Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Yvonne A R White; Dori C Woods; Yasushi Takai; Osamu Ishihara; Hiroyuki Seki; Jonathan L Tilly

doi:10.1038/nm.2669

Research Methods · Preclinical Research

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

White YA, Woods DC, Takai Y, Ishihara O, Seki H, Tilly JL

Published February 26, 2012 Nature medicine

DOI 10.1038/nm.2669 PMID 22366948 PMC PMC3296965

RRM Academy Synopsis

Young women's ovaries held rare cells that made oocyte-like cells

Ovarian tissue from six women aged 22 to 33 held rare dividing germ cells that formed oocyte-like cells in culture. When labeled human cells were injected into human ovarian tissue grafted onto mice, follicles with labeled oocytes (egg cells) formed in 1 to 2 weeks.

Key Findings

A DDX4 antibody sorted cells 5–8 µm wide from all six women's ovarian tissue: 1.7% ± 0.6% of viable cells sorted, versus 1.5% ± 0.2% in mouse ovaries (n = 15).
Human colonies of dividing cells appeared after 4–8 weeks in culture. Colony formation efficiency without feeder cells ranged from 0.18% to 0.40%.
Cultured human cells spontaneously formed large oocyte-like cells, with formation peaking 72 h after each passage. Chromosome-content analysis found 1n, 2n and 4n populations; fibroblast controls showed only 2n and 4n.
Human ovarian tissue injected with about 1.3 × 10³ GFP-labeled human cells was grafted into NOD-SCID mice (n = 40 grafts). Three grafts examined at 7 days held 15–21 GFP-positive oocytes each.
Five mice given GFP-labeled mouse cells without chemotherapy yielded 31 cumulus-oocyte complexes. Of these, 23 fertilized, and 8 of the 23 embryos were GFP-positive.

Interpretation

This laboratory study used cultured cells, mouse ovaries and human ovarian tissue grafted onto mice. The human tissue came from six women with Gender Identity Disorder, whose ovaries were removed for sex reassignment. The authors conclude it is reasonable to regard the cells as adult human oogonial stem cells. The authors note that the human endpoints were less complete for ethical, legal and technical reasons. The senior author declares patent interest and co-founded OvaScience.

RRM Context

Restorative reproductive medicine aims to restore ovulation in the ovaries a woman has. Clinicians do this by finding and treating the conditions that disrupt it. Studies of how ovaries make and keep oocytes add to what is known about ovarian aging.

Our editorial summary of this paper, not the article's abstract.

Abstract

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

Topics

Research Methods Preclinical Research Laboratory Methods

Research Methods › Preclinical Research › Laboratory Methods

Yvonne A R White, Dori C Woods, Yasushi Takai, Osamu Ishihara, Hiroyuki Seki, Jonathan L Tilly

Y White, D Woods, Y Takai, O Ishihara, H Seki, Jon Tilly, J Tilly

PMID 22366948 22366948 DOI 10.1038/nm.2669 10.1038/nm.2669 White et al. 2012, White 2012

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Young women's ovaries held rare cells that made oocyte-like cells

Key Findings

Interpretation

RRM Context

Abstract

Topics

By this author

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