Establishment of the need for oestrogen during implantation in non-human primates.

Deprivation of oestrogen during post-ovulatory mated cycles in proven fertile female bonnet monkeys by tamoxifen, aromatase inhibitor or oestrogen antiserum resulted in inhibition of pregnancy establishment in all three groups of animals. However, more than 85% of the animals became pregnant within three exposures to proven fertile males in the control group. These results suggest the requirement for oestrogen in pregnancy establishment in primates. Based on this conclusion, it is suggested that use of a suitable and potent anti-oestrogenic compound can be exploited as an alternative approach to contraception.

Immunobiology of a synthetic luteinizing hormone receptor peptide 21-41.

Immunization of adult male rabbits with a synthetic luteinizing hormone-receptor peptide (LH-RP; representing amino-acids 21-41 of the extracellular domain of the rat LH receptor) resulted in production of high-titer antibodies capable of interacting with particulate and cell-based LH receptors. The antibody produced was able to inhibit binding of 125I-labeled human chorionic gonadotropin (hCG) to a particulate sheep luteal LH receptor preparation by 40%-50%. Maximal inhibitory activity was correlated with high antibody titer. Immunocytometry revealed that the antibody could directly bind to cells having LH receptors, such as rat granulosa and Leydig cells. The antibodies recognized a 77-kilodalton membrane protein in Western blots of mouse testicular extracts. Interaction of endogenous Leydig cell LH receptor with the LH-RP antibody resulted in both hormone agonist and antagonistic activities. The hormone-mimicking activity (increase in serum testosterone over control) was confined only to the early phase of immunization when the antibody titer was low. Blockade of LH receptor during the later part of immunization resulted in a significant reduction in serum testosterone over controls and inhibition of spermatogenesis. DNA flow cytometry showed that a specific and significant inhibition of meiosis (transformation of primary spermatocytes to round and elongated spermatids P < .01) and spermiogenesis (transformation of round spermatids to elongated spermatids P < .0001) occurred following blockade of LH function.

Effect of long-term treatment with aromatase inhibitor on testicular function of adult male bonnet monkeys (M. radiata).

The role/need for estrogen in regulating testicular function of adult male bonnet monkeys (M. radiata) has been investigated by dosing orally a group of five normal males 2.5 mgs of CGP 47645, a long-acting nonsteroidal aromatase inhibitor (AI), once every 5 days for over 150 days. Such treatment resulted in a 10-fold increment in nocturnal serum testosterone (T) levels, which were sustained for 85 days of treatment, and a twofold increment in basal serum T levels was present throughout the 150 days of treatment. Analysis of ejaculated semen showed a marked reduction (approximately 90%) in sperm counts in four out of five monkeys between Days 55-85 of treatment. During this period, the motility score also was markedly reduced from a normal score of 3-5 to 0-2. Flow cytometric analysis of testicular germ cells obtained from biopsy tissue taken on Days 63 and 120 indicated a marked reduction only in elongating/elongated spermatid population (compared to Day 0 values), suggesting inhibition in spermiogenic process. Epididymal sperm maturation also seemed effected as sperm chromatin, on flow cytometric analysis for decondensability following exposure to 5 mM dithiotreitol, showed to be in a hypercondensed state. This study thus indicates that estrogen has an important role in providing normal testicular and sperm function in the primate.

Is there a true requirement for follicle stimulating hormone in promoting spermatogenesis and fertility in primates?

Although the role of follicle stimulating hormone (FSH) in regulating ovarian function is well accepted, its need in regulating testicular function of the adult continues to be debated. Sertoli cells of all mammals have FSH receptors and are known to regulate differentiation and transformation of germ cells to spermatozoa. However, there appear to be species and age differences in the way in which FSH regulates spermatogenesis. An attempt has been made in the current paper to examine critically the newer data available in support of and against the concept that FSH is required to regulate spermatogenesis and fertility of the primate. As there is no evidence to indicate that testicular function in monkeys and humans is regulated differently, the information available using the monkey as the experimental surrogate model is discussed in some depth. These are correlated, wherever feasible, to the newer information emerging from clinical studies. It appears from these studies that in the primate (including humans) FSH, besides promoting quantitative spermatogenesis leading to production of a normal number of spermatozoa, has a critical role in regulating spermiogenesis, the process that controls the formation of normal fertilizable mature spermatozoa. While the requirement for FSH in promoting fertility in the male monkey is reasonably well established, in humans the evidence currently available in favour of the concept is still circumstantial and more work needs to be done to establish the hypothesis beyond any doubt.

