Additive effects of nicotine and high-fat diet on hepatocellular apoptosis in mice: involvement of caspase 2 and inducible nitric oxide synthase-mediated intrinsic pathway signaling.

Smoking is a major risk factor for diabetes and cardiovascular disease and may contribute to nonalcoholic fatty liver disease (NAFLD). The health risk associated with smoking is exaggerated by obesity and is the leading causes of morbidity and mortality worldwide. We recently demonstrated that combined treatment with nicotine and a high-fat diet (HFD) triggers greater oxidative stress, activates hepatocellular apoptosis, and exacerbates HFD-induced hepatic steatosis. Given that hepatocellular apoptosis plays a pivotal role in the pathogenesis of NAFLD, using this model of exacerbated hepatic steatosis, we elucidated the signal transduction pathways involved in HFD plus nicotine-induced liver cell death. Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice daily IP injections of 0.75 mg/kg BW of nicotine or saline for 10 weeks. High-resolution light microscopy revealed markedly higher lipid accumulation in hepatocytes from mice received HFD plus nicotine, compared to mice on HFD alone. Addition of nicotine to HFD further resulted in an increase in the incidence of hepatocellular apoptosis and was associated with activation of caspase 2, induction of inducible nitric oxide synthase (iNOS), and perturbation of the BAX/BCL-2 ratio. Together, our data indicate the involvement of caspase 2 and iNOS-mediated apoptotic signaling in nicotine plus HFD-induced hepatocellular apoptosis. Targeting the caspase 2-mediated death pathway may have a protective role in development and progression of NAFLD.

Impairment of spermatogenesis in transgenic mice with selective overexpression of Bcl-2 in the somatic cells of the testis.

To explore the functional role of Bcl-2 in germ cell development, transgenic mice carrying 6 kilobases of the inhibin-alpha promoter were generated to express human bcl-2 gene product in the gonads. Although female transgenic mice demonstrated decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis, the adult males exhibited variable impairment of spermatogenesis. The degree of damage ranged from tubules with intraepithelial vacuoles of varying sizes to near atrophied tubules consisting of Sertoli cells and a few spermatogonia. Although there was no significant change in body weight, an approximately 34% decrease in testicular weights was noted in transgenic animals compared with wild-type mice. Gamete maturation, assessed by determining the percentage of tubules with advanced (steps 13-16) spermatids, was decreased to 44.4% of the values measured in the wild-type animals. The incidence of germ cell apoptosis increased 3.8-fold in the transgenic animals and was associated with a marked loss of germ cells. Electron microscopy of the testes further revealed large vacuoles in the Sertoli cell cytoplasm and dilations of the intracellular spaces between adjacent Sertoli cells, spermatid malformations, and increased germ cell apoptosis in the transgenic animals. There was no evidence of Sertoli cell death either by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay or electron microscopy. Leydig cell ultrastructure, cell size and numbers, and plasma levels of testosterone were not different between normal and the transgenic animals. Collectively, these results support the critical role of Bcl-2 in male germ cell development and are consistent with the gender-specific role of the Bcl-2 family members in reproduction.

Aging-related increased expression of inducible nitric oxide synthase and cytotoxicity markers in rat hypothalamic regions associated with male reproductive function.

UNLABELLED: We have previously demonstrated that the inducible nitric oxide synthase (iNOS) protein and total NOS activity increase in the hypothalamus and other regions of the male rat brain during aging. We have now tested the hypothesis that increased iNOS results in excessive nitric oxide (NO) and peroxynitrite production, and leads to increased apoptosis in CNS cells, including the GnRH and oxytocin hypothalamic neurons involved in the control of male reproductive function. Young (3-month-old) and old (24-month-old) male Brown Norway rats (n = 6) were perfused with 4% formalin. Adjacent coronal paraffin-embedded sections (5 microm) of preoptic area (POA), supraoptic nucleus (SON), paraventricular nucleus (PVN), and arcuate nucleus (ARC) of the hypothalamus were immunostained with antibodies for iNOS, neuronal NOS (nNOS), and nitrotyrosine (a marker of peroxynitrite formation). The intensity of immunostaining was measured using a densitometric image analysis system. Apoptosis was determined by the TUNEL assay. Double immunofluorescence staining with confocal laser scanning microscopy was used for co-localization studies. A significant increase in the iNOS immunostaining measured as optical density (OD) was found in the old compared to the young animals (SON: 0.32 +/- 0.02 vs. 0.23 +/- 0.03, p < 0.05; PVN: 0.34 +/- 0.03 vs. 0.07 +/- 0.05, p < 0.001; POA: 0.18 +/- 0.02 vs. 0.01 +/- 0.02, p < 0.001). Aging did not affect nNOS expression. Nitrotyrosine was elevated in the hypothalamic regions of old compared to young rats (SON: 0.32 +/- 0.05 vs. 0.10 +/- 0.04, p < 0.05; PVN: 0.32 +/- 0.04 vs. 0.13 +/- 0.03, p < 0.01; POA: 0.72 +/- 0.06 vs. 0.03 +/- 0.003, p < 0.001). Increased nitrotyrosine was accompanied by an elevation of the apoptotic index in the old rats (SON: 11.01 +/- 3.33 vs. 0.57 +/- 0.50, p < 0.001; PVN: 3.08 +/- 1.12 vs. 0.42 +/- 0.32; POA: 6.60 +/- 1.93 vs. 0.18 +/- 0.17, p < 0.01; ARC: 0.001 +/- 0.0001 vs. 4.33 +/- 2.33). iNOS staining co-localized with GnRH and oxytocin staining. IN CONCLUSION: The aging-related iNOS increased expression in the hypothalamus of the male rat affects regions known to control the synthesis and release of GnRH (POA, ARC) and oxytocin (PVN, SON), and the factors regulating penile erection (POA, and PVN). These observations suggest that iNOS may play a role in the reduction in GnRH and oxytocin neuronal secretion resulting in reproductive dysfunctions such as lowered serum testosterone, hypospermatogenesis, and diminished copulatory function in the aging male animal.

