Mouse Sperm Research Articles

Responses in organs, sperm, steroid hormones and CYP450 enzyme in male mice treated by quinestrol only or in conjunction with clarithromycin

Pest rodents persistently undermine crop yields and food security. Fertility control could be a viable alternative for managing rodent populations. This study investigates the antifertility effects of various concentrations of clarithromycin combined with 1.0 mg/kg quinestrol on male rodents to determine an effective contraceptive dose that minimizes quinestrol usage, addressing key concerns such as potential environmental residue, which may impact ecological balance, and poor palatability, which could reduce ingestion and limit the sterilant’s effectiveness. Male mice were divided into five groups and administered different doses of clarithromycin or clarithromycin and quinestrol for three consecutive days, while the control group received sunflower seed oil only. After seven days, organ weights, reproductive organ weights, sperm density, serum hormone levels, and CYP3A4 content in small intestinal and liver tissues were measured to assess persistent effects. Compared with the control group, all treatment groups had significant reductions in epididymal weight, seminal vesicle weight, and serum T and LH levels. Higher concentrations of clarithromycin (2 mg/kg and 10 mg/kg) significantly impacted reproductive metrics, including sperm density, organ weights, and serum LH and testosterone levels, though complete sterilization was not achieved, with more than 60 million cauda epididymal spermatozoa remaining. However, the combination demonstrated potential as an effective strategy for male fertility control. The combination of 2.0 mg/kg clarithromycin and quinestrol can mitigate organ enlargement seen with quinestrol alone. This combination also decreased total enzyme content, thereby diminishing quinestrol’s induction of CYP3A4, which may increase the sterilization effectiveness of the treatment.

Mammalian Ste-20-like Kinase 1/2 (MST1/2) Inhibitor XMU-MP-1: A Potential Compound to Improve Spermatogenesis in Mouse Model of Diabetes Mellitus

Background/Objectives: Spermatogenesis is a key process in male reproduction that, if it does not happen correctly, can lead to infertility, with diabetes being one of the most prevalent causes of spermatogenesis disruption. Currently, there is a lack of research examining the potential benefits of targeting cell proliferation to enhance spermatogenesis in this condition. XMU-MP1 has been identified as an inhibitor of MST1, a core component of the Hippo pathway, which is anticipated to promote proliferation and regeneration. This study aims to evaluate the effects of XMU-MP1 treatment on sperm and testicular characteristics in mice. Methods: We used the STZ-induced diabetic mouse model to investigate the impact of administering XMU-MP1 on testicular tissue and sperm parameters. This study compared the seminiferous tubules, specifically focusing on the diameter of the seminiferous tubule, the thickness of the seminiferous tubule epithelium, the ratio of the thickness of the seminiferous tubule epithelium to the diameter of the seminiferous tubules, and the lumen diameter of the seminiferous tubules. We also conducted a comparison of sperm parameters, including sperm concentration, progressive motility, total motility, total motility, and morphology. Results: XMU-MP1-treated mice had a larger spermatogenesis area and better sperm motility than control mice. Diabetic mice treated with XMU-MP1 also showed a trend toward improvements in the spermatogenesis area, sperm concentration, sperm motility, and sperm morphology, although these improvements were not statistically significant. Conclusions: XMU-MP1 serves as a potential compound to improve spermatogenesis in mice.

Disperse Red 1 azo dye: Consequences of low-dose/low-concentration exposures in mice and zebrafish

Color Index Disperse Red 1 (DR1), an azo dye widely used in the textile industry and released into aquatic environments, is genotoxic in somatic cells, but little is known concerning its effects on the reproductive system or the early stages of embryonic development. We have assessed the effects on the spermatozoa of male mice following oral exposure to the dye, at low doses, for 14 days. Measured endpoints were DNA damage (comet assay), miRNA-34c levels, and sperm number, morphology, and motility. Exposure caused decreased miRNA-34c levels. We have also examined dye effects on zebrafish embryos and larvae, which included developmental impairment, altered glutathione transferase activity, and effects on reactive oxygen species and lipid peroxidation levels.

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.

Female fertility and the mammalian egg's zona pellucida.

