Transplanted XY germ cells produce spermatozoa in testes of XXY mice.

XXY mouse has been characterized as an experimental model for men with Klinefelter's syndrome (XXY male phenotype). To test whether donor XY germ cells could proliferate and differentiate in the XXY testicular environment, donor testicular cells from adult (2-3 months old) and immature (10 days old) XY green fluorescence protein (GFP) transgenic mice were transplanted into the seminiferous tubules of adult (4-7 months old) and young (6 weeks old) XXY recipient mice respectively. Twelve weeks after transplantation, GFP positive spermatogonia were found in 21.74% (five out of 23) of adult XXY recipients who received adult donor cells. The GFP positive segments of seminiferous tubules were observed in 44.44% (four out of nine) young XXY recipients who received donor cells from 10 days old GFP mice. We found using immunohistochemistry and cell morphology that donor-derived GFP positive germ cells were spermatogonia, spermatocytes, round spermatids and spermatozoa in some of the seminiferous tubules of young XXY recipient mice. The results demonstrated that the donor XY germ cells were able to qualitatively complete spermatogenesis in some of the seminiferous tubules of XXY mice.

Thermal pain tolerance and pain rating in normal subjects: Gender and age effects.

BACKGROUND: Thermal detection thresholds and thermal pain thresholds are important in quantitative sensory testing. Although they have been well studied for assessing somatosensory function, the investigation of thermal pain tolerance has been insufficient. The aim of this study was to explore the characteristics of thermal pain tolerance and pain ratings in healthy subjects. METHODS: Cold pain tolerance (CPTol) and heat pain tolerance (HPTol) were tested in 213 healthy adults aged 18-81 years recruited from the local community. The thermal detection and thermal pain thresholds were also tested to investigate the association with pain tolerance. The visual analogue scale (VAS) was used for assessing pain severity immediately after the thermal pain and tolerance tests. RESULTS: The normality of the CPTol and HPTol was acceptable. Most participants rated the pain induced by the CPTol and HPTol testing as moderate. HPTol was lower in women than in men (p = 0.001), but CPTol did not differ between sexes. The pain ratings of CPTol and HPTol did not differ between sexes, but significant age effects were observed. The association of the tolerance temperature with pain ratings was weak, while those of pain ratings for CPTol and HPTol were strong (r = 0.87). CONCLUSIONS: Women were more sensitive to tolerance heat pain stimuli. Younger participants reported more pain for thermal pain and tolerance tests. SIGNIFICANCE: Thermal pain tolerance and pain rating for the thermal pain tolerance temperature depend on gender and age. Women are more sensitive to heat temperatures, young people rate more pain, and the pain ratings of heat and cold are strongly correlated.

Humanin protects against chemotherapy-induced stage-specific male germ cell apoptosis in rats.

Humanin (HN) has cytoprotective action on male germ cells after testicular stress induced by heat and hormonal deprivation. To examine whether HN has protective effects on chemotherapy-induced male germ cell apoptosis, we treated four groups of adult rats with (i) vehicle (control), (ii) HN, (iii) cyclophosphamide (CP); or (iv) HN+CP. To investigate whether the protective effects of HN on germ cells require the presence of Leydig cells, another four groups of rats were pre-treated with ethane dimethanesulfonate (EDS), a Leydig cell toxicant, to eliminate Leydig cells. After 3 days, when Leydig cells were depleted by EDS, we administered: (i) vehicle, (ii) HN, (iii) CP; or (iv) HN+CP to rats. All rats were killed 12 h after the injection of HN and/or CP. Germ cell apoptosis was detected by TUNEL assay and quantified by numerical count. Compared with control and HN (alone), CP significantly increased germ cell apoptosis; HN +CP significantly reduced CP-induced apoptosis at early (I-VI) and late stages (IX-XIV) but not at middle stages (VII-VIII) of the seminiferous epithelial cycle. Pre-treatment with EDS markedly suppressed serum and intratesticular testosterone (T) levels, and significantly increased germ cell apoptosis at the middle (VII-VIII) stages. CP did not further increase germ cell apoptosis in the EDS-pre-treated rats. HN significantly attenuated germ cell apoptosis at the middle stages in EDS pre-treated rats. To investigate whether HN has any direct effects on Leydig cell function, adult Leydig cells were isolated and treated with ketoconazole (KTZ) to block testosterone synthesis. HN was not effective in preventing the reduction of T production by KTZ in vitro. We conclude that HN decreases CP and/or EDS-induced germ cell apoptosis in a stage-specific fashion. HN acts directly on germ cells to protect against EDS-induced apoptosis in the absence of Leydig cells and intratesticular testosterone levels are very low.

