Germ Cell-specific Markers Research Articles

Isolation of Homogeneous Sub-populations of Spermatocytes from Mouse Testis.

Mammalian meiosis is a highly specialized cell division process, resulting in the production of genetically unique haploid cells. However, the molecular mechanisms governing meiosis remain largely unknown, primarily due to the difficulty in isolating pure sub-populations of spermatocytes. Definitive molecular, biochemical, and functional investigations of the meiosis process require the isolation of these individual homogeneous sub-populations of spermatocytes. Here, we present an approach that enables the purification of homogeneous spermatocytes from mouse testis at desired sub-stages. This approach consists of two strategic steps. The first is to synchronize spermatogenesis, aiming to minimize the diversity and complexity of testicular germ cells. The second involves utilizing mouse models with germ cell-specific fluorescent markers to differentiate the desired subtype from other cells in the testis. By employing fluorescence-activated cell sorting (FACS), this approach yields highly pure populations of spermatocytes at each sub-stage. When combined with other massively parallel sequencing techniques and in vitro cell culture methods, this approach will enhance our comprehension of the molecular mechanisms underlying mammalian meiosis and promote in vitro gametogenesis.

First study on repeatable culture of primordial germ cells from various embryonic regions with giant feeder cells in Japanese quail (Coturnix japonica)

Japanese quail (JQ, Coturnix japonica) is a farmed animal with a high economic value and has been used extensively as an avian model for research. Germline chimera production based on cryopreserved primordial germ cells (PGCs) is possible for conservation management of quail breeds as successful isolation has been reported of PGCs from their blood and gonads. However, the repeatable cultivation protocol has not been elucidated yet, which has hindered technological development. The current study characterized cultivation of pregonadal PGCs isolated from embryonic parts; embryonic blood (cPGCs), whole embryonic tissues (tPGCs), parts of tail buds (tbPGCs), and a mixture of blood and tail bud tissues (ctbPGCs). The results showed that the cultivation system required the presence of specific embryonic cells to act as a feeder for JQ-PGCs and that such a system facilitated more successful cultivation, as shown by the percentages of isolation and cultivation in tbPGCs (100%, 100%, respectively), tPGCs (60%, 55%, respectively), and ctbPGCs (60%, 30%, respectively), but not in cPGCs (0%) cultured on a mitomycin-treated JQ feeder cell-line. Once the co-culture system had been established, the PGCs could be propagated for at least 5 months. These PGCs expressed germ cell-specific markers (DAZL and CVH) and could colonize embryonic gonads. Conclusively, the isolation of pregonadal PGCs and their long-term cultivation in vitro requires a unique embryonic cell, giant cell feeder, that is indispensable for the proliferation of PGCs. Characterization of cell signaling sustaining a mutual interaction between the PGCs and the specific feeder cells will elucidate a superior environment for in vitro cultivation, as well as support the minimal transfer of used xenobiotics in chimera production.

Observation of the Expression of Dazl and Vasa Suggests that Chinese Soft-shelled Turtle Use the Inductive Mode of Primordial Germ Cell Specification

The origin, migration and differentiation of primordial germ cells (PGCs) have always been an important issue in developmental biology. At present, studies on the specification of PGCs in amniotes mainly focus on birds and mammals. Turtles are reptiles known as "living fossils", and their sex determination is influenced by both genetic and environmental factors, making them an ideal model animal for the study of vertebrate sex differentiation. However, the study of PGCs specification in turtles is very limited. Here we reported using RNA probes for the germ cell-specific markers Dazl and Vasa to examine growing oocytes, early embryos and adult ovaries in the Chinese soft-shelled turtle. The results shows that the expression of Dazl and Vasa could not be detected in the growing oocytes and early embryos, and the specification of germline begins around E7, indicating that the Chinese soft-shelled turtle do not contain germ plasm and belongs to the inductive specification.

