Effect of Punica granatum, Citrus limon and their combinations on the plasma Gonadotropins in female rabbits.

Fruits produce revitalizing effects, hence the impact of Punica granatum, Citrus limon and their combinations have been investigated on the plasma levels of gonadotropin, testosterone and sexual development capacity in female rabbits. Ninety female rabbits were randomly assigned into nine groups, each comprising of ten animals. One group was given saline and designated as control. Three groups were given P. granatum 2mL /kg, 5mL/kg, 8mL/kg, other three groups received C. limon 0.2mL/kg, 0.4mL/kg, 0.6mL/kg respectively, remaining groups received C. limon and P. granatum in combination i.e. 0.4mL/kg C. limon + 5mL/kg P. granatum and 0.2mL/kg C. limon + 8mL/kg P. granatum. Juices were administered once daily by mouth from day 0 of pups delivered to postnatal day15. Blood samples were gathered from ear vein at day11 and day15. There was significant increase in follicle stimulating hormone by P. granatum at 5 and 8mL/kg on day 11 and 15, by C. limon at 0.4 and 0.6mL/kg on day11, 0.4mL/kg at day15, by combination doses of C. limon and P. granatum 0.4 +5mL/kg at day 11, 0.4+5 mL/kg and 0.2 + 8mL/kg at day15. There was also significant increase in luteinizing hormone by P. granatum at 2, 5 and 8mL/kg and by C. limon 0.4mL/kg at day11. There was highly significant increase on day 11 in LH at combination doses of C. limon and P. granatum 0.4 + 5mL/kg. There was significant increase in testosterone level by P. granatum at 2, 5 and 8mL/kg on day 11 and 5mL/kg on day15 and highly significant increase at 2 and 8mL/kg. C. limon caused significant increase in testosterone at 0.4 mL/kg on day11, 0.2 and 0.6mL/kg on day 15 and highly significant increase at 0.4mL/kg on day15. Whereas combinations doses of C. limon and P. granatum at 0.4+5mL/kg caused highly significant increase in testosterone level as compare to control. Results of present study revealed increase in plasma gonadotropin and testosterone levels showing increase in sexual capacity of female rabbits which could be mainly accounted for high vitamin C and flavonoids contents of these juices.

Pharmacogenetics of G-protein-coupled receptors variants: FSH receptor and infertility treatment.

Infertility treatment may represent a paradigmatic example of precision medicine. Follicle-stimulating hormone (FSH) has been proposed as a valuable therapeutic option both in males and in females, even if a standardized approach is far to be established. To date, several genetic mutations as well as polymorphisms have been demonstrated to significantly affect the pathophysiology of FSH-FSH receptor (FSHR) interaction, although the underlying molecular mechanisms remain unclear. This review aims to highlight possible aspects of FSH therapy that could benefit from a pharmacogenetic approach, providing an up-to-date overview of the variability of the response to FSH treatment in both sexes. Specific sections are dedicated to the clinical use of FSH in infertility and how FSHR polymorphisms may affect the therapeutic endpoints.

Oral follicle-stimulating hormone agonist tested in healthy young women of reproductive age failed to demonstrate effect on follicular development but affected thyroid function.

OBJECTIVE: To assess the safety, pharmacokinetics, and pharmacodynamics of MK-8389. DESIGN: Double-blind, placebo-controlled, parallel-group, ascending dose study. SETTING: Two clinical research organizations. PATIENT(S): Healthy young women. INTERVENTION(S): Once-daily oral doses of MK-8389 or placebo for 14 days. MAIN OUTCOME MEASURE(S): Safety, including thyroid function tests (TFTs), pharmacokinetics, and follicular development (follicle size and number and serum E2 and inhibin B levels). RESULT(S): Treatment with MK-8389 was generally safe and well tolerated. An effect on TFTs was observed, which was transient and did not lead to clinical signs or symptoms but prevented dose escalation above 40 mg. MK-8389 was rapidly absorbed, slowly eliminated, and showed a large peak-to-trough ratio. No clinically meaningful effect was seen on follicle size and numbers, which was consistent with the low E2 levels. At doses >20 mg, inhibin B levels were increased, suggesting early follicular development at higher doses. CONCLUSION(S): Oral administration of MK-8389 demonstrated acceptable systemic exposure and was generally well tolerated. This study failed to demonstrate a clinically meaningful effect of MK-8389 on follicular development, whereas MK-8389 unexpectedly affected thyroid function. This study did not explore doses above 40 mg given the changes observed in TFTs, which may relate to high MK-8389 peak concentrations. CLINICAL TRIAL REGISTRATION NUMBER: EudraCT Number 2010-022396-57.

