Sperm Binding Research Articles

Specific expression of alternatively spliced genes in the turkey (Meleagris gallopavo) reproductive tract revealed their function in spermatogenesis and post-testicular sperm maturation

The tissue-specific profile of alternatively spliced genes (ASGs) and their involvement in reproduction processes characteristic of turkey testis, epididymis, and ductus deferens were investigated for the first time in birds. Deep sequencing of male turkey reproductive tissue RNA samples (n=6) was performed using Illumina RNA-Seq with 2 independent methods, rMATs and SUPPA2, for differential alternative splicing (DAS) event prediction. The expression of selected ASGs was validated using quantitative real-time reverse transcriptase-polymerase chain reaction. The testis was found to be the site of the highest number of posttranscriptional splicing events within the reproductive tract, and skipping exons were the most frequently occurring class of alternative splicing (AS) among the reproductive tract. Statistical analysis revealed 86, 229, and 6 DAS events in the testis/epididymis, testis/ductus deferens, and epididymis/ductus deferens comparison, respectively. Alternative splicing was found to be a mechanism of gene expression regulation within the turkey reproduction tract. In testis, modification was observed for spermatogenesis specific genes; the changes in 5' UTR could act as regulator of MEIG1 expression (a player during spermatocytes meiosis), and modification of 3' UTR led to diversification of CREM mRNA (modulator of gene expression related to the structuring of mature spermatozoa). Sperm tail formation can be regulated by changes in the 5' UTR of testicular SLC9A3R1 and gene silencing by producing dysfunctional variants of ODF2 in the testis and ATP1B3 in the epididymis. Predicted differentially ASGs in the turkey reproductive tract seem to be involved in the regulation of spermatogenesis, including acrosome formation and sperm tail formation and binding of sperm to the zona pellucida. Several ASGs were classified as cilia by actin and microtubule cytoskeleton organization. Such genes may play a role in the organization of sperm flagellum and post-testicular motility development. To our knowledge, this is the first functional investigation of alternatively spliced genes associated with tissue-specific processes in the turkey reproductive tract.

Velvet antler water extract protects porcine oocytes from lipopolysaccharide-induced meiotic defects.

Previous studies have demonstrated that lipopolysaccharide (LPS), as a central toxic factor of gram-negative bacteria, can induce oxidative stress and cellular inflammation to result in the impairment of female fertility in different organisms. Particularly, it has harmful effects on the oocyte quality and subsequent embryonic development. However, the approach concerning how to prevent oocytes from LPS-induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS-induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS-induced oxidative stress.

Effects of capacitating media and incubation time on collared peccary sperm quality, acrosome reaction, and binding ability

Context Several assisted reproduction techniques have been proposed for collared peccaries due to the interest in its conservation. However, there is little information about the requirements for sperm capacitation, an initial step in in vitro fertilisation. Aims We aimed to determine the optimal conditions for collared peccary sperm capacitation by comparing Tyrode’s albumin lactate pyruvate (TALP) or the same media plus heparin, caffeine, or their combination at different exposure times (1, 3, and 6 h). Methods The samples were evaluated for kinetic parameters, membrane functionality and integrity, mitochondrial activity, morphology, DNA integrity, reactive oxygen species (ROS) expression levels, capacitation, and sperm binding ability using swine oocytes. Key results Samples incubated with caffeine or heparin had a higher percentage of capacitated spermatozoa. The maximum percentage of capacitation was achieved after 3 h of incubation with either agent. Moreover, spermatozoa subjected to heparin capacitation showed better motility than when subjected to caffeine, and lower ROS expression levels after 1 h. No differences were observed among incubation times for the binding ability. Conclusions In summary, collared peccary spermatozoa can be capacitated with caffeine or heparin; however, heparin better maintains sperm motility and ROS expression levels. The co-incubation of gametes in a medium with heparin for 3 h could be efficient for in vitro fertilisation in collared peccaries. Implications This result will contribute to the development of assisted reproduction techniques for conservation and productivity of collared peccaries.

