Bovine ovarian stem cells differentiate into germ cells and oocyte‐like structures after culture in vitro

Abstract

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte-like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.

In Vitro Generation of Oocyte Like Cells and Their In Vivo Efficacy: How Far We have been Succeeded

“…A number of researchers have documented the presence of valuable stem cells in the ovaries that have been derived from different species including humans, porcine, mice, and bovines. [27,29,31,57]. Different approaches that have been adopted to isolate these cells include the use of ovarian cortex fragments [29,57], the scraping of the ovarian surface epithelium (OSE) [1,2], fluorescence-activated cell sorting (FACS) [5,6,23,58], magnetic-activated cell sorting (MACS) [5,33,34,59,60], and isolation on the basis of cell morphology [22,61].…”

“…A number of chemical constituents including hormones, proteins, sugars, anti-apoptotic factors, reactive oxygen species, cytokines, and growth factors are present in the FF, which directly or indirectly governs many important oocyte-specific processes [36]. Due to the presence of such vital constituents, different concentrations of FF derived from various species have been used as magical OLC differentiation inducers in many of the ovarian as well as non-ovarian stem cell sources that are derived from different organisms [7,8,11,13,14,25,[29][30][31]35,89]. In the literature, FF alone or in combination with other growth factors and cytokines has been used to induce OLCs in vitro.…”

“…In the last few decades, the in vitro development of oocyte-like cells (OLCs) with the ability to cure female reproductive disorders by neo-oogenesis and folliculogenesis has been the main focus of reproductive and stem cell scientists. Researchers have succeeded in deriving primordial germ-like cells (PGCLCs), follicle-like cells (FLCs), and OLCs, as well as in treating animal disease models by using embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and neonatal and adult ovarian stem cell sources from different species including humans [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], mice [17][18][19][20][21][22][23][24], porcine [25][26][27][28][29], bovines [30,31], and goats [32]. Ovaries are the primary source and reservoir of oocytes.…”

“…Attempts have been made to isolate cells from different parts of ovaries by using different methodologies. Researchers have succeeded in isolating cells from the ovarian surface epithelium (OSE) and the ovarian cortex, and they have also utilized whole ovaries by using mechanical and enzymatic degradation methods [1,22,23,29,31,[33][34][35]. By culturing these cells in specific media or inducing the ectopic expression of some transcriptional factors, OLCs that not only attained oocyte-like morphology but exhibited germ cell-specific marker expression both at messenger ribonucleic acid (mRNA) and protein levels have been generated [29].…”

“…During oogenesis (in vivo), a fluid-like substance enriched with a variety of molecules such as proteins, polysaccharides, steroid hormones, growth factors, reactive oxygen species, and many such valuable components is secreted by growing antral follicles that surround the developing oocyte and also helps in follicular maturation by providing a conducive microenvironment [36]. To mimic this supportive niche, many researchers have tried to enrich their differentiation media by using different concentrations (ranging from 5%-25%) of human-, bovine-, and porcine-derived follicular fluid (FF) alone or in combination with other differentiation inducers such as bone morphogenic protein (BMP) family members, retinoic acid (RA), and Activin-A, and succeeded in getting PGCLCs, FLCs or OLCs [12,29,31,37]. Stem cells have the ability to differentiate into many lineages when cultured under lineage-specific culturing conditions, for which selection of the stem cell source is equally important as the selection of the differentiation protocol.…”