Fertilizing Ability Of Spermatozoa Research Articles

Exosomes from uterine fluid promote capacitation of human sperm.

Extracellular vesicles (EVs) are membrane-bound vesicles containing various proteins, lipids, and nucleic acids. EVs are found in many body fluids, such as blood and urine. The release of EVs can facilitate intercellular communication through fusion with the plasma membrane or endocytosis into the recipient cell or through internalization of the contents. Recent studies have reported that EVs isolated from human endometrial epithelial cells (EECs) promote sperm fertilization ability. EVs from uterine flushing fluid more closely resemble the physiological condition of the uterus. However, it is unclear whether EVs derived directly from uterine flushing fluid have the same effect on sperm. This study aimed to research the effect of EVs from uterine flushing fluid on sperm. EVs were isolated from the uterine flushing fluid. The presence of EVs was confirmed by nanoparticle tracking analysis (NTA), Western blot, and transmission electron microscopy (TEM). EVs were incubated with human sperm for 2 h and 4 h. The effects of EVs on sperm were evaluated by analyzing acrosome reaction, sperm motility, and reactive oxygen species (ROS). The EVs fractions isolated from the uterine fluid were observed in cup-shaped vesicles of different sizes by TEM. All isolated vesicles contained similar numbers of vesicles in the expected size range (30-200 nm) by NTA. CD9 and CD63 were detected in EVs by western blot. Comparing the motility of the two groups incubated sperm motility significantly differed at 4 h. The acrosome reactions were promoted by incubating with EVs significantly. ROS were increased in sperm incubated with EVs. Our results showed EVs present in the uterine fluid. Acrosome reactions and ROS levels increased in human sperm incubated with EVs. EVs from uterine fluid can promote the capacitation of human sperm. The increased capacitation after sperm interaction with EVs suggests a possible physiological effect during the transit of the uterus.

Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

High-concentration bovine serum albumin enhances fertilization ability of cold-stored rat sperm.

Cold transport of the cauda epididymides is a useful technique for shipping laboratory rat sperm. Cold transport of rat sperm avoids potential risks of microbiological infection, animal escape or death, and animal welfare issues. Previously, we reported that a cold-storage solution containing dimethyl sulfoxide and quercetin maintained the fertility of cold-stored rat sperm. However, cold-stored rat sperm exhibited a decreased fertilization rate after 24-h storage. To recover the fertility of cold-stored sperm, we focused on the effects of bovine serum albumin (BSA), a cholesterol acceptor that induces sperm capacitation. We sought to determine the optimal concentration of BSA in fertilization medium based on the fertility of cold-stored rat sperm. High concentrations of BSA (40 mg/ml) enhanced the fertilization rate of cold-stored rat sperm and maintained sperm fertility for 144 h. Embryos derived from cold-stored and BSA-treated sperm normally developed into pups after embryo transfer. In summary, high BSA concentrations enhanced the fertility of cold-stored rat sperm and prolonged the storage period to 144 h, thereby expanding the transportable region for genetically engineered rats.

Analyzing the impact and mechanism of bisphenol A on testicular lipid metabolism in Gobiocypris rarus through integrated lipidomics and transcriptomics

Bisphenol A (BPA) is one of the most common environmental endocrine chemicals, known for its estrogenic effects that can interfere with male spermatogenesis. Lipids play crucial roles in sperm production, capacitation, and motility as important components of the sperm plasma membrane. However, limited research has explored whether BPA affects lipid metabolism in the testes of male fish and subsequently impacts spermatogenesis. In this study, we employed Gobiocypris rarus rare minnow as a research model and exposed them to environmentally relevant concentrations of BPA (15 μg/L) for 5 weeks. We assessed sperm morphology and function and analyzed changes in testicular lipid composition and transcriptomics. The results demonstrated a significant increase in the sperm head membrane damage rate, along with reduced sperm motility and fertilization ability due to BPA exposure. Lipidomics analysis revealed that BPA increased the content of 11 lipids while decreasing the content of 6 lipids in the testes, particularly within glycerophospholipids, glycerolipids, and sphingolipid subclasses. Transcriptomics results indicated significant up-regulation in pathways such as cholesterol metabolism, peroxisome proliferator-activated receptor signaling, and fat digestion and absorption, with significant alterations in key genes related to lipid metabolism, including apolipoprotein A-I, apolipoprotein C-I, and translocator protein. These findings suggest that BPA exposure can induce testicular lipid metabolism disruption in rare minnows, potentially resulting in abnormalities in rare minnow spermatogenesis.