Transcriptome analyses in infertile men reveal germ cell-specific expression and splicing patterns.

The process of spermatogenesis-when germ cells differentiate into sperm-is tightly regulated, and misregulation in gene expression is likely to be involved in the physiopathology of male infertility. The testis is one of the most transcriptionally rich tissues; nevertheless, the specific gene expression changes occurring during spermatogenesis are not fully understood. To better understand gene expression during spermatogenesis, we generated germ cell-specific whole transcriptome profiles by systematically comparing testicular transcriptomes from tissues in which spermatogenesis is arrested at successive steps of germ cell differentiation. In these comparisons, we found thousands of differentially expressed genes between successive germ cell types of infertility patients. We demonstrate our analyses' potential to identify novel highly germ cell-specific markers (TSPY4 and LUZP4 for spermatogonia; HMGB4 for round spermatids) and identified putatively misregulated genes in male infertility (RWDD2A, CCDC183, CNNM1, SERF1B). Apart from these, we found thousands of genes showing germ cell-specific isoforms (including SOX15, SPATA4, SYCP3, MKI67). Our approach and dataset can help elucidate genetic and transcriptional causes for male infertility.

Isolation of Female Germline Stem Cells from Mouse and Human Ovaries by Differential Adhesion.

Spermatogonial stem cell (SSC) counterparts known as female germline stem cells (fGSCs) were found in the mammalian ovary in 2004. Although the existence of fGSCs in the mammalian postnatal ovary is still under controversy, fGSC discovery encourages investigators to better understand the various aspects of these cells. However, their existence is not accepted by all scientists in the field because isolation of fGSCs by fluorescent activated cell sorting (FACS) has not been reproducible. In this study, we used differential adhesion to isolate and enrich fGSCs from mouse and human ovaries and subsequently cultured them in vitro. fGSCs were able to proliferate in vitro and expressed germ cell-specific markers Vasa, Dazl, Blimp1, Fragilis, Stella, and Oct4, at the protein level. Moreover, mouse and human fGSCs were, respectively, cultured for more than four months and one month in culture. Both mouse and human fGSCs maintained the expression of germ cell-specific markers over these times. In vitro cultured fGSCs spontaneously produced oocyte-like cells (OLCs) which expressed oocyte-relevant markers. Our results demonstrated that differential adhesion allows reproducible isolation of fGSCs that are able to proliferate in vitro over time. This source of fGSCs can serve as a suitable material for studying mechanisms underlying female germ cell development and function.

Dead-End (dnd) Gene Cloning and Gonad-Specific Expression Pattern in Starry Flounder (Platichthys stellatus).

Simple SummaryThe dead end (dnd) gene encodes an RNA-binding protein that plays a role for migration of primordial germ cells (PGCs) to the gonadal region during embryogenesis in vertebrates. Here, the starry flounder (Platichthys stellatus) dead end (psdnd) gene was characterized and its expression patterns were analyzed. Full-length psdnd mRNA was 1495 bp long, encoding 395 amino acids. psdnd was only expressed in gonadal tissues, we detected no psdnd expression in somatic cells. Furthermore, psdnd was strongly expressed during early embryogenesis. Our findings suggest that psdnd expression is gonad-specific and could therefore be used as a germ cell marker in starry flounder.dnd is a germline-specific maternal RNA expressed in various vertebrate classes, which encodes an RNA-binding protein that is essential for PGC migration. The purpose of this study is fundamental research about starry flounder dnd gene for germ cell marker development. In this study, we cloned and analyzed the expression levels of Platichthys stellatus dead end (psdnd) in various tissues and embryonic stages. The psdnd gene was isolated from starry flounder ovaries, cloned into a pGEM-t vector, and sequenced. Full-length of psdnd cDNA was 1495 bp long, encoding 395 amino acids. psdnd expression levels were investigated by real-time polymerase chain reaction (qRT-PCR) in various tissues and embryo developmental stages. psdnd transcripts were detected in the testes and ovaries, but not in somatic tissues. Embryonic psdnd expression levels were higher during early embryo development stages than during late embryogenesis; psdnd expression was highest at the 1 cell stage, then gradually decreased throughout the subsequent developmental stages. The spatial expression pattern was analyzed by whole-mount in situ hybridization (WISH). The psdnd transcripts migration pattern was similar with zebrafish (Danio rerio). Our results suggest that psdnd may function as a germ cell-specific marker.