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.

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.

Aging results in attenuated gonadotropin releasing hormone-luteinizing hormone axis responsiveness to glutamate receptor agonist N-methyl-D-aspartate.

Reproductive aging in the Brown Norway rat occurs because of testicular as well as hypothalamic-pituitary dysfunction. Excitatory amino acids (EAA) participate in the regulation of pulsatile secretion of hypothalamic GnRH and pituitary LH. In the present study, we studied the EAA-GnRH-LH axis for possible age-related alterations in prepubertal (35 days), young (3-4 months), middle-aged (12-13 months) and old (21-23 months) rats. In the first experiment, an intra-atrial cannula was implanted in rats of different ages to evaluate the pituitary response to small, physiological intravenous bolus administration of GnRH (0.5 or 1.0 nmol/100 g body weight). The results showed no age-related significant differences in in-vivo serum LH or FSH responsiveness to GnRH. In a second experiment, blood samples for the gonadotropins were withdrawn immediately before and 10 min after an iv injection of the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 5 mg/kg, a dose that induces a physiological LH pulse in young rats). Administration of NMDA induced significant increases in LH and prolactin in all groups of animals (P<0.05) and a significant FSH response in young and middle-aged but not old rats. NMDA-induced LH, FSH and prolactin release was higher (P<0.05) in prepubertal rats than in all other age groups. Compared with young rats, NMDA-induced increase in plasma LH and prolactin was lower (P<0.05) in old rats. In the third experiment, to ascertain whether this reduced LH response to NMDA in old rats was exerted at the hypothalamic level, the effects of NMDA on GnRH release in vitro from preoptic area-medial basal hypothalamus (POA-MBH) fragments were compared among rats of different ages. GnRH efflux in response to NMDA was significantly attenuated with increasing age. GnRH release in vitro was higher in prepubertal and lower in old than in young rats (P<0.05). Lastly, we measured amino acid concentrations in hypothalamic tissue (POA-MBH fragments). Prepubertal rats had higher levels of glutamate and taurine than young rats. Significant reductions in glutamate and gamma-aminobutyric acid (GABA) levels were found in old compared to young rats. In conclusion, these results showed that the hypothalamic NMDA-GnRH-LH axis was altered in old rats. The decreased hypothalamic content of some of the EAA and the reduced responsiveness of GnRH neurons to NMDA (both in vivo and in vitro) may contribute to an altered LH pulsatile secretion observed in old rats.

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.

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.

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.

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.

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.

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.

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.

Strength and functional performance of patients with limb-girdle muscular dystrophy.

This study was design to reach a better understanding of muscle strength, motor function and activity of daily living in patients of limb-girdle muscular dystrophy. Forty-eight patients diagnosed as cases of limb-girdle muscular dystrophy were included in this study. Manual muscle testing was used to evaluate muscle strength. The Brooke and Vignos scales were used to grade upper and lower extremities function, respectively, and the ability of daily living activity was measured by Barthel index. Our patients showed progressive symmetrical limb-girdle muscular weakness. Upon regression analysis we found that mean muscle strength was inversely related to disease duration (years) as follows: mean muscle strength = 0.6052 + (0.6309/disease duration). According to the Brooke functional scale, 89.6% of our patients were graded as 1-3 and 10.4% were graded as 5. On the Vignos functional scale, 79.1% of patients fell into the grades 1-5, one person (2.1%) in grade 6 and 18.8% in grade 9 category. The average Barthel index was 85.3 +/- 20.7. Mean muscle strength was significantly correlated with the average Barthel, Vignos and Brooke functional scales. Our study could offer the strength and functional performance of limb-girdle muscular dystrophy on natural history. The muscle strength declined in Taiwanese patients of limb-girdle muscular dystrophy in a typical pattern. Regression analysis showed that the strength was inversely related to disease duration. These findings demonstrate that most of our patients suffered from mild or moderate physical disability.

