Sperm Binding Research Articles

The C. elegans eggshell.

In all animals, oocytes are surrounded by an extracellular matrix upon fertilization. This matrix serves similar purposes in each animal. It functions to mediate sperm binding, to prevent polyspermy, to control the chemical environment of the embryo, and to provide physical protection to the embryo as it developes. The synthesis of the C. elegans matrix, or eggshell, begins when the oocyte enters the spermatheca and is fertilized by a single sperm. The process of eggshell synthesis is thought to take place during the completion of the maternal meiotic divisions such that the multi-layered eggshell is completed by anaphase II. The synthesis of the eggshell occurs in a hierarchical pattern in which the outermost layers are synthesized first in order to capture and retain the innermost layers as they form. Recent studies have revealed that the lipid-rich permeability barrier is distinct from the outer trilaminar eggshell. These new findings alter our previous understanding of the eggshell. This chapter aims to define each of the eggshell layers and the molecules that are known to play significant roles in their formation.

Comparative analysis of proteomes between diabetic and normal human sperm: Insights into the effects of diabetes on male reproduction based on the regulation of mitochondria-related proteins.

This study sought to identify sources of the reduced fertility of men with type 2 diabetes mellitus. Significant reductions in semen volume, sperm concentration, and total sperm count were observed in diabetic individuals, while transmission electron microscopy revealed that the structure of mitochondria in the tail of sperm from diabetic patients was damaged. Proteins potentially associated with these sperm defects were identified using proteomics. Isobaric tagging for relative and absolute quantitation labeling and high-performance liquid chromatography-tandem mass spectrometry allowed us to identify 357 proteins significantly differentially expressed in diabetic versus control semen (>1.2 or <0.83). According to gene ontology enrichment and pathway analyses, many of these differentially expressed proteins are associated with sperm function, including binding of sperm to the zona pellucida and proteasome function; of particular interest, half of these proteins were related to mitochondrial metabolism. Protein-interaction networks revealed that a decrease in Cystatin C and Dipeptidyl peptidase 4 in the mitochondria may be sources of the decreased motility of sperm from diabetic patients.