Susceptibility of sperm chromatin to acid denaturation in situ: a study in endogenous FSH-deprived adult male bonnet monkeys (Macaca radiata).

Acid denaturation of calf thymus DNA in vitro followed by acridine orange (AO) binding induced a 112% increase in the emission of red, a 58% decrease in green, and a consequential decrease in the ratio of green:red fluorescences from 1.7 to 0.9. This metachromatic property of AO on binding to DNA following acid denaturation was utilized to study the susceptibility of normal and ovine follicle-stimulating hormone (oFSH) actively immunized bonnet monkey spermatozoa voided throughout the year. For analyses, the scattergram generated by the emission of red and green fluorescences by 10,000 AO-bound sperm from each semen sample was divided into 4 quadrant zones representing percentage cells fluorescing high green-low red (Q1), high green-high red (Q2), low green-low red (Q3) and low green-high red. (Q4). Normal monkey sperm obtained during the months of July-December exhibited 76, 13, and 11% cells in Q2, Q3, and Q4 quadrants, respectively. However, during January-June, when the females of the species are markedly subfertile, noncycling, and amenorrhoeic, the spermatozoa ejaculated by the male monkeys exhibited 38, 39, and 23% sperm in Q2, Q3, and Q4, respectively, the differences being highly significant (p < .01-.001). FSH deprivation induced significant shifts in fluorescence emissions, from respective controls, with 39, 33, and 28% cells in Q2, Q3, and Q4, respectively, during July-December, and 15, 48, and 37% sperm in Q2, Q3, and Q4 quadrants, respectively, during January-June. It is postulated that the altered kinetics of germ cell transformations and the deficient spermiogenesis observed earlier following FSH deprivation in these monkeys may have induced the enhanced susceptibility to acid denaturation in sperm.

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.

PIP: Monthly intramuscular injection with an Alzet minipump of depot norethisterone enanthate and estradiol valerate (E-val) produced azoospermia in 13 adult male bonnet monkeys within 60-150 days. Although azoospermia was achieved earlier when E-val was added to the injection, this agent can be eliminated once azoospermia occurs. Addition of the depot form of testosterone (20 mg/month) restored the sexual behavioral response but did not reverse azoospermia. Serum testosterone levels were significantly reduced in all treatment groups. DNA flow cytometry revealed evidence for blockade in spermatogenesis after treatment. There were no changes in the serum lipid profile. The testosterone level returned to normal within 15 days after the end of treatment, but normal sperm counts were not observed for another 120 days. Since the steroid formulation investigated in this study has been used effectively in women for over a decade, tests of its contraceptive efficacy in men now seem warranted.

Development of male contraceptive vaccine--a perspective.

This paper reviews the recent advances that have occurred in the area of development of a male contraceptive vaccine. The vaccine candidates considered for review are hormone/hormone receptor-based proteins including luteinizing hormone-releasing hormone (LHRH)/LH, follicle stimulating hormone (FSH), as well as LH and FSH receptor proteins. The review also highlights the advances in our basic understanding of gonadotrophin action which have led to development of these vaccines. Focus is mainly on studies in the non-human primate which may be directly relevant to projected studies in the human. The data indicate that the vaccines are well tolerated by the primate (including the human based on limited data) and do not give rise to any known toxic symptoms or immediate health hazards. The response to the immunogen has been uniform and it may be possible to increase antibody titres as well as prolong the immune response by adding acceptable immune stimulators to the adjuvant cocktail and developing better immunization schedules or immunogen delivery systems. Contraceptive vaccines for the male are a feasible proposition and attention should now be focussed on evaluating carefully the bioefficacy of antibodies raised to recombinant ovine FSHbeta or FSH receptor protein fragments in both human and non-human primates. The advantage of the FSH/FSH receptor over the LHRH/LH-based vaccine lies in the fact that the former does not require an exogenous testosterone supplement to maintain accessory gland function, libido etc. The LHRH/LH-based vaccine results in azoospermia, while the FSH vaccine causes the production of low numbers of poor quality spermatozoa which are incapable of impregnating cycling females.