Development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing Fc to an FSH-ß subunit.

STUDY QUESTION: Does a novel long-acting recombinant human FSH, KN015, a heterodimer composed of FSHα and FSHß-Fc/Fc, offer a potential FSH alternative? SUMMARY ANSWER: KN015 had in vitro activity and superior in vivo bioactivity than recombinant human FSH (rhFSH), suggesting KN015 could serve as a potential FSH agonist for clinical therapy. WHAT IS KNOWN ALREADY: rhFSH has very short half-life so that repeat injections are needed, resulting in discomfort and inconvenience for patients. The longest-acting rhFSH available in clinics is corifollitropin alpha (FSH-CTP), but its half-life is not long enough to sustain the whole therapy period, and additional injections of rhFSH are needed. STUDY DESIGN, SIZE, DURATION: Plasmids containing FSHα, FSHß-Fc and Fc cDNA were transfected into Chinese hamster ovary (CHO) cells for KN015 production. The pharmacokinetics of KN015 was investigated in 6-week-old SD rats (n = 6/group) and healthy Cynomolgus monkeys in two different dose groups (n = 2/group). A series of experiments were designed for in vitro and in vivo characterization of the bioactivity of KN015 relative to rhFSH. PARTICIPANTS/MATERIALS, SETTING, METHODS: The purity and molecular weight of KN015 were determined by reducing and non-reducing SDS-PAGE. To measure KN015 half-life, sera were collected at increasing time points and the remaining FSH concentration was measured by enzyme-linked immunosorbent assay. To assess the bioactivity of KN015 versus rhFSH in vitro, firstly cAMP production was assessed in CHO cells expressing FSH receptor (FSHR) with the treatment of Fc/Fc, rhFSH or KN015 at eight different doses (0.03, 0.09, 0.28, 0.83, 2.5, 7.5, 22.5, 67.5 nM), and secondly cumulus oocyte complexes (COCs; n = 20/group) of ICR mice (primed-PMSG 44 h before sacrificed) were collected and cultured in medium containing 1.25 pM Fc/Fc, rhFSH or KN015 at 37°C and then germinal vesicle breakdown (GVBD) and COC expansion were observed at 4 and 16 h, respectively. The in vivo activity of KN015 was compared with rhFSH by ovary weight gain and ovulation assays. In the former, ovary weight gains in 21-day-old female SD rats, after a single subcutaneous injection of KN015, were compared with those after several injections of rhFSH over a range of doses (n = 8/group). Sera were harvested for estradiol (E2) analysis, and the ovaries were processed for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL), RT-PCR and western blot. In the latter, 26-day-old female SD rats (n = 8/group) were injected with different doses of KN015 or rhFSH, and were sacrificed at 24 h after an injection of hCG (20 IU/rat). Moreover, the molecular responses stimulated by KN015 or rhFSH in the ovary were also analyzed through detecting expression of the FSH target genes (Cyp19a1, Fshr and Lhcgr) and phosphatidylinositide 3-kinase (PI3K) pathway activation. MAIN RESULTS AND THE ROLE OF CHANCE: KN015 has a molecular weight of 82 kD and its half-life is 84 h in SD rats (10-fold longer than that of rhFSH) and 215 h in Cynomolgus monkeys. The EC50 value of the cAMP induction in CHO cells (KN015 versus rhFSH, 1.84 versus 0.87 nM), COC expansion and oocyte maturation assays showed KN015 had approximately half of rhFSH's activity in vitro. A single dose of KN015 (1.5 pmol/rat, 166.1 ± 19.7 mg, P < 0.01) stimulated significantly larger ovary weight gain than several injections of rhFSH (1.5 pmol/rat, 59.3 ± 28.1 mg, P < 0.01). The serum E2 level in the KN015 group was significantly higher than that in rhFSH group. The number of oocytes obtained by ovulation induction was comparable with or higher in the KN015 group than in the rhFSH group. KN015 was more effective than rhFSH in inducing FSH target genes (Cyp19a1, Fshr, Lhcgr) or activating the PI3K pathway in vivo. Moreover, a single injection of KN015 promoted granulosa cell proliferation and prevented follicle atresia to the same extent as several injections of rhFSH. LIMITATIONS, REASONS FOR CAUTION: All assays in this study were operated only in animals and clinical trials are needed to confirm they can be extrapolated to humans. WIDER IMPLICATIONS OF THE FINDINGS: KN015 is a valuable alternative to FSH and may have great potential for therapeutic applications. STUDY FUNDING/COMPETING INTERESTS: This study was supported by National Basic Research Program of China (2011|CB944504, 2012CB944403) and National Natural Science Foundation of China (81172473, 31371449). The authors have no conflicts of interest to declare.

