The Effect of Synthetic Polyamine BPA-C8 on the Fertilization Process of Intact and Denuded Sea Urchin Eggs.

Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.

Acrylamide impairs the developmental potential of germinal vesicle oocytes by inducing mitochondrial dysfunction and autophagy/apoptosis in mice.

Background: Acrylamide (ACR), an important endogenous contaminant in carbohydrate-rich foods, has been involved in various negative effects on multiple organ networks, including the reproductive system. Previous studies have reported that ACR affects oocyte quality and fertility. Purpose: This study aimed to explore the toxic effects and regulatory mechanisms of ACR on mouse germinal vesicle (GV) oocytes. Research Design: In this study, adult female mice were exposed to ACR at 10 mg/kg/day/body weight through their drinking water continuously for 4 weeks. Study Sample and Data Analysis: The mitochondrial function, autophagy/apoptosis, and development potential of GV oocytes were investigated. Results: The results showed that ACR reduced the oocyte diameter, sperm-binding ability, parthenogenetic activation and in vitro fertilization (IVF) rate, and development potential of pre-implantation embryos. We also found that ACR exposure disrupted chromatin configuration, mitochondrial distribution, and membrane potential (Δφm) of oocytes. Actin filament expression was significantly reduced in both the membrane and cytoplasm of mouse oocytes. Moreover, ACR exposure increased LC3-positive signals, early apoptosis rate, aberrant ATG3, ATG5, LC3, Beclin1, and mTOR mRNA expression. Conclusions: These results suggest that ACR exposure can affect the developmental potential of GV oocytes by inducing mitochondrial dysfunction, actin filament assembly, and autophagy/apoptosis.

2,4-DCP compromises the fertilization capacity of mouse oocytes.

2,4-DCP (2,4-dichlorophenol) is an environmental estrogen that is ubiquitously distributed in the environment and widely used to produce herbicides and pharmaceutical intermediates. Although 2,4-DCP is suspected to have endocrine disruption, the reproductive toxicity of 2,4-DCP in mammals has not been adequately assessed. In the present study, we examined the effect of 2,4-DCP on the fertility of mouse eggs. The data showed that oral administration of 2,4-DCP (180 mg/kg/day for 7 days) compromises the fertilization rate of mouse oocytes. To further analyze the mechanism by which 2,4-DCP decreases fertilization, the key regulators and events during fertilization of mouse eggs were investigated. We found that the dynamics of cortical granules (CGs) were disrupted by showing the redistribution of CG free domain in 2,4-DCP-administered oocytes. This abnormality perturbed the sperm binding site in the zona pellucida (ZP) and dramatically reduced the number of sperm binding to the ZP of 2,4-DCP-administered oocytes. In addition, the abundance of Juno, a sperm receptor on the egg membrane, was also decreased and its distribution was mislocated in 2,4-DCP-administered oocytes. Finally, we validated that the defects of fertilization participants and events caused by 2,4-DCP might be mediated by the increased level of reactive oxygen species-induced apoptosis of oocytes. Therefore, we demonstrate that 2,4-DCP compromises the fertilization ability of mouse oocytes via inducing oxidative stress.

Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model.

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically ß-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Methyl-beta cyclodextrin and creatine work synergistically under hypoxic conditions to improve the fertilization ability of boar ejaculated sperm.

Although successful fertilization is completed by only 150 sperm in the pig oviduct, more than 50,000 sperms are required to achieve a fertilization rate of more than 70% by pig in vitro fertilization (IVF). In this study, to improve the efficiency of pig IVF, the effects of hypoxic conditions and treatment with creatine and methyl-beta cyclodextrin (MßCD) on the glycolytic pathway were investigated. Under low O2 conditions, zig-zag motility was strongly induced within 30 min; however, the induction disappeared at 60 min. Although caffeine suppressed zig-zag motility under low O2 conditions, creatine induced and sustained zig-zag motility until 120 min. Additionally, pretreatment with MßCD for 15 min greatly enhanced zig-zag motility via ATP production in sperm incubated with creatine under low O2 conditions. Sperm pretreated with MßCD were used for IVF in medium containing creatine under low O2 conditions. A fertilization rate of approximately 70% was achieved with only 1.0 x 104 sperms/mL, and there were few polyspermic embryos. Therefore, our novel method was beneficial for efficient production of pig embryos in vitro. Moreover, the zig-zag motility may be a novel movement which boar capacitated sperm exhibit in the culture medium.

