Sperm Research Articles

Melatonin protects spermatogenic cells against DNA damage and necroptosis induced by atrazine

Atrazine (ATZ), a widely used triazine herbicide, has been shown to disrupt reproductive development in organisms. Melatonin (MLT) is a natural hormone and has been shown to have strong antioxidant properties. Due to its lipophilicity, it can cross biological barriers freely and act on germ cells directly. However, the mechanism through which melatonin affects atrazine-induced damage to male sperm cells remains unclear. This study aimed to investigate the effects of ATZ on spermatocyte development and to elucidate MLT's role in preventing ATZ-induced spermatogenesis failure. Pubertal mice were randomly divided into four groups: blank control group (Con), 5 mg/kg melatonin group (MLT), 170 mg/kg atrazine group (ATZ), and ATZ + MLT group. GC-1 cell culture was employed to access the in vitro effects of MLT and ATZ on spermatogonia. The results indicate that atrazine affected protein and metabolite composition, and reduced sperm viability, sperm motility (VAP, VSL and VCL) and levels of proteins related to spermatogenesis function in the mice testis. Melatonin alleviated the development of cellular DNA damage and necroptosis caused by atrazine both in vivo and in vitro. Moreover, we proposed that it was GC-1 cells developing necroptosis, but not other cell types in the testis. In conclusion, this study suggests that atrazine disrupts the development process, causing DNA damage in spermatozoa during spermatogenesis. Additionally, ATZ-induced necroptosis specifically targets spermatogenic cells. Notably, melatonin alleviates atrazine-induced necroptosis and DNA damage in spermatogenic cells. This study provides new insights into potential therapeutic strategies for atrazine-induced male infertility.

Treatment of human sperm with GYY4137 increases sperm motility and resistance to oxidative stress.

Hydrogen sulfide (H2S) has been shown to play a significant role in oxidative stress across various tissues and cells; however, its role in sperm function remains poorly understood. This study aimed to investigate the protective effect of GYY4137, a slow-releasing H2S compound, on sperm damage induced by H2O2. We assessed the effects of GYY4137 on motility, viability, lipid peroxidation and caspase-3 activity in human spermatozoa in vitro following oxidative damage mediated by H2O2. Spermatozoa from 25 healthy men were selected using a density gradient centrifugation method and cultured in the presence or absence of 10 μM H2O2, followed by incubation with varying concentrations of GYY4137 (0.625-2.5 μM). After 24 h of incubation, sperm motility, viability, lipid peroxidation, and caspase-3 activity were evaluated. The results indicated that H2O2 adversely affected sperm parameters, reducing motility and viability, while increasing oxidative stress, as evidenced by elevated lipid peroxidation and caspase-3 activity. GYY4137 provided dose-dependent protection against H2O2-induced oxidative stress (OS). We concluded that supplementation with GYY4137 may offer antioxidant protection during in vitro sperm preparation for assisted reproductive technology.

Behavioural thermoregulation prevents thermal stress in lizard sperm fertility

Global warming is threatening ectotherms, with strong repercussions on their population dynamics. Body temperature in ectotherm reptiles is crucial to perform all their biological functions, which are maximized within a narrow interval. When faced with new or adverse thermal conditions, reptiles will respond with distributional changes, behavioural adjustments to maintain their internal temperature, or by adapting to the new environment, otherwise, extinctions will occur. Higher temperatures may have negative repercussions, for example, shortening periods of activity, affecting embryo development during gestation or decreasing viability of sperm cells in males. Through behavioural thermoregulation, reptiles can compensate for environmental variations (Bogert effect). Furthermore, according to Janzen’s hypothesis, the physiological cost of responding to adverse thermal conditions will be low in species exposed to higher thermal overlap. Here, we analysed the effect of a change in the thermal regime on sperm cell viability in Sceloporus megalepidurus, a small viviparous lizard from central Mexico. We hypothesized that an active thermoregulator inhabiting temperate mountains is able to prevent the effects of thermal change on sperm cell viability. We found that the change in thermal regime did not modify sperm cell viability, nor does it affect the maturation of sperm cells in the epididymis. Our results support the Bogert effect and suggest that, despite the high temperatures and low thermal quality, S. megalepidurus can maintain its body temperature within an optimal range for sperm cell viability.

Protamine-Mediated Tangles Produce Extreme Deoxyribonucleic Acid Compaction.

