Effect of FSH receptor-binding inhibitor-8 on FSH-mediated granulosa cell signaling and proliferation.

Follicle-stimulating hormone is important for mammalian reproduction. It acts through specific receptors located on the plasma membrane of granulosa cells in ovaries and Sertoli cells in testes. The binding of follicle-stimulating hormone to its receptor activates intracytoplasmic signaling pathways leading to steroidogenesis. These steroids in turn regulate the follicle-stimulating hormone action from the anterior pituitary through exerting negative feedback effect. In addition to steroids, non-steroidal factors secreted by the ovaries are believed to modulate follicle-stimulating hormone action through autocrine/paracrine mode. One such low molecular weight peptide referred to as follicle-stimulating hormone receptor-binding inhibitor-8 purified from human follicular fluid has been extensively studied. Follicle-stimulating hormone receptor-binding inhibitor-8 has been shown to inhibit binding of follicle-stimulating hormone to its receptor. The present article describes the effect of follicle-stimulating hormone receptor-binding inhibitor-8 on follicle-stimulating hormone-induced signaling in rat granulosa cells. Follicle-stimulating hormone receptor-binding inhibitor-8 inhibited the follicle-stimulating hormone-induced cAMP, and the effect was observed to be mediated through the protein kinase A. Further, an inhibitory effect of follicle-stimulating hormone receptor-binding inhibitor-8 on the granulosa cell proliferation was evaluated using COV434 cell line which is derived from the human granulosa cell tumor. The effect of the peptide on the cell cycle analysis showed an increase in apoptotic population and the arrest of G1 phase. These findings suggest that follicle-stimulating hormone receptor-binding inhibitor-8 acts as a follicle-stimulating hormone antagonist and affects the follicle-stimulating hormone-mediated signaling and proliferation in the granulosa cells.

Interaction of follicle-stimulating hormone (FSH) receptor binding inhibitor-8: a novel FSH-binding inhibitor, with FSH and its receptor.

Follicle-stimulating hormone (FSH) receptor binding inhibitor (FRBI-8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI-8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI-8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI-8 with reported FSH-FSHR hormone binding (FSHR(HB)) domain complex using ZDOCK algorithm revealed that the FRBI-8 binds to FSHbetaL2-FSHR(HB) binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI-8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI-8(D6A)) had a higher binding affinity than the native FRBI-8. In vitro radioreceptor assay with FRBI-8(D6A) showed 50% lower IC(50) compared with the FRBI-8, confirming the in silico observations. Thus, the study reveals that both FRBI-8 and FRBI-8(D6A) interfered with the binding of FSH to its receptor.

Gene expression profiling during early folliculogenesis in the mouse ovary.

OBJECTIVE: To evaluate genes involved in ovarian primordial-to-primary follicle transition. DESIGN: Experimental animal study. SETTING: Research institute. ANIMAL(S): Day-2 and day-4 female Swiss mice. INTERVENTION(S): We conducted a complementary DNA array study using ovarian messenger RNAs from day-2 and day-4 mice. MAIN OUTCOME MEASURE(S): The expression profiles of 1,176 genes in neonatal mouse ovaries on day 2 and day 4, which contain primordial and primary follicles, respectively, were compared. RESULT(S): Twenty-six percent of genes were differentially expressed between day-2 and day-4 ovaries, with 19% being up-regulated and 7% down-regulated in day 4. Analysis of differentially expressed genes revealed that the primordial-to-primary follicle stage transition is associated with induction in the expression of mainly growth factors, immune-related factors, hormone and hormone receptors, and signal transducers. The transition is also associated with proliferation of granulosa cells and absence of apoptosis. In addition, our studies demonstrated that the primary follicles express estrogen receptor beta and are responsive to estrogen actions in vitro in terms of increase in the number of primary follicles and granulosa cell proliferation. CONCLUSION(S): The transition of primordial to primary follicles is associated with the participation of multiple pathways in regulating gene expression.

Nanovaccines: recent developments in vaccination.

In the past 100 years, vaccination has contributed immensely to public health by preventing a number of infectious diseases. Attenuated, killed or part of the microorganism is employed to stimulate the immune system against it. Progress in biotechnology has provided protective immunity through DNA vaccines. In recent years, nanovaccine is a novel approach to the methodology of vaccination. Nanomaterials are delivered in the form of microspheres, nanobeads or micro-nanoprojections. Painless, effective and safe needle-free routes such as the intranasal or the oral route, or patches of microprojections to the skin are some of the approaches which are in the experimental stage at present but may have a great future ahead in nanovaccination.

