Male Germ Cells Research Articles

IGF2BP1 stabilizes Akt2 mRNA to promote glucose metabolism and maintain spermatogonial proliferation.

Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), as a newly identified N6-methyladenosine (m6A) methylation reader, plays a key role in mRNA stability, alternative splicing, and translation. However, the function of IGF2BP1 and its target genes in the development of male germ cells remains unclear. In the present study, Western blotting, immunofluorescence, and other cellular methods were used to confirm whether IGF2BP1 is expressed throughout the male germline with high abundance in spermatogonia of mice, and whether its downregulation inhibits spermatogonia proliferation in vitro. Through RNA sequencing, dual luciferase reporter assays, RIP-qPCR, and mRNA stability experiments, it was identified that Akt2 is one of the target genes of IGF2BP1. By stabilizing Akt2 mRNA in an m6A-dependent manner, IGF2BP1 maintains spermatogonial proliferation. Furthermore, co-immunoprecipitation and mass spectrometry analyses revealed that PABPC1, DDX5, and FXR1 interact with IGF2BP1, and that the interaction between IGF2BP1 and PABPC1 promotes AKT2 expression. Finally, following the knockdown of both IGF2BP1 and AKT2, glucose and ATP levels in C18-4 and GC-1 spermatogonia cells were found to be reduced, indicating that IGF2BP1 regulates glucose metabolism homeostasis by stabilizing Akt2 expression, thereby influencing spermatogonia proliferation. Additionally, analysis of the cryptorchidism database from NCBI revealed that the expression of IGF2BP1 and AKT2 is significantly downregulated in cryptorchid patients with oligospermia or azoospermia. In conclusion, our study elucidates the role and underlying mechanism of IGF2BP1 in spermatogonia, providing a candidate target for male infertility.

Chromatin environment-dependent effects of DOT1L on gene expression in male germ cells

The H3K79 methyltransferase DOT1L is essential for multiple aspects of mammalian development where it has been shown to regulate gene expression. Here, by producing and integrating epigenomic and spike-in RNA-seq data, we decipher the molecular role of DOT1L during mouse spermatogenesis and show that it has opposite effects on gene expression depending on chromatin environment. On one hand, DOT1L represses autosomal genes that are devoid of H3K79me2 at their bodies and located in H3K27me3-rich/H3K27ac-poor environments. On the other hand, it activates the expression of genes enriched in H3K79me2 and located in H3K27me3-poor/H3K27ac-rich environments, predominantly X chromosome-linked genes, after meiosis I. This coincides with a significant increase in DOT1L expression at this stage and a genome-wide acquisition of H3K79me2, particularly on the sex chromosomes. Taken together, our results show that H3K79me2 positively correlates with male germ cell genetic program throughout spermatogenesis, with DOT1L predominantly inhibiting rather than activating gene expression. Interestingly, while DOT1L appears to directly regulate the (re)activation of X genes following meiotic sex chromosome inactivation, it also controls the timely expression of (autosomal) differentiation genes during spermatogenesis.

Порушення іонтранспортувальних процесів у сперматозоїдах неплідних чоловіків

During the study of the plasma membrane of spermatozoa and its organelles, a great variety of specifically localized ion channels, exchangers, and ATPases was discovered. Although the activity of ion channels and membrane transporters has been studied in detail, their involvement in the mechanisms leading to the dysfunction of male germ cells remains insufficiently elucidated. Numerous scientific works show that the absence of certain transport systems of the plasma membrane due to genetic mutations or their low activity leads to a decrease or loss of sperm motility, morphological changes that worsen the quality of sperm, and is the cause of male infertility. The review examines some ion transport systems that maintain resting membrane potential and ion homeostasis in spermatogenesis. It notes the possibility of using ion channels and membrane transporters as markers to establish the functionality of spermatozoa or as molecular targets for drugs in the treatment of male infertility.

