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

BACKGROUND: 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). METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Effect of endometrial cell-conditioned medium and platelet-rich plasma on the developmental competence of mouse preantral follicles: An in vitro study.

OBJECTIVE: The aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization. METHODS: In total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction. RESULTS: In the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups. CONCLUSION: Overall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

Comparative assessment of the efficiency of various decellularization agents for bone tissue engineering.

Bone regeneration can be possible through grafts or engineered bone replacement when bone defects are larger than the critical size. Decellularized bone extracellular matrix (ECM) is an alternative that is able to accelerate tissue regeneration, while decellularization protocols influence engineered bone quality. The objective of this study was to compare the quality of decellularized bone produced through different methods. Four decellularization methods were employed using (a) sodium lauryl ether sulfate (SLES), (b) sodium dodecyl sulfate (SDS) 0.5%, (c) SDS 1% and (d) trypsin/EDTA. All samples were then washed in triton X-100. DNA quantification, hematoxylin and eosin, and Hoechst staining showed that although DNA was depleted in all scaffolds, treatment with SLES led to a significantly lower DNA content. Glycosaminoglycan quantification, Raman confocal microscopy, alcian blue and PAS staining exhibited higher carbohydrate retention in the scaffolds treated with SLES and SDS 0.5%. Raman spectra, scanning electron microscopy and trichrom Masson staining showed more collagen content in SLES and SDS-treated scaffolds compared to trypsin/EDTA-treated scaffolds. Therefore, although trypsin/EDTA could efficiently decellularize the scaffolds, it washed out the ECM contents. Also, both MTT and attachment tests showed a significantly higher cell viability in SLES-treated scaffolds. Raman spectra revealed that while the first washing procedure did not remove SLES traces in the scaffolds, excessive washing reduced ECM contents. In conclusion, SLES and, to a lesser degree, SDS 0.5% protocols could efficiently preserve ultrastructure and ECM constituents of decellularized bone tissue and can thus be suggested as nontoxic and safe protocols for bone regeneration.

Chemoprotective effects of plasma derived from mice of different ages and genders on ovarian failure after cyclophosphamide treatment.

BACKGROUND: Premature ovarian failure is one of the major side effects of chemotherapy drugs. Blood plasma contains several factors that might lead to the repair of different tissues. OBJECTIVE: The chemoprotective effects of plasma derived from mice with different ages and genders were assessed on ovarian tissue in cyclophosphamide-treated mice. METHODS: Forty-two adult female mice were divided into six groups as follows: (A) control; (B) 0.9% sodium chloride as vehicle; (C) cyclophosphamide; (D) cyclophosphamide + young male blood plasma; (E) cyclophosphamide + old male blood plasma; (F) cyclophosphamide + young female blood plasma. Ovarian failure was induced by injecting cyclophosphamide. On the 1st day, three groups received simultaneous injections of 150 µL intraperitoneal and 70 µL intravenous plasma derived from mice of different ages and genders. Each plasma type (150 µL) was then injected intraperitoneally every other 3 days for 19 days. On day 21, the dissected ovaries were stained for stereological analysis. Also, estrogen and progesterone levels were measured. RESULTS: Cyclophosphamide had damaging effects on ovarian parameters and led to reduced hormone levels in comparison with the control group. However, treating with young female and, old male blood plasma, to a lesser degree, showed beneficial effects on the number of primordial follicles, pre-antral follicles, and granulosa cells. Also, these two treatments had protective effects on the volume of ovarian parameters as well as estrogen and progesterone levels in comparison with the cyclophosphamide group (P < 0.05). CONCLUSION: Plasma derived from mice of different ages and genders can ameliorate premature ovarian failure against the adverse effects of cyclophosphamide.

Effect of Different Concentrations of Forskolin Along with Mature Granulosa Cell Co-Culturing on Mouse Embryonic Stem Cell Differentiation into Germ-Like Cells

Background: Germ cell development processes are influenced by soluble factors and intercellular signaling events between them and the neighboring somatic cells. More insight into the molecular biology of the germ cell development from embryonic stem (ES) cells and investigation of appropriate factors, specifically those targeting differentiation processes, is of great importance. In this study, we established an in vitro model with higher ES cell differentiation rate to germ cells, using adenylate cyclase activator, forskolin. Methods: ES cells were first cultured for five days, leading to embryoid body (EB) formation. Subsequently, the EB were dissociated and cultured for an additional three days in different forskolin concentrations of 5, 20, and 50 µM, with or without granulosa cells (GC) co-culture. On the 8th day, we analyzed the expressions of 5 germ cell-specific markers using quantitative real-time-PCR technique along with cell viability assay by MTT test. Results: Our results showed that in the GC-free cultures, a 50-µM concentration of forskolin resulted in a significant increase in Mvh, Gdf9, Scp3, and Rec8 expression levels in comparison to the control. However, when the cells were co-cultured with the GCs, 20-µM concentration of forskolin could also increase the expression of those germ cell-specific marker genes. Furthermore, results from the MTT assay showed enhanced cell proliferation and survival at all three concentrations of forskolin, but 20-µM concentration was the most potent one. Conclusion: These data indicate that forskolin can stimulate differentiation and proliferation, dose-dependently; however, the influence of GCs co-culturing should not go unnoticed.

Effect of BMP4 preceded by retinoic acid and co-culturing ovarian somatic cells on differentiation of mouse embryonic stem cells into oocyte-like cells.

Bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling are the key regulators for germ cell and meiosis induction, respectively. Gonadal tissue also provides an appropriate microenvironment for oocyte differentiation in vivo. The current study aimed to determine whether mimicking in vivo niche is more efficient for oocyte differentiation from embryonic stem (ES) cells. Here, differentiation of mouse ES cells toward oocyte-like cells using embryoid body (EB) and monolayer protocols was induced in the presence (+BMP4) or absence (-BMP4) of BMP4. On day 5, each group was co-cultured with ovarian somatic cells in the presence or absence of RA (+RA or -RA) for an additional 14 days. Our results showed a significant increase in expression of meiotic markers in the +BMP4 condition in EB differentiation protocol. Further differentiation with ovarian somatic cells led to a subpopulation of oocyte-like cell formation. Compared to the controls, the +RA condition resulted in a significant elevation of the meiotic gene expression in contrast to Oct4 that significantly decreased in both protocols. In the cells pre-treated with BMP4 and then exposed to RA in the monolayer differentiation protocol, the gene expression levels of germ cell, Mvh, and maturation markers, Cx37, Zp2, and Gdf9, were also upregulated significantly. Therefore, it can be concluded that +BMP4 and +RA along with ovarian somatic cell co-culture improved the rate of in vitro oocyte differentiation.

Comparison of cell viability and embryoid body size of two embryonic stem cell lines after different exposure times to bone morphogenetic protein 4.

BACKGROUND: Activation of bone morphogenetic protein 4 (BMP4) signaling pathway in embryonic stem (ES) cells plays an important role in controlling cell proliferation, differentiation, and apoptosis. Adverse effects of BMP4 occur in a time dependent manner; however, little is known about the effect of different time exposure of this growth factor on cell number in culture media. In this study, we investigated the role of two different exposure times to BMP4 in cell viability, embryoid body (EB), size, and cavitation of ES cells. METHODS: Embryonic stem cells (R1 and B1 lines) were released from the feeder cell layers and were cultured using EBs protocol by using the hanging drop method and monolayer culture system. The cells were cultured for 5 days with 100 ng/mL BMP4 from the beginning (++BMP4) or after 48 h (+BMP4) of culture and their cell number were counted by trypan blue staining. The data were analyzed using non-parametric two-tailed Mann-Whitney test. P<0.05 was considered as significant. RESULTS: In EB culture protocol, cell number significantly decreased in +BMP4 culture condition with greater cavity size compared to the ++BMP4 condition at day 5 (P=0.009). In contrast, in monolayer culture system, there was no significant difference in the cell number between all groups (P=0.91). CONCLUSION: The results suggest that short-term exposure of BMP4 is required to promote cavitation in EBs according to lower cell number in +BMP4 condition. Different cell lines showed different behavior in cavitation formation.

Delayed BMP4 exposure increases germ cell differentiation in mouse embryonic stem cells.

Fate mapping studies have revealed that bone morphogenetic protein 4 (BMP4) signaling has a key role in segregation of primordial germ cells from proximal epiblast. Adding BMP4 to the culture media of embryonic stem (ES) cells could induce expression of germ cell markers; however, to provide a desired number of germ cells has remained a challenge. In the current study, we intended to establish an in vitro system to obtain reliable germ cells derived from ES cells. Differentiation was induced in ES cells via embryoid body (EB) and monolayer culture system. Cells were cultured with BMP4 from the beginning (++BMP4) or after 48 hours (+BMP4) of culturing for five days. The cultures were assessed for alkaline phosphatase (ALP) activity, expression of Oct4, Mvh and c-kit. In EB culture protocol, the expression of Mvh, Oct4 and ALP activity significantly increased in +BMP4 culture condition, but a significant down-regulation in the expression of germ cell markers was shown in ++BMP4 condition compared with the control group. Parallel differentiation experiments using monolayer culture system indicated the number of putative germ cells did not change. In the current study, we compared two differentiation methods (EB and monolayer) to achieve an optimal germ cell production. The EBs with a short exposure time period to BMP4, showing typical characteristics of germ cells. Therefore, our approach provides a strategy for the production of germline cells from ES cells.

A comparison of the multiple oocyte maturation gene expression patterns between the newborn and adult mouse ovary.

BACKGROUND: The interaction between follicular cells and oocyte leads to a change in gene expression involved in oocyte maturation processes. OBJECTIVE: The purpose of this study was to quantify the expression of more common genes involved in follicular growth and oocyte developmental competence. MATERIALS AND METHODS: In this experimental study, the expression of genes was evaluated with qRT-PCR assay in female BALB/c mice pups at 3-day of pre-pubertal and 8 week old virgin adult ovaries. The tissue was prepared by H&E staining for normal morphological appearance. The data were calculated with the 2-∆Ct formula and assessed using non-parametric two-tailed Mann-Whitney test. The p<0.05 was considered as significant. RESULTS: The data showed a significant increase in the level of Stra8 and GDF9 in adult compared with newborn mice ovaries (p=0.049). In contrast, a significant decrease in the level of Mvh, REC8, SCP1, SCP3, and ZP2 was observed in adult mice ovaries compared to those in the newborn mice ovaries (all p=0.049 except SCP1: p=0.046). There was no significant difference in the level of OCT4 and Cx37 expression between adult and newborn mice ovaries. CONCLUSION: The modifications in gene expression patterns coordinate the follicular developmental processes. Furthermore, the findings showed higher expression level of premeiotic gene (Stra8) and lower level of meiotic entry markers (SCP1, SCP3, and REC8) in juvenile than newborn mouse ovaries. This article extracted from Ph.D. thesis. (Nehleh Zarei fard).