Induction Of Sperm Capacitation Research Articles

394 Young Scholar Award Talk: Evaluation of new Markers to Predict Sperm Longevity and Bull Fertility

Abstract Bull fertility is currently evaluated through a breeding soundness exam (BSE) that can detect infertile/sterile bulls with high accuracy; however, subfertile bulls are often classified as satisfactory breeders. In the first study, Angus bulls (n=5) were used to evaluate the effect of different insemination doses (10, 20 and 40 million sperm/dose) on pregnancy/fixed-time AI (P/FTAI; cows n=4,866). The interaction between insemination dose and bull was not significant (P=0.53), and there was no effect of insemination dose on P/FTAI (P=0.31); however, there was an effect of bull on P/FTAI (P <0.01). Two bulls had high, one bull had average, and two bulls had low fertility. Post-thaw sperm analysis was performed by computer assisted sperm analysis (CASA) and flow cytometry; however, analysis performed could not explain field fertility differences. In a second study, Angus and Angus crosses bulls were used to investigate differences in sperm longevity between epididymal and ejaculated sperm. Epididymal and ejaculated semen was incubated at pH 5.8, 6.8, or 7.3. Proteins in epididymal fluid, seminal plasma, and proteins loosely attached to epididymal and ejaculated sperm were also investigated. It was identified that epididymal sperm maintained viability longer compared to ejaculated sperm regardless of media pH (P <0.05). Ejaculated sperm had increased longevity (total motility >20%) at pH 6.8 compared to pH 5.8 or 7.3. There were 458 unique proteins between all samples; 311 and 178 in epididymal fluid and seminal plasma, respectively, and 334 and 298 on epididymal and ejaculated sperm, respectively. Based on comparative proteomics, epididymal sperm energy metabolism is more glycolytic compared to ejaculated sperm. In addition, there were greater numbers of antioxidants available for epididymal sperm likely to maintain reactive oxygen species (ROS) at low concentrations to inhibit premature sperm activation. From this dataset, three proteins that are associated with cell-to-cell interaction were studied [plasma serine protease inhibitor (SERPINA5), dystroglycan (DAG1), and CD9]. Among dairy bulls, SERPINA5 and DAG1 were not associated with field fertility; thus, they are not potential fertility markers. The protein CD9 was evaluated to determine whether induction of sperm capacitation (in vitro) could be used to estimate/predict bulls fertility differences. Semen samples from first study were evaluated for total motility (CASA), and functional sperm analysis (flow cytometry) at pre-wash, post-wash, h 0, 3, 6, and 24 of incubation. Sperm analysis were not able to estimate fertility differences between high and average fertility bulls; however, low fertility bulls had decreased (P <0.05) viability, zinc signature 2, and zinc signature1 + 2, and had increased (P <0.05) dead CD9+ sperm. In summary, the use of an insemination dose of 10-40 million sperm can generate acceptable P/FTAI, and evaluation of CD9, viability and zinc signatures may increase the industry’s ability to estimate fertility of bulls that pass a BSE.

Heat inactivated follicular fluid induces release from oviduct explants of stallion sperm primed for fertilization

To date, conventional in vitro fertilization with equine gametes has been poorly successful; this is at least in part due to inadequate induction of sperm capacitation and hyperactivation. After insemination in vivo, a sperm reservoir is established in the mare’s oviduct in anticipation of oocyte arrival. Previously we showed that oviduct-bound spermatozoa display tail-associated protein tyrosine phosphorylation, a hallmark of capacitation. The current study aimed to determine how stallion sperm could be induced to detach from their binding to oviduct explants. To this end, oviduct explant-bound stallion spermatozoa were exposed to the various fluids and cells that they may contact in the oviductal reservoir. In experiment one, groups of five sperm-oviduct explants were incubated for 2h in 45ml droplets under oil containing (1) fluid flushed from the oviducts of mares slaughtered pre-ovulation; (2) fluid flushed from post-ovulation mare oviducts; (3) 100% follicular fluid (4); 10% follicular fluid diluted in modifiedWhitten’s medium; (5) modified-Whitten’s medium containing equine cumulus cells or (6) modified-Whitten’s medium containing mature (MII) equine oocytes. In experiment two (a) 100% heat-inactivated follicular fluid and (b) 10% heat-inactivated follicular fluid in modifiedWhitten’s medium was used. Detached spermatozoa were stained to assess protein tyrosine phosphorylation (fluoroscein-conjugated anti-phosphotyrosine antibody) and acrosome status (FITC-PNA), and their motility pattern was monitored. In experiment one, only minimal sperm release (<4 spermatozoa/oviduct explant) and very few sperm

