Goat Spermatogonial Stem Cells Research Articles

BLOC1S1 promotes proliferation of goat spermatogonial stem cells

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.

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.

Spliced X-box binding protein 1 (XBP1s) protects spermatogonial stem cells (SSCs) from lipopolysaccharide (LPS)-induced damage by regulating the testicular microenvironment

XBP1 is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum stress (ERS). Under stress conditions, the transcriptionally active form of XBP1 is generated by splicing of XBP1 mRNA by the ER-resident protein inositol-requiring enzyme-1α (IRE1α). This study aimed to investigate the role of XBP1 in male reproductive disorders. XBP1s-overexpressing goat spermatogonial stem cells (gSSCs) showed higher proliferative ability in vitro and in vivo. These cells also showed higher antioxidant capacity. In comparison, XBP1 knockdown significantly suppressed proliferation. Further analysis showed that XBP1 could stimulate the secretion of IL-6 from macrophages. Overall, the results indicate that XBP1s functions to enhance the proliferation ability and antioxidant capacity of gSSCs, potentially through a mechanism involving the regulation of gSSCs by macrophages.

Comparative Efficiency for in vitro Transfection of Goat Undifferentiated Spermatogonia Using Lipofectamine Reagents and Electroporation.

IntroductionSpermatogonial stem cells (SSC), also referred to as undifferentiated spermatogonia, are the germline stem cells responsible for continuous spermatogenesis throughout a male’s life. They are, therefore, an ideal target for gene editing. Previously, SSC from animal testis have been isolated and transplanted to homologous recipients resulting in the successful reestablishment of donor-derived spermatogenesis.MethodsEnhanced green fluorescent protein (eGFP) gene transfection into goat SSC was evaluated using liposomal carriers and electroporation. The cells were isolated from the prepubertal Galla goats testis cultured in serum-free defined media and transfected with the eGFP gene. Green fluorescing of SSC colonies indicated transfection.ResultsThe use of lipofectamineTM stem reagent and lipofectamineTM 2000 carriers resulted in more SSC colonies expressing the eGFP gene (25.25% and 22.25%, respectively). Electroporation resulted in 15% ± 0.54 eGFP expressing SSC colonies. Furthermore, cell viability was higher in lipofectamine transfection (55% ± 0.21) as compared to electroporation (38% ± 0.14).ConclusionThese results indicated that lipofectamine was more effective in eGFP gene transfer into SSC. The successful transient transfection points to a possibility of transfecting transgenes into male germ cells in genetic engineering programs.

Expression pattern of alkB homolog 5 in goat testis and its role in spermatogonial stem cells.

RNA N6-methyladenosine (m6A) is essential for many bioprocesses in many species, but its role in goat testis development remains elusive, especially alkB homolog 5 (ALKBH5), one of the m6A demethylases. To this end, nine healthy Haimen goats of different ages were chosen randomly to provide testes. The results showed that the expression level of ALKBH5 was increased significantly (P < 0.05) in the 9-month group compared with the 0-day and 3-month groups, and ALKBH5 was located in goat spermatocytes with the highest expression level compared with Leydig cells and Sertoli cells. Thus, pcDNA3.1-ALKBH5 was constructed to explore the influences of the ALKBH5 increase in goat spermatogonial stem cells (SSC) in vitro. The results showed that the expression level of ALKBH5 in SSC transfected with pcDNA3.1-ALKBH5 (OE_ALKBH5) was significantly increased (P < 0.001) compared with that in SSC transfected with pcDNA3.1-EGFP (EGFP). With ALKBH5 overexpression in SSC, flow cytometry analysis showed that cells at G1 phase were significantly reduced (P < 0.01), while cells at S phase significantly increased (P < 0.01), and cell apoptosis was inhibited. Accordingly, the mRNA degradation of CCND1, CCNE1, and BCL2 was suppressed with ALKBH5 overexpression in SSC after treatment with actinomycin D. Furthermore, the mRNA levels of pluripotency maintenance- and cell differentiation-associated genes were changed between the two groups. Overall, the results indicated the crucial role of ALKBH5 during Haimen goat testis development. The results of this study provide a theoretical basis and technical means for RNA methylation participating in goat testis development.

Eif2s3y Promotes the Proliferation of Spermatogonial Stem Cells by Activating ERK Signaling.