Immunization of male bonnet monkeys (M. radiata) with a recombinant FSH receptor preparation affects testicular function and fertility.

Immunization of proven fertile adult male monkeys (n=3) with a recombinant FSH receptor protein preparation (oFSHR-P) (representing amino acids 1-134 of the extracellular domain of the receptor Mr approximately 15KDa) resulted in production of receptor blocking antibodies. The ability of the antibody to bind a particulate FSH receptor preparation and receptors in intact granulosa cells was markedly (by 30-80%) inhibited by FSH. Serum T levels and LH receptor function following immunization remained unchanged. The immunized monkeys showed a 50% reduction (p<0.001) in transformation of spermatogonia(2C) to primary spermatocytes (4C) as determined by flow cytometry and the 4C:2C ratio showed a correlative change (R 0.81, p<0.0007) with reduction in fertility index (sperm counts X motility score). Breeding studies indicated that monkeys became infertile between 242-368 days of immunization when the fertility index was in the range of 123+/-76 to 354+/-42 (compared to a value of 1602+/-384 on day 0). As the effects observed are near identical to that seen following immunization with FSH it is suggestive that oFSHR-P can substitute for FSH in the development of a contraceptive vaccine.

Breeding of bonnet monkeys (Macaca radiata) in captivity.

The results of studies on the breeding of South Indian bonnet monkeys (macaca radiata) over 20 years at our institution are presented. The menstrual cycle and hormonal changes were similar to those reported for Macaca mulatta. It was noted that summer amenorrhoea could be eliminated by housing the monkeys in rooms supplied with humidified air. Although a fertility index (proportion of animals becoming pregnant within three exposures to a proven fertile male) of 60 to 65% was achieved by random breeding, an index of 80 to 85% was achieved by controlled breeding (prior monitoring of serum estradiol-17 beta concentration on days 7, 8, 9, and 10).

Responsiveness of human male volunteers to immunization with ovine follicle stimulating hormone vaccine: results of a pilot study.

A study of 140 days duration was performed to examine if human male volunteers (n = 5) respond to ovine follicle stimulating hormone (oFSH) immunization (administered adsorbed on Alugel on days 1, 20, 40 and 70) by producing antibodies capable of both binding and neutralizing bioactivity of human FSH. The kinetics of antibody production for both the immunogen (oFSH) and the cross-reactive antigen (hFSH) were essentially similar. The volunteers responded only to the first two immunizations. The boosters given on days 40 and 70 were ineffective, probably because of the presence of substantial amounts of circulating antibody to oFSH. Of the antibodies generated to oFSH, 25-45% bound hFSH with a mean binding affinity of 0.65 x 10(9) +/- 0.53 M(-1). The binding capacities at the time of high (30-80 days of immunization) and low (>110 days) titres were 346 +/- 185 and 10.5 +/- 5.8 ng hFSH/ml respectively. During the period of high titre, free serum FSH (value in normal males 1-5 ng/ml) was not monitorable. A 50 microl aliquot of the antiserum obtained from different volunteers between days 30 and 80 and on day 140 blocked binding of (125)I-labelled hFSH to its receptor by 82 +/- 9.7 and 53 +/- 12.2% respectively. The antibody produced was specific for FSH, and no significant change in the values of related glycoprotein hormones (luteinizing hormone/testosterone and thyroid stimulating hormone/thyroxine) were recorded. Seminal plasma transferrin, a marker of Sertoli cell as well as of seminiferous tubular function, showed marked reduction (30-90%) following immunization with oFSH. Considering that endogenous FSH remained neutralized for approximately one sperm cycle only (65 days), the reduction in sperm counts (30-74%) exhibited by some volunteers is encouraging. Immunization with oFSH did not result in any significant changes in haematology, serum biochemistry or hormonal profiles. There was no production of antibodies capable of interacting with non-specific tissues. It is concluded that it should be possible to obtain a sustained long-term blockade of endogenous FSH action in men by using oFSH as an immunogen. This is a prerequisite for obtaining significant reduction in the quality and quantity of spermatozoa produced, thus leading to infertility.

