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Reconstitution of ovarian function following transplantation of primordial germ cells.

ABSTRACT: Ovarian aging occurs earlier than somatic aging. We tested the hypothesis that ovarian functions could be artificially reconstructed by transplantation of primordial germ cells (PGCs). We compared various methods for transplantation of PGCs aggregated with gonadal somatic cells and showed that reconstituted ovaries exhibited folliculogenesis after transplantation of PGCs-aggregates into either kidney capsule or ovarian bursa. Neo-oogenesis occurred early after transplantation, as evidenced by the presence of prophase I meiocytes displaying homologous pairing. Moreover, endocrine function was recovered in ovariectomized recipients, including elevated levels of AMH and estradiol. Interestingly, folliculogenesis in the reconstituted ovaries failed to sustain past four weeks. Regardless of transplantation method, follicles diminished after 45 days, accompanied by increased apoptosis, and were undetectable after two months. Meanwhile, no replicative PGCs or prophase I meiocytes could be found. Together, transplantation of PGCs can effectively reconstitute ovarian functions but for limited time. These data suggest that PGCs do not undergo self-renewal but rapidly enter meiosis following transplantation. Global activation of primordial follicles in artificial ovaries can result in further rapid loss of germ cells. Methods for maintaining self-renewal and expansion in vivo of PGCs and controlling follicle activation will be essential for continuing maintenance of the functional reconstructed ovaries.

SUBMITTER: Zeng M

PROVIDER: S-EPMC5431110 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Reconstitution of ovarian function following transplantation of primordial germ cells.

Zeng Ming M Sheng Xiaoyan X Keefe David L DL Liu Lin L

Scientific reports 20170503 1

Ovarian aging occurs earlier than somatic aging. We tested the hypothesis that ovarian functions could be artificially reconstructed by transplantation of primordial germ cells (PGCs). We compared various methods for transplantation of PGCs aggregated with gonadal somatic cells and showed that reconstituted ovaries exhibited folliculogenesis after transplantation of PGCs-aggregates into either kidney capsule or ovarian bursa. Neo-oogenesis occurred early after transplantation, as evidenced by th ...[more]

PMID: 28469243

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Project description:BACKGROUND:Premature ovarian insufficiency (POI) is a common disease in reproductive women. The pathogenesis of POI is not clear, although it is known that it involves the disorder of oocyte differentiation and development. The introduction of reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) offers a unique opportunity to study many aspects of POI from cell differentiation in vitro that could ultimately lead to novel drug development and testing to help treat the disorder. METHODS:The fibroblasts from POI patients, including fragile X syndrome, abnormal karyotype (45, X; 45, X/46, XX; 45, XO and 47, XXX), and the gene mutation (FIGLA and GDF9) were reprogrammed to pluripotency status by retroviral transduction using defined factors. The morphology, growth characteristics, gene expression profiles, epigenetic status, and in vitro and in vivo differentiation potential of the POI-1-iPSCs (from fragile X syndrome) were analyzed. Then, POI-1-iPSCs were induced to differentiation into primordial germ cells (PGCs) with DNA methyltransferase inhibitors. RESULTS:The iPSCs were successfully generated from POI patients' fibroblasts. The formed iPS clones have the same characteristics of human ESCs. POI-1-iPSCs were successfully generated with germline competence. The POI-1-iPSCs, with genotypes of fragile X syndrome, can be induced to differentiation into PGCs with high efficiency under our culture system by DNA demethylation. This study proved that disease-specific iPSC lines derived from POI patients could be generated and successfully differentiated into PGCs. CONCLUSIONS:We established some novel, systemic cell models for the studying of the pathogenesis of POI patients. Second, DNA demethylation may accelerate the induction of human PGCs from iPSCs in vitro and the conclusion needs further exploration. This represents an important step in the novel approach for the study of the pathophysiology and potential egg resource for POI patients.

Project description:Research about potential use of stem cells for the development of germ line cells in vitro had been challenged. In the present study, we reported a novel protocol consisting of cocktail growth factor addition for germ cell differentiation followed by transfection. The cells were purificated based on the expression on the cell surface of a protein. This protein is not present in normal cells of mice and does not interfere with cellular function. This cell surface marker is efficiently recognized by monoclonal antibodies. Bone marrow mesenchymal stem cells derived primordial germ like cells were differentiated to spermatogonial stem like cells by inducer cocktail including Retinoic acid (RA)+Leukemia inhibitory factor (LIF)+Basic fibroblast growth factor (bFgF). Co-culture system was used as a feeder under differentiated cells. A 400 bp fragment of spermatogonia-specific Stra-8 locus was enough to direct gene expression to the germ line stem cells. Stra8-CD4HAglo construct was used for purification of premeiotic differentiated cells. Expression of pluripotency (Pou5F1, Nanog, c-Myc) and specific germ cell (Mvh, Piwil2, Stra-8) genes in each stage were analyzed. The purified cells expressed the known molecular markers of PGC-like cells such as Mvh, Piwil2 & Stra-8. The outcomes of qPCR showed that ratio pluripotency of genes expression in selective group significantly decreased (p≤0.05) in the initial differentiation process. This results showed that ratio of Pou5F1, Nanog, c-Myc, Mvh, Piwil2 & Stra-8 expression to purified PGC-like cells were 0.41, 0.204, 1.1, 0.003, 0.184 and 2.276, respectively. Treatment of cells with RA affected up regulation of Stra-8. Although, c-Myc gene as an oncogenic gene had significantly increased (p≤0.05) at the end of differentiation stage compared to initial phase of study, this level of expression could not be tumorgenic. qPCR results of the differentiation stage showed higher expression of Stra-8 in co-culture+ cocktail and co-culture groups, Also, there was a significant difference (p≤0.05) in the expression of Pou5F1 & Nanog. Our results suggest that selection and purification of PGC-like cells based on Stra-8 as a pre-meiotic marker is a useful tool for getting in vitro spermatogonial stem cell. This method facilitates identification of safely differentiated germ cells in vitro.

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Reconstitution of ovarian function following transplantation of primordial germ cells.

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Reconstitution of ovarian function following transplantation of primordial germ cells.

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