The extracellular calcium-sensing receptor promotes porcine egg activation via calcium/calmodulin-dependent protein kinase II.

Extracellular calcium is required for intracellular Ca2+ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium-sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R-568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R-568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation-dependent [Ca2+ ]i rise. When egg activation was conducted in intracellular Ca2+ chelator BAPTA-AM and NPS R-568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca2+ ]i effector p-CAMKII, and the presence of KN-93, an inhibitor of CAMKII, significantly reduced the CASR-mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca2+ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.

Progesterone induces sperm release from oviductal epithelial cells by modifying sperm proteomics, lipidomics and membrane fluidity.

The sperm reservoir is formed after insemination in mammals, allowing sperm storage in the oviduct until their release. We previously showed that physiological concentrations of progesterone (P4) trigger in vitro the sperm release from bovine oviductal epithelial cells (BOECs), selecting a subpopulation of spermatozoa with a higher fertilizing competence. Here, by using Western-Blot, confocal microscopy and Intact Cell MALDI-TOF-Mass Spectrometry strategies, we elucidated the changes derived by the P4-induced release on sperm cells (BOEC-P4 spz). Our findings show that, compared to controls, BOEC-P4 spz presented a decrease in the abundance of Binder of Sperm Proteins (BSP) -3 and -5, suggesting one mechanism by which spermatozoa may detach from BOECs, and thus triggering the membrane remodeling with an increase of the sperm membrane fluidity. Furthermore, an interesting number of membrane lipids and proteins were differentially abundant in BOEC-P4 spz compared with controls.

Porcine IVF embryo development and estrogen receptors are influenced by the concentration of percoll gradients during sperm selection.

This study aimed to evaluate the effect of three different concentrations of discontinuous gradients of percoll (90/45, 80/40, and 70/35) in the outcome of porcine in vitro fertilization (IVF) and its influence on further embryo development and quality. Embryo viability was assessed by the expression of estrogen receptors (E2 R) and cleaved caspase-3 (CC3). The highest percoll concentration (90/45) resulted in the lowest embryo production (24.9%) in comparison with 80/40 (37.5%) and 70/35 (40.0%), with the production being similar between the two lowest concentrations. The hatching rate for 90/45 (26.2%) was lower than for 80/40 (45.5%), and both were similar to the Group 70/35 (32.9%). The hatched embryos from the concentration 90/45 showed the lowest proportion of E2 R expression (3.6%), while the Groups 80/40 (22.6%) and 70/35 (39.3%) had a similar proportion of expression. The live embryos that did not hatch until Day 8 of culture presented a higher CC3 proportion for Group 90/45 (18.3%), in comparison with 80/40 (12.7%) and 70/35 (10.7%), with the latter two being similar. In conclusion, adjustments in percoll concentration used for sperm selection before porcine IVF can improve embryo production and competence for pregnancy recognition and establishment.

Melatonin rescues impaired penetration ability of human spermatozoa induced by mitochondrial dysfunction.

Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also 'rescued' the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.

Vitamin C protects carboplatin-exposed oocytes from meiotic failure.

