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The present study was designed to explore the steroidogenic responsiveness of ovine antral follicles of different sizes when cultured for varying time-periods with different doses of pregnenolone. Antral follicles of different sizes were... more
The present study was designed to explore the steroidogenic responsiveness of ovine antral follicles of different sizes when cultured for varying time-periods with different doses of pregnenolone. Antral follicles of different sizes were isolated from sheep ovaries and cultured in medium 199 with or without pregnenolone in the presence or absence of FSH for 1, 6, 10, and 24 hours at 37 C. The levels of progesterone and estradiol in the spent medium were estimated. In the absence of pregnenolone, steroid production by the follicles did not increase significantly beyond 1 hour of culture. However, in the presence of pregnenolone there was a significant increase in progesterone production at 6, 10, and 24 hours of culture compared to controls. Estradiol levels were unaffected. Addition of FSH in combination with pregnenolone failed to increase progesterone or estradiol levels beyond that seen with pregnenolone alone. These data demonstrate that short-term incubation of follicles is suf...
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Ovarian follicular fluid peptide (OFFP) has earlier been shown to inhibit maturation of large follicles and ovulation in mice. An active fraction hGF2, obtained following partial purification from human ovarian follicular fluid, was... more
Ovarian follicular fluid peptide (OFFP) has earlier been shown to inhibit maturation of large follicles and ovulation in mice. An active fraction hGF2, obtained following partial purification from human ovarian follicular fluid, was injected at a dose of 20 micrograms to 4-month-old immature marmosets, every alternate day for 2 months. The animal were autopsied at 4 and 6 months of age. Ovarian morphology indicated the appearance of large follicles at 6 months of age in the control group. In contrast, in hGF2 treated marmosets development up to preantral follicles could be seen at 6 months. Furthermore, only in 1 out of the 3 treated marmosets, presence of large preantral follicles was observed and in the remaining 4 animals only primary and secondary follicles were noted. These results reveal that OFFP suppresses follicular development and antrum formation in marmosets.
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Binding of FSH to ovarian cells was studied in PMSG primed immature Swiss mice, 48 h after PMSG treatment, FSH-binding was higher in the periphery than in the cumulus cells of the antral follicles. Binding of FSH to granulosa cells of... more
Binding of FSH to ovarian cells was studied in PMSG primed immature Swiss mice, 48 h after PMSG treatment, FSH-binding was higher in the periphery than in the cumulus cells of the antral follicles. Binding of FSH to granulosa cells of normal follicles was observed to be specific, 48 h after PMSG injection. No localization in the atretic follicles could be seen by autoradiography 72 h after priming.
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The activity of delta 5-3 beta hydroxysteroid dehydrogenase (delta 5-3 beta HSD) in pre-implantation mouse embryos was inhibited following their prior incubation with partially purified sheep ovarian follicular fluid peptide (OFFP). OFFP,... more
The activity of delta 5-3 beta hydroxysteroid dehydrogenase (delta 5-3 beta HSD) in pre-implantation mouse embryos was inhibited following their prior incubation with partially purified sheep ovarian follicular fluid peptide (OFFP). OFFP, 5 microliter, injected into the uterine lumen of day 4 pregnant mice impaired the ensuing implantation of embryos. Our observations suggest that OFFP may inhibit steroid metabolizing activity in pre-implantation embryos and thereby interfere with the process of implantation.
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Human ovarian follicular fluid protein has been partially purified and the active fraction designated as hGF2. Using specific polyclonal antiserum to hGF2, it was observed to be localized immunohistochemically in the granulosa cells of... more
Human ovarian follicular fluid protein has been partially purified and the active fraction designated as hGF2. Using specific polyclonal antiserum to hGF2, it was observed to be localized immunohistochemically in the granulosa cells of medium but not large follicles of human ovary. The hGF2 levels were estimated by ELISA in serum and follicular fluid of 10 gonadotropin-stimulated women recruited for IVF-ET programme. The results revealed a 3-fold increase in the concentration of hGF2 in follicular fluid compared to that in serum of these patients. These data indicate that the protein is secreted by granulosa cells and plays an important role in the regulation of follicular maturation and ovulation.
