In vitro development of mouse fetal germ cells into mature oocytes

Abstract

Little is known about the mechanisms underlying primordial follicular formation and the acquisition of competence to resume meiosis by growing oocytes. It is therefore important to establish an in vitro experimental model that allows one to study such mechanisms. Mouse follicular development has been studied in vitro over the past several years; however, no evidence has been presented showing that mature oocytes can be obtained from mouse fetal germ cells prior to the formation of primordial follicles. In this study, a method has been established to obtain mature oocytes from the mouse fetal germ cells at 16.5 days postcoitum (dpc). From the initiation of primordial follicular formation to the growth of early secondary follicles, ovarian tissues from 16.5 dpc fetal mice were cultured in vitro for 14 days. Subsequently, 678 intact secondary follicles were isolated from 182 mouse fetal ovaries and cultured for 12 days. A total of 141 oocytes inside antral follicles were matured in vitro, and 102 oocytes underwent germinal vesicle breakdown. We found that 97 oocytes were fertilized and 15 embryos were able to form morula-blastocysts. We also analyzed various genomic imprinting markers and showed that the erasure of genomic imprinting markers in the parental generation was also imposed on the oocytes that developed from fetal germ cells. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro.

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

“…Despite the presence of >100 growing oocytes per ovary, only four to six secondary follicles were successfully isolated ( Fig. 1 B and E), which is consistent with previous studies (1,11). An analysis of laminin-immunostained images (14) of the follicle basement membrane indicated that the hypoplastic follicles had incomplete laminin envelopes, unlike normal in vivo follicles, which develop a complete envelope (Fig.…”

“…However, mechanisms underlying the process are not fully elucidated. An approach to comprehensively resolve the mechanisms of oogenesis is to reconstitute the entire process of oogenesis in vitro (1,2).…”

“…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). However, many previous studies attempting in vitro oogenesis without the help of grafting have documented the occurrence of aberrant follicular formation and insufficient growth and ability of the oocytes after in vitro growth (IVG) (1,2,11). Thus, the crucial step remaining to create a connected "chain" of in vitro oogenesis is to eliminate the gap between what can be achieved using grafting and what has become possible by culture (i.e., IVG of oocytes from the primordial follicle stage) (3)(4)(5).…”

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).…”