Live Offspring Produced by Mouse Oocytes Derived from Premeiotic Fetal Germ Cells1

Abstract

Mature mouse oocytes currently can be generated in vitro from the primary oocytes of primordial follicles but not from premeiotic fetal germ cells. In this study we established a simple, efficient method that can be used to obtain mature oocytes from the premeiotic germ cells of a fetal mouse 12.5 days postcoitum (dpc). Mouse 12.5-dpc fetal ovaries were transplanted under the kidney capsule of recipient mice to initiate oocyte growth from the premeiotic germ cell stage, and they were recovered after 14 days. Subsequently, the primary and early secondary follicles generated in the ovarian grafts were isolated and cultured for 16 days in vitro. The mature oocytes ovulated from these follicles were able to fertilize in vitro to produce live offspring. We further show that the in vitro fertilization offspring were normal and able to successfully mate with both females and males, and the patterns of the methylated sites of the in vitro mature oocytes were similar to those of normal mice. This is the first report describing premeiotic fetal germ cells able to enter a second meiosis and support embryonic development to term by a combination of in vivo transplantation and in vitro culture. In addition, we have shown that the whole process of oogenesis, from premeiotic germ cells to germinal vesicle (GV)-stage oocytes, can be carried out under the kidney capsule.

Expression of growth differentiation factor 9, bone morphogenetic protein 15, and anti-Müllerian hormone in cultured mouse primary follicles

“…Over the past years, the growth of rodent (Eppig & O'Brien 1996, Parrott & Skinner 1999, Klinger & de Felici 2002, Obata et al 2002, Kezele & Skinner 2003, O'Brien et al 2003, Lee et al 2004, Nilsson & Skinner 2004, Shen et al 2006a, 2006b, 2007, cattle (Wandji et al 1996, Braw-Tal & Yossefi 1997, Fortune et al 1998, and primate (Wandji et al 1997), including human , Hreinsson et al 2002, Sadeu et al 2006 primordial follicles has been investigated in vitro. In these ovarian culture experiments, primordial follicles within newborn or neonatal mouse ovaries, ovarian cortex of fetal or adult bovine ovaries as well as fetal baboon ovaries, or fetal or adult human ovaries were able to initiate growth and to develop to primary or secondary follicles.…”

“…But generally, only a few growing primary follicles progressed to the secondary stage in cattle, primates, and humans. Meiotically competent oocytes have been mainly obtained after isolation and in vitro growth of mouse primary or secondary follicles as single functional units (Spears et al 1994, Cortvrindt et al 1996, Eppig & O'Brien 1996, O'Brien et al 2003, Lenie et al 2004, Kreeger et al 2005, Shen et al 2006b, 2007. Regarding the production of mature oocytes in vitro, there is scarce proof of concept in the non-rodent species (Hirao et al 1994).…”

Complete in vitro generation of fertile oocytes from mouse primordial germ cells

“…Thus far, the ectopic oogenesis from PGCs to fertile mature oocytes has been achieved only by means of grafting PGCs into other mice to facilitate key steps (7,8). Even PGC-like cells, originally produced from mouse embryonic stem and induced pluripotent stem cells, can develop into functional oocytes after reaggregation with gonadal somatic cells and grafting beneath the bursa (9, 10).…”

Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries

“…Our results from live cell imaging and de novo folliculogenesis experiments showed that Ddx4-expressing cells in postnatal mouse ovaries do not proliferate, and they do not contribute to oocytes during de novo folliculogenesis. Thus, in contrast to the results published by Zou et al (5) tested the ability of adult mouse ovaries to support the formation of new follicles when provided with female primordial germ cells (PGCs) that have well-documented potential for germ cell development (15,16). The majority of PGCs in mice migrate to the female gonad by 12.5 d post coitum (dpc) and serve as highly potent progenitors for germ cell development (15)(16)(17)(18)(19).…”

“…Thus, in contrast to the results published by Zou et al (5) tested the ability of adult mouse ovaries to support the formation of new follicles when provided with female primordial germ cells (PGCs) that have well-documented potential for germ cell development (15,16). The majority of PGCs in mice migrate to the female gonad by 12.5 d post coitum (dpc) and serve as highly potent progenitors for germ cell development (15)(16)(17)(18)(19). These mitotically active PGCs then undergo meiosis and actively interact with support cell progenitors to form primordial follicles (18,20).…”