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Environmental chemical (EC) exposure may be impacting male reproductive health. The translationally relevant biosolids treated pasture (BTP) sheep model was used to investigate gestational low-level EC mixture exposure on the testis of F1 male offspring. Adult rams from ewes exposed to BTP 1 month before and throughout pregnancy had more seminiferous tubules with degeneration and depletion of elongating spermatids, indicating “recovery” from previously reported testicular dysgenesis syndrome-like phenotype in neonatal and pre-pubertal BTP lambs. Expression of transcription factorsCREB1(neonatal) andBCL11AandFOXP2(pre-pubertal) were significantly higher in the BTP exposed testes, with no changes seen in the adults. IncreasedCREB1, which is crucial for testes development and regulation of steroidogenic enzymes, could be an adaptive response to gestational EC exposure to facilitate the phenotypic recovery. Overall, this demonstrates that testicular effects from gestational exposure to low-level mixtures of ECs can last into adulthood, potentially impacting fertility and fecundity.

Current declines in male reproductive health may, in part, be driven by anthropogenic environmental chemical exposure. Using the biosolid treated pasture (BTP) sheep model, this study examined the effects of gestational exposure to a translationally relevant mixture of environmental chemicals. At 8 weeks of age, ram lambs gestationally exposed to BTP were lighter than control, and their testes contained fewer germ cells and had a greater proportion of Sertoli-cell-only seminiferous tubules. Effects of biosolid exposure on the testicular transcriptome correlated with human testicular dysgenesis syndrome (TDS) patient data. Common differentially expressed genes indicated changes in apoptotic and mTOR signalling, which concurs with previous gene expression data from neonatal BTP lambs. Gene expression data and immunohistochemistry indicates increased HIF1α activation and nuclear localisation in BTP exposed animals, known to disrupt testosterone synthesis. Together, these results provide a potential mechanism for the pathogenesis of this phenotype, and TDS in humans.

Chemical approaches and small molecules are considered as an inseparable part of regenerative medicine and stem-cell-based therapies. Zinc (Zn) is a well-known trace element essential for normal spermatogenesis and has a regulatory role on the expression of several genes in different tissues of the body. This study determines and compares the effects of organic (acetate, ZnA) and inorganic Zinc (sulfate, ZnS) on the expression of a number of germ cell (GC)-specific genes in retinoic acid (RA)-treated ram bone marrow mesenchymal stem cells (BM-MSCs). First, BM-MSCs were treated with 10 μM RA for 21 days, real-time RT-PCR and immunocytochemistry (ICC) confirmed the expression of the markers in treated cells. Then, in vitro-derived GCs were treated with 1 μM of either ZnS or ZnA for one week. Treatment of in vitro-derived GCs with ZnA significantly downregulated all tested genes. ZnS treatment also induced a significant downregulation in Piwil2 and Vasa, and a moderate decrease in ITG b1 and Oct4 expression levels. Cells treated with ZnS or ZnA showed almost similar gene expression patterns. Zn treatments appeared to make in vitro-derived GCs return to a state similar to the untreated MSCs. We also studied the effects of simultaneous use of RA and one of zinc ions on BM-MSCs. The results showed that presence of organic or inorganic zinc in culture medium containing RA inhibited BM-MSCs to differentiate into GCs. This study showed a new dimension of zinc effect on stem cells which will be really helpful for establishment of novel strategies for Zn applications.

AIM Endometrial mesenchymal stem cells (eMSC) have a vital role in regeneration of endometrium during menstrual cycles. Since it has been suggested that (eMSC) likely play a role in uterine receptivity and establishment of pregnancy, we aimed to evaluate the expression levels of five most known receptivity markers-Integrin (ITG) β1, Rac1, HoxA11, ITGβ3 and Noggin-in eMSC of recurrent implantation failure (RIF) and non-RIF women. METHODS Human eMSC were isolated from menstrual blood (MB) of RIF and non-RIF women. The isolated eMSC characterized based on their morphological and behavioral characteristics, expression of MSC-specific surface CD markers and their capacity of differentiation into osteocytes and adipocytes. The expression levels of the five mentioned receptivity markers were analyzed with real time reverse transcription polymerase chain reaction. RESULTS Our findings revealed that RIF and non-RIF eMSC expressed all tested genes at different levels. ITGb1 expression in RIF eMSC was lower than its expression in non-RIF cells. On the other hand, all the other markers were expressed at higher levels in RIF eMSC than in non-RIF cells although only HOXA11 and ITG β3 showed statistically significant (P

