During late follicular growth, oestrus, fertilization and early embryonic development, the oviduct, under specific hormonal control, produces fluid and contributes secretory macromolecules that optimize the microenvironment for gamete maturation and transport, fertilization and early cleavage-stage embryonic development. This review describes the state of knowledge concerning the physiological and biochemical characterization of the major oviduct secreted protein, the oviduct-specific, oestrogen-dependent glycoprotein. The identification, localization within the oviduct, binding and association with oocytes, embryos and spermatozoa, hormonal control of gene expression, cloning, gene organization, protein sequences and post-translational modifications of oviduct-specific, oestrogen-dependent glycoprotein are discussed. Identification of biological functions for this glycoprotein, its interactions with spermatozoa, oocytes and embryos and its potential as a paracrine regulator of fertilization and development are also discussed.

During late follicular development and estrus, the mammalian oviduct undergoes specific physiological and biochemical modifications which contribute to an optimization of the microenvironment for fertilization and early cleavage-stage embryonic development. These changes appear to be hormonally regulated by ovarian steroids, most importantly, estrogen. The hundreds of macromolecules found within the oviductal lumen are contributed by selective serum transudation and active biosynthesis and secretion from nonciliated epithelial cells. Recent studies have indicated temporal and regional (infundibulum, ampulla and isthmus) differences in steady-state levels of specific mRNAs and in de novo protein synthesis and secretion by the oviduct. One protein synthesized de novo, the estrogen-dependent oviductal secretory glycoprotein (OSP), has been shown to be unique to the oviduct and is conserved across a number of mammalian species. This protein associates with the zona pellucida, perivitelline space and vitelline or blastomere membrane of ovulated eggs and preimplantation embryos. OSPs have been shown to enhance sperm binding and penetration in oocytes and may regulate development in early preimplantation embryos. Other regulatory molecules, protease inhibitors, growth factors, cytokines, binding proteins, enzymes and immunoglobulins have been identified in the oviductal microenvironment. The identification and potential roles for oviduct-secreted proteins will be reviewed and discussed. Current research focuses on continued identification and characterization of specific oviductal proteins and a determination of the molecular basis of their interactions with the oocyte, sperm or embryo.

Oviductal secretory products provide a biochemical environment important for establishment of pregnancy. A previous study identified three de novo-synthesized glycoproteins by one-dimensional SDS-PAGE as well as increased incorporation of [3H]Leu into secretory protein by whole oviduct and ampulla associated with proestrus, estrus, and metestrus only. Here, our objective was to further identify and characterize oviductal secretory proteins, specifically 115,000- and 85,000-Mr estrus-associated proteins (EAP). Two-dimensional SDS-PAGE resolved the 115,000-Mr protein into two proteins of 100,000 Mr, one basic and one acidic, and the 85,000-Mr protein into 75,000- and 85,000-Mr species (pI less than 4.0). Differential secretion of proteins between ampulla and isthmus was indicated. The 100,000-, 75,000-, and 85,000-Mr proteins were synthesized by ampulla during estrus but not by isthmus nor by uterine endometrium. De novo-synthesized EAP were labeled with glucosamine, Leu, and Met, and the 75,000-85,000-Mr proteins from ampulla and a 30,000-Mr family from isthmus were labeled with fucose. Inorganic [35S]sulfate labeled three EAP. Fractionation of culture medium by gel filtration demonstrated differences between products secreted by ampulla and isthmus and suggested that some EAP may be found as high-molecular weight forms in the native state. Results indicate that porcine oviductal tissue synthesizes specific EAP at the time of fertilization and early cleavage-stage embryonic development, that there are differences in the type and distribution of glycoproteins from ampulla and isthmus, and that post-translational modifications occur with the addition of glucosamine, fucose, and inorganic sulfate.

This study evaluated the effects of porcine oviduct-specific glycoprotein (pOSP) on in vitro fertilization (IVF), polyspermy, and development to blastocyst. Experiment 1 evaluated the effects of various concentrations (0-100 microgram/ml) of purified pOSP on fertilization parameters, including penetration, polyspermy, male pronuclear formation, and mean number of sperm penetrated per oocyte. Experiment 2 examined the ability of an anti-pOSP immunoglobulin G to inhibit the observed effects of pOSP on fertilization parameters. Experiments 3 and 4 examined various concentrations of pOSP (0-100 microgram/ml) on zona pellucida solubility and sperm binding, respectively. Lastly, experiment 5 assessed the effects of various concentrations of pOSP (0-100 microgram/ml) on the in vitro embryo cleavage rate and development to blastocyst. Pig oocytes matured and fertilized in vitro were used for all experiments. An effect of treatment (P < 0.05) was detected for pOSP on penetration, polyspermy, and mean number of sperm per oocyte. Concentrations for pOSP of 0-50 microgram/ml had no effect on sperm penetration rates; however, compared with the control, 100 microgram/ml significantly decreased the penetration rate (74% vs. 41%). Addition of 10-100 microgram/ml significantly reduced the polyspermy rate compared with the control (61% vs. 24-29%). The decrease in polyspermy achieved by addition of pOSP during preincubation and IVF was blocked with a specific antibody to pOSP. No effect of treatment was observed on zona digestion time relative to the control; however, the number of sperm bound to the zona pellucida was significantly decreased by treatment (P < 0.05). Compared with the control, all concentrations of pOSP examined reduced the number of sperm bound per oocyte (45 vs. 19-34). A treatment effect (P < 0.05) was observed for pOSP on embryo development to blastocyst but not on cleavage rates. Addition of pOSP during preincubation and fertilization significantly increased postcleavage development to blastocyst, but a synergistic stimulation on development was not detected when pOSP was included during in vitro culture. These results indicate that exposure to pOSP before and during fertilization reduces the incidence of polyspermy in pig oocytes, reduces the number of bound sperm, and increases postcleavage development to blastocyst.

