BLOC1S1 promotes proliferation of goat spermatogonial stem cells / 生物工程学报

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.

Loss of RBFOX2 inhibits meiotic initiation in male mice / 生物工程学报

Meiotic initiation is a critical step in gametogenesis. Recently, some genes required for meiotic initiation have been identified. However, meiosis-initiating factors and the underlying mechanisms are far from being fully understood. We have established a long-term culture system of spermatogonial stem cells (SSCs) and an in vitro model of meiotic initiation using mouse SSCs. Our previous study revealed that the RNA-binding protein RBFOX2 may regulate meiotic initiation, but the role and the mechanism need to be further elucidated. In this study, we constructed RBFOX2 knockdown SSC lines by using lentivirus-mediated gene delivery method, and found that the knockdown SSCs underwent normal self-renewal, mitosis and differentiation. However, they were unable to initiate meiosis when treated with retinoic acid, and they underwent apoptosis. These results indicate that RBFOX2 plays an essential role in meiotic initiation of spermatogonia. This work provides new clues for understanding the functions of RNA-binding proteins in meiotic initiation.

FOXP4 promotes proliferation of human spermatogonial stem cells / 亚洲男科学杂志(英文版)

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

Ubiquitin protein E3 ligase ASB9 suppresses proliferation and promotes apoptosis in human spermatogonial stem cell line by inducing HIF1AN degradation

BACKGROUND: Spermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis. Even though several regulators of SSC have been identified in rodents, the regulatory mechanism of SSC in humans has yet to be discovered. METHODS: To explore the regulatory mechanisms of human SSCs, we analyzed publicly available human testicular single-cell sequencing data and found that Ankyrin repeat and SOCS box protein 9 (ASB9) is highly expressed in SSCs. We examined the expression localization of ASB9 using immunohistochemistry and overexpressed ASB9 in human SSC lines to explore its role in SSC proliferation and apoptosis. Meanwhile, we used immunoprecipitation to find the target protein of ASB9 and verified its functions. In addition, we examined the changes in the distribution of ASB9 in non-obstructive azoospermia (NOA) patients using Western blot and immunofluorescence. RESULTS: The results of uniform manifold approximation and projection (UMAP) clustering and pseudotime analysis showed that ASB9 was highly expressed in SSCs, and its expression gradually increased during development. The immunohistochemical and dual-color immunofluorescence results displayed that ASB9 was mainly expressed in nonproliferating SSCs. Overexpression of ASB9 in the SSC line revealed significant inhibition of cell proliferation and increased apoptosis. We predicted the target proteins of ASB9 and verified that hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), but not creatine kinase B-type (CKB), has a direct interaction with ASB9 in human SSC line using protein immunoprecipitation experiments. Subsequently, we re-expressed HIF1AN in ASB9 overexpressing cells and found that HIF1AN reversed the proliferative and apoptotic changes induced by ASB9 overexpression. In addition, we found that ABS9 was significantly downregulated in some NOA patients, implying a correlation between ASB9 dysregulation and impaired spermatogenesis. CONCLUSION: ASB9 is predominantly expressed in human SSCs, it affects the proliferation and apoptotic process of the SSC line through HIF1AN, and its abnormal expression may be associated with NOA.

Rescue of male infertility through correcting a genetic mutation causing meiotic arrest in spermatogonial stem cells / 亚洲男科学杂志(英文版)

Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with an X-linked testis-expressed 11 (TEX11) mutation (Tex11

Characterization, isolation, and culture of spermatogonial stem cells in / 亚洲男科学杂志(英文版)

Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kit

Effects of different culture systems on the culture of prepuberal buffalo (Bubalus bubalis) spermatogonial stem cell-like cells in vitro

Mechanism underlying basic fibroblast growth factor and insulin-like growth factor-1 effects on proliferation and apoptosis of spermatogonial stem cells / 中国组织工程研究