Granulosa cell apoptosis induced by a novel FSH binding inhibitory peptide from human ovarian follicular fluid.

Pituitary gonadotropins, follicle-stimulating hormone and luteinizing hormone, are the key regulators of ovarian folliculogenesis; these are known to be directly or indirectly modulated by many intraovarian factors. Our group has identified and studied one such novel peptide from human ovarian follicular fluid. Its partial N-terminal eight amino acid sequence has been deduced, referred to as octapeptide (OP). OP induces follicular atresia in mice and interferes with normal ovarian function in non-human primates, this action being similar to the native peptide. Thus, in this study, an attempt has been made to elucidate the mechanism of action of the synthetic OP by studying the pathway of follicular atresia in mouse ovary. Changes in granulosa cells were studied using various apoptotic markers by flow cytometry and immunohistochemistry. An increase in apoptotic cell population in atretic- and peptide-treated groups was observed compared with normal controls. Interestingly, both these groups exhibited differences in the apoptotic pathway. Results showed that the mitochondrial pathway was predominant in the atretic group, whereas the Fas-FasL pathway was predominant in the peptide-treated groups. The ultrastructural study also showed apoptotic changes in the OP-treated and atretic groups; the pattern of apoptosis differed at the subcellular level.

Effect of octapeptide: FSH binding inhibitor on cyclicity of bonnet monkeys (Macaca radiata).

BACKGROUND: The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone play an important role in the development of ovarian follicles, and a number of autocrine/paracrine factors secreted by the ovary are known to directly or indirectly regulate gonadotropin action. The objective of the present study was to elucidate the effect of octapeptide (OP) on cyclicity and hormonal profile of bonnet monkeys, the menstruating Old World primates. STUDY DESIGN: Our group has purified one such factor from human ovarian follicular fluid, which inhibits the binding of FSH to the granulosa cells. N-terminal eight-amino-acid sequence of this peptide has been deduced, which is referred to as the OP. It has shown an antifertility effect in marmosets, the New World primates. In the present study, the bonnet monkeys were divided into two groups, namely, the treated group (n=5), which was administered with OP (250 mcg/kg body weight/day) intramuscularly during the follicular phase, and the control group (n=6), which was injected with vehicle (saline). Blood was collected every other day, and progesterone levels were estimated by enzyme-linked immunosorbent assay. RESULTS: Animals in the control group demonstrated normal plasma progesterone levels and exhibited normal cyclicity. On the other hand, in the treated group, progesterone levels decreased by 65.8%, as compared with that in pretreatment cycles. This probably disturbed the cyclicity, thus causing amenorrhea (73.0+/-6.7 days).

Effect of the human follicle-stimulating hormone-binding inhibitor and its N-terminal fragment on follicle-stimulating hormone-induced progesterone secretion by granulosa cells in vitro.

Intrafollicular factors play an important role in folliculogenesis. The follicle-stimulating hormone (FSH)-binding inhibitor (FSHBI), purified by our laboratory from human ovarian follicular fluid, has been shown to suppress ovulation and induce follicular atresia/apoptosis in mice as well as impair fertility in marmosets, the New World monkeys. The octapeptide, a peptide corresponding to the N-terminal region of human FSHBI (hFSHBI), has been synthesized and also shows FSHBI activity in vitro. In the present study, we have attempted to identify the mechanism of action of the peptide in granulosa cell cultures. Rat granulosa cell cultures were treated with varying concentrations of the octapeptide or partially purified hFSHBI (gel chromatography fraction hGF 2) in the presence or absence of human FSH (hFSH) and the amount of progesterone (P;4) secreted in the culture supernatants after 3 h/48 h was estimated. Both hGF2 and the octapeptide failed to alter basal levels as well as 8-bromo cAMP-induced P;4 production, while FSH-induced P 4 secretion was inhibited in a dose-dependent manner. These studies reveal that the octapeptide, a fragment of FSHBI, and the native protein have similar activity in vitro and both compounds alter FSH action at the receptor level upstream of cAMP formation.

Danazol-beta-cyclodextrin binary system: a potential application in emergency contraception by the oral route.

