Production of offspring from a germline stem cell line derived from prepubertal ovaries of germline reporter mice

Abstract

study hypothesis: We investigated whether DEAD-box polypeptide 4 (DDX4) positive cells from post-natal ovaries of germline lineage reporter mice can be isolated based on endogenously expressed fluorescent proteins and used to establish a cell line for producing offspring. what is known already: In recent years, female germline stem cells (FGSCs) have been isolated from the ovaries of post-natal mice by magnetic-activated cell sorting or fluorescence-activated cell sorting (FACS) relying on an antibody against DDX4. However, whether DDX4-positive cells from post-natal ovaries of germline lineage reporter mice can be established without using an antibody, as well as a cell line established for producing offspring, remains unknown. study design, samples/materials, methods: To obtain the expected offspring (Ddx4-Cre;mT/mG mice), Ddx4-Cre mice were crossed with mT/mG mice. In the ovaries of Ddx4-Cre;mT/mG mice, germ cells were destined to express enhanced green fluorescent protein (EGFP) while somatic cells still express tandem dimer Tomato (tdTomato). Therefore, the germ cells could be clearly distinguished from somatic cells by fluorescent proteins. Then, we investigated the pattern of fluorescent cells in the ovaries of 21-day-old Ddx4-Cre;mT/mG mice under a fluorescent microscope. Germ cells were sorted by FACS without using antibody and used to establish a FGSC line. The FGSC line was analyzed by DDX4 immunostaining, Edu (5-ethynyl-2 ′ -deoxyuridine) labeling, and RT-PCR for germ cell markers. Finally, the physiological function of the FGSC line was examined by transplanting FGSCs into the ovaries of sterilized recipients and subsequent mating.main results and the role of chance: Firstly, we have successfully isolated FGSCs from the ovaries of 21-day-old Ddx4-Cre;mT/mG mice based on endogenously expressed fluorescent proteins. FACS was used to separate the cells and 2.3% of all viable cells was EGFP-positive germ cells. Subsequently, a FGSC line was established that was doubly positive for DDX4 immunostaining and Edu labeling. The mRNA expression of several germ cell markers in this cell line, such as Ddx4, Deleted in azoospermia-like (Dazl), B lymphocyte-induced maturation protein-1 (Blimp1), Stella and Fragilis, was detected. Lastly, the FGSC line was proven to be functional under physiological conditions, as offspring were produced after transplanting FGSCs into ovaries of sterilized recipients and a subsequent mating.limitations, reasons for caution: The molecular mechanisms of proliferation and differentiation of FGSCs in vivo and in vitro still need to be elucidated.wider implications of the findings: Our results confirm that DDX4-positive cells can be separated from post-natal mouse ovaries and used to establish cell lines that are functional in producing offspring, and provide further evidence for the existence of post-natal FGSCs in mammals. The Ddx4-Cre;mT/mG mouse strain is an ideal model for the isolation, characterization and propagation of FGSCs and is a useful tool for fully elucidating the molecu...

Ovarian regeneration: The potential for stem cell contribution in the postnatal ovary to sustained endocrine function

“…Among the defining properties of OSCs are a stable karyotype and germline molecular profile following extended propagation, and, importantly, the ability to spontaneously initiate a differentiation program into oocytes following either culture in vitro or transplantation into ovarian tissue (Zou et al, 2009; Pacchiarotti et al, 2010; White et al, 2012; Ding et al, 2016). In mice, the oocytes formed from transplanted OSCs complete maturation to the metaphase-II stage of development, and can be fertilized yielding viable embryos and offspring (Zou et al, 2009; White et al, 2012; Xiong et al, 2015; Zhang and Wu, 2016). While a number of laboratories have independently successfully isolated OSCs using multiple methodologies, there remains some controversy as to the existence or biological significance of OSCs.…”

<p>Role of stem cells in fertility preservation: current insights</p>

“…21 Indeed, it was demonstrated that FGSCs isolated from mice ovaries maintained proliferative activity in vitro and led to offspring after transplantation to sterile mice. 21,[141][142][143][144][145] Their presence was also demonstrated in prepubertal rat 146 and pig 147 and adult pig 148 and human 149 ovaries. Indeed, when FGSCs isolated from adult minipig ovaries were infected by an EGFP lentivirus and injected into human ovarian cortex pieces, EGFP + oocytes were observed after 3 weeks in ovarian cortical xenografts.…”

Three-dimensional Genome Structure Reveals Distinct Chromatin Signatures of Mouse Female Germline Stem Cells During Development

“…FGSCs were isolated and cultured from the ovaries of Ddx4-Cre;mT/mG neonatal mice, as described previously 20 . After culture for at least 18 passages, the cells exhibited a characteristic morphology similar to that previously described for FGSCs 2,20 . The expression of female germline marker genes was determined by reverse-transcription polymerase chain reaction (RT-PCR).…”