The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

This study tested the hypothesis that murine oocytes participate in the establishment of granulosa cell phenotypic heterogeneity in preovulatory follicles. In these follicles, mural granulosa cells express LH receptors (LHR) and LHR mRNA, but expression of these molecules is low or undetectable in cumulus cells. Thus, the expression of LHR mRNA is a marker of the mural granulosa cell phenotype in preovulatory follicles. Cumulus cells expressed elevated steady-state levels of LHR mRNA when oocytes were microsurgically removed from oocyte-cumulus cell complexes, and this was prevented by paracrine factor(s) secreted by isolated oocytes. These factors also suppressed FSH-induced elevation of the level of LHR mRNA expression by mural granulosa cells isolated from small antral follicles, even when expression was augmented by culturing granulosa cells on components of basal lamina. Moreover, factor(s) secreted by oocytes suppressed the expression of LHR mRNA in mural granulosa cells isolated from preovulatory follicles already expressing elevated levels of these transcripts. The ability of oocytes to suppress the LHR mRNA expression by granulosa cells was developmentally regulated. Oocytes from preantral follicles and mature (metaphase II arrested) oocytes were less effective in suppressing expression than fully grown, germinal vesicle (GV)-stage oocytes. Furthermore, two-cell-stage embryos did not suppress LHR mRNA levels. Coculture of isolated oocytes with granulosa cells affected the synthesis of very few granulosa cell proteins detected by fluorography of two-dimensional gels after 35S-methionine labeling. Thus, oocyte suppression of FSH-induced LHR mRNA expression is specific in both the suppressing cell type and the effects on granulosa cells. It is concluded that the default pathway of granulosa cell differentiation produces the mural granulosa cell phenotype, as represented by the expression of LHR mRNA. This pathway is abrogated by oocytes. Thus, oocytes play a dominant role in establishing the fundamental heterogeneity of the granulosa cell population of preovulatory follicles.

Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogenreceptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells.oogenesis | primordial germ cells | follicle formation | oocytes | in vitro

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 microm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 microg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.