Sperm-oocyte Fusion Research Articles

Overexpression of PD-L1 causes germ cell failure and infertility via CRISP1/PD-L1 interaction in mouse epididymis.

Spermatogenesis is a highly complex process through which mature sperms are produced, and it requires three important stages; mitosis, meiosis and sperm formation. The expression of genes regulated by transcription factors at specific stages exerts important regulatory effects on the development process of germ cells. Male mice with overexpressed programmed death ligand 1 (PD-L1) (B7 homolog1) in the testis have infertility and abnormal sperm development, thereby exhibiting severe malformation and sloughing throughout spermatid maturation and collapsed and disorganized seminiferous epithelium structure. Furthermore, PD-L1 overexpression causes overexpression of cysteine-rich secretory protein 1 (CRISP1) in the epididymis and adversely affects or precludes sperm energization, sperm-pellucida binding and sperm-oocyte fusion. These findings suggest that CRISP1 and PD-L1 can interact with each other to induce male infertility and germ-cell dissociation.

Induction of acrosome reaction by 4-Br-A23187 alters the glycoproteomic profile of boar spermatozoa

Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 μM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion.

Oxidative Stress and Acrosomal Status of Human Spermatozoa Subjected to Hydrophobic Carbon Soot Treatments.

The fourth industrial revolution extensively reshapes the reality we are living in by blurring the boundaries of physical, digital and biological worlds. A good example is the previously unthinkable incursion of nanoscale waste materials, such as soot, into the technologies for assisted reproduction. Although the rapeseed oil soot may efficiently enhance the progressive motility of human spermatozoa, it is yet unknown whether this material induces undesirable oxidative stress and premature acrosome reaction, endangering the sperm-oocyte fusion and blastocyst formation. In an attempt to clarify this issue, we reveal that the three-hour incubation of human semen mixed with three main types of soot does not cause oxidative stress and spontaneous acrosome reaction of the sperm. These unique findings are attributed to synchronous elimination and stabilization of the oxidants via hydrogen bonding to the acidic groups of the soot (i.e., C=O and/or C-O-C) and electron donation by its basic chemical sites (i.e., C-OH and/or COOH). Moreover, the soot nanoparticles are electrostatically attracted by discrete positively charged areas on the sperm head, increasing its negative charge and in some cases interfering the acrosome reaction. Such novel mechanistic insights emphasize the credibility of rapeseed oil soot to confidently shift from the purely diagnostic and therapeutic phases in reproductive medicine to research dealing with the effect of carbon nanomaterials on the embryo development and implantation.

Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

Differential protein repertoires related to sperm function identified in extracellular vesicles (EVs) in seminal plasma of distinct fertility buffalo (Bubalus bubalis) bulls.

Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity.

#164 : In-Vitro Vitamin D Treatment on Human Sperm Improves Capacitation, Acrosome Reaction, and Protein Expression of Sperm-Oocyte Activation Factors (SOAF)

Background and Aims: Although many studies have been done to investigate the relationship between serum vitamin D and male infertility, the effect of in-vitro vitamin D treatment on sperm remains unclear, particularly the effect on sperm capacitation, hyperpolarisation, sperm-oocyte fusion and oocyte activation. This study aims to examine whether in-vitro vitamin D treatment on sperm can improve the condition of sperm parameters and further increase fertilisation and pregnancy rate. Method: Fifty men (N=50, fertile n=26; infertile men n=24, were recruited during their visit to fertility clinics for semen analysis. Each washed semen sample was aliquoted and divided into control and in-vitro vitamin D treatment groups. For in-vitro vitamin D treatment, semen samples were treated with 1nM in-vitro for 1 hour at 37°C. Intracellular calcium released, capacitation status and mitochondria membrane potential were measured after incubation in both the control and in-vitro vitamin D treatment groups. Furthermore, immunofluorescence of sperm-origin SOAF, Phospholipase C Zeta (PLC[Formula: see text]), was done to investigate fluorescence intensity. Results: Compared to the control, higher intracellular calcium release was noted following in-vitro vitamin D treatment, represented by higher Fluo-4AM staining (MD=25043, P=0.02). In the treatment group, sperm capacitation rate and CTCF were higher when Chlortetracyclin (CTC) (MD=4.7%, P=0.01) and immunofluorescence of phosphotyrosine (MD=0.03, P=0.01) were examined. More hyperpolarised mitochondria membranes in the treatment group showed by JC-1 staining (P=0.01) compared to the control group. The red-to-green, fluorescent ratio is 1.62 in the control group and 2.24 in the treatment group. Moreover, immunofluorescence of PLC[Formula: see text] shows that the treatment improves this protein’s expression (MD=8.013×10[Formula: see text], P=0.02). Thus, in-vitro vitamin D enhances the functionality of sperm by increasing intracellular calcium ions, sperm capacitation, hyperpolarisation and protein expression. Conclusion: Adding vitamin D in in-vitro fertilisation (IVF) in media culture is beneficial as capacitation, sperm membrane polarisation and SOAF protein expression were increased and therefore contributed to successful IVF.

