C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here we report identification of an uncharacterized protein C2CD6 as a novel subunit of the CatSper complex. C2CD6 contains a calcium-dependent membrane targeting C2 domain. C2CD6 associates with the CatSper calcium-selective core forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

Semen Viability Properties of Sexually Stimulated Boars on the Fertilization Capacity of Artificially Inseminated Large White Sows

Background: This study aimed to investigate the influences of sexual preparation on the effects of boars’ semen viability on the fertilization capacity of artificially inseminated sows. After all, boar sires more pigs than farrowed by a sow which the boar has been mated to improve reproductive parameters in response to AI. Methods: The semen viability of boars were studied during various sexual stimulations and analyzed during the study period. Sperm rich fractions were collected and separated at every level of sexual stimulation during the morning (08:30) and the afternoon (14:30) hours, respectively. Artificial insemination was performed following three levels of sexual preparations of boars (0 minutes of sexual restraint (MSR), 5 MSR and 10 MSR) before semen collection. Receptive sows were inseminated and evaluated for fertility traits using non-return rate, farrowing rate and litter size. Result: The non-return rate was recorded as a percentage of sows conceived after insemination over the total number of sows inseminated. The farrowing rate was recorded as a percentage of sows that farrows over the number of sows conceived and litter size as several live piglets per sow. Sexual desire was influenced by sexual preparations and significantly influenced the fertility of the artificially inseminated sows. This study is of practical significance to the animal breeder mainly because boars have greater influence than sows on the average litter size and live piglets. The study concludes that the use of at least 5 to 10 minutes of sexual restraint during the afternoon periods prior to semen collection and artificial insemination is found to be a practical method for optimizing sperm viability and fertility of sows in the intensive system.

PSXII-14 Estimation of phosphatidylserine positive sperm in fresh bull semen

Previous research demonstrated that phosphatidylserine (PS), a marker for apoptosis, plays a role in murine sperm fertilization capacity; however, less is known of the role of PS in bovine fertilization. The objective of this experiment was to determine the concentration of PS positive sperm on fresh bovine semen (n = 4). A breeding soundness evaluation was performed and electroejaculation was used to collect semen which was immediately evaluated for ejaculate density and gross motility through visual evaluation. Semen smears of semen mixed with an eosin-nigrosin strain were prepared on glass microscope slides. Semen was mixed with OptiXcell™ (IMV Technologies) semen extender, a 0.5 mL sample from each bull was washed with non-capacitating media and then incubated at 37.5°C 5% CO2 with capacitating media for 60 minutes. Samples were stained with Annexin V FITC and 7AAD for 15 minutes each. Samples were analyzed using a Flow Cytometer to determine the percentage of PS positive sperm. Bull 1 had a scrotal circumference of 37.5 cm, ejaculate density of 400–750 million sperm/mL, very good gross motility, 78% sperm with normal morphology, and 14.65% PS positive sperm. Bull 2 had a scrotal circumference of 37 cm, ejaculate density of 400–750 million sperm/mL, very good gross motility, 84% sperm with normal morphology, and 17.05% PS positive sperm. Bull 3 had a scrotal circumference of 39.5 cm, ejaculate density of 250–400 million sperm/mL, fair gross motility, 48% sperm with normal morphology, and 12.8% PS positive sperm. Bull 4 had a scrotal circumference of 38 cm, ejaculate density of 400–750 million sperm/mL, good gross motility, 68% of sperm with normal morphology, and 12.65% PS positive sperm. Our data demonstrate the possibility of identifying live PS positive sperm in fresh ejaculate from bulls. The possibility exists that PS plays a role in sperm oocyte fusion and fertilization in the bovine.