Immunocontraceptive Potential of Goat Zona pellucida as Monkey Population Control

A series of studies have explored the immunocontraceptive potential of zona pellucida-3 in local Indonesian goats. Goat zona pellucida-3 (gZP3) proteins have been identified and isolated. It has 82 kDa molecular weight and comprises 63.47% of the total goat zona pellucida protein. Immunofluorescence staining showed that gZP3 protein recognized goat sperm plasma membrane, whereas gZP3 antibodies recognized goat zona pellucida in native smear. In vitro fertilization study showed that gZP3 protein-supplemented in sperm capacitation medium and gZP3 antibody supplemented in oocyte maturation medium inhibited fertilization. It could be concluded that gZP3 was a fertilization receptor in a goat. Elisa and dot blot analysis showed that gZP3 protein was recognized by the serum of fertile unpregnant female mice. Therefore, it could be hypothesized that the gZP3 protein might be immunocontraceptive in mice. Immunization of mice with gZP3 proved that gZP3 prevented pregnancy and was dose-dependent. The contraceptive action of gZP3 was reversible after 14 estrous cycles. In vitro fertilization technique, it was proved that the antibody of gZP3 was able to prevent fertilization and decrease sperm binding rate on mice ova. The gZP3 nucleotide sequence has a homology at nearly 50% of the monkey ZP3 sequence (Macaca sp) but is foreign phylogenetically. The gZP3 protein has the potential to be developed as an immunocontraceptive substance for overpopulated monkeys to prevent agriculture disturbance in areas near forestry.

CRISPR/Cas9-mediated disruption of lipocalins, Ly6g5b, and Ly6g5c causes male subfertility in mice.

Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis-enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 family genes (Ly6g5b and Ly6g5c). These genes are evolutionarily conserved in mammals and form clusters on chromosomes 2 and 17 in the mouse, respectively. To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR/Cas9 system and analyzed their phenotype. We generated four lines of KO mice: Lcn9 single KO, the lipocalin family quadruple KO (Lcn5, Lcn6, Lcn8, and Lcn10), quintuple KO (Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9), and double KO of Ly6 family genes (Ly6g5b and Ly6g5c). Although the Lcn9 single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A/B that are required for ADAM3 maturation. Intriguingly, Ly6g5b and Ly6g5c double KO male mice also showed subfertility with reduced sperm ADAM3. These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability.

The impact of asymptomatic and mild COVID-19 on sperm characteristics

Background: Although the pandemic of the new coronavirus infection (COVID-19) is characterized by mostly mild and asymptomatic cases (more than 80%), it appears important to assess the potential risks for the male reproductive system, in particular, for the parameters of the sperm. Aim: To study the effect of a mild and asymptomatic course of the new coronavirus infection (COVID-19) on the male sperm's parameters. Methods: The study included 397 patients who applied for a spermogram. All the patients underwent a spermogram study according to the WHO protocol, with the additionally measured degree of sperm DNA fragmentation, acrosine activity, fructose level, zinc level, citric acid level, neutral-glucosidase activity. A test for sperm binding to hyaluronic acid (HBA test) was performed, and the serum antibodies (IgM and IgG) against SARS-CoV-2 were analyzed. The study was conducted twice with an interval of 35 months. Results: At the first examination, normozoospermia was found in 33.5% (n=133) of patients, pathospermia in 66.5% (n=264). All the patients had no antibodies against SARS-CoV-2 (COVID-19). When the analysis was repeated after 35 months, antibodies (IgG) against SARS-CoV-2 were detected in 144 patients: in 14.6% (n=21) the coronavirus infection was mild, and in 85.4% (n=123) it was asymptomatic. Those patients who received some type of a treatment (andrologic, anticovid, and/or other) were excluded from the further study. The subsequent analysis of the data from the untreated patients (131 patients without antibodies against SARS-CoV-2 and 93 patients with antibodies (IgG) against SARS-CoV-2) was performed using the Student's paired test. For all the parameters of the standard spermogram according to the WHO protocol, the found changes after the mild and asymptomatic form of COVID-19 were not statistically significant. Also, the changes in the degree of fragmentation of sperm DNA, the enzymatic activity of acrosine, and other estimated parameters of the sperm (levels of fructose, citric acid, zinc, and the activity of neutral-glucosidase) were not statistically significant. The results of the test for the spermatozoa's interaction with hyaluronic acid (HBA test) worsened and had statistically significant differences after the coronavirus infection in both the mild form (p=0.006) and asymptomatic form (p=0.001). There were no statistically significant changes in the sperm parameters (including those from the HBA test) in the patients who had not had the new coronavirus infection. Conclusions: No statistically significant data were obtained on the effect of mild and asymptomatic forms of the course of the new coronavirus infection on the parameters of the standard spermogram, on the degree of fragmentation of spermatozoa's DNA and the enzymatic activity of acrosin. Mild and asymptomatic forms of the new coronavirus infection course can negatively affect the interaction of spermatozoa with hyaluronic acid (HBA test), that indicates the impairment of the spermatozoa binding to the zona pellucida. One should pay attention to the HBA test when examining the sperm of patients who have suffered the new coronavirus infection.

