Search Results (23)

Background: Oxidative stress is a critical factor contributing to male infertility, impairing spermatogonial stem cells (SSCs) and disrupting normal spermatogenesis. This study aimed to isolate and characterize human SSCs and to investigate oxidative stress-related gene expression, protein interaction networks, and developmental trajectories involved in SSC function. Methods: SSCs were enriched from human orchiectomy samples using CD49f-based magnetic-activated cell sorting (MACS) and laminin-binding matrix selection. Enriched cultures were assessed through morphological criteria and immunocytochemistry using VASA and SSEA4. Transcriptomic profiling was performed using microarray and single-cell RNA sequencing (scRNA-seq) to identify oxidative stress-related genes. Bioinformatic analyses included STRING-based protein–protein interaction (PPI) networks, FunRich enrichment, weighted gene co-expression network analysis (WGCNA), and predictive modeling using machine learning algorithms. Results: The enriched SSC populations displayed characteristic morphology, positive germline marker expression, and minimal fibroblast contamination. Microarray analysis revealed six significantly upregulated oxidative stress-related genes in SSCs—including CYB5R3 and NDUFA10—and three downregulated genes, such as TXN and SQLE, compared to fibroblasts. PPI and functional enrichment analyses highlighted tightly clustered gene networks involved in mitochondrial function, redox balance, and spermatogenesis. scRNA-seq data further confirmed stage-specific expression of antioxidant genes during spermatogenic differentiation, particularly in late germ cell stages. Among the machine learning models tested, logistic regression demonstrated the highest predictive accuracy for antioxidant gene expression, with an area under the curve (AUC) of 0.741. Protein oxidation was implicated as a major mechanism of oxidative damage, affecting sperm motility, metabolism, and acrosome integrity. Conclusion: This study identifies key oxidative stress-related genes and pathways in human SSCs that may regulate spermatogenesis and impact sperm function. These findings offer potential targets for future functional validation and therapeutic interventions, including antioxidant-based strategies to improve male fertility outcomes. Full article

Black rockfish (Sebastes schlegelii) is a marine ovoviviparous teleost that exhibits significant sexual dimorphism, with females growing faster and reaching larger sizes than males. Establishing stable oogonial stem cells (OSCs) is critical for understanding germline stem cell dynamics and facilitating all-female breeding. In this study, we successfully isolated and cultured OSCs from S. schlegelii for 12 passages. These cells exhibited alkaline phosphatase activity, expressed germline marker genes (ddx4, cdh1, klf4), and maintained a diploid karyotype (2n = 48). Transcriptomic comparisons between early (P3) and late (P12) passages revealed significant metabolic dysfunction and cell cycle arrest in the late-passage cells. Specifically, the down-regulation of glutathione-related and glycolysis-related genes (gstm3, gstt1, mgst3, gsta1, gsta4, gsto1, gapdh) and key mitotic regulators (cdk1, chk1, cdk4, e2f3, ccne2, ccnb1) suggested that metabolic imbalance contributes to oxidative stress, resulting in cell cycle inhibition and eventual senescence. This study provides a marine fish model for investigating metabolism-cell cycle interactions in germline stem cells and lays the foundation for future applications in germ cell transplantation and all-female breeding. Full article

The germline stem cells (GSCs) in the male gonad of Drosophila can increase their division frequency in response to a demand for more sperm caused by repeated mating. However, the molecules and mechanisms regulating and mediating this response have yet to be fully explored. Here, we present the results of a transcriptome analysis comparing expression from the testis tips from non-mated and mated males. An overlapping set of 18 differentially expressed genes (DEGs) from two independent wild-type (wt) strains revealed that the majority of the DEGs encode secreted proteins, which suggests roles for them in cell–cell interactions. Consistent with a role for secretion in regulating GSC divisions, knocking down Signal Recognition Particle (SRP) components within the germline cells using RNA Interference (RNAi), prevented the increase in GSC division frequency in response to mating. The major class of DEGs encodes polypeptides below the size of 250 amino acids, also known as small proteins. Upon reducing germline expression of small proteins, males no longer increased GSC division frequency after repeated mating. We hypothesize that mating induces cellular interactions via small proteins to ensure continued GSC divisions for the production of sperm. Full article

