Nicotine induces senescence in spermatogonia stem cells by disrupting homeostasis between circadian oscillation and rhythmic mitochondrial dynamics via the SIRT6/Bmal1 pathway.

Infertility is intricately linked with alterations in circadian rhythms along with physiological decline and stem cell senescence. Yet, the direct involvement of circadian mechanisms in nicotine-induced injury to the testes, especially the senescence of spermatogonia stem cells (SSCs), is not well comprehended. This study revealed that nicotine exposure induced testis injury by triggering SSCs senescence along with the upregulation of senescence marker genes and senescence-associated secretory phenotype components. Moreover, nicotine treatment caused mitochondrial hyper-fusion, increased oxidative stress, and DNA damage. Exposure to nicotine was found to suppress the expression of sirtuin 6 (SIRT6), which accelerated the senescence of spermatogonia stem cells (SSCs). This acceleration led to increased acetylation of brain and muscle ARNT-like protein (Bmal1), consequently reducing the expression of Bmal1 protein. Conversely, the overexpression of Bmal1 alleviated mitochondrial hyper-fusion and senescence phenotypes induced by nicotine. Overall, this study unveiled a novel molecular mechanism behind nicotine-induced disorders in spermatogenesis and highlighted the SIRT6/Bmal1 regulatory pathway as a potential therapeutic target for combating nicotine-associated infertility.

Retinoic acid promotes formation of chicken (Gallus gallus) spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10 days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinß1, the specific marker protein of SSCs cells, and the integrinß1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinß1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

In vitro impact of genistein and mono(2-ethylhexyl) phthalate (MEHP) on the eicosanoid pathway in spermatogonial stem cells.

Perinatal exposures to endocrine disrupting chemicals (EDCs) alter the male reproductive system. Infants are exposed to genistein (GEN) through soy-based formula, and to Mono(2-ethylhexyl) Phthalate (MEHP), metabolite of the plasticizer DEHP. Spermatogonial stem cells (SSCs) are formed in infancy and their integrity is essential for spermatogenesis. Thus, understanding the impact of EDCs on SSCs is critical. Prostaglandins (PGs) are inflammatory mediators synthesized via the eicosanoid pathway starting with cyclooxygenases (Coxs), that regulate physiological and pathological processes. Our goal was to study the eicosanoid pathway in SSCs and examine whether it was disrupted by GEN and MEHP, potentially contributing to their adverse effects. The mouse C18-4 cell line used as SSC model expressed high levels of Cox1 and Cox2 genes and proteins, and eicosanoid pathway genes similarly to levels measured in primary rat spermatogonia. Treatments with GEN and MEHP at 10 and 100 µM decreased Cox1 gene and protein expression, whereas Cox2, phospholipase A2, prostaglandin synthases transcripts, PGE2, PGF2a and PGD2 were upregulated. Simultaneously, the transcript levels of spermatogonia progenitor markers Foxo1 and Mcam and differentiated spermatogonial markers cKit and Stra8 were increased. Foxo1 was also increased by EDCs in primary rat spermatogonia. This study shows that the eicosanoid pathway is altered during SSC differentiation and that exposure to GEN and MEHP disrupts this process, mainly driven by GEN effects on Cox2 pathway, while MEHP acts through an alternative mechanism. Thus, understanding the role of Cox enzymes in SSCs and how GEN and MEHP exposures alter their differentiation warrants further studies.

Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model.

Preserving the spermatogonial stem cells (SSCs) in long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium. Testicular torsion-a mouse model for long-term infertility-was used to transplant fresh SSCs (n = 6), fresh SSCs treated with PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6), and cryopreserved SSCs treated with PTX (n = 6). Eight weeks after germ cell transplantation, samples were assessed for proliferation, through evaluation of Ddx4 and Id4 markers, and differentiation via evaluation of C-Kit and Sycp3, Tnp1, Tnp2, and Prm1 markers. According to morphological and flow cytometry results, SSCs are able to form colonies and express Gfra1, Id4, α6-integrin, and ß1-integrin markers. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice. In the recipient testis, donor SSCs formed spermatogenic colonies and sperm. Respecting these data, adding pentoxifylline is a practical way to precisely cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup. However, more studies are needed to ensure its safety in the long term.

Autophagy modulation alleviates cryoinjury in murine spermatogonial stem cell cryopreservation.