BACKGROUND: As a necessary regulatory factor of in vitro cell culture, and in vivo cell growth and proliferation, growth factors have attracted much attention. OBJECTIVE: To investigate the effects of basic fibroblast growth factor (bFGF) combined with insulin-like growth factor-1 (IGF-1) on the proliferation and apoptosis of spermatogonial stem cells in mice. METHODS: Spermatogonial stem cells were isolated and cultured from the testis of 6-8 days old male Kunming mice and were identified. Spermatogonial stem cells were inoculated into the feeder layer of embryonic fibroblasts treated with mitomycin C, and were then divided into four groups. Control group was cultured with normal DMEM medium; bFGF and IGF-1 groups were cultured with DMEM medium containing 20 μg/L bFGF and 20 μg/L IGF-1, respectively; bFGF+IGF-1 group was cultured with DMEM medium containing 20 μg/L bFGF and 20 μg/L IGF-1. The proliferation activity of spermatogonial stem cells was detected by cell counting kit-8 assay and EDU staining. The growth cycle and apoptosis of spermatogonial stem cells were detected by flow cytometry. The expression levels of PCNA, Bax and Bcl-2 proteins were detected by western blot assay. RESULTS AND CONCLUSION: Compared with the control group, the absorbance values in the bFGF, IGF-1 and bFGF+IGF-1 groups were significantly increased. Compared with the bFGF and IGF-1 groups, the absorbance values in the bFGF+IGF-1 group were further increased (P < 0.05). EDU staining results showed the same conclusion as cell counting kit-8 assay results. The proportion of S+G2/M phase cells in the bFGF+IGF-1 group was significantly higher than that in the other three groups (P < 0.05). The proportion in the IGF-1 and bFGF groups was significantly higher than that in control group (P < 0.05). Compared with the control group, the number of apoptotic cells in the bFGF, IGF-1 and bFGF+IGF-1 groups was decreased. Compared with the bFGF and IGF-1 groups, the number of apoptotic cells in the bFGF+IGF-1 group was further decreased. Compared with the control group, the relative expression levels of Bax protein in the bFGF, IGF-1 and bFGF+IGF-1 groups were significantly decreased (P < 0.01), and the expression levels of Bcl-2 and PCNA proteins were significantly increased (P < 0.05). Compared with the bFGF and IGF-1 groups, the relative expression level of Bax protein in the bFGF + IGF-1 group was decreased further (P < 0.01), and the relative expression levels of Bcl-2 and PCNA proteins were increased further (P < 0.05). These results indicate that bFGF and IGF-1 can promote cell proliferation and inhibit cell apoptosis by up-regulating the expression of PCNA and Bcl-2 proteins and down-regulating the expression of Bax protein. The combination of bFGF and IGF-1 can achieve favorable effects.

Strawberry Notch 1 (SBNO1) promotes proliferation of spermatogonial stem cells via the noncanonical Wnt pathway in mice / 亚洲男科学杂志(英文版)

While it is known that spermatogonial stem cells (SSCs) initiate the production of male germ cells, the mechanisms of SSC self-renewal, proliferation, and differentiation remain poorly understood. We have previously identified Strawberry Notch 1 (SBNO1), a vertebrate strawberry notch family protein, in the proteome profile for mouse SSC maturation and differentiation, revealing SBNO1 is associated with neonatal testicular development. To explore further the location and function of SBNO1 in the testes, we performed Sbno1 gene knockdown in mice to study the effects of SBNO1 on neonatal testicular and SSC development. Our results revealed that SBNO1 is required for neonatal testicular and SSC development in mice. Particularly, in vitro Sbno1 gene knockdown with morpholino oligonucleotides caused a reduction of SSCs and inactivation of the noncanonical Wnt pathway, through Jun N-terminal kinases. Our study suggests SBNO1 maintains SSCs by promoting the noncanonical Wnt pathway.

Effect of Wuzi Yanzong Fang in Promoting Spermatogonial Stem Cells Proliferation in Aging Rats by Regulating miR-let-7-Imp Axis / 中国实验方剂学杂志

