Idania M. Alvarez

Secreted proteins of the oviduct.

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.

Effects of the Porcine Oviduct-Specific Glycoprotein on Fertilization, Polyspermy, and Embryonic Development In Vitro

Abstract 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 μg/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 μg/ml) on zona pellucida solubility and sperm binding, respectively. Lastly, experiment 5 assessed the effects of various concentrations of pOSP (0–100 μg/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 μg/ml had no effect on sperm penetration rates; however, compared with the control, 100 μg/ml significantly decreased the penetration rate (74% vs. 41%). Addition of 10–100 μg/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.

Synthesis and secretion of proteins by postpartum human oviductal tissue in culture

An explant culture system that used labelled leucine and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with fluorography was used to identify specific de novo synthesized and released polypeptides by the human postpartum oviduct. Both ampulla and isthmus tissue in culture exhibited de novo synthesis and release of a large number of polypeptide subunits. Immunoglobulins A and G appear to be the major proteins produced in the ampulla. In addition, two complexes of acidic (pI less than 5) polypeptide subunits are found primarily in ampulla culture medium. Two families of proteins (Mr 51,000 and 60,000) are released by the isthmus but appear to be minor in the ampulla cultures.

Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig

Recent identification of plasminogen activator inhibitor‐1 (PAI‐1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy. To examine PAI‐1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured. Culture media was collected and de novo synthesized PAI‐1 analyzed by 2D‐SDS‐PAGE, fluorography, and densitometry. To determine hormonal regulation of PAI‐1 synthesis and secretion, four groups of ovariectomized (OVX) cross‐bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured. Steady‐state mRNA levels of PAI‐1 were evaluated throughout the estrous cycle in cross‐bred gilts. To compare steady‐state PAI‐1 mRNA levels between cyclic and pregnant cross‐bred gilts, tissue was collected on days 0, 2, and 12. Quantitative analysis of steady‐state levels of PAI‐1 mRNA were analyzed by dot‐blot hybridization and densitometry. A greater (P < 0.01) synthesis and secretion of PAI‐1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed. No difference could be detected for PAI‐1 protein between breeds. The Large White had a greater (P < 0.05) secretion of PAI‐1 on day 2 of early pregnancy relative to other days examined. Whole oviductal tissue from cross‐bred gilts was found to have a significantly greater amount of PAI‐1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12. A significant effect of day and segment was detected for levels of PAI‐1 mRNA from cyclic and early pregnant cross‐bred gilts. PAI‐1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy. An interaction was detected for estrogen and progesterone on PAI‐1 mRNA (P < 0.05) and protein (P = 0.09). Estrogen was found to inhibit PAI‐1 protein synthesis and also inhibited progesterone‐mediated stimulation of PAI‐1 mRNA. Our results demonstrate expression of PAI‐1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage‐stage embryo. Mol. Reprod. Dev. 56:378–386, 2000.

Identification, characterization and localization of three proteins expressed by the porcine oviduct

At estrus, the oviduct undergoes endocrine-induced changes which provide an essential microenvironment for maturation of gametes, fertilization and embryonic development. Several oviduct expressed proteins which interact with gametes or embryos, including the oviduct-specific, estrogen-dependent glycoprotein (OGP), have been identified and characterized. The objective of the present study was to identify, characterize and localize other proteins expressed by the porcine oviduct during estrus that may function in an autocrine or paracrine manner to enhance fertilization and embryonic development. Oviducts were collected during the estrous cycle or early pregnancy, flushed and divided into functional segments, and portions of the infundibulum, ampulla and isthmus were fixed for immunocytochemical analysis or cultured. Culture media was semi-purified by heparin-agarose affinity chromatography, proteins were transferred to polyvinylidene fluoride (PVDF) membrane after two-dimensional (2D)-SDS-PAGE and three different proteins were identified, excised and subjected to N-terminal amino acid analysis. These proteins were identified as complement component C3b, the carboxy-terminal propeptide of alpha 1 (III) procollagen (PIIICP), and the heavy chain variable region of IgA. Electrophoresis and fluorography of media from Days 0 to 12 of early pregnancy or the estrous cycle revealed both spatial and temporal expression of C3b and IgA heavy chain but not PIIICP by the oviduct. Further, all three proteins were identified in oviduct fluid by electrophoresis, immunoblot or immunoprecipitation analysis. Complement component C3b and IgA heavy chain were immunolocalized in all three oviduct segments on all days; however, temporal and spatial differences were demonstrated. Staining was greater in the infundibulum and during estrus for all three identified proteins. In summary, three proteins expressed by the oviduct at estrus and during early pregnancy were identified; characterization and localization suggest they may play a critical role in protecting the luminal environment, participating in ECM remodeling and gamete interactions.