Bovine Fertility as Regulated by Sperm Binding Proteins: A Review

Bovine Fertility as Regulated by Sperm Binding Proteins: A Review

153 Fertilisation of Cattle Oocytes is Linked to Novel Waves of a Ca2+ Fluorophore in Cumulus Cells

During fertilization, multiple intracellular calcium (Ca2+) oscillations are initiated after sperm binding to the oocyte vitelline membrane. This Ca2+ signalling has been extensively studied in denuded mouse and Xenopus oocytes but minimally studied in larger mammals. Cows in particular are unusual, as the few studies on oocyte activation have observed fewer Ca2+ oscillations during fertilisation compared with mice. Furthermore, cattle intracytoplasmic sperm injection (ICSI) is inefficient, despite parthenogenetic activation occurring readily. We hypothesise that cumulus cells are important for cattle oocyte activation at fertilisation. Here, we assessed the behaviour of Ca2+oscillations in fertilising intact cattle cumulus–oocyte complexes (COC). Abattoir-derived cattle COC were matured and fertilised in vitro using Bovine Vitro Media Suite (IVF Vet Solutions). The COC were stained 3.5 h after insemination with the Ca2+ fluorescent probe Fluo4AM (5 μM, Molecular Probes Inc., Eugene, OR, USA) for 30 min, washed, and imaged every 5 min for 6 h in a Fluoview FV10i incubating time-lapse confocal microscope (Olympus) before being returned to culture. Embryo development was assessed at Day 8 to confirm fertilisation. Fluo4AM fluorescence intensity was assessed using FIJI ImageJ. Mean relative intensity over time was graphed for specific regions of interest and the area under graphs was calculated to quantify differences for comparison using a Mann-Whitney Test (mean ± SEM). Experiment 1 (4 reps of 10 COC) compared confirmed fertilised v. uninseminated; experiment 2 (2 reps of 10 COC) compared inseminated COC ± 10 μM BAPTA-AM (Ca2+ chelator, Sigma-Aldrich, St. Louis, MO, USA). There were distinct coordinated waves of differing Fluo4AM intensity in both the oocyte and the cumulus cells surrounding the confirmed fertilised oocytes. This contrasted to the random uncoordinated flashes of Fluo4AM fluorescence in the cumulus cells of the uninseminated oocytes. The fluorescence pattern in +BAPTA-AM COC matched the random flashes observed in the uninseminated group of experiment 1. The fluorescence in the media surrounding the COC immediately following the Fluo4AM waves spiked and then plateaued at a higher level of fluorescence. This was quantified by assessing the area under the graph for 1 h of the plateau following the fluorescence spike. There were no differences between confirmed fertilised (346.4 ± 41.62) and uninseminated groups (239.8 ± 32.08; P &gt; 0.05), but this was affected by differences in cumulus dispersal due to the presence or absence of sperm. Experiment 2 used BAPTA-AM to block oocyte activation with sperm present in both groups and showed a significant difference between the fluorescence increase in the media of the 2 groups (–BAPTA-AM: 311.2 ± 31.57, +BAPTA-AM: 201.4 ± 26.59; P &lt; 0.03). Although the physiological significance has yet to be determined, we have observed a novel Ca2+ wave in the cumulus cells that could be linked to oocyte activation in cattle. There was a significant increase in Fluo4AM fluorescence in the media surrounding the COC, which may indicate cumulus cells are releasing Ca2+ at the time of oocyte activation.

Effect of sorting boar spermatozoa by sex chromosomes on oviduct cell binding

This study examined the hypothesis that flow sorting sperm by sex chromosomes affects oviduct cell binding which would influence formation of the sperm reservoir in the oviduct. The sperm-rich fraction from boars (n = 5) was collected, sperm were stained with Hoechst 33342 and sorted. Sperm were sorted based on the presence of either an X or Y chromosome and placed into the following treatments: 1) sperm selected for the Y chromosome, 2) selected for the X, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were tested for oviduct cell binding within 12 h of sorting. Additionally, sperm were analyzed for motility characteristics, acrosome status, and binding to the two oviduct glycan motifs that bind porcine sperm, biantennary 6-sialylated N-acetyllactosamine on a mannose core (bi-SiaLN) and sulfated LeX trisaccharide (suLeX). The disaccharide found within both glycan motifs, N-acetyllactosamine (LacNAc), was used as a control. Sperm binding to oviduct cells was reduced by more than half in the three sorted samples when compared to the control sperm that were not sorted. The percentage of sperm that were motile 24 h after sorting was also decreased significantly in each of the sorted sample groups when compared to the unsorted control. In contrast, sorting did not decrease the percentage of sperm that bound purified soluble glycans or the location on sperm to which they bound. There was also no difference in sperm acrosome status among the four groups. In summary, sorting reduced sperm binding to the complex matrix around oviductal cell aggregates but sperm binding to purified soluble oviduct glycans was not affected. The requirement for higher affinity and motility to bind glycans immobilized on oviduct cells may explain this difference. The reduction in sperm fertility observed following sex-sorting may be explained partially by a reduced or altered ability to bind to the oviduct epithelium.