XXY male mice: an experimental model for Klinefelter syndrome.

Klinefelter syndrome (47,XXY) is the most common sex chromosome aneuploidy in men. Thus, it is important to establish an experimental animal model to explore its underlying molecular mechanisms. Mice with a 41,XXY karyotype were produced by mating wild-type male mice with chimeric female mice carrying male embryonic stem cells. The objectives of the present study were to characterize the testicular phenotype of adult XXY mice and to examine the ontogeny of loss of germ cells in juvenile XXY mice. In the first experiment the testicular phenotypes of four adult XXY mice and four littermate controls (40,XY) were studied. XXY mice were identified by either Southern hybridization or karyotyping and were further confirmed by fluorescence in situ hybridization. The results showed that the testis weights of adult XXY mice (0.02 +/- 0.01 g) were dramatically decreased compared with those of the controls (0.11 +/- 0.01 g). Although no significant differences were apparent in plasma testosterone levels, the mean plasma LH and FSH levels were elevated in adult XXY mice compared with controls. The testicular histology of adult XXY mice showed small seminiferous tubules with varying degrees of intraepithelial vacuolization and a complete absence of germ cells. Hypertrophy and hyperplasia of Leydig cells were observed in the interstitium. Electron microscopic examination showed Sertoli cells containing scanty amounts of cytoplasm and irregular nuclei with prominent nucleoli. The junctional region between Sertoli cells appeared normal. In some tubules, nests of apparently degenerating Sertoli cells were found. In the second experiment the ontogeny of germ cell loss in juvenile XXY mice and their littermate controls was studied. Spermatogonia were found and appeared to be morphologically normal in juvenile XXY mice. Progressive loss of germ cells occurred within 10 days after birth. This resulted in the absence of germ cells in the adult XXY mice. We conclude that a progressive loss of germ cells occurring in early postnatal life results in the complete absence of germ cells in adult XXY mice. The XXY mouse provides an experimental model for its human XXY counterpart, Klinefelter syndrome.

Redistribution of Bax is an early step in an apoptotic pathway leading to germ cell death in rats, triggered by mild testicular hyperthermia.

Programmed cell death occurs spontaneously during spermatogenesis and can be induced in a cell- and stage-specific manner by mild testicular hyperthermia. Studies using transgenic mice suggest the involvement of Bcl-2 proteins in regulating germ cell apoptosis. To delineate further the pathways involved, we examined the temporal changes in proapoptotic Bax and antiapoptotic Bcl-2 in rat testes after transient exposure to heat (43 degrees C for 15 min). Germ cell apoptosis, involving exclusively early (I-IV) and late (XII-XIV) stages, was activated within 6 h. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to perinuclear localization within 0.5 h of heating as assessed by immunocytochemical methods. In contrast, Bcl-2 is distributed both in the cytoplasm and nucleus in those cell types susceptible to heat-induced apoptosis. Despite the striking redistribution, Bax levels remained unchanged as determined by Western analysis; Bcl-2 levels increased significantly by 6 h after heat exposure. Reverse transcription-polymerase chain reaction analysis indicated no change in either Bax or Bcl-2 mRNA levels in response to heat, suggesting the involvement of post-transcriptional rather than transcriptional mechanisms mediating their activity. The marked subcellular redistribution of Bax prior to activation of apoptosis and the increase in Bcl-2 suggest an involvement of Bcl-2 family members in heat-induced apoptotic death of germ cells.

Long-term effects of triptolide on spermatogenesis, epididymal sperm function, and fertility in male rats.