Olive flounder (Paralichthys olivaceus) is a commercially important fish species in Japan, China, and the Republic of Korea. Despite numerous attempts to improve productivity, there have been no studies of in vitro germline stem cell (GSC) culture in this species. Here, olive flounder testicular and ovarian cell lines (OFT and OFO, respectively) were established and characterized. RT-PCR demonstrated that OFT and OFO expressed several gonadal somatic cell markers, including wt1 and fgf2, but lacked expression of germ cell markers, such as vasa, nanos2, and scp3. In addition, SNP analysis revealed that OFT originated from XY male P. olivaceus and OFO originated from XX female P. olivaceus. These results suggest that OFT was composed of Sertoli cells and OFO was composed of granulosa cells and theca cells. Finally, coculture of OFT or OFO with enriched male P. olivaceus GSCs isolated from the top 20% and 20–30% Percoll density gradient layers showed that GSCs were attached on both cell lines. In conclusion, we established P. olivaceus testicular and ovarian cell lines, which were expected to use for development of an in vitro GSC culture system. Full article

Stem cells are essential for the proper functioning of tissues, replacing damaged, senescent cells to ensure tissue regeneration. However, as age advances, the number of these stem cells can change, and their self-renewal abilities can become impaired, leading to disruption of homeostasis, loss of regenerative capacity, and, ultimately, deterioration of tissue function. In Drosophila testis, in addition to the germline and somatic cells involved in spermatogenesis, there are immune cells (hemocytes) with macrophage function. In our study, we aimed to investigate the role of hemocytes in maintaining germline stem cells throughout their lifespan. Our results show that in the absence of plasmatocytes and crystal immune cells, the number of germline stem cells (GSCs) and apoptotic germline cells also increases significantly during senescence, which may have detrimental effects on the differentiation processes of germline cells. The size of the hub increases in aged male testes. It is therefore conceivable that changes in the hub may induce dysfunction of differentiation processes. The fertility of aged immunodeficient animals is decreased. Furthermore, we show that the expression of the JAK/STAT signaling pathway, which is essential for the maintenance of the stem cell niche, is impaired in the lack of hemocytes. We found an increased expression of Socs36e, an inhibitor of JAK-STAT, which correlates with decreased JAK-STAT activity. Overexpression of Socs36e in the apical part of the germline led to a phenotype similar to the immunodeficient aged germline, where an increased GSC number and hub size were also observed. However, spermatogenesis was also disturbed in this case. Our study shows that hemocytes are required to regulate the number of GSCs. This regulation could be mediated through the JAK-STAT signaling pathway. These results may help to provide a more complex insight into the relationships between immune cells and stem cells. Full article

Drosophila spermatogenesis involves the renewal of germline stem cells, meiosis of spermatocytes, and morphological transformation of spermatids into mature sperm. We previously demonstrated that Ocnus (ocn) plays an essential role in spermatogenesis. The ValRS-m (Valyl-tRNA synthetase, mitochondrial) gene was down-regulated in ocn RNAi testes. Here, we found that ValRS-m-knockdown induced complete sterility in male flies. The depletion of ValRS-m blocked mitochondrial behavior and ATP synthesis, thus inhibiting the transition from spermatogonia to spermatocytes, and eventually, inducing the accumulation of spermatogonia during spermatogenesis. To understand the intrinsic reason for this, we further conducted transcriptome-sequencing analysis for control and ValRS-m-knockdown testes. The differentially expressed genes (DEGs) between these two groups were selected with a fold change of ≥2 or ≤1/2. Compared with the control group, 4725 genes were down-regulated (dDEGs) and 2985 genes were up-regulated (uDEGs) in the ValRS-m RNAi group. The dDEGs were mainly concentrated in the glycolytic pathway and pyruvate metabolic pathway, and the uDEGs were primarily related to ribosomal biogenesis. A total of 28 DEGs associated with mitochondria and 6 meiosis-related genes were verified to be suppressed when ValRS-m was deficient. Overall, these results suggest that ValRS-m plays a wide and vital role in mitochondrial behavior and spermatogonia differentiation in Drosophila. Full article