Objective: To explore the effect and mechanism of Wuzi Yanzong Fang (WZ) on proliferation of spermatogonial stem cells in aging rats by regulating miR-let-7-Imp axis.Method: A total of 40 18-month-old male SD rats were randomly divided into aging model group, and low, middle and high-dose WZ groups, with 10 rats in each group. Ten 2-month old rats were used as young control group. Low, middle and high-dose WZ groups were given by gastric WZ 0.4, 0.8, 1.6 g·kg-1 respectively. Young control group and aging model group were given normal saline for 4 months, which was suspended for 2 days every week. Rats were put to death after the final treatment with WZ, and then the testes were quickly removed from rats. The relative mRNA expression levels of miR-let-7 and Imp were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). The expressions and localization of p-JAK2/JAK2, phosphate(p)-signal transduction and transcriptional activators3 (STAT3)/STAT3 signaling pathway proteins were detected by Western blot and immunofluorescence. The numbers of spermatogonial stem cells and the expression levels of proliferating cell nuclear antigen (PCNA) were detected by immunofluorescence. The testicular tissue morphology was observed using htoxylin eosin (HE) staining.Result: Compared with young control group, the expression levels of miR-let-7 mRNA were significantly increased, while the expression levels of Imp mRNA were decreased in the aging model group(PPPPPPPConclusion: WZ effectively promote the proliferation of spermatogonial stem cells by regulating miR-let-7-imp axis in testis.

Strawberry Notch 1 (SBNO1) promotes proliferation of spermatogonial stem cells via the noncanonical Wnt pathway in mice / 亚洲男科学杂志(英文版)

While it is known that spermatogonial stem cells (SSCs) initiate the production of male germ cells, the mechanisms of SSC self-renewal, proliferation, and differentiation remain poorly understood. We have previously identified Strawberry Notch 1 (SBNO1), a vertebrate strawberry notch family protein, in the proteome profile for mouse SSC maturation and differentiation, revealing SBNO1 is associated with neonatal testicular development. To explore further the location and function of SBNO1 in the testes, we performed Sbno1 gene knockdown in mice to study the effects of SBNO1 on neonatal testicular and SSC development. Our results revealed that SBNO1 is required for neonatal testicular and SSC development in mice. Particularly, in vitro Sbno1 gene knockdown with morpholino oligonucleotides caused a reduction of SSCs and inactivation of the noncanonical Wnt pathway, through Jun N-terminal kinases. Our study suggests SBNO1 maintains SSCs by promoting the noncanonical Wnt pathway.

Enrichment and In Vitro Culture of Spermatogonial Stem Cells from Pre-Pubertal Monkey Testes

Spermatogonial stem cells (SSCs) are essential for spermatogenesis throughout the lifespan of the male. However, the rarity of SSCs has raised the need for an efficient selection method, but little is known about culture conditions that stimulate monkey SSC proliferation in vitro. In this study, we report the development of effective enrichment techniques and in vitro culturing of germ cells from pre-pubertal monkey testes. Testis cells were analyzed by fluorescence-activated cell sorting techniques and were transplanted into the testes of nude mice to characterize SSCs. Thy-1-positive cells showed a higher number of colonies than the unselected control after xenotransplantation. Extensive colonization of monkey cells in the mouse testes indicated the presence of highly enriched populations of SSCs in the Thy-1-positive sorted cells. Furthermore, monkey testis cells were enriched by differential plating using extracellular matrix, laminin, and gelatin, and then cultured under various conditions. Isolation of monkey testicular germ cells by differential plating increased germ cell purity by 2.7-fold, following the combinational isolation method using gelatin and laminin. These enriched germ cells actively proliferated under culture conditions involving StemPro medium supplemented with bFGF, GDNF, LIF, and EGF at 37 ℃. These results suggest that the enrichment and in vitro culture method proposed in the present study for harvesting a large number of functionally active monkey SSCs can be applied as the basis for efficient in vitro expansion of human SSCs.

Spermatogonial Stem Cells Protein Identification in In Vitro Culture from Non- Obstructive Azoospermia Patient

Background: Spermatogonial stem cells (SSCs) are classified as a unique adult stem cells that have capability to propagate, differentiate, and transmit genetic information to the next generation. Studies on human SSCs may help resolve male infertility problems, especially in azoospermia patients. Therefore, this study aims to propagate SSCs in-vitro with a presence of growth factor and detect SSC-specific protein cell surface markers. Methods: The sample was derived from non-obstructive azoospermic (NOA) patient. The disassociation of SSCs was done using trypsin. Specific cultures in serum-free media with added basic fibroblast growth factor (bFGF) were developed to support self-renewal division. This undifferentiated protocol was performed for 49 days. Cells were analysed on days 1, 7, 14, 21, and 49. Results: Human SSCs began to aggregate and form colonies after 14 to 21 days in specific culture. Then, the cells were successful expanded and remained stable for a duration of 49 days. Four specifics markers were identified using immunofluorescence in SSCs on day 49: ITGα6, ITGβ1, CD9, and GFRα1. Conclusion: This approach of using in vitro culture with additional growth factor is able to propagate SSCs from non-obstructive azoospermia patient via detection of protein cell surface markers.