Demonstration of complimentarity between monoclonal antibodies (MAbs) to human chorionic gonadotropin (hCG) and polyclonal antibodies to luteinizing hormone/hCG receptor (LH-R) and their use in better understanding hormone-receptor interaction.

We have earlier reported that polyclonal antisera raised in rabbits to a luteinizing hormone/chorionic gonadotropin receptor (LH-R) purified from sheep luteal tissue has antibodies exhibiting hormone agonistic and antagonistic activities. Western blot analysis showed this antibody (LHR-anti IgG) to be highly specific to sheep luteal LH receptor (LH-R) (Jeyakumar and Moudgal, 1991). Using this, along with a battery of mouse monoclonal antibodies (MAbs) to hCG, an attempt has been made to better understand the interaction of LH/hCG with its receptor. Of the eight hCG MAbs screened, three (B14/B7, B52/18 and A7/G4) were specific to the beta-subunit; while a second set of three (G10/F7, H9/E9 and B52/21) were specific to the alpha-subunit. Two additional MAbs (B52/28 and F9/G8) did not recognize individual subunits, but bound like the rest intact hCG. Both 125I hCG and 125I anti LHR-IgG bound specifically to ovine luteal membrane LH-R. Assuming that a certain degree of similarity should exist between hCG and LHR-anti IgG, different hCG MAbs were tested for their ability to block the binding of either 125I hCG or 125I LHR-anti IgG to sheep luteal LH-R. It appears that hCG and LH-R share a minimum of four sites that are complementary to each other and these are recognized by the hCG MAbs B14/B7, G10/F7, A7/G4, and H9/E9. Whereas two of the MAbs B14/B7 and G10/F7 blocked the binding of both 125I labeled hCG and LHR-anti IgG to the receptor, MAbs A7/G4 and H9/E9 only inhibited the binding of 125I LHR-anti IgG to the LH-R. Although individually B14/B7 and G10/F7 blocked the binding of 125I LHR-anti IgG to LH-R to a maximum extent of 43%, together they inhibited binding by as much as 80%. The ability of B14/B7 to inhibit binding of 125I LHR-anti IgG to the receptor was also significantly increased by the addition of A7/G4. Finally, by demonstrating direct binding of the immobilized hCG MAbs B14/B7, G10/F7, A7/G4, and H9/E9 to LHR-anti IgG, we have been able to establish that the receptor binding sites of hCG and LHR-anti IgG are complementary and that a set of four sites are recognizable by the hCG MAbs. From the degree of interaction, it appears that two sites recognized by MAbs B14/B7 and G10/F7 (representing a site each in the beta- and alpha-subunit of hCG) have a prominent role in the interaction of hCG with its receptor. Thus, this study has provided us with an opportunity to investigate the interaction of LH/hCG with its receptor by an indirect approach of monitoring the binding of their respective antibodies with each other.

Enhanced susceptibility of follicle-stimulating-hormone-deprived infertile bonnet monkey (Macaca radiata) spermatozoa to dithiothreitol-induced DNA decondensation in situ.

