes in the physiology of the developing testis that strongly influence the normal adult spermatogenesis. We analysed in this study the morphometry, the expression of the proliferation cell nuclear antigen (PCNA), cell pluripotency marker OCT-4, germ cell marker VASA and apoptosis in the developing testes of Lagostomus maximus, a rodent in which female germ line develops through abolished apoptosis and unrestricted proliferation. Morphometry revealed an increment in the size of the seminiferous cords with increasing developmental age, arising from a significant increase of PCNA-positive germ cells and a stable proportion of PCNA-positive Sertoli cells. VASA showed a widespread cytoplasmic distribution in a great proportion of proliferating gonocytes that increased significantly at late development. In the somatic compartment, Leydig cells increased at mid-development, whereas peritubular cells showed a stable rate of proliferation. In contrast to other mammals, OCT-4 positive gonocytes increased throughout development reaching 90% of germ cells in late-developing testis, associated with a conspicuous increase in circulating FSH from mid- to late-gestation. TUNEL analysis was remarkable negative, and only a few positive cells were detected in the somatic compartment. These results show that the South American plains viscacha displays a distinctive pattern of testis development characterized by a sustained proliferation of germ cells throughout development, with no signs of apoptosis cell demise, in a peculiar endocrine in utero ambiance that seems to promote the increase of spermatogonial number as a primary direct effect of FSH.     

Necrosis Is the Dominant Cell Death Pathway in Uropathogenic Escherichia coli Elicited Epididymo-Orchitis and Is Responsible for Damage of Rat Testis

Male infertility is a frequent medical condition, compromising approximately one in twenty men, with infections of the reproductive tract constituting a major etiological factor. Bacterial epididymo-orchitis results in acute inflammation most often caused by ascending canalicular infections from the urethra via the continuous male excurrent ductal system. Uropathogenic Escherichia coli (UPEC) represent a relevant pathogen in urogenital tract infections. To explore how bacteria can cause damage and cell loss and thus impair fertility, an in vivo epididymo-orchitis model was employed in rats by injecting UPEC strain CFT073 into the vas deference in close proximity to the epididymis. Seven days post infection bacteria were found predominantly in the testicular interstitial space. UPEC infection resulted in severe impairment of spermatogenesis by germ cell loss, damage of testicular somatic cells, a decrease in sperm numbers and a significant increase in TUNEL (+) cells. Activation of caspase-8 (extrinsic apoptotic pathway), caspase-3/−6 (intrinsic apoptotic pathway), caspase-1 (pyroptosis pathway) and the presence of 180 bp DNA fragments, all of which serve as indicators of the classical apoptotic pathway, were not observed in infected testis. Notably, electron microscopical examination revealed degenerative features of Sertoli cells (SC) in UPEC infected testis. Furthermore, the passive release of high mobility group protein B1 (HMGB1), as an indication of necrosis, was observed in vivo in infected testis. Thus, necrosis appears to be the dominant cell death pathway in UPEC infected testis. Substantial necrotic changes seen in Sertoli cells will contribute to impaired spermatogenesis by loss of function in supporting the dependent germ cells.     

Caspase activation throughout the first wave of spermatogenesis in the rat

Early in postnatal life, the first wave of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This may reflect an adjustment in the number of germ cells that can be adequately maintained by Sertoli cells. Two major pathways (intrinsic and extrinsic) are involved in the process of caspase activation and apoptosis in mammalian cells. The extrinsic pathway is characterized by the oligomerization of death receptors such as FAS or tumor necrosis factor, followed by the activation of caspase-8 and caspase-3. The intrinsic pathway involves the activation of procaspase-9, which in turn activates caspase-3. Extensive information is available concerning apoptotic inducers and their possible mechanisms in the adult rat. However, no data exist regarding the molecular and cellular mechanisms governing physiological cell death during puberty in the male rat. We have studied caspase activation throughout the first wave of spermatogenesis in the rat under physiological conditions, by combining the TUNEL procedure with the localization of active caspases in germ cells. We observed TUNEL-positive germ cells in rats of 5–40 days of age, the highest number being found in 25-day-old rats. TUNEL-positive and caspase-3-positive germ cells appeared as long chains of interconnected germ cells in 25-day-old rats. Caspase activation was assayed by either immunohistochemistry with antibodies against active caspase-3, -8, and -9, or by determining enzymatic activity in seminiferous tubules extracts. Both techniques showed activation of caspase-3, -8, and -9 in 25-day-old rats and low enzymatic activity at other ages. Confocal scanning laser microscopy indicated that active caspase-3, -8, and -9 co-localized with TUNEL-positive cells. Thus, caspase-3, -8, and -9 are active in apoptotic germ cells during the first wave of rat spermatogenesis. The extrinsic pathway of apoptosis may therefore play an important role in germ cell apoptosis during puberty in the rat.This work was financed by a research grant from FONDECYT (1040800) to R.D.M.     

Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis

Early in postnatal life the first phase of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This wave of germ cell death is thought to reflect an adjustment of germ cell numbers that can be adequately maintained by Sertoli cells. Caspase 2 is an initiator caspase whose activation has been found to stimulate apoptosis through the mitochondria. The present study investigates if germ cell apoptosis during the first phase of spermatogenesis involves activation of caspase 2. Germ cell apoptosis was found to peak at Postnatal Days (pnds) 15 and 16 in male C57BL/6 mice. Western blot analysis revealed that caspase 2 also increased in the testes at pnd 16. Immunolocalization of total caspase 2 showed staining of germ cells in the periphery of the seminiferous tubules as well as germ cells more centrally located in an area where apoptotic germ cells were observed. Cytoplasmic as well as nuclear staining was observed. Western blot analysis of cytoplasmic and nuclear proteins from pnd 16 testis revealed pro-caspase 2 in both fractions. Further Western blot analysis for caspase 2 detected an increase in the activation of caspase 2 at pnd 16 in proteins isolated from the cytoplasm but not from the nucleus. Proteins isolated from mitochondria from pnd 16 testes revealed an increase in pro-caspase 2 as well as activated caspase 2 corresponding with an increase in cytochrome c in cytoplasmic fractions. Injection of the caspase 2-specific inhibitor z-VDVAD-fmk directly into the testis significantly reduced the observed germ cell apoptosis at pnds 15 and 16. These results suggest that caspase 2 is present in germ cells in the murine testis in early postnatal life and increases in expression in correspondence to the initial wave of germ cell apoptosis. Caspase 2 also localizes to mitochondria, where it is correlated with a release of cytochrome c and germ cell apoptosis. Blockade of caspase 2 activation reduced the number of apoptotic germ cells in the initial wave of germ cell apoptosis, indicating that caspase 2 plays an important role upstream of the mitochondria in germ cell apoptosis during the first phase of spermatogenesis.     

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.     

Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice

The signaling events leading to apoptosis can be divided into two major pathways, involving either mitochondria (intrinsic) or death receptors (extrinsic). In a recent study, we have shown the involvement of the mitochondria-dependent apoptotic pathway in heat-induced male germ cell apoptosis in the rat. In additional studies, using the gld (generalized lymphoproliferation disease) and lprcg (lymphoproliferation complementing gld) mice, which harbor loss-of-function mutations in Fas L and Fas, respectively, we have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system is not required for heat-induced germ cell apoptosis in the testis. In the present study, we have found that the initiation of apoptosis in wild-type mice was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. The relocation of Bax is accompanied by sequestration of ultracondensed mitochondria into paranuclear areas of apoptotic germ cells, cytosolic translocation of mitochondrial cytochrome c and DIABLO, and is associated with activation of the initiator caspase 9 and the executioner caspase 3. Similar events were also noted in both gld and lprcg mice. Taken together, these results indicate that the mitochondria-dependent pathway is the key apoptotic pathway for heat-induced male germ cell death in mice.     

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.     

Hereditary defects in both germ cells and the blood-testis barrier system in as-mutant rats: evidence from spermatogonial transplantation and tracer-permeability analysis

The rat mutant allele as is located on chromosome 12. Homozygous (as/as) males show arrested spermatogenesis, mainly at the pachytene spermatocyte stage. It is not clear whether this defective spermatogenesis is caused by a failure in a somatic cell component that supports spermatogenesis or in the germ cell itself. Spermatogonial transplantation was performed to identify the genetically defective site in the as/as testis. In experiment 1, germ cells collected from as/as testes were transplanted into the testes of immunodeficient mice and normal rats. In experiment 2, normal rat germ cells were transplanted into as/as testes. The results of experiment 1 showed arrest of spermatogenesis at the pachytene spermatocyte stage, accompanied by a characteristic morphological feature, i.e., the formation of inclusion-like bodies in the cytoplasm, in both rat and mouse recipients. These results revealed the intrinsic effect of the mutant gene(s) on germ cells. In experiment 2, no restoration of spermatogenesis was detected in the recipient testes despite thorough histological examination. These results suggest that defects in a somatic cell component in as/as testes prevent the donor germ cells from colonizing and regaining their spermatogenetic ability. When the seminiferous epithelium of the as/as testis was examined by electron microscopy, no morphological abnormalities, including the formation of ectoplasmic specializations between adjacent Sertoli cells, were observed in the somatic cell components. However, when cytochrome c was applied as a tracer material, it penetrated the tight junctions between the Sertoli cells, indicating dysfunction of the blood-testis barrier in the as/as testis. The lack of restoration of spermatogenesis in the as/as testis after transplantation of normal germ cells may have been caused by the unfavorable environment in the seminiferous epithelium resulting from the incomplete barrier system between adjoining Sertoli cells. The gene(s) at the as locus may have a role in both germ cell differentiation and the establishment of the blood-testis barrier.     

The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis

The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages.     

TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-alpha and IFN-gamma markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-alpha-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-alpha and IFN-gamma on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.     

Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 1,2-dimethanesulfonate administration: relationship to Fas?