The future fertility of males with cancer may be irreversibly compromised by chemotherapy and/or radiotherapy. Spermatogonial stem cell transplantation is believed to be a way to restore fertility in men. However, the survival efficiency of transplanted cells is still low. Eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) located on the Y chromosome of male animals is a coding gene of eIF2γ which mainly functions in translation initiation. Recently, the emerging role of Eif2s3y in spermatogenesis has been emphasized in several studies. However, the underlying mechanism is still unclear. In addition, how Eif2s3y functions in large animals remains largely unknown. In this study, we obtained the CDS sequence of the Eif2s3y gene from the testis of dairy goats and found that this gene was highly expressed in the testis and was evolutionarily conserved among different species. Interestingly, overexpression of Eif2s3y promoted the proliferation of spermatogonial stem cells of dairy goats by activating the ERK signaling pathway. In animal experiments, overexpressing Eif2s3y promoted transplanted goat spermatogonial stem cells and produced more colonies after microinjection into the seminiferous tubules of infertile mice. In conclusion, our study highlights an undiscovered role of Eif2s3y in dairy goat reproduction. This finding may provide an important basis for future works regarding male spermatogenic cell restoration and represent a major advance toward surrogate sires becoming a tool for disseminating and regenerating germplasm in all mammals.

HT-2 toxin affects cell viability of goat spermatogonial stem cells through AMPK-ULK1 autophagy pathways

HT-2 toxin is widely found in moldy crops and is the major metabolite of T-2 toxin, which has been shown to exert various toxic effects in farm animals. However, little is known about the effects of HT-2 toxin on male reproduction, particularly spermatogenesis. This study aims to investigate the toxic effects of HT-2 toxin on goat spermatogonial stem cells (SSCs) and related autophagy-regulated mechanisms. Our results showed that HT-2 toxin exposure resulted in decreased cell viability and proliferation, disrupted SSCs self-renewal, and reduced germ cell-related gene expression. HT-2 toxin exposure also induced oxidative stress and cell apoptosis, as shown by ROS accumulation, increased antioxidant enzyme activity levels, decreased the mitochondrial membrane potential, and increased caspase-9 mRNA and Bcl/bax protein levels. Additionally, HT-2 toxin exposure increased the expression of the autophagy-inducing genes Atg5, Atg7 and Beclin1 and the number of autophagosomes, which indicated that HT-2 toxin induced autophagy in the goat SSCs. Moreover, we also examined a possible mechanism by which HT-2 toxin exposure induced higher expression of AMPK, mTOR and ULK at both the mRNA and protein levels. our results indicated that HT-2 toxin caused apoptosis and autophagy by activating AMPK-mTOR-ULK1 pathway, which further affected SSCs viability.

Effect of stage of puberty on culture characteristics of goat spermatogonial stem cells

The aim of experiment was to study the culture characteristics of spermatogonial stem cells (SSCs) obtained from pre-and post-pubertal goat testes. Six animals each of two age groups (3-6 months and 1.0-1.5 years) were included in the study. The testes were collected from slaughtered animals and after washing, morpho-biometric characters such as weight, length, mid circumference and volume of both the left and right testes were recorded. The isolated SSCs were enriched by double filtration through 80 and 60 μm nylon mesh filters. The filtrate was cultured overnight with SSC medium Dulbecco's modified eagle's medium / HamF-12 medium. The colonies were also characterized by alkaline phosphatase activity and demonstration of specific markers octamer binding transcription factor and protein gene product.All the morpho-biometrical parameters of left and right testes, except density, were significantly (P<0.01) different between the groups. The total number of SSCs isolated from pre-pubertal testes (6.85×106 cells/ml) was significantly (P<0.05) higher than post-pubertal testes (2.40×106 cells/ml). The colonies of SSCs inpre-pubertal culture appeared within seven days of culture whereas in post-pubertal SSCs, colonies appeared after 14 days of culture. The results demonstrated that the SSCs in prepubertal goat testes were higher in number, grew faster and formed colonies at least one week earlier compared to the SSCs of post-pubertal testes. Thus, the testes of pre-pubertal goats were a better source to study the physiological and regulatory mechanisms of SSCs.

