Comparative effects of retinoic acid, granulosa cells conditioned medium or forskolin in combination with granulosa cell co-culturing on mouse germ cell differentiation

Devising of an appropriate in vitro culture method for germ cells differentiation in the presence of soluble factors has attracted considerable attention, which results will provide new insight into reproductive biology. In this study, we compared the effects of forskolin, retinoic acid (RA) or granulosa cell-conditioned medium in the presence or absence of granulosa cell co-culturing on germ cell differentiation from embryonic stem cells (ESCs). Embryonic stem cells were differentiated using embryoid bodies (EBs) for 5 days, and then EB-derived cells were co-cultured with or without adult mouse granulosa cells using monolayer protocol and treated with 50 µM forskolin, 1 µM RA and 50% granulosa cell-conditioned medium for 4 days. Granulosa cell-conditioned medium significantly increased the levels of Scp3, Rec8, Mvh and Gdf9 expression in the granulosa cell co-culture method compared to untreated cells. A significant elevation of Stra8, Rec8 and Mvh was observed after treatment with RA in the absence of granulosa cells and there was no significant increase in the levels of expression of germ cell-specific genes after treatment with forskolin compared to control. Furthermore, forskolin and RA significantly increased viability and proliferation of germ-like cells, compared with granulosa cell-conditioned medium. Our study revealed that granulosa cell-conditioned medium and RA effectively can induce germ cell differentiation from ESCs, however combined application of granulosa cell-conditioned medium and co-culturing with granulosa cells had synergic effect on germ cell development in vitro as optimized protocol.


Introduction
The mouse primordial germ cells (PGCs) originate from epiblast by stimulation of extraembryonic ectoderm and migrate toward genital ridges where they continue cell proliferation [1]. The PGCs express a specific germ cell markers, Mvh, after colonization in gonadal ridges and reportedly the mutation of Mvh results in mouse infertility [2]. At this with each other and oocytes through gap junctions that permit metabolic and signaling molecules exchanges. Such a communication is critical for oocyte maturation [11,12]. In addition, the growth factors released by ovarian granulosa cells or STO mouse embryonic fibroblast cell line, into the medium (conditioned medium) activate signaling pathways leading to gamete development in vitro [13,14].
It was reported that different growth factors and morphogens could also induce germ cell differentiation from ESCs such as RA, bone morphogenetic protein 4 (BMP4), epidermal growth factor (EGF) and forskolin [7,[15][16][17]. Among these factors, RA, as a vitamin A-derived lipophilic molecule, was demonstrated to induce meiosis via up-regulation of Stra8 (stimulated by retinoic acid gene 8) expression [18]. Retinoic acid play a critical role in PGCs proliferation and can stimulate differentiation of germ-like cells from mouse ESCs at 10 − 6 M concentration [19][20][21]. Besides, previous research have shown that the initiation of meiosis was resulted from a transient increase in intracellular content of cAMP by forskolin which is an adenylate cyclase activator and dose dependently increases the cAMP in germ cells [22,23]. Forskolin and rolipram co-supplementation led to germ cell differentiation from ESCs in vitro [7]. In our previous study, we exposed the ESCs to different concentrations of forskolin. The data revealed that higher concentration (50 µM) of forskolin was optimum and more effective to induce germ cell differentiation ; compared with other forskolin concentrations [24]. Nevertheless, in vitro studies suggested that different exogenous factors are involved for derivation of germ cells from ESCs; more research are needed for optimization of in vitro germ cell differentiation mimicking in vivo physiological conditions and understanding of the essential mechanisms leading to germ-cell development [25,26].
Therefore, this study aimed to compare the effects of forskolin, RA and granulosa cells conditioned medium on the differentiation of ESCs into germ-like cells in the absence or presence of granulosa cells co-culture.