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Octapeptide (OP)/FSH-Receptor Binding Inhibitor-8 (FRBI-8), is a synthetic peptide corresponding to N-terminal sequence of purified fraction of Follicle Stimulating Hormone Binding-Inhibitor (FSHBI), isolated earlier from human ovarian... more
Octapeptide (OP)/FSH-Receptor Binding Inhibitor-8 (FRBI-8), is a synthetic peptide corresponding to N-terminal sequence of purified fraction of Follicle Stimulating Hormone Binding-Inhibitor (FSHBI), isolated earlier from human ovarian follicular-fluid. In order to avoid the repeated drug-administration, OP-loaded, polymeric polylactide (PLA) nanoparticle formulation (NP-OP), was developed using multiple-emulsion technique. This yielded an average particle size of 120 nm with 70% encapsulation-efficiency. In vitro release profile of NP-OP showed sustained release of OP for 21 days. In vivo anti-fertility studies were conducted in marmosets. Results indicated that control animals conceived in the same cycle while two of three treated animals failed to conceive in treatment cycle. The in vivo studies thus corroborate with in vitro release of OP, demonstrating its anti-fertility activity in 66% of animals.
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The present study was carried out to evaluate apoptosis in endometrium and to correlate these changes with the circulating levels of estradiol and progesterone in the mouse. Apoptosis was observed in various compartments of mouse uterus... more
The present study was carried out to evaluate apoptosis in endometrium and to correlate these changes with the circulating levels of estradiol and progesterone in the mouse. Apoptosis was observed in various compartments of mouse uterus i.e. stroma, glandular epithelium and luminal epithelium depending on the stage of cycle. Stromal cell apoptosis was observed during various stages of cyclicity except on estrus day. Luminal epithelial cells showed apoptotic changes during all stages of cyclicity except on diestrus day. During metestrus, apoptosis was observed in glandular and luminal epithelia as well as stromal cells. Steroid antagonists such as tamoxifen and onapristone altered the apoptotic changes in the uterus. The results suggest that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone.
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In the present study, changes in the immunohistochemical localization of endometrial estrogen receptor (ER) and progesterone receptor (PR) during various stages of the ovarian cyclicity in common marmoset, have been reported. Ovarian... more
In the present study, changes in the immunohistochemical localization of endometrial estrogen receptor (ER) and progesterone receptor (PR) during various stages of the ovarian cyclicity in common marmoset, have been reported. Ovarian cyclicity was monitored by estimating plasma estradiol and progesterone. During the early follicular phase, weak ER immunolocalization was observed in the endometrial stroma. During the late follicular phase under the influence of rising estradiol levels, stromal ER localization was intense. During the luteal phase, ER localization was absent in the stroma indicating that high concentrations of progesterone suppressed ER. PR localization was not observed in the stroma during the early follicular phase, while weak staining was seen in the stroma during the late follicular phase. PR localization was maximum during the mid luteal phase. However in marmoset, endometrial ER and PR localization was restricted only to the stroma. This unique feature may be due...
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Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries enhances apoptotic changes in ovarian granulosa cells of mice. To get an insight into the cell subpopulations responding to OFFP, the heterogeneity of granulosa cells was... more
Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries enhances apoptotic changes in ovarian granulosa cells of mice. To get an insight into the cell subpopulations responding to OFFP, the heterogeneity of granulosa cells was resolved. Subpopulations of granulosa cells were obtained from ovaries of immature mice treated with PMSG alone and autopsied 48 hr (control) and 72 hr after injection (atretic) and from animals injected OFFP 24 hr after PMSG injection and autopsied 24 hr later (OFFP treated) by separation on discontinuous Percoll gradient. Four fractions were collected and studied for their relative distributions and percent apoptotic cells measured by acridine orange staining. FSH binding to granulosa cell (sedimenting as a major) fraction was studied by radio receptor assay. There is a difference in densities in subpopulations of apoptotic cells induced by OFFP and those generated during the physiological process of atresia. This difference may be a reflection o...