Prior studies had suggested that triptolide, a diterpene triepoxide isolated from a Chinese medicinal plant, might be an attractive candidate as a post-testicular male contraceptive agent. Despite the promise that triptolide would not affect testis function, nagging concerns remained that a delayed onset of testicular effect might exist. The objectives of this study were to assess the effects of relatively longer treatment duration of triptolide on fertility, spermatogenesis, and epididymal sperm pathophysiology; and to evaluate the reversibility of these effects after the cessation of treatment. Adult male Sprague-Dawley rats were fed daily with either 30% gum acacia as a vehicle control (n = 12) or 100 microg/kg body weight (BW) of triptolide for 82 days (n = 12) followed by a recovery period of up to 14 weeks (n = 6). At the end of the treatment period, all rats treated with triptolide were sterile. Cauda epididymal sperm content decreased by 84.8% and sperm motility was reduced to zero. In addition, virtually all cauda epididymal sperm in the triptolide-treated group exhibited severe structural abnormalities. The most striking changes observed were head-tail separation, premature chromatin decondensation of sperm nuclei, a complete absence of the plasma membrane of the entire middle and principle pieces, disorganization of the mitochondrial sheath, and aggregation of many sperm tails. Longer treatment duration of triptolide also affected spermatogenesis, with marked variability in the response of individual animals. The degree of damage ranged from apparently normal-looking seminiferous tubules to flattened seminiferous epithelium lined by a single layer of cells consisting of Sertoli cells and a few spermatogonia. Affected tubules exhibited intraepithelial vacuoles of varying sizes, multinucleated giant cells, germ cell exfoliation, and tubular atrophy. Recovery occurred as early as 6 weeks after cessation of treatment. By 14 weeks, 4 out of 6 triptolide-treated males were fertile and the females that were impregnated by 3 out of 4 triptolide-treated male rats produced apparently normal litters. These results suggest that triptolide has 2 phenotypic effects on mature and maturing germ cells. The first action appears earlier and manifests mainly in epididymal sperm. The second action presumably is directly on germ cells in testis and causes a variable impairment of spermatogenesis that may not be completely reversible. It is unclear if the earlier effect is a delayed manifestation of subtle testicular injury or post-testicular action.

Posttesticular antifertility action of triptolide in the male rat: evidence for severe impairment of cauda epididymal sperm ultrastructure.

A variety of active diterpene epoxides, including the triptolide (isolated from Tripterygium wilfordii) have been reported to cause infertility in male rats. Previously, we showed that oral administration of triptolide at a dosage of 100 microg/kg per body weight for 70 days completely inhibited fertility in male rats, with little or no demonstrable detrimental effect on spermatogenesis and Leydig cell function as determined by testicular light microscopic appearance and serum and intratesticular testosterone levels. Despite the apparent absence of effects on the testes, cauda epididymal sperm were abnormal, with complete cessation of sperm motility and some reduction in sperm numbers. This study was undertaken to provide additional insight into the subcellular sites and possible mechanisms of action of this compound using ultrastructural analysis of the testes and epididymidis. The most striking effect of triptolide treatment was observed in sperm in the epididymis. In rats rendered infertile with 100 microg/kg per body weight of triptolide daily for 70 days, virtually all cauda epididymal sperm exhibited complete absence of plasma membrane over the entire middle and principal piece, premature decondensation of the nuclei, and disorganization of the mitochondrial sheath with many vacuolated mitochondria. No ultrastructural differences in the epididymal epithelium were observed between control and triptolide-treated rats. The testes appeared to be mildly affected after triptolide treatment but exhibited only subtle ultrastructural defects in the germ cells. The findings of severe impairment of cauda epididymal sperm ultrastructure, along with minimal discernible abnormalities in the fine structural cytology of the testes, further suggest that the site of action of this compound is posttesticular and may be confined to the cauda epididymal sperm. However, we cannot rule out an effect of triptolide that occurs during germ cell maturation but is delayed in its manifestation or triggered at the rete testis and epididymal level.

Testicular heat exposure enhances the suppression of spermatogenesis by testosterone in rats: the "two-hit" approach to male contraceptive development.