All mammalian eggs are surrounded by a relatively thick extracellular matrix (ECM) or zona pellucida (ZP) to which free-swimming sperm bind in a species-restricted manner during fertilization. The ZP consists of either three (e.g., Mus musculus) or four (e.g., Homo sapiens) glycosylated proteins, called ZP1-4. These proteins are unlike those found in somatic cell ECM, are encoded by single-copy genes on different chromosomes, and are well conserved among different mammals. Mammalian ZP proteins are synthesized as polypeptide precursors by growing oocytes that will become ovulated, unfertilized eggs. These precursors are processed to remove a signal-sequence and carboxy-terminal propeptide and are secreted into the extracellular space. Secreted ZP proteins assemble into long, crosslinked fibrils that exhibit a structural repeat due to the presence of ZP2-ZP3 dimers every 140 Å or so along fibrils. Fibrils are crosslinked by ZP1 and are oriented either perpendicular, parallel, or randomly to the plasma membrane of eggs depending on their position in the ZP. Free-swimming mouse sperm recognize and bind to ZP2 or ZP3 that serve as sperm receptors. Acrosome-intact sperm bind to ZP3 oligosaccharides and acrosome-reacted sperm bind to ZP2 polypeptide. ZP fibrils fail to assemble in the absence of either nascent ZP2 or ZP3 and results in mouse eggs that lack a ZP and female infertility. Gene sequence variations due to point, missense, or frameshift mutations in genes encoding ZP1-4 result in human eggs that lack a ZP or have an abnormal ZP and female infertility. These and other features of the mouse and human egg's ZP are discussed here.

Dysregulated miR-124 mediates impaired social memory behavior caused by paternal early social isolation.

Early social isolation (SI) leads to various abnormalities in emotion and behavior during adulthood. However, the negative impact of SI on offspring remains unclear. This study has discovered that paternal early SI causes social memory deficits and anxiety-like behavior in F1 young adult mice, with alterations of myelin and synapses in the medial prefrontal cortex (mPFC). The 2-week SI in the F1 progeny exacerbates social memory impairment and hypomyelination in the mPFC. Furthermore, the down-regulation of miR-124, a key inhibitor of myelinogenesis, or over-expression of its target gene Nr4a1 in the mPFC of the F1 mice improves social interaction ability and enhances oligodendrocyte maturation and myelin formation. Mechanistically, elevated levels of miR-124 in the sperm of paternal SI mice are transmitted epigenetically to offspring, altering the expression levels of miR-124/Nr4a1/glucocorticoid receptors in mPFC oligodendrocytes. This, in turn, impedes the establishment of myelinogenesis-dependent social behavior. This study unveils a novel mechanism through which miR-124 mediates the intergenerational effects of early isolation stress, ultimately impairing the establishment of social behavior and neurodevelopment.

Ascorbic acid 2-glucoside improves survival, quality, and fertility of frozen-thawed C57Bl/6J and C57Bl/6N mouse spermatozoa.

Ascorbic acid 2-glucoside (AA2G) is a stabilized form of ascorbic acid and a potent antioxidant. Ascorbic acid is present in the testes and epididymis and helps maintain the physiological integrity of reproductive organs. Its properties have been utilized to protect spermatozoa of different species from oxidative stress. Spermatozoa of C57Bl/6J and C57Bl/6N strains were frozen and analyzed, after thawing, by supplementing the capacitation medium with AA2G, both in the presence and absence of methyl-β-cyclodextrin (MBCD). The effect of treatment was evaluated by SCA System (Microptic) analyzing the velocity, vitality, morphology, and the DNA fragmentation. We also examined sperm capacitation (CTC), acrosome reaction (Coomassie Brillant Blue), and fertility (in vitro fertilization) of treated spermatozoa. AA2G improved sperm quality and fertility particularly in association with MBCD. We observed a significant increase of sperm motility, velocity, and vitality associated with an enhanced capacitation and acrosome reaction. These improvements resulted in a marked increase in in vitro fertilization success. Embryos obtained were cultured and reached normally the blastocyst stage. This study aimed to determine if AA2G could safeguard mouse spermatozoa during cryopreservation. We found a protective effect of AA2G that increased sperm survivability resulting in higher fertilization rate. This newly improved protocol shows potential for reanimating cryopreserved GEMMs stored in mouse biobanks and international repositories, such as the European Mouse Mutant Archive (EMMA). This can serve as a pivotal tool in fulfilling the 3Rs mission (replacement, reduction, and refinement), promoting ethical and humane research practices.

Autism-Related Cc2d1a Heterozygous Mice: Increased Levels of miRNAs Retained in DNA/RNA Hybrid Profiles (R-Loop).

