MARF1 regulates essential oogenic processes in mice.

Development of fertilization-competent oocytes depends on integrated processes controlling meiosis, cytoplasmic development, and maintenance of genomic integrity. We show that meiosis arrest female 1 (MARF1) is required for these processes in mammalian oocytes. Mutations of Marf1 cause female infertility characterized by up-regulation of a cohort of transcripts, increased retrotransposon expression, defective cytoplasmic maturation, and meiotic arrest. Up-regulation of protein phosphatase 2 catalytic subunit (PPP2CB) is key to the meiotic arrest phenotype. Moreover, Iap and Line1 retrotransposon messenger RNAs are also up-regulated, and, concomitantly, DNA double-strand breaks are elevated in mutant oocytes. Therefore MARF1, by suppressing levels of specific transcripts, is an essential regulator of important oogenic processes leading to female fertility and the development of healthy offspring.

Mouse oocytes regulate metabolic cooperativity between granulosa cells and oocytes: amino acid transport.

A search for genes expressed more highly in mouse cumulus cells than mural granulosa cells by subtraction hybridization yielded Slc38a3. SLC38A3 is a sodium-coupled neutral amino acid transporter having substrate preference for l-glutamate, l-histidine, and l-alanine. Detectable levels of Slc38a3 mRNA were found by in situ hybridization in granulosa cells of large preantral follicles, but levels were higher in all granulosa cells of small antral follicles; expression became limited to cumulus cells of large antral follicles. Expression of Slc38a3 mRNA in granulosa cells was promoted by fully grown oocytes from antral follicles but not by growing oocytes from preantral follicles. Fully grown oocytes were dependent on cumulus cells for uptake of l-alanine and l-histidine but not l-leucine. Fully grown but not growing oocytes secreted one or more paracrine factors that promoted cumulus cell uptake of all three amino acids but of l-alanine and l-histidine to a much greater extent than l-leucine. Uptake of l-leucine appeared dependent primarily on contact-mediated signals from fully grown oocytes. Fully grown oocytes also promoted elevated levels of Slc38a3 mRNA and l-alanine transport by preantral granulosa cells, but growing oocytes did not. Therefore, fully grown oocytes secrete one or more paracrine factors that promote cumulus cell uptake of amino acids that oocytes themselves transport poorly. These amino acids are likely transferred to oocytes via gap junctions. Thus, oocytes use paracrine signals to promote their own development via metabolic cooperativity with cumulus cells. The ability of oocytes to mediate this cooperativity is developmentally regulated and acquired only in later stages of oocyte development.

A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence.

The objective of this study was to improve the conditions for oocyte development in vitro beginning with the primordial follicles of newborn mice. Previous studies showed that oocytes competent of meiotic maturation, fertilization, and preimplantation could develop in vitro from primordial follicles. However, the success rates were low and only one live offspring was produced (0.5% of embryos transferred). A revised protocol was compared with the original protocol using oocyte maturation and preimplantation development as end points. The percentage of oocytes maturing to metaphase II and developing to the blastocyst stage was significantly improved using the revised protocol. In addition, we compared the production of offspring from two-cell stage embryos derived from in vitro-grown and in vivo-grown oocytes. Of 1160 transferred two-cell stage embryos derived from in vitro-grown oocytes, 66 (5.7%) developed to term and 7 pups (10.6%) died at birth. The remaining 59 pups (27 females, 32 males) survived to adulthood. By comparison, of 437 transferred two-cell stage embryos derived from in vivo-grown oocytes, 76 (17.4%) developed to term and 4 (5.3%) died at birth. The remaining 72 pups (35 females, 37 males) survived to adulthood. These studies provide proof of the principle that fully competent mammalian oocytes can develop in vitro from primordial follicles and present a significant advance in oocyte culture technology.

Toward the genetics of mammalian reproduction: induction and mapping of gametogenesis mutants in mice.

The genetic control of mammalian gametogenesis is inadequately characterized because of a lack of mutations causing infertility. To further the discovery of genes required for mammalian gametogenesis, phenotype-driven screens were performed in mice using random chemical mutagenesis of whole animals and embryonic stem cells. Eleven initial mutations are reported here that affect proliferation of germ cells, meiosis, spermiogenesis, and spermiation. Nine of the mutations have been mapped genetically. These preliminary studies provide baselines for estimating the number of genes required for gametogenesis and offer guidance in conducting new genetic screens that will accelerate and optimize mutant discovery. This report demonstrates the efficacy and expediency of mutagenesis to identify new genes required for mammalian gamete development.