The objectives of the study were to determine stage-specific changes in the kinetics of germ cell apoptosis induced by administration of exogenous testosterone (T) alone and to examine whether addition of a single testicular heat exposure would enhance the induction of germ cell apoptosis and the suppression of spermatogenesis by T. Adult male rats were implanted with 3-cm SILASTIC brand capsules (Dow Corning Corp.) containing T for up to 6 weeks. Intratesticular T levels declined to 2.9% of control values by 1 week and remained suppressed at 2, 3, and 6 weeks after T administration. The incidence of germ cell apoptosis (expressed as numbers per 100 Sertoli cells) was low in control rats (0-9.52). After T treatment, the mean incidence of apoptosis at stages VII-VIII increased significantly by 1 week (21.43 +/-3.33) and showed further increases by 6 weeks (56.30 +/- 7.47); apoptotic rates remained low at early (I-VI) and later (XII-XIV) stages. To test whether the combination of T with a single testicular heat exposure resulted in more complete suppression of spermatogenesis than either treatment alone, four groups of adult rats received one of the following treatments: 1) a subdermal empty polydimethylsilozane implant, 2) exposure to a single testicular heating (43 C for 15 min) applied on day 14, 3) 3-cm T implant, or 4) 3-cm T implant and a single testicular heat exposure (applied on day 14). All animals were killed at the end of 6 weeks. In the heat-treated group, testis weight and testicular sperm counts were decreased to 65.4% and 28.9% of control levels, respectively. The corresponding values in the T-treated group were 49.7% and 24.9% of control levels, respectively. Notably, addition of heat to T further reduced testis weight to 31.1% of control levels and testicular sperm counts to near zero. Histomorphometric analysis showed that all treatments reduced seminiferous tubular diameter and epithelial and luminal volume, with the greatest decrease after combined T and heat treatment. Heat exposure in animals bearing T implants markedly reduced the number of pachytene spermatocytes and round spermatids through apoptosis, resulting in tubules devoid of mature spermatids. Spermatogonia and preleptotene spermatocytes remained unaffected. These results clearly demonstrate that 1) exogenous T reduces intratesticular T and induces apoptosis mainly at stages VII-VIII within 1-6 weeks; 2) the combined treatment of T and heat markedly inhibits spermatogenesis, resulting in near azoospermia within 6 weeks; and 3) meiosis and spermiogenesis are the most vulnerable phases of spermatogenesis in response to T plus heat treatment. These findings suggest that a combination of hormonal treatment such as T and a physical agent (heat exposure) is more effective in suppressing spermatogenesis than either treatment alone. We hypothesize that combination of two antispermatogenic agents ("two hit") working at separate stages of the spermatogenic cycle will lead to greater male contraceptive efficacy.

Reproductive aging in the Brown Norway rat is characterized by accelerated germ cell apoptosis and is not altered by luteinizing hormone replacement.

Reproductive aging in the male Brown Norway (BN) rat is characterized by decreased Leydig cell steroidogenesis associated with seminiferous tubule dysfunction. This could be a result of a combination of a primary testicular defect and a secondary hypothalamic pituitary dysfunction. In the present study, we determined in the BN rat whether germ cell loss occurred via apoptosis. We then defined the age of onset of Leydig cell dysfunction and germ cell loss and examined whether chronic luteinizing hormone (LH) replacement would delay or prevent reproductive aging. Plasma hormone levels, testicular sperm concentrations, and germ cell apoptosis were studied in 6, 9, 12, 15, 18, and 21-month-old BN rats. Beginning at 15 months, testicular weight, sperm concentration, total sperm counts, plasma testosterone, LH, and inhibin decreased, whereas the proportion of regressed testes and plasma follicle-stimulating hormone (FSH) levels increased with aging. Accelerated germ cell apoptosis involving spermatogonia, preleptotene and pachytene spermatocytes, and spermatids was evident in some tubules of the relatively normal testes from 21-month-old rats. In the regressed testes, complete cessation of spermatogenesis occurred. The apoptotic index was higher in the testes of old (21-month-old) rats in particular at stages XII-XIV when compared with younger animals. Chronic LH replacement (0.5 microg i.p. twice per day) administered to 15-month-old BN rats for 6 months did not alter plasma hormone levels, testes weight, sperm concentration or content, or the germ cell apoptotic index. In the control group, 3 out of 10 testes were regressed, whereas in the LH-replaced group, only 1 out of 12 testes was regressed. We show in this study that early reproductive aging in the BN rat began at around 15 months. Germ cell loss associated with aging occurs via apoptosis. Replacement therapy with LH for 6 months does not decrease or delay the testicular dysfunction associated with aging. It is unlikely that hypothalamic-pituitary dysregulation is the major cause of testicular aging.

Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity.

