Effect of Ethanol Extract of Whole Plant of Trichosanthes cucumerina var. cucumerina L. on Gonadotropins, Ovarian Follicular Kinetics and Estrous Cycle for Screening of Antifertility Activity in Albino Rats

Ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina was evaluated for antiovulatory activity in adult rats. The ethanol extract at the doses 200 and 400mg/kg body weight (orally) affected the normal estrous cycle showing a significant increase in estrus and metestrus phases and decrease in diestrus and proestrus phases. The extract also significantly reduced the number of healthy follicles (Class I-Class VI) and corpora lutea and increased the number of regressing follicles (Stage IA, Stage IB, Stage IIA, and Stage IIB). The protein and glycogen content in the ovaries were significantly reduced in treated rats. The cholesterol level was significantly increased, whereas, the enzyme activities like 3b-HSD and 17b-HSD were significantly inhibited in the ovary of treated rats. Serum FSH and LH levels were significantly reduced in the treated groups were measured by RIA. In acute toxicity test, neither mortality nor change in the behavior or any other physiological activities in mice were observed in the treated groups. In chronic toxicity studies, no mortality was recorded and there were no significant differences in the body and organ weights were observed between controls and treated rats. Hematological analysis showed no significant differences in any of the parameters examined (RBC, WBC count and Hemoglobin estimation). These observations showed the antiovulatory activity of ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina in female albino rats.

Regulation of Testosterone Rhythmicity by Gonadotropins in Bonnet Monkeys (Macaca radiata)

In an attempt to study the factor(s) that regulates production of nychthemeral testosterone surges in adult male bonnet monkeys (Macaca radiata), serum levels of testosterone, LH, FSH, and prolactin were monitored during a 24 h period. Only prolactin showed a significant increment in its levels coincident with that of the testosterone surge. The relationship between LH and testosterone production was studied by 1) observing the responsiveness of testes, in terms of testosterone production, to one or two injections of oLH (1 mg/injection) given 12 h apart at 0900 and 2100 h; and 2) monitoring the effect on testicular testosterone production of LH antiserum injection given at 1000, 1700, and 2100 h. That each LH injection brought about an increment in testosterone level of equal magnitude suggests that the difference in responsiveness of the testes to unchanging levels of LH at morning and night hours is not due to any alteration in substrate availability at the two time intervals. The LH antiserum experiments indicate that irrespective of the time of its administration the nocturnal surge of testosterone, which normally occurs at 2200 h, is blocked. While the antiserum prevents a rise in testosterone level, it appears not to influence basal testosterone production. The results further show that even at 2100 h, when surge testosterone production is already initiated, the testis is still highly sensitive to lack of LH, antiserum injection bringing about within 2 h a significant reduction in testosterone levels (by 69% in experimentals vs 11% in controls).

Dynamics of Circulating Concentrations of Gonadotropins and Ovarian Hormones Throughout the Menstrual Cycle in the Bonnet Monkey: Role of Inhibin A in the Regulation of Follicle-Stimulating Hormone Secretion

In higher primates, increased circulating follicle-stimulating hormone (FSH) levels seen during late menstrual cycle and during menstruation has been suggested to be necessary for initiation of follicular growth, recruitment of follicles and eventually culminating in ovulation of a single follicle. With a view to establish the dynamics of circulating FSH secretion with that of inhibin A (INH A) and progesterone (P-4)secretions during the menstrual cycle, blood was collected daily from bonnet monkeys beginning day 1 of the menstrual cycle up to 35 days. Serum INH A levels were low during early follicular phase, increased significantly coinciding with the mid cycle luteinizing hormone (LH) surge to reach maximal levels during the mid luteal phase before declining at the late luteal phase, essentially paralleling the pattern Of P-4 secretion seen throughout the luteal phase. Circulating FSH levels were low during early and mid luteal phases, but progressively increased during the late luteal phase and remained high for few days after the onset of menses. In another experiment, lutectomy performed during the mid luteal phase resulted in significant decrease in INH A concentration within 2 hr (58.3 +/- 2 vs. 27.3 +/- 3 pg/mL), and a 2- to 3-fold rise in circulating FSH levels by 24 hr (0.20 +/- 0.02 vs. 0.53 +/- 0.14 ng/mL) that remained high until 48 hr postlutectomy. Systemic administration of Cetrorelix (150 mu g/kg body weight), a gonadotropin releasing hormone receptor antagonist, at mid luteal phase in monkeys led to suppression of serum INH A and P-4 concentrations 24 hr post treatment, but circulating FSH levels did not change. Administration of exogenous LH, but not FSH, significantly increased INH A concentration. The results taken together suggest a tight coupling between LH and INH A secretion and that INH A is largely responsible for maintenance of low FSH concentration seen during the luteal phase. Am. J. Primatol. 71:817-824, 2009.

