Differentiation Of Spermatogonial Cells Research Articles

The effect of epididymosomes on the development of frozen-thawed mouse spermatogonial stem cells after culture in a decellularized testicular scaffold and transplantation into azoospermic mice.

This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice. Epididymosomes were extracted and characterized using SEM and western blotting. After cryopreservation, thawed SSCs were cultured in a hydrogel-based three-dimensional (3D) culture containing 10 ng/mL GDNF or 20µg/mL epididymosomes. SSCs were assessed using the MTT assay, flow cytometry, and qRT-PCR after two weeks of culture. The isolated SSCs were microinjected into the efferent ducts of busulfan-treated mice. DiI-labeled SSCs were followed, and cell homing was assessed after two weeks. After 8 weeks, the testes were evaluated using morphometric studies and immunohistochemistry. The expression of PLZF, TGF-β, and miR-10b did not increase statistically significantly in the 3D + GDNF and 3D + epididymosome groups compared to the 3D group. Among the groups, the GDNF-treated group exhibited the highest expression of miR-21 (*P < 0.05). Caspase-3 expression was lower in the epididymosome-treated group than in the other groups (***P < 0.001). Compared to the 3D and negative control groups, the 3D + epididymosomes and 3D + GDNF groups showed an increase in spermatogenic cells. Immunohistochemical results confirmed the growth and differentiation of spermatogonial cells into spermatids in the treatment groups. The DTM hydrogel containing 20µg/mL epididymosomes or 10 ng/mL GDNF is a novel and safe culture system that can support SSC proliferation in vitro to obtain adequate SSCs for transplantation success. It could be a novel therapeutic agent that could recover deregulated SSCs in azoospermic patients.

The Expression Levels and Cellular Localization of Pigment Epithelium Derived Factor (PEDF) in Mouse Testis: Its Possible Involvement in the Differentiation of Spermatogonial Cells.

Pigment epithelium derived factor (PEDF) is a multifunctional secretory soluble glycoprotein that belongs to the serine protease inhibitor (serpin) family. It was reported to have neurotrophic, anti-angiogenic and anti-tumorigenic activity. Recently, PEDF was found in testicular peritubular cells and it was assumed to be involved in the avascular nature of seminiferous tubules. The aim of this study was to determine the cellular origin, expression levels and target cells of PEDF in testicular tissue of immature and adult mice under physiological conditions, and to explore its possible role in the process of spermatogenesis in vitro. Using immunofluorescence staining, we showed that PEDF was localized in spermatogenic cells at different stages of development as well as in the somatic cells of the testis. Its protein levels in testicular homogenates and Sertoli cells supernatant showed a significant decrease with age. PEDF receptor (PEDF-R) was localized within the seminiferous tubule cells and in the interstitial cells compartment. Its RNA expression levels showed an increase with age until 8 weeks followed by a decrease. RNA levels of PEDF-R showed the opposite trend of the protein. Addition of PEDF to cultures of isolated cells from the seminiferous tubules did not changed their proliferation rate, however, a significant increase was observed in number of meiotic/post meiotic cells at 1000 ng/mL of PEDF; indicating an in vitro differentiation effect. This study may suggest a role for PEDF in the process of spermatogenesis.

The Effect of 8-Weeks of Low-Intensity Swimming Training on Promyelocytic Leukemia Zinc Finger Protein and Spermatid Transition Nuclear Protein Gene Expression in Azoospermic Rats Model