Flow cytometry sex sorting affects bull sperm longevity and compromises their capacity to bind to oviductal cells

This study assessed the effect of flow cytometry sexing on sperm longevity and the capacity of sperm to bind to oviductal cells. Each ejaculate from four bulls was divided into two fractions: the first was immediately frozen as non sexed sperm (NS) and the second was sexed originating X- and Y-bearing sperm. The fourth treatment had sex-sorted X and Y sperm (XY) combined. Sperm from each group was assessed for sperm characteristics after thawing, after washing and at 2, 4, 8 and 12 h of incubation at 39 °C in 5% CO2 in air. For the binding test, sperm were incubated in sp-TALP medium for 30 min or 24 h with oviductal explants. Percentages of motility (58.1 ± 4.3 and 35.2 ± 4.4), progressive motility (46.1 ± 6.1 and 25.7 ± 4.8), mitochondrial membrane potential (79.2 ± 8.3 and 69.0 ± 6.3), plasma membrane stability (77.4 ± 4.6 and 19.4 ± 4.2), and live sperm with intact acrosome (57.2 ± 8.5 and 31.3 ± 7.9) were higher in NS than in XY, respectively (P < 0.05). Those differences were maintained for up to 8 h. The sexing process did not affect the sperm binding to the explants after 30 min. However, after 24 h, XY had less (6.7 ± 2.0) sperm bound to explants than NS (23.6 ± 7.2). In conclusion, even though XY was of lower quality than NS, the decreases in quality in both NS and XY groups were similar between groups during incubation. Moreover, the sex-sorting process affected the ability of sperm to remain bound to oviductal explants.

The protective role of melatonin in porcine oocyte meiotic failure caused by the exposure to benzo(a)pyrene.

Does melatonin restore the benzo(a)pyrene (BaP)-induced meiotic failure in porcine oocytes? Melatonin effectively inhibits the increased reactive oxygen species (ROS) level and apoptotic rate in BaP-exposed porcine oocytes to recover the meiotic failure. BaP, a widespread environmental carcinogen found in particulate matter, 2.5 µm or less (PM2.5), has been shown to have toxicity at the level of the reproductive systems. BaP exposure disrupts the steroid balance, alters the expression of ovarian estrogen receptor and causes premature ovarian failure through the rapid depletion of the primordial follicle pool. In addition, acute exposure to BaP has transient adverse effects on the follicle growth, ovulation and formation of corpora lutea, which results in transient infertility. Porcine oocytes were randomly assigned to control, BaP-exposed and melatonin-supplemented groups. BaP was dissolved in dimethylsulphoxide and diluted to a final concentration of 50, 100 or 250 μM with maturation medium, respectively. Melatonin was dissolved in the absolute ethanol and diluted with maturation medium to a final concentration of 1 nM, 100 nM, 10 μM and 1 mM, respectively. The in vitro cultured oocytes from each group after treatment were applied to the subsequent analysis. Acquisition of oocyte meiotic competence was assessed using immunostaining, fluorescent intensity quantification and/or immunoblotting to analyse the cytoskeleton assembly, mitochondrial integrity, cortical granule dynamics, ovastacin distribution, ROS level and apoptotic rate. Fertilization ability of oocytes was examined by sperm binding assay and IVF. BaP exposure resulted in the oocyte meiotic failure (P = 0.001) via impairing the meiotic apparatus, showing a prominently defective spindle assembly (P = 0.003), actin dynamics (P < 0.001) and mitochondrion integrity (P < 0.001). In addition, BaP exposure caused the abnormal distribution of cortical granules (P < 0.001) and ovastacin (P = 0.003), which were consistent with the observation that fewer sperm bound to the zona pellucida surrounding the unfertilized BaP-exposed eggs (P < 0.001), contributing to the fertilization failure (P < 0.001). Conversely, melatonin supplementation recovered, at least partially, all the meiotic defects caused by BaP exposure through inhibiting the rise in ROS level (P = 0.015) and apoptotic rate (P = 0.001). We investigated the negative impact of BaP on the oocyte meiotic maturation in vitro, but not in vivo. Our findings not only deeply clarify the potential mechanisms of BaP-induced oocyte meiotic failure, but also extend the understanding about how environmental pollutants influence the reproductive systems in humans. This study was supported by the National Natural Science Foundation of China (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.

Seminal plasma and cryopreservation alter ram sperm surface carbohydrates and interactions with neutrophils.

