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.

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.

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)

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.

Structure/function relationships in active and inactive hamster Leydig cells: a correlative morphometric and endocrine study.

Spermatogenesis can be turned on or off in the seasonally breeding golden (Syrian) hamster in a laboratory setting by exposure of animals to different photoperiod regimens. The present study provides the first detailed quantitative analysis of the subcellular features of hamster Leydig cells during active and inactive phases of spermatogenesis and correlates these features with the endocrine activity of the same animals. Conventional stereological principles and accepted morphometric techniques were used to determine changes in a variety of subcellular constituents of Leydig cells at the extreme phases of gonadal activity produced by maintaining adult hamsters in a long photoperiod (16 h of light, 8 h of darkness) or by exposing them to a short photoperiod (6 h of light, 18 h of darkness) for 12-13 weeks. Compared with Leydig cells from gonadally active animals, Leydig cells obtained from the regressed testis showed a significant reduction in the absolute volume of nearly all of its organelles, including nucleolus (77.0%), mitochondria (50.0%), total endoplasmic reticulum (ER) (86.4%; cisternal ER, 87.0%; tubular ER, 85.5%), lipid (84.2%), peroxisomes (82.5%), multi-vesicular bodies (71.9%), filament-rich area (95.5%), and Golgi complex (69.4%). There was also a significant reduction in organelle surface area, namely outer (76.5%) and inner (72.7%) mitochondrial membrane, total ER (85.0%), cisternal (85.3%) and tubular (84.4%) forms of ER, and Golgi complex (70.0%). The surface areas of the plasma membrane and nuclear membrane were also decreased by 58.3% and 33.7%, respectively. These results demonstrate that the short photoperiod-induced cellular inactivity of Leydig cells is associated with an overall diminution in the volume and surface area of all major organelles, not only those specifically associated with steroid biosynthesis. Virtually every structural parameter of the Leydig cell was significantly and positively correlated with plasma LH levels and with both plasma and testicular concentrations of testosterone. The total content of LH receptors per testis and per Leydig cell was reduced, but the concentration of receptors per unit area of the Leydig cell surface remained unchanged. Correlation of changes in hormonal status with alterations of all Leydig cell organelles suggests a general shut-down of cellular activity in the gonadally regressed animals, rather than a specific effect of pituitary hormones on selected cellular constituents.

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.

Correlative morphology and endocrinology of Sertoli cells in hamster testes in active and inactive states of spermatogenesis.

The seasonally breeding golden (Syrian) hamster, which exhibits photoperiod-dependent transitions between active and inactive states of spermatogenesis, was used as a model to study Sertoli cell structure in the two extreme phases of gonadal activity. The structural parameters of the Sertoli cell and its subcellular organelles were assessed using accepted stereological procedures during active and inactive states of spermatogenesis, and the results correlated with a battery of endocrine parameters obtained from the same animals. Short photoperiod-induced testicular involution was associated with a significant decrease in virtually all morphological parameters of the Sertoli cell, including a dramatic decrease in the volumes and surface areas of the Sertoli cells and their major subcellular organelles. Sertoli cell size and surface area were significantly and positively correlated with the testicular weight, volume of the seminiferous tubule, tubular lumena, tubule diameter, and germ cell numbers. Similar correlations were recorded between the number of germ cells and nearly all subcellular parameters of the Sertoli cell. Only those structural elements that are related to degredative processes (lysosomes and lipid) did not show significant volumetric differences between gonadally active and inactive animals. The observed changes in the structural parameters of the Sertoli cells were significantly correlated with the reduction in plasma levels of FSH, LH, and testosterone and intratesticular levels of testosterone. Exposure of hamsters to a short photoperiod was also associated with an increase in concentration (femtomoles per mg protein), but a decrease in the total content (femtomoles per testis) of testicular FSH receptors. The dissociation of changes in the content and concentration of FSH receptors appears to be related to changes in basal compartment plasma membrane surface areas of the Sertoli cells during testicular regression. The striking changes in Sertoli cell morphology between active and inactive states of spermatogenesis are structural manifestations of alterations in the function of these cells in response to the concomitant endocrine changes in the testis and indicate a virtual shut-down of Sertoli cell function during short photoperiod-induced testicular regression.