CBP (carboplatin) is a second-generation chemotherapeutic drug of platinum compound commonly applied in the treatment of sarcomas and germ cell tumours. Although it is developed to replace cisplatin, which has been proven to have a variety of side effects during cancer treatment, CBP still exhibits a certain degree of toxicity including neurotoxicity, nephrotoxicity, hematotoxicity and myelosuppression. However, the underlying mechanisms regarding how CBP influences the female reproductive system especially oocyte quality have not yet been fully determined. Here, we report that CBP exposure led to the oocyte meiotic defects by impairing the dynamics of the meiotic apparatus, leading to a remarkably aberrant spindle organisation, actin polymerisation and mitochondrial integrity. Additionally, CBP exposure caused compromised sperm binding and fertilisation potential of oocytes by due to an abnormal distribution of cortical granules and its component ovastacin. More importantly, we demonstrated that vitamin C supplementation prevented meiotic failure induced by CBP exposure and inhibited the increase in ROS levels, DNA damage accumulation and apoptotic incidence. Taken together, our findings demonstrate the toxic effects of CBP exposure on oocyte development and provide a potential effective way to improve the quality of CBP-exposed oocytes in vitro.

Melatonin protects against Fenoxaprop-ethyl exposure-induced meiotic defects in mouse oocytes.

Prolonged exposure of Fenoxaprop-ethyl (FE), a post-emergence herbicide, can cause serious damage to animals through food chain. Melatonin is synthesized by the pineal gland in mammals and believed to protect cells from oxidative stress damage. In this study, we aimed to investigate the effects of FE on mouse oocyte meiosis maturation and the protective roles of melatonin on FE-exposed oocytes by in vitro maturation model. FE exposure significantly caused defects of the first polar body extrusion, which could be protected by co-culture with melatonin. Furthermore, we examined the meiotic maturation details by performing the sperm binding, actin and tubulin immunofluorescence, ROS and apoptosis detection, and histone methylation assay. Our data showed that FE exposure to oocytes led to disrupted actin filament dynamics, mis-organized spindle, and reduced the sperm binding capacity. In addition, FE-exposure increased oxidative stress level and induced oocyte apoptosis. We also found that FE exposure resulted in histone methylation changes. Treatment with melatonin could significantly improve these phenotypes in oocytes exposed to FE. In conclusion, FE exposure can cause meiotic defects by disrupting the cytoskeletal integrality and inducing excessive ROS accumulation to initiate apoptosis in oocytes, while melatonin can reduce all these damages, suggesting that melatonin has protective effects on oocytes exposed to FE during meiotic maturation.

The importance of trace minerals copper, manganese, selenium and zinc in bovine sperm-zona pellucida binding.

SummarySperm-zona pellucida (ZP) binding is a necessary event for successful fertilization. The aim of this study was to determine the effect of trace minerals such as copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) on bovine spermatozoa binding to ZP. Sperm viability, functional membrane integrity, acrosomal status (AS), total antioxidant capacity (TAC) and sperm lipid peroxidation (LPO) were also evaluated. For the present study, in vitro fertilization (IVF) medium was supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), all minerals (Cu+Mn+Se+Zn), or tested without supplement (Control). Considerably more sperm bound to ZP when Cu, Se or Zn were added to the IVF medium, but there were no difference compared with the Control, Mn and Cu+Mn+Se+Zn groups. After 1 h of incubation, viability was increased by the addition of Cu, Mn and Se with respect to the Control but, after 2 h, viability was higher only with the addition of Mn to IVF medium. Functional membrane integrity improved in sperm treated with Cu. Acrosome integrity was higher in sperm treated with Zn after 1 h of incubation. LPO was significantly higher in sperm treated with Cu or Cu+Mn+Se+Zn. The mean TACs of sperm treated with Cu, Mn, Zn or Cu+Mn+Se+Zn were lower than in the Control. In conclusion, the results obtained in the present study determined that the presence of Cu, Se and Zn in the IVF medium increased the number of spermatozoa bound to the ZP, highlighting the importance of these minerals in the fertilization process.

Effects of dietary curcumin supplementation on seminal quality indices and fertility rate in broiler breeder roosters.