Exposure to anthropogenic environmental chemical mixtures could be contributing to the decline in male reproductive health. This study used the biosolid treated pasture (BTP) sheep model to assess the effects of exposure to low-dose chemical mixtures. Maternal BTP exposure was associated with lower plasma testosterone concentrations, a greater proportion of Sertoli cell-only seminiferous tubules, and fewer gonocytes in the testes of neonatal offspring. Transcriptome analysis highlighted changes in testicular mTOR signalling, including lower expression of two mTOR complex components. Transcriptomic hierarchical analysis relative to the phenotypic severity demonstrated distinct differential responses to maternal BTP exposure during pregnancy. Transcriptome analysis between phenotypically normal and abnormal BTP lambs demonstrated separate responses within the cAMP and PI3K signalling pathways towards CREB. Together, the results provide a potential mechanistic explanation for adverse effects. Exposure could lower gonocyte numbers through mTOR mediated autophagy, but CREB mediated survival factors may act to increase germ cell survival.

Background Mesenchymal stem cells (MSCs), have been suggested as a potential choice for treatment of male infertility. Yet, the effects of MSCs on regeneration of germinal epithelium of seminiferous tubules and recovery of spermatogenesis have remained controversial. In this research, we have evaluated and compared the fate of autologous bone marrow (BM)-MSCs during three different periods of time- 4, 6 and 8 weeks after transplantation into the testes of busulfan-induced infertile male rats. Methods Rats BM samples were collected from tibia bone under anesthesia. The samples were directly cultured in culture medium. Isolated, characterized and purified BM-MSCs were labeled with PKH26, and transplanted into the testes of infertile rats. After 4, 6 and 8 weeks, the testes were removed and underwent histological evaluations. Results Immunohistochemical analysis showed that transplanted BM-MSCs survived in all three groups. Some of the cells homed at the germinal epithelium and expressed spermatogonia markers (Dazl and Stella). The number of homed spermatogonia-like cells in 4-week testes, was more than the 6-week testes. The 8-week testes had the least numbers of homed cells (p

This is the first report on differentiation of mouse amniotic membrane mesenchymal stem cells (AM-MSCs) into male germ cells (GCs). AM-MSCs have the multipotent differentiation capacity and can be differentiated into various cell types. In the present study, AM-MSCs were induced for differentiation into GCs. AM-MSCs were isolated from mouse embryonic membrane by enzymatic digestion. AM-MSCs were characterized with osteogenic and adipogenic differentiation test and flow cytometric analysis of some CD-markers. AM-MSCs were induced to differentiate into GCs using a creative two-step method. Passage-3 AM-MSCs were firstly treated with 25 ng/ml bone morphogenetic protein 4 (BMP4) for 5 d and in continuing with 1 μM retinoic acid (RA) for 12 d (total treatment time was 17 d). At the end of the treatment period, real-time reverse transcription (RT)-PCR was performed to evaluate the expression of GC-specific markers-Itgb1, Dazl, Stra8, Piwil2, Mvh, Oct4, and c-Kit- in the cells. Moreover, flow cytometry and immunofluorescence staining were performed to evaluate the expression of Mvh and Dazl at protein level. Real-time RT-PCR showed that most of the tested markers were upregulated in the treated AM-MSCs. Furthermore, flow cytometric and immunofluorescence analyses both revealed that a considerable part of the treated cells expressed GC-specific markers. The percentage of positive cells for Mvh and Dazl was about 23 and 46%, respectively. Our results indicated that a number of AM-MSCs successfully differentiated into the GCs. Finally, it seems that AM-MSCs would be a potential source of adult pluripotent stem cells for in vitro generation of GCs and cell-based therapies for treatment of infertility.

