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

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      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 FACS-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically-active cells that exhibit a gene expression profile consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and spontaneously generate 35–50 µm oocytes, as determined by morphology, gene expression and attainment of haploid (1 n) 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, like adult mice, possess rare mitotically-active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

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      Most cited references 55

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      Blimp1 is a critical determinant of the germ cell lineage in mice.

      Germ cell fate in mice is induced in pluripotent epiblast cells in response to signals from extraembryonic tissues. The specification of approximately 40 founder primordial germ cells and their segregation from somatic neighbours are important events in early development. We have proposed that a critical event during this specification includes repression of a somatic programme that is adopted by neighbouring cells. Here we show that Blimp1 (also known as Prdm1), a known transcriptional repressor, has a critical role in the foundation of the mouse germ cell lineage, as its disruption causes a block early in the process of primordial germ cell formation. Blimp1-deficient mutant embryos form a tight cluster of about 20 primordial germ cell-like cells, which fail to show the characteristic migration, proliferation and consistent repression of homeobox genes that normally accompany specification of primordial germ cells. Furthermore, our genetic lineage-tracing experiments indicate that the Blimp1-positive cells originating from the proximal posterior epiblast cells are indeed the lineage-restricted primordial germ cell precursors.
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        Germline stem cells and follicular renewal in the postnatal mammalian ovary.

        A basic doctrine of reproductive biology is that most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. Here we show that juvenile and adult mouse ovaries possess mitotically active germ cells that, based on rates of oocyte degeneration (atresia) and clearance, are needed to continuously replenish the follicle pool. Consistent with this, treatment of prepubertal female mice with the mitotic germ-cell toxicant busulphan eliminates the primordial follicle reserve by early adulthood without inducing atresia. Furthermore, we demonstrate cells expressing the meiotic entry marker synaptonemal complex protein 3 in juvenile and adult mouse ovaries. Wild-type ovaries grafted into transgenic female mice with ubiquitous expression of green fluorescent protein (GFP) become infiltrated with GFP-positive germ cells that form follicles. Collectively, these data establish the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary.
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          Spermatogenesis following male germ-cell transplantation.

          In the adult male, a population of diploid stem-cell spermatogonia continuously undergoes self-renewal and produces progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. We report here that stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morpholigical characteristics and produced mature spermatozoa. This methodology, besides opening new avenues of basic research into spermatogenesis and stem-cell self-renewal, may prove useful as a tool for biomedical science and biotechnology.
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            Author and article information

            Affiliations
            [1 ]Vincent Center for Reproductive Biology, MGH Vincent Department of Obstetrics and Gynecology, Massachusetts General Hospital, and Department of Obstetrics, Gynecology and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02114, USA
            [2 ]Department of Obstetrics and Gynecology, Saitama Medical Center, Saitama Medical School, Saitama 350-0495, Japan
            Author notes
            [3 ]Correspondence should be addressed to J. L.T. ( jtilly@ 123456partners.org )
            [*]

            These authors contributed equally to this work

            Journal
            9502015
            8791
            Nat Med
            Nat. Med.
            Nature medicine
            1078-8956
            1546-170X
            31 January 2012
            26 February 2012
            01 September 2012
            : 18
            : 3
            : 413-421
            22366948
            3296965
            10.1038/nm.2669
            NIHMS349611

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            Funding
            Funded by: National Institute on Aging : NIA
            Award ID: R37 AG012279-17 || AG
            Categories
            Article

            Medicine

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