In sperm cells, protamine replaces histones to compact DNA 10-20 times more than in somatic cells. To characterize the extreme compaction, we employed confocal microscopy and optical tweezers to determine the conformations and stability of protamine-bound λ-DNA. Confocal images show increasing compaction of λ-DNA at increasing protamine concentration. In the presence of protamine, single λ-DNA molecules form tangles that withstand forces strong enough (∼55 pN) for strand separation and shorten the contour length by up to 40% even at high forces, as well as bends and loops that rupture at 10-40 pN forces. Strand separation nucleates tangles, implicating protamine interactions with DNA bases. Molecular dynamics simulations show that Arg sidechains of protamine each form hydrogen bonds with multiple bases, frequently in the form of a wedge between the two strands of DNA. Protamine may participate in both local and higher-order chromatin organization, leading to extreme compaction and global transcription silencing.

Alteration of the metabolite interconversion enzyme in sperm and Sertoli cell of non-obstructive azoospermia: a microarray data and in-silico analysis

Numerous variables that regulate the metabolism of Sertoli cells and sperm have been identified, one of which is sex steroid hormones. These hormones play a vital role in maintaining energy homeostasis, influencing the overall metabolic balance of the human body. The proper functioning of the reproductive system is closely linked to energy status, as the reproductive axis responds to metabolic signals. The aim of this study was to investigate the gene expression patterns of metabolite interconversion enzymes in testicular cells (Sertoli cells and spermatogonia) of non-obstructive azoospermia (NOA) patients, as compared to normal controls, to understand the molecular mechanisms contributing to NOA. We used microarray and bioinformatics techniques to analyze 2912 genes encoding metabolite interconversion enzymes, including methyltransferase, monooxygenase, transmembrane reductase, and phosphohydrolase, in both testicular cells and normal samples. In sperm, the upregulation of MOXD1, ACAD10, PCYT1A, ARG1, METTL6, GPLD1, MAOA, and CYP46A1 was observed, while ENTPD2, CPT1C, ADC, and CYB5B were downregulated. Similarly, in the Sertoli cells of three NOA patients, RPIA, PIK3C3, LYPLA2, CA11, MBOAT7, and HDHD2 were upregulated, while NAA25, MAN2A1, CYB561, PNPLA5, RRM2, and other genes were downregulated. Using STRING and Cytoscape, we predicted the functional and molecular interactions of these proteins and identified key hub genes. Pathway enrichment analysis highlighted significant roles for G1/S-specific transcription, pyruvate metabolism, and citric acid metabolism in sperm, and the p53 signaling pathway and folate metabolism in Sertoli cells. Additionally, Weighted Gene Co-expression Network Analysis (WGCNA) and single-cell RNA sequencing (scRNA-seq) were performed to validate these findings, revealing significant alterations in gene expression and cellular distribution in NOA patients. Together, these results provide new insights into the molecular mechanisms underlying NOA and identify potential therapeutic targets.

Impact of Vitamin C and Hydroxyurea on the Reproductive System Health in Male Rats

Hydroxyurea (HU) is a medication used to treat various diseases and is also being studied for its effects on spermatogenesis. Vitamin C (VC), an antioxidant, has been shown to protect sperm cells from oxidative stress, potentially improving fertility and sperm quality. In a study involving twenty-four adult male albino rats divided into four groups, Group 1 served as the control, Group 2 received 5 mg of vitamin C, Group 3 received 100 mg of hydroxyurea per body weight, and Group 4 received a combination of vitamin C and hydroxyurea to assess essential functions of the reproductive system, including hormone levels, antioxidant markers, oxidative stress indicators, histopathology, and identification of DNA damage. The study found that hydroxyurea significantly reduced testicular weight, SOD, CAT, and GSH while increasing FSH and MDA levels and causing abnormalities in sperm morphology. Hydroxyurea also caused apparent alterations in the histological structure of the testes and comet parameters. Rats treated with vitamin C showed a significant increase in absolute and relative epididymis weight compared to the control group. Moreover, vitamin C intervention reversed the adverse effects observed in rats fed hydroxyurea, indicating that low doses of vitamin C can protect against testicular damage.