Immunoneutralization of endogenous follicle-stimulating hormone (FSH) of adult male monkeys leads to oligospermia and infertility despite unchanged testosterone levels. The inability of these monkeys to impregnate despite repeated exposures to cycling females appeared to be due to abnormal alterations in the kinetics of germ cell transformations and deficient spermiogenesis. Here we investigated the stability of sperm chromatin in oFSH-immunized monkeys as a marker for spermiogenesis. The susceptibility of spermatozoa to in vitro decondensation induced by dithiothreitol (DTT, 0.05-50 mM) was studied by measuring the nuclear fluorescence of DTT-treated, ethidium bromide (EB)-stained sperm using flow cytometry. Changes in sperm morphology and binding of thiol-specific 14C-iodoacetamide (14C-IA) were also monitored under the same conditions. Sperm from the immunized monkeys decondensed at a lower concentration of DTT, bound more EB, and decondensed more extensively than those from control animals. The difference was apparent in sperm from all regions of the epididymis. Immunized monkey sperm also bound significantly more 14C-IA at all concentrations of DTT. Overall, the effective concentration of DTT required to elicit 50% of maximal decondensation (ED50) of epididymal and ejaculated sperm was significantly lower for the immunized monkeys than even the caput sperm of controls. These results suggest that FSH deprivation in monkeys results in production of sperm with limited potential for disulfide formation and reduced chromatin stability.

Immunization of male rabbits with sheep luteal receptor to LH results in production of antibodies exhibiting hormone-agonistic and -antagonistic activities.

Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAGE and ligand blotting) from sheep luteal membrane using human CG (hCG)-Sepharose affinity chromatography, were raised in three adult male rabbits (R-I, R-II and R-III). Each of the rabbits received 20-30 micrograms of the purified receptor in Freund's complete adjuvant at a time. Primary immunization was followed by booster injection at intervals. Production of receptor antibodies was monitored by (1) determining the dilution of the serum (IgG fraction) that could specifically bind 50% of 125I-LH/CG-R added and (2) analysing sera for any change in testosterone levels. Following primary immunization and the first booster, all three rabbits exhibited a 2.5- to 6.0-fold increase in serum testosterone over basal levels and this effect was spread over a period of time (approximately 40 days) coinciding with the rise and fall of receptor antibodies. The maximal antibody titre (ED50) produced at this time ranged from 1:350 to 1:100 to below detectable limits for R-I, R-II and R-III respectively. Subsequent immunizations followed by the second booster resulted in a substantial increase in antibody titre (ED50 of 1:5000) in R-I, but this was not accompanied by any change in serum testosterone over preimmune levels, suggesting that with the progress of immunization the character of the antibody produced had also changed. Two pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further experiments. IgG isolated from bleed I but not from bleed II antiserum showed a dose-dependent stimulation of testosterone production by mouse Leydig cells in vitro, thus confirming the in vivo hormone-mimicking activity of antibodies generated during the early immunization phase. The IgG fractions from both bleeds were, however, capable of inhibiting (1) 125I-hCG binding to crude sheep luteal membrane (EC50 of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimulated testosterone production by mouse Leydig cells in vitro, indicating the presence of antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The fact that bleed I-stimulated testosterone production could be inhibited in a dose-dependent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intrinsic to the bleed I antibody. The receptor antibody (bleed II) was also capable of blocking LH action in vivo, as rabbits passively (for 24 h with LH/CG-R antiserum) as well as actively (for 430 days) immunized against LH/CG-R failed to respond to a bolus injection of LH (50 micrograms). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to immunization with holo LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities.

Use of a specific aromatase inhibitor for determining whether there is a role for oestrogen in follicle/oocyte maturation, ovulation and preimplantation embryo development.

The specific role of oestrogen in follicular maturation, ovulation and early embryonic development was investigated using Fadrozole (CGS 16949A), a non-steroidal aromatase inhibitor, to block oestrogen synthesis specifically and effectively in experimental animals. Induced and normal cyclical follicular maturation as well as normal and hCG/LH-induced ovulation were relatively unaffected by significantly depleting oestrogen in all animals (hamsters, rabbits, monkeys) studied other than rats. Fadrozole treatment significantly reduced the number of healthy antral follicles produced and the ovulatory response to exogenous hCG of immature rats primed with pregnant mares' serum gonadotrophin. The effect was specific, in that exogenously administered oestrogen reversed the blockade. Depletion of oestrogen, starting early in pro-oestrus in hamsters, had no effect on ovulation, oocyte maturation and fertilization, as normal implantation sites were seen on day 6 after coitus. In rabbits, oestrogen depletion during the periovulatory phase affected oviductal morphology and function. Although fertilization was not impaired, early embryo development did not appear to be normal. In monkeys, oestrogen depletion during the follicular phase did not lead to a block of follicular maturation or ovulation but resulted in a significant reduction in secretion of cervical mucus. Administration of either Fadrozole or Tamoxifen during the early luteal phase in cyclic monkeys that were allowed to mate prevented implantation and this appears to be due to impaired fertilization or faulty embryo development. These results suggest that, although there is a clear requirement for oestrogen to support the reproductive cycle in the female, the need for oestrogen in regulating specific events is species dependent.