Melatonin Protects Goat Spermatogonial Stem Cells against Oxidative Damage during Cryopreservation by Improving Antioxidant Capacity and Inhibiting Mitochondrial Apoptosis Pathway

Spermatogonial stem cells (SSCs) are the only adult stem cells that pass genes to the next generation and can be used in assisted reproductive technology and stem cell therapy. SSC cryopreservation is an important method for the preservation of immature male fertility. However, freezing increases the production of intracellular reactive oxygen species (ROS) and causes oxidative damage to SSCs. The aim of this study was to investigate the effect of melatonin on goat SSCs during cryopreservation and to explore its protective mechanism. We obtained SSCs from dairy goat testes by two-step enzymatic digestion and differential plating. The SSCs were cryopreserved with freezing media containing different melatonin concentrations. The results showed that 10−6 M of melatonin increased significantly the viability, total antioxidant capacity (T-AOC), and mitochondrial membrane potential of frozen-thawed SSCs, while it reduced significantly the ROS level and malondialdehyde (MDA) content (P < 0.05). Further analysis was performed by western blotting, flow cytometry, and transmission electron microscopy (TEM). Melatonin improved significantly the enzyme activity and protein expression of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (P < 0.05), thereby activating the antioxidant defense system of SSCs. Furthermore, melatonin inhibited significantly the expression of proapoptotic protein (Bax) and increased the expression of antiapoptotic proteins (Bcl-2 and Bcl-XL) (P < 0.05). The mitochondrial apoptosis pathway analysis showed that the addition of melatonin reduced significantly the mitochondrial swelling and vacuolation, and inhibited the release of cytochrome C from mitochondria into the cytoplasm, thereby preventing the activation of caspase-3 (P < 0.05) and inhibiting SSC apoptosis. In addition, melatonin reduced significantly the autophagosome formation and regulated the expression of autophagy-related proteins (LC3-I, LC3-II, P62, Beclin1, and ATG7) (P < 0.05), thereby reversing the freeze-induced excessive autophagy. In summary, melatonin protected goat SSCs during cryopreservation via antioxidant, antiapoptotic, and autophagic regulation.

PAX7 promotes CD49f-positive dairy goat spermatogonial stem cells' self-renewal.

Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs' self-renewal regulation, especially in dairy goats. We isolated and enriched the CD49f-positive and negative dairy goat testicular cells by magnetic-activated cell sorting strategies. The RNA- sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f-positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When thePAX7 gene was overexpressed in dairy goat immortal mGSCs-I-SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f+ spermatogonia, and PAX7 can maintain the self-renewal of CD49f-positive SSCs.

EZH2 expression and its role in spermatogonial stem cell self-renewal in goats

Enhancer of zeste homolog 2 (EZH2) is a histone H3 lysine 27 (H3K27) methyltransferase that plays vital roles in mouse spermatogenesis. However, the expression pattern and role of EZH2 in goat spermatogonial stem cells (SSCs) is unknown. In the present study, we investigated EZH2 expression in the testis of postpubertal goats and its effect on the biological characteristics of goat SSCs. We found that EZH2 mRNA (P < 0.01) and protein (P < 0.05) expression was increased in the testes of postpubertal goats compared to that of prepubertal goats. Moreover, EZH2 was more highly expressed in goat SSCs than in Leydig cells (P < 0.01) and Sertoli cells (P < 0.01) as determined by qPCR, Western blot, and immunofluorescence. Compared to a negative control (NC), cell proliferation (P < 0.01) and viability (P < 0.01) were decreased in SSCs in which EZH2 was knocked down, and the G2/M phase of the cell cycle was blocked (P < 0.01), as determined by Edu staining, CCK-8 assay, and flow cytometry analysis. Additionally, the expression of CASP3, CASP9, and BAX was significantly increased (P < 0.01) while BCL2 expression was decreased (P < 0.01) in EZH2 knockdown SSCs. Notably, the expression of GDNF, a SSCs marker gene, and DAZL, a spermatogenesis-related gene, were significantly decreased (P < 0.01) while GFRA1 expression was significantly up-regulated (P < 0.01) in EZH2 knockdown SSCs. Our data suggest that EZH2 plays a pivotal role in the self-renewal of goat SSCs, and knockdown of EZH2 might impair spermatogenesis in goats.