Z-scores for comparative analyses of spermatogonial numbers throughout human development

To normalize age-dependent effects on standardized measures of spermatogonial quantity such as the number of spermatogonia per tubular cross-section (S/T) or fertility index. Published quantitative histologic data on human spermatogonial numbers were used to create Z-scores for reference means and tested on archived testicular tissue samples. Retrospective cohort study. The sample cohort comprised testicular samples from 24 boys with cancer diagnosis and 10 with Klinefelter syndrome, as part of the fertility preservation programs NORDFERTIL and Androprotect, as well as archived histologic samples from 35 prepubertal boys with acute lymphoblastic leukemia and 20 testicular biobank samples. None. Z-score values for S/T and fertility index on the basis of morphology and germ cell-specific markers (MAGEA4 and/or DDX4) were calculated, and the impact of cancer therapy exposure and genetic disorders on Z-score values was evaluated. The Z-scores for S/T values in the nontreated samples (-2.08 ± 2.20, n = 28) and samples treated with nonalkylating agents (-1.90 ± 2.60, n = 25) were comparable within ±3 standard deviations of the reference mean value but differed significantly from samples exposed to alkylating agents (-12.14 ± 9.20, n = 22) and from patients with Klinefelter syndrome (-11.56 ± 4.89, n = 8). The Z-scores for S/T were correlated with increasing cumulative exposure to alkylating agents (r = -0.7020). The Z-score values for S/T allow for the quantification of genetic and cancer treatment-related effects on testicular tissue stored for fertility preservation, facilitating their use for patient counseling.

Successful cryopreservation and regeneration of a partridge colored Hungarian native chicken breed using primordial germ cells

Primordial germ cells (PGCs) are the precursors of germline cells that generate sperm and ova in adults. Thus, they are promising tools for gene editing and genetic preservation, especially in avian species. In this study, we established stable male and female PGC lines from 6Hungarian indigenous chicken breeds with derivation rates ranging from 37.5 to 50 percent. We characterized the PGCs for expression of the germ cell-specific markers during prolonged culture in vitro. An in vivo colonization test was performed on PGCs from four Hungarian chicken breeds and the colonization rates were between 76 and 100%. Cryopreserved PGCs of the donor breed (Partridge color Hungarian) were injected into Black Transylvanian Naked Neck host embryos to form chimeric progeny that, after backcrossing, would permit reconstitution of the donor breed. For 24 presumptive chimeras 13 were male and 11 were female. In the course of backcrossing, 340 chicks were hatched and 17 of them (5%) were pure Partridge colored. Based on the backcrossing 1 hen and 3 roosters of the 24 presumptive chimeras (16.6%) have proven to be germline chimeras. Therefore, it was proven that the original breed can be recovered from primordial germ cells which are stored in the gene bank. To our knowledge, our study is a first that applied feeder free culturing conditions for both male and female cell lines successfully and used multiple indigenous chicken breeds to create a gene bank representing a region (Carpathian Basin).

The Germ Cell-Specific Markers ZPBP2 and PGK2 in Testicular Biopsies Can Predict the Presence as well as the Quality of Sperm in Non-obstructive Azoospermia Patients.