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Current study was carried out to identify the profile of newly synthesized and released proteins by human fallopian tube (hFT). Results indicated that hFT during menopause synthesised and released only 2-3 proteins as against several... more
Current study was carried out to identify the profile of newly synthesized and released proteins by human fallopian tube (hFT). Results indicated that hFT during menopause synthesised and released only 2-3 proteins as against several proteins ranging from molecular weight (MW) approximately 20 to approximately 130 kD during normal menstrual cycle. In vitro addition of estradiol-17 beta (E2) resulted in synthesis and release of a number of proteins including specific protein of MW 110-130 kD. Addition of progesterone (P) however, led to inhibition of protein synthesis and a combination of E2 and P negated the effect of the latter. An alteration in oviductal secretory protein-profile following addition of E2 in vitro were similar to that observed during normal menstrual cycle.
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Follicular development, ovulation and luteal function are controlled by gonadotrophins. However, recent evidence indicates that local factors are also responsible for the regulation of folliculogenesis. In addition to their endocrine... more
Follicular development, ovulation and luteal function are controlled by gonadotrophins. However, recent evidence indicates that local factors are also responsible for the regulation of folliculogenesis. In addition to their endocrine action on pituitary gonadotrophins, inhibin, activin and follistatin also have a paracrine role in follicular maturation. An ovarian follicular fluid peptide (OFFP) has been identified from sheep and humans. Purification of OFFP has been achieved by ultrafiltration and gel chromatography with further purification by fast performance liquid chromatography and reversed phase-high pressure liquid chromatography. OFFP is a small (< 5 kDa) peptide that competes with FSH in binding to granulosa cells in vitro and inhibits progesterone secretion from granulosa cells in culture. Immunohistochemical localization revealed the presence of OFFP mainly in granulosa cells of ovarian follicles. Furthermore, the peptide caused apoptosis in granulosa cells and induce...
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A protein fraction (GF2) was purified from sheep ovarian follicular fluid. Its action on 3 beta-HSD activity in the mouse granulosa cells was measured in an in vitro system. The fraction (GF2) caused dose-dependent depletion of the 3... more
A protein fraction (GF2) was purified from sheep ovarian follicular fluid. Its action on 3 beta-HSD activity in the mouse granulosa cells was measured in an in vitro system. The fraction (GF2) caused dose-dependent depletion of the 3 beta-HSD activity in granulosa cells and progesterone in the spent medium. A maximum inhibition of the activity was achieved after 30 min incubation of the granulosa cells with the GF2 fraction. Further purified HPLC fraction (Fr1) also inhibited 3 beta-HSD activity. In vivo administration of the GF2 fraction in normal cycling female mice also decreased the 3 beta-HSD activity in the granulosa cells of the ovarian follicles and plasma progesterone levels indicating the GF2 fraction to be a 3 beta-HSD inhibitor.
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A specific antiserum to ovine follicle stimulating hormone (AS/FSH) raised in rabbits and administered to prepubertal mice from days 5 to 35 of age, inhibited intermediate and type B spermatogonia. Development of spermatocytes,... more
A specific antiserum to ovine follicle stimulating hormone (AS/FSH) raised in rabbits and administered to prepubertal mice from days 5 to 35 of age, inhibited intermediate and type B spermatogonia. Development of spermatocytes, spermatids, and spermatozoa was affected, indicating a role for FSH in the initiation of spermatogenesis. Decrease in the testicular and epididymal weights, but not that of seminal vesicles and ventral prostate, demonstrated the specific action of AS/FSH. Reduction in the weights of the epididymis in AS/FSH-injected mice accompanied by the absence of spermatozoa in the lumen suggests a possible role for FSH in spermiation.