Identification and molecular modeling of novel endogenous activator proteins of Sirt-1: an in silico study

Sirt-1 is one of the most extensively studied mammalian Sirtuins that deacetylates histones and several non-histone proteins critical to cellular homeostasis. As a key sensor of cellular metabolism, it is regulated at multiple levels including transcriptional and post translational levels. As an allosteric enzyme, its activity is also modulated by ligands and certain endogenous proteins. The present study is an in silico approach to identify novel Sirt-1 binding proteins. Bioinformatic search for similarity in sequence, structure, and topology of binding region to Lamin-A, a known activator of Sirt-1, identified three proteins viz. Epididymis secretory sperm binding protein (ESSBP), xylosyltransferase 1 (XT-1), and Adenylyl cyclase 9 (ADCY-9). Molecular docking studies revealed binding of ESSBP and ADCY-9 to the N-terminal region of Sirt-1 while XT-1 docks at both N-terminal and C-terminal region of Sirt-1 with Z-Dock score better than Lamin-A; XT-1 and ADCY-9 showed better Z-Rank score as well. MD simulation studies for extended time followed by MM-PBSA analysis showed that the Sirt-1-protein complexes were stable with favourable binding energy and minimal change in RMSD relating to backbone structure and RMSF relating to residue fluctuations. Further, H-bond analysis showed only minimal changes in H bonding interactions. Docking of these proteins to Sirt-1 through interaction with several residues particularly to its N-terminal region spanning 1–243 residues, in a manner similar to the docking of the activator Lamin-A and different from the inhibitor DLBC-1 binding site, suggests that these proteins may also positively modulate Sirt-1 activity. Further experimental data would be required to validate the computational prediction and to understand its physiological role. Communicated by Ramaswamy H. Sarma

Alpha-lipoic acid supplementation restores the meiotic competency and fertilization capacity of porcine oocytes induced by arsenite.

Arsenite is known as a well-known endocrine disrupting chemicals, and reported to be associated with an increased incidence of negative health effects, including reproductive disorders and dysfunction of the endocrine system. However, it still lacks of the research regarding the beneficial effects of ALA on arsenite exposed oocytes, and the underlying mechanisms have not been determined. Here, we report that supplementation of alpha-lipoic acid (ALA), a strong antioxidant naturally present in all cells of the humans, is able to restore the declined meiotic competency and fertilization capacity of porcine oocytes induced by arsenite. Notably, ALA recovers the defective nuclear and cytoplasmic maturation of porcine oocytes caused by arsenite exposure, including the impaired spindle formation and actin polymerization, the defective mitochondrion integrity and cortical granules distribution. Also, ALA recovers the compromised sperm binding ability to maintain the fertilization potential of arsenite-exposed oocytes. Importantly, ALA suppresses the oxidative stress by reducing the levels of ROS and inhibits the occurrence of DNA damage along with apoptosis. Above all, we provide a new perspective for the application of ALA in effectively preventing the declined oocyte quality induced by environmental EDCs.

Human sperm TMEM95 binds eggs and facilitates membrane fusion

Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. Tmem95 knockout murine sperm can bind to, but do not fuse with, eggs. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion during fertilization.

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.

294 Awardee Talk: Porcine Sperm Storage in the Oviduct Before Fertilization: A Sweet Spot

Abstract Sperm storage in the female reproductive tract from sperm deposition until ovulation is conserved among a wide variety of animals allowing some asynchrony between mating and ovulation. Mammals, reptiles, birds, amphibians, and insects are among those that store sperm from a day or two to as long as a decade. We have studied the molecular basis of how sperm are retained and then released from the reservoirs in the female tract to fertilize oocytes, primarily in swine. Previous research by others implicated various oviduct glycans as receptors to retain sperm in the lower oviduct, the isthmus, but the complete structures of the glycans that bound sperm had not been identified. We used an array of hundreds of glycans to identify those that bound sperm and identified several glycans with sperm binding ability. Further inspection revealed that all sperm binding glycans contained at least one of two motifs, either a branched 6-sialylated structure or a Lewis X trisaccharide. Profiling all glycans produced by the isthmus showed that these motifs were abundant among the complex N-linked glycans of the isthmus. Further, they were found on the apical surface of the isthmic epithelium where they are available to bind sperm. These glycans suppressed the normal increase in intracellular free Ca2+ in sperm during capacitation and, when immobilized on beads, lengthened sperm lifespan, mimicking the effect of sperm of binding to the oviduct. Binding to oviduct glycans alters sperm metabolism in specific ways to prolong sperm viability. Sperm are released from immobilized glycans by progesterone, which induces hyperactivated motility, and by capacitation, which reduces the binding affinity of capacitated sperm for oviduct glycans. This information may be used to improve sperm lifespan in vivo or enhance sperm storage outside of the female tract.