Aims: One of the causes of infertility in men is the azoospermia disease, which is attributed to the lack of sperm in each sperm. The primary function of spermatogenesis is the maintenance, proliferation, and differentiation of spermatogonial cells. Thus, the present study aimed to investigate the changes in Promyelocytic Leukemia Zinc Finger (PLZF) and spermatid Transition Nuclear Protein (TNP) gene expression levels in an azoospermic rat model after 8 weeks of low-intensity aerobic training. Methods &amp; Materials: In this experimental study, 15 adult male Wistar rats were randomly divided into three groups of healthy control, with azoospermia, and exercise plus azoospermia after creating an azoospermia model. The patient plus exercise group performed a low-intensity swimming exercise 30 minutes a day, five days a week for 8 weeks, after the creation of the azoospermic rats. A One-way ANOVA test was used for data analysis. Findings: The results showed that a period of swimming exercise program in the exercise plus azoospermia group significantly reduced PLZF gene expression compared to the healthy control groups (P=0.001) and no significant increase to the azoospermia group (P=0.06). There was also a significant decrease in TNP gene expression levels in the exercise plus azoospermia group compared to the healthy control group (P=0.001) and a significant increase in the azoospermia group (P=0.057). Conclusion: Based on these Findings, it can be stated that the alteration of key molecules or signaling pathways and expression of the PLZF and TNP genes in the spermatogenesis process may increase infertility, but regular aerobic exercise, such as low-intensity swimming, helps to control the effects of infertility by increasing the maintenance and development of spermatogonial stem cells.

Cold seawater induces early sexual developmental stages in the BPG axis of European eel males

BackgroundThe impossibility of closing the life cycle of the European eel (Anguilla anguilla) in captivity troubles the future of this critically endangered species. In addition, the European eel is a highly valued and demanded resource, thus the successful closing of its life cycle would have a substantial economic and ecological impact. With the aim of obtaining the highest gamete quality, the study of the effects of environmental factors, such as temperature, on reproductive performance may prove valuable. This is especially true for the exposure to cold water, which has been reported to improve sexual development in multiple other Actinopterygii species.ResultsEuropean eel males treated with cold seawater (10 °C, T10) for 2 weeks showed an increase in the proliferation and differentiation of spermatogonial cells until the differentiated spermatogonial type A cell stage, and elevated testosterone and 11-ketotestosterone plasma levels. Transcriptomes from the tissues of the brain-pituitary-gonad (BPG) axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification, meiotic nuclear division, and others.ConclusionsCold seawater treatment had a clear effect on the activity of the BPG-axis of European eel males. In particular, our cold seawater treatment induces the synchronization and increased proliferation and differentiation of specific spermatogonial cells. In the transcriptomic results, genes related to thermoception were observed. This thermoception may have caused the observed effects through epigenetic mechanisms, since all analysed tissues further revealed differentially expressed genes involved in histone modification. The presented results support our hypothesis that a low temperature seawater treatment induces an early sexual developmental stage in European eels. This hypothesis is logical given that the average temperature experienced by eels in the early stages of their oceanic reproductive migration is highly similar to that of this cold seawater treatment. Further studies are needed to test whether a cold seawater treatment can improve the response of European eels to artificial hormonal treatment, as the results suggest.

Roles for osteocalcin in proliferation and differentiation of spermatogonial cells cocultured with somatic cells.

Spermatogonial cells (SCs) are key cells for spermatogenesis. These cells are affected by paracrine signals originated from nearby somatic cells, among them Leydig cells have receptors for osteocalcin, a hormone known for exerting positive roles in the promotion of spermatogenesis. The aim of this study was to evaluate roles for osteocalcin on SCs proliferative and differentiation features after coculture with Leydig cells. SCs and Leydig cells were isolated from neonate NMRI offspring mice and adult NMRI mice, respectively. SCs population were then enriched in a differential attachment technique and assessed for morphological features and identity. Then, SCs were cocultured with Leydig cells and incubated with osteocalcin for 4 weeks. Evaluation of proliferation and differentiation-related factors were surveyed using immunocytochemistry (ICC), Western blot, and quantitative real-time polymerase chain reaction (PCR). Finally, the rate of testosterone release to the culture media was measured at the end of 4th week. Morphological and flow cytometry results showed that the SCs were the population of cells able to form colonies and to express ID4, α6-, and β1-integrin markers, respectively. Leydig cells were also able to express Gprc6α as a specific marker for the cells. Incubation of SCs/Leydig coculture with osteocalcin has resulted in an increase in the rate of expressions for differentiation-related markers. Levels of testosterone in the culture media of SCs/Leydig was positively influenced by osteocalcin. It could be concluded that osteocalcin acts as a positive inducer of SCs in coculture with Leydig cells probably through stimulation of testosterone release from Leydig cells and associated signaling.

