Polymeric nanoparticle formulation of octapeptide (NP-OP): in vitro release and in vivo effect in common marmosets, Callithrix jacchus Linn.

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.

Identification of the region 285-309 of follicle stimulating hormone receptor as a bioneutralizing epitope.

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.

Fas and Fas ligand protein and mRNA in normal and atretic mouse ovarian follicles.

Apoptosis is the underlying mechanism of follicular atresia in the mammalian ovary. However, the apoptotic pathways governing this ovarian process are not completely elucidated. In the present study, expression of Fas and Fas ligand, the proximal members of the death receptor pathway, was evaluated in mouse ovarian follicles using immunofluorescence and in situ hybridization. Normal or atretic follicles were obtained from immature female Swiss mice after administration of 10 iu equine chorionic gonadotrophin for 48 or 72 h, respectively. Expression of both Fas and Fas ligand mRNA and protein was observed in granulosa cells of normal and atretic follicles. Although the oocytes of normal follicles failed to show any staining, those of atretic follicles stained intensely for Fas, indicating that the presence of Fas in the oocyte determines the fate of the follicle.

Expression of Fas and Fas ligand protein and mRNA in mouse oocytes and embryos.

During mammalian embryonic development, abnormal eggs and embryos are eliminated by apoptosis; however, the precise apoptotic pathways remain as yet unidentified. In the present study, expression of Fas and Fas ligand - the proximal members of the death receptor pathway, was evaluated in mouse preimplantation embryos by immunofluorescence and in situ hybridization techniques. Ovulated oocytes were collected from oviducts of cyclic mice on the day of oestrus (day 0), and one-cell, two-cell embryos and eight-cell morulae were collected from oviducts of mated animals on days 1, 2 and 3 of pregnancy, respectively. Blastocysts were flushed from the uterine horns on day 4. Expression of Fas and Fas L mRNAs and proteins was absent from embryos at days 0, 1 and 2. A marked increase in Fas and Fas L mRNA and protein expression was detected in all morphologically normal embryos on day 3 and day 4. In addition, embryos on days 3 and 4 were positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining; however, absence of caspase 8 and 3 and intense localization of proliferating cell nuclear antigen confirmed the proliferative status of these embryos. Furthermore, TUNEL staining was absent in postimplantation embryonic sections obtained on day 6. The results of the present studies thus indicate an equilibrium between proliferation and apoptosis in the preimplantation embryo.

A detailed study of the L2beta long-loop region of human chorionic gonadotrophin suggests it to be spatially close to, but not part of, the receptor-binding site.

An in-depth study of the L2beta long-loop region of human chorionic gonadotrophin (hCG), earlier identified to be a conformational bioneutralization epitope and receptor-binding site of the hormone, was carried out. The linear 38-57 hCGbeta peptide and the corresponding cyclic disulphide peptide were synthesized and antipeptide antibodies developed. Binding studies with antibodies to the linear peptide, and with hCGbeta, hCG and human LH suggest that part of the region is buried at the alpha/beta interface and part exposed in hCG. Observation of the surface exposure of residues 47-53 from the crystal structure of hCG was confirmed by epitope mapping studies of the region. The region is not unique to hCG as a majority of the antibodies to both the linear and cyclic peptides did not exhibit the required specificity. Competitive inhibition studies with the linear and cyclic peptides failed to show inhibition of radiolabelled hCG binding to its receptors. However, both the antipeptide antibodies were able to bioneutralize the hormone in an in vivo assay. Taken together, these results seem to indicate that the L2beta long-loop region is not a receptor-binding site of hCG but spatially close to it.

Apoptosis in endometrium of mouse during estrous cycle.

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.

Potential researchable areas in ARTs--oocyte maturation and embryo development.

Infertility is a commonly encountered situation occurring equally in both sexes. In vitro fertilization and embryo transfer (IVF-ET) and other assisted reproductive technologies (ARTs) have enhanced the possibilities for successful treatment to tackle infertility. However, ARTs currently face limitations due to the fact that although success rate is high for the initial stages such as ovulation induction and fertilization, it dwindles progressively so that the success rate of a take home baby is as low as 15-20%. Research centred around various stages in an IVF programme is therefore necessary to devise protocols that ensure a higher success rate. This review takes a look at the potential areas currently under research in the field of ARTs, such as, in vitro oocyte maturation, oocyte/embryo cryopreservation, embryo culture, preimplantation genetic diagnosis. Their applications, in clinical conditions such as cancer, have been discussed.

Leukaemia inhibitory factor in the endometrium of the common marmoset Callithrix jacchus: localization, expression and hormonal regulation.