Utilizing TP53 hotspot mutations as effective predictors of gemcitabine treatment outcome in non-small-cell lung cancer

TP53 mutations are recognized to correlate with a worse prognosis in individuals with non-small cell lung cancer (NSCLC). There exists an immediate necessity to pinpoint selective treatment for patients carrying TP53 mutations. Potential drugs were identified by comparing drug sensitivity differences, represented by the half-maximal inhibitory concentration (IC50), between TP53 mutant and wild-type NSCLC cell lines using database analysis. In addition, clinical data from NSCLC patients were collected to evaluate both their TP53 status and their response to gemcitabine, thereby facilitating further validation. Subsequently, NSCLC cell lines with different TP53 status (A549 and H1299) were subjected to gemcitabine treatment to investigate the association between TP53 mutations and gemcitabine response. According to the dataset, NSCLC cell lines carrying TP53 mutations displayed heightened sensitivity to gemcitabine. From a clinical standpoint, patients exhibiting TP53 hotspot mutations demonstrated prolonged overall survival upon gemcitabine treatment. In vitro, overexpressing various hotspot TP53 mutations significantly sensitized H1299 cells to gemcitabine. Moreover, the knockdown of TP53 in A549 cells notably augmented sensitivity to gemcitabine treatment, as evidenced by cell viability and reproductive cell death assays. Conversely, the overexpression of wild-type TP53 in H1299 cells led to an increased resistance against gemcitabine. Gemcitabine is a treatment option for patients with non-small cell lung cancer (NSCLC) who carry TP53 hotspot mutations. This potential effectiveness might arise from its ability to disrupt DNA damage repair processes, leading to G2/M phase cell cycle arrest or an augmentation of mitotic abnormalities, eventually cause cell death. As a result, when planning treatment strategies for NSCLC patients possessing TP53 hotspot mutations, gemcitabine should be considered to incorporate into the indication.

TRAPPC2l Participates in Male Germ Cell Development by Regulating Cell Division

ABSTRACTTRAPPC2L is a core subunit of the Transport Protein Particle (TRAPP) complex, which is involved in vesicle transport and autophagy. Mutations in Trappc2l gene are associated with neurodevelopmental disorders, characterised by severe neurodevelopmental delays and varying degrees of muscle abnormalities. In this study, we found that the knockout of Trappc2l did not cause developmental abnormalities in both male and female mice. However, the male mice were completely infertile. Histological examination revealed that germ cell syncytial structures with multiple nuclear were formed in Trappc2l knockout mice from embryonic day 17.5 (E17.5) and the number and size of these structures gradually were increased at later developmental stages. The germ cells were completely lost at 2 weeks after birth. Further study found that germ cell syncytial structures were most likely formed by abnormal cell division but not cell fusion. We also found that meiosis‐associated genes Stra8 and Sycp3 were expressed in Trappc2l‐deficient germ cells during the embryonic stage. Our study demonstrated that Trappc2l is essential for germ cell development in male mice which is probably involved in keeping the mitotic quiescent state of male germ cells during the embryonic stage.

Chromatin remodeler CHD4 establishes chromatin states required for ovarian reserve formation, maintenance and male germ cell survival.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades due to the maintenance of meiotic arrest in non-growing oocytes (NGOs) residing in primordial follicles. Unknown is how the chromatin state of NGOs is established to enable long-term maintenance of the ovarian reserve. Here, we show that a chromatin remodeler, CHD4, a member of the Nucleosome Remodeling and Deacetylase (NuRD) complex, establishes chromatin states required for formation and maintenance of the ovarian reserve. Conditional loss of CHD4 in perinatal mouse oocytes results in acute death of NGOs and depletion of the ovarian reserve. CHD4 establishes closed chromatin at regulatory elements of pro-apoptotic genes to prevent cell death and at specific genes required for meiotic prophase I to facilitate the transition from meiotic prophase I oocytes to meiotically-arrested NGOs. In male germ cells, CHD4 establishes closed chromatin at the regulatory elements of pro-apoptotic genes, allowing germ cell survival. These results demonstrate a role for CHD4 in defining a chromatin state that ensures germ cell survival, thereby enabling the long-term maintenance of both female and male germ cells.