Participation of WD repeat-containing protein 54 (WDR54) in rat sperm-oocyte fusion through interaction with both IZUMO1 and JUNO

Fertilization is a complex process that depends on the fusion of the cell membrane of sperm with that of oocyte, and it involves sperm-oocyte recognition, binding, and fusion, which are mediated by multiple proteins. Among those proteins, IZUMO1 and its receptor JUNO have been identified as essential factors for sperm-oocyte recognition and fusion. However, the interaction between IZUMO1 and JUNO alone does not lead to cell membrane fusion, suggesting the involvement of additional proteins in sperm-oocyte membrane fusion. In this study, we have discovered that a protein called WDR54, which consists of WD-repeat modules, is located on the cell membrane of sperm, as well as on the cell membrane and in the cytoplasm of the oocyte. We have found that WDR54 is involved in sperm-oocyte fertilization. When sperm and oocyte were treated with anti-WDR54 ascites, the in vitro fertilization (IVF) rate significantly decreased. Furthermore, our research has shown that WDR54 interacts with both IZUMO1 and JUNO, and it colocalizes with IZUMO1 on the surface of the sperm head and with JUNO on the oocyte surface. Through structural analysis of the putative complexes of WDR54-IZUMO1 and WDR54-JUNO, we infer that these three proteins could form a complex of JUNO-WDR54-IZUMO1-JUNO (referred to as the “JWIJ complex”) on the oocyte surface. Our findings suggest that WDR54 is an important factor involved in sperm-oocyte adhesion and fusion. This discovery provides new insight into the mechanisms of mammalian sperm-oocyte adhesion and fusion.

O-179 Fertilization topologies that occur seldom or not at all in nature give rise to distinct functional classes of mouse embryos