1. Decreased semen quality is an underlying contributor to age-related subfertility in broiler breeder roosters. This study investigated the effects of dietary curcumin (derived from turmeric) supplementation as an antioxidant source on semen quality and fertility in broiler breeder roosters. 2. Twenty-eight Ross 308 roosters were randomly allotted to four groups with seven birds in each and were fed a standard diet supplemented with different levels of curcumin at 0 (C0), 10 (C10), 20 (C20) and 30 (C30) mg/bird per day from 48 through to 61 weeks of age. Body weight and semen quality traits were evaluated on a weekly basis and seminal concentrations of malondialdehyde (MDA) as a measure of antioxidation status were quantified at one-week intervals during the first 11 weeks of the trial (48-59 weeks of age). Semen samples from last 2 weeks (60 and 61 weeks of age) were used to artificially inseminate to assess the sperm-egg penetration (SP) in perivitelline membrane and fertility rates. 3. Except for body weight and ejaculate volume, other characteristics, including semen concentration, total sperm production, progressive motility and plasma membrane integrity were linearly improved by the increasing levels of curcumin supplementation (P < 0.01). However, dietary curcumin levels were linearly and quadratically associated with decreased seminal concentration of MDA (P < 0.01 and P < 0.03), percentage of abnormal sperm (P < 0.01 and P < 0.07) and increased plasma membrane functionality (P < 0.01 and P < 0.04), respectively. The SP holes in perivitelline membrane were increased in a linear and quadratic manner in response to increasing levels of curcumin (P < 0.01). Moreover, fertility rate was linearly improved (P < 0.01) as the dosage of curcumin increased, and resulted in 8, 12 and 14% improvements in the birds fed C10, C20 and C30, compared to C0, respectively. 4. In conclusion, the results showed that increasing levels of dietary supplementation of curcumin was associated with beneficial effects on semen quality indices and fertility rate in aged broiler breeder roosters.

Bovine epididymal spermatozoa treatment for in vitro fertilization: Heparin accelerates fertilization and enables a reduction in coincubation time.

This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P<0.05) than that in the EJ (19%) and EP- (42%) groups. At 12 and 18 h, FR remained higher in the EP+ group, and a gradual increase in polyspermy was observed. The use of a P or PS gradient yielded a similar BR on D7 (54% and 52%), which was higher than the rate obtained using the washing method (37%). The embryos produced by EP and selected in a P or PS gradient resulted in a sex deviation in favor of male embryos (P>0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient.

Acrosome reaction-inducing substance triggers two different pathways of sperm intracellular signaling in newt fertilization.

The acrosome reaction is induced in the sperm of Cynops pyrrhogaster immediately in response to a ligand protein called acrosome reaction-inducing substance (ARIS) in the egg jelly at fertilization, whereas a spontaneous acrosome reaction occurs time-dependently in correlation with the decline of sperm quality for fertilization. The ARIS-induced acrosome reaction was recently found to be mediated by TRPV4 in association with the NMDA type glutamate receptor, although the intracellular mediators for the acrosome reaction are largely unclear. In the present study, spontaneous acrosome reaction was significantly inhibited by Ni2+, RN1734, and diltiazem, which blocks Cav3.2, TRPV4 or TRPM8, and the cyclic nucleotide-gated channel, respectively. In contrast, expression of Ca2+-activated transmembrane and soluble adenylyl cyclases was detected in the sperm of C. pyrrhogaster by reverse transcription-polymerase chain reaction. Activator of transmembrane or soluble adenylyl cyclases (forskolin or HCO 3-) independently promoted spontaneous acrosome reaction, while an inhibitor of each enzyme (MD12330A or KH7) inhibited it only in the sperm with high potential for spontaneous acrosome reaction. An inhibitor of protein kinase A (H89) inhibited spontaneous acrosome reaction in a manner independent of sperm potential for spontaneous acrosome reaction. Surprisingly, KH7 significantly inhibited ARIS-induced acrosome reaction, but its effect was seen in a small percentage of sperm. H89 had no effect on ARIS-induced acrosome reaction. These results suggest that C. pyrrhogaster sperm possess multiple intracellular pathways for acrosome reaction, involving Ca2+ permeable channels, adenylyl cyclases and PKA, and that two pathways having distinct dependencies on adenylyl cyclases may contribute to ARIS-induced acrosome reaction at fertilization.