Studies on follicular atresia: role of gonadotropins and gonadal steroids in regulating cathepsin-D activity of preovulatory follicles in the rat

Preovulatory follicular atresia was studied using pregnant mare serum gonadotropin (PMSG)-primed rats (15 IU/rat) which were deprived of hormonal support either by allowing the metabolic clearance of the PMSG or by injecting a specific PMSG antiserum (PMSG a/s). Atresia was monitored by an increase in lysosomal cathepsin-D activity and a decrease in the receptor activity of the granulosa cells (GC) isolated from the preovulatory follicles. It was shown that the increase in lysosomal activity and the decrease in receptor activity seen at 96 h after PMSG (or PMSG plus PMSG a/s) could be arrested both by follicle stimulating hormone (FSH) and luteinizing hormone (LH). Injection of cyanoketone or clomiphene citrate together with FSH/LH prevented this 'rescue' suggesting a role for estrogens in the regulation of atresia. Although the administration of estradiol-17 beta (20 micrograms/rat) together with PMSG a/s could show a 'rescue effect' in terms of reduction in cathepsin-D activity the gonadotropin receptor activities of these granulosa cells were not restored. The injection of dihydrotestosterone (DHT) to 48 h PMSG-primed rats induced atresia as noted by an increase in cathepsin-D activity. However, the exogenous administration of FSH along with DHT prevented this atretic effect suggesting that DHT is not having a direct effect on atresia. Determination of androgen: estrogen content of the granulosa cells and an analysis of the individual profile of androgen and estrogen revealed that the increase in cathepsin-D activity could be correlated only with the decrease in GC estrogen content. This along with the observation that GC showed a loss of estrogen synthesis well before the increase in cathepsin-D activity strongly points out that the lack of estrogen rather than an increase in androgen is the principle factor responsible for the atresia of preovulatory follicles in the rat.

Pituitary gonadotropins regulate spermatogonial differentiation and proliferation in the rat

The relative regulatory roles of the pituitary gonadotropins, luteinizing hormone and follicle stimulating hormone in the spermatogonial proliferation has been studied using specific antibodies against these hormones in the immature rats. Immunoneutralization of luteinizing hormone for 7 days resulted in significant reduction in tetraploid cells and total absence of haploid cells, while there was a relative increase in the diploid population. This was also accomopanied by a decrease in spermatogonial proliferation as indicated by a decrease in [H-3] thymidine incorporation into DNA by purified spermatogonia. Administration bf follicle stimulating hormone als for 7 days also caused a significant decrease in the rate of spermatogonial proliferation. Withdrawal of follicle stimulating hormone led to a significant reduction in tetraploid and haploid cells However interestingly, it failed to totally abolish the appearance of these cells. Administration of testosterone (3mg/day/rat) for 2 days along with the gonadotropin a/s could partially reverse the effect on spermatogonial proliferation. It is concluded that (i) both luteinizing hormone and follicle stimulating hormone are involved in spermatogonial proliferation, (ii) lack of testosterone consequent of the neutralization of luteinizing hormone prevented the entry of spermatogonial cells into meiosis, (iii) testosterone may be involved in spermatogonial proliferation providing a mitotic signal and (v) both follicle stimulating hormone and testosterone act synergistically and lack of any one of the hormones results in impairment of spermatogonial proliferation.

Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSH beta (AsFSH beta) and LH beta (AsLH beta) subunit cDNAs are 345, 363, 387 and 414 bp in length, and encode mature peptides of 115, 121, 129 and 138 aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSH beta and AsLH beta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSH beta and AsLH beta) in mature sturgeon pituitaries, but only AsFSH beta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSH beta was expressed in 4-year-old individuals, whereas in females, AsFSH beta was just expressed in 5-year-old individuals. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Serum concentrations of gonadotropins and steroid hormones of Neophocaena phocaenoides asiaeorientalis in middle and lower regions of the Yangtze River

As a relatively isolated and unique freshwater population, the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) is the most endangered subpopulation of this species. The objective of this study was to improve our understanding of their reproductive endocrinology by measuring (radioimmunoassay) serum concentrations of FSH, LH, estradiol (E-2), progesterone (P-4) and testosterone (T-2) in free-ranging animals. Blood samples were collected from 66 Yangtze finless porpoises (41 males and 25 females) captured in the middle and lower reaches of the Yangtze River. Based on significant variation of serum T-2 concentrations in males with body length (BL) > 138 cm, we inferred that they were mature; in these animals, there were significant seasonal variations in serum T-2 concentrations, with the highest concentrations in March and April (502.0 +/- 319.8 ng/dL, mean +/- S.D.) and the lowest in December (79.4 +/- 83.2 ng/dL). Serum T-2 concentrations positively correlated with serum concentrations of LH and weakly correlated with serum concentrations of E-2, whereas there was a significant negative correlation between serum LH and FSH concentrations in males > 138 cm. The smallest apparently pregnant female porpoise had a BL of 130 cm. Serum P-4 concentrations ranged from 13.2 to 112.4 ng/mL (43.9 +/- 28.3 ng/mL) in pregnant females, and fluctuated under 1.0 ng/mL in non-pregnant females with BL > 130 cm. Serum LH concentrations were significantly higher in non-pregnant females > 130 cm versus those females < 130 cm. To our knowledge, this is the first study of endocrine-related maturity and seasonal breeding characteristics of the Yangtze finless porpoise. (c) 2006 Elsevier Inc. All rights reserved.

Molecular characterization and phenotypic expression of mutations in genes for gonadotropins and their receptors in humans.

Gonadotropin hormones undergo important dynamic changes during life. Their rise during puberty stimulates gonadal steroid secretion, triggering the development of secondary sexual characteristics and the acquisition of fertility. The full spectrum of possible mutations and polymorphisms in the human gonadotropins and in their receptor genes has been described in recent years. Patients harboring these mutations display a very wide range of phenotypes affecting all aspects of the reproductive axis. An important insight provided by the careful study of these patients lies in the striking gender differences in the phenotypes associated with a given mutation. As a result, the careful study of these rare patients has allowed us to better define the respective roles of luteinizing hormone and follicle-stimulating hormone in normal human pubertal development and in the achievement of full fertility potential in either males or females. In this work, we describe briefly the known mutations in the genes for both gonadotropins and their receptors, and discuss their genotype/phenotype correlations in light of these important gender differences.

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.

Involvement of pituitary gonadotropins, gonadal steroids and breeding season in sex change of protogynous dusky grouper, Epinephelus marginatus (Teleostei: Serranidae), induced by a non-steroidal aromatase inhibitor

Two experiments were performed using the aromatase inhibitor (AI) letrozole (100mg/kg) to promote sex change, from female-to-male, in protogynous dusky grouper. One experiment was performed during the breeding season (spring) and the other at the end of the breeding season (summer). During the spring, AI promoted sex change after 9weeks and the sperm produced was able to fertilize grouper oocytes. During the summer, the sex change was incomplete; intersex individuals were present and sperm was not released by any of the animals. Sex changed gonads had a lamellar architecture; cysts of spermatocytes and spermatozoa in the lumen of the germinal compartment. In the spring, after 4weeks, 11ketotestosterone (11KT) levels were higher in the AI than in control fish, and after 9weeks, coincident with semen release, testosterone levels increased in the AI group, while 11KT returned to the initial levels. Estradiol (E2) levels remained unchanged during the experimental period. Instead of decreasing throughout the period, as in control group, 17 α-OH progesterone levels did not change in the AI-treated fish, resulting in higher values after 9weeks when compared with control fish. fshβ and lhβ gene expression in the AI animals were lower compared with control fish after 9weeks. The use of AI was effective to obtain functional males during the breeding season. The increase in androgens, modulated by gonadotropins, triggered the sex change, enabling the development of male germ cells, whereas a decrease in E2 levels was not required to change sex in dusky grouper. © 2013 Elsevier Inc.

