Human GV oocytes generated by mitotically active germ cells obtained from follicular aspirates.

Xinbao Ding,Guishu Liu,Xin Ni,Changqing Wu,Ning Hui,Jian Wang,Bo Xu,Meirong Du,Xiaoming Teng,Ji Wu

doi:10.1038/srep28218

Abstract

Human female germline stem cells (FGSCs) have opened new opportunities for understanding human oogenesis, delaying menopause, treating infertility, and providing a new strategy for preserving fertility. However, the shortage of adult human ovaries tissues available impedes their future investigations and clinical applications. Here, we have established FGSC lines from scarce ovarian cortical tissues that exist in follicular aspirates (faFGSCs), which are produced and discarded in in vitro fertilization centers worldwide. The faFGSCs have characteristics of germline stem cells involved in the gene expression profile, growth characteristics, and a normal karyotype consistent with that of FGSCs obtained from ovarian cortexes surgically removed from patients (srFGSCs). Furthermore, faFGSCs have developmental potentials including spontaneous differentiation into oocytes under feeder-free conditions, communicating with granulosa cells by gap junctions and paracrine factors, entering meiosis after RA induction, as well as forming follicles after injection into human ovarian cortical tissues xenografted into adult immunodeficient female mice. Lastly, we developed a strategy guiding FGSCs differentiated into germinal vesicle (GV) stage oocytes in vitro and revealed their developmental mechanisms. Our study not only provides a new approach to obtain human FGSCs for medical treatment, but also opens several avenues to investigate human oogenesis in vitro.

Highlights

In line with previous studies[11,24], we demonstrated that DDX4 and KI67 double-positive cells presented in human ovarian cortex, suggesting that they might be FGSCs (Fig. 1A,B)
To characterize the srFGSCs, we determined the expression of DDX4, IFITM3, OCT4, STELLA, DAZL, BLIMP-1, STRA8, SYCP3, C-KIT, FIGLA, GDF9, GJA4, ZP1-3, NANOG, SOX-2 (SRY-box containing gene 2), and REX-1 in srFGSCs using reverse transcription polymerase chain reaction (RT-PCR) analysis
These results suggested that the srFGSC line was consistent with the previously reported morphology and germline lineage properties of human FGSCs obtained from healthy reproductive-age women[13]

Summary

Procedure of Sample Taken

Mice and healthy reproductive-age women using fluorescence-activated cell sorting (FACS) with the same antibody[13]. The oocytes from these follicles could grow, suggesting a new source of oocytes[30] These findings, together with the discoveries on FGSCs, lead us to hypothesize that small pieces of ovarian cortical tissues containing FGSCs could be captured from FAs. In humans, oogonia begin meiosis at approximately embryonic week 9, and form primordial follicles enclosed by pre-granulosa at approximately week 2031. Very little is known about how RA and GCs regulate the differentiation of human FGSCs. In this study, we have developed a method to isolate and culture FGSCs from scarce ovarian cortical tissues that exist in FAs (hereinafter referred to as faFGSCs) after oocyte retrieval and evaluated their capacities including, differentiation into oocytes, communicating with GCs, entering meiosis, and forming follicles associated with ovarian somatic cells. Our study provides a new approach to obtain human FGSCs for medical treatment, and opens several avenues to investigate human oogenesis in vitro

Results

Discussion

Methods