The Sperm Protein Spaca6 is Essential for Fertilization in Zebrafish

Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, yet its function has not been investigated outside of mammals. Here we show that zebrafish spaca6 encodes for a sperm membrane protein which is essential for fertilization. Zebrafish spaca6 knockout males are sterile. Furthermore, Spaca6-deficient sperm have normal morphology, are motile, and can approach the egg, but fail to bind to the egg and therefore cannot complete fertilization. Interestingly, sperm lacking Spaca6 have decreased levels of another essential and conserved sperm fertility factor, Dcst2, revealing a previously unknown dependence of Dcst2 expression on Spaca6. Together, our results show that zebrafish Spaca6 regulates Dcst2 levels and is required for binding between the sperm membrane and the oolemma. This is in contrast to murine sperm lacking SPACA6, which was reported to be able to bind but unable to fuse with oocytes. These findings demonstrate that Spaca6 is essential for zebrafish fertilization and is a conserved sperm factor in vertebrate reproduction.

How Cold Shock Affects Ploidy Level and Early Ontogenetic Development of the Sterlet, A. ruthenus L.

The objective of the present research was to study the effect of cold shock (3 °C and 6 °C) on fertilized eggs of the sterlet, Acipenser ruthenus L. Cold shock was applied for various durations (30, 60 and 90 min) and the ploidy levels, survival, and genotypes of the treated embryos/larvae were recorded. Analysis of ploidy levels confirmed the presence of diploid, triploid, and mosaic (1n/2n, 2n/3n, and 1n/2n/3n) genotypes in experimental groups, while it was strictly diploid in control groups. Microsatellite genotyping confirmed both the incidence of polyspermy and retention of the 2nd polar body in experimental groups. However, patterns of inheritance in all diploid offspring in experimental and control groups revealed classical Mendelian disomic inheritance. Interestingly, the observed mosaic sterlets had normal morphology and were alive. However, some larvae had abnormal morphology which may be due to haploid syndrome. In all treatment groups (treatments: 3 °C–30 min; 3 °C–60 min; 3 °C–90 min; 6 °C–60 min), where the percentage of polyploid/mosaic larvae were high, the mortality was also high. Whereas, in the control groups (where there were only diploid (2n) larvae), the mortality was relatively low.

A new animal model signifying a decisive bridge between innate immunity and the pathognomonic morphological characteristics of Type 1 Diabetes

Available animal models for Type 1 Diabetes (T1D) show limited similarities with the human disease and have no predictive value in screening for effective intervention therapies. Heat-inactivated bacteria instilled in the ductal compartment of the pancreas in healthy rats rapidly cause periductal inflammation and accumulation of mainly granulocytes and monocytes in the exocrine pancreas and in the peri-islet area mimicking the acute pancreatic inflammation in subjects with recent onset T1D. After three weeks, the triggered exocrine inflammation had vanished and pancreases showed normal morphology. However, a distinct accumulation of both CD4+ and CD8+ T cells within and adjacent to affected islets was found in one third of the rats, mimicking the pathognomonic insulitis in T1D. As in human T1D, the insulitis affected a fraction of all islets and was observed only in certain lobes of the pancreases. The presented animal model for T1D will allow detailed mechanistic studies to unravel a previously unknown interplay between bacteria-activated innate immunity and an acquired cellular immunity forming the immunopathological events described in humans at different stages of T1D.Summary StatementThe results presented signify a previously unknown decisive bridge between innate immunity and formation of the pathognomonic immunopathological events described in subjects with recent onset T1D.

Single-Larva RNA Sequencing Identifies Markers of Copper Toxicity and Exposure in Early Mytilus californianus Larvae

One of the challenges facing efforts to generate molecular biomarkers for toxins is distinguishing between markers that are indicative of exposure and markers that provide evidence of the effects of toxicity. Phenotypic anchoring provides an approach to help segregate markers into these categories based on some phenotypic index of toxicity. Here we leveraged the mussel embryo-larval toxicity assay in which toxicity is estimated by the fraction of larvae that exhibit an abnormal morphology, to isolate subsets of larvae that were abnormal and thus showed evidence of copper-toxicity, versus others that while exposed to copper exhibited normal morphology. Mussel larvae reared under control conditions or in the presence of increasing levels of copper (3–15 μg/L Cu2+) were physically sorted according to whether their morphology was normal or abnormal, and then profiled using RNAseq. Supervised differential expression analysis identified sets of genes whose differential expression was specific to the pools of abnormal larvae versus normal larvae, providing putative markers of copper toxicity versus exposure. Markers of copper exposure and copper-induced abnormality were involved in many of the same pathways, including development, shell formation, cell adhesion, and oxidative stress, yet unique markers were detected in each gene set. Markers of effect appeared to be more resolving between phenotypes at the lower copper concentration, while markers of exposure were informative at both copper concentrations.

