Investigation of a thiazolidinone derivative as an allosteric modulator of follicle stimulating hormone receptor: evidence for its ability to support follicular development and ovulation.

FSH signalling through its cognate receptor is critical for follicular development and ovulation. An earlier study had documented thiazolidinone derivatives to activate FSH receptor expressed in CHO cells and rat granulosa cells; however development of this compound for clinical use was halted for unobvious reasons. The objective of the current study is to extend the previous investigations in detail on the ability of thiazolidinone derivative (henceforth referred to as Compound 5) to activate FSH signalling and learn the barriers that preclude development of this derivative for clinical purposes. Our results demonstrate that the Compound 5 in a dose-dependent manner stimulated cAMP production, activated AKT and ERK signalling pathways and induced estradiol production in cultured rat granulosa cells. Compound 5 also caused dose-dependent increase in estradiol production from human granulosa cells. In increasingly more complex in vitro systems, Compound 5 was able to induce the expansion of mouse cumulus-oocyte-complex and support in vitro development of mouse preantral follicle to preovulatory stage and release of oocyte from the follicle. In vivo, the compound stimulated preovulatory follicular development and ovulation in immature rats. Pharmacokinetic and safety investigations reveal poor oral availability and genotoxicity. Together, our results document Compound 5 to act as a FSHR allosteric modulator but have poor pharmacological properties for development of an oral FSH receptor modulator.

Interleukin-6: an autocrine regulator of the mouse cumulus cell-oocyte complex expansion process.

Ovulation has long been regarded as a process resembling an inflammatory response. Recent studies indicate that genes associated with innate immune responses were also expressed during the ovulation process. Because the innate immune genes are induced in cumulus cell oocyte complexes (COCs) later than the inflammation-associated genes, we hypothesize that COC expansion is dependent on specific sequential changes in cumulus cells. Because IL-6 is a potent mediator of immune responses, we sought to determine what factors regulate the induction of Il6 mRNA in COCs and what impact IL-6 alone would have on COC expansion. We found that the levels of Il6 mRNA increased dramatically during COC expansion, both in vivo and in vitro. Moreover, IL-6, together with its soluble receptor (IL-6SR), could bypass the need for either amphiregulin and/or prostaglandin E2 to induce the expansion of COCs. This ability of IL-6/IL-6SR to induce COC expansion was blocked by the inhibitors to p38MAPK, MAPK kinase 1/2, and Janus kinase. More importantly, when COCs were in vitro maturated in the presence of IL-6, they had a significantly higher embryo transfer rate than the ones without IL-6 and comparable with in vivo matured oocytes. IL-6/IL-6SR activated multiple signaling pathways (Janus kinase/signal transducer and activator of transcription, ERK1/2, p38MAPK, and AKT) and progressively induced genes known to impact COC expansion, genes related to inflammation and immune responses, and some transcription factors. Collectively, these data indicate that IL-6 alone can act as a potent autocrine regulator of ovarian cumulus cell function, COC expansion, and oocyte competence.

Leukemia inhibitory factor induces cumulus expansion in immature human and mouse oocytes and improves mouse two-cell rate and delivery rates when it is present during mouse in vitro oocyte maturation.

OBJECTIVE: To examine the role of leukemia inhibitory factor (LIF) during in vitro maturation (IVM) on human and mice cumulus expansion and mice oocyte competence by in vitro fertilization (IVF), culture, and embryo transfer (ET). DESIGN: Prospective animal and human study. SETTING: Serono laboratories and IVF clinic. PATIENT(S): Healthy women volunteers and 8-week-old female mice. INTERVENTION(S): Cumulus compacted human and mice oocytes were matured in IVM media with and without recombinant follicle-stimulating hormone (FSH) and with and without LIF. Mice IVM oocytes with and without 0.2 IU/mL of recombinant FSH; or with and without recombinant FSH + LIF (0.1, 1.0, 1000.0 ng/mL) and ovulated oocytes were in vitro fertilized and cultured. We transferred 395 blastocysts to the uterine horn of 2.5-day pseudopregnant female mice. MAIN OUTCOME MEASURE(S): Cumulus expansion in human and mice oocytes, and two-cell rate, blastocyst rate, and delivered rate of live pups in mice. RESULT(S): In human and mouse oocytes, LIF induced cumulus expansion. When 1000 ng/mL of LIF was added in combination with recombinant FSH, a statistically significant increase in cleavage rate, embryo development rate, and birth rate was observed when compared with oocytes matured with FSH alone. CONCLUSION(S): Leukemia inhibitory factor induced cumulus expansion similarly in human and mouse cumulus-oocyte complexes, and recombinant FSH plus LIF supplementation during mouse IVM significantly improved oocyte competence as measured by cleavage rate, blastocyst development, and birth rate.

Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content.

Cysteamine and beta-mercaptoethanol supplementation of in vitro maturation (IVM) medium has been found to increase intracellular glutathione (GSH) content in oocytes and to improve embryo development and quality in several species. The objective of this experiment was to study the effect of cysteamine and beta-mercaptoethanol added during IVM of sheep oocytes on GSH synthesis and embryo development. Furthermore, we examined if cysteamine addition (hence GSH production) had an effect on the reduction of the intracellular peroxide content. We matured oocytes obtained from ovaries collected at a slaughterhouse in vitro in the presence of 0, 50, 100, and 200 microM cysteamine (Experiment 1) or with 0, 50, 100, and 200 microM beta-mercaptoethanol (Experiment 2). Following fertilization and embryo development, there was a increasing level of morula and blastocyst development in the presence of cysteamine, reaching significance in the presence of 200 microM (P < 0.05). However, beta-mercaptoethanol did not influence on the rate of embryo development. GSH levels were measured in oocytes matured in the presence or absence of 200 microM cysteamine (Experiment 3) or 50 microM beta-mercaptoethanol (Experiment 4), with or without buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Results demonstrated that for both cysteamine and beta-mercaptoethanol, intracellular GSH levels increased against control values (P < 0.01), which was abolished in the presence of BSO. Finally, we reduced intracellular peroxide levels, as measured by the relative fluorescence of the intracellular peroxide probe, carboxy-H2DCFDA, in the presence of either 200 microM cysteamine or 50 microM beta-mercaptoethanol (Experiment 5). These results demonstrate that cysteamine, but not beta-mercaptoethanol, when present during IVM, stimulates sheep embryo development; both cysteamine and beta-mercaptoethanol stimulate GSH synthesis; the increase in intracellular GSH is associated with a decrease in peroxide levels within oocytes.