Expression Of Dazl Research Articles

Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium.

In vitro gamete derivation has been proposed as an interesting strategy for treatment of infertility, improvement of genetic traits, and conservation of endangered animals. Spermatogonial stem cells (SSCs) are primary candidates for in vitro gamete derivation; however, recently, mesenchymal stem cells (MSCs) have also been proposed as candidates for germ cell (GCs) differentiation mainly due to their transdifferentiating capacity. The objective of the present study was to compare the potential for GC differentiation of bovine peripheral blood-derived MSCs (PB-MSCs) and SSCs under the effect of conditioned medium (CM) derived from Sertoli cells (SCs/CM). Samples were collected every 7 days for 21 days and analyzed for pluripotent, GC, and MSC marker expression. The absence of OCT4 and the increased (p < 0.05) expression of NANOG seems to play a role in SSC differentiation, whereas the absence of NANOG and the increased expression (p < 0.05) of OCT4 may be required for PB-MSC differentiation into GCs. SSCs cultured with SCs/CM increased (p < 0.05) the expression of PIWIL2 and DAZL, while PB-MSCs cultured under the same condition only increased (p < 0.05) the expression of DAZL. Overall, the patterns of markers expression suggest that PB-MSCs and SSCs activate different signaling pathways after exposure to SCs/CM and during differentiation into GCs.

Association of DAZL expression with risk of recurrence in clinical stage 1 testicular germ cell tumors.

523 Background: One model for testicular germ cell tumor (TGCT) formation posits that these tumors arise from a failure of germ cell determination characterized by persistent pluripotency and apoptotic escape. Risk assessment for relapse in clinical stage 1 (CS1) TGCT is dictated by clinical factors; molecular biomarkers to improve patient stratification for post-orchiectomy care are lacking. In this study, we examined the relationship between protein expression of pluripotency and apoptosis factors and risk of relapse in clinical stage 1 TGCT. Methods: Orchiectomy specimens from 73 patients treated for clinical stage 1 TGCT at one institution (27 seminoma, 46 non-seminoma) were subjected to immunohistochemistry (IHC) to assess protein expression of DAZL, POU5F1, BAK1, DDX4, NANOG, and PMAIP1. Samples were dichotomized as negative or positive for target protein expression. Patients with CS1 TGCT managed with surveillance were annotated for baseline clinical characteristics and whether they experienced relapse within two years of orchiectomy. Exact logistic regression estimated odds ratios (OR) and 95% confidence intervals were calculated for the association between protein expression and relapse. Results: At two years, 17% of patients in our cohort experienced relapse. DAZL expression (score &gt; 0) was detected in 43% of seminoma samples and 13% of embryonal carcinomas. After adjusting for histology, tumors negative for DAZL expression were more likely to recur within two years (adjusted OR for recurrence 0.08, 95% CI 0 – 0.81, p = 0.03). The direction of this relationship was consistent in seminoma when adjusted for tumor size (adjusted OR 0.14, 95% CI 0-1.83, p = 0.18) and non-seminoma after adjustment for embryonal predominance and lymphovascular invasion (adjusted OR 0.53, 95% CI 0-3.10, p = 0.30). No notable findings were observed for POU5F1, BAK1, DDX4, NANOG, and PMAIP1. Validation studies in two independent cohorts from other institutions are ongoing. Conclusions: The absence of DAZL expression by IHC is associated with a greater risk of relapse in CS1 TGCT. As DAZL promotes germ cell commitment by suppressing the pluripotency program, absence of DAZL expression may promote an oncogenic germ cell phenotype and more aggressive tumor behavior. Validation in larger patient cohorts is required to credential DAZL as a biomarker for clinical stage 1 relapse on surveillance.