To assess the role of three testis-specific genes including ZPBP2, PGK2, and ACRV1 in the prediction of sperm retrieval result and quality of retrieved sperm by microdissection testicular sperm extraction (micro-TESE) in non-obstructive azoospermia (NOA) patients. This was a case-control study including 57 testicular samples of NOA patients including 32 patients with successful sperm retrieval (NOA+) and 25 patients with failed sperm retrieval (NOA-), and 9 samples of men with normal spermatogenesis in the testes as the positive control (OA). We investigated the expression of candidate genes by RT-qPCR and germ cell population patterns by DNA flow cytometry in testicular biopsy samples. The association between PGK2 expressions with the quality of retrieved spermatozoa was also evaluated. The RT-qPCR data revealed a significantly higher expression of ZPBP2 and PGK2 in the NOA+ in comparison to NOA- group (P = 0.002, and P = 0.002, respectively). Flow cytometry results revealed that the haploid cell percentage was significantly higher in NOA+ vs. NOA- group (P = 0.0001). In samples with a higher percentage of haploid cells, expression levels of ZPBP2 and PGK2 were higher (P = 0.001). The PGK2 expression was significantly associated with retrieved sperm quality (P = 0.01). Our results contribute to the search for the biomarkers for predicting the presence of testicular sperm and would be useful to avoid unnecessary multiple micro-TESE. Overall, the expression pattern of the ZPBP2 and PGK2 may be useful in predicting sperm recovery success and quality of retrieved sperm in NOA patients.

Identification of a novel germ cell marker MnTdrd from the oriental river prawn Macrobrachium nipponense.

Germ cell-specific genes play an important role in establishing the reproductive system in sexual organisms and have been used as valuable markers for studying gametogenesis and sex differentiation. Previously, we isolated a vasa transcript as a germ cell marker to trace the origin and migration of germ cells in the oriental river prawn Macrobrachium nipponense. Here, we identified a new germ cell-specific marker MnTdrd RNA and assessed its temporal and spatial expression during oogenesis and embryogenesis. MnTdrd transcripts were expressed in high abundance in unfertilized eggs and embryos at cleavage stage and then dropped significantly during late embryogenesis, suggesting that MnTdrd mRNA is maternally inherited. In situ hybridization of ovarian tissue showed that MnTdrd mRNA was initially present in the cytoplasm of previtellogenic oocyte and localized to the perinuclear region as the accumulation of yolk in vitellogenic oocyte. Whole-mount in situ hybridization of embryos showed that MnTdrd-positive signals were only localized in one blastomere until 16-cell stage. In the blastula, there were approximately 16 MnTdrd-positive blastomeres. During embryonized-zoea stage, the MnTdrd-positive cells aggregated as a cluster and migrated to the genital rudiment which would develop into primordial germ cells (PGCs). The localized expression pattern of MnTdrd transcripts resembled that of the previously identified germ cell marker vasa, supporting the preformation mode of germ cell specification. Therefore, we concluded that MnTdrd, together with vasa, is a component of the germ plasm and might have critical roles in germ cell formation and differentiation in the prawn. Thus, MnTdrd can be used as a novel germ cell marker to trace the origin and migration of germ cells.

Production of germline chimeric quails by transplantation of cryopreserved testicular cells into developing embryos

The germplasm is a resource and tool for the conservation of genetic diversity in animals, including birds. Securing germplasm is limited in most bird species due to difficulties in semen collection and germ cell isolation, lack of germ cell-specific markers, and in vitro culture systems. Here, we report the production of germline chimeric quails by transplant of cryopreserved testicular cells (TCs) into the developing embryo.The testicular germ cell properties were maintained after freeze-thaw, with no significant reduction in cell viability irrespective of storage length. Cryopreserved TCs were transferred into Hamburger Hamilton (HH) stage 14–17 quail embryos, and were demonstrated to migrate into the embryonic gonads with similar efficiency to freshly isolated TCs. Twenty of 81 recipient embryos yielded hatchlings from cryopreserved TCs and the germline transmission efficiency was similar to that of freshly isolated cells. In conclusion, cryopreserved adult quail TCs are capable of (de)differentiation into functional gametes in recipient quail gonads and can generate donor TCs-derived progenies. This system is feasible for the isolation of sufficient germplasm resources from various bird species for conservation purposes.

