Pilar Coy

Oviduct-specific glycoprotein and heparin modulate sperm–zona pellucida interaction during fertilization and contribute to the control of polyspermy

Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm–oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con- and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of ≈1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm–ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OF-exposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoprotein–heparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy.

Roles of the oviduct in mammalian fertilization

The oviduct or Fallopian tube is the anatomical region where every new life begins in mammalian species. After a long journey, the spermatozoa meet the oocyte in the specific site of the oviduct named ampulla and fertilization takes place. The successful fertilization depends on several biological processes that occur in the oviduct some hours before this rendezvous and affect both gametes. Estrogen and progesterone, released from the ovary, orchestrate a series of changes by genomic and nongenomic pathways in the oviductal epithelium affecting gene expression, proteome, and secretion of its cells into the fluid bathing the oviductal lumen. In addition, new regulatory molecules are being discovered playing important roles in oviductal physiology and fertilization. The present review tries to describe these processes, building a comprehensive map of the physiology of the oviduct, to better understand the importance of this organ in reproduction. With this purpose, gamete transport, sperm and oocyte changes in the oviductal environment, and other interactions between gametes and oviduct are discussed in light of recent publications in the field.

Susceptibility of Bovine Germinal Vesicle-Stage Oocytes from Antral Follicles to Direct Effects of Heat Stress In Vitro

Abstract Delineation of maternal versus direct effects of heat stress in reducing development at the germinal vesicle (GV) stage is challenging, because oocytes spontaneously resume meiosis after removal from antral follicles. The use of S-roscovitine (inhibitor of p34cdc2/cyclin B kinase) to hold bovine oocytes at the GV stage without compromising early embryo development was previously validated in our laboratory. The objective of the present study was to assess the direct effects of an elevated temperature commonly seen in heat-stressed dairy cows on cumulus-oocyte complexes (COCs) held at the GV stage using 50 μM S-roscovitine. During roscovitine culture, GV-stage COCs (antral follicle diameter, 3–8 mm) were cultured at 38.5 or 41°C. Thereafter, oocytes were removed from roscovitine medium and allowed to undergo in vitro maturation, fertilization, and culture. Zona pellucida hardening (solubility to 0.5% pronase), nuclear stage (Hoechst 33342), cortical granule type (lens culinaris agglutinin-fluorescein isothiocyanate [FITC]), and early embryo development were evaluated. Culture of GV-stage COCs at 41°C increased the proportion that had type III cortical granules and reduced the proportion that progressed to metaphase II after in vitro maturation. Effects of 41°C on zona pellucida hardening, fertilization (penetration, sperm per oocyte, pronuclear formation, and monospermic and putative embryos), and cleavage of putative zygotes were not noted. However, culture of GV-stage COCs at 41°C for 6 h decreased the proportion of 8- to 16-cell embryos, whereas 41°C for 12 h reduced blastocyst development. In summary, antral follicle COCs are susceptible to direct effects of elevated body temperature, which may account in part for reduced fertility in heat-stressed cows.

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1 x 10(6) to 10 x 10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.

Characteristics and seasonal variations in the semen of Murciano-Granadina goats in the Mediterranean area

An experiment was carried out in the Murcia valley in the Mediterranean basin, with nine 13-month-old Murciano-Granadina male goats to determine the characteristics and seasonal variations of semen collected twice a week with an artificial vagina. The semen was characterized by an ejaculate volume of 1.05±0.01 ml (mean±SE), and a concentration of (3.49±0.04) × 109 sperm ml−1. The total number of spermatozoa per ejaculate was (3.63±0.06) × 109; 86.81±0.52% were active, 7.91±0.21% had abnormal morphology and 6.91±0.15% had a damaged acrosome. Seasonal differences (P<0.05) were observed for all semen parameters studied. Semen of superior quality was collected in the latter half of summer and throughout the autumn. The magnitude of these seasonal effects, however, was not sufficient to prevent the bucks being used for breeding throughout the year.

Hardening of the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in the pig and cow.

One of the proposed mechanisms of polyspermy block is an increased resistance of the zona pellucida (ZP) to proteolytic digestion (ZP hardening) as a consequence of cortical granule exocytosis that occurs soon after fertilization. However, evidence is available that the zonae pellucidae of freshly ovulated pig and cow oocytes harden considerably before fertilization. It was thought that such pre-fertilization ZP hardening could be involved in the control of polyspermy, and its lack in the oocytes matured in vitro could be one of the reasons for the extremely high incidence of polyspermy in pig in vitro fertilization (IVF). To test this hypothesis, two different types of cross-linking reagents were employed and their effects on ZP hardening and IVF efficiency were examined. The sulfhydryl-reactive cross-linkers produced a slight hardening of ZP (P<0.001) of treated oocytes compared with control oocytes, and totally inhibited sperm penetration into pig oocytes after IVF. In the cow, sperm penetration into eggs was reduced to 10%. It is proposed that formation of disulfide bonds in ZP or blocking of SH groups in the oocyte plasma membrane proteins prevents sperm penetration. An amine-reactive cross-linker was then assayed and produced strong ZP hardening, increasing the incidence of monospermy in both pig and cow oocytes after fertilization. When the cross-linker concentration was optimized, a 45% improvement for pig IVF efficiency was reached. It is proposed that the observed physiological ZP hardening is a mechanism to control polyspermy, differentially affecting various mammalian species and can be imitated by the use of amine-reactive cross-linkers during IVF.