OCT-4 and DAZL expression in benign and malignant breast lesions: a prospective case control study

Purpose: The impact of cancer stem cells on breast cancer tumor biology has been poorly investigated during the last years. In the present study we sought to identify the expression of OCT-4 and DAZL, two prominent stem cell biomarkers, on malignant breast lesions. Methods: A retrospective analysis of patients that underwent simple or radical mastectomy was performed. Immunohistochemical expression of OCT-4 and DAZL was sought, and real time PCR was also applied in deparaffinized tissue samples retrieved from the pathology bank of our hospital. Results: Eighteen women aged 43-84 years old (median 57 years) were included in the present study. Of those, 10 women had invasive ductal carcinoma, 4 women had in situ ductal carcinoma and 4 women had invasive lobular carcinoma. Immunohistochemistry of tissue samples did not reveal expression of OCT-4, whereas DAZL was expressed in only 3 samples. Real time PCR was available in 13 samples as the control gene (G6PD) was not expressed in 5 samples. The 13 studied breast cell samples showed positive expression for the Oct-4 gene. In the same population the expression of DAZL varied significantly with 9 samples showing complete absence of expression and 4 providing a small signal in the last cycles, indicating barely detectable expression. The expression was significant in lobular carcinoma cases compared to cases with invasive or in situ ductal carcinoma. Conclusion: The findings of our study indicate the difficulty of retrieving reliable tissue samples following the process of deparaffinization and points towards the need for prospective case control studies that will be based on fresh tissue samples for the analysis of stem cell markers. Nevertheless, it also indicates a potentially increased expression of stemness markers in lobular carcinoma samples compared to ductal cancer cases, an observation that requires further research in the future.

In-vitro generation of follicle-like structures from human germ cell-like cells derived from theca stem cell combined with ovarian somatic cells

BackgroundThe objective of this study was to induce the differentiation of human theca stem cells (hTSCs) into germ cell-like cells (hGCLCs) and assess their developmental progression following in vitro 3D culture with ovarian somatic cells within the follicle-like structures. To achieve this, the hTSCs were isolated from small antral follicles of three patients of varying ages and were then seeded in a differentiation medium for 40 days. The differentiated hGCLCs were subsequently aggregated with somatic ovarian cells (cumulus cells and hTSCs) in a ratio of 1:10 and cultured in a growth medium in a suspension culture dish. In addition to examining the morphologies, sizes, and viabilities of the differentiated hGCLCs, this study also analyzed the expression of DAZL and GDF9 proteins within the follicle-like structures.ResultsAfter 12 days, the hTSCs began to differentiate into hGCLCs, with their shapes changing from spindle-shaped to spherical. The sizes of hGCLCs increased during the differentiation period (from 25 μm to 50 μm). The survival rate of the hGCLCs after differentiation and in vitro development in primordial follicle-like structures was 54%. Unlike hTSCs, which did not express the DAZL protein, the hGCLCs and follicle-like structures successfully expressed DAZL protein (P-value < 0.05). However, hGCLCs poorly expressed the GDF9 protein. Further, the culture of hGCLCs in primordial follicle-like structures significantly increased GDF9 expression (P-value < 0.05).ConclusionIn conclusion, our study demonstrated that 3D cultures with ovarian somatic cells in follicle-like structures caused the successful differentiation of reproducible hGCLCs from hTSCs derived from three patients of different ages. Moreover, this method not only enhanced the in vitro development of hGCLCs but also presented a novel approach for co-culturing and developing in vitro oocyte like cells, ultimately leading to the production of artificial follicles.

A83-01 and DMH1 effects in the zebrafish spermatogonial niche: Unraveling the roles of TGF-β and BMP signaling in the Fsh-mediated spermatogonial fate

Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-β superfamily. On the other hand, the specific roles of TGF-β and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-β (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-β or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-β inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-β and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-β and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-β subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-β (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors.

DMRT1 regulates human germline commitment

Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.