Temporal and stage-specific changes in spermatogenesis of rat after gonadotropin deprivation by a potent gonadotropin-releasing hormone antagonist treatment.

GnRH antagonists (GnRH-As) rapidly and reversibly inhibit testicular functions in a variety of experimental models as well as man. Their potential for human male contraception is currently being tested in many centers, including our own. This study was undertaken to provide comprehensive quantitative information on the testes and to document the temporal and stage-specific changes in the kinetics of germ cell degeneration in rats treated daily with the Nal-Glu GnRH-A (1250 micrograms/kg body wt) for up to 4 weeks. Plasma levels of testosterone (T) and the concentrations of testicular T declined to 20.7% and 5.4% of control values, respectively, by 1 week and remained suppressed throughout the treatment period. Preleptotene and pachytene spermatocytes, and step-7 and step-19 spermatids at stage VII were the first germ cells to degenerate soon after (1 week) GnRH-A treatment. Germ cell counts at stage VII also revealed a significant (P < 0.05) reduction in number of preleptotene (25.6%), pachytene (35.4%), and step-7 spermatids (29.1%) in comparison with controls. The number of homogenization-resistant advanced spermatids decreased by 70%. A further progressive loss of spermatogenic activity occurred with time. Treatment with GnRH-A for 4 weeks caused advanced spermatids to decline to nearly undetectable, Step-7 spermatids to decline to 17.7% of the normal level, and the P and PL to decline to 28.6% and 67.7%, respectively, of control values. The number of Sertoli cells and A1 spermatogonia remained unchanged throughout the experimental period. The effects of GnRH-A treatment on spermatogenesis were identical to that of hypophysectomy. These results suggest that: 1) early deprivation of gonadotropins and/or intratesticular T by GnRH-A treatment is followed by stage-specific degeneration of germ cells; 2) pituitary secretions other than LH and FSH have little primary influence on spermatogenesis during early regression; and 3) the GnRH-A-treated rat would be an excellent animal model for studying the targeted effects of LH, FSH, and T on the regulation of spermatogenesis.

Reproductive aging in the male brown-Norway rat: a model for the human.

Previous studies in the Sprague-Dawley rat have demonstrated that male reproductive aging is primarily a neuroendocrine dysfunction characterized by decreased pulsatile LH secretion leading to low serum testosterone (T) levels, whereas sperm production is relatively well maintained. In contrast to the Sprague-Dawley rat, the Brown-Norway (BN) rat has a longer life span, does not become obese, and experiences fewer age-related tumors of the endocrine or reproductive system, thus providing a disease-free model for studying male reproductive aging. We studied the changes in serum hormone levels and related these alterations to testicular T production, Leydig cell morphometry, and spermatogenesis in young (6 months), aging (22 months), and old (30 months) BN rats. Low serum T levels were associated with decreased LH levels in the 22-month-old (T, 0.58 +/- 0.08 ng/ml; LH, 0.45 +/- 0.06 ng/ml) and 30-month-old rats (T, 0.63 +/- 0.10 ng/ml; LH, 0.34 +/- 0.04 ng/ml) compared to those in young rats (T, 1.35 +/- 0.30 ng/ml; LH, 0.79 +/- 0.10 ng/ml). In vitro Leydig cell T production, basally and after stimulation by LH, was similar in young and old rats. The total testicular T content was lower in 30- than in 6-month-old rats. Testicular morphometry showed smaller Leydig cell volume in the old (857 +/- 97 microns3) than in the young rats (1387 +/- 103 microns3), although their number per testis remained unchanged (6 months, 22.7 +/- 1.6 x 10(6)/testis; 30 months, 25.2 +/- 3.1 x 10(6)/testis). In contrast, a marked (68.4%) reduction in the total number of Sertoli cell per testis was noted in the 30-month-old rats. The proportion of the testis occupied by seminiferous tubules was also reduced in the old rats. Significant (P < 0.05) age-related reductions occurred in tubule diameter, length of tubules, and volume of tubules and their lumens. The testicular sperm concentration and total sperm production were significantly reduced in the 22- and 30-month-old rats. These changes in seminiferous tubule function in the old rats were associated with low serum and testicular inhibin and high serum FSH levels. We conclude that aging in the reproductive axis of the BN rat is manifested by 1) lower serum T levels due to decreased pituitary LH stimulation of endogenous T production, and 2) decreased seminiferous tubule function accompanied by elevated FSH levels indicative of a primary testicular disorder.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.