Proteomic changes in human spermatozoa during in vitro capacitation and acrosome reaction in normozoospermia and asthenozoospermia.

The cellular and molecular mechanisms of the events that help spermatozoa acquire their fertilizing capability during capacitation and acrosome reaction are not completely understood. This study was performed with a postulation that the identification of sperm proteins and their changes during in vitro capacitation and acrosome reaction will unravel unknown molecular aspects of fertilization that impact male fertility. Spermatozoa collected from sequential conditions, that is, separation of ejaculated spermatozoa by Percoll gradient centrifugation, in vitro capacitation, and acrosome reaction were processed for tandem mass spectrometric analysis, followed by protein identification, label-free quantitation, and statistical analysis. Collectively, a total of 1088 sperm proteins were identified. In comparison to ejaculated spermatozoa, 44 and 141 proteins were differentially expressed in capacitated and acrosome reacted spermatozoa, respectively. A large number of proteins were found downregulated, including clusterin, pyruvate dehydrogenase E1 component, semenogelin-1 and 2, heat shock protein 90, beta-microseminoprotein, and keratin. It was expected as sperm-membrane-associated proteins are removed during capacitation. There were significant proteomic alterations in asthenozoospermia compared to normozoospermia; however, variation was more noticeable among proteins of acrosome reacted spermatozoa and those released during the acrosome reaction. The processes enriched among downregulated proteins in asthenozoospermia included acrosome assembly, binding of spermatozoa to zona pellucida, nucleosome assembly, flagellated sperm motility, protein folding, oxidative phosphorylation, tricarboxylic acid cycle, chromatin silencing, gluconeogenesis, glycolytic process, and glycolysis. The dynamic information generated about proteomic alterations in spermatozoa during capacitation and acrosome reaction and their variability in asthenozoospermia will contribute not only to enhancing our understanding of processes that prepare spermatozoa to acquire fertilization capability but also help in deciphering novel factors of male infertility.

The vertebrate- and testis- specific transmembrane protein C11ORF94 plays a critical role in sperm-oocyte membrane binding

Sperm-oocyte membrane fusion is necessary for mammalian fertilization. The factors that determine the fusion of sperm with oocytes are largely unknown. So far, spermatozoon factor IZUMO1 and the IZUMO1 counter-receptor JUNO on the oocyte membrane has been identified as a protein requiring fusion. Some sperm membrane proteins such as FIMP, SPACA6 and TEME95, have been proved not to directly regulate fusion, but their knockout will affect the fusion process of sperm and oocytes. Here, we identified a novel gene C11orf94 encoding a testicular-specific small transmembrane protein that emerges in vertebrates likely acquired via horizontal gene transfer from bacteria and plays an indispensable role in sperm-oocyte binding. We demonstrated that the deletion of C11orf94 dramatically decreased male fertility in mice. Sperm from C11orf94-deficient mice could pass through the zona pellucida, but failed to bind to the oocyte membrane, thus accumulating in the perivitelline space. In consistence, when the sperm of C11orf94-deficient mice were microinjected into the oocyte cytoplasm, fertilized oocytes were obtained and developed normally to blastocysts. Proteomics analysis revealed that C11orf94 influenced the expression of multiple gene products known to be indispensable for sperm-oocyte binding and fusion, including IZUMO1, EQTN and CRISP1. Thus, our study indicated that C11ORF94 is a vertebrate- and testis-specific small transmembrane protein that plays a critical role in sperm binding to the oolemma.