Exogenous gonadotropins have little impact on follicular but considerable effect on serum cytokine concentrations – a comparison between Natural Cycle and stimulated IVF using a multiplexed assay platform

Introduction: Throughout follicular growth and subsequent corpus luteum formation the leukocyte number increases and follicular vascularisation changes. These processes are enhanced under exogenous stimulation with gonadotropins. Cytokines released by leukocytes contribute to further recruitment and vascularisation of the follicle, and they play an important role in regulating ovarian steroidogenesis by influencing theca and granulosa–lutein cell function. Changes in cytokine and vascular endothelial growth factor (VEGF) concentrations in the ovary as a consequence of gonadotropin stimulation may negatively influence oocyte quality. In this project we have compared the intrafollicular production of inflammatory cytokines and growth factors between natural IVF cycles (NC) and classical, gonadotropin-stimulated IVF cycles (gsIVF). Material and Methods: Serum on the day of oocyte retrieval and follicular fluid (FF) were collected in 37 NC and 39 gsIVF cycles. Thirteen women within this population underwent one NC and one gsIVF cycle each. A total of 14 cytokines from Bio-Plex panels I and II were determined in matched serum and FF samples using Luminex xMAP technology on the Bio-Plex(R) platform, using the serum protocol. Results: Tumour necrosis factor-alpha, RANTES, eotaxin and interferon-gamma-induced protein-10 levels were lower in FF than in serum, and thus not further investigated. Interleukin (IL)-6, -8, -10, -15, -18, monocyte chemotactic protein-1 (MCP-1), VEGF and leukaemia inhibitory factor (LIF) showed higher median concentrations in FF than in serum, indicating possible ovarian production. Moreover, most of these showed higher evels in the gsIVF than in the NC groups in the serum, but not in the follicular fluid. IL-8 was reduced in gsIVF cycles. Conclusion: The fact that serum but not FF levels of the studied cytokines were higher in the stimulated than in the natural cycles can be attributed to the increased number of active follicles present after controlled ovarian stimulation.

Exogenous gonadotropins do not increase the blood-follicular transportation capacity of extra-ovarian hormones such as prolactin and cortisol.

BACKGROUNDS In vitro fertilization involves high dosage gonadotropin stimulation, which apparently has some negative impact on follicular endocrine function. As chorionic gonadotropin stimulation has been shown to increase the blood-follicular permeability in animal models, this raises the question if such an effect also applies to gonadotropins in humans, possibly affecting the endocrine follicular milieu. FINDINGS Follicular fluid and serum were collected at the time of follicular aspiration in in vitro fertilisation without (Natural cycle IVF, n = 24) and with (conventional gonadotropin stimulated IVF, n = 31) gonadotropin stimulation. The concentration of the extra-ovarian hormones prolactin and cortisol were analysed by immunoassays. RESULTS Median serum prolactin and cortisol concentrations were 12.3 ng/mL and 399 nmol/L without versus 32.2 ng/mL and 623 nmol/L with gonadotropin stimulation. The corresponding concentrations in follicular fluid were 20.6 ng/mL and 445 nmol/L versus 28.8 ng/ml and 456 nmol/L for prolactin and cortisol. As a consequence, mean follicular fluid:serum ratios were significantly reduced under gonadotropin stimulation (prolactin p = 0.0138, cortisol p = 0.0001). As an enhanced blood-follicular permeability and transportation, induced by gonadotropin stimulation, would result in increased instead of decreased follicular fluid:serum ratios as found in this study, it can be assumed that this does not affect extra-ovarian protein and steroid hormones as illustrated by prolactin and cortisol. CONCLUSIONS The model of serum follicular fluid:serum ratio of hormones, produced outside the ovaries, did not reveal a gonadotropin induced increased blood-follicular transportation capacity. Therefore it can be assumed that the effect of gonadotropins on follicular endocrine function is not due to an increased ovarian permeability of extra-ovarian hormones.