Germ cell apoptosis, which occurs normally during spermatogenesis, increases after testosterone withdrawal from the testis. The molecular mechanism by which this occurs remains uncertain. The Fas system has been implicated as a possible key regulator of apoptosis in various cells: binding of Fas ligand (FasL), a type II transmembrane protein, to Fas, a type I transmembrane receptor protein, triggers apoptosis in cells expressing Fas. Recently, Fas has been localized to germ cells, and FasL to Sertoli cells, within the rat testis. We hypothesized that Fas protein content would rise in response to reduced levels of testosterone as part of a suicide pathway that would result in germ cell apoptosis. To test this hypothesis, ethane 1,2-dimethanesulfonate (EDS), a Leydig cell toxicant, was used to kill Leydig cells and thus reduce intratesticular testosterone levels in Sprague Dawley rats. Apoptosis was examined in situ and biochemically, and Fas protein content in the testis was monitored by Western blot analysis. We show that EDS injection results in the following sequence of events: apoptotic death of Leydig cells by a mechanism that does not involve Fas; reduced testosterone; increased testicular Fas content; and germ cell apoptosis. These results suggest that Fas may play a role in the apoptotic death of germ cells that results from reduced intratesticular testosterone levels, and that testosterone may play a role in germ cell survival via its suppression of Fas.     

Effect of testosterone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Previous studies have shown that androgens have direct inhibitory effects on steroidogenesis in active Leydig cells. It is not clear what effect androgens have on inactive Leydig cell either through direct action on the cell itself or indirectly through stimulation of Sertoli cell activity. The hpg mouse has undetectable levels of circulating gonadotrophins and the gonads fail to develop post-natally. The effect of androgen treatment on testicular steroidogenesis and morphology was examined in these animals. Treatment with testosterone propionate for two weeks significantly increased testicular and seminal vesicle weight. Seminiferous tubules showed marked development in androgen-treated animals, indicating increased Sertoli cell activity, but the abnormal Leydig cell morphology of the hpg testis was unchanged. Androgen production per testis in vitro was low in control hpg animals and remained unaffected by treatment with androgen. Similarly, the pattern of [3H]pregnenolone metabolism was not significantly affected by androgen treatment. The androgen content of the testis was higher in androgen-treated animals but this could be accounted for by uptake of administered steroid from the circulation. It is concluded that androgens have no direct trophic effect on Leydig cells and that stimulation of Sertoli cell activity is not, in itself, sufficient to affect Leydig cell function.     

In situ demonstration of both TUNEL-labeled germ cell and Sertoli cell in the cimetidine-treated rats

Cimetidine has caused dysfunction in the male reproductive system. In the rat testis, intratubular alterations and loss of peritubular tissue due to peritubular myoid cell death by apoptosis have been recently shown. Thus, the aim of this study is to evaluate which cells of the seminiferous epithelium have been affected and/or died by apoptosis after the treatment with cimetidine. For this purpose, an experimental group containing five male albino Wistar rats received intraperitoneal injections of cimetidine (50 mg/kg body weight) during 52 days. The testes were fixed with 4% buffered formaldehyde and were embedded in paraffin. For detection of DNA breaks (apoptosis) in the cells of the seminiferous epithelium, the testicular sections were treated by the TUNEL method (Apop-Tag Plus Peroxidase Kit). In the tubules affected by cimetidine, altered peritubular tissue, including the presence of TUNEL labeling in the myoid peritubular cells, were usually found. In these tubules, the seminiferous epithelium exhibited low density of germ cells and TUNEL-positive labeling in the germ cells of the basal compartment. The concomitant staining in both germ cells of the basal compartment and late spermatids suggest a sensitivity of these cells in the damaged tubules. Besides germ cells, TUNEL-positive Sertoli cells were also found in the injured seminiferous tubules. Thus, a relationship between dying germ cells and Sertoli cell damage and/or death must be considered in tubules where peritubular tissue has been affected by toxicants.     

ATRA and KL promote differentiation toward the meiotic program of male germ cells.

While it is known that Retinoic Acid (RA) induces meiosis in mouse female fetal gonads, the mechanisms which regulate this process during spermatogenesis are poorly understood. We show that the All trans RA derivative (ATRA) and Kit Ligand (KL) increase meiotic entry of postnatal mouse spermatogonia in vitro without synergism. Competence to enter meiosis is reached by spermatogonia only at the stage in which they undergo Kit-dependent divisions. Besides increasing Kit expression in spermatogonia, ATRA also upregulates KL expression in Sertoli cells. Both ATRA and KL increase the expression of Stimulated by Retinoic Acid Gene 8 and Dmc1, an early meiotic marker. A specific Kit tyrosine kinase inhibitor prevents the increase in the number of meiotic cells induced by both the two factors, suggesting that they converge on common Kit-dependent signalling pathways. Meiotic entry induced by ATRA and KL is independent from their ability to affect germ cell viability, and is mediated by the activation of PI3K and MAPK pathways through Kit autophosphorylation. ATRA-induced phosphorylation of the two downstream kinases is mediated by a non-genomic mechanism.

These data suggest that RA may control the timing of meiosis by influencing both the somatic and the germ cell compartment of the postnatal testis through the activation of the KL/Kit system.