Insights into male germ cell apoptosis due to depletion of gonadotropins caused by GnRH antagonists

The role of pituitary gonadotropins in the regulation of spermatogenesis has been unequivocally demonstrated, although, the precise mechanism of this regulation is not clearly understood. Previous studies have shown that specific immunoneutralization of LH/testosterone caused apoptotic cell death of meiotic and post-meiotic germ cells while that of FSH resulted in similar death of meiotic cells. In the present study, the death process of germ cells has been characterized by depleting both FSH and testosterone by administering two different potent GnRH antagonists, Cetrorelix and Acyline to both rats and mice. Pro-survival factors like Bcl-2 and Bcl-x/l were unaltered in germ cells due to GnRH antagonist treatment, although a significant increase in several pro-apoptotic markers including Fas and Bax were evident at both protein and RNA levels. This culminated in cytochrome C release from mitochondria and eventually increase in the activity of caspase-8 and caspase-3. These data suggest that both extrinsic and intrinsic apoptotic death pathways are operative in the germ cells death following decrease in FSH and testosterone levels. Multiple injections of GnRH antagonist resulted in complete disappearance of germ cells except the spermatogonial cells and discontinuation of the treatment resulted in full recovery of spermatogenesis. In conclusion our present data suggest that the principal role of FSH and testosterone is to maintain spermatogenic homeostasis by inhibiting death signals for the germ cells.     

Ejaculate-hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster ova and zona-intact domestic cat oocytes

Electroejaculate traits and circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were analyzed in adult leopard cats (Felis bengalensis), a rare felid species indigenous to east Asia. The ability of leopard cat sperm to bind and penetrate zona-free hamster ova and zona-intact domestic cat oocytes in vitro was examined as a means of testing sperm function. The influence of culture media [Biggers, Whitten, Whittingham (BWW) vs. modified Krebs Ringer bicarbonate (mKRB)], seminal plasma removal, and swim-up separation on sperm motility, sperm morphology, and oocyte penetration also were assessed. Sperm treatments included dilution of raw semen (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU). The percentage of oocytes penetrated (penetration rate) and the number of penetrated sperm/oocyte (penetration index) were determined. Ejaculates from each male consisted of at least a 50% sperm motility rating, and hormone concentrations in individual males were unrelated to any ejaculate trait measured concurrently on the same day. The SU technique improved (P less than 0.05) percent sperm motility and the proportion of structurally normal sperm compared to DR and NS treatments. Leopard cat spermatozoa were capable of binding to and penetrating hamster ova and domestic cat oocytes; however, penetration was influenced by culture medium and seminal processing. In the hamster assay, a higher (P less than 0.05) penetration rate and penetration index were achieved when mKRB was used for gamete incubation instead of BWW. NS processing also increased (P less than 0.05) overall penetration compared to DR and SU. In the cat oocyte assay, zona penetration rate was similar (P greater than 0.05) in the DR, NS, and SU aliquots; however, the zona penetration index was increased (P less than 0.05) by the NS compared to the DR and SU treatments. This study 1) provides baseline ejaculate and endocrine norms for the leopard cat, 2) demonstrates that leopard cat sperm undergo nuclear decondensation in hamster ova and penetrate zona-intact domestic cat oocytes, 3) indicates that seminal plasma removal enhances leopard cat sperm fertilizing ability and ovum penetration, and 4) suggests that heterologous oocyte penetration is effective for assessing factors influencing fertilization and sperm function in this nondomestic felid.     

Hormonal responses to gonadotrophin releasing hormone during testicular development in male African green monkeys

Reproductive development in male African green monkeys was characterized by evaluating both luteinizing hormone (LH) and testosterone (T) before and after gonadotrophin releasing hormone (GnRH) stimulation in relation to the physical maturation of the testis. There were LH responses to GnRH at all ages studied, but the failure of some animals to respond at earlier ages suggested developmental changes in the responsiveness of the pituitary. The T secretion developed progressively but did not reach adultlike characteristics until approximately 44 months of age, at which time sperm could be demonstrated in ejaculated semen.     