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with a highly variable expression of phenotypes (restricted interest or activity and repetitive behavior in communication and social interactions), genes (mutation), markers (alteration of transcription) and pathways. Loss of function of the CC2D1A gene appears to primarily affect the brain, leading to a range of behavioral problems in humans. In our study published in 2020, we found that the expressions of miR-19a-3p, miR-361-5p, miR-150-5p, miR-3613-3p, miR-126-3p and miR-499a-5p were downregulated in the serum samples of autistic patients, their families and mouse models (Cc2d1a +/- and valproic acid treated males). Here, acquired non-Mendelian hereditary character in a genetically defined mouse model of autism (Cc2d1a +/-) correlates with the transcriptional alteration of five miRNAs. We seek to test the hypothesis that miRNA levels vary by changes in RNA/DNA structure during development, thereby creating transcription alteration and cell memory. Behavioral tests were conducted on the offspring of Cc2d1a (+/-) mutant and control mice, such as novel object, social interaction, marble burying and tail suspension behavior. Two RNA fractions were isolated from mouse hippocampal tissues and sperm cells via standard TRIzol extraction: free RNA and the fraction of RNA bound to DNA in the form of a DNA/RNA hybrid (R-loop). The expression levels of miR-19a-3p, miR-361-5p, miR-150-5p, miR-126-3p and miR-499a-5p were investigated by quantitative real-time RT-PCR. We report differences in the distribution of five miRNAs in the hippocampus between male and female mice, particularly in colonies of Cc2d1a (+/-) mice. Furthermore, the number of miRNAs engaged in the DNA/RNA hybrid fraction is generally higher in the mutant pedigree than in the control group. On the other hand, in sperm, both fractions are at lower levels than in controls. R-loops contribute to the physiology and pathology of organisms including human disease. Here, we report a variation in five miRNA levels between gender and tissue. Our results suggest that the transcription levels of these five miRNAs are directly regulated by their RNA.

Depletion of the paternal gut microbiome alters sperm small RNAs and impacts offspring physiology and behavior in mice

The paternal environment prior to conception has been demonstrated to influence offspring physiology and behavior, with the sperm epigenome (including noncoding RNAs) proposed as a potential facilitator of non-genetic inheritance. Whilst the maternal gut microbiome has been established as an important influence on offspring development, the impact of the paternal gut microbiome on offspring development, health and behavior is largely unknown. Gut microbiota have major influences on immunity, and thus we hypothesized that they may be relevant to paternal immune activation (PIA) modulating epigenetic inheritance in mice. Therefore, male C57BL/6J mice (F0) were orally administered non-absorbable antibiotics via drinking water in order to substantially deplete their gut microbiome. Four weeks after administration of the antibiotics (gut microbiome depletion), F0 male mice were then mated with naïve female mice. The F1 offspring of the microbiome-depleted males had reduced body weight as well as altered gut morphology (shortened colon length). F1 females showed significant alterations in affective behaviors, including measures of anxiety and depressive-like behaviors, indicating altered development. Analysis of small noncoding RNAs in the sperm of F0 mice revealed that gut microbiome depletion is associated with differential expression of 8 different PIWI-interacting RNAs (piRNAs), each of which has the potential to modulate the expression of multiple downstream gene targets, and thus influence epigenetic inheritance and offspring development. This study demonstrates that the gut-germline axis influences sperm small RNA profiles and offspring physiology, with specific impacts on offspring affective and/or coping behaviors. These findings may have broader implications for other animal species with comparable gut microbiota, intergenerational epigenetics and developmental biology, including humans.

Stress increases sperm respiration and motility in mice and men

Semen quality and fertility has declined over the last 50 years, corresponding to ever-increasing environmental stressors. However, the cellular mechanisms involved and their impact on sperm functions remain unknown. In a repeated sampling human cohort study, we identify a significant effect of prior perceived stress to increase sperm motility 2-3 months following stress, timing that expands upon our previous studies revealing significant stress-associated changes in sperm RNA important for fertility. We mechanistically examine this post-stress timing in mice using an in vitro stress model in the epididymal epithelial cells responsible for sperm maturation and find 7282 differentially H3K27me3 bound DNA regions involving genes critical for mitochondrial and metabolic pathways. Further, prior stress exposure significantly changes the composition and size of epithelial cell-secreted extracellular vesicles that when incubated with mouse sperm, increase mitochondrial respiration and sperm motility, adding to our prior work showing impacts on embryo development. Together, these studies identify a time-dependent, translational signaling pathway that communicates stress experience to sperm, ultimately affecting reproductive functions.