Study on effect of hair coat color on various reproductive attributes of Cholistani service bulls

The objective of the present study was to assess the possible effect of hair coat color on various orchidometric, seminal and andrological attributes of black-coated and brown-coated Cholistani service bulls. Adult Cholistani bulls (n= 8) were grouped as black-spotted (n= 4) and brown-spotted (n=4). Semen collection was performed once weekly (2 ejaculates/collection) using artificial vagina (AV) throughout the study period (October-May). Semen samples were evaluated for various physical attributes. Body weight and orchidometric attributes of the bulls were attained using calipers. Blood sampling was performed twice weekly and serum testosterone level was deduced through automated analyzer. The scrotal circumference, Scrotal Skin Fold Thickness and Paired Testicular Volume were significantly higher (P<0.05) in black-spotted Cholistani bulls as compared to brown-spotted bulls. Similarly, the ejaculatory volume (5.8±0.1 mL) and individual sperm motility (63.3±1.4%) were significantly higher (P<0.05) in black-spotted bulls. Percentage of live spermatozoa, and spermatozoa with normal morphology and acrosome was also higher in black-spotted bulls though statistically non-significant (P>0.05). Amongst post-thaw seminal attributes, percentage of live spermatozoa, and spermatozoa with normal morphology and acrosome were significantly higher (P<0.05) in black-spotted bulls. Similarly, serum testosterone level in black-spotted Cholistani bulls (6.9±0.3 ng/mL) was significantly higher (P<0.05) compared to brown-spotted bulls (5.4±0.3 ng/mL). The black-spotted Cholistani service bulls showed substantially better orchidometric and seminal attributes allied with higher serum level of testosterone.

Is there a relationship between serum vitamin D and semen parameters? A cross-sectional sample of the Iranian infertile men

Background Recent studies suggest that serum vitamin D may be associated with semen parameters. In the present cross-sectional study, we attempted to investigate the association between serum vitamin D levels and semen parameters among Iranian sub-fertile men. Results A total of 350 infertile men recruited for this cross-sectional study using a simple random sampling method with a mean age of 34.77 years old, body mass index of 26.67 kg/m2, serum vitamin D of 20.17 ng/ml, semen volume of 3.82 mL, sperm count of 44.48 (106/mL), sperm total motility of 38.10 %, and morphologically normal sperm of 7.0 %. After controlling for potential confounders, serum vitamin D was positively associated with semen volume (β = 0.63, 95 % CI: 0.06, 1.20), sperm count (β = 14.40, 95 % CI: 4.56, 24.25), sperm total motility (β = 18.12, 95 % CI: 12.37, 23.86), and sperm normal morphology (β = 1.95, 95 % CI: 1.07, 2.83). Conclusions The present findings suggest that higher serum vitamin D levels are positively associated with higher semen volume, sperm count, sperm total motility, and normal morphology rate. These findings, however, do not specify a cause-and-effect relationship, and there is a need for further research in this area to understand whether vitamin D supplementation can improve semen parameters.

The sperm protein Spaca6 is essential for fertilization in zebrafish

Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, yet its function has not been investigated outside of mammals. Here we show that zebrafish spaca6 encodes for a sperm membrane protein which is essential for fertilization. Zebrafish spaca6 knockout males are sterile. Furthermore, Spaca6-deficient sperm have normal morphology, are motile, and can approach the egg, but fail to bind to the egg and therefore cannot complete fertilization. Interestingly, sperm lacking Spaca6 have decreased levels of another essential and conserved sperm fertility factor, Dcst2, revealing a previously unknown dependence of Dcst2 expression on Spaca6. Together, our results show that zebrafish Spaca6 regulates Dcst2 levels and is required for binding between the sperm membrane and the oolemma. This is in contrast to murine SPACA6, which was reported not to be required for sperm-egg membrane binding but necessary for fusion. These findings demonstrate that Spaca6 is essential for zebrafish fertilization and is a conserved sperm factor in vertebrate reproduction.