Effect of time and dose of recombinant follicle stimulating hormone agonist on the superovulatory response of sheep.

The objective of this study was to determine the superovulatory potential of a single-chain analog of human FSH (Fcα) when administered to ewes either 3 days before, or coincident with, simulated luteolysis (pessary removal [PR]). A total of 40 animals were randomly assigned to receive Fcα at doses of 0.62, 1.25, or 2.5 IU/kg of body weight (bwt) 3 days before PR or 0.31, 0.62, 1.25, or 2.5 IU/kg of bwt at PR. Control ewes received protein without FSH activity. Blood samples were collected during the periovulatory period and ovarian tissue was collected 11 days after PR. Ovulation rate did not differ from the control group in ewes receiving the smallest doses of Fcα (0.31 and 0.62 IU/kg). However, a significant superovulatory response was noted in sheep receiving Fcα at doses of 1.25 and 2.5 IU/kg and this response was comparable in animals receiving the largest dose levels of Fcα at, or 3 days before, PR. The interval between PR and the LH surge was significantly extended and the LH surges were less synchronous in animals receiving Fcα at PR when compared with animals receiving the potent FSH agonist 3 days before PR. Taken together, these data indicate that the human single-chain gonadotropin with FSH activity promotes superovulation in ewe lambs in the breeding season. A single injection of the recombinant gonadotropin 3 days before luteolysis synchronizes the LH surge. The use of the single-chain analog of FSH in assisted reproduction for domestic animals is likely to be of practical significance as an alternative to conventional gonadotropins in superovulation protocols in livestock species.

Discovery of substituted benzamides as follicle stimulating hormone receptor allosteric modulators.

Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.

Effects of genistein on gonadotropic cells in immature female rats.

The effects of genistein on pituitary gonadotropic cells of immature female rats were examined and compared to actions of the synthetic estrogen, 17α-ethynylestradiol. Immature female rats received 50mg/kg/bw of genistein in dimethylsulfoxide (DMSO) subcutaneously (s.c.) daily for 3 days at 18, 19 and 20 days of age. A second group was injected with 1µg/kg of 17α-ethynylestradiol in olive oil in the same schedule. The genistein control group received DMSO only, while 17α-ethynylestradiol controls were given sterile olive oil only. Changes in cell number per mm(2), cell volume and volume density of follicle-stimulating (FSH) and luteinizing (LH) immunolabeled cells were evaluated by morphometry and stereology. Genistein induced significant increases in the number of FSH cells (by 21%) and LH cells (by 20%) per mm(2) compared to corresponding controls. Volumes of FSH and LH cells were significantly increased by 19.7% and 20% and their volume densities by 20% and 20.2%, respectively. Estradiol markedly affected gonadotropes in the same manner, but to a greater extent. It can be concluded that genistein acted as an estrogenic agonist in the pituitaries of immature female rats, and as such, stimulated gonadotropic cells.

Are circulating gonadotropin isoforms naturally occurring biased agonists? Basic and therapeutic implications.