Aquaporin-3a Dysfunction Impairs Osmoadaptation in Post-Activated Marine Fish Spermatozoa.

Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca2+ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon.

Development of a Novel Male Reproductive Toxicity Assessment Method to Predict Male-mediated Effects on the Next Generation

Less than 10% of the candidate drug compounds are associated with male reproductive toxicity. Genetic and/or epigenetic information on sperm may be crucial for fetal development. Therefore, developmental toxicity, such as paternally transmitted birth defects, is possible if genetic abnormalities in the male germ line persist and accumulate in the sperm during spermatogenesis. First, this study provides an overview of chemical and male reproductive toxicity, which may lead to developmental toxicity from the perspective of male reproduction. Second, we demonstrate methods for evaluating male reproductive toxicity to anticipate male-mediated developmental toxicity. We developed a novel staining technique for evaluating sperm quality, as well as a noninvasive imaging analysis of male reproductive toxicity. The former is a mammalian male germ cell-specific staining method using reactive blue 2 dye (RB2), as previously confirmed in human sperm, and a method for detecting the early-stage DNA fragmentation in a single nucleus from mouse spermatozoa using single-cell pulsed-field gel electrophoresis. The latter is a new, ready-to-use, and compact magnetic resonance imaging (MRI) platform utilizing a high-field permanent magnet to evaluate male reproductive toxicity. The histopathological analysis supported the suitability of the MRI platform. The present study, for the first time, revealed a rapid, noninvasive evaluation of male reproductive toxicity in vivo using compact MRI. These novel toxicity assessments can help predict male-mediated developmental toxicity, contributing to accelerated drug discovery and drug repositioning.

The Sperm-Associated Antigen 11A (Spag11a) Knockout Mice Display Sub-Fertility and Perturbations in the Sperm Proteome.

Spermatogenesis and sperm maturation that occur in the testis and epididymis respectively are crucial for fertility. Factors secreted by the testicular and epididymal epithelial cells govern the processes of gametogenesis and maturation. Proteins encoded by the sperm-associated antigen 11a (Spag11a) gene are implicated as having a possible role in sperm maturation. However, studies that demonstrate their definite role in fertility and sperm function using knockout models have not yet reported. In this study, Spag11a knockout mice were generated, genotyped and the reproductive parameters (fecundity, sperm count, capacitation, and acrosome reaction) and sperm proteome were determined. Litter size and sperm count were decreased in the Spag11a knockout mice when compared to the wild-type controls. Spermatozoa from the knockout mice were able to undergo capacitation. However, acrosome reaction did not occur in sperm obtained from knockout mice. Structural abnormalities in the head and tail structures were evident in the spermatozoa of knockout mice. Perturbations in the expression of sperm proteins that are involved in gametogenesis were evident. The subfertility observed in Spag11a knockout mice could be a manifestation of lower sperm count, impaired acrosome reactions, and disturbances in the sperm proteome. The results of this study lend further support to the role of Spag11a gene in male gamete function.

Silymarin mitigates toxic effects of cyclophosphamide on testicular tissue and sperm parameters in mice

Cyclophosphamide, a chemotherapy drug, increases oxidative stress in sperm and testicular tissue. This study evaluated the effect of silymarin, a potent antioxidant, on the quality of sperm and testicular tissue in mice treated with cyclophosphamide. NMRI adult male mice were divided into four groups: control; cyclophosphamide (intraperitoneal injection, 100 mg/kg, once a week); cyclophosphamide + silymarin; and silymarin (intraperitoneal injection, 200 mg/kg, every other day). After a 35-day treatment period, the caudal region of the epididymis was examined for sperm parameters, the right testis was used for stereological studies, and the left testis was used to assess biochemical factors. The data were statistically analyzed using SPSS software, one-way ANOVA and Tukey's test. In the cyclophosphamide group, there was a significant reduction in the mean total volume of testicular tissue, the average volume of seminiferous tubules and their components, and the average volume of interstitial tissue. Additionally, there was a notable decrease (p < 0.001) in the average number of Leydig cells, Sertoli cells, and sperm parameters. The mean concentration of testosterone hormone (p < 0.05) and total antioxidant capacity (TAC) level (p < 0.01) also significantly decreased, while the malondialdehyde (MDA) level increased significantly (p < 0.05). However, these adverse changes were mitigated in the cyclophosphamide + silymarin group compared to the cyclophosphamide group. Our results showed that silymarin as an antioxidant can mitigate the adverse effects of cyclophosphamide on testicular tissue and sperm parameters.