Platelet-derived growth factor-BB and epidermal growth factor promote dairy goat spermatogonial stem cells proliferation via Ras/ERK1/2 signaling pathway

Spermatogonial stem cells (SSCs) have been used for the production of transgenic animals and for the recovery of male fertility. However, the proliferation of SSCs in vitro is still immature, and the mechanisms and pathways involved in the proliferation of SSCs are not clear. Here, the effects of platelet-derived growth factor-BB (PDGF-BB) and epidermal growth factor (EGF) on the proliferation of dairy goat SSCs in vitro were detected. The results showed that 20 ng/ml PDGF-BB or 25 ng/ml EGF was the optimum concentration, and that the BCL2 in the experimental groups was significantly higher than that in the control (P < 0.05), while BAX and BAD were dramatically downregulated (P < 0.05). The pERK1/2 in the experimental groups was about 3–5 times higher than that in the control. After the specific MEK1/2 inhibitor was added, BCL2 was reduced significantly (P < 0.001), while BAX and BAD were upregulated (P < 0.001). The expression of pERK1/2 decreased by 10%–30%. We speculated that these two growth factors may be mediated through the Ras/ERK1/2 signaling pathway to regulate the expression of pERK1/2 protein, and thus enhance the resistance of SSCs to apoptosis. However, further studies are needed to verify this hypothesis.

Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions.

In the present study, we used a serum-free culture media to propagate goat putative spermatogonial stem cells (SSCs) and evaluated the effect of crucial growth factors on relative expression of some SSC markers and self-renewal related genes. The enriched SSCs were cultured on a homologous Sertoli cell feeder layer in KO-DMEM supplemented with 10% KOSR. Putative SSC colonies emerged between day 6 and 10 which were then characterized by the expression of numerous spermatogonial and pluripotency related markers. After 15 days of subculture, the relative mRNA expression study revealed that 40 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of BCL6B, ID4, PLZF, and UCHL1. Moreover, the supplementation of GDNF + bFGF up-regulated the expression of PLZF and BCL6B. UCHL1 expression was higher after addition of GDNF + LIF while, THY1 overexpressed in response to the addition of GDNF + CSF1. These results demonstrated that the goat SSCs were efficiently propagated using a KOSR based serum-free media and the growth factor supplementation markedly influences their gene expression profile.

180 Increasing GfrA1-Positive Spermatogonial Stem Cell Population of Goat

Spermatogonial stem cells (SSC) form the basis of spermatogenesis and continuous fertility in male. Their meagre population in the testis is a hindrance in the in vitro study of biological activity of these cells. The objective of the present study was to isolate and characterise goat SSC and increase their number during in vitro culture by different methods. Two goat testes (3 to 4 months of age) were collected from the slaughterhouse and transported to the laboratory. The testes were washed and seminiferous tubules were collected and minced in the laminar flow hood. The seminiferous tubules were washed twice with PBS to remove spermatozoa and subjected to double enzymatic digestion (collagenase, 1 mg mL−1, hyaluronidase, 1 mg mL−1, trypsin, 0.05%, and DNaseI, 10 µg mL−1 for 45 min and second digestion with same set of enzymes except trypsin for 30 min). The isolated cells were filtered sequentially through nylon mesh filters of pore size 70 and 40 µm. The cells were plated on DSA-lectin coated dishes for 4 h and the unattached cells were cultured on a Sertoli cell feeder layer prepared by treating with mitomycin-C for 3 h. The cells were cultured in DMEM/F-12 supplemented with human recombinant growth factors (glial cell-derived neurotrpic factor, 10 ng mL−1, fibroblast growth factor FGF, 10 ng mL−1, epidermal growth factor, 20 ng mL−1), 10% fetal bovine serum, and antibiotics. The expression of pluripotency markers (Oct4, Nanog, Sox2) and SSC-specific markers (Thy1, GfrA1, and Uchl1) in the SSC colonies was determined by RT-PCR and immunostaining, after in vitro culture of 3 weeks. The SSC population was enhanced by differential plating, Percoll density gradient (on Day 1) and SSC passaging (by passaging SSC colonies on Day 20). The cells were tagged with GfrA1 antibody and their population was tested by flow cytometry. The SSC colonies started appearing after 7 days and continued to grow in size and number until 3 weeks. The SSC colonies were positive for the pluripotency markers Oct4, Nanog, and Sox2 by RT-PCR and immunostaining. The SSC were also positive for the SSC-specific markers Thy1, GfrA1, and Uchl1 by RT-PCR and immunostaining. Flow cytometry showed that the GfrA1-positive population in the SSC enriched by the differential plating was 11.23%, Percoll density gradient was 23.57%, and by passaging of SSC colonies, after picking and trypsinising with 0.05% trypsin, was 91.23%. In vitro culture of the SSC enriched by these methods also revealed that the number of SSC colonies appearing in the cells enriched by passaging was higher than the other methods. From the results of present study, we conclude that SSC are positive for markers of pluripotency and SSC-specific markers. The SSC population can be enhanced to a very high level following SSC passaging, which is an inexpensive method and does not require expensive instruments like fluorescence- or magnetic-activated cell sorting.