Noninversion Variants in Sporadic Hemophilia A Originate Mostly from Females

F8 gene inversion variants originate in male germ cells during spermatogenesis. Our recent study revealed that de novo variants (DNVs) caused F8 noninversion variants (NIVs) in sporadic hemophilia A (HA). Here, we conducted a direct clinical determination of sex differences in the origin of sporadic HA-NIV according to the data of two new HA-NIV families, as well as of the families demonstrated in the previous study. Of the 126 registered families with HA, 23 were eligible for inclusion. We conducted a linkage analysis with F8 gene markers and an amplification refractory mutation system–quantitative polymerase chain reaction to confirm the origin of the sporadic NIVs (~0% mutant cells) or the presence of a mosaic variant, requiring further confirmation of the origin in the parent. Two sporadic DNV events were determined. One event occurred in grandparents, consisting of five maternal grandmothers and seven maternal grandfathers, who were the origins; their respective daughters became carrier mothers who gave birth to probands. The other event included 11 mothers, who were the origins exclusively; their respective sons became probands. In this study, we found that sporadic HA-NIVs originate mostly from females (16 out of 23). Our study contributes to a better understanding of the genetic pathogenesis of HA.

An extra X chromosome: dissecting its role in male fetal germ cell maturation.

An extra X chromosome: dissecting its role in male fetal germ cell maturation.

Advanced 3Dimentional engineered microenvironment to improve of in vitro spermatogenesis: narrative review.

Induction of in vitro spermatogenesis may be helpful in the treatment of infertility in azoospermic individuals and those undergoing chemotherapy. Different cultivation systems have been implemented to achieve this aim. This review study aimed to investigate the application of three-dimensional culture in the induction of in vitro spermatogenesis. Relevant studies published in English were identified using PubMed using a range of search terms related to the core focus on tissue engineering of male reproductive systems, in vitro spermatogenesis, germ cell preservation, 3D culture systems for in vitro spermatogenesis, a 3D culture of testis tissue with were last updated in end of 2023. Searches were not restricted to a particular time frame or species, although the emphasis within the review is on regenerative medicine in mammalian male fertility preservation and in vitro spermatogenesis. Spermatogenesis is one of the most complicated cellular differentiation processes in the body. Significant attempts have been made to control spermatogenesis to drive differentiation of male germ stem cells toward mature sperm. Current research efforts focus on providing appropriate microenvironmental conditions to support the process of in vitro spermatogenesis by applying the principles of cell transplantation, material science, and bioengineering. Regenerative medicine may open a new avenue to patients for restoration and maintenance of normal function in spermatogenesis. The techniques reviewed are still in development, and this paper can become the primary reference for a large body of scientists developing advanced tissue engineering for male germ cells or developing the next generation of reproductive medicine.

Utilization of the QuPath open-source software platform for analysis of mammalian spermatogenesis.

The adult mammalian testis is filled with seminiferous tubules, which contain somatic Sertoli cells along with germ cells undergoing all phases of spermatogenesis. During spermatogenesis in postnatal mice, male germ cells undergo at least 17 different nomenclature changes as they proceed through mitosis as spermatogonia (=8), meiosis as spermatocytes (=6), and spermiogenesis as spermatids (=3) [1-6]. Adding to this complexity, combinations of germ cells at each of these stages of development are clumped together along the length of the seminiferous tubules. Due to this, considerable expertise is required for investigators to accurately analyze changes in spermatogenesis in animals that have spontaneous mutations, been genetically modified (transgenic or knockout/knockin (KO/KI)), or been treated with pharmacologic agents. Here, we leverage our laboratory's expertise in spermatogenesis to optimize the open source "Quantitative Pathology & Bioimage Analysis" (QuPath) software platform for automated analyses of germ and somatic cell populations in both the developing and adult mammalian testis.

A novel SEPT12 mutation, T96I, is associated with sperm head and annulus defects.