Selective regulation of aspartyl intramembrane protease activity by calnexin

Signal peptide peptidase-like 2c (SPPL2c) is a testis-specific aspartyl intramembrane protease that contributes to male gamete function both by catalytic and non-proteolytic mechanisms. Here, we provide an unbiased characterisation of the in vivo interactome of SPPL2c identifying the ER chaperone calnexin as novel binding partner of this enzyme. Recruitment of calnexin specifically required the N-glycosylation within the N-terminal protease-associated domain of SPPL2c. Importantly, mutation of the single glycosylation site of SPPL2c or loss of calnexin expression completely prevented SPPL2c-mediated intramembrane proteolysis of all tested substrates. By contrast and despite rather promiscuous binding of calnexin to other SPP/SPPL proteases, expression of the chaperone was exclusively required for SPPL2c-mediated proteolysis. Despite some impact on the stability of SPPL2c most presumably due to assistance in folding of the luminal domain of the protease, calnexin appeared to be recruited rather constitutively to the protease thereby boosting its catalytic activity. In summary, we describe a novel, highly specific mode of intramembrane protease regulation, highlighting the need to systematically approach control mechanisms governing the proteolytic activity of other members of the aspartyl intramembrane protease family.

Ethnopharmacological insights: hyperoside from Marsdenia latifolia (Benth.) K.Schum. mitigates reproductive toxicity in male Wistar rats induced by manganese exposure

This study provides novel findings on the ameliorative effect of hyperoside isolated from Marsdenia latifolia leaves (HIGLL) in reproductive health challenged by manganese toxicity. The study investigated the efficacy of HIGLL on male fertility in rats exposed to manganese chloride (MnCl2). The rats received either MnCl2 (30 mg/L) or in combination with HIGLL (100 mg/kg or 200 mg/kg). MnCl2 reduced fluid intake, organ-body weight, body weight; sperm count, sperm viability, sperm density, sperm motility, semen viscosity, daily sperm production, testicular sperm number; testosterone, follicle-stimulating and luteinising hormones, oestradiol, testosterone/oestradiol ratio; G6PDH, ALP, glucose, NO, acid phosphatase, sialic acid, 17-β-HSD, superoxide dismutase, CAT, GST, GSH, and T-SH without modifying the semen pH and volume, while it correspondingly raised the abnormalities associated with morphology of sperm cells in head, neck, and tail; H2O2 and lipid peroxidation levels. HIGLL abrogated the damaged histoarchitecture of the testes and epididymis caused by MnCl2. HIGLL can serve as a therapeutic agent in the management of male reproductive disorders related to oxidative stress and endocrine disruption.

Protective Effects of Anthocyanin and α-Tocopherol Against Titanium Dioxide Nanoparticle-Induced DNA Damage in Human Sperm Cells

Protective Effects of Anthocyanin and α-Tocopherol Against Titanium Dioxide Nanoparticle-Induced DNA Damage in Human Sperm Cells

GhGME31D identified to regulate AsA activation in response to alkali stress from GME gene family implications in cotton

Vitamin C, also referred to as ascorbic acid (AsA), is recognized for its capacity to cure and avert scurvy, and it is crucial for regular human growth and development. In various crops, AsA participates in stress response mechanisms mediated by abscisic acid and has been discovered to have a crucial function in the morphogenesis, growth, development, and production of male gametes in plants. GDP-D-mannose 3′,5′-epimerase (GME) is essential in the synthesis of vitamin C. Our research identified 91, 83, 51, and 46 genes, respectively, found in G. barbadense (GbGMEs), G. hirsutum (GhGMEs), G. arboretum (GaGMEs), and G. raimondii (GrGMEs). Plants resulting from VIGS infection with GhGME31D clearly showed yellowing, water loss and wilting of leaves and black spots on stems. Measurement of MDA and AsA levels indicated that the plants were more damaged. This indicates that AsA has a substantial impact on plant growth and development.

Cryopreservation of human spermatozoa or testicular tissue for fertility preservation

Loss of reproductive capacity due to treatments for malignant or non-malignant diseases or even as aresult of diseases themselves significantly impacts patients' quality of life. Cryopreservation of sperm from ejaculate is awell-established procedure for preserving the fertility of these patients and thus improving their quality of life in the long term. If cryopreservation of sperm from ejaculate is not possible, either because ejaculation cannot occur or no sperm can be found in the ejaculate, the preferred treatment option is (microsurgical) testicular sperm extraction (mTESE). Testicular sperm and ejaculated spermatozoa can be cryopreserved and later used for intracytoplasmic sperm injection (ICSI) treatment. The use of cryopreserved sperm for fertility treatment does not carry an increased risk of malformations in the offspring. If gonadotoxic therapy is necessary in pre- or early pubertal boys, the only option to preserve fertility in the long term is to cryopreserve spermatogonial stem cells from testicular tissue as part of the Androprotect© network. This is an experimental approach which has been available since 2012 acrossGermany and which is accompanied by intensive scientific work ( www.androprotect.de ).