This study explored the potential of beta-cyclodextrin to improve the aqueous solubility and dissolution of danazol, investigated a simple and less expensive method for preparation of a danazol-beta-cyclodextrin binary system, and explored the potential application of a danazol-beta-cyclodextrin binary system as a single-dose emergency contraceptive. Phase solubility analysis indicated formation of a first-order soluble complex with stability constant 972.03 M(-1), while Job's plot affirmed 1:1 stoichiometry. The hyperchromic shift in the UV-Vis spectrum of danazol in the presence of beta-cyclodextrin indicated solubilization capability of beta-cyclodextrin for danazol. The extrinsic Cotton effect with a negative peak at 280.7 nm confirmed the inclusion of danazol in the asymmetric locus of beta-cyclodextrin. (1)H-nuclear magnetic resonance analysis suggested that the protons of the steroidal skeleton of danazol display favorable interactions with the beta-cyclodextrin cavity. The danazol-beta-cyclodextrin binary system was prepared by kneading, solution, freeze-drying, and milling methods. The extent of the enhancement of dissolution rate was found to be dependent on the preparation method. Dissolution studies showed a similar relative dissolution rate (2.85) of the danazol-beta-cyclodextrin binary system prepared by the freeze-drying and milling (in the presence of 13% moisture) methods. In a mouse model, the danazol-beta-cyclodextrin binary system at 51.2 mg/kg (equivalent to a 400-mg human dose) showed 100% inhibition of implantation when given postcoitally. Moreover, the danazol-beta-cyclodextrin binary system is safe up to 2000 mg/kg in the mouse (15.52 g/70 kg human) as a single oral dose. Thus, the danazol-beta-cyclodextrin binary system could serve as a new therapeutic application: an oral emergency contraceptive at a physiologically acceptable single dose.

Differential gene expression in transition of primordial to preantral follicles in mouse ovary.

In the mammalian ovary, early follicular development is gonadotropin independent. Interaction between the oocyte and granulosa cells possibly plays an important role in transition of primordial to preantral stage. However, the molecular and cellular control of early follicular development and cell-cell interaction is complex and poorly understood. In the present study, we examined gene expression in primordial, primary and preantral follicle by cDNA arrays using Day 2, Day 4 and Day 6 neonatal mouse ovaries that contain the various developmental stages of these follicles, respectively. The results revealed that 30% of the genes were differentially expressed in Day 4 ovaries containing primary follicles as compared to D2 neonatal ovaries. The data were confirmed by the expression of Growth Differentiation Factor-9 in the oocytes of primary and preantral follicles. Also, Stem Cell Factor was localized in the granulosa cells of primary and preantral follicles. Electron microscopic studies of Day 6 ovaries showed projections from granulosa cells and microvilli from oocytes in the follicle during the transition from the primary to preantral stage. Further, initiation of gap junctions were observed at ultrastructure level and corroborated with the expression of specific gap junction protein, connexin 43 in preantral follicles of the ovaries. These results infer that primordial follicles are quiescent while the major activities of cell-cell communication and the production of local paracrine factors, are initiated in primary and preantral follicles of the mouse ovary. These preliminary observations may contribute to the elucidation of molecular and cellular pathways involved in follicle transition.

Application of nanotechnology in biomedicine.

Nanotechnology is the development of engineered devices at the atomic, molecular and macromolecular level in nanometer range. Nanoparticles have potential application in medical field including diagnostics and therapeutics. Nanotechnology devices are being developed for diagnosis of cancer and infectious diseases which can help in early detection of the disease. Advances in nanotechnology also proved beneficial in therapeutic field such as drug discovery, drug delivery and gene/protein delivery. Nanoparticles can be constructed by various methodology so that effect can be targeted at desired site. In this review, some of the applications of nanoparticles in medicine as diagnostics and therapeutics which can be employed safely at the clinical level have been described. On other hand, as the particles become generally smaller their likehood of causing harm to the lung increases. Therefore, there is a need to study safety of nanoparticles.

Postnatal development and reproductive performance of F1 progeny exposed in utero to an ayurvedic contraceptive: Pippaliyadi yoga.