Abstract Study question Would new embryo properties emerge if fertilization was imposed on oocytes in regions, such as the animal pole, where it does not take place naturally? Summary answer Oocyte fertilization at the animal pole, vegetal pole or equator results in 2-cell embryos with distinguishable transcriptomes and functional peculiarities. What is known already There is fertilization bias in embryos used in basic and clinical research, because: 1) sperm–oocyte fusion hardly occurs at the surface above the meiotic spindle – the animal pole; 2) the region opposite the spindle - vegetal pole - is poorly accessible to sperm due to tiny perivitelline space. These regions are also avoided during intracytoplasmic sperm injection (ICSI) for fear of damaging the spindle or losing the sperm nucleus into the 2nd polar body. Pole materials are held non-essential for mouse development, but this conclusion relies on the 2nd polar body, which is an unreliable topological marker. Study design, size, duration Oocytes were rotated using a micromanipulator fitted with Nomarski optics, using the meiotic spindle as a landmark. Between 9:30 and 10:30 am single sperm heads were microinjected (ICSI) at the animal or vegetal pole (treatments) vs. the naturally prevalent equatorial region (control). This way two fertilization topologies and possibly also two classes of zygotes were created that are otherwise seldom or not at all represented in natural fertilization, in vitro insemination or conventional ICSI. Participants/materials, setting, methods Metaphase II oocytes were collected from 8-week-old B6C3F1 mice stimulated with 10 I.U. eCG+hCG. Sperm heads from a single batch of cryopreserved CD1 semen were deposited via ICSI in the cortex at the animal pole, vegetal pole or half-way between poles i.e. equatorially. Zygotes were cultured in KSOM(aa) and analyzed (triplicate or more) for: cleavage rates, transcriptomes at the 2-cell stage (RNAseq), blastocyst germ layers (immunostaining for trophectoderm, primitive endoderm, epiblast), and postimplantation development. Main results and the role of chance Although full development was supported irrespective of ICSI site, embryos clustered by site, as revealed by single-cell RNAseq of 21, 21 and 13 two-cell embryos whose oocytes were fertilized at the animal pole, vegetal pole or equator, respectively. When examining the sister blastomeres together, 462 genes of the shared transcriptome were differently expressed between ICSI sites, with the equatorial class contributing most to the difference (adj.p&amp;lt;0.05, Wilcoxon test). This was true also when examining the sister blastomeres separately: interblastomere differences of the equatorial class exceeded those of the pole classes (72% vs. 14% of differently expressed genes). Ontology analysis of the differently expressed genes using Enrichr pointed at the endomembrane system – an acquaintance of oocyte polarity studies (PMID 10545249; PMID 29746690). Follow-up of 2-cell embryos to blastocysts revealed that sister blastomere contribution to each germ layer was less balanced in the equatorial class, as measured by linear correlation of cell numbers (e.g. equatorial R2=0.00 vs. polar R2&amp;gt;0.23 for the epiblast). Summing up, it is difficult to reconcile these data with a mainstream view that fertilization at the animal pole is harmful. Rather they support that the topology of fertilization defines functional classes of 2-cell embryos with distinguishable transcriptomes. Limitations, reasons for caution This is an animal study. Mouse ICSI uses mercury-loaded piezo-driven needles, human ICSI does not. The higher consistency of using the one and same batch of cryopreserved spermatozoa was traded off against lower developmental rates. Single-cell resolution posed a limit on RNAseq depth. Results need confirmation in other mouse strains. Wider implications of the findings The higher blastomere similarity observed after ICSI at the vegetal pole compared to ICSI at the equator is not consistent with the prevalent model of first zygotic cleavage that is driven by the topology of the two apposing pronuclei. Trial registration number not applicable

P-074 In-vitro vitamin D treatment on human sperm improves sperm motility, capacitation, and oocyte activation

Abstract Study question Does in-vitro vitamin D treatment on fertile and infertile human sperm improves sperm motility and alter protein markers involved in capacitation and oocyte-activation. Summary answer In-vitro vitamin D treatment enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm motility and capacitation status. What is known already Many studies have been done to investigate the relationship between vitamin D and male infertility. It is known that low serum vitamin D in human contribute to male infertility. However, the effect of in-vitro vitamin D treatment on sperm remains unclear, particularly on the effect of vitamin D following ejaculation into female reproductive tract. This includes processes involved in fertilisation such as capacitation, hyperpolarization, oocyte activation and sperm-oocyte fusion. Therefore, we examined whether in-vitro vitamin D treatment on sperm can improve the sperm parameters, hence will then improve fertilisation and pregnancy. Study design, size, duration Randomised controlled trial with prospective interventions (in-vitro vitamin D treatment). Based on power analysis, 40 subjects both fertile and infertile men who came for semen analysis were recruited at University Malaya Medical Centre from January 2022 to January 2023. All men were consented to participate in this study. Each washed semen sample were aliquoted to serve as control and receive in-vitro vitamin D treatment. Participants/materials, setting, methods Total of 40 samples were included for final analysis. All semen samples were washed and treated with 1nM vitamin D in-vitro for 45 minutes at 37ºC. Motility, intracellular calcium released, capacitation status, mitochondria membrane potential and other basic semen parameters were measured after in-vitro vitamin D treatment for fertile and infertile samples. Furthermore, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ), was done to investigate fluorescence intensity per cell using Image J. Main results and the role of chance In both fertile and infertile sample group, total motility is increased after in-vitro vitamin D treatment (MD = 8.727%, P &amp;lt; 0.001). Fluo-4 AM stain shows there are increase of intracellular calcium release after in-vitro vitamin D, with mean difference of Corrected Total Cell Fluorescent (CTCF) for Fluo-4 AM (MD = 25043, P = 0.0228. Chlortetracyclin (CTC) test for capacitation status and immunofluorescence of phosphotyrosine shows there are increase CTCF (MD = 0.02595, P = 0.0142) after in-vitro vitamin D treatment. Staining of JC-1 shows there are more hyperpolarised mitochondria membrane in in-vitro vitamin D treatment group compare to the control group. Ratio of red to green fluorescent is 0.639 in control group and 2.22 in in-vitro vitamin D treatment group. Moreover, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ) show in-vitro vitamin D improve the expression of these protein. Mean difference of CTCF between control and vitamin D treatment group for PLCζ is 8.013x 10-3, P = 0.0226. Thus, in-vitro vitamin D enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm capacitation status and sperm membrane hyperpolarization. Limitations, reasons for caution Although power analysis is used to calculate sample size, however bigger sample size always needed to increase the accuracy. Moreover, patients’ sample are examined and treated after 3 hours due to limited collection room in the facility. Wider implications of the findings Vitamin D can be used as a tool in in-vitro fertilisation (IVF) procedure and can be added to sperm preparation medium due to its implication in increasing capacitation and hyperpolarised sperm membrane to increase chance of successful IVF. Trial registration number not applicable