Multipotent male germline stem cells (mGSCs) from neonate porcine testis

ABSTRACT Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, during which unlimited spermatozoa is produced daily derived from SSCs in the testis throughout life of the male. Germline stem (GS) cells can be isolated from spermatogonia, which shared the characteristics of SSCs and embryonic stem cells (ESCs), and can be passaged stably in vitro. The study of GS cells contributes to understanding spermatogenesis process. However, little is known about the GS cells in domestic animals. Here, we report the successful establishment of a serum- and feeder-free system for multipotent male GS cells (mGSCs) from postnatal porcine testis. These cells expressed pluripotent markers, such as Oct4, Nanog, C-myc, and germline-specific markers including Vasa, CD90, CD49f, Gfrα1, Plzf and Dazl. Then we assayed the developmental potential of these cells in vitro. The porcine multipotent male germline stem cells (pmGSCs) can form embryoid bodies (EBs) by suspension culture. Immunofluorescence analysis showed that the EBs differentiated into neuron-specific enolase (NSE, ectoderm), α-actin (mesoderm), and Pdx1 (endoderm) positive cells. These cells induced by 10-6 M retinoic acid (RA) could be differentiated into spermatid-like cells which were positive for Acrosin. The pmGSCs has been cultured over 14 passages. Thus, we have established a long-term culture system for pmGSCs. This culture system provides a platform for the study of porcine mGSCs.

Influence mechanism of glial cell line-derived neurotrophic factor on the proliferation of spermatogonial stem cells / 中华泌尿外科杂志

Objective To investigate the molecular mechanisms of glial cell derived neurotrophic factor in promoting proliferation of spermatogonial stem cell.Methods RNAi expression vectors,targeted at GDNF,were constructed and transfected into SSCs from 5 to 7 days old mice.The SSCs with highest effectiveness of GDNF interfere was set as study group.And the SSCs without GDNF interfere was considered as control group.The ELISA method was used to compare the proliferative rate between study group and control group.Flow cytometry,RT-PCR were used to detect the expression of GDNF,RTKs,Fyn and FAK's mRNA,and the apoptosis of SSCs.Results From 1 to 4 days after transinfection,the absorbable A value in study group was 0.45 ± 0.02,0.68 ± 0.03,1.12 ± 0.03,2.24 ± 0.04,respectively.Meanwhile,the same item in control group was 0.46 ± 0.03、0.73 ± 0.02、1.32 ± 0.05、1.15 ± 0.06,respectively (P ＜ 0.05).There were significant different between experiment groups (25.43 ± 1.91) ％ and control group (5.61 ± 0.16)％ in the apoptosis rates of SSCs (P ＜ 0.05).Significant differences were noted between experimental group and control group(P ＜ 0.05).The mRNA expression rates of GDNF was (12.32 ± 1.22) ％ in study group and (54.25 ± 1.34)％ in control group (P ＜0.01).The mRNA expression rates of RTKs and Fyn and FAK in study group and control group were (16.24 ± 1.35)％ vs (45.35 ± 1.37)％,(18.32 ±1.34)％ vs (38.37 ± 1.55)％,(20.04 ± 1.65)％ vs (43.27 ± 1.28)％,respectively (P ＜0.05).Conclusions The glial cell line derived neurotrophic factor was important in course of SSCs' proliferation,which may up-regulating the expression of RTKs,Fyn and FAK.

Preliminary Isolation and Purification of Spermatogonial Stem Cells / 中国医师杂志

Objective To obtain a simple method to isolate and purify spermatogonia stem cells. Methods The testis of newborn mouse were digested by modified two step digest method, and the type A spermatogonia cells were separated by different adhesive time with Sertoli cells. Results High concentration (95%) purified type A spermatogonia cells were acquired, and the spermatogonia transplanting experiment demonstrated the efficiency of the method.Conclusions This method is easy and efficient to isolation and purification of spermatogonial stem cells.