Factors affecting the developmental competence of mouse oocytes grown in vitro: follicle-stimulating hormone and insulin.

This study was undertaken to test the hypothesis that FSH treatment of cultured oocyte-granulosa cell complexes promotes acquisition of competence to complete preimplantation embryo development. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in serum-free medium, supplemented with insulin (5 microgram/ml), transferrin (5 microgram/ml), and selenium (5 ng/ml) and containing a highly potent preparation of FSH (0-5 ng/ml). Oocytes were matured and fertilized in vitro and embryos cultured to determine the frequency of development to the blastocyst stage. There was no effect of FSH on oocyte size, general morphology, or competence to resume meiosis. However, addition of FSH to medium containing insulin had a deleterious effect on the percentage of mature oocytes competent to develop to the blastocyst stage. Deletion of insulin from the medium for culture of oocyte-granulosa cell complexes prevented the deleterious effect of FSH, but FSH still did not promote acquisition of competence to complete preimplantation development. Culture of oocyte-granulosa cell complexes with FSH resulted in elevated expression of LH receptor (LHR) mRNA by granulosa cells and stimulated the production of functional LHRs, whether or not insulin was present. However, FSH-induced expression of LHR mRNA reached a maximum steady-state level by 4 days of culture in the presence of insulin, but this level was not reached until 10 days of culture without insulin. Granulosa cells encompassing growing mouse oocytes in vivo do not express LHR mRNA. Thus, expression of LHR mRNA by granulosa cells closely associated with growing oocytes in vitro indicates inappropriate or ambiguous development. In conclusion, conditions occurring during oocyte growth can have profound detrimental effects on oocyte developmental competence to complete preimplantation development, even when oocyte growth, general morphology, and competence to resume meiosis appear unaffected

New mouse genetic models for human contraceptive development.

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets

Oocyte control of granulosa cell development: how and why.

Interaction between oocytes and granulosa cells is complex and involves both gap junctions and paracrine signalling factors. Oocyte development in antral follicles is highly dependent on communication with cumulus cells, a subset of granulosa cells that is intimately associated with oocytes. Cumulus cells express characteristics distinct from the mural granulosa cells of preovulatory follicles. The thesis of this paper is that, without the influence of oocytes, the pathway of granulosa cell differentiation in antral follicles leads to the establishment of the mural granulosa cell phenotype. Oocytes in antral follicles abrogate that pathway of granulosa cell differentiation and promote the development of the cumulus cell phenotype. Oocytes may do this in order to control their own microenvironment by regulating differentiation of the supporting cells that are in direct communication with them. Possibly, some aspects of the mural granulosa cell phenotype are antagonistic to, or insufficient for, supporting the final stages of oocyte development. We present evidence that oocytes control their environment by suppressing differentiation of the mural granulosa cell phenotype and promoting differentiation of the cumulus cell phenotype. They achieve this suppression via the secretion of labile paracrine signalling factors. Errors in this regulatory mechanism, whether instigated by defects in the production of oocyte-derived ligands or granulosa cell responses to them, may result in the production of oocytes unable to undergo embryo development or that undergo abnormal follicular development

Abstract 4292: Indole-3-carbinol (I3C) enhances efficacy of Gemcitabine in leiomyosarcoma

Abstract Introduction: Adjuvant Gemcitabine have recently been shown to improve progression free survival in some types of leiomyosarcoma. Adequate cellular uptake of the drug remains a challenge. We aimed to evaluate the role of nucleoside transporter activator (I3C) in increasing the in vitro efficacy of gemcitabine in the treatment of leimyosarcoma. Methods: Leiomyosarcoma cells (SKLMS) were incubated with DMEM+10% FBS + 1% Antibiotics. Dose response curves were generated for gemcitabine and I3C to identify the IC-50 values. Triplicates of cells were then plated in 96 well plates (3000 cells/well) and allowed to adhere. The adherent cells were further treated with gemcitabine in the presence or absence of I3C and compared with controls (DMSO treatment). MTS cell viability assays were done and absorbance was monitored in a micro-plate reader. Flow cytometry was performed with Propidium Iodide (PI) staining. Scratch assays were done to assess cell migration. Results: Viability assay showed significant increase in the cytotoxicity of gemcitabine in presence of I3C compared to controls. Flow cytometry data showed gemcitabine alone treatment has arrested the cell cycle at G0/G1 phase without increase of cell death. On the contrary, use of gemcitabine in combination with I3C increased the cell death by at least three folds compared to control as well as gemcitabine alone treated cell population. Scratch assay over a 16 hour period showed that the rate of cellular migration significantly dropped by 1.5 folds upon treatment with gemcitabine combined with I3C compared to gemcitabine alone. Western blots showed increased expression of membrane transporter hENT-1 with I3C treatment. Conclusion: The use of I3C enhanced cellular uptake, transport and cytotoxicity of gemcitabine in SKLMS cells. Further studies are warranted and underway to elaborate on intracellular mechanisms of action to optimize the use of I3C as a potential adjunct in preclinical and clinical leiomyosarcoma treatment. Note: This abstract was not presented at the meeting. Citation Format: Sujit Suwal, Alexandra Moran, Ana Paz-Mejia, Lorena Flor, Omar Picado, Basem Azab, Jonathan Trent, Fiorella Pendola, Alan S. Livingstone, Danny Yakoub. Indole-3-carbinol (I3C) enhances efficacy of Gemcitabine in leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4292. doi:10.1158/1538-7445.AM2017-4292</jats:p

The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes.

Mammalian oocytes are held in prophase arrest by an unknown signal from the surrounding somatic cells. Here we show that the orphan Gs-linked receptor GPR3, which is localized in the oocyte, maintains this arrest. Oocytes from Gpr3 knockout mice resume meiosis within antral follicles, independently of an increase in luteinizing hormone, and this phenotype can be reversed by injection of Gpr3 RNA into the oocytes. Thus, the GPR3 receptor is a link in communication between the somatic cells and oocyte of the ovarian follicle and is crucial for the regulation of meiosis