Effects of adriamycin on cell differentiation and proliferation in rat testis

ABSTRACT Although adriamycin (ADR) is used to treat many cancers, it can be toxic to healthy organs including the testis. We investigated the effects of ADR on pluripotency in rat testis. Testicular damage was induced by either cumulative or single dose single dose administration of ADR in Wistar albino rats. Rats were divided randomly into three groups: untreated control, cumulative dose ADR group (2 mg/kg ADR every three days for 30 days) and single dose ADR group (15 mg/kg, single dose ADR). Testicular damage was evaluated and seminiferous tubule diameters were measured using light microscopy. Expression levels of Oct4, Sox2, Klf4, c-Myc, Utf1 and Dazl were assessed by immunohistochemistry and real time PCR. Serum testosterone levels were measured using ELISA assay. Histopathologic scores were lower and mean seminiferous tubule diameters were less compared to the ADR groups. Oct4, Sox2, Klf4 and Utf1 expressions were decreased significantly in spermatogenic cells of both cumulative and single dose ADR groups compared to the control group. We found that c-Myc expression in spermatogenic and Leydig cells were increased significantly in both ADR groups compared to the control group. Dazl expression was decreased in the cumulative adriamycin group compared to the control group, but increased in the single dose ADR group compared to both the control and cumulative ADR groups. Serum testosterone levels were decreased in both ADR groups compared to the control group. Our findings suggest that ADR is detrimental to regulation and maintenance of pluripotency in rat testis.

A genome-wide screen reveals new regulators of the 2-cell-like cell state.

In mammals, only the zygote and blastomeres of the early embryo are totipotent. This totipotency is mirrored in vitro by mouse '2-cell-like cells' (2CLCs), which appear at low frequency in cultures of embryonic stem cells (ESCs). Because totipotency is not completely understood, we carried out a genome-wide CRISPR knockout screen in mouse ESCs, searching for mutants that reactivate the expression of Dazl, a gene expressed in 2CLCs. Here we report the identification of four mutants that reactivate Dazl and a broader 2-cell-like signature: the E3 ubiquitin ligase adaptor SPOP, the Zinc-Finger transcription factor ZBTB14, MCM3AP, a component of the RNA processing complex TREX-2, and the lysine demethylase KDM5C. All four factors function upstream of DPPA2 and DUX, but not via p53. In addition, SPOP binds DPPA2, and KDM5C interacts with ncPRC1.6 and inhibits 2CLC gene expression in a catalytic-independent manner. These results extend our knowledge of totipotency, a key phase of organismal life.

MiR-101–5p overexpression suppresses the proliferation of goat spermatogonial stem cells by targeting EZH2

MicroRNAs (miRNAs), as post-transcriptional gene mediators, regulate the biological characteristics of spermatogonial stem cells (SSCs), including proliferation, differentiation and apoptosis. However, the potential roles and mechanisms by which miR-101–5p affected the biological characters of goat SSCs have not been fully elucidated. Herein, we reported that miR-101–5p overexpression decreased cell viability (P < 0.01), arrested cell cycle in the G1 phase (P < 0.05), and aggravated apoptosis of goat SSCs (P < 0.01) compared with negative control (NC), as determined by CCK-8 assay and flow cytometry analysis. Additionally, PCNA protein expression was attenuated by miR-101–5p overexpression (P < 0.05). Notably, the expression of SSCs specific genes Oct4 (P < 0.05), PLZF (P < 0.01) and DAZL (P < 0.01) were decreased in miR-101–5p overexpressed SSCs. Furthermore, the dual luciferase reporter assay showed that, when co-transfected with miR-101–5p mimics, the relative luciferase activity of EZH2 wide-type (WT) was inhibited (P < 0.05) compared with the transfection of EZH2 mutant (MUT). EZH2 expression was negatively correlated with miR-101–5p expression in goat SSCs. Collectively, our data implicates that miR-101–5p overexpression aggravates cell apoptosis, and suppresses cell proliferation of goat SSCs via targeting EZH2, which may impair spermatogenesis.

Efficacy of Cellular Therapy for Azoospermia in Animal Models: A Systematic Review and Meta-Analysis