Species of the genus Drosophila have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are under-investigated. Here, we performed an analysis of hybrid female sterility by crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis and molecular, cellular, and genetic approaches, we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Because of the differential expression of genes encoding piRNA pathway components, rhino and deadlock, the functional RDC^mel complex in hybrid ovaries was not assembled. However, the activity of the RDC^sim complex was maintained in hybrids independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, we found no evidence that their activity can be considered the main cause of hybrid sterility. We revealed a complicated pattern of Vasa protein expression in the hybrid germline, including partial AT-chX piRNA targeting of the vasa^sim allele and a significant zygotic delay in vasa^mel expression. We arrived at the conclusion that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species. Full article

The dynamic process of Drosophila spermatogenesis involves asymmetric division, mitosis, and meiosis, which ultimately results in the production of mature spermatozoa. Disorders of spermatogenesis can lead to infertility in males. ADAR (adenosine deaminase acting on RNA) mutations in Drosophila cause male infertility, yet the causative factors remain unclear. In this study, immunofluorescence staining was employed to visualize endogenous ADAR proteins and assess protein levels via fluorescence-intensity analysis. In addition, the early differentiation disorders and homeostatic alterations during early spermatogenesis in the testes were examined through quantification of transit-amplifying region length, counting the number of GSCs (germline stem cells), and fertility experiments. Our findings suggest that deletion of ADAR causes testicular tip transit-amplifying cells to accumulate and become infertile in older male Drosophila. By overexpressing ADAR in early germline cells, male infertility can be partially rescued. Transcriptome analysis showed that ADAR maintained early spermatogenesis homeostasis through the bone-morphogenetic-protein (BMP) signaling pathway. Taken together, these findings have the potential to help explore the role of ADAR in early spermatogenesis. Full article

Stem cell maintenance and differentiation can be regulated via the differential activity of transcription factors within stem cells and their progeny. For these factors to be active, they need to be transported from their site of synthesis in the cytoplasm into the nucleus. A tissue-specific requirement for factors involved in nuclear importation is a potential mechanism to regulate stem cell differentiation. We have undertaken a characterization of male sterile importin alpha 1 (Dα1) null alleles in Drosophila and found that Dα1 is required for maintaining germline stem cells (GSCs) in the testis niche. The loss of GSCs can be rescued by ectopic expression of Dα1 within the germline but the animals are still infertile, indicating a second role for Dα1 in spermatogenesis. Expression of a Dα1 dominant negative transgene in GSCs confirmed a functional requirement for Dα1 in GSC maintenance but expression of the transgene in differentiating spermatogonia did not exhibit a phenotype indicating a specific role for Dα1 within GSCs. Our data indicate that Dα1 is utilized as a regulatory protein within GSCs to facilitate nuclear importation of proteins that maintain the stem cell pool. Full article

Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants—c.1138G>A (p.G380R) and c.1948A>G (p.K650E)—which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor’s age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline. Full article

Introduction: Previously, it was assumed that genetic influence played a minor role in acute myeloid leukemia (AML). Increasing evidence of germline mutations has emerged, such as DDX41 germline mutation associated with familial AML. Case presentation: A 64-year-old male patient presented with reduced exercise tolerance and shortness of breath. Following confirmation of AML diagnosis, the patient was enrolled into the AMLSG-30-18 study with a requirement for allogenic stem cell transplantation. The sister was initially selected as a fully HLA-matched donor. However, the family history showed risks for familial AML. Due to the striking family history, further diagnostic steps were initiated to detect a germline mutation. Methods: Using NGS in the patients’ bone marrow AML sample, a DDX41 mutation with a VAF of 49% was detected, raising the possibility of a germline mutation. DNA from cheek swabs and eyebrows were tested for the presence of the DDX41 mutation in all siblings. Results: DDX41 germline mutation was detected in 5 out of 6 siblings. The sister was excluded as a related donor and the search for an unrelated donor was initiated. Conclusion: Obtaining family history of cancer patients plays a crucial role in oncology. If a germline mutation is suspected, further family work-up should be initiated. Full article

