A role for nocturnal serum testosterone surge in regulating spermatogenesis in the adult non-human primate

Adult male bonnet monkeys exhibit nychthemeral rhythms in testosterone (T) secretion but the precise role of this heightened level of T secretion in regulating spermatogenesis is not known. Intranasal administration of microdoses (500 mu g or 250 mu g/day) of Norethisterone (IN-NET) to adult monkeys (n = 6) at 1600 h each day selectively and completely suppressed the nocturnal surge levels of serum T. Concomitant with this was a significant reduction (P<0.01) in serum LH but not FSH levels. DNA flow cytometric analysis of testicular biopsy tissue showed by week 10 of IN-NET treatment an arrest in meiotic transformation of primary spermatocytes (4C) to round/elongate (1C/HC) spermatids and by week 20 there was a complete absence of 4C, 1C and HC cells (with a relative accumulation in 2C cells). The accumulated meiotic (4C) cells at week 10 showed an increase (>80%, P<0.01) in coefficient of variation and a decrease in intensity of DNA-bound ethidium bromide fluorescence, parameters characteristic of degenerating 'apoptotic' subpopulation of germ cells. While two monkeys exhibited acute oligozoospermia 4 became azoospermic by 20 weeks of IN-NET treatment. A complete, qualitative reversal in the regressive changes in spermatogenesis and near-normal sperm output were apparent at the end of a 20-week recovery phase. These data demonstrate that prolonged, selective suppression of nocturnal surge levels of serum T secretion exerts a primary effect on meiosis in spermatogenesis leading to oligo/azoospermic status in adult bonnet monkeys.

The plant Banjauri (Vicoa indica) exhibits antifertility activity in adult female bonnet monkeys (Macaca radiata)

The antifertility activity of the plant Vicoa indica was tested in proven fertile bonnet monkeys. The dry powder of the whole plant was fed to the cycling monkeys on day 1 to 14 of menstrual cycle or day 9 to 14 of cycle or on day 2 to 5 after delivery and the fertility was evaluated in the following cycle in cycle fed monkey or after weaning the young one in the post-partum fed monkeys. Results indicated that while feeding in the post-partum monkeys did not confer any protection against pregnancy feeding during day 1 to 14 of cycle, protected from pregnancy. The monkeys did not become pregnant even after exposure to the proven fertile male monkeys for 13 ovulatory cycles while all the vehicle fed monkeys became pregnant within 3 cycles.

Use of norethisterone and estradiol in mini doses as a contraceptive in the male : Efficacy studies in the adult male bonnet monkey (Macaca radiata)

Administration of norethisterone (NET) or NET + estradiol benzoate using an Alzet minipump or as once-a-month intramuscular injection of their depot forms, NET-enanthate (NET-EN) and estradiol valerate (E-val), resulted in azoospermia in all monkeys (n = 13) within 60 to 150 days of treatment. Although addition of depot form of testosterone (T, 20 mg/month) to the regimen restored the behavioral response typical of a normal male, it did not reverse the azoospermic state. Serum T (heightened nocturnal) levels were significantly reduced (> 85%, p < 0.001) in all the treated groups. Evidence for blockade in spermatogenesis following treatment was obtained by DNA flow cytometry. Following withdrawal of treatment, the T level was restored to normalcy within 15 days but 120 days more were required for the animals to exhibit normal sperm counts. In conclusion, the efficacy of once-a-month injection of relatively low doses of NET-EN + E-Val to bring about azoospermia in monkeys, in a relatively short time, has been demonstrated. As the results are uniform and reproducible, it appears desirable that this steroid regimen be tested in man for its contraceptive efficacy.