Quantitative evaluation of testicular biopsies from men with unilateral torsion of spermatic cord.

Torsion of the spermatic cord is not uncommon among young men. Various abnormalities in the histology of the contralateral testis have been reported due to unilateral torsion of the spermatic cord. We quantitatively estimated the germ cells from three groups of men: normal individual (Group I), men with unilateral torsion of short duration (Group II), and men with unilateral torsion of long duration or some other condition such as varicocele or intermittent torsion to the contralateral testis (Group III). No significant difference in the number of spermatogonia, spermatocytes, and spermatids of Groups I and II patients was observed. This observation indicates that there was no pre-existing morphophysiologic defect in the testis of Group II patients. Severe damage in the contralateral testis was noted in Group III patients. This indicates that if a damaged testis is retained in the body for a long time, the contralateral testis may be affected. Contralateral testis may also be affected by intermittent torsion or varicocele.

Unilateral torsion of spermatic cord in men: effect on Leydig cell.

In our previous studies, we reported that short-term unilateral spermatic cord torsion had no adverse effect on the germ cells and the Sertoli cell in the contralateral testis of men. As an extension of our earlier investigations on the testicular pathophysiology in humans after unilateral spermatic cord torsion, the present study was undertaken to assess the Leydig cell function employing both fine structural and morphometric analysis in patients with short-term spermatic cord torsion. Bilateral testicular biopsy samples obtained from 4 men (15-19 years) with short-term unilateral torsion of the spermatic cord and from a control group of 6 men (15-40 years) were used in the present investigation. No appreciable difference in the Leydig morphology was noted between the biopsy samples from control and the contralateral testes. This was substantiated by morphometric analysis. The present study clearly indicates that patients with unilateral torsion of the spermatic cord may not essentially have bilateral testicular abnormalities, as suggested by the previous investigators. This report, thus lends further support to our earlier contention that alteration in microcirculation is quite likely the earliest and possibly the most significant contributor to the contralateral testicular damage in man after ipsilateral spermatic cord torsion.

Effects of diazacholesterol dihydrochloride (SC-12937), an avian antifertility agent, on rat testis.

The present study was undertaken to evaluate the effectiveness of an avian chemosterilant, 20, 25-diazacholesterol dihydrochloride (SC-12937), on the rat testis. Adult male rats were injected intraperitoneally with 10 mg (Group 1) or 30 mg (Group 2) of SC-12937/kg/d or with vehicle alone (Group 3) for 10 days, and were killed 24 hours after the last injection. A wide range of variation in the appearance of affected seminiferous tubules was observed in the testis of SC-12937-treated rats at both dose levels. This ranged from apparently normal-looking seminiferous tubules to almost completely atrophied tubules with no cells. Affected tubules exhibited intraepithelial vacuoles of varying size, multinucleated giant cells, germ cell exfoliation, and tubular atrophy. The presence of severely damaged and entirely normal seminiferous tubules adjacent to one another in the same section was noteworthy. The changes appeared to be dose-related. A greater number (34.6%) of affected tubules were observed in rats receiving 30 mg of SC-12937 compared with the ones receiving 10 mg of this compound (19.6%). The Sertoli cells also were affected by this drug and exhibited cytoplasmic vacuolation, a marked increase in the accumulation of lipid droplets and myeloid bodies. Necrotic Sertoli cells also were observed in the severely affected tubules. The possible mechanism of antispermatogenic action of SC-12937 in rats has been discussed briefly.