Rat epididymal sperm exhibit on dithiothreitol treatment in vitro quantifiable differences in patterns of light scatter, uptake of 14C-iodoacetamide and binding of ethidium bromide to DNA.

The extent to which chromatin of rat caput (CAP), corpus (COR), cauda (CAU) spermatozoa undergo condensation and compaction is known to be a function of progressive increase in the formation of inter- as well as intra-protamine disulphide bridges during their transit through the epididymis. Relative compaction undergone by the nuclear chromatin of these sperm populations was studied by monitoring their susceptibility to in vitro decondensation induced by varying concentrations (0, 0.01, 1, 5, 10, 50 mM) of disulphide reducing agent, dithiothreitol (DTT) after an initial exposure to 0.01% papain. Following this treatment and staining with the nucleic acid specific fluorochrome, ethidium bromide (EB), it was observed that irrespective of the epididymal region from which they were collected, spermatozoa exhibited DTT dose-dependent (a) increase in nuclear size as seen under fluorescence microscopic examination, (b) decrease in flow cytometrically quantifiable light scatter parameters--forward scatter (FSc, 'nuclear size') and side scatter (SSc, nuclear 'granularity'), (c) increase in individual cell EB binding when analyzed by DNA flow cytometry, and (d) increase in thiol specific 14C-iodoacetamide (14C-IA) uptake. The decrease in both FSc and SSc occurring in spite of actual increase in nuclear size has been attributed to increase in translucency of spermatozoan nuclei consequent to decondensation. The FSc, SSc and EB bindability were studied by monitoring both the channels of maximal cell concentration detected in the flow cytograms as well as by digitally quantitating the numbers of cells within specific channels (1-64, 65-128, 129-192 and 193-256) of the flow cytogram. The latter indicated a measure of the variability in the response of populations of sperm within each sample to DTT induced decondensation. At any given concentration of DTT, especially between 5-10 mM, the differences observed between sperms of different regions were consistent and significant (P < 0.01-P < 0.001), maximal changes being shown by CAP and minimal by CAU sperm, COR sperm appearing in between. The effective concentration of DTT required to elicit 50% of maximal (i.e. that exhibited by CAP sperm when taken as 100%) effect (ED50) varied significantly among CAP, COR and CAU sperms for each of the parameters studied (P < 0.01-P < 0.001). It is concluded that the differences observed among the three epididymal sperm populations are due to differences in the extent of susceptibility to decondensation in vitro and that this is dependent upon the variation in the -S-S-content of their chromatin during different stages of epididymal transit. All the parameters used (with the exception of fluorescence microscopy) can be quantified and as all of them show a similar dose dependency to DTT treatment, any one of these parameters can be conveniently used to determine the mature/immature status of the sperms voided. Application of such a method to determine the quality of sperms voided by man appears feasible.

Blockade of estrogen synthesis with an aromatase inhibitor affects luteal function of the pseudopregnant rat.