We designed a study to induce differentiation of Oct4-GFP (expression of Green Fluorescent Protein of oct4) embryonic stem cells (ESCs) by embryoid body (EB) culture system into germ cells (GCs) using retinoic acid (RA) and evaluated the expression level of (Fkbp6, Mov10l1, 4930432K21Rik, and Tex13) in differentiated cells. The expression levels of four GC-related genes, Oct4, Mvh, Scp3, and Stra8, was determined by quantitative real-time polymerase chain reaction (q-RT-PCR). Immunostaining and flow cytometry were used as additional tests to confirm q-RT-PCR findings. A significant increase occurred in the expression of meiotic markers and specific genes, Fkbp6 (p = 0.00), Mov10l1 (p = 0.01), and Tex13 (p = 0.00) in ESCs treated with RA (+RA) compared with the controls (-RA). Oct4 expression was decreased in all studied groups. The expression levels of 4930432K21Rik, Mvh, Stra8, and Scp3 in the +RA group was higher than that of the -RA group. Flow cytometry analysis showed that mean number of Mvh-positive cells in the +RA group was greater as compared with ESCs, -RA and EB7 groups (p = 0.00). Downregulation of Oct4 as a pluripotency factor as well as the expression of meiosis markers, this hypothesis is raised that ESCs are differentiated by RA, and have been introduced into the zygote/pachytene of first meiosis as GC-like cells.

Several studies have proposed that in vitro generation of germ cells (GCs) from stem cells can be considered a future option for infertility treatment. Mesenchymal stem cells (MSCs) have the capability to differentiate into male GCs with the use of inducers such as retinoic acid. Transforming growth factor-beta 1 (TGFb1) has been shown to play important roles in male fertility and spermatogenesis. Bone morphogenic protein 4 (BMP4) and BMP8b are also involved in the derivation of primordial GCs (PGCs) from epiblast cells. Therefore, this study aims to determine whether TGFb1, BMP4 and BMP8b can initiate transdifferentiation of MSCs into GCs in vitro and to determine the type of changes that occur in the expression of GC-specific markers. In this study, we have divided passage-3 ram bone marrow (BM)-MSCs into three main groups (BMP4, BMP8b and TGFb1) which were separately treated with 10 ng/ml TGFb1, 100 ng/ml BMP4 and 100 ng/ml BMP8b for a period of 21 days. We have evaluated the ability of these groups to differentiate into GCs by assessing expressions of GC-specific markers with reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), immunocytochemistry, morphological changes and alkaline phosphatase (ALP) activity. Our results showed that BMP4 and BMP8b induced PGCs properties in some BM-MSCs and TGFb1 formed spermatogonial stem cells (SSCs) and spermatogonia-like cells in BM-MSCs culture. The important results of this study provide the basis for additional studies to determine the exact mechanism of GCs differentiation and possibly solve the problem of infertility.

Mesenchymal stem cells (MSCs) have the capacity to differentiate into germ cells (GCs). This research, for the first time, has evaluated the fate of in vitro MSC-derived GCs generated by three different induction methods and compared them after transplantation into the testes of rams. Passage-3 ram bone marrow (BM)-MSCs were divided into three treatment groups: (1) 14-d treatment with 10 μM retinoic acid (RA; RA14), (2) 21-d treatment with 10 μM RA (RA21), and (3) 21-d treatment with 10 ng/ml transforming growth factor beta-1 (TGFb1). After confirmation of the existence of germ-like cells in the culture, the treated cells were labeled and transplanted into the testes of ram lambs. After 2 mo, we conducted histological evaluations of the rams’ testes. Results showed that in vitro-derived GCs from all treatment groups survived in the testes. Some of these GCs homed at the basement membrane of seminiferous tubules and formed colonies. The homed cells and cell colonies were similar to testicular native spermatogonia and expressed PGP9.5. TGFb1 exhibited the highest efficiency for in vitro production of GCs as well as the highest capability for homing and colony formation in the testes. RA21 was less efficient than TGFb1, particularly in colony formation. RA14 was the weakest group. No further differentiation of the transplanted GCs was observed. From our results, it could be concluded that a 21-d treatment period of BM-MSCs with TGFb1 is the most efficient method for in vitro generation of spermatogonia-like cells that survive, home, and form colonies in the testes.