Expression of genes associated with sperm-oocyte interaction in the stallion reproductive tract

Sperm-oocyte fusion is an essential function during the fertilization process. Membrane fusion occurs as a result of a complex series of intermolecular interactions between the sperm and the oocyte. Proteins involved in this interaction include izumo sperm-egg fusion 1 (IZUMO1), sperm acrosome associated 6 (SPACA6), transmembrane protein 95 (TMEM95), fertilization influencing membrane protein (FIMP) and sperm-oocyte fusion required 1 (SOF1), ADAM metallopeptidase domain 2 (ADAM2), cysteine rich secretory protein 1-3 (CRISP1-3), cation channel sperm associated 1-4 (CATSPER1-4) and polycystin family receptor for egg jelly (PKDREJ) (Rivera and Swanson, 2022. Front. Cell Dev. Biol., 10:827-827454) on the male gamete, and izumo 1 receptor (JUNO) and cluster of differentiation 9 (CD9) on the female gamete (Inoue et al., 2021. eLife; 10:e66313). The IZUMO1-JUNO interaction is essential for gamete fusion and the block of polyspermy (Bianchi et al., 2014: Nature, 508:483-487). Other proteins called dendrocyte expressed seven transmembrane protein (DC-STAMP) and homologue domain-containing 1 and 2 (DCST1/2) are also required for sperm-egg fusion in mice (Inoue et al., 2021. eLife; 10:e66313). The objective of this study was to identify and quantify the expression of genes associated with sperm-oocyte interaction during the fertilization process in tissue samples from the testis, head, body and tail of the epididymis. Tissues were collected after castration from three light-breed reproductively normal stallions(5-13 years). mRNA was extracted and libraries were generated using Next Generation Sequencing. FPKM of the genes of interest was compared between epididymal head, body and tail, and testis (control tissue) using in silico analysis (online databases). Expression of CRISP3, SPACA6, ADAM2, CATSPER1, CATSPER2, CATSPER3, CATSPER4, GLPR1L1, IZUMO1, PKDREJ, TMEM95, DCST1 and DCST2 genes were identified in the testis and all segments of the epididymis of the horse. All these genes, except for CRISP1, were up-regulated in the testis compared with the three segments of the epididymis, while CRISP1 was down-regulated in the testis compared with the epididymis. In this study, JUNO gene was expressed in the testis (FPKM 48.15), and was downregulated in the head, body and tail of the epididymis (FPKM 2.3, 3.2 and 2.9, respectively). In summary, genes encoding for proteins associated with sperm-oocyte interaction were more strongly expressed within the testis, suggesting that these proteins are acquired during spermatogenesis or spermiogenesis. Meanwhile, CRISP1 may be acquired during sperm transport along the epididymis. The expression of JUNO gene within the stallion reproductive tract is striking since the expression has not been reported in mice (RPKM 0) and human testis (RPKM 0.003). JUNO is a receptor only found on the oocyte membrane. The specific cells that express these genes, and their function within the stallion reproductive tract needs further studies.