BACKGROUND: Cryopreservation can expand the usefulness of spermatogonial stem cells (SSCs) in various fields. However, previous investigations that have attempted to modulate cryoinjury-induced mechanisms to increase cryoprotective efficiency have mainly focused on apoptosis and necrosis. OBJECTIVES: This study aimed to establish an effective molecular-based cryoprotectant for SSC cryopreservation via autophagy modulation. MATERIALS AND METHODS: To determine the efficacy of autophagy modulation, we assessed the recovery rate and relative proliferation rate and performed western blotting for the determination of autophagy flux, immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization, and spermatogonial transplantation for in vivo SSC functional activity. RESULTS: The results showed that a basal level of autophagy caused a higher relative proliferation rate (pifithrin-µ 0.01 µM, 184.2 ± 11.2%; 3-methyladenine 0.01 µM, 175.3 ± 10.3%; pifithrin-µ 0.01 µM + 3-methyladenine 0.01 µM, P3, 224.6 ± 22.3%) than the DMSO control (100 ± 6.2%). All treatment groups exhibited normal characteristics, suggesting that these modulators could be used as effective cryoprotectants without changing the properties of the undifferentiated germ cells. According to the results of the in vivo spermatogonial transplantation assay, the colonies per total number of cultured SSCs was significantly higher in the pifithrin-µ 0.01 µM (1596.7 ± 172.5 colonies), 3-methyladenine 0.01 µM (1522.1 ± 179.2 colonies), and P3 (1727.5 ± 196.5 colonies) treatment groups than in the DMSO control (842.8 ± 110.08 colonies), which was comparable to that of the fresh control (1882.1 ± 132.1 colonies). DISCUSSION: A basal level of autophagy is more essential for resilience in frozen SSCs after thawing, rather than the excessive activation or inhibition of autophagy. CONCLUSION: A basal level of autophagy plays a critical role in the pro-survival response of frozen SSCs after thawing; herein, a new approach by which SSC cryoprotective efficiency can be improved was identified.

Astaxanthin Relieves Busulfan-Induced Oxidative Apoptosis in Cultured Human Spermatogonial Stem Cells by Activating the Nrf-2/HO-1 pathway.

Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10µM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways.

Estradiol modulated differentiation and dynamic growth of CD90+ spermatogonial stem cells toward Sertoli-like cells.

Mouse CD90+ SSCs were enriched using the MACS technique and incubated with different doses of estradiol, ranging from 0.01 ng/mL to 500 µg/mL, for 7 days. The viability of SSCs was determined using an MTT assay. The combined effects of estradiol plus Sertoli cell differentiation medium on the orientation of SSCs toward Sertoli-like cells were also assessed. Using immunofluorescence imaging, we monitored protein levels of Oct3/4 after being exposed to estradiol. In addition, protein levels of testosterone, TF, and ABP were measured using ELISA. The expression of Sertoli cell-specific genes such as SOX9, GATA4, FSHR, TF, and ESR-1 and -2 was monitored using real-time PCR assay, and the effects of 14-day injection of estradiol on sperm parameters and Oct3/4 positive progenitor cells in a model of mouse were determined. Data showed that estradiol increased the viability of mouse SSCs in a dose-dependent manner compared to the control (p < 0.05). Along with these changes, cells displayed morphological changes and reduced Oct3/4 transcription factor levels compared to the control SSCs. 7-day incubation of SSCs with estradiol led to the up-regulation of SOX9, GATA4, FSHR, TF, and ESR-1 and -2, and levels of testosterone, TF, and ABP were increased compared to the control group (p < 0.05). The in-vivo examination noted that estradiol reduced sperm parameters coincided with morphological abnormalities (p < 0.05). Histological examination revealed pathological changes in seminiferous tubules and reduction of testicular Oct3/4+ progenitor cells. In conclusion, estradiol treatment probably can induce Sertoli cell differentiation of SSCs while exogenous administration leads to testicular progenitor cell depletion and infertility in long term.

Differential RA responsiveness among subsets of mouse late progenitor spermatogonia.