Male germline stem cells (mGSCs), also known as spermatogonial stem cells (SSCs), are the fundamental seed cells of male animal reproductive physiology. However, environmental influences, drugs, and harmful substances often pose challenges to SSCs, such as population reduction and quality decline. With advancements in bioengineering technology and biomaterial technology, an increasing number of novel cell culture methods and techniques have been employed for studying the proliferation and differentiation of SSCs in vitro. This paper provides a review on recent progress in 3D culture techniques for SSCs in vitro; we summarize the microenvironment of SSCs and spermatocyte development, with a focus on scaffold-based culture methods and 3D printing cell culture techniques for SSCs. Additionally, decellularized testicular matrix (DTM) and other biological substrates are utilized through various combinations and approaches to construct an in vitro culture microenvironment suitable for SSC growth. Finally, we present some perspectives on current research trends and potential opportunities within three areas: the 3D printing niche environment, alternative options to DTM utilization, and advancement of the in vitro SSC culture technology system. Full article

Plasticizers give flexibility to a wide range of consumer and medical plastic products. Among them, phthalate esters are recognized as endocrine disruptors that target male reproductive functions. With this notion, past studies designed and produced alternative plasticizers that could replace phthalates with limited toxicity to the environment and to male reproductive functions. Here, we focused on one reproductive cell type that was not investigated in past studies—spermatogonial stem cells (SSCs)—and examined in vitro the effects on 22 compounds (seven plasticizers currently in use and 15 newly synthesized potential alternative plasticizers) for their effects on SSCs. Our in vitro compound screening analyses showed that a majority of the compounds examined had a limited level of toxicity to SSCs. Yet, some commercial plasticizers and their derivatives, such as DEHP (di-(2-ethylhexyl) phthalate) and MEHP (mono-(2-ethylhexyl) phthalate), were detrimental at 10⁻⁵ to 10⁻⁴ M. Among new compounds, some of maleate- and fumarate-derivatives showed toxic effects. In contrast, no detrimental effects were detected with two new compounds, BDDB (1,4 butanediol dibenzoate) and DOS (dioctyl succinate). Furthermore, SSCs that were exposed to BDDB and DOS in vitro successfully established spermatogenic colonies in testes of recipient mice after transplantation. These results demonstrate that SSC culture acts as an effective platform for toxicological tests on SSC function and provide novel information that two new compounds, BDDB and DOS, are alternative plasticizers that do not have significant negative impacts on SSC integrity. Full article

Graves’ disease, characterized by hyperthyroidism resulting from loss of immune tolerance to thyroid autoantigens, may be attributable to both genetic and environmental factors. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a means to induce immunotolerance via an artificial immune environment. We present a male patient with severe aplastic anemia arising from a germline SAMD9L missense mutation who successfully underwent HSCT from his HLA-haploidentical SAMD9L non-mutated father together with nonmyeloablative conditioning and post-transplant cyclophosphamide at 8 years of age. He did not suffer graft-versus-host disease, but Graves’ disease evolved 10 months post-transplant when cyclosporine was discontinued for one month. Reconstitution of peripheral lymphocyte subsets was found to be transiently downregulated shortly after Graves’ disease onset but recovered upon antithyroid treatment. Our investigation revealed the presence of genetic factors associated with Graves’ disease, including HLA-B*46:01 and HLA-DRB1*09:01 haplotypes carried by the asymptomatic donor and germline FLT3 c.2500C>T mutation carried by both the patient and the donor. Given his current euthyroid state with normal hematopoiesis, the patient has returned to normal school life. This rare event of Graves’ disease in a young boy arising from special HSCT circumstances indicates that both the genetic background and the HSCT environment can prompt the evolution of Graves’ disease. Full article

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics Chromium^TM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (A_pale and A_dark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, A_pale and A_dark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs. Full article