Estrogens and cell multiplication in the adenohypophysis of the male rat: in vivo and in vitro studies

A single dose (1 microgram) of oestradiol sub-cutaneously injected to an immature male rat promotes a transitory increase of the pituitary mitotic activity, the maximum of which is reached between 32 and 48 hours ; the observed fluctuations are similar to those previously described for the thymidine kinase activity. In these conditions, the concentration of blood prolactin remains unaltered, as were those of LH and FSH. It follows that hyperplasy of the pituitary can be quickly induced by doses of oestrogen that do not affect significantly the hormone release. Using Moxestrol, a synthetic oestrogen not bound by the oestradiol plasma binding protein, we show that in the very young rat, the in vivo responsiveness of the pituitary increases and reaches its maximum by day 17. This results can be tentatively related to the ontogeny of the oestradiol receptors in the pituitary described by others ; all our attempts to induce the thymidine kinase in cultured glands remained unsuccessful.     

The fine structural localization of testicular phosphatases in man: The control testis

Summary Electron microscopic cytochemistry was used to determine the localization of five phosphatase enzymes—glucose-6-phosphatase, inosine diphosphatase, thiamine pyrophosphatase, acid phosphatase, and adenosine triphosphatase—in control human testes. Glucose-6-phosphatase occurred in the endoplasmic reticulum and nuclear envelope of Sertoli cells, Leydig cells and primitive spermatogonia, but was not observed in more advanced spermatogenic cells. The presence of glucose-6-phosphatase activity paralleled the presence of glycogen in spermatogenic cells, i.e., both occurred in type AL and AD spermatogonia but not in type AP or B spermatogonia or in more advanced spermatogenic cells. Inosine diphosphatase activity was found in the endoplasmic reticulum, nuclear envelope, and Golgi complex of Sertoli cells and all spermatogenic cells except late spermatids. Additionally, inosine diphosphatase activity was localized at the junctions between Sertoli cells and late spermatids, but was not associated with any other plasma membrane. Thiamine pyrophosphatase reaction product was found in the Golgi bodies of Sertoli cells and in spermatogenic cells through immature spermatids. Neither inosine diphosphatase nor thiamine pyrophosphatase was observed in the Golgi bodies of spermatids during acrosomal formation. Acid phosphatase activity was found in lysosomes of spermatogonia, spermatocytes, and spermatids, in lysosomes of Leydig cells, and in lysosomes, lipofuscin bodies, and Golgi cisternae of Sertoli cells. It is thought that Sertoli lysosomes play a role in the phagocytosis of degenerating germ cells; however, the role of spermatogenic or Leydig lysosomes is unknown. Adenosine triphosphatase activity occurred at the interfaces between two spermatogonia, and between Sertoli cells and spermatogonia, but was not observed in the spaces between two Sertoli cells, two spermatocytes, two spermatids, or between Sertoli cells and spermatocytes, or between Sertoli cells and spermatids.Supported in part by a grant from the U.S. Atomic Energy Commission (AT-(40-1)-4002).     

Effects of aromatase inhibitors and different doses of testosterone on gonadotropins in one year old male Atlantic salmon (Salmo salar)

The effects of different doses of testosterone (T), the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD) and 4-hydroxy-4-androstene-3,17-dione (4OH), and the combined treatment of T and ATD on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at the onset of puberty in juvenile Atlantic salmon males were investigated. T always increased pituitary LH. Also, ATD increased pituitary LH, though to a lesser extent than T. However, ATD combined with T diminished pituitary LH levels compared to T alone, indicating an aromatase-dependent positive feedback of T on LH in immature males. 4OH, which was less effective than ATD as an aromatase inhibitor, increased LH content. ATD treatment resulted in increased pituitary FSH levels, similar to those of mature controls. Positive effects of ATD on plasma FSH were found, indicating the presence of an aromatase-dependent negative feedback. The 4OH effects on FSH levels were inconsistent. T exerted both positive and negative effects on pituitary FSH and testes growth, depending on dose and season, with the positive effects being more pronounced with the low doses and the negative effects with the high doses. The treatment of T combined with ATD did not affect the positive effect of T alone on pituitary and plasma FSH, indicating the presence of an aromatase-independent positive feedback on FSH. There was a positive correlation between FSH and gonadosomatic index, especially during summer when gonadal development occurs.     