Infertility affects around 8%-12% of reproductive-aged couples and is a major health concern. Both genetic and environmental factors influence male infertility. SEPTIN12 is a crucial testis-specific gene essential for the final differentiation of male germ cells and is strongly linked to male infertility due to numerous detected mutations. The present study identified a novel SEPTIN12 T96I mutation that causes male infertility. Immunofluorescence staining and transmission electron microscopy (TEM) analysis of T96I sperm revealed co-localization of SEPT12 and SEPT7 in the obliquely positioned annulus. In addition, the sperm carrying the T96I mutation demonstrated large nuclear vacuoles, irregular swelling, and decondensation of the acrosomal cap. The overexpression of SEPT12 T96I in NT2/D1 cells impaired the formation of SEPT7 filaments, emphasizing the significance of SEPT12 filaments for sperm morphology and function. Our results demonstrate the importance of SEPTIN12 T96I in male infertility and offer valuable insights for future detection in infertile men.

Icariin targets PDE5A to regulate viability, DNA synthesis and DNA damage of spermatogonial stem cells and improves reproductive capacity.

Icariin is a pure compound derived from Epimedium brevicornu Maxim, and it helps the regulation of male reproduction. Nevertheless, the role and underlying mechanisms of Icariin in mediating male germ cell development remain to be clarified. Here, we have demonstrated that Icariin promoted proliferation and DNA synthesis of mouse spermatogonial stem cells (SSCs). Furthermore, surface plasmon resonance iron (SPRi) and molecular docking (MOE) assays revealed that phosphodiesterase 5A (PDE5A) was an important target of Icariin in mouse SSCs. Mechanically, Icariin decreased the expression level of PDE5A. Interestingly, hydrogen peroxides (H2O2) enhanced the expression level of phosphorylation H2A.X (p-H2A.X), whereas Icariin diminished the expression level of p-H2A.X and DNA damage caused by H2O2 in mouse SSCs. Finally, our in vivo animal study indicated that Icariin protected male reproduction. Collectively, these results implicate that Icariin targets PDE5A to regulate mouse SSC viability and DNA damage and improves male reproductive capacity. This study thus sheds new insights into molecular mechanisms underlying the fate decisions of mammalian SSCs and offers a scientific basis for the clinical application of Icariin in male reproduction.

Expression of NKp46 and other activating inhibitory receptors on uterine endometrial NK cells in females with various reproductive failures: A review.

Uterine endometrial natural killer (uNK) cells represent major leukocytes in the mid-secretory phase of the cell cycle, and their number is further increased during early pregnancy. The activating and inhibitory receptors expressed on their surface mediate various functions of uNK cells, such as cytotoxicity, cytokine production, spiral artery remodeling, and self-recognition. This study reviewed the most recent information (PubMed database, 175 articles included) regarding the activating and inhibitory receptors on uNK cells in human females with healthy pregnancies and the evidence indicating their significance in various reproductive failures. Numerous studies have indicated that the natural cytotoxic receptors, killer cell immunoglobulin-like receptors, and C-type lectin receptors, particularly those expressed on uNK cells, play crucial roles in successful pregnancy. As studies on human uNK cells are limited owing to the low availability of fertile samples, and the extrapolation of animal models has certain limitations, the invivo role of uNK cells has not yet been fully elucidated. However, immunotherapies focusing on modulating uNK cell function have been controversial in terms of pregnancy outcomes. Further research is required to elucidate the role of uNK cells in reproduction.

CEP44 and CCDC15 label the spermatozoa proximal and atypical distal centrioles.

The centrosome is a conserved characteristic of eukaryotic and human cells but is highly specialized in reproductive cells. The spermatozoan centrosome includes a slightly modified proximal centriole, an atypical distal centriole, and specialized pericentriolar material, including striated columns and capitellum. We investigated the localization of canonical centriolar proteins CEP44 and CCDC15 in human spermatozoa. We found that CEP44 localizes mainly at the proximal centriole and distal centriole bases relative to centrin. CCDC15 colocalizes with centrin in both the proximal centriole and distal centriole. These findings further our understanding of the spermatozoan centrosome composition.

Transformation of sperm structure in Octopus vulgaris: From spermatogenesis to spermatophoric release.