Changes in testicular function following specific deprivation of LH in the adult male rabbit

Sexually mature male rabbits actively immunized against highly purified ovine LH (oLH) were used as a model system to study the effects of endogenous LH deprivation (and therefore testosterone) on spermatogenesis as well as pituitary FSH secretion. Immunization against oLH generated antibody titres capable of cross-reacting and neutralizing rabbit LH and this resulted in a significant reduction (P<0.01) in serum testosterone levels by 2-4 weeks of immunization. A significant increase in circulating FSH concentration (from a basal level of similar to 1 ng to 60-100 ng/ml; P<0.01) was observed within 4-6 weeks of immunization, perhaps a consequence of the negative feedback effect of the lack of testosterone. The effect of LH deprivation on spermatogenesis assessed by DNA flow cytometry and histological analyses of testicular biopsy tissue revealed that lack of testosterone primarily results in a rapid reduction and complete absence of round (1C) and elongated (HC) spermatids. The immediate effect of LH/testosterone deprivation thus appears to be at the step of meiotic transformation of primary spermatocytes (4C) to 1C. A significant reduction (>80%; P<0.01) in the 4C population and a relative accumulation (>90%; P<0.01) in spermatogonia (2C) was also observed, suggesting a need for testosterone during the transformation of 2C to 1C. In all but one of the rabbits, both qualitative and quantitative recovery in spermatogenesis occurred during the recovery phase, even at a time when only a marginal increase in serum testosterone (compared with the preimmunization) levels was observed as a result of a rapid decline in the cross-reactive antibody titres. These results clearly show that LH/testosterone deprivation in addition to primarily affecting the meiotic step also regulates the conversion of 2C to 4C during spermatogenesis.

Hemiorchidectomy leads to dramatic and immediate alterations in pituitary follicle-stimulating hormone secretion and the functional activity of the remaining testis in the adult male bonnet monkey (Macaca radiata)

The aim of the present study was to examine the effect of hemiorchidectomy (HO) on serum FSH, LH, testosterone (T), and inhibin (INH) concentrations as well as on the testicular volume (TV) and on changes in the kinetics of germ cell turnovers in the remaining testis of adult male bonnet monkeys. Blood samples collected at 2200 h at various times before and after HO and testicular biopsies obtained at different periods were subjected to hormone analysis and DNA flow cytometry. Though serum T levels were lowered (p < 0.05) at 12 h after HO, T levels rapidly returned to intact control concentrations by Day 5. While serum LH remained unaltered, serum FSH increased markedly within 2 days of HO and remained significantly (p < 0.05) elevated over the next 90 days. Though serum INH showed a significant decrease (p < 0.05) by 15 min of HO, it returned to approximately 80% of intact levels within one week. The TV of the remaining testis showed maximal increment by Day 30 (p < 0.05) of HO. DNA flow cytometric analysis 24 days after HO showed increases (p < 0.05) in spermatogonia (2C) and primary spermatocytes (4C). These cell types by Day 45 had transformed to round (1C) and elongate (HC) (by 38%, p < 0.001) spermatids. Overall spermatogenesis (conversion of 2C to 1C and HC) showed significant enhancement at Days 110 and 175, suggesting that the spurt in spermatogenic activity is not confined to a single spermatogenic cycle.

Correlation of seasonal changes in sperm output with endocrinological changes in the adult male bonnet monkey, Macaca radiata

We have examined the monthly variations in sperm output and attempted to correlate the profiles of endocrine hormones secreted with the sperm counts throughout the ,year in the adult male bonnet monkey. As previously reported, there was a distinct spurt in sperm output beginning September through December months. A concomitant increase in serum testosterone and prolactin concentrations were also noted during September through November (mid and post-monsoon season). Although there was a marked increase in gonadotropin releasing hormone stimulated testosterone secretion, the peak testosterone concentrations post gonadotropin releasing hormone injection did not vary significantly (P>0.05) throughout the year. Basal serum follicle stimulating hormone concentrations did not vary significantly (P>0.05) during April to June months compared to September-November months. Serum inhibin concentration remained unaltered throughout the year, except in the month of March. The results of this study provide evidence for annual rhythms in prolactin and testosterone secretion and a distinct seasonality in the sperm output of the adult male bonnet monkey, but the pituitary responsiveness to exogenous gonadotropin releasing hormone remains unaltered throughout the year. Because of the existence of seasonality as noted in the present study, future studies which utilize the adult male bonnet monkey as an experimental model need to take into consideration the seasonal effects on reproductive function in this species.