Treatment with cholesterol just after thawing maintains the fertility of bull sperm.

Freezing and thawing diminish sperm motility and fertility by disrupting the cholesterol balance in sperm plasma and organelle membranes. The aim of this study was to elucidate the mechanisms through which exogeneous cholesterol treatment enhances the quality of frozen-thawed bull sperm. The incorporation of cholesterol was investigated using boron-dipyrromethene (BODIPY)-cholesterol, and BODIPY signals were detected not only in the plasma membrane but also in the midpiece region immediately after thawing. The positive signal of cholesterol in the midpiece region was inhibited by a scavenger receptor class B Type I (SR-BI) inhibitor, block lipid transport 1 (BLT-1). To comprehend the role of exogenous cholesterol in the functions of the plasma membrane, propidium iodide (PI)/Annexin V and peanut agglutinin lectin (PNA) staining were performed. The results showed that treatment with exogenous cholesterol increased the number of acrosome-intact sperm and decreased the number of sperm with damage to the plasma membrane. Moreover, since BODIPY signals were also observed in the midpiece region, mitochondrial function was evaluated using a flux analyzer and a flow cytometer with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) staining, revealing an increase in the number of sperm with high-mitochondrial activity and oxygen consumption. Finally, to assess sperm fertility, computer-assisted sperm analysis (CASA) and IVF were carried out. Sperm velocities and fertilization rates in IVF were significantly enhanced by the addition of cholesterol just after thawing. Thus, the treatment with cholesterol after thawing protected the plasma membrane from the stress of thawing and maintained mitochondrial function, thereby preserving the fertilization ability of frozen-thawed bull sperm for conventional IVF and artificial insemination (AI). Therefore, the application of cholesterol just after thawing is a promising option for improving the fertility of frozen-thawed sperm.

General and comparative aspects of endometritis in domestic species: a review.

Endometritis is a leading cause of sub- and infertility in domestic animal species. The healthy uterus is colonized by commensal bacteria, viruses, and yeast/fungi that represent the non-pathogenic microbiota. A shift in the number or type of organisms accompanied by immune dysfunction, however, may trigger uterine infection and inflammation. Metritis is associated with inflammation of all uterine layers (endometrium, myometrium, and perimetrium) whereas endometritis is a more superficial inflammation involving solely the endometrium. Endometritis generally occurs at two time points in domestic animal species, post-partum and post-mating. Post-partum endometritis may chronically persist, either as a low-grade disease that often manifests as a vaginal discharge but not a systemic illness (in some species termed clinical endometritis) or sometimes subclinical where features are only detected by endometrial sampling. Contamination of the uterus at the time of mating occurs by direct deposition of semen (ejaculated or artificially inseminated) into the uterus. Improper drainage of the ejaculatory fluid or an inadequate immune response may result in persistent mating-induced endometritis. Both post-partum and post-mating endometritis interferes with fertility by creating a suboptimal environment for embryo development and placentation and chronic endometritis may have an impact on sperm survival and fertilisation ability. In the post-partum animal, there may also be changes in milk production and maternal behaviour which can affect offspring health and survival. Preventive strategies for endometritis largely depend on monitoring their known risk factors, which are sometimes specific with regard to the species. Effective, non-antibiotic therapy for endometritis is not available to date. Overall, extensive research has been performed in cattle and horses to unravel key aspects of endometritis, but in sows and bitches, the available literature is scant. Thus, the need and opportunity to investigate the condition vary considerably among domestic species and necessitate their comparative assessment. This article reviews general and comparative aspects of the diagnosis and classification, pathogenesis, preventive strategies, and therapeutics of endometritis in domestic species with a specific focus on cows, mares, sows, and bitches.

Identification of IQCH as a calmodulin-associated protein required for sperm motility in humans

Sperm fertilization ability mainly relies on proper sperm progression through the female genital tract and capacitation, which involves phosphorylation signaling pathways triggered by calcium and bicarbonate. We performed exome sequencing of an infertile asthenozoospermic patient and identified truncating variants in MAP7D3, encoding a microtubule-associated protein, and IQCH, encoding a protein of unknown function with enzymatic and signaling features. We demonstrate the deleterious impact of both variants on sperm transcripts and proteins from the patient. We show that, invitro, patient spermatozoa could not induce the phosphorylation cascades associated with capacitation. We also provide evidence for IQCH association with calmodulin, a well-established calcium-binding protein that regulates the calmodulin kinase. Notably, we describe IQCH spatial distribution around the sperm axoneme, supporting its function within flagella. Overall, our work highlights the cumulative pathological impact of gene mutations and identifies IQCH as a key protein required for sperm motility and capacitation.