Culture of adult mouse granulosa cells
Animal experimental procedures was approved by the Ethics Committee of Shiraz University of Medical Sciences and conducted in compliance with the ARRIVE guidelines. Adult BALB/C mice were obtained from the Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
The granulosa cells were derived from mouse ovaries and used for co-culturing with embryoid bodies (EBs)-derived cells and collection of granulosa cell-conditioned medium, using Sedes and Campbell protocols [28,29]. Briefly, after gonadotropin administration, the animals were sacrificed and the ovaries were isolated from animals and trimmed of extra ovarian tissue in phosphate buffered saline (PBS). The trimmed ovaries were incubated in EGTA-BSA solution for 20 min at 37 °C, followed by centrifugation at 1200 rpm for 5 min. Then, the samples were exposed to hypertonic sucrose solution containing 0.5 M Sucrose with 1.8 mM EGTA and 0.2% BSA for 5 min at 4 °C and centrifuged at 1200 rpm for 5 min. The cumulus-oocyte complexes and granulosa cells were isolated from ovaries in DMEM/F12 (Gibco) using insulin-gauge needles under stereomicroscope and then washed three times with medium. The live cells were counted using trypan blue exclusion dye method and cultured in a DMEM/F12 supplemented with 15% FBS, 1% penicillin/streptomycin, and 2 mM L-Glutamine. Debris and oocytes were omitted after sequential washing and subculturing. Upon reaching confluence, granulosa cells were incubated with 10 µg/ml mitomycin C at 37 °C for 1 h to inactivate proliferation. Then, the granulosa cells were dissociated and transferred into gelatin-pre-coated 24-well plate at the density of 5 × 10 4 cells /well. Granulosa cellconditioned medium was also collected after 24 h cell culture and filtered to remove the cell debris.

Embryonic stem cells differentiation
The ESCs were dissociated and isolated from feeder cells. Then, the ESCs were diluted at 1 × 10 5 cells/mL in LIF-free media containing 10% charcoal-stripped FBS (Biowest). The hanging drop protocol was used for germ cell differentiation from ESCs. The undifferentiated ESCs were seeded in 20 µL of ESC suspension (2000 cells) onto a lid of 10 cm petri dishes filled with 10 mL sterile PBS. After 48 h, the spherical cell aggregates (EBs) were transferred into 3 cm low attachment plates (Corning) at the culture media containing 50 ng/mL BMP4 for 3 days. After 5 days of EB formation, EBs were trypsinized and dissociated cells were seeded into pre-gelatinated 24-well culture plate containing DMEM/F12 media (Gibco), supplemented with 10% charcoal-stripped FBS, 1% penicillin/streptomycin, 2 mM L-Glutamine, 0.1 mM nonessential amino acids, 0.1 mM β -mercaptoethanol as monolayer culture system. Germ cell differentiation from EB-differentiated cells was induced using either treatment with 1 µM RA (Sigma), 50 µM forskolin (Sigma) or 50% granulosa cell-conditioned medium for an additional 4 days. Those four groups (control, forskolin, RA or conditioned medium group) were co-cultured with or without granulosa cells at a density of 5 × 10 4 or 10 5 cells/24well, respectively. All differentiation protocols were carried out 9 days for all experimental groups, and after that, differentiated cells were collected for further analysis. Moreover, in co-culture method, granulosa cells were only cultured in basal culture medium of control for gene expression assessment.

Quantitative RT-PCR analysis
At the end of experiments, total RNA from differentiated cells was extracted by trizol reagent (Invitrogen). To avoid DNA contamination, RNA was treated with DNase I (Gibco) before cDNA synthesis. cDNA was synthesized in a reaction volume of 20 µL by RevertAid first strand cDNA synthesis Kit (Fermentas). Q RT-PCR was performed using Taq Man PCR Master Mix (Applied biosystems), containing 150 nMol of each Forward (F) and Reverse (R) primers ( Table 1).The thermal cycling parameters were primary denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 1 min. Amplification reactions were analyzed on an Applied Biosystem's ABI 7500 instrument. The relative amounts of gene expression were normalized and calculated based on β actin, as a housekeeping gene, using 2 −∆∆Ct method.