Evidence for follicle-stimulating hormone mediation in the hemiorchidectomy-induced compensatory increase in the function of the remaining testis of the adult male bonnet monkey (Macaca radiata)

Hemiorchidectomy (HO) in the adult male bonnet monkey results in a selective increase in circulating concentrations of FSH and testosterone, and this is accompanied by compensatory increase in sperm production by the remaining testis. We investigated the possible role of increased FSH concentration that occurs after HO in the compensatory increase in the activity of the remaining testis. Of eight adult male bonnet monkeys that underwent HO, four received i.v. injections every other day for 30 days of a well-characterized ovine FSH antiserum (a/s) that cross-reacts with monkey FSH. The remaining four males received normal monkey serum (NMS) as control treatment in a protocol similar to that employed for ais-treated males. Blood samples were collected between 2100 and 2200 h before and 1/2, 1, 3, 5, 7, 14, 22, and 29 days after HO. Testicular weight, number of 3 beta-hydroxy steroid dehydrogenase-positive (3 beta-HSD+) cells, and DNA flow cytometric analysis of germ cell populations were obtained for testes collected before and at the termination of NMS or ais treatment. In NMS-treated males, circulating serum FSH concentrations progressively increased to reach a maximal level by Day 7 after HO (1.95 +/- 0.3 vs. 5.6 +/- 0.7 ng/ml on Days -1 and 7, respectively). Within 30 min of ais injection, FSH antibodies were detected in circulation, and the antibody level was maintained at a constant level between Day 7 and end of treatment (exhibiting 50-60% binding to I-125-hFSH). Although circulating mean nocturnal serum testosterone concentration showed an initial decrease, it rose gradually to pre-HO concentrations by Day 7 in NMS-treated males. In contrast, nocturnal mat serum testosterone concentrations in a/s-treated males remained lower than in NMS-treated controls (p < 0.05) up to Day 22 and thereafter only marginally increased. Testicular weights increased (p < 0.05) over the pre-HO weight in NMS- but not in ais-treated males. After HO, the number of 3 beta-HSD+ cells (Leydig cells) was markedly increased but was significantly (p < 0.05) higher in NMS-treated males compared to a/s-treated males. A significant (p < 0.05) reduction in the primary spermatocyte population of germ cells was observed in ais-treated compared to NMS-treated males. These results suggest that the increased FSH occurring after HO could be intimately involved in increasing the compensatory functional activity of the remaining testis in the male bonnet monkey.

Transcription factor erect wing (EWG) is involved in indirect flight muscle patterning, development and maintenance in Drosophila

Muscle development is a multistep process which includes myoblast diversification, proliferation, migration, fusion, differentiation and growth. A hierarchical exhibition of myogenic factors is important for dexterous execution of progressive events in muscle formation. EWG (erect wing) is a transcription factor known to have a role in indirect flight muscle development (IFM) in Drosophila. We marked out the precise spatio-temporal expression profile of EWG in the myoblasts, and in the developing muscles. Mutant adult flies null for EWG in myoblasts show variable number of IFM, suggesting that EWG is required for patterning of the IFM. The remnant muscle found in the EWG null flies show proper assembly of the structural proteins, which implies that some myoblasts manage to fuse, develop and differentiate normally indicating that EWG is not required for differentiation program per se. However, when EWG expression is extended beyond its expression window in a wild type background, muscle thinning is observed implying EWG function in protein synthesis inhibition. Mis-expression studies in wing disc myoblasts hinted at its role in myoblast proliferation. We thus conclude that EWG is important for regulating fusion events which in turn decides the IFM pattern. Also IFM in EWG null mutants show clumps containing broken fibres and an altered mitochondrial morphology. The vertebrate homolog of EWG is nuclear respiratory factor1 (NRF1) which is known to have a function in mitochondrial biogenesis and protection against oxidative stress. Gene expression for inner mitochondrial membrane protein, Opa1-like was found to be absent in these mutants. Also, these flies were more sensitive to oxidative stress, indicating a compromised mitochondrial functioning. Our results therefore demonstrate that EWG functions in maintaining muscles’ structural integrity by ensuing proper mitochondrial activity.

Roles of the troponin isoforms during indirect flight muscle development in Drosophila

Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, a majority of the myofibrillar proteins in the indirect flight muscles (IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.