Can we Make SpermFAST? Improving Sperm Function to Increase Fertilisation Rates after in vitro Fertilisation

Background: Intracytoplasmic sperm injection (ICSI) usage ([Formula: see text]64% of autologous cycles) has increased in Australia as an attempt to avoid low fertilisation and total fertilisation failure after standard in vitro fertilization (IVF). ICSI and to a lesser extent IVF, bypass key sperm maturation (hyperactivation, capacitation/acrosome reaction) events that naturally occur in the female tract and are vital for successful fertilisation. Current commercial sperm preparation media are not fully designed to induce these changes in sperm prior to insemination. Aim: To improve fertilisation rates following IVF by increasing sperm capacitation and hyperactivation between sperm preparation and insemination ultilising a new sperm medium (SpermFAST). Method: Sperm from 12 consenting normospermic men were incubated in either G-IVF+ (Vitrolife) or SpermFAST (UoA/Monash IVF) following a direct swim-up. Measures of capacitation, hyperactivation, sperm binding, acrosome reaction and oxidative DNA damage were assessed. Further, sperm from male CBAF1 mice (N=8) were incubated in either G-IVF+ or SpermFAST prior to IVF insemination. Fertilisation rates, embryo development, blastocyst cell numbers and DNA damage were assessed. Results: Incubation of human sperm in SpermFAST increased tyrosine phosphorylation (15.8% vs 9.5%, P[Formula: see text]0.05), hyperactive motility (38.3% vs 14.9%, P[Formula: see text]0.01), sperm binding (73.1% vs 47.7%, P[Formula: see text]0.01), while having no impact on sperm acrosomal status or oxidative DNA damage levels. Following IVF in the mouse, sperm incubated in SpermFAST increased fertilisation rates (94% vs 88%, P[Formula: see text]0.05), blastocyst total cell (92.2% vs 77.4%, P[Formula: see text]0.05), inner cell mass (14.9% vs 18.9%, P[Formula: see text]0.01) and epiblast cell numbers (3.7% vs 1.6%, P[Formula: see text]0.01), while the proportion of DNA damaged cells decreased in blastocysts (2.3% vs 4.8%, P[Formula: see text]0.001). Conclusion: Sperm function and fertilisation rates are improved when the sperm medium better mimics the environment of the female reproductive tract during natural conception. Improving IVF culture media to better meet the physiological needs of sperm could potentially improve outcomes following IVF.

OP-21 - Age-related alterations in the surface morphology of zona pellucida prevent sperm binding to oocyte and affect fertilization.

Mammalian fertility with aging is thought to decrease due to an increase in aneuploidy, abnormal mitochondrial function, and changes in the expression of oocytes. Although previous studies have reported that naturally aged over 8-month-old female mice have lower fertility, the mechanism of egg quality deterioration is still unknown. In this study, we focus on the effects of aging on ovarian tissue and fertility using young mice (2 months old) and aged mice (8 months old) and clarify the mechanism of age-related deterioration of egg quality. When we examined the fertility by natural mating, the number of pups produced by aged mice was significantly decreased. Histological analysis of ovaries displayed a decrease in the number of remaining follicles, and ovulated oocytes ovulated number by superovulation treatment was also significantly reduced in aged mice. Examination of the fertilization ability in vitro revealed a significant decrease in the fertilization rate in aged mice, whereas there was no difference in gene expression in MII eggs by RNA-seq. These results suggest that the age-related decrease in fertilization ability of mouse oocytes is not due to gene expression in the oocytes themselves, but to inhibition of sperm binding to surface of the zona pellucida.

Symposium 1-1 - Immunocontraceptive vaccine development using zona pellucida (ZP)

The zona pellucida (ZP) is an extracellular matrix surrounding ovarian oocytes, ovulated eggs and preimplantation embryos. Its constituent glycoproteins are expressed in a tissue-specific manner. Since the 1970s, ZP had been assumed to be a promising candidate for immunocontraceptive vaccines, because anti-ZP antibody blocked fertilization by prevention of sperm binding to ZP in vitro. Biochemical and immunological studies revealed that ZP is composed of glycoproteins and possesses strong antigenicity. It was thus considered that ZP could surely satisfy the requirements for effective contraceptive vaccines. Contraceptive vaccines for human use should be highly effective and reversible as well as safe. However, early animal experiments showed that infertility by ZP immunization resulted from ovarian impairment. Use of immunocontraceptive vaccines with naturally occurring ZP protein has thus been limited to wildlife population control. Application of recombinant ZP proteins was expected to provide safe and irreversible vaccines but ovarian pathology could not be avoided. As a next stage, contraceptive vaccine development continued through DNA engineering, whereby the antigens were not necessarily based on native amino acid sequences of ZP. Artificial DNA constructs coding multiple promising peptides were also prepared with GnRH and promiscuous T-cell epitopes. However, their efficacy was insufficient. Recently, single chain fragment for variable regions (scFv) has been proposed as a future strategy. The scFv is expected to bind directly to ZP in the inoculated animals by skipping the antibody production process. In conclusion, intensive studies and advances in technology have not achieved ideal targets for ZP-based immunocontraceptive vaccines at this moment. We are anticipating future scientific progress.