Assessment of acute, subacute genetic toxicities and immunomodulatory activity of palm (Trachycarpus fortunei) buds

Ethnopharmacology relevancePalm buds are a natural green resource of the forest, which are not only rich in nutrients but contain a large number of phenolic acids and flavonoids, among other components. It has a variety of biological activities such as antioxidant and uterine smooth muscle stimulation.Aim of the study: To evaluate the safety of palm buds for use as a nutraceutical product and food by evaluating the toxicity, subacute toxicity and genotoxicity of the young palm buds. Also studied for its immune-enhancing activity. Materials and methodsAcute toxicity tests were performed in mice using the maximum tolerance method, and the manifestations of toxicity and deaths were recorded after administration of 10,000 mg/mL for 14 consecutive d (days) of observations. To assess subacute toxicity, mice were treated with palm buds (750, 1500, or 3000 mg/mL) daily for 28 days. The teratogenicity of palm buds was assessed by the Ames test, the mouse bone marrow cell micronucleus test, and the mouse spermatozoa malformation test. In addition, we evaluated the immune-enhancing ability of palm buds by the mouse carbon profile test, delayed-type metamorphosis reaction, and serum hemolysin assay. ResultsIn the acute toxicity study, the Median Lethal Dose (LD50) was greater than 10,000 mg/kg bw in both male and female rats. There were also no deaths or serious toxicities in the subacute study. The no-observed-adverse-effect level (NOAEL) was 3000 mg/kg bw. However, the mice's food intake decreased after one week. The medium and high dose groups had a reducing effect on body weight in mice of both sexes. In addition, the changes in organ coefficients of the liver, kidney and stomach in male mice were significantly higher in the high-dose group (3.23 ± 0.35, 0.75 ± 0.05, 0.57 ± 0.05 g) than in the control group (2.94 ± 0.18, 0.58 ± 0.05, 0.50 ± 0.02 g). Hematological analyses showed that all the indices of the rats in each palm sprout dose group were within the normal range. The results of blood biochemical indicators showed that there was a significant reduction in TP in the blood of male mice in the high-dose group (44.6 ± 7.8 g/L) compared to the control group (58.3 ± 15.1 g/L). In histopathological analysis, none of the significant histopathological changes were observed. The results of the immunological experiment in mice showed that the liver coefficient and thymus coefficient of the high-dose group (8400 mg/kg) were significantly lower than the control group. There was no remarkable difference in auricle swelling between each dose palm bud group (1400, 2800, or 8400 mg/kg) and the control group. The anti-volume number of the high-dose group was significantly increased. ConclusionPalm buds have non-toxic effects in vivo and have little effect on non-specific and cellular immunity in the test mice within the dose range of this experiment. The immunoenhancement in mice is mainly achieved through humoral immunity. In conclusion, our results suggest that palm buds are safe for use as healthcare products and food.

Treatment of cryopreserved bovine sperm with calcium ionophore A23187 increases in vitro embryo production

After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest.

GGNBP2 regulates histone ubiquitination and methylation in spermatogenesis

ABSTRACT Gametogenetin binding protein 2 (GGNBP2) was indispensable in normal spermatids for transformation into mature spermatozoa in mice, and when Gametogenetin binding protein 2 is bound to BRCC36 and RAD51, the complex participates in repairing DNA double-strand breaks (DSB) during the meiotic progression of spermatocytes. Ggnbp2 knockout resulted in the up-regulation of H2AK119ubi and down-regulation of H2BK120ubi in GC-2 cells (mouse spermatogonia-derived cell line) and postnatal day 18 testis lysate. Our results also demonstrated that Gametogenetin binding protein 2 inducedASXL1 to activate the deubiquitinating enzyme BAP1 in deubiquitinating H2A, while Gametogenetin binding protein 2 knockout disrupted the interaction between ASXL1 and BAP1, resulting in BAP1 localization change. Furthermore, the Gametogenetin binding protein 2 deletion reduced H2B ubiquitination by affecting E2 enzymes and E3 ligase binding. Gametogenetin binding protein 2 regulated H2A and H2B ubiquitination levels and controlled H3K27 and H3K79 methylation by PRC2 subunits and histone H3K79 methyltransferase. Altogether, our results suggest that Ggnbp2 knockout increased DNA damage response by promoting H2A ubiquitination and H3K27trimethylation (H3K27me3) and reduced nucleosome stability by decreasing H2B ubiquitination and H3K79 dimethylation (H3K79me2), revealing new mechanisms of epigenetic phenomenon during spermatogenesis. Gametogenetin binding protein 2 seems critical in regulating histone modification and chromatin structure in spermatogenesis.