The luteotropic action of estrogen (E) was investigated using immature pseudopregnant rat as the model and CGS 16949A (Fadrozole hydrochloride), a potent aromatase inhibitor (AI), to block E synthesis. Aromatase activity could be inhibited by administering CGS 16949A (50 micrograms/day/rat) via a mini osmotic Alzet pump (model 2002) for 3 days during pseudopregnancy. This resulted in significant reduction of serum (40%, P < 0.05) and intraovarian 70.6%, P < 0.001) estradiol-17 beta (E2) levels. The serum and intraovarian progesterone (P4) levels as analyzed on day 4 of pseudopregnancy were also reduced by > or = 50% (for both, P < 0.01). Simultaneous administration of estradiol-3-benzoate (E2B) via an Alzet pump during the AI treatment period at a dose of 1 microgram/day could completely reverse the AI induced reduction in P4 secretion. The luteal cells of experimental rats depleted of E in vivo showed a significantly reduced response upon incubation with hCG or dbcAMP in vitro (P < 0.05 and 0.001, respectively). Addition of E2 (500 pg/tube) at the time of in vitro incubation was able to partially increase the responsiveness to hCG. The luteal cell LH/hCG receptor content and the affinity of hCG binding to the receptor remained unchanged following AI treatment in vivo. Both esterified and total cholesterol content of luteal cells of rats treated with AI in vivo was significantly high (P < 0.05) suggesting that E lack results in an impairment in cholesterol utilization for steroidogenesis. The results clearly show that E regulates luteal function in the pseudopregnant rat by acting at a non-cAMP mediated event and this perhaps involves facilitation of cholesterol utilization at the mitochondrial level for P4 synthesis.

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 approximately 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 4C. 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.

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 a/s-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 a/s 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 a/s 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 125I-hFSH).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies on the effects of specific immunoneutralization of endogenous FSH or LH on testicular germ cell transformations in the adult bonnet monkey (Macaca radiata).

PROBLEM: It is yet to be determined clearly whether the two hormones FSH and T act synergistically in the same cell type--the Sertoli cells--to control overall spermatogenesis or influence independently the transformation of specific germ cell types during spermatogenesis in the adult mammal. METHOD: Adult male bonnet monkeys specifically deprived of either FSH or LH using immunoneutralization techniques were monitored for changes in testicular germ cell transformation by DNA flow cytometry. RESULTS: FSH deprivation caused a significant reduction ( > 40%; P < 0.05) in [3H] thymidine incorporation into DNA of proliferating 2C (spermatogonial) cells, a marked inhibition ( > 50%) in the transformation of 2C to primary spermatocytes (4C) and a concomitant, belated reduction (50%) in the formation of round spermatids (1C). In contrast, specific LH/T deprivation led to an immediate arrest in the meiotic transformation of 4C to 1C/HC leading to an effective and significant block ( < 90%; P < 0.01) in sperm production. CONCLUSION: Thus, LH rather than FSH deprivation has a more pronounced and immediate effect as the former primarily blocks meiosis (4C --> 1C/HC) which controls production of spermatids. These data provide evidence for LH/T and FSH regulating spermatogenic process in the adult primate by primarily acting at specific germ cell transformation steps.

Is there a role for estrogen in follicular maturation in the primate?

The present study focusses attention on the effects of blocking estrogen synthesis, during follicular phase, on follicular maturation in the adult female bonnet monkey (Macaca radiata). Administration of cycling females (n=4) with an aromatase inhibitor CGS 16949A (Al) by Alzet mini-pump (2,5 mg/day) from day 3 of cycle resulted in significant reduction in basal (by 53%) and surge levels of estrogen (by 70%) but this had no effect on follicular maturation, ovulation and luteal function as assessed by serum hormone profiles as well as laparotomy. This lack of need for estrogen for completion of follicular maturation process was confirmed by administering cycling monkeys hFSH (25 IU/day) from day 3 till day 8 of the cycle along with (5 mg Al/day) or without Al (n=3/group). Administration of AI resulted in suppression of FSH induced increase in serum estrogen (by 100%) and elevation in circulating androstenedione. Aromatase inhibitor treatment had no effect on either the number of follicles developed or their size relative to control. Testing the ability of both granulosa and thecal cells, removed on day 9 of treatment cycle, to respond to gonadotropinsin vitro showed no change indicating that cellular development and maturation of follicular cells had occurred normally. It is concluded that follicular maturation in the primate can occur even when increase in estrogen synthesis is blocked.