JUNO, the receptor of sperm IZUMO1, is expressed by the human oocyte and is essential for human fertilisation.

STUDY QUESTION: Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process? SUMMARY ANSWER: JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion. WHAT IS KNOWN ALREADY: Fusion of gamete membranes is the culminating event of the fertilisation process, but its molecular mechanisms are poorly understood. Until now, three molecules have been shown to be essential: CD9 tetraspanin in the oocyte, Izumo1 protein on the sperm and Juno, its corresponding receptor on the oocyte. Oocyte CD9 and sperm IZUMO1 have been identified in human gametes and their interaction is also well-conserved among several mammalian species. The presence of JUNO on human oocytes, however, has not yet been reported, nor has its role in fertilisation been investigated. STUDY DESIGN, SIZE, DURATION: We selected an anti-human JUNO antibody in order to investigate the presence of JUNO on the oocyte membrane surface and studied its potential involvement in gamete membrane interaction during fertilisation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Monoclonal antibodies against human JUNO (anti-hJUNO mAb) were produced by immunisation of mice with HEK cells transfected with the putative human JUNO sequence (HEK-hJUNO). These antibodies were used for immunostaining experiments and in vitro fertilisation assays with human gametes (GERMETHEQUE Biobank). MAIN RESULTS AND THE ROLE OF CHANCE: Three hybridoma supernatants, verified by immunostaining, revealed specifically HEK-hJUNO cells. The three purified monoclonal antibodies, FJ2E4 (IgG1), FJ8E8 (IgG1) and FJ4F5 (IgG2a), recognised the soluble recombinant human JUNO protein and, in a western blot of HEK-hJUNO extracts, a protein with an expected MW of 25 kDa. In addition, soluble recombinant human IZUMO protein inhibited the binding of anti-hJUNO mAbs to cells expressing hJUNO. Using these anti-hJUNO mAbs in immunostaining, we identified the presence of JUNO protein at the plasma membrane of human oocytes. Furthermore, we revealed a progressive expression of JUNO according to oocyte maturity. Finally, we showed that human zona-free oocytes, inseminated in the presence of anti-hJUNO mAb, were not fertilised by human sperm. These results suggest that, as seen in the mouse, JUNO is indeed involved in human gamete membrane fusion during fertilisation. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In accordance with French bioethics laws, functional tests were performed using zona-free oocytes, which of course does not fully encompass all normal in vivo physiological conditions. However, these in vitro tests do provide direct information regarding sperm-oocyte membrane interactions. WIDER IMPLICATIONS OF THE FINDINGS: Mechanisms of gamete fusion appear to be homologous between mice and humans. However, some differences do exist and analysing the human mechanisms is essential. In fact, this is the first report describing the presence of JUNO on human oocytes and its involvement in human fertilisation. This discovery allows further examination of the understanding of molecular mechanisms that drive gamete fusion: a crucial challenge at a time when infertility affects 16% of reproductively active couples. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Agence Nationale pour la Recherche, Grant no. ANR-13-BVS5-0004, and by Association Institut du Cancer et d'Immunogénétique (ICIG). There are no competing interests.

Effects of chlorogenic acid and caffeic acid on the quality of frozen-thawed boar sperm.