The strength and functional performance in patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a slowly progressive myopathy with autosomal dominant inheritance remarkable for its early involvement of facial musculature. The purpose of our study was to assess the rate of strength deterioration, functional condition and performance of activity of daily living of patients with FSHD in Taiwan. Twenty patients diagnosed with FSHD were included in this study. Manual muscle testing (MMT) was used to evaluate muscle strength. The Brooke and Vignos scales were used to assess upper and lower extremity function respectively, and the capability of the activity of daily living was measured by Barthel index. The result of the strength testing was characterized by the presence of a progressive asymmetrical muscular weakness in patients with FSHD. The mean muscular strength of the right extremity was weaker than its left counterparts (p < 0.05) and the shoulder muscle group was the weakest. According to the Brooke functional scale, 20% of our patients were graded as 1, 30% as grade 2, and 50% as grade 3. On the Vignos functional scale, 50% of patients fell into grade 1, 10% in grade 2, and 40% in grades 3-5. Vignos scale was significantly correlated with mean muscle strength (p < 0.05). The average value of Barthel index was 97.8 +/- 4.7. The muscle strength decline in this Taiwanese of FSHD population was more severe in shoulder girdle area. The mean muscle strength of the right extremity was weaker than the left. Most of our patients suffered from mild or moderate physical disability. Finding of these Taiwanese FSHD population is similar to those reported elsewhere in the world.

Knee isokinetic strength and body fat analysis in university students.

Many factors such as anthropometric variables influence strength performance. This study is to determine the relationship between knee isokinetic strength and body composition, and to compare the gender differences. Test-retest reliability had been performed within one week for all measurement methods before the formal study. Fifty-eight 20-25 year-old university students, 32 females and 26 males, participated in this study. Isokinetic strength of the knee flexion and extension was measured at two angular velocities of 60 degrees/sec and 120 degrees/sec. Body composition was measured by bioelectrical impedance analysis (BIA) and skinfold caliper. The others variables including height, body weight, body mass index (BMI), and waist to hip ratio were measured or calculated. The results showed that the intra-class correlation coefficients for isokinetic knee strength were between 0.83 and 0.93, and body composition and anthropometric variables were between 0.83 and 0.98. Isokinetic knee strength was significantly correlated with body height, body weight, BMI, waist and hip ratio and percent of body fat estimated by skinfold caliper (r = -0.56 to 0.64). The correlation between isokinetic strength with percent of body fat estimated by BIA (r = -0.60 to -0.74; p < 0.001) and with fat free mass (r = 0.64 to 0.78; p < 0.001) was even higher. Although male subjects had significantly greater mean values in body height, body weight, waist to hip ratio and isokinetic strength than female subjects, the MANCOVA showed that the effect of gender on knee isokinetic strength would be eliminated when the covariant variable, the percent of body fat measured by BIA and BMI was controlled in the analysis model. In conclusion, knee isokinetic strength was significantly negatively correlated with proportion of fat and positively correlated with fat free mass. The magnitude of strength difference between males and females could be explained by differences in body fat proportion and BMI in this study. Therapist would take the body fat composition, fat free mass, and BMI into consideration in knee muscle strength measurement. Less body fat and higher BMI will contain more fat free mass that produces more muscle strength.

Testosterone replacement ameliorates nonalcoholic fatty liver disease in castrated male rats.

Nonalcoholic fatty liver disease is common in developed countries and is associated with obesity, metabolic syndrome, and type 2 diabetes. T deficiency is a risk factor for developing these metabolic deficiencies, but its role in hepatic steatosis has not been well studied. We investigated the effects of T on the pathogenesis of hepatic steatosis in rats fed a high-fat diet (HFD). Adult male rats were randomly placed into four groups and treated for 15 weeks: intact rats on regular chow diet (RCD), intact rats on liquid HFD (I+HFD), castrated rats on HFD (C+HFD), and castrated rats with T replacement on HFD (C+HFD+T). Fat contributed 71% energy to the HFD but only 16% of energy to the RCD. Serum T level was undetectable in castrated rats, and T replacement led to 2-fold higher mean serum T levels than in intact rats. C+HFD rats gained less weight but had higher percentage body fat than C+HFD+T. Severe micro- and macrovesicular fat accumulated in hepatocytes with multiple inflammatory foci in the livers of C+HFD. I+HFD and C+HFD+T hepatocytes demonstrated only mild to moderate microvesicular steatosis. T replacement attenuated HFD-induced hepatocyte apoptosis in castrated rats. Serum glucose and insulin levels were not increased with HFD in any group. Immunoblots showed that insulin-regulated proteins were not changed in any group. This study demonstrates that T deficiency may contribute to the severity of hepatic steatosis and T may play a protective role in hepatic steatosis and nonalcoholic fatty liver disease development without insulin resistance.