P-028 Loss of the m6A methyltransferase METTL5 impairs spermiogenesis and male fertility

Abstract Study question Whether and how METTL5 regulates germ cell development during spermatogenesis. Summary answer METTL5 is required for spermiogenesis. Loss of METTL5 resulted in teratozoospermia and male infertility via reduced translation of acrosome and flagellum formation proteins. What is known already The roles of m6A modifications on mRNA in spermatogenesis have been extensively studied. It was reported that METTL5 knockout mice showed the brain development defect and sterility of 16-week male mice. However, the detailed mechanism of METTL5 affecting male fertility remains elusive. Study design, size, duration Mettl5 KO mice were kindly gifted from Prof. Shuibin Lin of The First Affiliated Hospital of Sun Yat-sen University. Heterozygotes of Mettl5 mice were used to generate Mettl5 homozygous knockout mice. The phenotype of KO mice was assessed. Ribosomal sequencing, proteomics analysis and further validation of protein translation were performed to explore the mechanism. Participants/materials, setting, methods Fertility assessment, sperm parameter analyses, sperm nuclear morphology analysis, Transmission electron microscopy (TEM), tissue Collection and histological analysis, protein extraction and western blot analysis, immunofluorescence studies, cDNA synthesis and qRT-PCR, in vitro fertilization (IVF) were used in this study. Main results and the role of chance Here we reported that methyltransferase-like 5 (METTL5) is involved in spermiogenesis as a methyltransferase mediating m6A modification on rRNA. Mettl5 knockout mice were infertile with teratozoospermia. The acrosome in the sperm head was absent with reduced sperm motility. Furthermore, the fertilization ability of sperm in the IVF experiment failed. Mechanistically, the level of rRNA m6A modification was significantly decreased in the testes of Mettl5 KO mice. The translational efficiency and protein levels of acrosome and flagellum formation proteins such as FSIP2, ODF2, GK2, PGK2, and AKAP4 were significantly reduced when METTL5 was depleted. Limitations, reasons for caution The METTL5 mutation in the patient with teratozoospermia was not examined in the present study Wider implications of the findings The rRNA m6A modification is also involved in regulating spermatogenesis by METTL5. This study highlights the critical role of rRNA epigenetic modifications during spermatogenesis and provides novel theoretical explanations for the m6A modifications. Trial registration number Not applicable

P-095 Increased sperm high DNA stainability is associated with a diminished chance of fertilization with IVF cycles: a retrospective study using a propensity score-matching analysis