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The present study identifies uterine fluid (UF) proteins that display differential abundance during the embryo-permissive phase in nonconception and conception cycles in rats. UF samples were collected from nonpregnant rats in the... more
The present study identifies uterine fluid (UF) proteins that display differential abundance during the embryo-permissive phase in nonconception and conception cycles in rats. UF samples were collected from nonpregnant rats in the proestrous (n=17) and metestrous (n=18) phases and also from pregnant (n=17) and pseudopregnant (n=17) rats on day 4 post coitus. UF protein profile in the metestrous phase was compared with that in the proestrous phase. Similarly, UF protein profile of the pregnant rats was compared with that of the pseudopregnant rats. Two-dimensional PAGE, followed by densitometric analysis of the paired protein spots, revealed differential abundance of 44 proteins in the metestrous phase, compared with that in the proestrous phase. Of these, 29 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight or liquid chromatography-tandem mass spectrometry. Functional groups such as proteases, protease inhibitors, and oxidoreductases were enriched in differentially abundant proteins. Total protease activity in UF was found to be significantly (P&amp;amp;lt;0.05; t-test) higher in the proestrous phase, compared with that in the metestrous phase. Furthermore, 41 UF proteins were found to be differentially abundant in pregnant rats, compared with pseudopregnant rats. Of these, 11 proteins could be identified. Immunoblotting analysis confirmed significantly higher (P&amp;amp;lt;0.05; t-test) abundance of β-actin, Rho-specific guanine nucleotide dissociation inhibitor alpha (Rho-GDIα), and peroxiredoxin-2 and -6 in the metestrous phase, compared with that in the proestrous phase. Compared with pseudopregnant rats, pregnant rats had significantly higher (P&amp;amp;lt;0.05; t-test) levels of UF β-actin and Rho-GDIα. Furthermore, these proteins could be detected in the culture supernatants of endometrial epithelial cell lines, thereby providing an evidence of their secretion from endometrial epithelial cells. Data obtained from the study expand our knowledge on the uterine milieu that favours embryo implantation.
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Structure-function relationship studies of the follicle stimulating hormone and its receptor assume importance as this hormone is essential for folliculogenesis and spermatogenesis in females and males, respectively. The interaction... more
Structure-function relationship studies of the follicle stimulating hormone and its receptor assume importance as this hormone is essential for folliculogenesis and spermatogenesis in females and males, respectively. The interaction between the hormone and the receptor is complex and not well understood. In vitro studies using synthetic peptides from the extracellular domain of the receptor and corresponding antipeptide antibodies have suggested that the 285-309 region is surface-oriented. Antipeptide antibodies to this region also inhibit hormone-receptor interaction in a dose-dependent manner and the mechanism of inhibition appears to be competitive in nature. To test this hypothesis in an animal model, antibodies to peptide 285-309 from rat follicle stimulating hormone receptor (FSHR) were developed and characterized. These antibodies were able to detect FSHR in rat ovaries by immunohistochemistry. Further, these antibodies were administered into adult female rats and their effect on fertility status was monitored. These antibodies were found to neutralize the biological activity of endogenous receptor, which resulted in the induction of infertility in the treated animals. Thus, bioneutralization of FSHR has been achieved by targeting its region 285-309 in an in vivo system.
Research Interests: Immunology, Western blotting, Antibodies, Pregnancy, Female, and 11 moreAnimals, Reproductive Immunology, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Rats, Radioligand Assay, In Vitro Studies, Biological activity, Female Infertility, Epitopes, Enzyme Linked Immunosorbent Assay, and Follicle stimulating hormone
The crucial role played by follicle stimulating hormone (FSH) in regulating both male and female reproduction and the possibilities of developing contraceptive methods for males by blocking the function of the hormone, makes it important... more
The crucial role played by follicle stimulating hormone (FSH) in regulating both male and female reproduction and the possibilities of developing contraceptive methods for males by blocking the function of the hormone, makes it important to delineate the hormone-specific bioneutralization epitopes of human follicle stimulating hormone (hFSH) on its beta-subunit. Predictive methods were used to identify the potential surface-oriented regions of hFSH-beta. Peptides corresponding to these regions, i.e. 31-52, 66-75 and 86-95 hFSH-beta, were synthesized, anti-peptide antibodies were elicited in rabbits and the properties of these antisera to bind hFSH and neutralize its biological activity were assessed. Anti-31-52 hFSH-beta antisera bound hFSH specifically, whereas anti-66-75 and anti-86-95 hFSH-beta antisera did not show any detectable binding, proving the region 31-52 hFSH-beta to be a specific antigenic determinant of hFSH. The bioneutralizing abilities of the anti-peptide antibodies were assessed by measuring the hFSH-induced progesterone secretion by rat granulosa cells in vitro. Antibodies to 31-52 and 66-75 hFSH-beta neutralized the bioactivity of hFSH, but anti-86-95 hFSH-beta antibodies did not. Furthermore, the three linear peptides and two disulphide looped peptides of 31-52 hFSH-beta and 86-95 hFSH-beta were also subjected to the in-vitro granulosa cell assay. The linear peptides 31-52 hFSH-beta and 66-75 hFSH-beta and the cyclic 31-52 hFSH-beta disulphide loop peptide significantly inhibited the hFSH-induced progesterone secretion by rat granulosa cells, but the linear 86-95 hFSH-beta peptide and the corresponding cyclic disulphide loop peptide did not. The results clearly show that the regions 31-52 and 66-75 of hFSH-beta harbor bioneutralization epitopes of the hormone. The studies also indicate that cyclization of the linear 31-52 hFSH-beta peptide greatly enhances receptor recognition and that the region 66-75 hFSH-beta may also be involved in hormone-receptor interaction.