C2CD6 is required for assembly of the CatSper calcium channel complex and fertilization

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here we report identification of an uncharacterized protein C2CD6 as a novel subunit of the CatSper ion channel complex. C2CD6 contains a calcium-dependent membrane targeting C2 domain. C2CD6 interacts with the CatSper calcium-selective core forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. Interestingly, transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm in vitro. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 may regulate CatSper assembly, target the CatSper complex to flagellar plasma membrane, and function as a calcium sensor. The identification of C2CD6 as an essential subunit may facilitate the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

P–567 Re-establishment of fertilization competency of the oocyte via CRISPR/dCas9 epigenome edition technology

Study question Is it possible to increase the decreased levels of the sperm-oocyte binding protein, Juno, to restore the fertilization capacity of the oocyte, via the use of the CRISPR/dCas9 activation system? Summary answer JUNO domain (in the oocyte) suppressed with melamine was opened using the CRISPR/dCas9 system and sperm-oocyte binding and fertilization capability have been restored. What is known already Melamine is a chemical that is widely used in the manufacture of laminates, plastics, etc. Evidence reveals that long-term exposure to melamine could damage reproductive systems in mammals leading to infertility. Izumo1 is the only cell surface protein expressed on sperm that is known to be essential for sperm-egg interaction in vivo. It was in vitro shown that high-dose feeding of melamine to female mice led to a significant decrease of JUNO on the plasma membrane of eggs. CRISPR/dCas9 system can provide the gene activation or repression to activate the target gene via Sam and Tet1 based systems. Study design, size, duration Six different gRNAs were designed for the transfection of oocytes. Six-week-old mice were fed with melamine (50mg/kg/day) for 2 weeks via gavage. Melamine gavage, water gavage, and control groups (n = 15 /each group) were created. CRISPR activation plasmids (SAM) were given by piezo microinjection into the GV oocytes (n = 100 oocytes/each group). Fertilization capacity was evaluated by sperm binding assay, qPCR, Western blotting, and IF staining. Two technical replicates were used in molecular studies. Participants/materials, setting, methods 293T cells were transfected (dCas9 SAM plasmids+gRNA) with Fugene. Mice randomly were assigned to 3 groups (n = 15), as each was given orally a dose of 50mg/kg/d of melamine, only water or no water via gavage. Microinjection of plasmids was performed. Post-injection, oocytes were incubated until MII stage. For binding and fertilization evaluation, motile sperms were incubated with oocytes, and pronuclei were checked. Juno and IZUMO1 levels were evaluated by qPCR, Western blotting, and IF staining. Main results and the role of chance As the SAM system is more efficient compared to the Tet1 system when tested in 293t cells, the SAM system was used for mouse experiments. As a result of qPCR performed in oocytes collected at the end of gavage, it was observed that the JUNO expression levels were decreased by 40 folds in melamine fed mice (p < 0.05). The decrease in the level of JUNO protein was demonstrated by IF stainings, and a decrease in the oocyte count along with abnormal uterine shapes was also observed in this group. IZUMO1 expression in motile sperms was demonstrated by qPCR before sperm binding assay and the position of the IZUMO 1 domain before the acrosome reaction was demonstrated by IF stainings. Sperm binding assay has demonstrated a 70% reduction in fertilization competency of melamine-treated oocytes (p < 0.05). SAM plasmids and JUNO gRNA were given to oocytes by piezo injection. By sperm binding experiments conducted to evaluate fertilization capacities after microinjection, it was shown that the fertilization capacity was restored by 75% (p < 0.05). Re-gaining of Juno expression in the oocytes was supported by a 60 fold increase in qPCR results. Recovery of JUNO protein expression in the oocytes was also demonstrated by IF stainings. Limitations, reasons for caution There is no known promoter region for the JUNO gene in the mouse. Therefore, we designed gRNAs targeting possible promoter regions. However, we have used two activation systems(SAM and Tet1) that are widely used to open a closed gene, but other activation systems (acetylation, etc.) can also be tried. Wider implications of the findings: This study is valuable since: -it presents a possible cure for unsuccessful fertilization in related cases. -it possibly reveals melamine’s unknown way of action. - it presents a new approach as a sperm-binding assay to be used in IVF clinics since Juno can also be expressed in somatic cell lines. Trial registration number non-clinical trials

Effects of Pre-ovulatory Follicular Fluid of Repeat Breeder Dairy Cows on Bovine Fertility Transcriptomic Markers and Oocytes Maturation and Fertilization Capacity