Initiation of spermatogonial differentiation in the mouse testis begins with the response to retinoic acid (RA) characterized by activation of KIT and STRA8 expression. In the adult, spermatogonial differentiation is spatiotemporally coordinated by a pulse of RA every 8.6 days that is localized to stages VII-VIII of the seminiferous epithelial cycle. Dogmatically, progenitor spermatogonia that express retinoic acid receptor gamma (RARG) at these stages will differentiate in response to RA, but this has yet to be tested functionally. Previous single-cell RNA-seq data identified phenotypically and functionally distinct subsets of spermatogonial stem cells (SSCs) and progenitor spermatogonia, where late progenitor spermatogonia were defined by expression of RARG and Dppa3. Here, we found late progenitor spermatogonia (RARGhigh KIT-) were further divisible into two subpopulations based on Dppa3 reporter expression (Dppa3-ECFP or Dppa3-EGFP) and were observed across all stages of the seminiferous epithelial cycle. However, nearly all Dppa3+ spermatogonia were differentiating (KIT+) late in the seminiferous epithelial cycle (stages X-XII), while Dppa3- late progenitors remained abundant, suggesting that Dppa3+ and Dppa3- late progenitors differentially responded to RA. Following acute RA treatment (2-4 h), significantly more Dppa3+ late progenitors induced KIT, including at the midpoint of the cycle (stages VI-IX), than Dppa3- late progenitors. Subsequently, single-cell analyses indicated a subset of Dppa3+ late progenitors expressed higher levels of Rxra, which we confirmed by RXRA whole-mount immunostaining. Together, these results indicate RARG alone is insufficient to initiate a spermatogonial response to RA in the adult mouse testis and suggest differential RXRA expression may discriminate responding cells.

Cryopreservation and Transplantation of Spermatogonial Stem Cells.

Cryopreservation as a method that enables long-term storage of biological material has long been used for the conservation of valuable zebrafish genetic resources. However, currently, only spermatozoa of zebrafish can be successfully cryopreserved, while protocols for cryopreservation of eggs and embryos have not yet been fully developed. Transplantation of germline stem cells (GSCs) has risen as a favorable method that can bypass the current problem in cryopreservation of female genetic resources and can lead to reconstitution of fish species and lines through surrogate production. Here, we describe essential steps needed for the cryopreservation of spermatogonial stem cells (SSCs) and their utilization in the conservation of zebrafish genetic resources through SSC transplantation and surrogate production.

Effect of a Freezing Medium Containing Melatonin on Markers of Pre-meiotic and Post-meiotic Spermatogonial Stem Cells (SSCs) After Transplantation in an Azoospermia Mouse Model Due to Testicular Torsion.

Spermatogonial stem cells (SSCs) are essential to the initiation of spermatogenesis. Cryopreservation, long-term maintenance, and auto-transplantation of SSCs could be a new treatment for infertility. The aim of this study was to add melatonin to the basic freezing medium and to evaluate its effect on the efficiency of the thawed SSCs after transplantation into the testicles of azoospermic mice. SSCs were isolated from newborn NMRI mice, and the cells were enriched to assess morphological features. The thawed SSCs were evaluated for survival, apoptosis, and ROS level before transplantation, and the proliferation (MVH and ID4) and differentiation (c-Kit, SCP3, TP1, TP2, and Prm1) markers of SSCs were examined using immunofluorescence, western blot, and quantitative real-time polymerase chain reaction (PCR) after transplantation. It was found that the survival rate of SSCs after thawing was significantly higher in the melatonin group compared with the cryopreservation group containing basic freezing medium, and the rate of apoptosis and level of ROS production also decreased significantly in the cryopreservation group with melatonin (p < 0.05). The expression of proliferation and differentiation markers after transplantation was significantly higher in the cryopreservation group with melatonin compared to the cryopreservation group (p < 0.05). The results suggest that adding melatonin to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production and apoptosis and improve the transplantation efficiency of SSCs after cryopreservation, which will provide a significant suggestion for fertility protection in the clinic.

SCF Improves In Vitro Differentiation of SSCs Through Transcriptionally Up-regulating PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 Genes.

Several lines of evidence strongly suggest that retinoic acid (RA) and stem cell factor (SCF)/c-Kit signal transduction pathways are involved in the differentiation of spermatogonial stem cells (SSCs). This study was aimed to investigate the effect of RA and SCF on in vitro differentiation of SSCs via evaluation of the mRNA expression of meiosis-specific genes in cultured testicular tissues. Testicular tissue samples were obtained from bilaterally vasectomized rats and also healthy adult rats and then were cultured for 25, 30, and 35 days on different conditions. The cultured testicular pieces were sectioned and stained with PAS to histological analysis. The total RNA was extracted from cultured testicular samples, and the expression of ACR, PRTM1, SYCP3, STRA8, c-KIT, PIWIL2, and OCT4 genes at mRNA level was quantified using real-time polymerase chain reaction (qPCR) procedure. After 1-month surgery, bilateral testicular weight showed a significant decrease in vasectomized adult rats compared with healthy adult rats (P < 0.05). Reduction in the diameter of the seminiferous tubules and depletion of advanced germinal elements in vasectomized rats compared with healthy adult rats were also observed. Our findings also demonstrated that the mRNA expression level of PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 genes in cultured testicular tissues significantly up-regulated in experimental group II compared with the control group (P < 0.001). Our findings lead us to conclude that SCF improves in vitro differentiation of SSCs in the OA rats, at least partially, by transcriptionally upregulating PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 genes.