The gonadotropins, luteinizing hormone, human chorionic gonadotropin and follicle-stimulating hormone, are key regulators of reproduction. As a result of this function, they have been the focus of research for many years. Isolated or recombinant proteins have been successfully used therapeutically for the treatment of infertility; and, in the case of compounds that block gonadotropin activity, for their potential utility in contraception. Until recently, selective small molecules modulating gonadotropin receptor activity have proven difficult to identify. The gonadotropins are glycoproteins that are released into the plasma as differently glycosylated isoforms and bind to specific G protein-coupled receptors. The degree of glycosylation on the gonadotropins has been shown to be important for the biological activities of these hormones and is differentially regulated depending on the steroidal status. Recent data from the study of glycosylated variants of LH, hCG and FSH have revealed that these isoforms have distinct signaling properties that allow for gonadotropin pleiotropic signals to be transduced effectively at the level of the receptor. Thus, glycosylated variants of the gonadotropins behave as biased agonists. Recently, newly developed, small molecule, synthetic allosteric compounds have been identified that are capable of mimicking this biased signaling. This opens the door to development of orally available, drug-like therapies for reproductive disorders that offer similar pleiotropic richness as that offered by the complex, endogenous hormones.

Discovery of selective luteinizing hormone receptor agonists using the bivalent ligand method.

Two series of dimeric ligands for a G-protein-coupled receptor were prepared that differ by the interconnecting spacer system. Biological evaluation revealed that both dimeric series exhibit unique biological properties relative to their monomeric counterparts.The luteinizing hormone receptor (LHR), the follicle-stimulating hormone receptor (FSHR), and the thyroid-stimulating hormone receptor (TSHR) belong to the glycoprotein hormone receptor (GpHR) family. A prominent feature of all endogenous glycoprotein ligands is that they share an identical alpha subunit and acquire their selectivity from the unique beta subunit. Recent developments in pro-fertility research have led to the discovery of several low-molecular-weight agonists for the luteinizing hormone/choriogonadotropin receptor that bind to the transmembrane (TM) region of the LHR. Interestingly, some of these agonists are also able to activate the FSHR. Several research groups have shown that ligand dimerization presents a powerful tool to increase the subtype selectivity for structurally related G-protein-coupled receptors. In this work, we applied the dimerization strategy to GpHRs and explored the effect on receptors with closely related TM regions. Two series of dimeric ligands were prepared that differ in the interconnecting spacer system. Biological evaluation revealed that both series exhibit unique selectivity properties for the LHR, originating from either decreased potency or a decreased efficacy toward the FSHR.

Development and characterization of a long-acting recombinant hFSH agonist.

BACKGROUND: Fusion of the carboxyterminal peptide (CTP) of hCG to FSH results in a follitropin agonist with an extended half-life, presumably due to the four O-oligosaccharides on the CTP. Alternatively, an rhFSH analogue containing additional N-linked carbohydrate is described in this report. METHODS: A DNA sequence containing two N-oligosaccharide signal sequences was ligated into a vector containing hFSHbeta- and alpha-subunit encoding cDNA, and expressed in CHO-K1 cells. In-vitro bioactivity of the single-chain hormone was assessed in CHO cells expressing the hFSH receptor. Pharmacokinetic values were derived from serial serum assays of the analogue in immature female rats following a single i.v. injection. In-vivo bioactivity was assessed by measuring ovarian weight gain 3 days post-injection. RESULTS: rhFSH-N2 and native rhFSH induced comparable levels of cAMP in vitro. t(1/2) for native rhFSH, rhFSH-CTP and rhFSH-N2 were 3.7, 7.1 and 7.3 h respectively. Rats receiving rhFSH-N2 had a mean +/- SD ovarian weight 3 days post-i.v. injection (22 +/- 3.6 mg) significantly greater than rats receiving rhFSH and saline (16.7 +/- 1.5 and 15.3 +/- 0.47 mg respectively, P < 0.05). CONCLUSIONS: rhFSH-N2 has prolonged half-life and increased bioactivity compared with native rhFSH. This rhFSH agonist, and other analogues containing additional N-oligosaccharides may have important clinical applications.

The effect of FSH on male germ cell survival and differentiation in vitro is mimicked by pentoxifylline but not insulin.