The current study aimed to determine the effects of the preovulatory follicular fluid (FF) of normal heifer (NH) and repeat breeder cows with subclinical endometritis (SCE) or without (nSCE) on oocyte maturation (Experiment 1) and fertilization rates (Experiment 2). Moreover, the pattern of gene expression of cumulus oocyte-complexes was evaluated in Experiment 1. In Experiment 1, nuclear maturation in the nSCE group was higher, compared to that in the SCE group (P = 0.05). In addition, the oocyte nuclear maturation in the normal heifer was significantly higher, in comparison to that of SCE groups (P < 0.05). Furthermore, the mean percentage of normal oocyte fertilization was higher in the nSCE group, compared to that in the SCE group (P < 0.05). The expressions of growth differentiation factor, GDF9; steroidogenic acute regulatory, StAR and follicle-stimulating hormone receptor, FSHr in the NH group were significantly higher, compared to those in SCE and nSCE groups (P < 0.05). Moreover, the expressions of all genes in the nSCE group were not significant, in comparison to those in the SCE group (P > 0.05). The supplementation of oocyte maturation medium with FF from pre-ovulatory follicles of repeat breeder cows resulted in less oocyte maturation and cumulus cell expansion. In conclusion, the lower fertility in RB cows could be ascribed to the lower oocyte maturation rate and less expression of GDF9, StAR, and FSHr in the cumulus-oocyte complexes.

Mutant Zp1 impedes incorporation of ZP3 and ZP4 in the zona pellucida, resulting in zona absence and female infertility in rats

The zona pellucida (ZP) plays vital roles in reproductive processes including oogenesis, fertilization and preimplantation development. Both human and rat ZP consist of four glycoproteins, called ZP1, ZP2, ZP3 and ZP4. Our previous research reported a novel Zp1 mutation in cases of human infertility, associated with an abnormal phenotype involving the absence of the zona pellucida. Here, we developed a homologous rat strain to investigate the pathogenic effect. The ovaries of homozygous (Zp1MT/MT) females possessed both growing and fully grown oocytes; the oocytes completely lacked a zona pellucida, but ZP1 was detectable inside the cytoplasm. Only 1-2 eggs were recovered from oviducts of superovulated Zp1MT/MT females, while ≈ 21 eggs were recovered from superovulated Zp1WT/WT females. The eggs of Zp1MT/MT females were not surrounded by a zona pellucida and lost their fertilization capacity in vitro. Zp1MT/MT females mated with wild-type males failed to become pregnant. Studies in 293 T cells showed that mutant Zp1 resulted in a truncated ZP1 protein, which might be intracellularly sequestered and interact with wild-type ZP3 or ZP4. Our results suggest that the Zp1 point mutation led to infertility and loss of the ZP in oocytes in rats.

Impact of Mitochondrial Genetic Variants in ND1, ND2, ND5, and ND6 Genes on Sperm Motility and Intracytoplasmic Sperm Injection (ICSI) Outcomes

Sperm mitochondrial dysfunction causes the generation of an insufficient amount of energy needed for sperm motility. This will affect sperm fertilization capacity, and thus, most asthenozoospermic men usually require assisted reproductive techniques. The etiology of asthenozoospermia remains largely unknown. The current study aimed to investigate the effect of mitochondrial genetic variants on sperm motility and intracytoplasmic sperm injection (ICSI) outcomes. A total of 150 couples from the ICSI cycle were enrolled in this study. One hundred five of the male partners were asthenozoospermic patients, and they were subdivided into three groups according to their percentage of sperm motility, while forty-five of the male partners were normozoospermic. Genetic variants were screened using direct Sanger’s sequencing in four mitochondrial genes (nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase 1 (ND1), NADH dehydrogenase 2 (ND2), NADH dehydrogenase 5 (ND5), and NADH dehydrogenase 6 (ND6)). We identified three significant variants: 13708G>A (rs28359178) in ND5, 4216T>C (rs1599988) in ND1, and a novel 12506T>A in ND5 with P values 0.006, 0.036, and 0.013, respectively. The medians of sperm motility, fertilization rate, embryo cleavage score, and embryo quality score were significantly different between men showing 4216T>C, 12506T>A, 13708G>A and wild type, Mann-Whitney P values for the differences in the medians were < 0.05 in all of them. The results from this study suggest that 13708G>A, 12506T>A, and 4216 T>C variants in sperm mitochondrial DNA negatively affect sperm motility and ICSI outcomes.