Derivation of oocyte-like cells from putative embryonic stem cells and parthenogenetically activated into blastocysts in goat

Germ cells are responsible for the propagation of live animals from generation to generation, but to surprise, a steep increase in infertile problems among livestock poses great threat for economic development of human race. An alternative and robust approach is essential to combat these ailments. Here, we demonstrate that goat putative embryonic stem cells (ESCs) were successfully in vitro differentiated into primordial germ cells and oocyte-like cells using bone morphogenetic protein-4 (BMP-4) and trans-retinoic acid (RA). Oocyte-like cells having distinct zonapellucida recruited adjacent somatic cells in differentiating culture to form cumulus-oocyte complexes (COCs). The putative COCs were found to express the zonapellucida specific (ZP1 and ZP2) and oocyte-specific markers. Primordial germ cell-specific markers VASA, DAZL, STELLA, and PUM1 were detected at protein and mRNA level. In addition to that, the surface architecture of these putative COCs was thoroughly visualized by the scanning electron microscope. The putative COCs were further parthenogenetically activated to develop into healthy morula, blastocysts and hatched blastocyst stage like embryos. Our findings may contribute to the fundamental understanding of mammalian germ cell biology and may provide clinical insights regarding infertility ailments.

Deletion of the mouse X-linked Prame gene causes germ cell reduction in spermatogenesis.

Preferentially expressed antigen in melanoma (PRAME) is cancer/testis antigen and a transcriptional repressor, inhibiting the signaling of retinoic acid through the retinoic acid receptor (RAR) for promoting cell proliferation and preventing cell apoptosis in cancer cells. The role of PRAME in testis and germline is unknown. We report here the generation and characterization of an X-linked Prame conditional knockout (cKO) mouse. Although fertile, the testis size (p < .01) and sperm count (p < .05) of the Prame cKO mice were significantly reduced by 12% at 4 months of age compared with the Prame floxed mice. Histological, immunofluorescence with germ cell-specific markers and terminal deoxynucleotidyl transferase dUTP nick end labeling analyses of testis cross-sections at postnatal day 7 (P7), P14, P21, P35, P120, and P365 indicated a significant increase in apoptotic germ cells at P7 and P14 and an increase in abnormal seminiferous tubules at P21 and P35. Germ cells were gradually lost resulting in two different phenotypes in the Prame cKO testes: Sertoli-cell-only for some of the affected tubules in young mice (at P35) and germ cell arrest at spermatogonia stage for other affected tubules in mature mice. Both phenotypes were a consequence of disruption in RAR signaling pathway by the depletion of Prame at a different time point during the first and subsequent rounds of spermatogenesis. The results suggest that Prame plays a minor, but important role in spermatogenesis and different paralogs in the Prame gene family may be functionally and partially redundant.

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 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 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 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.

Germ cell differentiation of mouse embryonic stem cells can be influenced by the culture medium

ABSTRACTAlthough embryonic stem (ES) cells can differentiate into germ cells, little is known about the influence of culture media on this process. We investigated the effect of two culture media on the capacity of ES cells to differentiate into germ cells using embryoid body (EB) and monolayer culture protocols. Germ cell differentiation was induced in mouse ES cells under four experimental conditions: EB/Dulbecco’s modified Eagle’s medium (EB/DMEM), EB/knockout Dulbecco’s modified Eagle’s medium (EB/KO-DMEM), monolayer/Dulbecco’s modified Eagle’s medium (monolayer/DMEM), and monolayer/knockout Dulbecco’s modified Eagle’s medium (monolayer/KO-DMEM). After incubation for 6 days, quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess expression of the germ cell markers, Mvh, Oct4, Rec8, Scp1, Scp3 and Stra8. Also, Oct4 and Mvh expressions at the protein level were assessed using immunocytochemistry; we evaluated alkaline phosphatase activity in addition to cell number and viability. Germ cell-specific marker expression was increased significantly in cells differentiated in KO-DMEM for both EB and monolayer protocols; the highest level was in cultures using the EB protocol. The highest cell proliferation rate was observed using the monolayer/KO-DMEM protocol and the lowest using the EB/DMEM protocol. Generally, KO-DMEM exhibited the greatest impact on germ cell differentiation and cell proliferation. Optimization of germ cell differentiation of ES cells requires careful selection of culture medium.

