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Copper ionophore NSC319726 has attracted researchers' attention in treating diseases, particularly cancers. However, its potential effects on male reproduction during medication are unclear. This study aimed to determine whether NSC319726 exposure affected the male reproductive system. The reproductive toxicity of NSC319726 was evaluated in male mice following a continuous exposure period of 5 weeks. The result showed that NSC319726 exposure caused testis index reduction, spermatogenesis dysfunction, and architectural damage in the testis and epididymis. The exposure interfered with spermatogonia proliferation, meiosis initiation, sperm count, and sperm morphology. The exposure also disturbed androgen synthesis and blood testis barrier integrity. NSC319726 treatment could elevate the copper ions in the testis to induce cuproptosis in the testis. Copper chelator rescued the elevated copper ions in the testis and partly restored the spermatogenesis dysfunction caused by NSC319726. NSC319726 treatment also decreased the level of retinol dehydrogenase 10 (RDH10), thereby inhibiting the conversion of retinol to retinoic acid, causing the inability to initiate meiosis. Retinoic acid treatment could rescue the meiotic initiation and spermatogenesis while not affecting the intracellular copper ion levels. The study provided an insight into the bio-safety of NSC319726. Retinoic acid could be a potential therapy for spermatogenesis impairment in patients undergoing treatment with NSC319726., (© 2024. The Author(s).)

The self-renewal of spermatogonial cells (SCs) provides the foundation for life-long spermatogenesis. To date, only a few growth factors have been used for the culture of SCs in vitro, and how to enhance proliferation capacity of SCs in vitro needs further research. This study aimed to explore the effects of periostin (POSTN) on the proliferation of human SCs. GC-1 spg cells were cultured in a medium with POSTN, cell proliferation was evaluated by MTS analysis and EdU assay, and the Wnt/β-catenin signaling pathway was examined. Thereafter, the proliferations of human SC were detected using immunofluorescence and RT-PCR. In this study, we found that CM secreted by human amniotic mesenchymal stem cells (hAMSCs) could enhance the proliferation capacity of mouse GC-1 spg cells. Label-free mass spectrometry and ELISA analysis demonstrated that high level of POSTN was secreted by hAMSCs. MTS and EdU staining showed that POSTN increased GC-1 spg cell proliferation, whereas CM from POSTN-silenced hAMSCs suppressed cell proliferation capacity. Then POSTN was found to activate the Wnt/β-catenin signaling pathway to regulate the proliferation of GC-1 spg cells. XAV-939, a Wnt/β-catenin inhibitor, partially reversed the effects of POSTN on GC-1 spg cell proliferation. We then analyzed human SCs and found that POSTN promoted human SC proliferation in vitro. These findings provide insights regarding the role of POSTN in regulating SC proliferation via the Wnt/β-catenin signaling pathway and suggest that POSTN may serve as a cytokine for male infertility therapy., (© 2021. Society for Reproductive Investigation.)

Background: Mammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival., Methods: We investigated the novel role of β-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, β-estradiol, or the combination of β-estradiol and the estrogen receptor inhibitor ICI 182780., Results: In the system with purified human fetal Sertoli cells (MIS+/c-Kit-/AP-), β-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), β-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by β-estradiol and was completely reversed by the combination of β-estradiol and ICI 182780., Conclusion: Here we report, for the first time, that β-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Spermatogonial stem cell transplantation (SSCT) is a strategy that has demonstrated to be feasible to restore spermatogenesis in animal models when it is performed shortly after the gonadotoxic onset to destroy their endogenous germ cells. However, in the case of boys subjected to fertility preservation, future transplantations will be performed with a delay of many years. In order to study how timing of SSCT affects donor-derived spermatogenic recovery in mice, we compared the percentage of spermatogenic tubule cross-sections within testes of 59 C57BL/6NCrl mice distributed in 6 groups: group 1, untreated mice controls (n = 9); group 2, mice that received a single dose of busulfan 40 mg/kg (n = 10); group 3, mice that received two additional doses of busulfan 10 mg/kg every 5 weeks (n = 10); group 4 (SSCT-A), mice subjected to a standard SSCT performed 5 weeks after a single injection of busulfan 40 mg/kg (n = 10); group 5 (SSCT-B), mice subjected to a delayed SSCT performed 15 weeks after a single injection of busulfan 40 mg/kg (n = 10); and group 6 (SSCT-C), mice subjected to a delayed SSCT with two additional doses of busulfan 10 mg/kg every 5 weeks (n = 10). Spermatogenic recovery in standard SSCT-A and SSCT-C groups ranged between 22.29 and 22.65%, compared with a lower recovery rate of 11.54% showed in the SSCT-B group. However, donor contribution resulted higher in standard SSCT-A, representing a 69.71% of cross-sections, compared with the rest of conditions ranging from 34.69 to 35.42%. Overall, we concluded that a delay in the SSCT from the gonadotoxic onset decreases the efficiency of donor-derived spermatogenic recovery in mice.