Abstract Study question To investigate the association of high DNA stainability (HDS) with fertilization, embryonic development and clinical outcome after IVF treatment. Summary answer Increased HDS levels might be associated with decreased fertilization of IVF cycle. What is known already The HDS parameter of sperm suggests loosening and weakening of chromatin condensation structure resulting from the lack of full histone-to-protamine exchange. Elevated HDS may negatively correlate with sperm quality, including sperm morphology and concentration. Study design, size, duration Couples that underwent IVF cycles from January 2016 to December 2020 were retrospectively studied, including a total of 2604 target cycles after IVF treatment consisting of 676 cycles in the HDS &amp;gt; 15% group and 1928 cycles in the HDS ≤ 15% group. In addition, 548 cycles undergoing fresh IVF-embryo transplantation treatment consisting of 151 cycles in the HDS &amp;gt; 15% group and 397 in the HDS ≤ 15% group were included. Participants/materials, setting, methods HDS was assessed by sperm chromatin structure assay method. Couples were classified into HDS &amp;gt; 15% group and HDS ≤ 15% group based on the HDS thresholds recommended by the manufacturer’s instructions. After controlling the bias between groups by the propensity score-matching method, the Mann-Whitney U test or Chi-squared test was used to evaluate the differences between groups. Regression analysis was used to assess the effect of HDS levels on fertilization, embryo quality, and clinical outcome of IVF cycles Main results and the role of chance No significant differences were observed between the HDS &amp;gt; 15% group and HDS ≤ 15% group regarding the number of fertilized oocytes (10.00 [6.00, 15.00] vs. 10.00 [6.00, 15.00], P = 0.230), the number of 2 pronuclei zygotes (8.00 [4.00, 12.00] vs. 8.00 [5.00, 12.00], P = 0.311), the number of cleaved zygotes (10.00 [5.00, 15.00] vs. 10.00 [6.00, 15.00], P = 0.301), and the number of high-quality embryos on day 3 (5.00 [2.00, 9.00] vs. 5.00 [3.00, 9.00], P = 0.729). However, linear regression analysis showed that HDS levels negatively impacted fertilization (adjusted B value = −0.015, 95% CI: −0.028 ∼ −0.002, P = 0.022) and 2 pronuclei formation (adjusted B value = −0.015, 95% CI: −0.029 ∼ 0.000, P = 0.046). The implantation rate (47.1% vs. 50.2%, P = 0.418), clinical pregnancy rate (60.9% vs. 64.5%, P = 0.440), ongoing pregnancy rate (83.7% vs. 86.7%, P = 0.475), early miscarriage rate (16.3% vs. 13.3%, P = 0.475), late miscarriage rate (1.1% vs. 3.5%, P = 0.232), and live birth rate (82.6% vs. 83.2%, P = 0.896) were not significantly different between groups. Linear regression analysis showed that HDS did not affect the implantation rate. Binary logistic regression analysis also showed that HDS did not affect clinical pregnancy, ongoing pregnancy, early miscarriage, late miscarriage, and live birth. Limitations, reasons for caution The main weakness of this study was the nature of the retrospective design, and additionally, patients were not randomized. Wider implications of the findings Although our results are not entirely new, it can be considered as reliable because 2604 IVF cycles and 548 fresh IVF-ET cycles were included and subsequently controlled the bias between groups This study suggested that HDS might be an indicator in predicting the fertilization ability of sperm in IVF treatment. Trial registration number not applicable

P-056 Oxidative stress and human semen quality: new results for diagnostic process of male infertility

Abstract Study question Is oxidative stress (OS) evaluated in human spermatozoa a predictive marker of better semen quality? Summary answer CellROX®Orange is a new fluorescent probe able to detect Reactive Oxygen Species (ROS) identifying a viable oxidized sperm fraction related to a better sperm performance. What is known already OS, defined as an unbalance between ROS production and antioxidant defences, is considered one of the causes of male infertility. OS evaluation in spermatozoa represents an important goal of research in this field because this parameter could be helpful to predict sperm fertilization ability to improve Assisted Reproductive Technology outcomes. By using different probes and methods for OS evaluation in spermatozoa or in semen, several studies were performed demonstrating a negative role of ROS on sperm functions. Such studies were not conclusive for the small number of included subjects, the high variability in the cohorts and the lack of validation. Study design, size, duration An observational study was conducted on 121 semen samples from patients undergoing routine semen analysis for couple infertility in the Andrology Laboratory of Careggi University Hospital of Florence from September 2021 to March 2022. Washed and Swim-up selected spermatozoa were incubated with two fluorescent probes, CellROX® Orange and Dihydroethidium (DHE), and revealed by flow cytometry. Participants/materials, setting, methods After routine semen analysis, the percentage of oxidized spermatozoa was evaluated with CellROX® Orange and DHE, and then correlated with standard semen parameters and sperm DNA fragmentation (sDF, evaluated by TUNEL/PI). Sperm kinematic parameters and hyperactivated motility were also assessed by C.A.S.A. system. Furthermore, CellROX® Orange positivity and phosphatidylserine membrane exposure (determined by Annexin V staining) or Caspase 3 and 7 activities (measured by FLICA™) were concomitantly evaluated. Main results and the role of chance We demonstrated that CellROX® Orange is able to detect hydrogen peroxide only in viable spermatozoa and the percentage of CellROX® Orange positive spermatozoa were positively associated with semen quality and negatively with sDF. To confirm these results, we performed the same experiments by using DHE, another probe which reveals hydrogen peroxide and superoxide anion in both viable and unviable cells. Similarly to CellROX® Orange, a positive correlation with semen quality and a negative one with sDF were found in viable spermatozoa. Conversely, in unviable spermatozoa, opposite associations were observed. These results indicate that oxidative status revealed by the two probes is related to a better sperm quality. To further investigate this possibility, we double labelled spermatozoa with CellROX® Orange and Annexin V (a marker of early signs of apoptosis) as well as with CellROX® Orange and FLICA™ (that detects caspase activity, a late sign of apoptosis). We found that CellROX® Orange mostly identifies spermatozoa without apoptotic features. Furthermore, we evaluated CellROX® Orange positivity in Swim-up selected spermatozoa, finding significantly higher levels in these samples respect to unselected samples, demonstrating once again that this probe identifies spermatozoa with better quality. Limitations, reasons for caution Our results indicate that within the viable oxidized spermatozoa there are cells with physiological and non-physiological intracellular ROS levels, however, they do not explain at what levels ROS cease to be functional and become deleterious for spermatozoa. Wider implications of the findings ROS evaluation in viable spermatozoa with the commercially available probes CellROX® Orange and DHE, allows the identification of the oxidized sperm fraction related to a sperm better performance. Therefore, the two probes may be useful to determine such fraction, likely improving the diagnostic process of male infertility. Trial registration number not applicable