Spermatozoa deposited vaginally must navigate the physical, chemical and immune barriers of the cervix to reach the site of fertilisation. Characteristics that favour successful cervical transit remain largely unknown beyond the obvious factors of motility and viability. Epididymal and cryopreserved ram spermatozoa demonstrate poor cervical transit, for unknown reasons. We hypothesised that seminal plasma exposure and cryopreservation alter the surface sugars of these sperm populations and, consequently, their interaction with immune cells, both potential factors for successful cervical transit. The carbohydrate profiles of epididymal, ejaculated and frozen-thawed ram spermatozoa were assessed by flow cytometry and western blotting using lectins for galactose, sialic acid, N-acetylglucosamine and mannose. Seminal plasma exposure and cryopreservation caused significant changes to the relative amounts of surface sugars detected by flow cytometry and lectin blotting. Immune cell interaction was characterised using a neutrophil-binding assay. Seminal plasma acted as a robust protective mechanism, limiting binding of spermatozoa, whereas the media used for cryopreservation caused a significant disruption to opsonin-mediated binding. We were unable to demonstrate a link between changes to surface sugars and neutrophil susceptibility. Seminal plasma and cryopreservation clearly alter the sperm glycocalyx, as well as the interaction of spermatozoa with immune cells.

Species-specific and differential expression of BSP-5 and other BSP variants in normozoospermic and asthenozoospermic buffalo (Bubalus bubalis) and cattle (Bos taurus) seminal plasma

Binder of sperm-5 (BSP-5) is one of the fertility-associated proteins of cattle seminal plasma. Binding of sperm to the oviductal epithelium is mediated by BSP group of proteins. However, it is not clear, whether this protein is also involved in sperm motility. In the present study, attempts were made to characterize BSP-5 protein in both normozoospermic (NS) and asthenozoospermic (AS) Murrah buffalo (n = 18; Bubalus bubalis), Holstein Friesian (n = 8, Bos taurus) and Jersey cattle (n = 8; Bos taurus) bull seminal plasma and also study its expression pattern in these species. 1-D Western blot demonstrated three major BSP-5 immunoreactive protein bands (24.2 kDa, 20.5 kDa, and 12.3 kDa) in buffalo seminal plasma. Of these, the intensities of 24.2 and 20.5 kDa protein bands reduced significantly (P ≤ 0.05) in seminal plasma of AS group compared to that of NS group. On the contrary, the expression of 12.3 kDa protein band did not vary significantly between the groups. In Holstein Friesian seminal plasma, at least six BSP-5 immunoreactive protein bands (25.1, 23.6, 19.5, 13.8, 13.1 and 12.3 kDa) could be detected. Of these, the intensities of 23.6, 13.8/13.1 and 12.3 kDa protein bands decreased (P = 0.058, 0.111, 0.053) in AS group bulls compared to NS bulls. Holstein Friesian bull seminal plasma demonstrated a BSP-5 immunoreactive duplex protein band of 13.8/13.1 kDa, which was not evident in buffalo seminal plasma. In 2-D Western blot, a train of five BSP-5 immunoreactive duplex protein spots (Mr 21.0–27.6 kDa, pI of ∼3.9–5.1) was detected. Mass spectrometry of one of the representative duplex spot confirmed that these were BSP-5 and BSP-3 proteins, respectively. Indirect immunofluorescence studies showed that BSP-5 is primarily localized to the mid-piece/mitochondrial region of buffalo spermatozoa. To conclude, the findings of the present study could establish the significance and association of BSP-5 proteins in sperm motility and how their level differ in semen from two different clinical groups of buffalo bull (NS vs. AS). Further, the study also demonstrated that the expression pattern of BSP-5 and other BSP variants in seminal plasma of bulls is species-specific.