The application of the improved 3D rat testicular cells co-culture model on the in vitro toxicity research of HZ1006

The aims of the present research are to further validate the application of the improved three-dimensional (3 D) rat testicular cell co-culture model to evaluate the effects of various reprotoxic chemicals on the function of the main somatic cells, as well as on spermatogonial cell differentiation and even spermatogenesis, and to investigate the specific toxicant mechanisms in testes treated with HZ1006, a hydroxamate-based a hydroxamate-based histone deacetylase inhibitor (HDACI). Based on the characteristics of HZ1006, the appropriate exposure duration (8, 16, or 24 days), dosage (0, 3.125, 6.25, 12.5, or 25 μM) and toxic endpoints suitable for detection were selected in the experiments. The results showed inhibition of cell proliferation, reduced testosterone levels, and decreased spermatogonial cell meiosis-specific gene expression, as well as decreased protein levels of androgen receptor (AR) and decreased expression of the AR target gene PSA, accompanied by inhibition of Hdac6 expression after HZ1006 exposure in the 3 D rat testicular cell co-culture model. These findings indicate that the improved 3 D rat testicular cell co-culture model we have established has the potential to become a new testicular toxicity test system that can be used to test toxic characteristics and mechanisms of new compounds and has good application prospects, although more research on the model is required.

Mrhl Long Noncoding RNA Mediates Meiotic Commitment of Mouse Spermatogonial Cells by Regulating Sox8 Expression.

Long noncoding RNAs (lncRNAs) are important regulators of various biological processes, including spermatogenesis. Our previous studies have revealed the regulatory loop of mrhl RNA and Wnt signaling, where mrhl RNA negatively regulates Wnt signaling and gets downregulated upon Wnt signaling activation. This downregulation of mrhl RNA is important for the meiotic progression of spermatogonial cells. In our present study, we identified the transcription factor Sox8 as the regulatory link between mrhl RNA expression, Wnt signaling activation, and meiotic progression. In contrast to reports from other groups, we report the expression of Sox8 in germ cells and describe the molecular mechanism of Sox8 regulation by mrhl RNA during differentiation of spermatogonial cells. Binding of mrhl RNA to the Sox8 promoter is accompanied by the assembly of other regulatory factors involving Myc-Max-Mad transcription factors, corepressor Sin3a, and coactivator Pcaf. In the context of Wnt signaling, Sox8 directly regulates the expression of premeiotic and meiotic markers. Prolonged Wnt signaling activation in spermatogonial cells leads to changes in global chromatin architecture and a decrease in levels of stem cell markers.

Punicalagin Mollifies Lead Acetate-Induced Oxidative Imbalance in Male Reproductive System.

Punicalagin (PU) is a known antioxidant. The present study examined PU to protect against lead-induced oxidative stress (OS) testicular damage in mice. Significant increase in lipid peroxidation (LPO) after intraperitoneal injection of lead acetate (LA) indicated enormous generation of reactive oxygen species (ROS). Lead-induced OS has a direct effect on the differentiation of spermatogonial cells, showing a significant decline in sperm count. Supplementation of PU significantly changes values of LPO and glutathione (GSH) with a concomitant increase in sperm count, a marked decrease in the abnormal sperms, and a decline in the morphologically abnormal sperm population. Moreover, the histopathological evaluation of testes and epididymides showed severe changes in mice treated with LA. PU significantly induced nuclear factor erythroid-2 related factor 2-like 2 (Nrf2) expression and phase II enzymes, and data suggest that PU may inhibit OS through Nrf2 activation. The fertility test proved that PU might play an important role in male infertility treatment, especially in the type of infertility induced by OS.