Pippaliyadi yoga or pippaliyadi vati is an ayurvedic contraceptive used in India since ancient times. It is a combination of powdered fruit berries of Embelia ribes Burm.f. (Myrsinaceae), Piper longum L. (Piperaceae) and borax in equal proportion. Though the contraceptive potential is known since ancient times, no systematic developmental toxicity studies have been carried out. The present study was carried out to evaluate the postnatal developmental toxicity and the reproductive performance of the progeny exposed in utero to pippaliyadi. Pippaliyadi yoga was obtained from National Institute for Pharmaceutical Education and Research (NIPER), India and the developmental toxicity was studied by administering three doses, viz. 140, 300 and 700 mg/(kg day) to gravid females from day 6 to day 16 of gestation. Pippaliyadi did not have any adverse developmental effects with low doses, however, with the five times higher dose, a decrease in body weight of the pups was observed. The reproductive performance of the progeny born to mothers treated with pippaliyadi was not significantly affected. The present study suggests that in utero exposure to pippaliyadi does not have any adverse effect on the postnatal development and reproductive performance of the F(1) progeny.

Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus).

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets (Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3'-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets (n=14) and also in ovariectomized marmosets (n=13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E+P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)--a proliferation marker and steroid receptors, ERalpha and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E+P group as compared to other groups of ovariectomized animals (P<0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERalpha and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E+P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.

Expression of steroid hormone receptors, proliferation and apoptotic markers in primate endometrium.

In humans, circulating levels of steroid hormones vary during the menstrual cycle, with oestrogen elevated in the proliferative phase and progesterone increased during the secretory phase. In humans, oestrogen levels also increased during the mid-secretory phase, but this is not observed in non-human primates. In marmosets, the New World monkeys, plasma oestrogen and progesterone levels exhibit similar profiles as those found in Old World monkeys, however, these animals do not menstruate. Proliferative and apoptotic changes occur at specific phases of the menstrual cycle, suggesting regulation by steroid hormones. It was, therefore, of interest to compare expression of steroid hormone receptors, proliferation and apoptotic markers in the uterine endometrium of marmosets and humans. Localization of oestrogen receptor-alpha (ER-alpha) was observed in glandular epithelial cells during the proliferative phase in marmosets and in human endometria. A decrease in ER-alpha and progesterone receptor (PR-A) was observed during the mid luteal phase in both species although a postovulatory oestradiol peak is observed in human but not in marmoset. A decrease in PR-A in the late secretory phase was seen in marmoset as well as in human endometria. The proliferation marker PCNA was enhanced in the proliferative phase in both species, but it was increased in late secretory phase only in marmosets. Apoptosis as revealed by a TUNEL assay was moderate in early stage in both species. Surprisingly, apoptosis, as well as the localization of the pro-apoptotic Bcl-2 family member, Bax, was optimal in marmosets during the mid-secretory phase when plasma progesterone levels were high. On the other hand, in the human, apoptosis was maximal by TUNEL assay in late secretory phase, but Bax protein was highest in the mid-secretory phase. Thus, Bax may be initiating apoptosis in the endometrial glands as well as in the stroma. Although the pattern of steroid receptor expression in endometria of marmosets and humans are similar, proliferation and apoptosis markers appear to be regulated by other factors along with steroid hormones.

Circulating auto-antibodies against the zona pellucida and thyroid microsomal antigen in women with premature ovarian failure.

Premature ovarian failure (POF) is a disorder of multicausal etiology leading to infertility in women. Development of ovarian auto-antibodies is a causative factor in most POF cases, but no consensus on the ovarian antigenic determinants has been reached till date. In the present study, sera from 15 POF cases, seven normally cycling women and eight menopausal women were studied by immunohistochemistry (IHC) for the presence of anti-ovarian antibodies. 10 of the 15 POF sera (66.6%) presented with anti-ovarian antibodies (Ao). Of these, two demonstrated antibodies to the zona pellucida (ZP) as well as strong immunoreactivity to granulosa cells (Azg), while the remaining eight exhibited anti-ZP antibodies with negligible staining in granulosa cells (Az). The antibodies showed cross-reactivity with ZP from various species such as human, sheep, marmoset, pig and mouse. Among various murine tissues, the antibodies cross-reacted only with thyroid and not with uterus, spleen, kidney, liver, adrenal, pancreas and pituitary. Five of the eight Az individuals presented with significant titres of anti-thyroid antibodies (Azt). In the control group, one menopausal control presented with reactivity to both ZP and GC, the autoimmunity possibly being a consequence of surgical trauma; while one normally cycling woman tested positive for anti-thyroid antibodies. The IHC results were confirmed by ELISA using heat-solubilized isolated ZP (SIZP) as the antigen. Out of seven Ao samples assessed by ELISA, five reacted with SIZP. Preincubation of these five samples with varying concentrations of SIZP demonstrated a dose-dependent decrease in reactivity in ELISA and abolished staining in IHC, confirming the specificity of auto-antibodies to ZP in the POF group. Our results thus suggest that ZP is an important ovarian antigen in autoimmune POF.