Oviduct-specific glycoprotein (OGP) displays estrus-associated regional and temporal differences in expression and localizes to the zona pellucida, perivitelline space, and plasma membrane of oviductal oocytes and embryos, suggesting that it may have a role in regulation of fertilization and/or early embryonic development. The aims of this study were to evaluate the effect of exogenous OGP on in vitro fertilization (IVF) and embryo development in the pig using a defined serum-free culture system. In vitro-matured porcine oocytes were incubated with homologous OGP (0, 1, 10, 20, and 40 microg/ml) for 3 h and then washed prior to IVF. Exposure of oocytes to 10 or 20 microg/ml porcine OGP (pOGP) significantly reduced the incidence of polyspermy compared with the control (P < 0.01) while maintaining high penetration rates. When oocytes, spermatozoa, or both were preincubated with 10 microg/ml pOGP prior to IVF, the incidence of polyspermy was similarly reduced (P < 0.01) by all three treatments without affecting penetration rates. The ability of spermatozoa to undergo calcium ionophore-induced acrosome reaction was similar with or without exposure to pOGP. However, significantly fewer spermatozoa (P < 0.01) bound to the zona pellucida when oocytes were preincubated with pOGP. To evaluate the effect of pOGP on embryo development, embryos were cultured in pOGP-supplemented medium for 48 h or 144 h. Both transient and continuous exposure to pOGP significantly enhanced cleavage and blastocyst formation rate compared with the control (P < 0.01). These data demonstrate that exposure of either in vitro-matured oocytes or spermatozoa to pOGP decreased polyspermy and spermatozoa binding while maintaining high penetration rates of pig oocytes fertilized in vitro. Furthermore, pOGP exerted an embryotrophic effect independent of effects demonstrated on spermatozoa and oocytes at fertilization.

The objectives of the present study were to develop an antibody probe to the porcine estrogen-dependent oviductal glycoproteins and to determine, by use of immunogold electron microscopy, whether these glycoproteins become associated with oviductal and uterine oocytes and early embryos. Polyclonal antibody, prepared using the M(r) 75,000-85,000 glycoprotein, separated from other proteins by two-dimensional SDS-PAGE, specifically recognized all three estrogen-dependent glycoproteins (acidic 75,000-85,000 M(r); acidic 100,000 M(r); basic 100,000 M(r)). In ampullary tissue collected from ovariectomized and estrogen-treated gilts and from gilts at Day 1 of estrus, gold particles were clustered over putative secretory granules restricted to the apical region of secretory epithelial cells. While follicular oocytes did not react with immunoreactive colloidal gold, oviductal and uterine unfertilized oocytes were found to be densely and uniformly labeled by colloidal gold throughout the zona pellucida, associated with flocculent material in the perivitelline space, and associated with microvilli and vitelline membrane. Similarly, in oviductal (1-4-cell) and unhatched uterine (4-cell/blastocyst) embryos, colloidal gold particles were distributed throughout the zona pellucida, heavily associated with flocculent material in the perivitelline space, and associated with the plasma membrane of the blastomeres. Immunoreactive colloidal gold remained detectable within Day 7 hatched uterine embryos, but not with embryos from later days. These results further support the proposal that porcine estrogen-dependent oviductal glycoproteins are released into the oviductal lumen, become associated with oviductal and uterine oocytes and early embryos, and are retained by oocytes and early embryos in the uterus.

No abstract

The objective of the present study was to identify and characterize in a limited manner the major de novo oviductal secretory proteins (OSP) synthesized and released by the porcine oviduct. Oviductal tissue was collected on various days of the estrous cycle (EC) and early pregnancy (EP) and cultured in a modified minimal essential medium supplemented with 100 muCi L-[3H]-leucine. Oviductal secretory activity, as measured by the rate of incorporation of 3H-leucine (dpm/mg wet tissue weight) into nondialyzable macromolecules, was greatest (P less than .01) between days 0 and 2 and reached its lowest levels on days 10 to 15. There was no difference between left and right side or pregnancy status. This increased rate of incorporation at proestrus and estrus is temporally associated with elevated levels of estrogen. Incorporation rate for ampulla was greater than for the isthmus. Analysis of oviductal culture medium by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography revealed three protein bands of relative molecular weight (Mr) 335,000, 115,000, and 85,000, which were associated with proestrus, estrus, and metestrus and were not detectable on other days. All three proteins also incorporated 3H-glucosamine. The 115,000 Mr band was the major 3H-glucosamine-labeled protein. Two protein bands (Mr 60,000 and 20,000) were expressed with increasing progesterone during diestrus. Other de novo synthesized protein bands appear to be present throughout the EC and EP with little modulation by estrogen or progesterone. Thus, this study demonstrates that for the porcine oviduct, the increase in the incorporation rate of 3H-leucine into OSP by both whole oviduct and ampulla and de novo synthesis and secretion of three glycoproteins, Mr 335,000, 115,000, and 85,000, were associated with proestrus and estrus when events such as fertilization and early cleavage stages of embryo development occurred.