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Research Interests: Endocrinology, Animal Production, Crystal structure, Mice, Female, and 14 moreAnimals, Epitope mapping, Male, Clinical Sciences, Software Requirement Specification, Analysis of Variance, Radioligand Assay, Veterinary Sciences, Rabbits, Uterus, Protein Binding, Biological Assay, Luteinizing Hormone, and Enzyme Linked Immunosorbent Assay
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Human chorionic gonadotrophin (hCG)-like activity has been reported in mouse and rabbit blastocysts. The presence of this hCG-like activity seems to be essential for nidation of the pre-implantation embryo. Binding sites for hCG were... more
Human chorionic gonadotrophin (hCG)-like activity has been reported in mouse and rabbit blastocysts. The presence of this hCG-like activity seems to be essential for nidation of the pre-implantation embryo. Binding sites for hCG were localised on day 4 mouse embryos by immunohistochemical techniques. Presence of hCG-like activity was confirmed by cytotoxicity test. The number of implantation sites was significantly reduced on day 8 of pregnancy. Prior treatment to day 4 mouse embryos with hCG antiserum, for 1 h in utero or in vitro and their subsequent transfer to uteri of synchronised pseudopregnant mice resulted in impaired implantation of embryos, compared to controls treated with normal rabbit serum (NRS). These results suggest that hCG-like activity present on the pre-implantation embryo may have a significant role in implantation of the embryo.
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ABSTRACT
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A low molecular weight peptide has been partially purified from sheep follicular fluid. It inhibited FSH binding to granulosa cells from ovarian follicles of common marmosets (Callithrix jacchus). When injected into cycling marmosets... more
A low molecular weight peptide has been partially purified from sheep follicular fluid. It inhibited FSH binding to granulosa cells from ovarian follicles of common marmosets (Callithrix jacchus). When injected into cycling marmosets during the follicular phase, it reduced the area under the curve (AUC) of circulating progesterone. The peptide also shortened the luteal phase in all marmosets during the treatment cycle compared to the pretreatment control cycle. These results indicate that the ovarian follicular fluid peptide inhibited FSH binding to granulosa cells thereby probably resulting in decreased progesterone secretion (AUC) from these cells and subsequently inducing luteal insufficiency.
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Injection of antiserum to ovine FSH on the day of proestrus could inhibit ovulation in the next cycle. Inhibin preparation purified from sheep ovaries failed to affect ovulation in mice. Suppression of FSH by this preparation of inhibin... more
Injection of antiserum to ovine FSH on the day of proestrus could inhibit ovulation in the next cycle. Inhibin preparation purified from sheep ovaries failed to affect ovulation in mice. Suppression of FSH by this preparation of inhibin during early pregnancy reversed the mitotic index. Administration of inhibin during the peri-implantation period terminated pregnancy in mice. The inhibin preparation decreases FSH in circulation which in turn may possibly reduce ovarian estrogens, thereby inhibiting pregnancy.