The transmembrane domain of Frey1 harbors a transplantable inhibitory motif for intramembrane proteases

Although aspartic intramembrane-cleaving proteases (I-CLIPs) are crucial switches of multiple signaling pathways and involved in several devastating diseases, little is known about their physiological regulation. We have recently identified Frey regulator of sperm-oocyte fusion 1 (Frey1) as an inhibitory protein of Signal Peptide Peptidase-like 2c (SPPL2c), a member of this protease family. Employing structure modeling along with cell-based inhibition and interaction studies, we identify a short motif within the Frey1 transmembrane domain essential for inhibition of SPPL2c. Intriguingly, this motif can be transplanted to the SPPL2c substrate PLN, thereby transforming it into an inhibitor of this enzyme. It can be adopted for the generation of Notch1-based γ-Secretase inhibitors demonstrating its versatile use among aspartic I-CLIPs. In summary, we describe a mechanism of aspartic I-CLIP inhibition which allows the targeted generation of specific inhibitors of these enzymes and might enable the identification of endogenous negative regulators of these enzymes.

Mitochondria in Human Fertility and Infertility.

In human spermatozoa and oocytes (and their surrounding granulosa cells), mitochondria carry out important functions relating to human fertility and infertility. Sperm mitochondria are not transmitted to the future embryo, but are closely related to the generation of energy needed for sperm movement, capacitation, and acrosome reactions, as well as for sperm-oocyte fusion. On the other hand, oocyte mitochondria produce energy required for oocyte meiotic division and their abnormalities can thus cause oocyte and embryo aneuploidy. In addition, they play a role in oocyte calcium metabolism and in essential epigenetic events during the oocyte-to-embryo transition. They are transmitted to the future embryos and may thus cause hereditary diseases in the offspring. Due to the long life span of the female germ cells, the accumulation of mitochondrial DNA abnormalities often causes ovarian aging. Mitochondrial substitution therapy is the only way of dealing with these issues nowadays. New therapies based on mitochondrial DNA editing are under investigation.

Cytochalasin D restores nuclear size acting on F-actin and IZUMO1 localization in low-quality spermatozoa.

In spermatozoa, the nuclear F-actin supports the acroplaxome, a subacrosomal structure involved in the correct exposure of several acrosomal membrane proteins; among them, the glycoprotein IZUMO1 is the major protein involved in sperm-oocyte fusion. Nuclear F-actin is also involved in sperm head shaping and chromosome compartmentalization. To date, few notions regarding the bivalent role of F-actin on sperm chromatin organization and IZUMO1 positioning have been reported. In our work, we characterized subcellular organization of F-actin in human high- and low-quality spermatozoa (A- and B-SPZ), respectively, showing that F-actin over-expression in sperm head of B-SPZ affected IZUMO1 localization. A correct IZUMO1 repositioning following in vitro induction of F-actin depolymerization, by cytochalasin D treatment, occurred. Interestingly, F-actin depolymerization was also associated with a correct acrosome repositioning, thus to favor a proper acrosome reaction onset, with changes in sperm nuclear size parameters and histone acetylation rate reaching high-quality conditions. In conclusion, the current work shows a key role of F-actin in the control of IZUMO1 localization as well as chromatin remodeling and acetylation events.

Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin Treatment

Vitrification of oocytes is crucial for embryo biotechnologies, germplasm cryopreservation of endangered and excellent female animals, and the fertility of humans. However, vitrification significantly impairs the fertilization ability of oocytes, which significantly limits its widely used application. JUNO protein, a receptor for Izumo1, is involved in sperm-oocyte fusion and is an indispensable protein for mammalian fertilization, and its abundance is susceptible to vitrification. However, it is still unclear how vitrification reduces the fertilization capacity of bovine oocytes by affecting JUNO protein. This study was designed to investigate the effect of vitrification on the abundance and post-translational modifications of JUNO protein in bovine oocytes. Our results showed that vitrification did not alter the amino acid sequence of JUNO protein in bovine oocytes. Furthermore, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis results showed that vitrification significantly reduced the number and changed the location of disulfide bonds, and increased the number of both phosphorylation and glycosylation sites of JUNO protein in bovine oocytes. Finally, the fertilization capacity and development ability of vitrified oocytes treated with 200 pg JUNO mRNA microinjection and cholesterol-loaded methyl-β-cyclodextrin (CLC/MβCD) were similar to those of fresh oocytes. In conclusion, our results showed that vitrification of bovine oocytes did not alter the protein sequence of JUNO, but induced post-translational modifications and changed protein abundance. Moreover, the fertilization and development ability of vitrified bovine oocytes were improved by the combination treatment of JUNO mRNA microinjection and CLC/MβCD.