24-hour changes in circulating prolactin, follicle-stimulating hormone, luteinizing hormone, and testosterone in young male rats subjected to calorie restriction

This work analyzes the effect of calorie restriction on the 24 h variation of pituitary-testicular function in young male Wistar rats by measuring the circulating levels of prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Control animals were provided an equilibrium calorie diet and the experimental animals a calorie-restriction diet equivalent to 66% of food restriction for four weeks starting on day 35 of life. Different groups of control and experimental rats were killed at 6 h intervals around the clock, beginning 1 h after light on (HALO). Compared to the control animals, the mean secretion of prolactin was augmented and that of LH and testosterone decreased in calorie-restricted rats, whereas FSH release remained unchanged. Significant changes in the 24 h secretory pattern of circulating prolactin, LH, and testosterone occurred in the calorie-restricted rats. These include the appearance of a second maximum of plasma prolactin at 21 HALO, blunting of the LH peak seen at 13 HALO, and phase-shift of the testosterone peak from 13 HALO in controls to 17 HALO in calorie-restricted rats. The significant positive correlation between individual LH and testosterone levels found in controls was no longer observed in calorie-restricted rats. Availability of nutrients presumably affects the mechanisms that modulate the circadian variation of the pituitary-gonadal axis in growing male rats.     

Effect of a gonadotropin-releasing factor vaccine on follicle-stimulating hormone and luteinizing hormone concentrations and on the development of testicles and the expression of boar taint in male pigs

The objective of this study was to determine the effect of using a gonadotropin-releasing factor (GnRF) vaccine on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in plasma, the size of testicles, and the expression of boar taint in male pigs. Vaccinated pigs were compared with surgically castrated pigs and entire males. Pigs were randomly assigned to three treatment groups: surgically castrated during the first week of life (T01, n = 274), immunized twice during the fattening period with a GnRF vaccine, the first when 13 to 14 wk of age and the second when 20 to 21 wk of age (T02, n = 280), and entire males (T03, n = 56). From a subgroup of both T01 and T02 and from all pigs of group T03, blood samples were collected immediately before second vaccination (T02) and again before slaughter at either 24 to 25 or 26 to 27 wk of life to determine the plasma concentrations of LH and FSH. Testicles were removed after slaughter and their size was determined. Meat and fat samples from all pigs of T02 and T03 as well as 25% of the pigs of T01 were examined with the cold cooking and fat melting test. Immediately before the second vaccination (T02 only), LH and FSH concentrations were not significantly different between T02 and T03. However, LH and FSH concentrations were significantly higher in T01 compared with T02 and T03. Before the first slaughter date, LH and FSH concentrations were significantly lower in T02 than in T03. Testicle size was significantly lower in T02 compared with that in T03. In T02, 98% (235 of 239) of the samples were rated negative for boar taint by the cooking test, whereas in T03, 94% (48 of 51) were rated positive. In the fat melting test, 97% of T02 were rated negative and 3% (7 pigs) were rated positive, including the pigs tested positive in the cold cooking test. In T03, 94% were rated positive. All pigs (7 of 239) in T02 that were positive for boar taint in the cooking or melting test and that were tested had androstenone and skatole concentrations in backfat below threshold levels of 1 μg/g and 0.2 μg/g, respectively.     

Thymoquinone ameliorated elevated inflammatory cytokines in testicular tissue and sex hormones imbalance induced by oral chronic toxicity with sodium nitrite

Scientific evidence illustrated the health hazards of exposure to nitrites for prolonged time. Nitrites affected several body organs due to oxidative, inflammatory and apoptosis properties. Furthermore, thymoquinone (TQ) had curative effects against many diseases. We tried to discover the impact of both sodium nitrite and TQ on inflammatory cytokines contents in testicular tissues and hormonal balance both in vivo and in vitro. Fifty adult male SD rats received 80 mg/kg sodium nitrite and treated with either 25 or 50 mg/kg TQ daily by oral-gavage for twelve weeks. Testis were removed for sperms’ count. Testicular tissue homogenates were used for assessment of protein and gene expression of IL-1β, IL-6, TNF-α, Nrf2 and caspase-3. Serum samples were used for measurement of testosterone, LH, FSH and prolactin. Moreover, all the parameters were measured in human normal testis cell-lines, CRL-7002. Sodium nitrite produced significant decrease in serum testosterone associated with raised FSH, LH and prolactin. Moreover, sodium nitrite significantly elevated TNF-α, IL-1β, IL-6, caspase-3 and reduced Nrf2. TQ significantly reversed all these effects both in vivo and in vitro. In conclusion, TQ ameliorated testicular tissue inflammation and restored the normal balance of sex hormones induced by sodium nitrite both in vivo and in vitro.     