Immunocytochemistry
For germ cell differentiation induction in the absence of granulosa cells, a smear from single cells on cover glass slides (Roth) was prepared. However, for co-culture groups, granulosa cell and then dissociated EBs were grown on cover glass slides and the adherent differentiated cells were assessed. Then, after differentiation induction, the cells of each group were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS) for 15 min and non-specific binding sites were blocked in PBS solution containing 1% Triton-X-100 (Merk), 1% BSA (Sigma) and 10% goat serum (Gibco) for 30 min at room temperature. Incubation with primary antibodies including, rabbit anti-Stra8 (1:200, abcam) and rabbit anti-Mvh (1:200, Santa Cruz Biotechnology) was performed at 4ºC. After several washes, the samples were incubated with FITC-conjugated donkey anti-rabbit (Santa Cruz), 1:100 (4 µg/ml) secondary antibodies for 45 min at room temperature. They were counterstained with Hoechst 33,343 (Chemicon) for 5 min and mounted using antifade solution. All experiments were visualized under ziess fluorescent microscope and at random fields were captured at 400× magnification with selecting the auto-exposure bracketing icon.

Cell viability assay
To determine cell viability of differentiated cells, tetrazolium dye (MTT, Sigma) colorimetric assay was used. Accordingly, the cells were incubated with 500 µl of 5 mg/ ml MTT for 4 h, followed by elution of the dye with 200 µl DMSO (Sigma). Then, absorbance was measured at 595 nm wavelength by a microplate reader (Bio photometer plus Eppendorf).

Statistical analysis
Statistical analyses were performed using One-way ANOVA followed by LSD post-hoc test in SPSS 22 software for windows (SPSS Chicago USA). The graphs were plotted with prism v6.05 software (GraphPad software, SanDiego, CA, USA). Data were presented as mean ± standard deviation (SD) of triplicate experiments (n = 3). A P-value of less than 0.05 was considered to be statistically significant.
conditioned media. The expression of pre-meiotic marker, Stra8, was significantly decreased in differentiated cells cocultured with granulosa cells and treated with conditioned medium compared to the control (P < 0.05; Fig. 1B). The granulosa cells conditioned medium significantly increased the levels of meiotic markers Scp3 (P < 0.0001; Fig. 1D), Rec8 (P < 0.001; Fig. 1 F), and enhanced the germ cell

Germ cells marker expressions
The qRT-PCR for germ cells specific genes including Stra8, Scp3, Rec8, Mvh and Gdf9 was performed after treatment of EBs-derived differentiated cells with forskolin, RA and Immunohistochemistry also confirmed the gene expression data for Stra8 and Mvh. Higher intensity reactions against Mvh antibody were exhibited in the differentiated cells after treatment with RA or conditioned medium in the absence or presence of granulosa cell co-culturing, respectively (Fig. 3). Intense reaction to Stra8 antibody (Fig. 4) were also showed in RA culture condition compared with the other groups cultured in the absence of granulosa cells. However, RA supplementation had no influence on Stra8 expression like the other groups in co-culture conditions.

Morphology of differentiated cells
The morphology of differentiated cells was assessed after treatment with 50 µM forskolin, 1 µM retinoic acid and 50% conditioned medium (Fig. 5). The colonies of round cells with the size range of 10-15 µM were observed in all the groups co-cultured with granulosa cells. There were higher numbers of round cells in conditioned medium group and the round cells were morphologically considered as germlike cells (Fig. 5D). However, there were no round cells in the absence of granulosa cells while small differentiated cells were observed (Fig. 5B).

