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.

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.

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.

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.