This study investigated the efficacy of stem cell transplantation for azoospermia, a major cause of male infertility. We conducted a systematic meta- analysis to assess the therapeutic effectiveness of stem cell transplant, using different transplant methods, injection sites, and stem cell types, and the reliability of this approach in different animal species. PubMed, the Cochrane Library, and Embase were searched for studies published from January 2006 to February 2022 that evaluated the use of stem cell transplant to treat azoospermia. We included 18 studies and conducted the analyses using Review Manager 5.2 software. Expression of the meiosis-related genes Vasa, Scp3, and Dazl and the average hematoxylin and eosin- positive staining area were improved after stem cell transplant. Subgroup analyses by mode of transplant showed higher expression of Scp3 and Dazl in the xenotransplant group. Although subgroup analyses by injection site showed that the seminiferous tubule group showed the most significant effect on Scp3 expression, spermatogenesis and repair of damaged testis were induced in the tunica albuginea group. The testicular torsion group also induced high levels of Scp3. Another subgroup analysis by stem cell type showed that umbilical cord mesenchymal stem cells promoted the highest expression of meiosis-related genes and successfully induced spermatogenesis and the repair of damaged testis. Urine-derived stem cells, spermatogonial stem cells, and amniotic fluid-derived stem cells showed significantly therapeutic effects; however, more studies are needed for definitive conclusions. Subgroup analyses by type of azoospermia animal model indicated that the use of stem cell transplant in rat or mouse models had an obvious therapeutic effect, but no significant therapeutic effect was seen in azoospermia hamsters. The meta-analysis confirmed that stem cell transplant can effectively treat azoospermia in animal models. Xenotransplant is shown to enhance the therapeutic effects of stem cell transplant on azoospermia.

Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression.

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.

Cryopreservation of testicular cell in striped catfish (Pangasianodon hypophthalmus) and its effects on apoptosis, germ-cell specific gene expression and germ cell transplantability

Cryopreservation of testicular tissue would provide a useful tool to maintain spermatogonia, a precursor of gametes, for fish regeneration by germ cell transplantation. This study aimed to develop a cryopreservation method for spermatogonia by freezing the testicular tissue of striped catfish (Pangasianodon hypophthalmus) and testing whether cryopreserved spermatogonia exhibit transplantability. Freezing cryomedium containing L-15 medium and 1.3 M DMSO yielded the highest spermatogonia recovery rate after thawing. Supplementation with glucose and non-permeating cryoprotectants was not necessary to improve the recovery rate. We determined that an optimised equilibration time for cryopreserved testes in cryomedium included 60 min of equilibrium on ice before slow freezing (−1 °C/min), and subsequent thawing at 10 °C for 4–8 min. An Annexin V affinity assay and flow cytometry analysis were performed to identify apoptosis in cryopreserved testicular cells. Our results showed that cryoinjury of spermatogonia during cryopreservation could lead to apoptosis. Additionally, to determine the characteristics of cryopreserved spermatogonia at the molecular level, RT-qPCR was performed to quantify the expression of cxcr4b, dazl, dnd1, dnd2, nanos1, nanos2, and vasa. The results showed that compared to fresh testicular cells, frozen cells exhibited lower expression levels of cxcr4b and dazl. However, there were no significant differences in dnd1, dnd2, nanos1, and nanos2 expression levels. Although the expression level of vasa was lower in cryopreserved testes, both fresh and cryopreserved testicular cells showed positive vasa expression using in situ hybridisation. Furthermore, cryopreserved spermatogonia were transplanted into allogeniec recipient larvae and compared with fresh spermatogonia. Although the incorporation rate was lower than that of fresh spermatogonia, cryopreserved spermatogonia could be incorporated into the genital ridge of recipient larvae, demonstrating the transplantability of cryopreserved spermatogonia. Taken together, a method for cryopreservation of testicular tissue of striped catfish was optimised, and the cryopreserved testicular cells retained several spermatogenic stem cell characteristics, as well as transplantability.

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.