Capacidade de ligação dos espermatozoides do epidídimo de equinos às células da tuba uterina cultivadas in vitro

Muitas biotecnologias estão sendo desenvolvidas visando a conservação do material genéticode garanhões de alto valor zootécnico. Dentre estas pode-se destacar a colheita deespermatozoides do epidídimo de animais que sofreram algum trauma ou enfermidade queimpossibilitem a colheita do sêmen, óbito ou eutanásia. Porém essas células espermáticas nãoentram em contato com o plasma seminal, importante por conter proteínas que participam deprocessos relacionados à proteção e ligação dos espermatozoides aos reservatóriosespermáticos na tuba uterina. Neste sentido, sugere-se que ocorram alterações bioquímicasnas células espermáticas do epidídimo. Objetivou-se com esta revisão, estudar as principaisdiferenças morfofuncionais entre os espermatozoides provenientes do epidídimo e doejaculado de equinos

BaP exposure causes oocyte meiotic arrest and fertilization failure to weaken female fertility.

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant and carcinogen that is frequently found in particulate matter, with a diameter of ≤2.5 μm (PM2.5). It has been reported to interrupt the normal reproductive system, but the exact molecular basis has not been clearly defined. To understand the underlying mechanisms regarding how BaP exposure disrupts female fertility, we evaluated oocyte quality by assessing the critical regulators and events during oocyte meiotic maturation and fertilization. We found that BaP exposure compromised the mouse oocyte meiotic progression by disrupting normal spindle assembly, chromosome alignment, and kinetochore-microtubule attachment, consequently leading to the generation of aneuploid eggs. In addition, BaP administration significantly decreased the fertilization rate of mouse eggs by reducing the number of sperm binding to the zona pellucida, which was consistent with the premature cleavage of N terminus of zona pellucida sperm-binding protein 2 and precocious exocytosis of ovastacin. Furthermore, BaP exposure interfered with the gamete fusion process by perturbing the localization and protein level of Juno. Notably, we found that BaP exposure induced oxidative stress with an increased level of reactive oxygen species and apoptosis in oocytes and thereby led to the deterioration of critical regulators and events during oocyte meiotic progression and fertilization. Our data document that BaP exposure reduces female fertility via impairing oocyte maturation and fertilization ability induced by oxidative stress and early apoptosis in murine models.-Zhang, M., Miao, Y., Chen, Q., Cai, M., Dong, W., Dai, X., Lu, Y., Zhou, C., Cui, Z., Xiong, B. BaP exposure causes oocyte meiotic arrest and fertilization failure to weaken female fertility.

Effect of supplementation of recombinant Regucalcin in extender on cryopreservation of spermatozoa of water buffalo (Bubalus bubalis).

Elevated intracellular calcium concentration and oxidative damage are two major factors contributing to the poor fertility of cryopreserved spermatozoa. Regucalcin (RGN), also known as Senescence marker protein-30 (SMP-30), is a calcium-binding protein with multiple roles that include calcium homeostasis, anti-oxidative, anti-apoptosis, and anti-proliferation. In Drosophila, RGN is reportedly a putative cold-tolerance gene and a cytoprotective role for RGN against intracellular calcium elevation and oxidative stress was reported in P19 cell lines. Given that RGN has anticapacitatory effect and abundant in the male reproductive tract, we hypothesized that it may play a cryoprotective role for spermatozoa. We investigated this by including RGN, at three different concentrations (20, 40, and 60 μg/ml), as a supplement for Tris-egg yolk-based semen extender. Post-thaw metrics of progressive motility, acrosome integrity, and zona pellucida binding of spermatozoa were evaluated for three ejaculates of three clinically normal, breeding Murrah buffaloes. A concentration of 40 μg/ml of recombinant RGN supplemented during sperm freezing resulted in significant increases in the post-thaw progressive motility of spermatozoa (50.6 ± 3.5% vs 40.6 ± 2.6%; p < 0.01), acrosome integrity (53.3 ± 7.4 vs 75.6 ± 6.8; p < 0.05), and zona pellucida binding (31.6 ± 14.0 vs 191.9 ± 12.3 bound spermatozoa; p < 0.01) compared to control conditions without RGN. Thus, ∼1 μM recombinant RGN, which retains the ability to bind calcium, has a cryoprotective effect for buffalo spermatozoa in extender.