Antifertility effect of an octapeptide, a fragment of FSH binding inhibitor, in the common marmoset (Callithrix jacchus).

The follicle-stimulating hormone (FSH) binding inhibitor (FSHBI) has been identified as one of the factors present in follicular fluid exerting autocrine/paracrine effects on FSH actions in the ovary. Our group has isolated FSHBI from human ovarian follicular fluid and deduced its partial amino acid sequence from the N-terminal region. A synthetic peptide corresponding to this sequence also demonstrated FSH binding inhibitory activity in vitro. The objective of the present study was to elucidate the effect of the octapeptide on ovarian cyclicity and pregnancy in the common marmoset. For the study, three groups of postpartum marmosets were treated with the octapeptide during the follicular phase. Administration of the octapeptide from days 6-10 postpartum (pp) predominantly induced luteal insufficiency in two of seven and two of five marmosets at a dose of 100 microg/day (group 1) and 300 microg/day (group 2), respectively. This was confirmed by the presence of small regressing corpora lutea on day 13 pp. Pregnancy was terminated prematurely in one animal from each of these groups. The treatment impaired fertility by 43% and 60% in the marmosets of groups 1 and 2, respectively. In view of the fact that FSH levels peak on day 2 and day 6 of the follicular phase in marmosets, a third group was administered 200 microg/day octapeptide from days 1-8 pp. The treatment induced luteal insufficiency in one out of four marmosets, while premature termination of pregnancy occurred in two other marmosets of this group, demonstrating a 75% effect on pregnancy. Thus, treatment of marmosets with the octapeptide, a fragment of the FSHBI, predominantly induced luteal insufficiency as well as resorption of the fetus leading to impairment of fertility.

Stem cell research: its relevance to reproductive biology.

Stem cells provide an excellent model system to understand the differentiation, development and functioning of gonads, and further use of these cells in transplantation or cell-based therapies. Embryonic germ cells present as a better source of pluripotent stem cells. The germ cells are specialized cells, which differentiate into sperm or oocytes. Spermatogonial stem cells are the only stem cells in the adult mammalian body that can be recognized and studied at cellular level with respect to proliferation and differentiation. In the present study, basic process of spermatogenesis, testicular niche and molecular regulation of spermatogenesis and density regulation has been discussed. Research on oogonial stem cells has recently been encouraged due to the demand for oocytes for various research purposes. Mechanism of regulation of follicle formation, oocyte attrition and follicle development and atresia are only partially understood. Hence, the stages of development, its interaction with the neighbouring somatic cells during each developmental stage and the molecular regulation underlying it has been reviewed. These studies will result in establishment of treatment of ovarian disorders, and in identifying cure for infertility that occurs due to ovarian pathophysiology. Indian scenario in terms of stem cell research and its benefits is also discussed.

Expression of bcl(xs) and c-myc in atretic follicles of mouse ovary.

At birth, in the human female, one million follicles are present, out of which only 450 ovulate during the reproductive lifespan while the remainder of the 99.9% follicles degenerate. It is therefore important to understand the regulation of follicular atresia. Immature mice injected with pregnant mare serum gonadotrophin (PMSG) were used as a model for follicular atresia. In this model, we demonstrated by various methods, including the TdT-mediated dUTP nick end labelling (TUNEL) technique, that granulosa cells in atretic follicles undergo apoptosis. In eukaryotic cells, apoptosis is regulated by genes such as bcl(2) and c-myc. Regulation of apoptosis in the ovary by these proteins/genes was studied using the mouse model. bcl(2) is anti-apoptotic; however, bcl(xs), a member of the bcl(2) family, is apoptotic. Immunohistochemical localization of bcl(xs) in the granulosa cells of atretic follicles suggested its role in follicular atresia. Expression of c-myc was studied by in-situ hybridization in the mouse follicular atresia model. In the atretic follicles, expression of c-myc in granulosa cells was observed. Thus our studies indicate that bcl(xs) regulates granulosa cell apoptosis, while c-myc may also play a role in cell death during follicular atresia.