Short term exposure of the testis to heat causes degeneration of germ cells. However, the mechanisms underlying this process are poorly understood. The major objectives of this study were to determine whether the heat-induced loss of germ cells in the adult rat occurs via apoptosis, to document its stage-specific and cell-specific distribution, and to examine whether intratesticular testosterone (T) plays any role in the stage specificity of heat-induced germ cell death. Testes of adult male Sprague-Dawley rats were exposed to 22 C (control) or 43 C for 15 min. Animals were killed on days 1, 2, 9, and 56 after heat exposure. Germ cell apoptosis was characterized by DNA gel electrophoresis and in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling assay. The incidence of germ cell apoptosis [apoptotic index (AI)] was quite low in control rats (AI = 0.04-0.1). Mild hyperthermia within 1 or 2 days resulted in a marked activation (AI = 4.7-5.6) of germ cell apoptosis predominantly at early (I-IV) and late (XII-XIV) stages. Stages V-VI and VII-VIII were relatively protected from heat-induced apoptosis. Spermatocytes, including pachytenes at stages I-IV and IX-XII, diplotene and dividing spermatocytes at stages XIII-XIV, and early (steps 1-4) spermatids, were most susceptible to heat. On day 9, the majority of the tubules were severely damaged and displayed only a few remaining apoptotic germ cells. By day 56, spermatogenesis was completely recovered, and the incidence of germ cell apoptosis was compatible with the control levels. To determine whether intratesticular T plays a role in protecting germ cells at stages VII-VIII against heat-induced cell death, adult rats were exposed to local testicular heating on day 2 or were given a daily sc injection of GnRH antagonist (GnRH-A) for 4 days with and without a single exposure of testes to heat applied on day 2. By day 4, the incidence of increased germ cell apoptosis at stages other than VII-VIII were not different between heat-treated and GnRH-A- plus heat-treated groups, whereas the control group and the group treated with GnRH-A alone showed minimal apoptosis. GnRH-A addition to heat resulted in a further increase in apoptosis (by 3.2-fold) at stages VII-VIII over the values measured in the heat-treated group, and it became comparable to that at all other stages. Collectively, these results provide evidence that 1) heat induces germ cell apoptosis in a stage-specific and cell-specific fashion; and 2) intratesticular T plays a pivotal role in protecting germ cells at stages VII-VIII against heat-induced cell death. However, the possible involvement of various other factors, including growth factors, thermoprotectants, cytokines, and various death-related proteins, in protecting germ cells against heat-induced apoptosis cannot be ruled out.

Hormonal and genetic control of germ cell apoptosis in the testis.

Programmed cell death is an evolutionarily conserved cell death process that plays a major role during normal development and homeostasis. In many cases, the ordered execution of this internal death programme leads to typical morphological and biochemical changes that have been termed apoptosis. The crucial role of this mode of cell death in the pathogenesis of diverse human diseases including cancer, acquired immunodeficiency syndrome, neurodegeneratives disorders, atherosclerosis and cardiomyopathy is now supported by a wealth of data. In adult mammals, including humans, germ cell death is conspicuous during normal spermatogenesis and plays a pivotal role in sperm output. Withdrawal of gonadotrophins and testosterone further enhances the degeneration of germ cells in the testis. The availability of a quantitative method for analysing the testicular DNA fragmentation and in situ methods to localize specific germ cells undergoing apoptosis, either spontaneously or in response to a variety of death triggering signals, opens new avenues in the understanding of the significance of germ cell apoptosis during normal and abnormal states of spermatogenesis. A growing body of evidence demonstrates that both spontaneous (during normal spermatogenesis) and accelerated germ cell death triggered by deprivation of the gonadotrophic support or moderately increased scrotal temperature in adult rats occur almost exclusively via apoptosis. Although there has been spectacular progress in the understanding of the molecular mechanisms of apoptosis in various systems other than spermatogenesis, elucidation of the biochemical and molecular mechanisms by which germ cell apoptosis is regulated has only just begun. It is likely that germ cell apoptosis is controlled in a cell-type specific fashion, but the basic elements of the death machinery may be universal. In addition, there is increasing evidence that homozygous disruption of a number of genes in mice results in infertility through accelerated germ cell apoptosis. Manipulation of spermatogenesis by survival factor(s) deprivation or increases in extrinsic death signals in loss-of-function or gain-of-function mouse models provides a basis for further attempts to define the intrinsic regulation of various death-related genes by external death signals. Such information is crucial for effective management of male factor infertility as well as more targeted approaches to male contraception.

Triptolide: a potential male contraceptive.

The antifertility effect of triptolide and other related compounds, isolated from Tripterygium wilfordii, has been demonstrated in male rats. The exact sites and mechanism of action of triptolide remain unknown. Our objectives were to determine whether triptolide at selected dose levels that induce infertility has any detrimental effects on the testes and to determine the sites and the possible mechanisms of its action. Groups of six adult male Sprague-Dawley rats were given oral administration of either vehicle (control group) or triptolide (50 or 100 microg/kg body weight) daily for 35 or 70 days. Body weight gain was normal in all treated groups. All six rats treated with a high dosage of triptolide were infertile during the second (63-70 days) mating trial. A lower dose (50 microg) of triptolide gave intermediate fertility values. Plasma levels of luteinizing hormone, follicle-stimulating hormone, testosterone, and intratesticular testosterone were not significantly different between control and triptolide-treated groups. Cauda epididymal sperm content was decreased by 68% and the motility, which averaged 58.2% in the control rat, was reduced to almost zero. No effects of triptolide were observed on testis and accessory organs weight, volumes of tubular lumen and the total Leydig cells, tubule diameter, and the number of Sertoli cells, spermatogonia, preleptotene (PL), and pachytene (P) spermatocytes. There were, however, modest but significant decreases in tubule volume and the number of round spermatids at stages VII-VIII. No changes in the germ cell apoptotic index measured at stages VII-VIII and XIV-I were noted between controls and rats rendered infertile with a high dose of triptolide. Thus, triptolide, at a dose level that induces complete infertility in the adult rats, has minimal adverse effects on the testes and acts primarily on the epididymal sperm making triptolide an attractive lead as a post-testicular male contraceptive.