A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3 -/- testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3 -/- males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis.

Optimization of tissue culture systems able to complete male germ cell maturation to post-meiotic stages is considered as an important matter in reproductive biology. Considering that hypoxia is one of the factors limiting the efficiency of organ culture, the aim of this study was to use isolated seminiferous tubules (STs), having more surface and less thickness, in an organotypic culture system in order to improve oxygen diffusion and reduce hypoxia. The mechanically separated STs embedded in agarose or alginate and 1-3-mm 3 testicular tissue fragments of 3 adult mice were separately placed on the flat surface of agarose gel that was half-soaked in the medium. Survival and differentiation of germ cells using PLZF and SCP3 markers, identity of Sertoli cell using GATA4, cell proliferation with the Ki67 marker, and ST integrity using a ST scoring were evaluated up to 36 d at different culture times, each corresponding to the duration of one spermatogenic cycle. We observed a significantly reduced ST integrity in STs embedded in agarose or alginate on day 9 (versus tissue fragments p ≤ 0.05). There was no difference in the number of PLZF-positive cells between groups, but the number of SCP3 (in all-time points) and GATA4-positive cells was significantly higher in the culture of embedded STs. Although embedding STs can be useful for the progress of in vitro spermatogenesis, it makes them sensitive to degeneration. Further improvements are required to modify the air-liquid interface method to maintain ST integrity.

Course administration streptozotocin to male C57Bl/6 mice induces a complex of symptoms typical of type 1 diabetes mellitus: hyperglycemia and insulin deficiency, focal inflammatory infiltration of the pancreas, destructive changes in the Langerhans islets, damage to the insular apparatus (reduced number of PDX1 + cells and insulin expression by the secreting cells). Male reproductive disorder are serious complications of type 1 diabetes mellitus. In "diabetic" mice, interstitial edema with inflammatory infiltration and microvascular disorders in the testicular tissue are observed, the number of endothelial precursors (CD45 - /CD31 + ) and the total number and percentage of motile spermatozoa decreased, immature spermatogenic epithelium cells are desquamated of into the lumen of the tubules. Disturbances in the proliferation and differentiation of various spermatogonial stem cell populations (c-kit - /CD90 + , c-kit + /CD90 + , and CD51 - /CD24 + /CD52 + ) in diabetes can be explained by the inhibitory influence of inflammatory factors on testosterone-producing Leydig cells.

The properties of spermatogonial stem cells, endothelial progenitor cells, and the epithelial progenitors of C57Bl/6 mice under conditions of metabolic disorders were studied using the model of busulfan-induced suppression of spermatogenesis and in vitro culture technique. Spermatogonial stem cells CD117-CD90 + and epithelial progenitors CD45-CD31-Sca-1 + CD49f + derived from the testes of mice with metabolic disturbances demonstrated 17- and 28-fold increase in the respective cell mass and generated cell colonies in vitro. In contrast, spermatogonial stem cells with immune phenotype CD51-CD24 + CD52 + had reduced selfrenewal capacity. Spermatogonial stem cells CD117-CD90 + and CD117 + CD90 + as well as endothelial progenitors CD45-CD31 + derived from the testes of donor mice with metabolic disorders demonstrated high transplantation capacity in C57Bl/6 mouse testes damaged by cytostatic busulfan.