Melatonin alleviates LPS-induced endoplasmic reticulum stress and inflammation in spermatogonial stem cells.

Orchitis is one of the leading causes of male animal infertility and is associated with inflammatory reactions caused by the bacterium. It has been reported that there is a mutual coupling effect between endoplasmic reticulum stress (ERS) and inflammatory response. Our studies showed that lipopolysaccharide (LPS) could cause testicular damages, apoptosis, ERS, and inflammatory responses in spermatogonial stem cells (SSCs); ERS-related apoptosis proteins were activated and the expression of ERS genes was significantly upregulated; meanwhile, the expression of Toll-like receptor 4 and inflammation factors was apparently increased with LPS treatment. Moreover, melatonin (MEL) could rescue testicular damage, and significantly inhibited the expression of ERS-related apoptosis genes, ERS markers, and inflammatory factors in SSCs and MEL played repairing and anti-infection roles in LPS-induced testicular damage. Therefore, MEL may be used as a drug to prevent and control bacterial infections in male reproductive systems. However, the specific molecular mechanism of MEL to resist ERS and inflammatory response remains to be further studied.

The IL-27 component EBI-3 and its receptor subunit IL-27Rα are essential for the cytoprotective action of humanin on male germ cells†.

Humanin (HN) is a mitochondrial-derived peptide that protects many cells/tissues from damage. We previously demonstrated that HN reduces stress-induced male germ cell apoptosis in rodents. HN action in neuronal cells is mediated through its binding to a trimeric cell membrane receptor composed of glycoprotein 130 (gp130), IL-27 receptor subunit (IL-27R, also known as WSX-1/TCCR), and ciliary neurotrophic factor receptor subunit (CNTFR). The mechanisms of HN action in testis remain unclear. We demonstrated in ex-vivo seminiferous tubules culture that HN prevented heat-induced germ cell apoptosis was blocked by specific anti-IL-27R, anti-gp130, and anti-EBI-3, but not by anti-CNTFR antibodies significantly. The cytoprotective action of HN was studied by using groups of il-27r-/- or ebi-3-/- mice administered the following treatment: (1) vehicle; (2) a single intraperitoneal (IP) injection of HN peptide; (3) testicular hyperthermia; and (4) testicular hyperthermia plus HN. We demonstrated that HN inhibited heat-induced germ cell apoptosis in wildtype but not in il-27r-/- or ebi-3-/- mice. HN restored heat-suppressed STAT3 phosphorylation in wildtype but not il-27r-/- or ebi-3-/- mice. Dot blot analyses showed the direct interaction of HN with IL-27R or EBI-3 peptide. Immunofluorescence staining showed the co-localization of IL-27R with HN and gp130 in Leydig cells and germ cells. We conclude that the anti-apoptotic effects of HN in mouse testes are mediated through interaction with EBI-3, IL-27R, and activation of gp130, whereas the role of CNTFR needs further studies. This suggests a multicomponent tissue-specific receptor for HN in the testis and links HN action with the IL-12/IL-27 family of cytokines.

Meiotic susceptibility for induction of sperm with chromosomal aberrations in patients receiving combination chemotherapy for Hodgkin lymphoma.