Preimplantation Developmental Competence of Bovine and Porcine Oocytes Activated by Zinc Chelation.

After sperm-oocyte fusion, intracytoplasmic rises of calcium (Ca) induce the release of zinc (Zn) out of the oocyte (Zn sparks). Both phenomena are known to play an essential role in the oocyte activation process. Our work aimed to explore different protocols for activating bovine and porcine oocytes using the novel zinc chelator 1,10-phenanthroline (PHEN) and to compare developmental rates and quality to bovine IVF and parthenogenetic ionomycin-induced embryos in both species. Different incubation conditions for the zinc chelator were tested, including its combination with ionomycin. Embryo quality was assessed by immunofluorescence of SOX2, SOX17, OCT4, and CDX2 and total cell number at the blastocyst stage. Even though blastocyst development was achieved using a zinc chelator in bovine, bypassing calcium oscillations, developmental rates, and blastocyst quality were compromised compared to embryos generated with sperm-induced or ionomycin calcium rise. On the contrary, zinc chelation is sufficient to trigger oocyte activation in porcine. Additionally, we determined the optimal exposure to PHEN for this species. Zinc chelation and artificial induction of calcium rise combined did not improve developmental competence. Our results contribute to understanding the role of zinc during oocyte activation and preimplantation embryo development across different mammalian species.

Changes in cortical endoplasmic reticulum clusters in the fertilized mouse oocyte†.

Oocytes from many invertebrate and vertebrate species exhibit unique endoplasmic reticulum (ER) specializations (cortical ER clusters), which are thought to be essential for egg activation. In examination of cortical ER clusters, we observed that they were tethered to previously unreported fenestrae within the cortical actin layer. Furthermore, studies demonstrated that sperm preferentially bind to the plasma membrane overlying the fenestrae, establishing close proximity to underlying ER clusters. Moreover, following sperm-oocyte fusion, cortical ER clusters undergo a previously unrecognized global change in volume and shape that persists through sperm incorporation, before dispersing at the pronuclear stage. These changes did not occur in oocytes from females mated with Izumo1 -/- males. In addition to these global changes, highly localized ER modifications were noted at the sperm binding site as cortical ER clusters surround the sperm head during incorporation, then form a diffuse cloud surrounding the decondensing sperm nucleus. This study provides the first evidence that cortical ER clusters interact with the fertilizing sperm, indirectly through a previous unknown lattice work of actin fenestrae, and then directly during sperm incorporation. These observations raise the possibility that oocyte ER cluster-sperm interactions provide a competitive advantage to the oocyte, which may not occur during assisted reproductive technologies such as intracytoplasmic sperm injection.

Introduction to the pathways involved in the activation and regulation of sperm motility: A review of the relevance of ion channels

To participate in sperm–oocyte fusion, spermatozoa need to be motile. In the testes, spermatozoa are immotile, although these gametes acquire the capacity for motility during the transit through the epididymis. During the period of epididymal transport from the male genital tract to the female genital tract, spermatozoa exhibit various types of motility that are regulated by complex signalling and communication mechanisms. Because motility is very dynamic, it can be affected by small changes in the external or internal environment of spermatozoa within a very short time. This indicates that regulatory membrane proteins, known as sperm ion channels, are involved in the regulation of sperm motility. Research results from studies, where there was use of electrophysiological, pharmacological, molecular and knock-out approaches, indicate ion channels are possibly involved in the regulation of sperm membrane polarisation, intracellular pH, motility, energy homeostasis, membrane integrity, capacitation, hyperactivity, acrosome reaction and fertilisation processes. In this review, there is summarisation of the key functions that ion channels have in the regulation, initiation, maintenance, and modulation of sperm motility. In addition, in this review there is highlighting of novel insights about the pathways of ion channels that are activated in spermatozoa while these gametes are located in the oviduct leading to the fertilisation capacity of these cells.