Double sex and mab-3 related transcription factor 1 regulates differentiation and proliferation in dairy goat male germline stem cells.

The protein encoded by double sex and mab-3 related transcription factor 1 (Dmrt1) gene contains a double sex/mab-3 domain, which was considered as one of the most conservative structures in sex determination. However, its effect on spermatogenesis of dairy goat spermatogonial stem cells (SSCs) remains to be clarified. For the first time, the roles of Dmrt1 in spermatogenesis of livestock are highlighted. Here, we investigated the expression pattern of Dmrt1 in the testes of dairy goats. Dmrt1 primarily located in undifferentiated SSCs. Moreover, Dmrt1 enhanced differentiation and proliferation of mGSCs. On the contrary, the level of meiosis was down-regulated, as Dmrt1 determines whether SSCs undergo mitosis and spermatogonial differentiation or meiosis. In the busulfan-treated mice testes, Dmrt1 repair germ cell damage was emphasized as well. Our results exposed that Dmrt1 maintenance mGSCs in two ways: facilitating proliferation and self-renewal of SSCs; and reducing the inflammatory response caused by reproductive injury. These findings identify a central role for Dmrt1 in controlling population stability and injury restoring of SSCs.

Lin28a promotes self-renewal and proliferation of dairy goat spermatogonial stem cells (SSCs) through regulation of mTOR and PI3K/AKT.

Lin28a is a conserved RNA-binding protein that plays an important role in development, pluripotency, stemness maintenance, proliferation and self-renewal. Early studies showed that Lin28a serves as a marker of spermatogonial stem cells (SSCs) and promotes the proliferation capacity of mouse SSCs. However, there is little information about Lin28a in livestock SSCs. In this study, we cloned Capra hircus Lin28a CDS and found that it is evolutionarily conserved. Lin28a is widely expressed in different tissues of Capra hircus, but is expressed at a high level in the testis. Lin28a is specifically located in the cytoplasm of Capra hircus spermatogonial stem cells and may also be a marker of dairy goat spermatogonial stem cells. Lin28a promoted proliferation and maintained the self-renewal of GmGSCs-I-SB in vivo and in vitro. Lin28a-overexpressing GmGSCs-I-SB showed an enhanced proliferation rate, which might be due to increased PCNA expression. Moreover, Lin28a maintained the self-renewal of GmGSCs-I-SB by up-regulating the expression of OCT4, SOX2, GFRA1, PLZF and ETV5. Furthermore, we found that Lin28a may activate the AKT, ERK, and mTOR signaling pathways to promote the proliferation and maintain the self-renewal of GmGSCs-I-SB.

In vitro production of functional haploid sperm cells from male germ cells of Saanen dairy goat

The dairy goat is an important economic animal. Studies on in vitro differentiation of dairy goat spermatogonial stem cells (SSCs) could understand the molecular mechanisms for detecting mammalian sperm generation and innovative transgenetics. This study established an in vitro differentiation system for Saanen dairy goat SSCs. After 35 days incubation, single flagellum sperm-like cells with late round spermatid (Sa2) morphologically and the ability to express sperm-specific protein acrosin is detected. DNA ploid analysis showed that addition of testosterone to the culture system significantly improved the differentiation efficiency (P < 0.05) of haploid cells, and stimulated the post-meiotic gene (Tnp1, Tnp2 and Prm1) expression. A green fluorescent protein expressed in the morula was observed after transfection of the green fluorescent protein (GFP) vector with a round spermatid microinjection into the oocyte at metaphase II-stage. This study optimized activated method of the goat oocytes injected with round spermatids in vitro. This method increases the reconstructed embryonic development rate. These results suggest that testosterone boosts the efficiency of haploid cell differentiation in Saanen dairy goat testicular cells in the in vitro differentiation system. After optimization of activated and injection methods, fertility and embryonic were improved.