Male haploid cells through direct spherification

To obtain de novo male gametes capable of inducing full preimplantation blastocyst development using a novel three-dimensional (3D) culture system. Mouse embryonic stem cells (mESCs) were spherified by plunging in sodium alginate followed by calcium chloride, delineating a 3D environment that simulates the seminiferous tubule. As a control, mESCs cultured on two-dimensional plates were used. Plates and spheres containing mESCs from both methods were exposed to Activin-A, bFGF, and KSR followed by exposure to BMP4, LIF, SCF, and EGF to promote differentiation into male germ-like cells. Cells were assessed for VASA, DAZL, and BOULE on days 3 and 10. Cells were later injected into activated oocytes and monitored using time-lapse imaging on days 15, 22, 29, and 36. Control conceptuses generated using mature epididymal spermatozoa were also monitored via time-lapse imaging. On day 3, cells differentiated on plates expressed VASA at 1% and DAZL at 29%. In spheres, VASA was expressed at a rate of 15% and DAZL at a rate of 45% (P<.001). On day 10, cells differentiated on plates had VASA expression of 7%, DAZL of 23%, and BOULE of only 0.5%. Cells differentiated into spheres expressed VASA at a rate of 20%, DAZL at 43%, and BOULE at 10% (P<.001). Subsequent differentiation in spheres on day 3 exhibited a DAZL (expressed in spermatogonia) expression of 43% and a VASA (further spermatogenesis progression) expression of 15%. On day 10, DAZL and VASA expressions were reassessed and increased to 45% and 18%, respectively. BOULE, a marker expressed solely in postmeiotic spermatocytes, was expressed at 8%, whereas acrosin was expressed in spermatids at 2%. On day 15, VASA expression plateaued at 17%, BOULE peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, BOULE dropped to 2%, and acrosin remained stable at 7%. On day 36, VASA expression remained at 13%, whereas BOULE and acrosin expression decreased to 0% and 1%, respectively. The control cohort attained 88.4% fertilization and 76.9% blastocyst rates. De novo gametes achieved fertilization rates of 35.0%, 61.1%, 81.8%, and 50.0% on days 15, 22, 29, and 36, respectively. Neogametes-generated blastocyst rates were 5.0%, 16.7%, 36.4%, and 8.3% for days 15, 22, 29, and 36, respectively. Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allogeneic/xenogeneic transplantation. The decreased overall marker expression and the reduced blastocyst development indicated that intrasphere germ cell differentiation correlated with the length of mouse spermatogenesis at approximately 30 days. Future experiments will be conducted to confirm the reproducibility of our findings and the eventual generation of offspring.

Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC).

Although spermatogonial stem cells (SSC) constitute primary candidates for in vitro germ cell (GC) derivation, they are scarce and difficult to maintain in an undifferentiated state. Alternatively, mesenchymal stem cells (MSC) are also candidates for GC derivation due to their simplicity for culture and multipotential for transdifferentiation. The aim of the present study was to compare the GC differentiation potentials of bull peripheral blood-derived MSC (PB-MSC) and SSC using an in vitro 3D co-culture system with Sertoli cells (SC). Samples of PB-MSC or SSC co-cultures with SC were collected on days 0, 7, 14 and 21 and analyzed for pluripotency, GC and mesenchymal marker expression. Co-culture of PB-MSC+SC resulted in down-regulation of NANOG and up-regulation of OCT4 at day 7. In comparison, co-culture of SSC+SC resulted in consistent expression of NANOG, OCT4 and SOX2 at day 14. During co-culture, SSC+SC increased the expression of DAZL, PIWIL2, FRAGILIS and STELLA and activated the expression of STRA8, whereas co-culture of PB-MSC+SC only increased the expression of DAZL and PIWIL2. Thus, co-culture of bull PB-MSC+SC and SSC+SC in 3D SACS results in differential expression of pluripotency and GC markers, where bull SSC display a more robust GC differentiation profile compared to PB-MSC.

Expression of scp3 and dazl reveals the meiotic characteristics of the olive flounder Paralichthys olivaceus†.