Chlorogenic acid (CGA) and caffeic acid (CA) are potent antioxidants that are mostly found in coffee beans. This study aimed to investigate the effects of CGA and CA supplementation during semen freezing on the quality of frozen-thawed boar spermatozoa. The antioxidants CGA and CA were added to a semen extender to achieve final concentrations of 50, 100, 200 and 400 µM. Supplementation of 100 µM CGA and CA yielded a significantly higher percentage of sperm viability (increased by 8%-10%) and plasma membrane integrity (increased by 4%-6%) than the control groups without the antioxidants at 0 and 3 hr after thawing (p < 0.05). At a concentration of 100 µM, CGA and CA also yielded beneficial effects on total and progressive sperm motility. Increases of CGA and CA concentrations to more than 200 µM did not enhance any sperm quality parameters. When the sperm penetrability and oocyte development by spermatozoa frozen with CGA and CA were evaluated, CGA and CA supplementations had no positive effects on the percentages of total fertilization, monospermic fertilization, cleavage and blastocyst formation. In conclusion, the supplementation of 100 µM CGA and CA during sperm freezing improved certain sperm parameters including motility, viability and plasma membrane integrity.

Progesterone stimulates the long-distance migration of capacitated ram spermatozoa through viscous media under geotactic condition.

Forward progressive motility of spermatozoa is an essential prerequisite for reproductive success, and sperm navigation is assisted by guidance mechanisms that may depend on micro-environmental factors. In the present study, we performed an integrated analysis of long-distance ram sperm migration in vitro that combined two environmental factors (10 µM progesterone and a geotactic effect) and the physiological status of the cells (capacitation treatment). A penetration assay was used in which spermatozoa had to travel 20 mm in a viscous medium (two media of differing viscosity: acrylamide and hyaluronic acid) through a tube device. The number of migrating spermatozoa, the physiology of the cells (motility analyzed using a CASA system; acrosomal status, viability and active mitochondria evaluated by flow cytometry; DNA fragmentation index calculated by quantitative PCR) and the morphometry of sperm heads (performed using an image analysis system) were evaluated after long-distance sperm migration. Ram sperm capacitation significantly stimulates cell migration through viscous media under geotactic conditions, and this effect is enhanced by progesterone induction. The rheological characteristics of viscous media have a marked impact on ram sperm migration, and acrylamide more favorably facilitates navigation over a large distance. The migrating spermatozoa are morphologically better adapted (high ellipticity) for displacement in viscous media and exhibit remarkably depleted mitochondrial membrane potential.

Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ.

In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/µl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

Chemoenzymatic Synthesis of Asymmetrical Multi-Antennary N-Glycans to Dissect Glycan-Mediated Interactions between Human Sperm and Oocytes.

Complex N-glycans of glycoproteins of the zona pellucida (ZP) of human oocytes have been implicated in the binding of spermatozoa. The termini of these unusual bi-, tri-, and tetra-antennary N-glycans consist of the tetrasaccharide sialyl-Lewisx (SLex ), which was previously identified as the minimal epitope for sperm binding. We describe here the chemoenzymatic synthesis of highly complex triantennary N-glycans derived from ZP carrying SLex moieties at the C-2 and C-2' arm and a sialyl-Lewisx -Lewisx (SLex -Lex ) residue at the C-6 antenna and two closely related analogues. The compounds were examined for their ability to inhibit the interaction of human sperm to ZP. It was found that the SLex -Lex moiety is critical for inhibitory activity, whereas the other SLex moieties exerted minimal effect. Further studies with SLex -Lex and SLex showed that the extended structure is the more potent inhibitor. In addition, trivalent SLex -Lex and SLex were prepared which showed greater inhibitory activity compared to their monovalent counterparts. Our studies show that although SLex can inhibit the binding of spermatozoa, presenting this epitope in the context of a complex N-glycan results in a loss of inhibitory potential, and in this context only SLex -Lex can make productive interactions. It is not the multivalent display of SLex on a multi-antennary glycan but the presentation of multiple SLex -Lex on the various glycosylation sites of ZP that accounts for high avidity binding.