The regenerative potential of stem and progenitor cells from ischemic testes of C57Bl/6 mice was studied in vitro (cell culture) and in vivo (mouse model of busulfan-induced suppression of spermatogenesis). Spermatogonial stem cells with phenotypes CD117 - CD90 + and CD51 - CD24 + CD52 + from ischemic testes demonstrated 33-fold and 7-fold increments of cell mass and generated colonies in vitro. Epithelial (CD45 - CD31 - Sca-1 + CD49f + ) and endothelial (CD45 - CD31 + ) precursors exhibited lower self-renewal capacity. On day 30 after injection of stem and progenitor cells from ischemic testes to the rete testis zone of the testes of busulfantreated animals, an increase in the count of CD117 - CD90 + spermatogonial stem cells, total count, and mobile sperm count in the testes of recipient mice was observed. In addition, we observed an increase in Sca-1 + cell count, recovery of the spermatogenic epithelium in the seminiferous tubules, and appearance of immature Leydig cells in "busulfan" testes; the level of tissue testosterone and fertility index also increased.

Effectiveness of the granulocyte colony-stimulating factor immobilized by using electronbeam synthesis nanotechnology was investigated on the model of experimental testicular failure caused by the toxic effect on stem spermatogonia. Administration of the drug to experimental paclitaxel-treated animals increased the number of sources of the proliferative pool of spermatogenesis and its productivity. The effectiveness of immobilized granulocyte colony-stimulating factor was based on its ability to stimulate reparative regeneration of the spermatogenic tissue, which manifested in a decrease in spermatogenic layer maturity and increase in the number of microenvironment cells. Effectiveness of the immobilized form of the drug was superior to that of non-immobilized form.

Licorice (glycyrrhiza uralensis) is known as an herb with detoxication, and it has been widely used in clinical prescription of Oriental herbal medicine. Studies on the effects of licorice in the reproductive system were very rare, especially in spermatogenesis. In order to elucidate the effects of licorice on spermatogonial proliferation and spermatocyte differentiation during neonatal mice spermatogenesis, the organ culture model of testis tissue from neonatal C57BL/6N mice (born 6 d) was established. Then, in the presence of licorice extract (LE), the proliferation activity of spermatogonia was identified with the positive rate quantitative analysis of 5-bromo-2-deoxyuridine (BrdU) and anti-proliferating cell nuclear antigen (PCNA) antibody by immunohistochemical staining. The results showed that, compared to the control group, the percentage of positive cells by BrdU staining enhanced dramatically and that the expression of PCNA protein increased significantly in the spermatogonia from the LE group and showed a concentration-dependent manner (P < 0.05). This indicated that the LE can significantly promote the proliferation of spermatogonia in the spermatogenesis of neonatal mice. Furthermore, proteins related to spermatocyte differentiation, synaptonemal complex protein 3 (SCP3) and meiotic recombinant protein Spo11, were detected by immunohistochemical staining. The results showed that the differentiated spermatocyte in the LE group was significantly increased compared with that of the control group and showed a concentration-dependent manner (P < 0.05). The above results suggested that the LE can significantly accelerate the proliferation of spermatogonia and the differentiation of spermatocytes in the testicular tissue of the neonatal mice, which may be a potential drug for male infertility.

A decrease in the total number of sperm cells and reduction of spermatogonium population were observed upon cytostatic damage of spermatogenous tissue caused by single paclitaxel administration. It was established that the paclitaxel-induced damage to the testicular tissue is underlain by reduction of its regenerative potential consisting in depletion of regional precursor pool. Changes in functional activity of progenitor cells were caused not only by direct action of paclitaxel, but also by suppression of the secretory function of the tissue microenvironment.

Population of spermatogonia was reduced in 2, 3, and 6 months after single intravenous injection of antitumor drug paclitaxel in maximum tolerated dose (MTD). The count of Sertoli cell increased in 3 months after the start of the experiment. The maturity of the seminiferous tubule epithelium was lower than in intact rats. Spermatogenesis productivity did not differ from that in intact animals 6 months after start of the experiment. These data indicate that regeneration of the spermatogenous tissue after paclitaxel treatment is realized via renewal of the spermatogenic epithelium, but considering the amount of spermatogonial cell population, the recovery rate would be low.