Three-dimensional decellularized amnion membrane scaffold promotes the efficiency of male germ cells generation from human induced pluripotent stem cells

Cells grow differently in conventional 2D cell culture than when they grow in the physiological microenvironment. In this study, we developed a 3D cell culture model for generating male germ cells from human iPSCs using a human decellularized amnion membrane (DAM) scaffold. To this end, human iPSCs were generated using retroviral vectors and characterized for pluripotency properties by immunofluorescence assay, flow cytometry, ALP staining, cytogenetic assay, and differentiation capacity. The iPSCs were used for investigating male germ cells differentiation efficiency in both conventional 2D culture and 3D-DAM scaffold. The expression of male germ cell markers was evaluated at day 21 of differentiation using immunofluorescence assay, flow-cytometry, and RT-qPCR. The results indicated a successful reprogramming of human foreskin fibroblast cells into pluripotent iPSCs. The reprogrammed cells were positive for pluripotency markers and differentiated into the three germ layers. During male germ cell differentiation, the cells tend to aggregate and form colony-like structures in both 2D and 3D conditions. However, significant expression of VASA, DAZL, PLZF, STELLA, and NANOS3 markers and more efficient haploid male germ cell production were observed in the 3D condition when compared to the 2D model. Considering the effect of the 3D-DAM scaffold in prompting male germ cell-specific markers and increased efficiency of germ cell differentiation in 3D culture, it appears that DAM scaffold is a useful tool for in vitro studies of human germ cell development and ultimately future clinical application.

High-resolution analysis of germ cells from men with sex chromosomal aneuploidies reveals normal transcriptome but impaired imprinting

BackgroundThe most common sex chromosomal aneuploidy in males is Klinefelter syndrome, which is characterized by at least one supernumerary X chromosome. While these men have long been considered infertile, focal spermatogenesis can be observed in some patients, and sperm can be surgically retrieved and used for artificial reproductive techniques. Although these gametes can be used for fertility treatments, little is known about the molecular biology of the germline in Klinefelter men. Specifically, it is unclear if germ cells in Klinefelter syndrome correctly establish the androgenetic DNA methylation profile and transcriptome. This is due to the low number of germ cells in the Klinefelter testes available for analysis.ResultsHere, we overcame these difficulties and successfully investigated the epigenetic and transcriptional profiles of germ cells in Klinefelter patients employing deep bisulfite sequencing and single-cell RNA sequencing. On the transcriptional level, the germ cells from Klinefelter men clustered together with the differentiation stages of normal spermatogenesis. Klinefelter germ cells showed a normal DNA methylation profile of selected germ cell-specific markers compared with spermatogonia and sperm from men with normal spermatogenesis. However, germ cells from Klinefelter patients showed variations in the DNA methylation of imprinted regions.ConclusionsThese data indicate that Klinefelter germ cells have a normal transcriptome but might present aberrant imprinting, showing impairment in germ cell development that goes beyond mere germ cell loss.

Spermatogenesis Recovery Potentials after Transplantation of Adipose Tissue-Derived Mesenchymal Stem Cells Cultured with Growth Factors in Experimental Azoospermic Mouse Models.