Cell viability
In the present study, cell viability and proliferation were assessed by MTT. The cells differentiated by forskolin were significantly viable and could proliferate (P < 0.05). Meanwhile, the proliferation rate and viability of differentiated specific gene Mvh in the co-culture with granulosa cells in comparison to the untreated group (P < 0.0001; Fig. 2B). In contrast, the conditioned medium significantly decreased the expression of Mvh in non-co-culture condition (P < 0.001; Fig. 2 A). The Gdf9 gene expression was significantly higher in germ cells differentiated by conditioned medium than RA and forskolin in the absence of granulosa cells (P < 0.05; Fig. 2 C). A 2-fold increase in Gdf9 was detected after treatment with conditioned medium in differentiated cells with granulosa cells co-culture (P < 0.05; Fig. 2D).
Retinoic acid significantly increased the levels of meiotic marker Stra8 in differentiated cells without granulosa cells (P < 0.0001; Fig. 1 A). However, a non-significant decrease of Stra8 expression was detected after treatment with RA in the co-culture with granulosa cells (P = 0.0502; Fig. 1B). The up-regulation of Rec8 (P < 0.0001; Fig. 1B) and Mvh (P < 0.0001; Fig. 2 A) was observed after treatment with RA and in the absence of granulosa cells co-culture. RA also enhanced the levels of Scp3 (P < 0.0001; Fig. 1D) and Rec8 (P < 0.0001; Fig. 1 F) expression with granulosa cells coculture. In RA treated group, the Rec8 expression level significantly increased in differentiated cells without granulosa cells (P < 0.0001; Fig. 1E). Conversely, RA significantly decreased the Scp3 gene expression in germ-like cells without co-culture condition (P < 0.0001; Fig. 1 C).
The results showed no significant increase in the levels of Stra8, Scp3, Rec8, Mvh and Gdf9 gene expression in differentiated cells treated with 50 µM forskolin in co-culture condition or without granulosa cells, compared with RA and conditioned medium. Gdf9 in differentiated cells after treatment with forskolin, retinoic acid or conditioned medium in the absence or presence of granulosa cell co-cultures. The data are in mean ± standard deviation (SD) (n = 3) *P < 0.05, ** P < 0.01, *** P < 0.001 these have not yet been reported. It was reported that cumulus cell-conditioned medium induces the up regulation of specific germ cell markers in germ cell-like cells differentiated from buffalo ESCs [13]. Granulosa cells-conditioned medium contains various factors released by granulosa cells into culture medium such as steroids, cytokines and growth factors such as EGF-like proteins, insulin-like growth factors and fibroblast growth factors [32,33]. These factors provide the optimal activation of various signaling pathways needed for germ cell differentiation [34]. To induce germ cell differentiation, Qing et al. co-cultured mouse EBs with ovarian granulosa cells, and found the elevation of specific germ cell markers. Conversely, no further development was observed by granulosa cell-conditioned medium [35]. Such studies lend support to our results, which demonstrated that combined effects of conditioned medium and co-culture with granulosa cells directly differentiate ESCs cell into round germ-like cells and enhance germ cell specific markers.
In our experiment, one µM RA increased the levels of germ cell -specific gene expression in differentiated cells cells significantly increased by RA (P < 0.05). Nevertheless, the results demonstrated that the effects of conditioned medium on cell viability and proliferation was not significant (Fig. 5E).

Discussion
It is known that presence of exogenous factors involves in germ cell differentiation from ESCs, proliferation and entering into meiosis [1,30]. However, studying the effect of different soluble factors in the absence or presence of feeder cells provides a great foundation for assessing the molecular and cellular mechanisms of germ cell development.
In the current study, we showed that granulosa cell-conditioned medium could increase the levels of the germ cell markers Scp3, Rec8, Mvh and Gdf9 in the cells co-cultured with granulosa cells. Some studies have confirmed the positive effects of conditioned medium or direct co-culture methods on germ cell differentiation from stem cells, separately [5,13,24,31]. However, the effect of combination of  previous studies were concluded that forskolin stimulate germ cell differentiation, there were no considerable germ cells specific gene expressions in differentiated cells by forskolin, compared with RA and conditioned medium.
In the present study, both RA and forskolin were able to significantly increase proliferation rate in the cells differentiated from EScs. RA and forskolin are able to sustain the survival and cell proliferation in mouse PGCs in the absence of somatic cells support [43]. Conversely, our results revealed that the effect of conditioned medium on cell viability and proliferation during differentiation induction was not notable. However, other studies reported that conditioned medium has a crucial role in cell survival and proliferation [46,47]. Kobayashi et al. showed that presence of growth factors into STO-condition medium is critical for primordial germ cell-like cells proliferation and expression of PGC markers [14]. As in our study, significant elevation of the expression of post-meiotic germ cell marker Gdf9 gene was found in differentiated cells treated with conditioned medium, it seems that cells in condition medium respond to differentiation signals instead to proliferation decision [48].