Auditory stimulation of reproductive function in male Rufous-winged Sparrows, Aimophila carpalis

Prolonged exposure to conspecific song stimulates gonadal function and reproductive hormone secretion in female birds but few studies have investigated the physiological effects of conspecific song exposure on males outside of short-term, aggressive interactions. We exposed male Rufous-winged Sparrows, Aimophila carpalis, either to conspecific song (CS Song), to heterospecific song (Black-throated Sparrow, Amphispiza bilineata; HS Song), or to no recorded song (No Song) for 59 consecutive days (two h per day). Birds were exposed to short days (8L:16D) for the first 21 days of treatment and were then transferred to long days (13L:11D) for the remaining 38 days. During long day exposure, CS Song birds experienced faster growth of testes than HS Song and No Song birds. HS Song birds also grew their testes faster than No Song birds. Plasma luteinizing hormone (LH) and testosterone did not differ between CS Song and No Song birds. However, plasma LH was higher in HS Song birds compared to other groups. There were no differences in hypothalamic immunocytochemical labeling for gonadotropin-releasing hormone, its precursor proGnRH, or gonadotropin-inhibitory hormone, nor were there differences in two song control nuclei volumes (HVC and RA) between CS Song and No Song treatment groups. Furthermore, we found no effect of heterospecific song on free-living Rufous-winged Sparrow aggressive behaviors. These data indicate that long-term exposure to auditory stimuli, such as song, can influence the reproductive system of male songbirds and different types of auditory stimuli can have differential effects on reproductive function.     

Deletion of MgcRacGAP in the male germ cells impairs spermatogenesis and causes male sterility in the mouse

MgcRacGAP (RACGAP1) is a GTPase Activating Protein (GAP), highly produced in the mouse embryonic brain and in the human and mouse post-natal testis. MgcRacGAP negatively controls the activity of Rac and Cdc42, which are key molecular switches acting on the microtubule and actin cytoskeleton and controlling various cell processes such as proliferation, adhesion and motility. Previous studies demonstrated that MgcRacGAP plays a critical role in the cytokinesis of somatic cells; hence homozygous inactivation of the gene in the mouse and mutation in Caenorhabditis elegans led to embryonic lethality due to the inability of MgcRacGAP-null embryos to assemble the central spindle and to complete cytokinesis.     

Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue

Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank’s balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen–thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen–thawed-graft groups (p < 0.05). Fresh grafts and frozen–thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p > 0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p < 0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p < 0.05). The typical damage observed in the frozen–thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes.     

Effects of photoperiod and temperature on sexual recrudescence in the male weakfish,Cynoscion regalis

Synopsis Male weakfish,Cynoscion regalis, were collected in the south-west portion of Delaware Bay from April through August of 1992. Histological examination of testes collected from these specimens was used as a baseline for comparison with laboratory data. Weakfish testes were found to be of the unrestricted, continuous spermatogenic type and spermatogenesis was apparently prenuptial. The effects of photoperiod and temperature on gonadal maturation were studied in the laboratory using fish collected in June and July 1991. These fish were exposed to two months of simulated winter conditions prior to assignment to one of four experimental photoperiod/temperature regimes. The treatments included combinations of long day (LD,15 h light) or short day (SD, 9 h light) and high (HT, 20° C) or low (LT, 13° C) water temperatures. The four treatment groups were LD/HT, LD/LT, SD/HT, SD/LT. The LD/HT group was the only one to mature fully, expressing increased plasma androgen levels, increased gonadosomatic index (GSI) and advanced spermatogenesis, as well as hypertrophy of the sonic muscles, a seasonally expressed secondary sexual characteristic of male weakfish. High temperatures promoted the later stages of spermatogenesis, which were apparently inhibited by low temperatures. The presence of an endogenous annual cycle is suggested by the partial maturation of the testes and sonic muscles in all treatment groups, regardless of photoperiod/temperature regime.     

