T.D. Nandedkar

Danazol-β-cyclodextrin binary system: A potential application in emergency contraception by the oral route

This study explored the potential of β-cyclodextrin to improve the aqueous solubility and dissolution of danazol, investigated a simple and less expensive method for preparation of a danazol-β-cyclodextrin binary system, and explored the potential application of a danazol-β-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 Jobs plot affirmed 1∶1 stoichiometry. The hyperchromic shift in the UV-Vis spectrum of danazol in the presence of β-cyclodextrin indicated solubilization capability of β-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 β-cyclodextrin.1H-nuclear magnetic resonance analysis suggested that the protons of the steroidal skeleton of danazol display favorable interactions with the β-cyclodextrin cavity. The danazol-β-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-β-cyclodextrin binary system prepared by the freeze-drying and milling (in the presence of 13% moisture) methods. In a mouse model, the danazol-β-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-β-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-β-cyclodextrin binary system could serve as a new therapeutic application: an oral emergency contraceptive at a physiologically acceptable single dose.

Steroidogenesis in sheeo 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.

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

Luteal deficiency caused by human ovarian follicular fluid peptide in post-partum marmosets, Callithrix jacchus

A peptide purified from human ovarian follicular fluid (hOFFP) was administered during the follicular phase of post-partum marmosets. Four of the five animals treated with hOFFP, ovulated as evidenced by the presence of ovarian stigmata at the time of laparotomy. However, only 2 of these animals became pregnant, one had a normal delivery while the other aborted. A shortening of the luteal phase was observed in the other three animals and all animals conceived in subsequent cycles. The results indicated that treatment with the ovarian follicular fluid peptide resulted in impairment of fertility as a result of luteal insufficiency.

Uterine secretome and its modulation in rat (Rattus norvegicus)

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<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<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<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.

Shortening of luteal phase in common marmosets by sheep ovarian follicular fluid peptide

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.

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.

Inhibition of 3 β-hydroxysteroid dehydrogenase (3 β-HSD) activity in the granulosa cells of mouse ovarian follicles by follicular fluid peptide

A protein fraction (GF2) was purified from sheep ovarian follicular fluid. Its action on 3 β-HSD activity in the mouse granulosa cells was measured in an in vitro system. The fraction (GF2) caused dose-dependent cep emotion of the 3 β-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 β-HSD activity. In vivo administration of the GF2 fraction in normal cycling female mice also decreased the 3 β-HSD activity in the granulosa cells of the ovarian follicles and plasma progesterone levels indicating the GF2 fraction to be a 3 β-HSD inhibitor.

Effect of inhibin on ovulation and implantation in mice

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.

Ovarian Follicular Fluid Peptide (OFFP): Effects on Follicular Maturation, Ovulation and Luteinization in Mice and Marmosets

Intraovarian concentrations of steroids and non-steroidal factors have recently been reported by Demoalin and Franchimont (1985) to play an important role in deciding the fate of the follicle. We have attempted to partially purify and characterize a peptide from sheep follicular fluid (OFFP). The pooled follicular fluid was chromatographed on Sephadex G-100 and later on Sephadex G-25 column (Kadam et al., 1984). The bioactive fraction (GF2) inhibited maturation of large follicles and ovulation in cycling mice. It also decreased plasma progesterone (P) levels but did not affect FSH levels significantly in these mice (Nandedkar et al., 1986). It reduced 33 hydroxysteroid dehydrogenase (33 HSD) activity in mouse granulosa cells. Further, the purified Fraction 1 on high performance liquid chromatography also inhibited 3 3 HSD activity in mouse granulosa cells. The trypsin digestion and heat inactivation of the peptide indicated the heat labile nature of OFFP (Kadam et al., 1984).