Zona pellucida protein B2 messenger ribonucleic acid expression varies with follicular development and granulosa cell location

The freshly ovulated ovum in avian species is surrounded by a protein layer called the inner perivitelline layer (IPVL). The IPVL contains zona pellucida proteins and 6 distinct zona pellucida genes have been identified (ZPA, ZPB1, ZPB2, ZPC, ZPD and ZPX1) in the chicken. In the present research, the expression of the mRNA for ZPA, ZPB2, and ZPX1 was investigated in 2 lines of turkey hens selected for either increased egg production (E line) or increased body weight (F line). Theca and granulosa cell expression of the mRNA for ZPA and ZPB2 was also investigated in hierarchical and prehierarchical follicles from broiler breeder hens. Granulosa tissue was collected from F1 through F4 and F1 through F10 follicles in E line and F line hens, respectively. A one cm2 section of the granulosa layer around the germinal disc (GD) and an equivalent sized nongerminal disc (NGD) area was also collected from the F1 and F2 follicles from other hens from each genetic line. Granulosa and theca tissue was collected from hierarchical and prehierarchical follicles of broiler breeder hens. Total RNA was extracted from the samples. Minor groove-binding probes and primers for detecting ZPA, ZPB2, and ZPX1, were made for real-time PCR analyses. Expression of ZPA, ZPB2, and ZPX1 was detected in all follicle sizes from both genetic lines of hens. No significant differences in ZPA and ZPX1 mRNA expression were detected between the GD and NGD granulosa cells. However, the expression of the mRNA for ZPB2 was significantly greater in the GD granulosa cells when compared to the NGD granulosa cells in F1 and F2 follicles from E line and F line hens. In broiler breeder hens, the mRNA expression of ZPA and ZPB2 was greatest in the smallest prehierarchical follicles. The results suggest that higher expression of ZPB2 in the germinal disc area may be important for the preferential binding of sperm to this region of the IPVL.

Anandamide exerts a suppressive effect on sperm binding to oviduct explants through CB1 receptors in the water buffalo (Bubalus bubalis)

An endocannabinoid system comprising of Anandamide (AEA) and its receptor has been shown to play a role in sperm acquisition of fertilizing potential and sperm-oviduct interaction. In the present study, we assessed the effect of sperm pre-treatment with AEA or co-incubation of sperm-oviduct explants with AEA in the presence or absence of CB1 receptor antagonist (SR141716A) on sperm-oviduct binding in the water buffalo. Cryopreserved spermatozoa from 3 Murrah buffalo bulls (3 ejaculates from each bull) were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 1h. Then, sperm-oviduct explants were stained with a fluorescent stain (JC-1) and sperm binding index (BI – No. of bound spermatozoa/unit area of oviduct explants) was assessed. The results indicate that BI decreased significantly (P<0.05) when spermatozoa were either pre-treated with AEA (14.16±0.87) or sperm-oviduct explants were co-incubated with AEA (16.27±0.86) at 1nM concentration compared to the control group (29.12±2.17), however such effect was not observed when AEA was used at 1μM concentration. Incorporation of SR141716A in the incubation medium inhibited the suppressive effect of AEA on BI. It was concluded that AEA, at 1nM concentration, decreased the number of spermatozoa bound to the oviduct explants and the suppressive effect of AEA on sperm-oviduct binding was inhibited by CB1 receptor antagonist suggesting that the effect of AEA was mediated through CB1 receptor in the water buffalo.