Spontaneous germ cell apoptosis in humans: evidence for ethnic differences in the susceptibility of germ cells to programmed cell death.

Spontaneous death of certain classes of germ cells has been shown to be a constant feature of normal spermatogenesis in a variety of mammalian species, including the human. Recent studies on various animal models have demonstrated that apoptosis is the underlying mechanism of germ cell death during normal spermatogenesis. Withdrawal of gonadotropins and/or testosterone further accelerates the germ cell apoptosis. We examined the involvement of apoptosis in the spontaneous loss of germ cells in men. Testicular samples obtained at autopsy from 5 Chinese and 9 non-Hispanic Caucasian men were analyzed. To identify individual germ cells undergoing apoptosis, we used a modified terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling technique that detects germ cell apoptosis with high sensitivity and specificity. Testicular sections from all 14 subjects exhibited spontaneous occurrence of germ cell apoptosis involving spermatogonia, spermatocytes, and spermatids (apoptotic indexes, 1.6 +/- 0.4 2.5 +/- 0.6, and 5.5 +/- 1.2, respectively). The incidence of spermatogonial (2.8 +/- 0.8 vs. 1.0 +/- 0.2) as well as spermatid (9.3 +/- 2.1 vs. 8.4 +/- 0.9) apoptosis was higher in Chinese than in Caucasian men. A higher incidence of spermatocyte apoptosis was also noted for Chinese (4.4 +/- 1.4) compared to Caucasian (1.9 +/- 0.4) men, but the difference was not statistically significant. These results suggest that germ cell death during normal spermatogenesis in men occurs via apoptosis and provide evidence for ethnic differences in the inherent susceptibility of germ cells to programmed cell death. Our data may also help to explain the greater efficacy of testosterone-induced spermatogenic suppression to azoospermia observed in Asian compared to non-Asian men.

Significance of apoptosis in the temporal and stage-specific loss of germ cells in the adult rat after gonadotropin deprivation.

The major objectives of the present study were to document the temporal and stage-specific acceleration of germ cell apoptosis in adult rats after selective suppression of pituitary gonadotropins by GnRH antagonist (GnRH-A) treatment, and to examine the possibility that apoptosis is the sole mechanism of germ cell death in response to hormonal deprivation. Groups of adult male rats were given a daily injection of a vehicle for 14 days or GnRH-A (1.25 mg/kg BW) for 2, 5, 7, and 14 days. Analysis of testicular apoptotic DNA fragmentation revealed a detectable increase at Day 5 and a maximal increase at 14 days after treatment. In situ analysis of germ cell apoptosis fully corroborated the observed increase in the degree of DNA fragmentation with time and also revealed a stage-related activation of apoptosis of specific germ cells. A low incidence (0.06-0.09) of germ cell apoptosis (expressed as numbers per Sertoli cell) was detectable at stages I, IX-XI, and XII-XIV in control rats. Mean incidence of apoptotic germ cells specifically at stages VII-VIII increased significantly (0.40 +/- 0.06) by Day 5 and increased another 2.2-fold (over the 5-day treatment values) on Day 7 after GnRH-A treatment as compared to values in controls, where no apoptosis was detected. Significantly increased incidence of apoptosis at stages IX-XI (0.37 +/- 0.05) over control values (0.07 +/- 0.01) was noted by Day 7. Within the study paradigm, the highest number of dying cells occurred by Day 14, at which time a modest but significant (p < 0.05) increase in the incidence of apoptosis was also noted at stages I, II-IV, V-VI, and XII-XIV in comparison with control values. Stages VII-VIII and IX-XI still exhibited the higher number of cells undergoing apoptosis (0.97 +/- 0.22, and 1.03 +/- 0.22, respectively). Comparison between rates of apoptosis and cell degeneration measured at stages VII-VIII demonstrated an intimate association (r = 0.94; p < 0.001) between apoptosis and germ cell loss, strongly supporting the concept that germ cell death (at these stages) after removal of hormonal support in the adult rat occurs almost exclusively via apoptosis.

Separation of germ cell apoptosis from toxin-induced cell death by necrosis using in situ end-labeling histochemistry after glutaraldehyde fixation.

In situ end-labeling of fragmented DNA is routinely being used to detect apoptotic cells in various tissues including the testis. In this study, we examined the influence of various fixatives (neutral buffered formalin, paraformaldehyde, and glutaraldehyde) on the testicular structural integrity and immunoreactivity of fragmented DNA in apoptotic germ cells of the adult rat. Accelerated apoptosis of germ cells was induced in the adult rate by gonadotropin deprivation. Visualization of apoptotic DNA fragmentation in individual germ cells was achieved by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. Glutaraldehyde fixation significantly improved the in situ detection of apoptotic germ cells while maintaining excellent morphological preservation. The labeling is also specific for apoptosis since necrotic germ cells show no specific signals. Fixed tissues could be processed for electron microscopy for further characterization of germ cell death using morphological criteria. Thus, glutaraldehyde fixation is advantageous for recognition of apoptotic germ cells with high sensitivity and specificity on a cell-by-cell basis. It should also be applicable to detect apoptosis in other cells and tissues.