Heat shock protein family D member 1 in boar spermatozoa is strongly related to the litter size of inseminated sows

BackgroundSperm quality evaluation is the logical first step in increasing field fertility. Spermatozoa contain cytoplasmic organelles and biomolecules known as sperm-intrinsic factors, which play key roles in sperm maturation, sperm-oocyte fusion, and embryo development. In particular, sperm membrane proteins [e.g., arginine vasopressin receptor 2, beta-actin, prohibitin, and heat shock protein family D member 1 (HSPD1)] and RNA could be used as functional indicators of male fertility. We sought to clarify the effects of differential mRNA expression of selected genes on several fertilisation parameters, including sperm motility, motion kinematics, capacitation, and litter size, in a porcine model.ResultsOur results demonstrated that HSPD1 expression was significantly correlated with male fertility, as measured by the litter size of inseminated sows. The expression of HSPD1 mRNA was linked to sperm motility and other motion kinematic characteristics. Furthermore, HSPD1 had a 66.7% overall accuracy in detecting male fertility, and the high-litter size group which was selected with the HSPD1 marker had a 1.34 greater litter size than the low-litter size group.ConclusionsOur findings indicate that HSPD1 might be a helpful biomarker for superior boar selection for artificial insemination, which could boost field fertility.

The Role of Sperm Proteins IZUMO1 and TMEM95 in Mammalian Fertilization: A Systematic Review.

Gamete membrane fusion is a critical cellular event in sexual reproduction. In addition, the generation of knockout models has provided a powerful tool for testing the functional relevance of proteins thought to be involved in mammalian fertilization, suggesting IZUMO1 and TMEM95 (transmembrane protein 95) as essential proteins. However, the molecular mechanisms underlying the process remain largely unknown. Therefore, the aim of this study was to summarize the current knowledge about IZUMO1 and TMEM95 during mammalian fertilization. Hence, three distinct databases were consulted—PubMed, Scopus and Web of Science—using single keywords. As a result, a total of 429 articles were identified. Based on both inclusion and exclusion criteria, the final number of articles included in this study was 103. The results showed that IZUMO1 is mostly studied in rodents whereas TMEM95 is studied primarily in bovines. Despite the research, the topological localization of IZUMO1 remains controversial. IZUMO1 may be involved in organizing or stabilizing a multiprotein complex essential for the membrane fusion in which TMEM95 could act as a fusogen due to its possible interaction with IZUMO1. Overall, the expression of these two proteins is not sufficient for sperm–oocyte fusion; therefore, other molecules must be involved in the membrane fusion process.

Boar spermadhesin AWN: novel insights in its binding behavior and localization on sperm.

As a major spermadhesin first found in the seminal plasma (SP) of boars, AWN is described to fulfil a variety of reproduction related tasks. Although being the best investigated boar spermadhesin, information about its interaction with membranes is inconsistent. In this regard, previous reports locate AWN either inside or on the surface of sperm cells and at different regions, depending on the method and antibody used. Here, we localize native AWN (natAWN) in/on epididymal, ejaculated, capacitated, and acrosome-reacted boar sperm using epifluorescence and electron microscopy as well as an analysis of potential lipid-binding partners of natAWN and recombinant AWN (recAWN). By applying a custom-made anti-AWN antibody, localization of AWN in the equatorial segment (EQS) of ejaculated, capacitated, and acrosome-reacted boar sperm was discovered. Electron microscopy showed that AWN is localized both on the sperm surface and on the cytoplasmic side of the plasma membrane and in close vicinity to the nuclear and both acrosomal membranes of sperm. Analysis of epididymal sperm indicated migration of AWN from the retral postacrosomal part to the EQS during the epididymal passage. In contrast to hypotheses claiming a specific association of AWN to phosphatidylethanolamine (PE) and in line with our previous study describing an interaction with phosphatidic acid (PA), the current results show a rather electrostatically driven binding mechanism of AWN to negative lipids. In conclusion, this work provides new insights into the arrangement of AWN in the EQS, which suggest a possible role in sperm-oocyte fusion.