Glycosidases in porcine follicular fluid and their effect on zona pellucida-AWN 1 spermadhesin interaction

Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-l-fucosidase, α-d-galactosidase, β-d-galactosidase, β-D-N-acetylhexosaminidase, and α-d-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-d-mannosidase, which was increased (P < 0.001), and β-d-galactosidase, which was decreased (P < 0.001) at neutral pH, and of α-d-galactosidase and β-N-acetylhexosaminidase, which were decreased (P < 0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both β-d-galactosidase and to a lesser degree α-d-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.

Lactadherin is a candidate oviduct Lewis X trisaccharide receptor on porcine spermatozoa.

A recent study has demonstrated that porcine spermatozoa recognize with high affinity carbohydrate structures containing Lewis X motifs. Sperm adhesion to Lewis X is proposed to mediate sperm binding to the oviduct epithelium to form a reservoir. The objective of this study was to identify Lewis X-binding proteins from porcine spermatozoa as candidate receptors for oviduct glycans. To identify low-abundance proteins typically masked by proteins originating from seminal fluid, Lewis X candidate receptors were enriched from cauda epididymal boar spermatozoa. Plasma membrane preparations from cauda epididymal spermatozoa were subjected to RP-HPLC and glycan blotting assays to isolate and detect proteins that bind Lewis X. Following bottom-up LC-MS/MS analysis, among the two bands that bound sulfated Lewis X, ADAM5, which spermatozoa, was confidently identified. ADAM family members have been established as contributors to sperm entry into the oviduct. A second sulfated Lewis X-binding protein identified was the peripheral membrane protein lactadherin (also known as P47, SED1 and MFG-E8 in different species). The interaction between Lewis X and lactadherin was functionally important because competitive inhibition by soluble recombinant lactadherin reduced sperm binding to the oviduct epithelium. Furthermore, far-western blotting demonstrated that purified lactadherin could bind oviduct cells. In summary, these findings reveal that, in addition to the previously reported glycan affinity of accessory gland proteins that adhere to spermatozoa, multiple proteins intrinsic to spermatozoa have affinity for a specific oviduct glycan. Further, in addition to binding to the zona pellucida, lactadherin is now implicated in binding to oviduct glycans to promote formation of the sperm reservoir.

A new media without animal component for sperm cryopreservation: motility and various attributes affecting paternal contribution of sperm.

Our aim was the development of a safe sperm cryopreservation New Media (NM), composed of consistent and reproducible components devoid of any animal origin, and evaluation of NM in terms of its effect on sperm structure and function as compared to regularly used yolk media (TYM) (Irvine Scientific). We evaluated patient semen samples and cryopreserved them in duplicates in either NM or TYM. The samples were cryopreserved for either a short term of 1week or long term of 1month prior to thawing. The parameters investigated include sperm motility via computer-assisted semen analysis (CASA), sperm concentration, and sperm biomarkers that promote paternal contribution of spermatozoa to fertilization including hyaluronic acid binding, chromatin maturity, apoptotic markers, cytoplasmic retention, and sperm DNA integrity. As compared to TYM, NM was equally capable of sperm cryopreservation with both short-term and long-term storage in media, and after freeze-thaw and gradient processing of sperm. HA binding of sperm was comparable post thaw in both NM and yolk media. There are also no differences observed between the samples cryopreserved in NM or TYM in terms of their aniline blue staining, CK immunocytochemistry, caspase 3 immunostaining, or DNA nick translation. NM has the advantage of being xeno-free, yet in preservation of sperm motility and other sperm attributes, the NM is as effective as the TYM.

Melatonin protects oocyte quality from Bisphenol A-induced deterioration in the mouse.