Anti‐Bacterial Factors Secreted From Cumulus Cells of Ovulated COCs Enhance Sperm Capacitation During In Vitro Fertilization

The aim of this study was to find immune-related genes expressed in cumulus cells of ovulated cumulus oocyte complexes (COCs) and to clear the functional roles during fertilization process. Ovulated COCs were collected from oviduct 16hr after the hCG injections followed by eCG priming. The cumulus cells were used for RT-PCR or western blotting study. COCs were also used for in vitro fertilization study. Cramp, Trf, Lyz2, S100a8, and S100a9 were expressed in cumulus cells during ovulation process. The protein levels of CRAMP or transferrin were detected in ovulated COCs and then secreted into hyaluronan-rich matrix. The high dose of these factors reduced the proliferative activity of E. coli; however, the lower levels of them significantly increased the rate of fertilization in in vitro via the induction of sperm capacitation. Cumulus-secreted anti-bacterial factors act on sperm to induce sperm capacitation.

Anandamide Induces Sperm Release from Oviductal Epithelia through Nitric Oxide Pathway in Bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.

The Cumulus Cell-Secreted Factor Neuregulin 1 Maintains the Cumulus Cell Function in Ovulated COCs To Enhance Fertilization and Developmental Competence in Porcine Oocytes.

It has been known that cumulus cells support oocyte meiotic and cytoplasmic maturation. Our previous study showed that the EGF like factors (amphiregulin, epiregulin and betacellulin) are expressed in granulosa cells and cumulus cells and act on cumulus cells to induce cumulus cell-oocyte complex (COC) expansion and oocyte maturation in mice and in pigs. Additionally, when ovulated mouse COCs were cultured with amphiregulin, this factor activated/phosphorylated the EGF receptor (ErbB1) and maintained cumulus expansion and the expression of specific genes when expression of endogenous amphiregulin is decreased markedly. Moreover, we have shown recently that the maintenance of the COC matrix and the secretion of specific chemokines from cumulus cells enhance successful fertilization via supporting sperm motility and induction of sperm capacitation. These data suggested that additional ligand members for the ErbB family are required to act to maintain cumulus cell function after ovulation. However, there is little information about what mechanisms regulate cumulus cells in oviductal COCs. Therefore, this study was designed to determine the kinetic expression patterns of ErbB family and the ligand in cumulus cells in vivo and in vitro, and then activate the ErbB signaling pathway to stimulate oocyte maturation in COCs in culture. For this, intact follicles and cumulus cells were collected at early, antral and ovulatory stages of follicle, or from COCs in the oviduct at specific time intervals following eCG and hCG treatment of gilts. FSH/eCG increased levels of EGF receptor and ErbB3 protein in cumulus cells, whereas ErbB2 remained constant and ErbB4 was down regulated. Immuno-histochemistry localized ErbB3 to cumulus cells after eCG and hCG stimulation. Because we found high expression of neuregulin 1 (Nrg1) mRNA in cumulus cells from our rat COC micro-array database, and because RT-PCR analyses showed that Nrg1 expression in cumulus cells of porcine COCs was increased after hCG stimulation and reached maximum levels after ovulation, we sought to determine if NGR1 might regulate cumulus cell functions via ErbB3. When porcine COCs were cultured in vitro with FSH alone, the expression of Nrg1 in cumulus cells did not increase. Therefore, to determine if NRG1 might enhance the actions of FSH, NRG1 was added to FSH-containing medium at 24 hr of culture and COC expansion and oocyte maturation were analyzed after an additional 20-hr culture (total 44 hr culture period). The addition of NRG1 strongly induced the phosphorylation of ErbB3 but not ErbB1, which resulted in the activation of both ERK1/2 and PI 3-kinase-PKB pathways in cumulus cells. The COCs exhibited improved cumulus cell survival (no apoptosis) and sustained expansion. The developmental competence of fertilized oocytes to the blastocyst stage was also enhanced by NGR1. From these results, we show for the first time that NRG1 is a novel COC secreted EGF-like factor that acts via autocrine mechanisms to activate ErbB3 expressed on cumulus cells within COCs after ovulation, and thereby helps maintain cumulus cell function to improve oocyte developmental competence, maybe due to suppress oocyte aging. Supported in part by JSPS-18688016 (MS). (poster)

Effects of epigallocatechin-3-gallate (EGCG) on in vitro maturation and fertilization of porcine oocytes