Conclusion
This study showed that treatment with granulosa cell-conditioned media or retinoic acid effectively accelerate germ like cells differentiation from ESCs however the combinatorial application of granulose cell-derived feeder layer and conditioned media seems to be more optimal culture condition for germ cells development in vitro. Also, forskolin and RA are potent stimulators of germ cell proliferation and viability.
Author contributions Z.M. and N.Z. performed the experiments and was involved in the collection, analysis, and interpretation of data and manuscript drafting. S.B. and N.Z. conceived the original idea and supervised the project. In addition, S.B. and N.Z. interpreted the data and revised the manuscript. All authors read and approved the final manuscript.
Funding The project was assigned as grant number 7912 in the office of the Vice Chancellor of Research Affairs, Shiraz University of Medical Sciences.
Data Availability All data generated or analyzed during this study are in the absence of granulosa cells co-culture. Nayernia et al. (2006) showed that this concentration of RA stimulates high Stra8 gene expression in germ cells differentiated from ESCs [20]. It was also revealed that RA regulates germ cell differentiation in mouse ESCs through a BMP/Smad dependent pathway in the absence of granulosa cells and RA could induce the up-regulation of germ cell markers such as Stra8, Mvh and Rec8 in differentiated cells [36]. In the present study, the level of Stra8 decreased in the presence of both RA and co-culture condition which could be explained by up-regulation of cytochrome p450 26B1 enzyme (Cyp26b1) in adult mouse ovaries which strongly degrade RA and inhibit the Stra8 gene expression [37]. In contrast, RA enhanced the up-regulation of Stra8 in ESCs derived germ cells with newborn ovarian granulosa cells coculture in the presence of FSH [15]. Our qRT-PCR analysis revealed that RA increased the level of Scp3 in co-culture condition. Chen et al. (2014) is also consistent with our findings, which shows that co-treatment of 1 µM RA and conditioned medium derived from human granulosa cells could increase the expression of SCP3 in ESCs-derived germ cells [6]. However, our results show that Scp3 exhibits lower expression after treatment with RA in the absence of granulosa cells co-culture. Jørgensen et al. (2015) found a significant increase in Stra8 expression in primordial germ cells directly treated with RA for 24 h but they reported that RA had no significant effect on SCP3 transcript levels [38]. Interestingly, a study has shown that expression levels of Scp3 increased in fetal ovary treated with retinoic acid [3]. This finding may account for essential role of granulosa cells to promote levels of Scp3 expression in differentiated cells by RA.
In addition, the results indicate that meiotic marker Rec8 was highly expressed by RA induction. Rec8 is required for the localization of late recombinational repair proteins, synapsis and sister chromatid cohesion during meiosis in spermatocytes and RA induces Rec8 expression in germ cells of postnatal testes independently of Stra8 [39,40]. It was demonstrated that 1µM RA is able to stimulate the expression of Rec8 directly in mouse primordial germ cells [41] in agreement with the present study findings.
The current study showed that the elevation of germ cell markers by forskolin treatment was not noticeable when compared with conditioned medium and RA. It is suggested that forskolin is effective in meiotic progression of germ cells in gonads. Forskolin is also known to stimulate PGCs proliferation via the robust activation of cAMP signaling [23, 42,43]. Previous studies reported that cocktail of forskolin with RA effectively induced meiosis in the differentiated germ cells from ESCs. [44,45]. Furthermore, it was shown that the combination of forskolin and rolipram leads to PGCs differentiation from mouse ESCs [7]. Although

Conflict of interest
The authors declare that they have no conflict of interest.
Ethics approval This study was approved by the Ethics Committee of Shiraz University of Medical Sciences.