Effect of glial cell line-derived neurotrophic factor on mouse spermatogonial stem cells in vitro

Objective To investigate the effect of glial cell line-derived neurotrophic factor(GDNF)on proliferation and differentiation of mouse spermatogonial stem cells in vitro.Methods The percoll discontinue density gradient centrifugation,followed by removing contaminated somatic cells through adhesion to plastic dishes,was used to purify the spermatogonial stem cells of Kunming mice.Mouse spermatogonial stem cells were confirmed by immunofluorescence and flow cytometry.The cells were divided into control groups and exprimental groups.The spermatogonial stem cells were cultured on sertoli cells.GDNF was added to medium of DMEM/F12.The growth of spermatogonial stem cells was determined by ELISA.The cell cycle of spermatogonial stem cells was determined by flow cytometry.Sperm was allied with ovum by intracytoplasmic sperm injection(ICSI).The chromosome figure and quantity were analysed after 3 days.Results A values of spermatogonial stem cells in experimental group were 0.362±0.031,0.448±0.028,0.502±0.062,0.556±0.045,0.621±0.072 in 3,6,9,12,15 days respectively;in control group,they were 0.365±0.045,0.377±0.053,0.402±0.071,0.432±0.019,0.461±0.037 respectively.Significant differences were noted between experimental group and controI group(P＜0.05).S period's quantities of DNA synthesizing were 20.86,26.34,31.23,37.54,28.02 in 3,6,9,12,15 days respectively,while they were 1.69,1.73.2.56,4.85,1.82 in 3,6,9,12,15 days in control group.Significant differences were noted between experimental group and control group(P＜0.05).The dual DNA could be found when sperm was integrated ovum after 3 days.Conclusions ICSI could identify spermatogial stem cells and translate into spermic cells in vitro.GDNF could improve the proliferation and differenciation of the spermatogonial stem cells in vitro. Key words: Spermatogonia; Glial cell line-derived neurotrophic factor; Cell culture; Mice

Mouse testicular gene expression pattern differences between spermatogonial stem cell proliferative and differential stages

Objective To detect the mouse testicular gene expression pattern differences be-tween spermatogonial stem cell (SSC) proliferative and differential stages and study the molecular reg-ulation mechanism in SSC proliferation and differentiation. Methods With the interval of 24 days, male Kunming mice were injected intraperitoneally with two doses of busulfan (10 mg/kg) to establish spermatogenesis regeneration models. 36 k Mouse Genome Array was used to detect the differential gene expression profiles between the stages of SSC proliferation and differentiation. Bioinforrnsties analysis was conducted in GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Ge-nomes) pathway to describe the potential roles that may play in spermatogonial stern cells behavior regulation. Results Nine hundred and eleven differential expression genes were identified by gene arrays in mice testes, consisting of 608 up-regulated and 303 down-regulated in SSC proliferation stage and SSC differentiation stage. The differential expression genes were classified by their biological process, molecular function and cellular component, respectively. Alterations with statistical signifi-cance (P<0.05)appeared in 84 KEG(;signal pathways, including Notch and Wnt signaling pathways which had been proved to be important for stem cell maintenance. Fifty-six differential expression genes were selected as genes related to stem cells, among which 40 genes were up-regulated, including some stem cell biomarkers(such as Cd9, StraS, hgbl-, Oct4 and Thyl)and some growth factors(such as Fgf2, Pdgfa and Csfl). Conclustion The regulation of SSC proliferation and differentiation involves inmany differentially expressed genes in various signal pathways. This study provides a molecular basis for the elucidation of the molecular mechanism behind self-renewal and differentiation of spermatogonial stem cells. Key words: Spermatogonia stem cells; Proliferation; Differentiation; Gene array

Cyclic AMP and genetic sterility of a male restricted (Hre/plus) mutant of Rattus.

Restricted (H re /+) male rats marked by a coat color pattern have normal testes at birth. By 9 days postpartum, testes of the mutant animals are smaller than normal and by approximately 90 days of age the animals are sterile. The genetically sterile testes are totally devoid of spermatogonial cells, spermatocytes, spermatids, and spermatozoa, with only Sertoli cells remaining in the seminiferous tubules. Cyclic AMP concentrations in the whole testes (and the seminiferous tubules) of the mutant males are approximately 10–35% greater than in testes of control males when tested at intervals from 5 to 120 days of age. The possible role of excess cyclic AMP in reducing the rate of mitotic division of spermatogonial cells while enhancing differentiation of spermatogonial cells into spermatozoa is discussed. Such a change in the respective rates of mitotic and meiotic divisions would ultimately deplete the mutant testes of all spermatogonial cells.