The beneficial properties of green tea and especially of its principal active polyphenol, epigallocatechin-3-gallate (EGCG), have led to an increased demand for dietary supplements with highly enriched EGCG concentrations. In order to investigate the possible reproductive-related consequence of EGCG supplementation, the effects of this catechin on in vitro maturation (IVM) and fertilization (IVF) of oocyte, using the pig as experimental model, were examined. In the first series of experiments EGCG, at concentrations ranging from 0 to 25 microg/ml, was added during in vitro maturation of pig oocytes. EGCG had no effect on nuclear maturation of pig oocytes and on fertilization traits considered after IVF at any of the doses tested. By contrast, a significant (p<0.05) decrease in the number of embryos that developed to blastocysts following parthenogenetic activation was recorded when 25 microg/ml EGCG was added to IVM medium; in addition this catechin concentration significantly (p<0.05) inhibited progesterone production by cumulus cells after 48 h of culture. When induction of sperm capacitation was performed in presence of EGCG, a significantly lower percentage of spermatozoa showing a Hsp70-capacitated pattern and a significant reduction of sperm H(2)O(2) production were evident at a concentration of 25 microg/ml EGCG (p<0.05). During gamete coincubation EGCG reduced, in a dose response manner, the number of reacted spermatozoa suspended in fertilization medium and increased the number of sperm bound to ZP. Supplementation of 10 microg/ml EGCG during IVF significantly increased the fertilization rate while higher EGCG concentrations (25 microg/ml) decreased the percentage of fertilized oocytes (p<0.05). In conclusion, our data suggest that high EGCG concentrations could affect in vitro maturation and fertilization in pig; it cannot be totally excluded that excessive EGCG concentrations could induce reproductive-related consequences also in vivo.

Effect of Monosaccharide L-fucose and Polysaccharide Fucoidan on Sperm ??L-fucosidase Activity and Relation to Sperm-oocyte Interaction in Pig

Carbohydrate-protein interactions are known to be important in gamete interactions. Several evidence indicated that a fucose-containing sulfated polysaccharide fucoidan was potential inhibitor of fertilization in vitro and thus fucose seemed to be part of the recognition signal of gamete interaction in mammals. In recent investigation we found that α-L-fucosidase activity was present in boar spermatozoa and it was related to sperm binding to and penetration into zona pellucida (ZP) in vitro. The objective of this study was to determine the effects of monosaccharide L-fucose and polysaccharide fucoidan on sperm α-L-fucosidase activity and relation to sperm-oocyte interaction in pig. Results indicated that the activity of sperm α-L-fucosidase was largely inhibited (62%) when sperm suspension was treated with monosaccharide L-fucose. It also significantly inhibited the number of sperm binding to ZP (32%) and penetration into zona-intact oocytes (72%), but did not inhibit penetration into zona-free oocytes when fertilization medium contained L-fucose. The chlorotetracycline (CTC) assessment showed that L-fucose did not affect induction of sperm capacitation and acrosome reaction. In contrast, the activity of sperm α-L-fucosidase was not inhibited when sperm suspension was treated with polysaccharide fucoidan but sperm-ZP binding was greatly inhibited (85%) and completely blocked sperm penetration into zona-intact or zona-free oocytes. The CTC assessment showed that fucoidan increased the F pattern and decreased the AR pattern sperm. These results suggested that the different inhibitory mechanisms were present between monosaccharide L-fucose and polysaccharide fucoidan on sperm-oocyte interaction, the inhibition effect of α-L-fucose on sperm binding and penetrating into ZP caused sperm α-L-fucosidase inhibited by α-L-fucose.

Epithelial cell coculture and the induction of sperm capacitation: Kervancioglu M.E.; Djahanbakhch O.; Aitken R.J. GBR Fertil Steril 1994 61/6 (1103–1108)

Epithelial cell coculture and the induction of sperm capacitation: Kervancioglu M.E.; Djahanbakhch O.; Aitken R.J. GBR Fertil Steril 1994 61/6 (1103–1108)

Epithelial cell coculture and the induction of sperm capacitation

To investigate the influence of cultured human oviductal epithelial cells on the movement characteristics of human spermatozoa. Human spermatozoa were cultured with monolayers of human epithelial or Vero cells or conditioned media derived from these cell types. The viability and movement characteristics of the cells was subsequently analyzed at 4, 24, and 48 hours. University hospital and Medical Research Council laboratories. Volunteer donors. Movement characteristics of human spermatozoa. The presence of both Vero and oviductal epithelial cells, but not conditioned media, had a general promoting effect on sperm survival, significantly enhancing sperm viability and motility for up to 48 hours of culture. In addition, the presence of oviductal epithelial cells had a specific, significant stimulatory effect on sperm capacitation, enhancing the incidence of hyperactivated motility after 4, 24, and 48 hours of culture. Significantly, this effect was not observed with cocultures containing Vero cells. The coincubation of human spermatozoa with human oviductal epithelial cells provides a convenient system for the induction and analysis of sperm capacitation.