In the male brown-Norway (BN) male rat, reproductive aging is associated with decreased LH-pulse amplitude and area.

The Brown-Norway (BN) rat has been proposed as a rodent model for the study of human male reproductive aging. As in man, reduction in serum or plasma testosterone (T) and both testicular (primary) and hypothalamic-pituitary (secondary) reproductive dysfunction have been associated with aging in male BN rats. However, the presence of secondary testicular failure in this rodent, as indicated by low serum luteinizing hormone (LH) levels, needs further corroboration. The present study was designed to determine whether age-related differences in the pulsatile patterns of pituitary LH and follicle-stimulating hormone (FSH) secretion occur in gonad-intact male BN rats. Three age groups were examined: young (3-4 months), middle aged (12-13 months), and old (21-22 months). Using intra-atrial cannulae, serial 5-minute blood samples were withdrawn from conscious, unrestrained animals. Plasma LH concentrations were determined by a supersensitive immunofluorometric assay (FIA) and FSH and T by radioimmunoassay (RIA). Mean T levels were different among groups (young > middle age > old). In young rats, T levels were higher in the late morning/early afternoon than in the late afternoon: this variation was not found in older rats. Mean FSH concentrations were higher in the old than in the middle-aged and young rats. Significant differences in mean LH levels were not found among groups. Compared to young rats, shortened pulse interval and reduced area of pulses characterized the secretory pattern of both gonadotropins in old rats. In addition, LH-pulse amplitude and total area of LH pulses were also significantly lower in old than in young rats. Besides the well-recognized primary testicular failure that occurs in the old BN rat, this study confirms a hypothalamic-pituitary deficiency that makes this rodent model ideal for studying human male reproductive aging.

Early effects of vasectomy on testicular structure and on germ cell and macrophage apoptosis in the hamster.

This study provides quantitative information on the early (up to 3 months) effects of vasectomy on apoptosis in the hamster testis. Groups of five adult male golden hamsters were either bilaterally vasectomized or sham-operated and sacrificed at intervals of 3, 6, and 12 weeks after surgery. In all three postvasectomy groups, testis weight and testicular and plasma testosterone (T) levels were not different from controls. Spermatogenic alterations, ranging from tubules with mild intraepithelial vacuoles to almost completely atrophied tubules, were detected in samples of 1 of 5 testes both at 3 and 12 weeks after vasectomy. Histometric analysis of testicular tissues at 3, 6, and 12 weeks in the postvasectomy groups showed no discernible effect of vasectomy on the absolute volumes of seminiferous tubules, tubular lumen, and total Leydig cells when compared to respective controls. In situ analysis of germ-cell apoptosis, characterized by 3'-end-labeling immunocytochemistry, revealed a significant increase (2.5-fold) in germ-cell apoptosis at stages XIII-I, involving primarily the dividing spermatocytes after 3 weeks of vasectomy. Apoptotic index was not changed from sham-operated animals at 6 and 12 weeks postvasectomy. Interestingly, a very high incidence of macrophage apoptosis was detected in the samples of three out of five testes in the 12 weeks postvasectomy group (39.3%) compared to that of controls (0.8%). These results demonstrate that vasectomy has little or no detrimental effect on the morphologic characteristics of the spermatogenesis or intratesticular concentrations of testosterone in the majority of the animals studied up to 12 weeks postsurgery, although vasectomy transiently (3 weeks postsurgery) activated germ-cell apoptosis, involving dividing spermatocytes at stages XIII-I.

Involvement of apoptosis in the induction of germ cell degeneration in adult rats after gonadotropin-releasing hormone antagonist treatment.

In adult mammals, including man, germ cell death is conspicuous during spermatogenesis and plays a pivotal role in sperm output. The possible mechanism(s) underlying this phenomenon remains, however, poorly understood. Apoptosis is a programmed physiological process by which cells die either spontaneously or in response to changes in the levels of specific physiological stimuli. Previously, we reported that early deprivation of gonadotropin and testosterone by GnRH antagonist (GnRH-A) treatment is followed by a stage-specific degeneration of germ cells in the testis. In this study, we examined the possible involvement of apoptosis in the induction of germ cell degeneration. Adult male rats were given a daily injection of Nal-Glu GnRH-A (1 mg/kg BW) for 0 (control), 2, or 5 days. The onset of germ cell degeneration was assessed by high resolution light microscopy as well as by a germ cell degeneration assay. The occurrence of apoptosis was characterized by 1) detection of internucleosomal DNA fragmentation after agarose gel electrophoresis, and 2) direct immunoperoxidase detection of digoxigenin-labeled genomic DNA in specific cell types. The earliest morphological signs of germ cell degeneration involving preleptotene and pachytene spermatocytes, step 7 spermatids (at stage VII), and step 19 spermatids (at both stages VII and VIII) were detected 5 days after the commencement of GnRH-A treatment. The onset of germ cell degeneration was further accompanied by a significant increase in the degree of low mol wt DNA fragmentation. In situ detection of apoptosis within this time frame fully corroborates the observed stage-related degeneration of specific germ cells. These results suggest that apoptosis provides the basic mechanism by which germ cells die in the testis in response to a lack of hormonal stimulation.