Identification and Localization of Plasminogen Activator Inhibitor-1 Within the Porcine Oviduct

Abstract The porcine oviduct synthesizes de novo and secretes a number of proteins into culture medium, many of which are unidentified. The objectives of the present study were to 1) semipurify and identify a Mr 45 000 secreted protein of the oviduct, 2) examine its synthesis within the three functional segments (infundibulum, ampulla, and isthmus), and 3) evaluate its distribution throughout the oviduct. Oviductal tissue was collected during early pregnancy, divided into functional segments, and subsequently cultured. Medium was collected, and the Mr 45 000 protein was concentrated by gel-filtration chromatography. The semipurified protein was transferred onto a polyvinylidene fluoride membrane and subjected to N-terminal amino acid analysis. The 26-amino acid sequence was 96% identical to that of pig plasminogen activator inhibitor (PAI)-1. Analysis by 1-dimensional SDS-PAGE and fluorography of rabbit anti-human PAI-1-immunoprecipitated product confirmed PAI-1. Subsequent 2-dimensional SDS-PAGE and fluorographic analyses of media revealed greater PAI-1 synthesis by the isthmus than by the ampulla or infundibulum. PAI-1 was immunolocalized throughout the oviduct and was heavily concentrated in the apical region of epithelial cells. Immunogold electron microscopy localized PAI-1 within putative secretory granules in the epithelial apical region and also associated with cilia in the isthmus. Isthmic PAI expression suggests a crucial role in protecting the preimplantation embryo from proteolytic degradation as well as in regulation of extracellular matrix turnover and remodeling.

De novo synthesis of glucocorticoid hormone regulated inner ear proteins in rats.

Changes of rat inner ear de novo protein synthesis in response to dexamethasone (DEX), a synthetic glucocorticoid, have been analyzed by high resolution two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-SDS-PAGE) and fluorography. Two proteins (M(r) 41,000 and 35,000) were amplified and one protein (M(r) 47,000) was suppressed by DEX in a cochlear culture medium. In the culture medium conditioned by vestibular tissue, three proteins (M(r) 67,000, 57,000 and 50,000) were amplified after DEX administration. In cochlear and vestibular tissues, glucocorticoid-responsive protein synthesis was down-regulated by DEX, including two proteins (M(r) 39,000 and 35,000) in the cochlea and five proteins (M(r) 80,000, 64,000, 59,000, 56,000 and 40,000) in the vestibule. The regulation of these inner ear proteins by DEX suggests that glucocorticoid may play an important role in normal inner ear microhomeostasis, as well as in the treatment of some inner ear disorders.

Canine conceptus appearance and de novo protein synthesis in relation to the time of implantation

Abstract Characterization of proteins secreted by the endometrium and the conceptus, and timing of the secretion relative to endocrine patterns, ovulation and implantation is essential for understanding mechanisms in maternal recognition of pregnancy. Plasma estradiol-17β, progesterone and serum LH were assayed in samples taken daily during proestrus, estrus and diestrus in 8 adult bitches. The day of the LH peak (DL 1) was accepted as the first day on which LH concentrations exceeded the preceding days value by 2 SD. Embryos were flushed from uteri collected on DL 16 to 20, and incubated with ( 35 S) methionine in MEM; nascent polypeptides in the medium were characterized by 2-dimensional SDS-PAGE. Some attached embryos were first observed on DL 20, and all embryos were attached on DL 22. Major radiolabeled proteins recognized on fluorograms were canine protein 1 (cP1) (Mr = 86,000, pI 5), cP2 (Mr = 42–44,000, pI 5.5−5), cP4 (Mr = 30−28,000, pI 5), cP6 (Mr = 20–25,000 pI 5.5minus;5) and the canine conceptus protein 7 (cCP7) (Mr = 18,000, pI 4). All the proteins were found in samples taken from DL 19 to 20. Proteins cP1 through cP6 were identified as produced by the embryos in this study. They were found to migrate on 2D-SDS-PAGE in a manner judged to be identical to proteins that had been produced by the endometrium. Canine Conceptus Protein 7 was found to be secreted prior to implantation, only by blastocysts.