The Association Between Abstinence Period And Semen Parameters: Results In Normal Samples And Different Sperm Pathology

Purpose: The length of sexual abstinence seems to influence sperm quality. However, few data on the relevance of abstinence time in pathological sperm samples are available. With our study, we look for associations of abstinence length and semen quality. Methods: We studied semen samples from 4423 men undergoing fertility evaluation. Sperm concentration, percentage of progressively motile spermatozoa, total motile sperm count, percentage of spermatozoa with normal morphology, were compared after each day and 0-2, 3-7 and >7 days of abstinence.Results: We found that a longer abstinence time was related to higher sperm concentration in normal semen samples (P<0.001) and in semen samples with any sperm pathology (P<0.001, P=0.004) with the exception of oligozoospermia (P=0.125). Longer abstinence time was also associated with significantly reduced progressive motility in normal samples (P<0.001) and in cases of teratozoospermia (P<0.001). In normal samples a higher percentage of sperm were morphologically normal after a shorter abstinence period (P=0.03); in oligoasthenoteratozoospermia (OAT) samples, this was the case after a longer abstinence period (P=0.013).Conclusion: A longer abstinence time is associated with higher sperm concentration, whereas sperm motility is optimal after shorter abstinence times; results on morphology are controversial. The recommendation on abstinence time needs to be adjusted in relation to the parameter that needs to be improved.

Atrazine Induces the Cytotoxicity of Ovarian Granulosa Cells via Inhibition of Nrf2-Mediated Defense Response

Atrazine (ATR) is a commercial herbicide, which is widely used worldwide. Diaminochlorotriazine (DACT) is the main metabolite of ATR, which poses potential risks to ecosystem and health. However, the mechanism of ATR and DACT on ovarian granulocyte cells (GCs) is still not clear. To investigate the toxic effect of ATR and DACT in quail ovarian GCs. We established primary ovarian GCs as models. GCs were cultured with ATR (20, 100, and 250 µM) and DACT (20, 100 and 200 µM). Which showed normal morphology and uniform size in GCs of the control group, ovarian GCs are specifically identified, in which cell purity was above 90%. The half-inhibitory concentrations (IC50) of ATR and DACT were 261.20 µM and 214.17 µM, respectively. High doses of ATR and DACT caused changes in the ultrastructure of mitochondria, leading to oxidative stressand changes in apoptosis-related indicators, ATR and DACT activated Nrf2-mediated defense system-related factors. Our research showed that ATR and DACT are cytotoxic to GCs. ATR induced oxidative stress in ovarian GCs and activated Nrf2-mediated antioxidant signaling pathway to alleviate the toxicity of ATR. These results suggested useful evidence to investigate the female reproductive toxicity of ATR and DACT.

DNA Methylation Differences Between Zona Pellucida-Bound and Manually Selected Spermatozoa Are Associated With Autism Susceptibility

Children conceived through intracytoplasmic sperm injection (ICSI) have been reported to have a higher risk of many abnormalities and disorders, including autism and intellectual disability, which may be due to bypassing of the natural sperm selection process during ICSI. Zona pellucida (ZP)-bound spermatozoa (ZPBS) have normal morphology and nuclear DNA. Using these spermatozoa for ICSI results in better outcomes compared with conventional ICSI. However, differences besides morphology that exist between sperm selected by ZP and by an embryologist and whether these differences affect the risk of autism in offspring after ICSI are unclear. To explore these questions, we compared genome-wide DNA methylation profiles between ZPBS and manually selected spermatozoa (MSS)using single-cell bisulfite sequencing. Global DNA methylation levels were significantly lower in ZPBS than in MSS. Using gene ontology (GO) analysis, genes overlapping differentially methylated regions (DMRs) were enriched in biological processes involving neurogenesis. Furthermore, we found that 47.8% of autism candidate genes were associated with DMRs, compared with 37.1% of matched background genes (P&lt;0.001). This was mainly because of the high proportion of autism candidate genes with bivalent chromatin structure. In conclusion, bivalent chromatin structure results in large differences in the methylation of autism genes between MSS and ZPBS. ICSI using MSS, which increases the risk of methylation mutations compared with ZPBS, may lead to a higher risk of autism in offspring.