Improvements in survival rates with gonad-sparing protocols for childhood and adolescence cancer have increased the optimism of survivors to become parents after treatment. Findings in rodents indicate that chromosomal aberrations can be induced in male germ cells by genotoxic exposures and transmitted to offspring and future generations with effects on development, fertility and health. Thus, there is a need for effective technologies to identify human sperm carrying chromosomal aberrations to assess the germ-line risks, especially for cancer survivors who have received genotoxic therapies. The time-dependent changes in the burden of sperm carrying structural chromosomal aberrations were assessed for the first time in a cancer setting, using the AM8 sperm FISH protocol which simultaneously detects abnormalities in chromosomal structure and number in sperm. Nine Hodgkin lymphoma (HL) patients provided 20 semen samples before, during, and after NOVP therapy (Novantrone, Oncovin, Velban and Prednisone) and radiation therapy that produced scattered gonadal doses from <0.05 to 0.6 Gy. Late meiosis was found to be the most sensitive to NOVP treatment for the production of sperm with chromosomal abnormalities, both in structure and number. Earlier stages of spermatogenesis were less sensitive and there was no evidence that therapy-exposed stem cells resulted in increased frequencies of sperm with abnormalities in chromosomal structure or number. This indicates that NOVP therapy may increase the risks for paternal transmission of chromosomal structural aberrations for sperm produced 32 to 45 days after a treatment with these drugs and implies that there are no excess risks for pregnancies conceived more than 6 months after this therapy. This clinical evaluation of the AM8 sperm FISH protocol indicates that it is a promising tool for assessing an individual's burden of sperm carrying chromosomal structural aberrations as well as aneuploidies after cancer therapy, with broad applications in other clinical and environmental situations that may pose aneugenic or clastogenic risks to human spermatogenesis.

Effects of selenium on the proliferation and apoptosis of sheep spermatogonial stem cells in vitro.

The objective of this study was to investigate effects of selenium (Se) on proliferation and apoptosis of sheep spermatogonial stem cells (SSC) in vitro. The SSC were assigned to five treatment groups (0, 2.0, 4.0, 8.0 and 16.0 µmol/L Se). After treatment with Se for 96 h, cell proliferation and apoptosis were evaluated. The relative abundance of P53 mRNA transcript and protein, cell cycle and apoptosis-related genes were detected using real-time PCR and Western blot quantifications, respectively. The results indicate there were the least cell proliferation rates in the Se16.0 group. Treatments with relatively greater Se concentrations (8.0 and 16.0 µmol/L) resulted in a greater percentage of apoptotic cells, which was consistent with the relative abundances of P53, P21, P27 and pro-apoptosis mRNA transcripts. There were relatively greater ROS concentrations in the control, Se8.0 and Se16.0 groups. Compared with the control group, treatment with the Se concentration of 16.0 µmol/L resulted in an increased abundance of P53, P21, P27 and BAX proteins. Treatment with Pifithrin-α suppressed the increase in abundance of P53 and P21 proteins induced by the relatively greater concentration of Se (16.0 µmol/L), however, did not result in a change in abundances of P27 and BAX proteins. These results indicate the regulatory functions of Se on proliferation and apoptosis of sheep SSC is associated with the P21-mediated P53 signalling pathway. The P27 and BAX proteins have limited functions during the apoptotic process of SSC induced by the relatively greater concentrations of Se.

Moderate hypoxia modulates ABCG2 to promote the proliferation of mouse spermatogonial stem cells by maintaining mild ROS levels.

The aim of this study was to investigate the effects of different oxygen (O2) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O2 concentrations (1%, 2.5%, 5%, and 20% O2) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O2 concentrations for 7 days, and there were no major morphologic differences between the O2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O2 treatment group. Compared with other O2 concentrations, the number of cells in G0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O2. Moreover, the intracellular ROS levels gradually increased with increasing O2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O2 was higher than in the other O2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro.

Cyclooxygenase 2 (COX2) expression and prostaglandin synthesis in neonatal rat testicular germ cells: Effects of acetaminophen and ibuprofen.