Octocrylene exposure impairs mouse oocyte quality by inducing spindle defects and mitochondria dysfunction

One of organic ultraviolet (UV) filters, Octocrylene (OCL), is mainly used in various cosmetic products, which is being frequently detected in soil, sediment, aquatic systems and food chain. There is evidence confirmed the reproductive toxicity of OCL in Japanese medaka. However, less was known about the effects of OCL exposure on oocyte quality. Here, we investigated the impacts of OCL on mouse oocyte maturation and quality by exposing oocytes to OCL in vitro at concentrations of 8, 22, 30, 40 and 50 nM. The results showed that OCL markedly reduced mouse oocyte germinal vesicle breakdown (GVBD) at 50 nM and polar body extrusion (PBE) rates at 40 and 50 nM. OCL exposure further disrupted spindle assembly and chromosome alignment, finally inducing aneuploid. Mitochondrial function was also damaged by OCL exposure, leading to ROS overproduction and apoptosis in oocytes. Moreover, OCL treatment impaired the distribution of cortical granules and sperm binding ability of oocytes. In summary, these data demonstrated that OCL could disturb the oocyte meiotic maturation and reduce oocyte quality.

Quantitative mRNA and Protein Analyses of ZPB1, ZPB2, and ZPC in Different Genetic Strains of Broiler Breeders

The avian inner perivitelline layer (IPVL), containing the zona pellucida (ZP) family of proteins, surrounds the ovulated ovum. Sperm binding at the germinal disc (GD) region of the IPVL initiates fertilization in avian species, and the amount of sperm binding at the GD reflects female fertility. RT-PCR and quantitative Western blot analyses were used to determine differences in ZP protein mRNA expression and protein concentration between GD and nongerminal disc (NGD) regions among four genetic strains of broiler breeders. Both the mRNA expression and protein concentration of chicken ZPB2 was greater in the GD region, compared to the NGD region, and ZPB2 protein was more abundant in the small prehierarchical follicles. Chicken ZPC mRNA, but not ZPC protein, was greater in the NGD region compared to the GD region, and hepatically expressed ZPB1 protein was more concentrated in the NGD region. Testosterone increased the expression of both ZPC mRNA and protein in cultured granulosa cells from prehierarchical follicles. The current research is the first to investigate the relative amount of ZP proteins between the GD region and NGD region in chicken IPVL. The results provide further evidence that ZPB2 may be critical for avian sperm binding at the GD region of the IPVL.

Development of homologous hemizona assay as a potential predictor of fertility in buffalo bulls: A preliminary approach

Although, myriads of tests are routinely used, no single test can accurately predict fertilization potential of semen. The hemizona assay (HZA) has advantages in two ways: (a) it determines multitude traits of sperm and (b) it is a controlled sperm function test. In the present study, we developed homologous HZA in buffaloes to predict bull fertility. In this experiment, bulls with fertility rate 53.3% and 48.5% were used as control, whereas bulls with fertility rate 32.6% and 32.2% were used as test semen samples. For HZA, matching buffalo hemizonae were co-incubated with processed buffalo sperm for 4 h. The number of sperms bound to the outer surface of hemizona was determined. No significant difference was observed in sperm binding for co-incubation of same bull sperm with matching hemizona (p < .05). Significant difference in sperm binding to matching hemizona was seen, while two halves were incubated with control and test semen, respectively (p < .05). Hemizona assay index (HZAI) of test bull semen has been determined from percentage of test-sperm bound to the matching hemizona in comparison to control-sperm. For finding relation, HZAI was correlated with respective fertility rates of semen samples, and it was found that a significant positive correlation was present with r=0.83, p < .1. A regression equation of Y=1.39X - 55.8 (where Y=pregnancy rate of test semen sample and X=HZAI of test semen sample) was presented to predict fertility rates of unknown semen samples. Thus, HZA can be used as a potential predictor of buffalo bull 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.