Comparative Investigation of the Mechanisms of Calcium Response in Human and Murine Spermatozoa

Calcium signaling is a principal method of signal transduction in cells of non-excitable tissues. In both mouse and human sperm, it can be induced in response to progesterone, manifesting as oscillations or single peaks and followed by the acrosomal reaction. However, the molecular mechanisms of progesterone activation may vary between species. In this study, we aim to compare the calcium signaling mechanisms in human and mouse spermatozoa. We investigated the calcium response in mouse sperm activated by progesterone. We employed spectrofluorometry to quantify the rise in calcium concentration in response to progesterone in Fura-2 loaded mouse sperm cells in suspension. Our experiments demonstrated that mouse sperm cells respond to 50 μM progesterone with a peak 120 ± 35 s wide and 0.8 ± 0.3 μM high. Based on literature data, a scheme for the induction of calcium signaling was constructed, suggesting an intermediate stage with the synthesis of a certain prostanoid (possibly PGE2) and activation of mouse sperm by this prostanoid through a G-protein-coupled receptor. Based on the obtained reaction scheme, two computational models were developed: a point model and a three-dimensional model. As with human sperm, the point model provided only a qualitative description of calcium responses, whereas the three-dimensional model produced the shape of the calcium peak and the frequency of calcium oscillations in response to progesterone that were similar to the experimentally obtained values. Using in silico analysis, it was shown that in mouse sperm, the spatial distribution of signaling enzymes regulates the type and form of the calcium response. We conclude that the presence of time delays due to the diffusion and spatial distribution of calcium signaling enzymes regulates the calcium response in both human and mouse sperm.

Selection of functional sperm by using hyaluronic acid modified magnetic microbeads and an electromagnetic manipulation system.

In recent years, the incidence of infertility has increased year by year. Assisted reproductive technology (ART) is one of the effective strategies to treat infertility. In the process of ART, commonly used methods for sperm separation have shortcomings and there is still room for improvement. In this study, a functional sperm selection strategy was established based on hyaluronic acid (HA) modified magnetic microbeads (MBs) and a supporting two-dimensional electromagnetic manipulation device system. Due to the modification of HA on the surface of MBs, the HA-MBs have the ability to target and bind to specific receptors on the sperm membrane to form a sperm-MB complex. A disulfide linker was introduced to connect HA and MBs. After modifying HA and connecting it with the disulfide linker, the sperm of sperm-MBs can be released under the combined effect of hyaluronidase and reduced GSH with the disulfide bond broken and HA degraded. A two-dimensional electromagnetic manipulation system was introduced to generate a magnetic field and control the directional movement of the sperm-MB complex under the guidance of an inverted microscope. The free MBs can also be removed after the sperm is released. Furthermore, the mouse sperm selected through this strategy can achieve normal insemination via ICSI and the obtained blastocysts have normal morphology and developmental milestones. This strategy has potential to be developed into an automated screening solution for the screening of functional sperm for assisted reproductive technology.

Optimization of spermatozoa analysis in mice: A comprehensive protocol

Sperm quality is critical to predict reproductive alterations caused by immunological factors or toxicant agents. Yet, no detailed protocol has been published focusing on analyses of sperm parameters in mice. Our aim was to evaluate the most efficient diluent for mice sperm analyses and to optimize the sperm morphology classification, through the comparison of different staining methods. The diluents assessed were PBS (baseline), HTF, DMEM, 1 % BSA in PBS and 9 % skimmed powdered milk diluted in PBS. Spermatozoa were evaluated for vitality, motility, and morphology, smears were stained with Papanicolaou, HE, Giemsa, and Rapid staining. Sperm vitality and total motility reached better scores in milk based and DMEM diluents. HE raised up as an effective option since its combination with any of the diluents we tested, resulted in a fair staining, which was appropriated to evaluate mice spermatozoa. Finally, based on WHO manual, we have updated the current morphological classification for mice sperm, since we have detailed the head defects as well as included midpiece and tail defects on it. Taken together, we presented a useful, low cost, and reliable method to assess sperm morphology that could be employed worldwide by laboratories dedicated to study reproductive biology on mice model.