In vitro production of pig embryos: a point of view

Porcine embryos have become raw materials for different programmes of reproductive biotechnology and the in vitro production of embryos has some advantages over in vivo production in gene transfer programmes and for xenotransplantation. Despite this promising future, several problems limit the success of the in vitro production (IVP) of viable porcine embryos. Porcine IVP has not been fully developed because of several problems associated with different techniques, such as incomplete final maturation status after in vitro maturation, a high incidence of polyspermy after in vitro fertilization and a low development rate and poor quality of blastocysts at the end of culture. The results could be improved with studies comparing in vivo and in vitro conditions, standardization of techniques for sperm processing, testing new additives in the culture media and developing intracytoplasmic sperm injection procedures. The first objective of the present article is to summarize the main studies published on the subject. Second, we provide a guide for researchers starting work on the IVP of pig embryos, making special mention of first papers and the most recent achievements for each of the different techniques. Third, we provide suggestions for future experiments designed to improve the results of each technique.

Effects of oviductal fluid on the development, quality, and gene expression of porcine blastocysts produced in vitro

In mammals, fertilization and early pre-implantation development occur in the oviduct. Previous results obtained in our laboratory have identified specific molecules in the oviduct that affect porcine sperm-egg interactions. The aim of the present study was to determine whether the contact between oocytes and oviductal fluid also affect embryo development, quality, and gene expression. In vitro matured porcine oocytes were exposed to bovine oviductal fluid (bOF) for 30 min prior to fertilization. Cleavage and blastocyst development rates were significantly higher from bOF-treated oocytes than from untreated oocytes. Blastocysts obtained from bOF-treated oocytes had significantly greater total cell numbers than those obtained from untreated oocytes. Using real-time PCR, grade 1 (very good morphological quality) and grade 2 (good morphological quality) blastocysts were analyzed for gene transcripts related to apoptosis (BAX, BCL2L1), mitochondrial DNA (mtDNA) transcription/replication (POLG, POLG2, and TFAM), blastomere connection and morula compaction (GJA1), and blastocyst formation and pluripotency (POU5F1). We found that the entire set of genes analyzed was differentially expressed between grade 1 and 2 blastocysts. Furthermore, bOF treatment reduced the ratio of BAX to BCL2L1 transcripts and enhanced the abundance of TFAM transcripts in grade 2 blastocysts. Not only do these findings demonstrate that factors within the bOF act on porcine oocytes both quickly and positively, but they also suggest that such factors could promote embryo development and quality by protecting them against adverse impacts on mtDNA transcription/replication and apoptosis induced by the culture environment.

What controls polyspermy in mammals, the oviduct or the oocyte?

A block to polyspermy is required for successful fertilisation and embryo survival in mammals. A higher incidence of polyspermy is observed during in vitro fertilisation (IVF) compared with the in vivo situation in several species. Two groups of mechanisms have traditionally been proposed as contributing to the block to polyspermy in mammals: oviduct‐based mechanisms, avoiding a massive arrival of spermatozoa in the proximity of the oocyte, and egg‐based mechanisms, including changes in the membrane and zona pellucida (ZP) in reaction to the fertilising sperm. Additionally, a mechanism has been described recently which involves modifications of the ZP in the oviduct before the oocyte interacts with spermatozoa, termed “pre‐fertilisation zona pellucida hardening”. This mechanism is mediated by the oviductal‐specific glycoprotein (OVGP1) secreted by the oviductal epithelial cells around the time of ovulation, and is reinforced by heparin‐like glycosaminoglycans (S‐GAGs) present in oviductal fluid. Identification of the molecules contributing to the ZP modifications in the oviduct will improve our knowledge of the mechanisms of sperm‐egg interaction and could help to increase the success of IVF systems in domestic animals and humans.

Determination of glycosidase activity in porcine oviductal fluid at the different phases of the estrous cycle.

Sperm-oocyte binding and gamete-oviductal epithelium interactions are carbohydrate-mediated events occurring in the oviductal fluid (OF). Thus, knowledge about the activities of glycosidases (enzymes catalyzing hydrolytic cleavage of terminal sugar residues) in this milieu would help us understand the molecular mechanisms involved in these events. This work was carried out to investigate the glycosidase activity, protein content, and volume of OF collected from gilts and sows. Oviducts were classified into four phases of the estrous cycle (early follicular, late follicular, early luteal, and late luteal) based on the appearance of the ovaries. OF was aspirated, centrifuged, measured for volume, and frozen until assay. Substrates conjugated to 4-methylumbelliferyl were used to screen the activities of seven different glycosidases at physiological pH (7.2). alpha-L-Fucosidase and beta-N-acetyl-glucosaminidase activities increased at the late follicular phase to decrease after ovulation. beta-D-Galactosidase, alpha-D-mannosidase, and beta-N-acetyl-galactosaminidase showed higher activities at the early follicular phase, which decreased after ovulation. N-Acetyl-neuraminidase and alpha-D-galactosidase did not show activity at any phase of estrous cycle neither in sows nor in gilts at pH 7.2, although it did at acidic pH (4.4) in the follicular and luteal phase samples. Total protein also changed during the cycle showing the maximum secretion at the late follicular phase (2118.6+/-200.7 microg/oviduct). The highest volumes of OF were collected from the oviducts at the late follicular phase (50.7+/-1.3 microl/oviduct). These results indicate that OF from sows and gilts shows glycosidase activity varying throughout the estrous cycle suggesting a role of these enzymes in carbohydrate-mediated events.