Purpose: This study aims to proliferate spermatogonial stem cells (SSCs) and compare the in-vitro effects of laminin and growth factors on the proliferation of adult human SSC., Methods: Isolated testicular cells were cultured in DMEM supplemented with 5 % fetal calf serum (FCS). During the culture, enriched spermatogonial cells were treated with a combination of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and mouse leukemia inhibitory factor (LIF) in the presence or absence of human placental laminin-coated dishes. Cluster assay was performed during culture. Presence of spermatogonia was determined by an ultrastructural study of the cell clusters, reverse transcription polymerase chain reaction (RT-PCR) for spermatogonial markers and xenotransplantation to the testes of busulfan-treated recipient mice. Statistical significance between mean values was determined using statistical ANOVA tests., Results: The findings indicated that the addition of GDNF, bFGF, EGF and LIF on laminin-coated dishes significantly increased in-vitro spermatogonial cell cluster formation in comparison with the control group (p ≤ 0.001). The expression of spermatogonial markers was maintained throughout the culture period. Furthermore, a transplantation experiment showed the presence of SSC among the cultured cells. In addition, a transmission electron microscopy (TEM) study suggested the presence of spermatogonial cells of typical morphology among the cluster cells., Conclusions: It can be concluded that human SSCs obtained from non-obstructive azoospermic (NOA) patients had the ability to self-renew in the culture system. This system can be used for the propagation of a small number of these cells from small biopsies.

The aim of this study was to clarify effects of inflammation on spermatogenesis in LPS-administered mice. ICR mice were treated by intraperitoneal injection for 7 days with either physiological saline (control) or 0.1 mg lipopolysaccharide (LPS)/kg body weight/day. Control mice were killed at 24 h after the last injection and the LPS-treated group after 24 h or 1, 3, or 5 weeks. Sperm concentration and motility in the cauda epididymis were examined as well as immunohistochemical localization of Fas and FasL and germ cell apoptosis. Sperm concentration and motility markedly fluctuated in LPS-treated mice. Increase of apoptotic cells was common in all post-LPS treatment groups, with a peak at 24 h after LPS injection. In contrast to the lack of Fas immunoreactivity in control testes, LPS-treated groups demonstrated Fas in many germ cells, especially in spermatocytes and spermatids. Immunoreactivity for FasL, on the other hand, was positive for some Sertoli cells, Leydig cells, and germ cells in both control and LPS-treated groups at all time points. The results suggest that the Fas/FasL system mediates apoptosis of germ cells in LPS-treated mice testes. LPS-administered mice thus provide a good experimental model for the study of transient disruption of spermatogenesis.

Massive apoptosis of pubertal male germ cells is important for the development of functional spermatogenesis in the adult testis. Although the trigger(s) for male germ cell loss at puberty remain undefined, we have hypothesized that transforming growth factor-betas (TGF-betas) play an active role. Here we demonstrate that the three mammalian TGF-beta isoforms, TGF-beta1, TGF-beta2 and TGF-beta3, induce distinct apoptosis of pubertal spermatogonia and spermatocytes in a dose-dependent manner. Induction of male germ cell death by activation of caspase-3 was most pronounced with TGF-beta2 compared to TGF-beta1 and TGF-beta3. Furthermore, we found colocalization of activated caspase-3 with apoptotic protease-activating factor-1 (Apaf-1) in apoptotic germ cells, thus indicating the importance of the intrinsic mitochondrial pathway in TGF-beta-induced apoptosis. The specificity of the TGF-beta effects was proven by addition of recombinant latency-associated peptide against TGF-beta1 (rLAP-TGF-beta1) which completely abolished TGF-beta1-induced and TGF-beta3-induced germ cell apoptosis. Although TGF-beta2-triggered germ cell death also was significantly reduced by rLAP-TGF-beta1, inhibition was not maximal. Our results suggest that the three TGF-beta isoforms induce apoptosis of pubertal male germ cells via the mitochondrial pathway in vitro and are thus likely candidates involved in the excessive first wave of apoptosis of male germ cells during puberty.

Mitotic index of type B spermatogonia in intact animals is characterized by a circadian rhythm. Mitotic index increased and its circadian rhythm disappeared after pinealectomy. Treatment with Epithalamin for 14 days restored the circadian rhythm. The circadian biorhythm of spermatogonia proliferation suggests the presence of circadian rhythm of spermatogenesis in general and its regulation by the pineal gland.

The quantitative micronucleus test showed that the natural dipeptide carnosine increases the count of aberrant spermatogonia and round spermatids in the testes in SAMR1 mice resistant to accelerated aging by 64 and 85%, respectively, compared to the control. However, this agent did not modify the incidence of chromosome mutations in spermatogenic cells in SAMP1 mice predisposed to accelerated aging.