Temporal and stage-specific effects of recombinant human follicle-stimulating hormone on the maintenance of spermatogenesis in gonadotropin-releasing hormone antagonist-treated rat.

Despite considerable attention to the hormonal regulation of spermatogenesis, the role of FSH in adult mammals remains controversial. This is mainly due to the unavailability until recently of FSH preparations free of contaminating LH and discrepant results in various species. Using LH-free recombinant human FSH (rhFSH), we sought to determine if FSH is able to maintain spermatogenesis in rats in which both LH and FSH, but not other pituitary hormones, are markedly suppressed by GnRH-A treatment. Groups of five adult SD rats were given daily sc injections of vehicle, Nal-Glu-GnRH-A (1.25 mg/kg BW) or GnRH-A + 10 IU rhFSH for up to 4 weeks. In agreement with our previous report, GnRH-A treatment for 1 week led to a significant (P < 0.05) reduction in testis weight (26.6%) and in the number of specific germ cells involving preleptotene (27.7%) and pachytene spermatocytes (36.7%) and step 7 spermatids (30.3%) at stage VII of the seminiferous epithelial cycle. The number of advanced spermatids declined by 44.3%. Concomitant administration of rhFSH for 1 week resulted in a significant increase in testicular weight, tubular areas at stage VII-VIII, and in the absolute volumes of seminiferous tubules and their lumens compared to GnRH-A alone. Most importantly, FSH replacement to GnRH-A-treated rats fully attenuated the early (1 week) GnRH-A-induced reduction in germ cell numbers at stage VII as well as the number of advanced (steps 17-19) spermatids, and effectively prevented GnRH-A-induced reduction in the number of pachytene spermatocytes and step 7 spermatids for 2 weeks. In addition, FSH replacement to GnRH-A-treated rats was able to increase the number of B spermatogonia available for entry into meiosis and maintain the number of preleptotene spermatocytes throughout the treatment period. The observed beneficial effects of rhFSH on spermatogenesis in GnRH-A-treated rats are most likely not due to the stimulation of the Leydig cell function (via paracrine interaction between Sertoli and the Leydig cells), because FSH addition to GnRH-A had no discernible effect on intratesticular or plasma T levels, accessory organs weight, and the total volume of the Leydig cells when compared with GnRH-A alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Time course of recovery of spermatogenesis and Leydig cell function after cessation of gonadotropin-releasing hormone antagonist treatment in the adult rat.

This study examined the time course of recovery of spermatogenesis and its relationship to the temporal changes in circulating levels of gonadotropin and testosterone (T) and intratesticular T levels after cessation of treatment with a potent GnRH antagonist (GnRH-A). Adult male rats were given a daily sc injection of Nal-Glu-GnRH antagonist (1250 micrograms/kg BW) for 4 weeks and killed in groups of five 0, 1, 2, 3, 4, and 6 weeks after discontinuation of treatment. After cessation of treatment, plasma FSH levels returned to control values by 6 weeks, whereas LH levels returned to control values within 1 week. Both circulating as well as intratesticular levels of T returned to normal levels by 3 and 4 weeks, respectively. Interestingly, a rebound in both FSH and intratesticular T, but not in plasma T, beyond control levels occurred early in the recovery phase. The total volume of Leydig cells, which was only 15% of control values, increased 4.3-fold within 1 week and was not significantly different from control values (92% recovery) by 2 weeks posttreatment. Enumeration of earlier phases of germ cells as well as homogenization-resistant advanced (steps 17-19) spermatids revealed a progressive increase in germ cell numbers with time. Complete restoration of the numbers of preleptotene spermatocytes, pachytene spermatocytes, step 7 spermatids, and advanced spermatids occurred 1, 3, 4, and 6 weeks, respectively, after termination of GnRH-A treatment. There was also a complete reversal of GnRH-A-induced changes in testicular weight, tubule diameter, and volume of seminiferous tubules and their lumens by 6 weeks posttreatment, paralleling the recovery of spermatogenesis. These results suggest that 1) complete recovery of spermatogenesis and various other testicular parameters can be achieved in GnRH-A-treated rats after cessation of treatment; 2) the progression of various germ cells during the recovery period follows the normal time schedule of germ cell development; and 3) the recovery of spermatogenesis is preceded by supranormal levels of FSH and intratesticular T. These findings further emphasize the suitability of antagonistic analogs of GnRH for male fertility control.