Olive flounder Paralichthys olivaceus is an important cultured marine fish. We found that the meiosis marker scp3 and its intrinsic regulator dazl were mainly expressed in the gonads. During the ovarian differentiation, scp3 signal was detected first in pre-meiotic oogonia at 60-mm total length (TL) and then in primary oocytes at 80- and 100-mm TL, with a sharp increase in scp3 expression level observed at 80- and 100-mm TL. Dazl signal was detected in primordial germ cells at 30-mm TL and oogonia at 60-mm TL, but no significant change of expression was observed. During the testicular differentiation period, scp3 and dazl expression remained at low levels, and scp3 signal was weakly detected in spermatogonia at 80-mm TL, whereas dazl signal was not found. During the ovarian developmental stages, the highest expression levels of scp3 and dazl were detected at stages I and II, respectively, and strong signals of scp3 and dazl were detected in primary oocytes and oocytes at phases I and II. In the testis, the high expression of scp3 and dazl was detected at stages II-IV and II-III, respectively. Scp3 signal was weakly observed in pre-meiotic spermatogonia at stages I and II and strongly detected in primary spermatocytes at stages III-V. Dazl was detected in the nuclei of spermatogonia and spermatids at stages II-IV. Furthermore, scp3 expression in the ovary could be promoted by 17α-ethynylestradiol and tamoxifen, whereas dazl expression could be downregulated by tamoxifen.

Establishment of a Spermatogonial Stem Cell Line with Potential of Meiosis in a Hermaphroditic Fish, Epinephelus coioides.

Spermatogonial stem cells (SSCs) are unique adult stem cells capable of self-renewal and differentiation into sperm. Grouper is a protogynous hermaphroditic fish farmed widely in the tropical and subtropical seas. In this study, we established an SSC line derived from adult testis of orange-spotted grouper, Epinephelus coioides. In the presence of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), the cells could be maintained with proliferation and self-renewal over 20 months and 120 passages under in vitro culture conditions. The cells exhibited strong alkaline phosphatase activity and the characteristics of SSCs with the expression of germ cell markers, including Vasa, Dazl, and Plzf, as well as the stem cell markers Nanog, Oct4, and Ssea1. Furthermore, the cultured cells could be induced by 11-ketotestosterone treatment to highly express the meiotic markers Rec8, Sycp3, and Dmc1, and produce some spherical cells, and even sperm-like cells with a tail. The findings of this study suggested that the cultured grouper SSC line would serve as an excellent tool to study the molecular mechanisms behind SSCs self-renewal and differentiation, meiosis during spermatogenesis, and sex reversal in hermaphroditic vertebrates. Moreover, this SSC line has great application value in grouper fish aquaculture, such as germ cell transplantation, genetic manipulation, and disease research.

MiR-106b enhances human mesenchymal stem cell differentiation to spermatogonial stem cells under germ cell profile genes involved in TGF-b signaling pathways.

Mesenchymal stem cells can be differentiated into tissue-specific cells. MicroRNAs (miRNAs) regulate the translation of mRNAs involved in the growth and development of a variety of cells, including primordial germ cells (PGCs). This study evaluated male germ cell differentiation from human MSCs by miR-106b. The MSCs were obtained from human adipose tissue. The differentiation of MSCs into PGCs was accomplished by transfection of a lentiviral vector expressing miR-106b. MSCs were treated with bone morphogenic factor 4 as a control and also as a putative inducer of PGC differentiation. PGC was differentiated into spermatogonial-like cells by retinoic acid. Moreover, Dazl, Plzf, Stra8, Gfra, and Thy1 gene expressions were investigated using real-time PCR. Our results showed that Dazl, Plzf, and Stra8 genes that were treated with BMP4 and miR-106b did not show any significant difference, meaning that miR-106b, like BMP4, is able to differentiate PGC cells from MSCs. In spermatogonial-like cells, Thy1 was significantly unregulated in both the miR-106b and BMP4 groups. Our findings showed that miR-106b regulates the differentiation of MSCs into PGCs. miR-106b influences on the expression of Dazl, Plzf, and Stra8 genes in PGC and Gfra, Stra8, and Thy1 genes.

Spermatogenic differentiation of spermatogonial stem cells on three-dimensional silk nanofiber scaffold