Effect of different serum concentrations on short term in vitro culture of goat testicular cells

Proper in vitro culture and proliferation of spermatogonial stem cells (SSCs) are key tools to achieve functional potentials of SSCs. The fresh serum extracted has been successfully used to stimulate growth of spermatogonial stem cells in vitro. However, there is no report on the optimal concentration of serum for in vitro culture of goat SSCs. This study investigated this issue. Retrieved testicular cells were cultured in DMEM in presence of different 0 to 15% serum for 7 days and examined on 3rd and 7th day of culture for colony formation, cell viability, immunocytochemistry and quantitative gene expression analyses. The maximum numbers of colonies were observed in presence of 1% serum on days 3 and 7 of culture. Differential viable staining revealed that most cells were non-viable in complete absence of serum (0%). Immunocytochemistry showed that cells positive for pan cytokeratin were observed in colonies developed in presence of 1, 5, 10 and 15% serum but not 0%, suggesting the presence of epithelial cells within the developing colonies. The colonies developed in the presence of 1 and 5% serum had remarkable alkaline phosphatase activity. The maximum expressions of Plzf, Bcl6b and Vasa were observed in colonies developed in presence of 1% serum, with a significant difference to all the other groups. No significant difference was observed for the expression of Vimentin between different treatment groups. The results of this study indicate that for maintenance of SSCs, 1% serum required for maintaining viability while allowing proliferation and maintenance of SSCs like colonies.

MiR-204 Regulates the Proliferation of Dairy Goat Spermatogonial Stem Cells via Targeting to Sirt1.

The regulation of spermatogonial stem cell (SSC) proliferation and self-renewal is a complex process. Several studies on the microRNA regulation of mammalian spermatogenesis have been reported. Here, we predicted miRNA targeting of Sirt1, and a dual luciferase experiment confirmed that miR-204 interacted with the Sirt1 3'-untranslated region (3'-UTR). The expression of miR-204 and Sirt1 in dairy goat testicles was investigated, and the results showed that the expression pattern of Sirt1 was similar to that of miR-204 in the temporal-spatial distribution. The over-expression of Sirt1 in goat SSCs can promote SSCs' self-renewal gene expression and cell proliferation. Furthermore, miRNA sequencing results showed that Sirt1 had a higher expression level in dairy goat CD49f(+) and CD90(+) SSCs, but the expression level of miR-204 was lower. In an in vitro assay, Sirt1 was significantly down-regulated in dairy goat SSCs when transfected with miR-204 mimics, indicating that Sirt1 was a target of miR-204 in the dairy goat. On the basis of the results of RT-qPCR, fluorescence-activated cell sorting (FACS), and western blotting, we found that the over-expression of Sirt1 in goat SSCs can promote cellular proliferation and change self-renewal and pluripotent gene expression. Thus, miR-204 was involved in the regulation of dairy goat SSCs proliferation via Sirt1.

Derivation, enrichment and characterization of goat (Capra hircus) spermatogonial stem cells from pre-pubertal testes

Isolation of goat spermatogonial stem cells (gSSC) from pre-pubertal testes (3 to 6 months of age) by double enzymatic digestion is reported. The isolated cells were further enriched for spermatogonial stem cell population by filtration through 80- and 60-μm nylon mesh filters, followed by differential plating on DSA-Lectin coated dishes. After overnight incubation, the unattached cells (putative gSSCs) were cultured on sertoli cell feeder layers in SSC medium, composed of DMEM supplemented with 10% FBS, GDNF (Glial cell line derived neurotrophic factor, 40 ng mL-1), bFGF (Basic fibroblast growth factor, 10 ng mL-1) and EGF (Epidermal growth factor, 10 ng mL-1). After 10-15 days, the putative SSCs started to develop and the cultures were maintained till the colonies stopped growing. The colonies were characterized for their affinity to Dolichos biflorus Agglutinin (DBA), alkaline phosphatase (AP) activity and various spermatogonial stem cell markers (PLZF, THY1, UCHL1, BCL6B and ID4). The developed colonies were positive for DBA, AP and all other markers, which is an indication of their being spermatogonial stem cells. In the present study, goat SSCs have been successfully isolated and characterized and the culture conditions will be improved further for developing more efficient and long term in vitro spermatogonial stem cell culture system in goat.