Bisphenol A (BPA) has been reported to adversely affect the mammalian reproductive system in both sexes. However, the underlying mechanisms regarding how BPA disrupts the mammalian oocyte quality and how to prevent it have not been fully defined. Here, we document that BPA weakens oocyte quality by impairing both oocyte meiotic maturation and fertilization ability. We find that oral administration of BPA (100μg/kg body weight per day for 7days) compromises the first polar body extrusion (78.0% vs 57.0%, P<.05) by disrupting normal spindle assembly, chromosome alignment, and kinetochore-microtubule attachment. This defect could be remarkably ameliorated (76.7%, P<.05) by concurrent oral administration of melatonin (30mg/kg body weight per day for 7days). In addition, BPA administration significantly decreases the fertilization rate of oocytes (87.2% vs 41.1%, P<.05) by reducing the number of sperm binding to the zona pellucida, which is consistent with the premature cleavage of ZP2 as well as the mis-localization and decreased protein level of ovastacin. Also, the localization and protein level of Juno, the sperm receptor on the egg membrane, are strikingly impaired in BPA-administered oocytes. Finally, we show that melatonin administration substantially elevates the in vitro fertilization rate (63.0%, P<.05) by restoring above defects of fertilization proteins and events, which might be mediated by the improvement of oocyte quality via reduction of ROS levels and inhibition of apoptosis. Collectively, our data reveal that melatonin has a protective action against BPA-induced deterioration of oocyte quality in mice.

An intact acrosome is required for the chemotactic response to progesterone in mouse spermatozoa.

Mammalian sperm become fertilization-competent in the oviduct, during a process known as capacitation that involves the acquisition of the ability to exocytose the acrosome but also the chemotactic responses-both of which contribute to successful fertilization. Chemotaxis is used by spermatozoa to orient and to locate the egg; the acrosome reaction facilitates sperm binding to and fusing with the egg membrane. Mammalian spermatozoa are able to sense picomolar concentrations of progesterone, which drives chemotactic behavior. The state of the acrosome during the chemotactic response, however, is unknown. Genetically modified mouse spermatozoa were employed in a chemotaxis assay under fluorescence microscopy to evaluate their acrosome status while swimming, allowing us to elucidate the acrosome integrity of sperm responding to progesterone-induced chemotaxis. We first showed that wild-type mouse spermatozoa chemotactically respond to a gradient of progesterone, and that the genetic modifications employed do not affect the chemotactic behavior of sperm to progesterone. Next, we found that acrosome-intact, but not acrosome-reacted, spermatozoa orient and respond to picomolar concentrations of progesterone and that chemotaxis normally occurs prior to the acrosome reaction. Our results suggest that premature commitment to acrosome exocytosis leads to navigation failure, so proper control and timing of the acrosome reaction is required for fertilization success and male fertility.

ZP3 is Required for Germinal Vesicle Breakdown in Mouse Oocyte Meiosis

ZP3 is a principal component of the zona pellucida (ZP) of mammalian oocytes and is essential for normal fertility, and knockout of ZP3 causes complete infertility. ZP3 promotes fertilization by recognizing sperm binding and activating the acrosome reaction; however, additional cellular roles for ZP3 in mammalian oocytes have not been yet reported. In the current study, we found that ZP3 was strongly expressed in the nucleus during prophase and gradually translocated to the ZP. Knockdown of ZP3 by a specific siRNA dramatically inhibited germinal vesicle breakdown (GVBD) (marking the beginning of meiosis), significantly reducing the percentage of MII oocytes. To investigate the ZP3-mediated mechanisms governing GVBD, we identified potential ZP3-interacting proteins by immunoprecipitation and mass spectrometry. We identified Protein tyrosine phosphatase, receptor type K (Ptprk), Aryl hydrocarbon receptor-interacting protein-like 1 (Aipl1), and Diaphanous related formin 2 (Diaph2) as potential candidates, and established a working model to explain how ZP3 affects GVBD. Finally, we provided preliminary evidence that ZP3 regulates Akt phosphorylation, lamin binding to the nuclear membrane via Aipl1, and organization of the actin cytoskeleton via Diaph2. These findings contribute to our understanding of a novel role played by ZP3 in GVBD.