The present study describes the differentiation process of male germ cells in Octopus vulgaris, the morphology of sperm in the testis and spermatophore, and the sperm released after the spermatophoric reaction. During spermatogenesis, the male sperm cell gradually elongates from a round shape, with cytoplasm shifting toward the head and the acrosome forming. Additionally, in the spermatid stage, the flagellum develops within the posterior nuclear channel and extends outside the cytoplasm. The sperm is composed of a head and a tail. The head is approximately 17.9 μm long and consists of a highly electron-dense nucleus and a helical acrosome. The tail is divided into three parts: the mid-piece, principal-piece, and end-piece. The mid-piece forms a mitochondrial sheath with 7-8 mitochondria surrounding a "9+2" axoneme. The principal-piece is composed of an axoneme, outer dense fibers, and fibrous sheath, while the end piece lacks outer dense fibers or fibrous sheath. The sperm in the testis and spermatophore, and the sperm released after the spermatophoric reaction have the same structure. However, in the sperm located in the testis and spermatophore, the structure of the acrosome is unclear due to the presence of cytoplasm in the head. In contrast, sperm released after the spermatophoric reaction lack their cytoplasm, revealing the helical acrosome. This unique sperm morphology, adapted for internal fertilization, is thought to be advantageous for fertilization and long-term storage within the female reproductive system.

External pressure induced the dysfunction of Sertoli cells via the Fas/FasL signaling pathway

Cryptorchidism, a condition where the testis fails to fully descend into the scrotum during development, is associated with elevated environmental temperatures and pressures, leading to male infertility and germ cell tumors. Factors such as oxidative stress and high temperatures contribute to infertility in cryptorchidism. This study aims to explore how external pressure affects Sertoli cells and discover new mechanisms affecting spermatogenesis in cryptorchidism. Sertoli cells were subjected to various pressure levels (0 mmHg, 25 mmHg, 50 mmHg, 100 mmHg) and durations (0 h, 2 h, 4 h) using an enzyme-linked immunosorbent assay (ELISA) to measure androgen binding protein (ABP) and inhibin B (INH B) secretion. Cell morphology changes were observed using immunofluorescence; apoptosis rates were measured with terminal-deoxynucleotidyl transferase mediated nick end labelling (TUNEL) assay and flow cytometry; ultrastructural variations were examined via transmission electron microscopy; and the expression of apoptosis-related proteins (Fas, FasL, caspase 3, and caspase 8) was analyzed through immunohistochemistry, real-time polymerase chain reaction (real-time PCR), and western blotting. The results showed that elevated pressure suppressed ABP and INH B secretion from Sertoli cells. Structural changes were observed under pressure, including cytoskeleton loosening and nuclear fragmentation. Apoptosis rates increased with higher pressure levels. Ultrastructural analysis revealed chromatin changes, apoptotic bodies, and mitochondrial alterations. Increased expressions of Fas and FasL were detected, along with elevated levels of caspase 3 and caspase 8. The caspase 8 inhibitor blocked pressure-induced apoptosis and caspase 3 activation, while the cytochrome C inhibitor did not show the same effect. Our findings suggested that external pressure induces apoptosis of Sertoli cells via the Fas/FasL signaling pathway, potentially contributing to male infertility associated with cryptorchidism.

Impact of Oncological Treatments on Reproductive Capacity: Alternatives to Minimize Risks and the Role of DEX Immunotherapy

Fertility preservation in cancer patients is an increasingly relevant challenge due to the gonadotoxic effects of conventional treatments, such as chemotherapy and radiotherapy. This article reviews current options for fertility preservation and explores the potential of DEX immunotherapy as a less invasive alternative. Initially, an overview of cancer incidence in fertile age and the importance of addressing reproductive concerns in this population group is presented. Subsequently, the negative impacts of conventional treatments on reproductive capacity are analyzed and strategies to minimize these risks, such as reducing treatment intensity and using cryopreservation techniques, are discussed DEX immunotherapy stands out as a promising option, with a lower risk of gonadotoxicity compared to traditional treatments. This immunological approach allows to specifically target cancer cells, reducing damage to reproductive cells and offering better fertility preservation in young patients. Finally, the article addresses future research needs and practical guidelines for clinicians, highlighting the importance of a multidisciplinary approach that integrates fertility preservation options in cancer treatment. It is concluded that, although DEX immunotherapy is a viable alternative, it is essential to promote further research and develop specific clinical protocols to maximize its effectiveness and safety in terms of long-term reproductive health