BackgroundNano-fibrous scaffolds provide a three-dimensional matrix that guides sufficient orientation of seeded cells similar to a natural niche. In this research, we designed a silk scaffold to improve the differention of mouse spermatogonial stem cells to spermatogenic cell lines. Spermatogonial stem cells were collected from neonatal mouse (2–6 days) testes (n=60) using a two steps mechanical and enzymatic method. Cells were seeded on a silk scaffold and were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 15 % fetal bovine serum and 1000 units/ml leukemia inhibitory factor, and incubated at 32°C in a humidified atmosphere of 5% CO2 in air. SEM technique was done for confirmation of seeding cells.In this study two major groups (i.e., 2D and 3D culture groups) of 30 mice each. Isolated testicular cells from each group were cultured in the absence of silk scaffold or the presence of silk scaffold.For induction of differentiation, seeded cells on a scaffold were exposed to 1 μM and 50 ng/ml BMP-4. The specific spermatogenic genes, e.g.; VASA, DAZL, PLZF, and Piwil2, were assessed via real-time PCR and immunocytochemistry techniques. P values less than 0.05 were assumed significant. All experiments were performed at least three times.ResultsSEM analysis confirmed the homogeneity of fabricated silk scaffold and average diameter of 450 nm for nanofibers fibers. Silk scaffold induces attachment of SSCs in comparison to the monolayer group. Spermatogonia stem cell colonies were observed gradually after 1 week of culture. Electrospun scaffold supports the differentiation of SSCs to spermatogenic lines. Dates of real-time PCR showed that the expression of meiotic markers, VASA, DAZL, and Piwil2 as related to specific spermatogenic genes, had a significant upregulation in cell-seeded silk scaffold compared to the control group (P < 0.05).Immunocytochemistry founding approved the expression of specific spermatogenic markers; DAZL and PLZF were higher in the experiment group compared to the control (P < 0.05).ConclusionIt is concluded silk scaffold induces spermatogenic differentiation of mouse spermatogonial stem cells in vitro.

Overexpression of bmp4, dazl, nanos3 and sycp2 in Hu Sheep Leydig Cells Using CRISPR/dcas9 System Promoted Male Germ Cell Related Gene Expression.

Simple SummaryMale germ cell development plays a crucial role in male reproduction, and gene expression also presents an essential regulatory role in its development. Many studies have been devoted to the induction and differentiation of pluripotent stem cells into germ cells in vitro. However, the culture system for pluripotent stem cells from domestic animals is not stable, especially in sheep. Our study attempted to transdifferentiate sheep somatic cells into germ cells in vitro by the overexpression of key germ cell related genes, with the aim of perfecting the construction of germ cell research models in vitro. Therefore, we explored the expression pattern of four crucial genes, bmp4, dazl, nanos3 and sycp2, in Hu sheep testicular development, and investigated the potential efficiency of overexpression of the four candidate genes using the CRISPR/dcas9 system in Leydig cells. We revealed that the overexpression of bmp4, dazl, nanos3 and sycp2 can promote the expression of male germ cell related genes. To the best of our knowledge, this is the first study to construct an overexpression induction system using CRISPR/dcas9 technology, and to induce sheep somatic cells into germ cells in vitro.Male germ cells directly affect the reproduction of males; however, their accurate isolation and culture in vitro is extremely challenging, hindering the study of germ cell development and function. CRISPR/dcas9, as an efficient gene reprogramming system, has been verified to promote the transdifferentiation of pluripotent stem cells into male germ cells by editing target genes. In our research, we explored the expression pattern of the germ cell related genes bmp4, dazl, nanos3 and sycp2 in Hu sheep testicular development and constructed the overexpression model using the CRISPR/dcas9 system. The results indicated that four genes showed more expression in testis tissue than in other tissues, and that bmp4, dazl and sycp2 present higher expression levels in nine-month-old sheep testes than in three-month-olds, while nanos3 expressed the opposite trend (p < 0.05). In addition, the expression of four potential genes in spermatogenic cells was slightly different, but they were all expressed in sheep Leydig cells. To verify the potential roles of the four genes in the process of inducing differentiation of male germ cells, we performed cell transfection in vitro. We found that the expression of the germ cell related genes Prdm1, Prdm14, Mvh and Sox17 were significantly increased after the overexpression of the four genes in Leydig cells, and the co-transfection effect was the most significant (p < 0.05). Our results illustrate the crucial functions of bmp4, dazl, nanos3 and sycp2 in Hu sheep testis development and verified the effectiveness of the overexpression model that was constructed using the CRISPR/dcas9 system, which provided a basis for further male germ cell differentiation in vitro.