P-106 The inhibition of sperm SIRT1 enhances fertilization and modifies subsequent embryonic epigenetic quality

Abstract Study question Is SIRT1 present in sperm and thus involved in fertilization and/or sperm epigenetic code? Do the SIRT1-driven epigenetic marks be transmitted to the progeny? Summary answer SIRT1 localized at the sperm head and influences fertilization and sperm epigenetic code via its deacetylation activity. SIRT1-modulated epigenome is transmitted to zygotes and blastocysts. What is known already The addition or removal of acetyl group from lysine residua (i.e. acetylation) post-translationally modulates proteins through the action of acetylases and deacetylases, respectively. SIRT1 (also Sirtuin 1) is capable to deacetylate a number of proteins, including histones, with roles in male reproduction. Testicular SIRT1 deficit leads to subfertility and increases number of abnormal and immotile spermatozoa that fail to fertilize. However, neither sperm SIRT1 presence nor its function has been investigated despite the significance of acetylation in the events around fertilization. Moreover, the epigenetic mode of action of sperm SIRT1 highlights its relevance for the success of the pre-implantation embryo. Study design, size, duration Mouse sperm of 14-18-weeks-old males of CD-1 x C57Bl6 hybrid strain were isolated from the epididymis. Sperm were incubated under capacitating conditions in presence of sirtinol (5 µM), a selective SIRT1 inhibitor, or the vehicle (0.1% v/v DMSO) for 1 h in 37 °C and 5% CO2. Participants/materials, setting, methods Motility and acrosome reaction was evaluated as previously stated (Ritagliati et al., 2018). Moreover, sperm proteins were extracted into RIPA-buffer and acetylated proteins were depicted by western blot. Finally, in vitro fertilization and embryo culture were performed. Above parameters defining IVF outcomes (i.e., fertilization, cleavage, and blastocyst rate), immunocytochemistry of acetylated histone 4 in the 16th residua of lysine (H4K16) in zygotes and blastocyst was quantified. Main results and the role of chance SIRT1 was localized in the sperm head, apparently at the perinuclear theca and perforatorium. Inhibition of SIRT1 leads to the increase of lysine acetylation (2.28 vs 1, fold change values) and hyperactivation (16.55 ± 0.79 vs 10.84 ± 0.79 %sperm), the enhanced motility that facilitates sperm to penetrate the oocyte, as well as the acrosome reaction (28.69 ± 0.41 vs 17.98 ± 0.41 %sperm). Subsequently, these spermatozoa were able to better fertilize oocytes in vitro (90.5 ± 7.21 vs 66.25 ± 7.21, %). Interestingly, zygotes derived from sirtinol-pre-treated spermatozoa developed to 2-cell embryos at a higher rate than those fertilized by untreated spermatozoa (79.33 ± 7.21 vs 67.00 ± 7.21, %). Moreover, we observed significant hyperacetylation of H4K16 in the male pronuclei of zygotes and embryos derived from sirtinol-treated sperm (8.19 ± 1.02 vs 6.10 ± 2.09 and 45.55 ± 13.96 vs 27.62 ± 13.96, integrated density of the signals, respectively). Limitations, reasons for caution Although acetylation of H4K16 was accumulated in male pronuclei, we do not know which subset of genes was affected. Therefore, we only can assure that SIRT1-driven epigenetic marks are transmitted to the offspring, but their consequences should be further elucidated. For knowledge transfer to humans, a verification study is required. Wider implications of the findings the reduction in testicular SIRT1 expression is an age-related phenomenon. Therefore, our results support SIRT1 as the basis for new interventions against age-related male infertility. Moreover, due to the high reactivity of SIRT1 to its inactivation, SIRT1 becomes a target to pharmacologically enhance sperm fertilization ability in assisted reproductive techniques. Trial registration number Animal procedures were conducted in accordance with Act No. 246/1992 Coll. on the Protection of Animals Against Cruelty under the supervision of the Animal Welfare Committee of the Ministry of Education, Youth and Sports of the Czech Republic, approval ID MSMT-11925/2016-3.

