The present study was designed to explore the steroidogenic responsiveness of ovine antral follic... 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...
Experimental and Clinical Endocrinology & Diabetes, 1990
Ovarian follicular fluid peptide (OFFP) has earlier been shown to inhibit maturation of large fol... 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.
Binding of FSH to ovarian cells was studied in PMSG primed immature Swiss mice, 48 h after PMSG t... 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.
The activity of delta 5-3 beta hydroxysteroid dehydrogenase (delta 5-3 beta HSD) in pre-implantat... 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.
Human ovarian follicular fluid protein has been partially purified and the active fraction design... 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.
Octapeptide (OP)/FSH-Receptor Binding Inhibitor-8 (FRBI-8), is a synthetic peptide corresponding ... 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.
The present study was carried out to evaluate apoptosis in endometrium and to correlate these cha... 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.
In the present study, changes in the immunohistochemical localization of endometrial estrogen rec... 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...
Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries enhances apoptotic changes in... 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...
The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopi... more The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopian tube was investigated by immunohistochemical localization with specific monoclonal antibodies. Nuclear immunostaining was observed. Intense PR immunostaining was seen in tissues obtained at mid cycle and luteal stages of the normal menstrual cycle. On the other hand, enhanced staining for ER was seen in early follicular phase and mid cycle. Menopausal tissues showed negligible staining for both ER and PR. The ER and PR were characterized for their molecular size, anatomical distribution and levels during the menstrual cycle and in menopause. ER protein was present throughout the cycle and also during menopause. Western blot analysis revealed two forms of ER approximately 66 kDa and a truncated from approximately 49 kDa in hFT. Presence of A [approximately 90 kDa] and B [approximately 120 kDa] isoforms of human PR was detected. Follicular and early luteal tissue possessed relatively hi...
Current study was carried out to identify the profile of newly synthesized and released proteins ... 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.
Journal of reproduction and fertility. Supplement, 1996
Follicular development, ovulation and luteal function are controlled by gonadotrophins. However, ... 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...
The present study was designed to explore the steroidogenic responsiveness of ovine antral follic... 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...
Experimental and Clinical Endocrinology & Diabetes, 1990
Ovarian follicular fluid peptide (OFFP) has earlier been shown to inhibit maturation of large fol... 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.
Binding of FSH to ovarian cells was studied in PMSG primed immature Swiss mice, 48 h after PMSG t... 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.
The activity of delta 5-3 beta hydroxysteroid dehydrogenase (delta 5-3 beta HSD) in pre-implantat... 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.
Human ovarian follicular fluid protein has been partially purified and the active fraction design... 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.
Octapeptide (OP)/FSH-Receptor Binding Inhibitor-8 (FRBI-8), is a synthetic peptide corresponding ... 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.
The present study was carried out to evaluate apoptosis in endometrium and to correlate these cha... 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.
In the present study, changes in the immunohistochemical localization of endometrial estrogen rec... 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...
Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries enhances apoptotic changes in... 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...
The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopi... more The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopian tube was investigated by immunohistochemical localization with specific monoclonal antibodies. Nuclear immunostaining was observed. Intense PR immunostaining was seen in tissues obtained at mid cycle and luteal stages of the normal menstrual cycle. On the other hand, enhanced staining for ER was seen in early follicular phase and mid cycle. Menopausal tissues showed negligible staining for both ER and PR. The ER and PR were characterized for their molecular size, anatomical distribution and levels during the menstrual cycle and in menopause. ER protein was present throughout the cycle and also during menopause. Western blot analysis revealed two forms of ER approximately 66 kDa and a truncated from approximately 49 kDa in hFT. Presence of A [approximately 90 kDa] and B [approximately 120 kDa] isoforms of human PR was detected. Follicular and early luteal tissue possessed relatively hi...
Current study was carried out to identify the profile of newly synthesized and released proteins ... 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.
Journal of reproduction and fertility. Supplement, 1996
Follicular development, ovulation and luteal function are controlled by gonadotrophins. However, ... 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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