In the present study, changes in the immunohistochemical localization of leukaemia inhibitory factor (LIF) in the endometrium during various phases of ovarian cyclicity of the common marmoset have been reported. LIF was absent during the early and late follicular phases. LIF was observed mainly in the cytoplasm of the endometrial glands during the early luteal phase, reached maximum intensity during the mid-luteal phase and declined again during late luteal phase. In-situ hybridization also showed a similar cyclic pattern in the expression of LIF. Stromal cells only showed signals for LIF during the mid-luteal phase. In ovariectomized marmosets, graded dosages of oestradiol alone failed to induce the appearance of LIF protein. Progesterone treatment following oestradiol priming, however, induced distinct glandular localization of LIF, indicating that LIF is a progesterone-dependent protein. Thus endometrial LIF is under maternal control and is secreted in response to the increased progesterone concentrations in circulation. It is possible that high concentrations of LIF during mid-luteal phase may prepare the endometrium for blastocyst implantation in marmosets.

Localization of estrogen and progesterone receptors in the endometrium of common marmosets Callithrix jacchus.

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 to the characteristic reproductive profile of this nonmenstruating species and needs to be studied further. Thus it can be hypothesized that in the marmoset endometrium, steroid hormone mediated effects possibly occur directly in the stroma and are then transmitted to the epithelium by autocrine/paracrine action of growth factors and cytokines.

Subpopulations of physiological and ovarian follicular fluid peptide induced apoptotic cells.

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 of different granulosa cell subpopulations involved in peptide response or differences in phases as the cells transit from normal to apoptotic phenotype. FSH binding to granulosa cells from OFFP treated animals was significantly less than those from control and atretic group.

Characterization and localization of estrogen and progesterone receptors of human fallopian tube.

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 high concentration of immunoreactive PR whereas it was almost undetectable in menopausal tissues. These results suggests that ER and PR are regulated by the changing ovarian steroid hormones.

Effect of estrogen and progesterone on newly synthesised and released human fallopian tube specific proteins.

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.

Autoimmune disorders of the ovary.

Infertility and ovarian disorders may occur not only due to imbalance in the steroid hormones but also due to the alteration in the immune function. A number of autoimmune disorders of the ovary, such as polycystic ovarian syndrome, premature ovarian failure and autoimmune oophoritis, have been documented. Both the cell mediated and humoral immune responses play an important role in these disorders. Cytokines secreted by the immune cells could have an adverse effect on ovarian steroidogenesis whereas the production of pathogenic T cells could lead to autoimmune disease of the ovary. Autoantibodies to granulosa and theca cells as well as zona pellucida of the oocyte have also been documented in women indicating that autoimmunity plays an important role in infertility. Thus the endocrine and the immune system work hand in hand to regulate ovarian function.

Identification of bioneutralization epitopes of human follicle stimulating hormone in the regions 31-52 and 66-75 of its beta-subunit.

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.

Regulation of follicular maturation by human ovarian follicular fluid peptide.

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 induced follicular atresia. OFFP may act indirectly on oocytes via its effect on granulosa cells. The peptide from ovarian follicular fluid appears to have an important autocrine or paracrine role in the regulation of folliculogenesis.

Steroidogenesis in sheep ovarian antral follicles in culture: time course study and supplementation with a precursor.

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 sufficient for the secretion of steroids into the culture medium and supplementation of the culture medium with an immediate precursor is essential for optimal steroidogenesis in vitro.

Immunoendocrine interrelationship--effect of ovariectomy on spleen and thymus in mouse.

Increase in the weights of spleen were observed 15 days after ovariectomy in adult as well as immature mice. However, no corresponding change was recorded in the splenocyte count. Morphological alterations in the shape of germinal centres was noted after ovariectomy in adult and immature mice. Increase in weight of the thymus could be demonstrated in adult mice only, with a concomitant enlargement of cortical and medullary region.

Interference with ovulation and luteal function by human ovarian follicular fluid peptide in bonnet monkeys, Macaca radiata.

Partially purified human ovarian follicular fluid peptide (hGF2) was administered during follicular phase in 5 bonnet monkeys. In control as well as hGF2-injected animals, ovulation was synchronized by treatment with FSH, Pergonal and hCG. All the 5 control bonnet monkeys showed plasma estradiol peak followed by increased progesterone levels. In 4 out of 5 hGF2-injected animals, plasma progesterone levels were drastically reduced. Plasma estradiol levels were decreased only in 3 animals in which amenorrhea was observed. These data reveal that hGF2 injection disrupted ovarian function in bonnet monkeys.

Localization and detection of ovarian follicular fluid protein in follicles of human ovaries.

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.