Advanced SPR Sensor for Human Sperm Analysis: Leveraging Silver and Nanomaterials for Enhanced Performance

Advanced SPR Sensor for Human Sperm Analysis: Leveraging Silver and Nanomaterials for Enhanced Performance

Epigenetic aging and fecundability: the Norwegian Mother, Father and Child Cohort Study.

Is there an association between male or female epigenetic age acceleration (EAA) or deceleration (EAD) and fecundability? We do not find compelling evidence of an association between EAA or EAD and fecundability. Prior research has shown that female accelerated epigenetic aging is associated with unfavorable clinical fecundity outcomes and use of in vitro fertilization, and that epigenetic aging in sperm cells is associated with unfavorable sperm parameters. Studies of epigenetic aging and fecundability among individuals who conceive naturally are lacking. This study is based on the Norwegian Mother, Father and Child Cohort Study (MoBa), a population-based pregnancy cohort which recruited pregnant couples between 1999 and 2008. We used data from 1657 couples (women and men) with planned naturally conceived pregnancies and available blood samples. Methylation levels were measured in DNA from blood samples taken recruitment (at ∼18 gestational weeks) from pregnant women and their partners using the Illumina Methylation EPIC Array. To obtain a measure of EAA/EAD, we performed a linear regression of each of seven different established epigenetic biomarkers (DNAmAge by Horvath, DNAmAge by Hannum et al., PhenoAge by Levine et al., DunedinPoAm by Belsky et al., DunedinPACE by Belsky et al., DNAmTL by Lu et al., and GrimAge by Lu et al.) against chronological age. We fitted proportional probability regression models to obtain fecundability ratios (FRs) for each standard deviation increase in epigenetic aging, and obtained crude and adjusted (for body mass index, smoking, and education level) estimates. Results were evaluated at a false discovery rate (FDR) of 5%. We evaluated all models for non-linear associations using categories of epigenetic age where appropriate. Although the DunedinPACE clock in males demonstrated slightly increasing fecundability with increasing EAA (adjusted FR 1.05 per one standard deviation increase in EAA, 95% CI 1.00-1.10), this was not robust when evaluated at an FDR of 5%. We found evidence of non-linearity between biological aging and fecundability in two models in females and three models in males, but non-linear associations were weak and conflicting. As MoBa is a pregnancy cohort, our findings may not be generalizable to all couples attempting conception. Fecundability is a couple-level measure, and any impacts of epigenetic aging in each partner may be obscured by effects of the other partner. Our findings contrast with those of prior studies, which have indicated an association between EAA and unfavorable clinical fertility outcomes in populations using fertility treatments, possibly due to less important effects of epigenetic aging among couples who conceive naturally. More research is needed on the association between blood-based EAA and clinical fertility parameters in both sexes. The study was supported by the Research Council of Norway through its Medical Student Research Program funding scheme (project number 271555/F20), its Centres of Excellence funding scheme (project number 262700), and a grant from the Women's Health Program (320656). Co-funding was also received from the Strategic Research Council (SRC), FLUX consortium, decision numbers 345130 and 345131; the National Institute on Aging (R01AG075208); grants to the Max Planck-University of Helsinki Center from the Max Planck Society (decision number 5714240218), Jane and Aatos Erkko Foundation, Faculty of Social Sciences at the University of Helsinki, and Cities of Helsinki, Vantaa, and Espoo; and the European Research Council; and the European Research Council (ERC Synergy, BIOSFER, grant number 101071773, and the Horizon 2020 research and innovation program, grant number 947684). The authors declare no conflicts of interest. N/A.