Effects of gold on testicular steroidogenic and gametogenic functions in immature male albino rats

The present study was undertaken to evaluate the effects of gold chloride, a metallic earth salt, on steroidogenic and gametogenic functions of testis in immature rats. Immature rats of Wistar strain, were injected (s.c.) with gold chloride at the dose of 0.3 mg and 0.5 mg/kg body weight/day for 26 days. All the treated animals along with the vehicle-treated controls were sacrificed 24 hours after last injections. Testicular steroidogenic activity was evaluated by measuring the activities of two steroidogenic key enzymes, Delta5-3beta-hydroxysteroid dehydrogenase (Delta5-3beta-HSD) and 17-beta hydroxysteroid dehydrogenase (17-beta HSD). Gametogenic capacity was determined by counting the number of germ cells at stage VII of seminiferous cycle. Plasma levels of testosterone (T) was measured by radioimmunoassay (RIA). Administration of gold chloride at a dose of 0.3 mg/ kg body weight for 26 days led to insignificant changes of testicular Delta5-3beta-HSD,17beta-HSD activities and gametogenesis along with plasma T. In contrast 0.5 mg gold chloride treatment for 26 days caused a significant increase in plasma T (p < 0.001) along with stimulation of testicular Delta5-3beta-HSD activity (p < 0.001) and 17beta-HSD activity (p < 0.001). Gametogenic activity exhibited a significant increase in the number of step 7 spermatids (7Sd) (p < 0.001) at stage VII of seminiferous cycle when compared to control. The results of our experiment suggest that gold chloride treatment might be associated with significant stimulatory effects on testicular activities. Furthermore, since hormonal changes, altered steroidogenic enzymes and gametogenic activities were evident to a specific dose of gold chloride treatment, our data may have some clinical implication on the stimulation of fertility.     

Effects of intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion in American black bears (Ursus americanus)

Eight adult American black bears were used to evaluate the effects of chemical castration by intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion. Treatment did not affect testicular dimensions and did not result in decreased resting or GnRH-stimulated testosterone secretion. Multifocal hyperchoic areas in the testicular parenchyma were observed on ultrasound examination, and white foci were observed on gross pathology examination after zinc gluconate treatment. Histologically, there were normal seminiferous tubules containing either round or elongated spermatids, along with abnormal tubules in all bears after treatment. Vacuolation of the seminiferous epithelium, sloughing of germ cells into the tubules' lumen, presence of multinuclear giant cells, and reduced height of the seminiferous epithelium with missing generations of germ cells were commonly observed. The most severe testicular changes were multifocal and included fibrosis, complete degeneration of the seminiferous epithelium with shrinkage of the tubule, and sperm stasis. Epididymal sperm reserve was 982.74 ± 654.16 × 10⁶ sperm (mean ± SEM) and motile sperm were observed in the epididymis of all but one of the bears. In conclusion, although intratesticular zinc gluconate treatment in black bears resulted in testicular degenerative changes detected by ultrasound and histology examinations, sperm production was not completely ablated. We inferred that normal fertility might have been compromised, but treatment unlikely resulted in sterility.     

Effects of sub-chronic aluminum chloride on spermatogenesis and testicular enzymatic activity in male rats

Aims

The aim of this study was to determine the effects of sub-chronic aluminum chloride (AlCl₃) on spermatogenesis and testicular enzymatic activity in male rats.

Main methods

Forty Wistar male rats were randomly divided into four groups: control group (CG, 0), low-dose group (LG, 64.18 mg/kg BW AlCl₃), mid-dose group (MG, 128.36 mg/kg BW AlCl₃) and high-dose group (HG, 256.72 mg/kg BW AlCl₃). The rats were orally administered with AlCl₃ for 120 days. At the end of the experiment, the contents of Al, Fe, Cu and Zn, the enzyme activities of testicular acid phosphatase (ACP), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), lactate dehydrogenase isoenzyme (LDH-x), the sperm count and the sperm malformation rate were examined.

Key findings

The results showed that the Al and Cu contents, sperm count and the enzyme activities of testicular ACP, SDH, LDH and LDH-x decreased, while the Zn and Fe contents and sperm malformation rate increased in AlCl₃-treated rats.

Significance

It suggests that sub-chronic AlCl₃ disorders the balance of trace element and decreases the spermatogenesis and the activities of testicular enzymes, indicating that AlCl₃ has adverse effect on the testicular function in male rats.