Stagnation of blood in the microcirculatory vessels in the testes of men with varicocele.

This study reports on the stagnation of blood within the microcirculatory vessels of the testes of patients with varicocele. Both fine structural and quantitative studies were carried out on testicular biopsies from 14 men with varicocele and a control group of three men. Arterioles, capillaries, and venules were completely filled with blood in all affected testes. Enlarged pores were also noticed between the endothelial cells of these affected vessels. Lumen diameters of the arterioles were significantly decreased in the affected testis compared to controls. No change in the overall diameter of the arterioles and venules was noted. Significant thickening of the limiting membrane was also noted in the affected testis. It was concluded that the stagnation of blood in the microcirculatory vessels may cause local hypoxia and ischemia, which lead to spermatogenic disorders.

Stagnation of blood in the microvasculature of the affected and contralateral testes of men with short-term torsion of the spermatic cord.

Bilateral testicular biopsies from four men with a short duration (3 hours 10 minutes to 4 hours 30 minutes) of unilateral spermatic cord torsion and testicular biopsies from six men with irreversible brain death were used for the present investigation. Extensive light and electron microscopic studies and quantitative analyses of all biopsy materials were performed. The torsioned testes revealed variable degrees of damage to the seminiferous tubules, including germ cell disorganization and sloughing of immature germ cells. Ninety-five percent of the blood vessels from the biopsied tissue specimens were clogged with blood cells. The seminiferous tubules of the contralateral testis had normal germ cell arrangements and counts. However, 88% of the microvessels from the tissue biopsied from the contralateral testes were packed with blood cells, whereas only 10% of the blood vessels in the control biopsy specimen were clogged with blood cells. At the electron microscopic level, fewer tight junctions and enlarged pores were found between the endothelial cells of the affected vessels, and microvilli were completely absent from these endothelial cells. The clogging caused by blood cells in the affected vessels was so severe that no space was found between the membrane of the endothelial cell and the membrane of the blood cells. It has been suggested that local clogging by blood is responsible for the initiation of degenerative changes in the testes of men with unilateral torsion of the spermatic cord.

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.

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.

Germ cell degeneration in the contralateral testis of the guinea pig with unilateral torsion of the spermatic cord. Quantitative and ultrastructural studies.

This study evaluated the long term effects of unilateral torsion of the spermatic cord on the contralateral testis of guinea pigs, employing both fine structural and quantitative studies. Young, adult Hartley strain guinea pigs were divided into six experimental groups (12 animals per group). The first three groups consisted of 36 animals in which unilateral torsion was surgically induced. In group I (torsion maintained), unilateral torsion of the spermatic cord was maintained until the day of sacrifice; in group II (torsion and untwist), torsion of the spermatic cord was maintained for 8 to 12 hours, then the spermatic cord was untwisted and the testis was retained until the day of sacrifice. In group III (torsion and orchiectomy), testes were removed after 8 to 12 hours of spermatic cord torsion. The second three groups consisted of 36 animals: group IV (unilateral orchiectomy), group V (unilateral sham operation), and group VI (pentobarbital injection alone), which served as controls. One half of the animals from each group were killed after 4 months and the other half were killed after 8 months. The most frequently observed histologic changes in the contralateral testes of the experimental animals were focal disorganization and exfoliation of immature germ cells into the lumen. Severe damage, with almost complete absence of germ cells, was noted only in an occasional tubule. Quantitative evaluation of the germ cells of the contralateral testis revealed significant loss of germ cells in groups I, II, and III after 4 months, and in groups I and II after 8 months.(ABSTRACT TRUNCATED AT 250 WORDS)