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21st to July 20th) and at the end (November 20th to December 21st) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6–19 rams at two very distant points in the mating season (July as Early Breeding Season –EBS– and November as Late Breeding Season –LBS–). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations.

CpG DNA methylation changes during epididymal sperm maturation in bulls

BackgroundDuring epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions.ResultsCaput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda.ConclusionsSpecific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Improvement of rooster semen freezability and fertility rate after sericin supplementation in freezing semen extender.

Semen cryopreservation result in decreased sperm parameters and fertilization ability. Sericin exhibits antioxidant activity by reducing lipid peroxidation resulting from free radicals, which can potentially improve cryopreservation outcomes. The present study aimed to examine the efficacy of various sericin concentrations supplemented with a rooster semen-freezing extender on post-thaw semen quality and fertilizing ability of sperm after cryopreservation. Semen samples were collected from 40 roosters (5 reps), then were pooled, and divided into four groups by the levels of sericin supplementation (0%, 0.25%, 0.50%, and 0.75%) in a freezing extender. Semen suspensions were loaded in medium straw (0.5 mL) and cryopreserved with the traditional liquid nitrogen vapor method. Post-thawed semen was evaluated for sperm motility, sperm viability, and lipid peroxidation. Also, the fertility test was determined. The results showed that supplementation of the freezing extender with 0.50% to 0.75% sericin resulted in greater total motility and progressive motility and lower malondialdehyde levels than the other groups after cryopreservation (p<0.05). However, the viability of 0.75% decreased compared with the value of 0.50% sericin supplementation (p<0.05). Moreover, the fertility and hatchability of total eggs were significantly higher in the 0.50% sericin group than in the other groups (p<0.05). In conclusion, 0.50% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved rooster semen.

Effects of Dietary Cholesterol Regulation on Spermatogenesis of Gobiocypris rarus Rare Minnow.

Cholesterol is an important component of cell membranes, and also a precursor for the synthesis of sex hormones, playing an important role in reproduction. However, few studies have focused on cholesterol and reproductive health. To investigate the toxic effects of different cholesterol levels on the spermatogenesis of rare minnows, we regulate the cholesterol content in fish by feeding them a high-cholesterol diet and cholesterol inhibitor pravastatin, and cholesterol levels, sex hormone (T and 11KT) levels, testis histology, sperm morphology and function, and the expression of genes related to sex hormone synthesis were investigated. The research findings indicate that increasing cholesterol levels significantly increases the liver weight and hepatic-somatic index, as well as the total cholesterol and free cholesterol levels in the testis, liver, and plasma of rare minnow, while inhibiting cholesterol has the opposite effect (p < 0.05). However, both increasing and decreasing cholesterol levels can suppress rare minnow testicular development, as evidenced by a decrease in testis weight, lowered gonadosomatic index, suppressed sex hormone levels, and reduced mature sperm count. Further exploration revealed that the expression of sex hormone synthesis-related genes, including star, cyp19a1a, and hsd11b2, was significantly affected (p < 0.05), which may be an important reason for the decrease in sex hormone synthesis and consequent inhibition of testicular development. At the same time, the fertilization ability of mature sperm in both treatment groups significantly decreased. Scanning electron microscopy and fluorescence polarization tests showed that reducing cholesterol levels significantly increased the rate of sperm head cell membrane damage, while both increasing and decreasing cholesterol levels led to a reduction in sperm cell membrane fluidity, which may be the main reason for the decrease in sperm fertilization ability. This study demonstrates that both increasing and decreasing the levels of cholesterol are detrimental to the fish spermatogenesis, providing fundamental information for the study of fish reproduction and also a reference for the causes of male reproductive dysfunction.