Recombinant Follicle-Stimulating Hormone and Luteinizing Hormone Enhance Mitochondrial Function and Metabolism in Aging Female Reproductive Cells

Ovarian aging significantly impacts female fertility, with mitochondrial dysfunction emerging as a key factor. This study investigated the effects of recombinant follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on mitochondrial function and metabolism in aging female reproductive cells. Human granulosa cells (HGL5) were treated with FSH/LH or not. Mitochondrial function was assessed through various assays, including mitochondrial mass, membrane potential, ROS levels, and ATP production. Mitochondrial dynamics and morphology were analyzed using MitoTracker staining. Cellular respiration was measured using a Seahorse Bioenergetics Analyzer. Metabolic reprogramming was evaluated through gene expression analysis and metabolite profiling. In vivo effects were studied using aging mouse oocytes. FSH/LH treatment significantly improved mitochondrial function in aging granulosa cells, increasing mitochondrial mass and membrane potential while reducing ROS levels. Mitochondrial dynamics showed a shift towards fusion and elongation. Cellular respiration, ATP production, and spare respiratory capacity were enhanced. FSH/LH-induced favorable alterations in cellular metabolism, favoring oxidative phosphorylation. In aging mouse oocytes, FSH/LH treatment improved in vitro maturation and mitochondrial health. In conclusion, FSH/LH supplementation ameliorates age-related mitochondrial dysfunction and improves cellular metabolism in aging female reproductive cells.

Antioxidant activity and protective effect of hydroxy derivatives of chalcones for sterlet (Acipenser ruthenus, Linnaeus, 1758) sperm.

The aim of this work is to study the effect of adding hydroxy derivatives of chalcones to the basic cryomedium on the ability of sterlet sperm to utilize superoxide and hydrogen peroxide, the intensity of lipid peroxidation of male fish germ cells, and their viability both before cryopreservation and after 3 days of freezing at liquid nitrogen temperature. The ability of phenolic derivatives of chalcones to increase the superoxide dismutase and catalase activities of sterlet sperm and to reduce the intensity of lipid peroxidation has been established. The antioxidant activity of the derivatives exceeds the effect of Trolox, which inhibits the functioning of the enzyme component of the antioxidant protection of fish sperm and promotes lipid peroxidation of fish sperm before cryopreservation. A beneficial effect of hydroxy derivatives of chalcones on the motility parameters of thawed sperm has been shown, indicating their ability to increase the cryoresistance of sperm in such a promising aquaculture species as sterlet.

The Expression Regulation and Cancer-Promoting Roles of RACGAP1.

RACGAP1 is a Rho-GTPase-activating protein originally discovered in male germ cells to inactivate Rac, RhoA and Cdc42 from the GTP-bound form to the GDP-bound form. GAP has traditionally been known as a tumor suppressor. However, studies increasingly suggest that overexpressed RACGAP1 activates Rac and RhoA in multiple cancers to mediate downstream oncogene overexpression by assisting in the nuclear translocation of signaling molecules and to promote cytokinesis by regulating the cytoskeleton or serving as a component of the central spindle. Contradictorily, it was also reported that RACGAP1 in gastric cancer could inactivate Rac and RhoA. In addition, studies have revealed that RACGAP1 can be a biomarker for prognosis, and its role in reducing doxorubicin sensitivity poses difficulties for treatment, while the current drug targets mainly focus on its downstream molecule. This article mainly reviews the expression regulation of RACGAP1 and its cancer-promoting functions through oncogene expression mediation and Rho-GTPase activation.