A method for determining potential parental contamination: linkage disequilibrium-based log-likelihood ratio analysis for IVF-PGT

BackgroundAt present, embryologists are attempting to use conventional in vitro fertilization (cIVF) as an alternative to intracytoplasmic sperm injection (ICSI) for preimplantation genetic testing (PGT). However, the potential parental contamination origin of sperm cells and cumulus cells is considered the main limiting factor in the inability of cIVF embryos to undergo PGT.MethodsIn this study, we established an IVF-PGTA assay for parental contamination tests with a contamination prediction model based on allele frequencies and linkage disequilibrium (LD) to compute the log-likelihood ratio (LLR) under competing ploidy hypotheses, and then verified its sensitivity and accuracy. Finally, comparisons of the effectiveness of SNP-based analysis and LLR-based IVF-PGTA among 40 cIVF embryos was performed, based on both statistical analysis of the parental contamination rate and chromosomal ploidy concordance rate between TE biopsy and ICM isolations.ResultsWith IVF-PGTA assay, biopsies with 10% maternal contamination could be detected accurately, and contamination caused by sperm cells could be eliminated completely. Utilizing LLR-based or single Nucleotide Polymorphism (SNP) -based analyses, our comprehensive examination of 40 clinically discarded fresh cIVF embryos revealed an absence of paternal contamination. Strikingly, the LLR-based analysis uniquely revealed a mere instance of 24% maternal contamination within the trophectoderm cell (TE) biopsy of 5* embryo. Furthermore, it was solely through this analysis that embryo (9-F) was identified as a triploid of paternal origin.ConclusionsIn this study, we developed a new bioinformatics analysis method for identifying parental contamination during IVF-PGT, especially for couples with nonmale factor infertility.

The effect of myo-inositol antioxidant activity on human sperm parameters and DNA damage in ultra-rapid and conventional freezing methods

Male fertility preservation is still challenged by cell damage induced during sperm cryopreservation and impaired sperm structure and function. Sperm ultra-rapid freezing, despite a higher protective effect compared to conventional freezing method, is still associated with suboptimal sperm cryosurvival and needs to be modified to increase its efficiency in sperm protection. Sperm freezing media supplemented with antioxidants can improve sperm parameters following freezing-warming process. In this study, we aimed to investigate the effect of employing ultra-rapid freezing and myo-inositol on sperm cryosurvival. Thirty semen samples with normal sperm parameters were collected and each one was divided into four portions to cryopreserve by conventional freezing, ultra-rapid freezing, conventional freezing + myo-inositol 2 mg/ml, and ultra-rapid freezing + myo-inositol 2 mg/ml. Sperm samples warmed after at least 24 h of freezing and sperm cryosurvival were analyzed by evaluation of sperm motility, viability, morphology and DNA fragmentation index (DFI). Freezing method had a significant influence on post-thaw sperm DFI and morphology (p < 0.05) and the interaction between freezing method and antioxidant supplementation significantly affected sperm morphology (p < 0.05). The highest percentage of sperm normal morphology and minimal DFI was achieved using ultra-rapid freezing supplemented by myo-inositol antioxidant compared to other groups (P < 0.05). The highest sperm DNA damage after freezing-warming was observed following the conventional freezing method. In conclusion, sperm freezing method was identified as factor strongly influencing sperm DFI and morphology after thawing/warming. Sperm samples can be rapidly frozen using the modified freezing media supplemented by myo-inositol without impacting sperm DNA and morphology.

Successful formation of sperm cells from transplanted primordial germ cells in sterile interspecific avian recipients.

Primordial germ cells (PGCs) are stem cells, from which only gametes develop. In birds, the female sex is heterogametic, thus female gene conservation necessitates preservation of PGCs. PGC transplantation can generate germline chimeras in a host organism and develop into gametes. However, competition between host and transplanted PGCs hinder efficiency of germline chimera generation. We hypothezised that in sterile hybrid recipients with no germ cells of its own, transplanted donor PGCs may exclusively form gametes. Advantages of sterile hybrids as host for PGCs is compliant with many national regulations on genetically modified organisms and technically simpler procedure than the use of busulphan. Therefore, we investigated whether sterile interspecific hybrids may be suitable as recipients for supporting donor PGCs by injecting green fluorescent protein-labelled chicken PGCs into 3-day-old Guinea fowl and domestic fowl hybrid embryos and monitoring PGC development. The injected PGCs colonized almost 100% of the recipient gonads and produced mature spermatozoa after 44 weeks. However, gamete production in these hybrids was initiated much slower than in domestic fowls. This delay may be caused by suboptimal hormonal regulation of gametogenesis in the hybrids. Our results suggest that sterile interspecific hybrids may be suitable hosts for PGCs for efficient gamete production.