Objective Approximately 1% of the male population suffers from obstructive or non-obstructive azoospermia. Previous in vitro studies have successfully differentiated mesenchymal stem cells (MSCs) into germ cells. Because of immune- modulating features, safety, and simple isolation, adipose tissue-derived MSCs (AT-MSCs) are good candidates for such studies. However, low availability is the main limitation in using these cells. Different growth factors have been investigated to overcome this issue. In the present study, we aimed to comparatively assess the performance of AT-MSCs cultured under the presence or absence of three different growth factors, epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF), following transplantation in testicular torsion-detorsion mice Materials and Methods This was an experimental study in which AT-MSCs were first isolated from male Naval Medical Research Institute (NMRI) mice. Then, the mice underwent testicular torsion-detorsion surgery and received bromodeoxyuridine (BrdU)-labeled AT-MSCs into the lumen of seminiferous tubules. The transplanted cells had been cultured in different conditioned media, containing the three growth factors and without them. The expression of germ cell-specific markers was evaluated with real-time polymerase chain reaction (PCR) and western-blot. Moreover, immunohistochemical staining was used to trace the labeled cells. Results The number of transplanted AT-MSCs resided in the basement membrane of seminiferous tubules significantly increased after 8 weeks. The expression levels of Gcnf and Mvh genes in the transplanted testicles by AT-MSCs cultured in the growth factors-supplemented medium was greater than those in the control group (P<0.001 and P<0.05, respectively). The expression levels of the c-Kit and Scp3 genes did not significantly differ from the control group. ConclusionOur findings showed that the use of EGF, LIF and GDNF to culture AT-MSCs can be very helpful in terms of MSC survival and localization.

Organic and inorganic zinc show similar regulatory effects on the expression of some germ cell specific markers induced in bone marrow mesenchymal stem cells after treatment with retinoic acid

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.

Induction of Mouse Peritoneum Mesenchymal Stem Cells into Germ Cell-Like Cells Using Follicular Fluid and Cumulus Cells-Conditioned Media.

The peritoneum mesothelium lines body cavities and has the same origin as ovarian surface epithelium with probable existence of peritoneum mesenchymal stem cells (PMSCs). In the present research, PMSCs were isolated from peritoneum and differentiated into ovarian cell-like cells using human follicular fluid (HFF) and human cumulus-conditioned medium (HCCM). Anterior abdominal wall and intestinal peritoneum explants were used for cells isolation and cultured in Dulbecco's modified Eagle's medium. After passage 3, purified PMSCs were assessed for morphology, proliferation rate, and cell viability. Then, isolated PMSCs underwent two characterization procedures: (1) differentiation to mesodermal lineage and (2) expression of mesenchymal (CD90 and CD44) and epithelial cell (CK19) markers. The characterized PMSCs were differentiated into ovarian cell-like cells using 10% HFF and 50% HCCM for 21 days, and the expressions of oocyte (Zp3, Gdf9), germ cell (Ddx4, Dazl), granulosa cell (Amh), and theca cell (Lhr) markers were assessed using real-time polymerase chain reaction and immunocytofluorescence assay. Both anterior abdominal wall and intestinal peritoneum mesenchymal stem cells (AP-MSCs and IP-MSCs) showed mesenchymal characters and differentiated to adipocyte and osteocyte. AP-MSCs expressed mesenchymal- and epithelial cell-specific markers more than IP-MSCs and showed an analytically better proliferation rate. The induced AP-MSCs and IP-MSCs were expressed as germ and oocyte cell-specific markers, and this expression increased in the third week of culture. In both groups of AP-MSCs and IP-MSCs, the expressions of Gdf9, Zp3, Ddx4, Dazl, and Amh genes under just HCCM induction showed upregulation significantly on the 21st day of culture compared with day 0. But in protein synthesis of all mentioned genes, both HFF and HCCM had equal induction effect on the 21st day of culture against the 0th day. In addition, LHR was not expressed in any groups. Finally, in both characterization and differentiation procedures, the AP-MSCs respond to inducers better than IP-MSCs.