BACKGROUND: In infants, fever is often treated with acetaminophen or ibuprofen, two antipyretic and analgesic drugs inhibiting cyclooxygenases (COXs), enzymes catalyzing prostaglandin production. Infancy represents a critical developmental period when neonatal germ cells/gonocytes differentiate to spermatogonial stem cells required for spermatogenesis. OBJECTIVES: (a) Determine the expression of Cox2 and associated genes in postnatal day (PND)3 rat gonocytes compared to spermatogonia. (b) Examine whether acetaminophen or ibuprofen disrupts neonatal gonocyte functions. (c) Determine whether neonatal gonocytes produce prostaglandins and whether this process is altered by acetaminophen and ibuprofen. MATERIALS AND METHODS: The expression of Cox2 and related genes was determined by gene arrays and qPCR. Cox2 protein levels were determined by immunocyto/histochemistry and immunoblots. The effects of acetaminophen and ibuprofen on PND3 gonocyte viability, apoptosis, proliferation, and differentiation were examined alone and with a proliferation cocktail or differentiation factor. Prostaglandins were examined by immunocyto/histochemistry and LC-MS. RESULTS: Cox2 and related genes are highly expressed in gonocytes and spermatogonia. Acetaminophen and ibuprofen did not affect gonocyte survival or apoptosis, but they increased gonocyte proliferation. Ibuprofen significantly reduced RA-induced Stra8 expression, indicating an inhibitory effect on differentiation. Ibuprofen combined with RA decreased Cox2 mRNA and protein expression. PGE2 and PGF2α were produced by neonatal gonocytes and decreased by acetaminophen and ibuprofen. DISCUSSION: The concomitant decrease of Stra8 expression, Cox2 expression, and PGE2 and PGF2a production in gonocytes co-treated with RA suggests that Cox2 plays a role in PND3 gonocyte differentiation. The effects of acetaminophen and ibuprofen on proliferation suggest a negative relationship between Cox2 and proliferation. Treating neonates with acetaminophen or ibuprofen could disrupt gonocyte development, leading to adverse reproductive effects. CONCLUSION: Understanding COX2 role in neonatal gonocytes and the potential risk of acetaminophen and ibuprofen treatment of infants may help prevent male reproductive pathologies.

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini-pig.

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.

C89 Induces Autophagy of Female Germline Stem Cells via Inhibition of the PI3K-Akt Pathway In Vitro.

Postnatal female germline stem cells (FGSCs) are a type of germline stem cell with self-renewal ability and the capacity of differentiation toward oocyte. The proliferation, differentiation, and apoptosis of FGSCs have been researched in recent years, but autophagy in FGSCs has not been explored. This study investigated the effects of the small-molecule compound 89 (C89) on FGSCs and the underlying molecular mechanism in vitro. Cytometry, Cell Counting Kit-8 (CCK8), and 5-ethynyl-2'-deoxyuridine (EdU) assay showed that the number, viability, and proliferation of FGSCs were significantly reduced in C89-treated groups (0.5, 1, and 2 µM) compared with controls. C89 had no impact on FGSC apoptosis or differentiation. However, C89 treatment induced the expression of light chain 3 beta II (LC3BII) and reduced the expression of sequestosome-1 (SQSTM1) in FGSCs, indicating that C89 induced FGSC autophagy. To investigate the mechanism of C89-induced FGSC autophagy, RNA-seq technology was used to compare the transcriptome differences between C89-treated FGSCs and controls. Bioinformatics analysis of the sequencing data indicated a potential involvement of the phosphatidylinositol 3 kinase and kinase Akt (PI3K-Akt) pathway in the effects of C89's induction of autophagy in FGSCs. Western blot confirmed that levels of p-PI3K and p-Akt were significantly reduced in the C89- or LY294002 (PI3K inhibitor)-treated groups compared with controls. Moreover, we found cooperative functions of C89 and LY294002 in inducing FGSC autophagy through suppressing the PI3K-Akt pathway. Taken together, this research demonstrates that C89 can reduce the number, viability, and proliferation of FGSCs by inducing autophagy. Furthermore, C89 induced FGSC autophagy by inhibiting the activity of PI3K and Akt. The PI3K-Akt pathway may be a target to regulate FGSC proliferation and death.

Differentiation of spermatogonial stem cells by soft agar three-dimensional culture system.

Since proliferation and differentiation of spermatogonial stem cells (SSCs) in culture system provide successful transplantation in this study, culture of human SSCs was compared to SACS (soft agar culture system), gelatin and control groups. The cells were isolated from seminiferous tubules of non-azoospermia patients (NOA) and cultured in DMEM for 3 weeks. The presence of SSCs in culture system was confirmed by immunocytochemistry of GFR-α1 and ITGα6 antibodies. The proliferated cells were cultured in three mentioned groups in the presence of retinoic acid and Sertoli cells conditioned medium for another 2 weeks. The number of colonies in the SACS group was significantly higher than two other groups. Before 2 weeks of culture, only Oct4 expression was observed in testicular cells (2.32 ± 0.25). After 2 weeks, the expression of Oct4 in the gelatin group was higher than that of the SACS group on day 7. The maximum expression of Stra8 was observed in SACS and gelatin groups after 7 days, but its expression was significantly decreased after 14 days of culture (p < .05). The expression of Scp3 and Acrosin genes were higher after 14 days in the SACS group compared to other groups. SACS has positive effects on proliferation and differentiation of hSSCs.