Sperm and egg in vitro storage effects on artificial fertilization and hatching in common carp (Cyprinus carpio L.)

This is the first report showing fertilization of short-term stored eggs (19 °C) with short-term stored diluted sperm (0–2 °C) in common carp (Cyprinus carpio). From 5 males, fresh (control) and 7-day stored sperm were diluted with common carp extender. After collection of eggs from nine females, the eggs were immediately pooled into three groups from females 1–3, 4–6 and 7–9, according to the eggs' quality based on visual criteria. They were placed in beakers, covered and incubated at 19 °C (for each pool, 10 g of eggs from each female were used). The 7-day short-term storage sperm was incubated at 20 °C for 20 min and pooled before being used for fertilization. The eggs were fertilized with fresh and short-term storage sperm after 1, 3 and 6 h post storage of eggs and activated with hatchery water and Perchec solution. Sperm motility and velocity (50–54% and 80–115 µm/s) of 7-day short-term storage sperm at 0–2 °C were ensured by additional incubation of sperm at 20 °C for 20 min before being activated. Principal component analysis showed that the sorting of eggs of different females into three groups according to sperm quality, fertilization ability and production of malformed larvae was successful. It is recommended that overripe common carp eggs are stored for a maximum of 1 h. A period of 3 h can be used for better quality eggs and exceptionally, up to 6 h for very high-quality eggs. Higher larvae malformation was found in the 6 h aged eggs together with fertilized short-term storage and fresh sperm. In all quality groups of eggs, the effect of different sperm, activation medium and egg storage time on the level of malformations was observed. The activation solution for carp did not show any positive fertilization and hatching effects.

SiO2 Nanoparticles Suspension Exposures with Marine Invertebrates: Genotoxicity Response

The volume of nanoparticles (NPs) used in various sectors of human life is steadily increasing. Due to their unique properties, silicon dioxide nanoparticles (SiO2 NPs) are widely used in industrial production. However, their ecotoxicological effect on marine biota is extremely poorly understood. To highlight this problem, experiments were conducted with short-term SiO2 NPs exposure to the sand dollar Scaphechinus mirabilis sperm, and a 6-day exposure to the adult bivalve mollusk Mytilus trossulus. SiO2 NPs exposure caused an increase in the concentration of Si in the digestive gland of the mollusk, resulting in DNA damage increase. Exposure to all NPs concentrations caused DNA damage to increase but did not affect the sperm fertilization ability. It was found that the DNA is a target in both chronic and acute SiO2 NPs exposure.

Cathepsin B plays a role in spermatogenesis and sperm maturation through regulating autophagy and apoptosis in mice.

Spermatogenesis and sperm maturation are complex and highly ordered biological processes. Any failure or disorder in these processes can cause defects in sperm morphology, motility, and fertilization ability. Cathepsin B (CTSB) is involved in the regulation of a variety of pathological processes. In the present study, we found that CTSB was abundantly expressed in the male reproductive system, however, the specific role of CTSB in regulating spermatogenesis and sperm maturation remained elusive. Hence, we generated Ctsb -/- mice using CRISPR/Cas9 technology. In Ctsb -/- mice, sperm count was significantly decreased while the level of morphologically abnormal sperm was markedly increased. Additionally, these mice had significantly lower levels of progressive motility sperm and elevated levels of immobilized sperm. Histological analysis showed slight vacuolization in the testis epithelium, as well as the loss of epididymal epithelium cells. Further investigation showed that autophagic activity was inhibited and apoptotic activity was increased in both the testis and epididymis of Ctsb -/- mice. Together, our findings demonstrate that CTSB plays an important role in spermatogenesis and sperm maturation in mice.