Recent studies have shown that mesenchymal stem cells (MSCs), under appropriate conditions, can differentiate into cell types including germ cells (GCs). These studies also show that MSCs without any induction express some GC-specific genes innately. Moreover, one report suggests that female MSCs have a greater tendency to differentiate into female instead of male GCs. Therefore, for the first time, this study attempts to assay and determine the differences between the expression levels of some important GC-specific genes (Stra8, Vasa, Dazl, Stella, Piwil2, Oct4, Fragilis, Rnf17 and c-Kit) in male and female bone marrow (BM)-MSCs of rats. BM sampling of the rate was performed by a newly established method. We cultured rat BM samples, then characterized male and female MSCs according to their adhesion onto the culture dish, their differentiation potential into bone, cartilage and fat cells, and phenotype analysis by flow cytometry. The expression of GC-specific genes and their expression levels were evaluated with reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Our results showed that Dazl and Rnf17 did not express in the cells. The majority of examined genes, except Piwil2, expressed at almost the same levels in male and female MSCs. Piwil2 had higher expression in male MSCs which was probably related to the more prominent role of Piwil2 in the male GC development process. Male BM-MSCs appeared more prone to differentiate into male rather than female GCs. Additional research should be performed to determine the exact role of different genes in the male and female GC development process.

Sertoli cells (SCs), one of the most important components of seminiferous tubules, are vital for normal spermatogenesis and male fertility. In recent years, numerous in vitro studies have shown the potential and actual activities of SCs. However, pure SCs are necessary for various in vitro studies. In this study, we have evaluated the efficiency of the starvation method for SC purification as compared with the washing method. Seminiferous tubule-derived cells (STDCs) of rats' testes underwent two different techniques for SC purification. In the first group (washing group), the medium was changed every 3-4 d, and cells were washed twice with phosphate-buffered saline that lacked CaC12 and MgSO4 (PBS(-)) before the addition of fresh medium. In the second group (starvation), the medium was changed every 7-8 d. Primary culture (P0), passage 1 (P1), and passage 2 (P2) cells were analyzed for the expression of SC-specific genes, vimentin, Wilm's tumor 1 (WT1), germ cell gene (vasa), Leydig cell marker, 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3), and a marker of peritubular myoid cells, alpha smooth muscle actin (αSma), by reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR. Gene expression analysis showed that P0 cells expressed all tested genes except Hsd17b3. The starvation method caused significant downregulation of vasa and αSma expression in P0, P1, and P2 cells, whereas vimentin and WT1 were upregulated. In contrast, the washing method was less effective than the starvation method for the removal of germ and pretubular myoid cells (p 0.001). Totally, the results have revealed that although washing is the only common technique for elimination of contaminant cells in SC cultures, starvation has a stronger effect and is a suitable, affordable technique for SC purification. We propose that starvation is an efficient, inexpensive method that can be used for purification of SCs in animal species.

Background: Bone morphogenetic protein 4 (BMP4) is a significant signaling molecule that involves in initiating of differentiation and performs multifunctional effects on embryonic stem cells (ESCs) and embryos. Objective: The goal of the present study was to evaluate an in vitro differentiation model of mouse embryonic stem cells into germ cells, using BMP4. Materials and Methods: in this experimental study, we used Oct4-GFP mouse ESCs to form embryoid body (EB) aggregations for two days. Then, single cells from EB were cultured for four days with BMP4. Using MTT assay and gene expression levels for evaluation of Mvh and Riken by real-time RT-PCR of six concentrations, 12.5 ng/ ml BMP4 was determined as an optimized dose. Then, the expression level of Fkbp6, Mov10l1, 4930432K21Rik, Tex13, Mvh, Scp3, Stra8, Oct4 were evaluated. Flow cytometry and immunostaning were used to confirm the findings of the real-time RT-PCR. Results: In the +BMP4 group, the genes encoding Riken (p≤0.001) and Mvh (p≤0.001) were found to be increased with significant differences compared with the control group. Mov10l1 (p=0.22), Tex13 (p=0.10), Fkbp6 (p=0.90), Scp3 (p=0.61) and Stra8 (p=0.08) were up-regulated without significance differences compared with control group. Flow cytometry analysis showed that the mean number of Mvh-positive cells in the +BMP4 group was greater when compared with ESCs, -BMP4 and EB groups (p=0.03, p≤0.001, p=0.02, respectively). Conclusion: Down-regulation of Oct4, expression of germ cells genes and meiosis markers expression raise this hypothesis that ESCs were differentiated by BMP4, and may be introduced into the first meiosis as germ cell-like cells.