High concentrations of FSH have been shown to boost in-vitro differentiation of germ cells from men with normal spermatogenesis and from some patients with in-vivo maturation arrest. This study shows that the differentiation-promoting effect of FSH is connected to protection against germ cell apoptosis and that both effects can be mimicked by the intracellular cyclic AMP (cAMP)-elevating drug pentoxifylline. On the other hand, a high concentration of insulin, supposed to act at the insulin-like growth factor I receptor, did not exert any effect either on differentiation or apoptosis of germ cells in vitro. These data show that the in-vitro effects of supraphysiological concentrations of FSH on human spermatogenesis are mediated by the classical FSH signal transduction pathway involving cAMP as a second messenger. Pentoxifylline may thus be useful as an alternative means for intracellular cAMP elevation in men with high circulating FSH concentrations leading to desensitization of the FSH receptor.

Generating FSH antagonists and agonists through immunization against FSH receptor N-terminal decapeptides.

Follicle-stimulating hormone (FSH) via interaction with G-protein coupled specific receptors plays a central role in the control of gametogenesis in mammals of both sexes. In females, FSH is crucial for follicle growth, follicle maturation and ovulation. FSH receptors, together with luteinizing hormone-chorionic gonadotropin and thyrotropin receptors belong to a subfamily of structurally related receptors within the seven transmembrane receptor family. Among several other regions, the N-terminus of these receptors is believed to be responsible for important specific hormone-receptor contact sites. Recombinant filamentous phages displaying at their surface three overlapping N-terminal decapeptides of the FSH receptor, peptides A18-27, B25-34 and C29-38 were constructed. Ewes and female mice were immunized against the three FSH receptor (FSHR) recombinant phages. Immunoglobulins purified from immunized animals were analyzed for their biochemical properties on a Chinese hamster ovary cell line expressing the porcine FSH receptor. AntiA and antiB immunoglobulins (IgGs) behave as antagonists for 125I-FSH binding and for FSH-dependent cAMP production, while antiC IgGs did not compete for hormone binding. By contrast, antibodies against the C29-38 peptide displayed FSH agonist activity and stimulated the FSH receptor, whereas antiA and antiB IgGs did not. Furthermore, when the FSHR phages were used as peptidic vaccines, they induced a reversible inhibition of ovulation rate in ewes, and impaired fertility in female mice.

Follicle stimulating hormone and its receptor: future perspectives.

In this report, we review our present effort in the field of molecular reproductive endocrinology: to identify a small molecular weight follicle stimulating hormone (FSH) agonistic molecule. To achieve this goal we require a number of molecular tools. We have cloned and expressed the human gonadotrophin, FSH and the human FSH receptor and developed a reliable high throughput assay. We have also proposed a model to explain FSH receptor activation and from that model, begun to create small molecules predicted to induce FSH signal transduction without binding to the extracellular domain of the membrane protein. In this report, we summarize our efforts to date and discuss our future research efforts in this area.

Developments in human recombinant follicle stimulating hormone technology: are we going in the right direction?

Recent developments in recombinant DNA technology have enabled the large scale production of human recombinant follicle stimulating hormone (rFSH); and this compound has recently been introduced to the market. Understanding of the structure-function relationship of FSH isohormones is crucial in understanding discussions on the standardization procedures of gonadotrophin preparations, potential differences in clinical efficacy of the various gonadotrophin preparations and in comprehending future developments (long-acting and short-acting forms, and rFSH preparations with altered isohormone profiles). Differences between immunoreactive and bioactive serum FSH concentrations have been observed following the administration of rFSH. Accordingly, the isohormone distribution of rFSH is similar to, but not identical with, natural human FSH. Issues relevant to daily practice discussed in this review include: the total absence of urinary contaminants allowing for the safe s.c. administration of the compound. Production is independent from urine, and the capacity can be adjusted according to clinical needs. The relationship between serum oestradiol concentrations and number and size of follicles observed by ultrasound may change when rFSH is combined with gonadotrophin-releasing hormone (GnRH) agonist, due to low serum lueinizing hormone (LH) concentrations. In the case of endogenous serum LH concentrations within the normal range, exogenous administration of LH is redundant. In the near future, rFSH preparations with altered bioactivity will be available.