Connexin-43 is a promising target for lycopene preventing phthalate-induced spermatogenic disorders

IntroductionMale infertility is a multifactorial pathological condition and may be a harbinger of future health. Phthalates are ubiquitous environmental contaminants that have been implicated in the global decline in male fertility. Among them, di-(2-ethylhexyl) phthalate (DEHP) is the most prevalently used. Lycopene (LYC) is a possible preventive and therapeutic agent for male infertility owing to its antioxidant properties. The blood-testis barrier (BTB) is formed between Sertoli cells where it creates a unique microenvironment for spermatogenesis. ObjectivesWe hypothesize that phthalate caused male infertility and LYC plays an important role in phthalate-induced male fertility disorders. MethodsHematoxylin-eosin (H&E) staining, ultrastructure observation, and fluorescence microscopy were used to examine the morphological changes. RNA-Seq, and western blotting were conducted to detect gene and protein levels. Routine testing for sperm morphology and sperm-egg binding assay were conducted to examine the morphological structure and function of sperm. Cell scratch assay and transepithelial electrical resistance (TER) were used to detect cell migration capacity and barrier integrity. ResultsIn vivo experiments, we showed that LYC prevented DEHP-induced impairment of BTB integrity, which provided a guarantee for the smooth progress of spermatogenesis. LYC improved DEHP-induced change in sperm parameters and fertilization ability. Subsequent in vitro experiments, LYC alleviated MEHP-induced disruption of intercellular junctions in mouse Spermatogonia cells (GC-1 cells) and mouse Sertoli cells (TM4 cells). In MEHP-induced BTB impairment models of Sertoli cells, treatment with LYC or overexpressing connexin-43 (Cx43) promoted cell migration capacity and normalized BTB integrity. Cx43 knockdown inhibited cell migration capacity and perturbed BTB reassembly in LYC preventing DEHP-induced BTB impairment. ConclusionOur study provides evidence for the role of LYC in phthalates-induced spermatogenic disorders and points to Cx43 as a potential target for male fertility.

Heparin and Progesterone Exert Synergistic Effects to Improve the In-Vitro Fertilization Rate of Bovine Sperm Bound to Oviduct Cell Aggregates from the Isthmus.

Simple SummaryAfter mating, sperm starts its long journey with the ultimate goal of fertilizing the oocyte. Inside the oviduct, sperm is attached to the surface of epithelial cells. The intact and good-quality sperm are elected and stored. Many infertility-related problems are related to the short life span of the sperm and/or the delay of the capacitation process of sperm attached to the oviduct. Thus, the elongation of the life span of the sperm would be very helpful in overcoming such problems. We hereby aimed to evaluate the fertilization ability of sperm that bind to cell aggregates oviduct (infundibulum-ampulla-isthmus), and assess the effect of heparin and or progesterone on the in-vitro fertilization ability of sperm co-incubated with isthmus cells aggregates. The sperm bound to isthmus aggregates improved the rate of in-vitro fertilization compared to infundibulum and ampulla. Moreover, pre-treatment of mature oocytes with heparin and progesterone plays a coactive role that improves the in-vitro fertilization of sperm bound to cell aggregates from isthmus. In conclusion, binding to isthmus aggregates improves the in-vitro fertilization of bovine sperm. Additionally, heparin together with progesterone, exerts a synergistic action that improves the in-vitro fertilizing potential of sperm attached to isthmus aggregates.After the copulation process, some sperm start the long journey with an ultimate goal of fertilizing the oocyte. Inside the oviduct, sperm are stored, waiting for the ovulated oocyte where they bind to the apical surface of the oviduct cells, which in turn hold sperm to form a sperm nest. The essential functions of the sperm reservoir include attaching spermatozoa to oviduct epithelial cells, selecting intact, good-quality sperm with an end result of extending sperm life expectancy. The current study aimed to evaluate the fertilization ability of sperm that bind to cell aggregates from different parts of the oviduct (infundibulum-ampulla-isthmus), and to assess the effect of heparin and or progesterone (P4) on the in-vitro fertilization ability of sperm co-incubated with cell aggregates from the isthmus. In-vitro fertilization was identified as a cleaved oocyte to two cells or more. The sperm bound to cell aggregates from the isthmus improved the rate of in-vitro fertilization (48.09%) compared to aggregates from the infundibulum (36.90%) and ampulla (37.61%). Moreover, pre-treatment of mature COCs with heparin (40 μg/mL) and P4 (80 nanomolar) play a coactive role that improves the in-vitro fertilization ability of sperm bound to cell aggregates from isthmus (63.33%), compared to 42.61% in the absence of cells aggregates. In conclusion, binding to cell aggregates from isthmus improves the in-vitro fertilization ability of Bovine sperm. Additionally, heparin, together with P4, exerts a synergistic action that improves the in-vitro fertilizing potential of sperm attached to cell aggregates from the isthmus of the bovine oviduct.