Dietary supplementation with rosemary essential oil improves genital characteristics, semen parameters and testosterone concentration in Barki rams

BackgroundMale reproductive performance is an essential part of sheep production; therefore, the use of natural antioxidants to improve sperm quality and maintain reproductive performance in males is very important. Hence, oral administration of rosemary essential oil (REO) was investigated to improve the fertility rate, including the ultrasonographic testes, epididymal tail and genital glands, as well as semen parameters and testosterone concentration. Sixty animals were splitted into two groups, each with 30 rams; the rosemary group (C + REO) received 2 mg/kg/bw and the control group (C-REO). Ultrasound images and blood samples were collected at 15, 30 and 45 days of the REO treatment.ResultsThe testis and epididymal tail ultrasonographic assessments demonstrated a significant enhancement in the C + REO group compared to the C-REO group. However, the rams in the C + REO group showed significant improvements in the pampini-form plexus, seminal vesicle, Cowper's and prostate genital glands compared to the C-REO group. The data showed that the sperm cell concentration (× 109/ml) and individual motility (%) were significantly improved in the C + REO group. Furthermore, ejaculate volume (ml) in the C + REO group was significantly higher than that in the C-REO group. While the animals treated with REO did not improve live spermatozoa (%), it reduced the abnormalities of spermatozoa (%) compared to the C-REO group. Also, the C + REO group significantly increased the testosterone concentration more than the C-REO group.ConclusionIt can be concluded that supplementation with 2 mg/kg/bw REO improves genital characteristics, semen parameters and testosterone concentration in Barki rams.

Selenium efficiency in protecting sperm quality and testicular parameters of South African indigenous Zulu rams exposed to heat stress.

The study investigated selenium's (Se) efficiency in preserving South African Zulu rams' sperm quality and testicular parameters when they were exposed to heat stress. Indigenous Zulu rams (20) between 2 and 5 years old were allocated into four groups, namely the Se, testicular heat stress (THS), selenium plus testicular heat stress (SeTHS), and control. Each group comprised five rams; the groups were balanced according to the rams' body weight and scrotal circumference. The Se and SeTHS groups received sodium selenite orally bi-weekly for 5 months. To induce heat stress, testicular heat insulation bags were wrapped around the testes of the rams receiving the THS and SeTHS treatments for 49 days. Semen was collected from the rams weekly from the third month onward; the first two months were for Se & THS acclimatization. In addition, testicular measurements were taken bi-weekly. Analysis of variance was used to analyze the sperm quality data. Duncan's multiple range test was used to compare the groups' data for significant differences. The results showed that the Se-supplemented rams' scrotal circumference was smaller (p < 0.05) compared with the other groups. The Se, SeTHS, and control groups demonstrated similar total sperm motility; in contrast, the THS and SeTHS groups recorded low and high total sperm motility, respectively, compared with other treatment groups (p < 0.05). The semen from the rams that received THS without Se displayed a significantly higher number of immotile sperm cells (p < 0.05) and poor sperm quality, including total and progressive motility, and kinematic parameters when compared with other treatments, suggesting that Se protects sperm against THS. We concluded that selenium protected some sperm parameters (TSM, PSM, MV, VCL, VSL) of THS- treated rams while others did not improve (RV, NSM, C, STR).

Real-Time Imaging of Calcium Dynamics in Human Sperm After Precise Single-Cell Stimulation.

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.

Testis-Specific Gene C7orf61 Is Involved in Mouse Sperm-Egg Fusion.

Chromosome 7 open reading frame 61 (C7orf 61) was a testis-specific gene, and may be involved in the process of spermatogenesis. This study aimed to investigate the expression of C7orf61 in the testis and determine its role in spermatogenesis. Reverse transcription-quantitative polymerase chain reaction, Western blot and immunofluorescence were performed to evaluate the expression characteristics of C7orf61 in mice and humans. In vitro fertilization assay was used to determine the role of the C7ORF61 protein in sperm-egg fusion. The results demonstrated that C7orf61 was a testis-specific gene; the C7ofr61 mRNA expression level sharply increased in the fourth postnatal week and gradually increased until the adult stage. The C7ORF61 protein was located throughout the subacrosomal area and close to the nucleus in both mouse and human sperm. The incubation with the C7ORF61 antibody significantly decreased the fertilization rate of mouse eggs. The present findings suggested that the C7ORF61 protein might be involved in sperm-egg fusion, and could serve as a useful target for contraceptives. However, further research is still needed to know the detailed molecularmechanismofitsrole.