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.

Effects of the porcine oviduct-specific glycoprotein on fertilization, polyspermy, and embryonic development in vitro.

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.

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.

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.

Identification and localization of plasminogen activator inhibitor-1 within the porcine oviduct.

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 M(r) 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 M(r) 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.

Identification and characterization of proteins synthesized de novo and secreted by the reproductive tract of the American alligator, Alligator mississippiensis.

The objectives of this study were to identify, characterize and examine differences in proteins synthesized de novo and secreted by different regions of the reproductive tract of the American alligator, Alligator mississippiensis, during three reproductive (vitellogenic, gravid, post-clutch) and one non-reproductive state. After capture, alligators from lakes in north central Florida were anaesthetized, the reproductive tract excised aseptically, the size of any follicle determined, and different functional regions of the tract dissected out and partitioned for explant culture. Analysis of the biosynthetic activity indicated regional variations within the tract, differences among reproductive groups and region by status interactions. When oviductal regions were considered regardless of reproductive status, the greatest incorporation of [3H]Leu into secreted nondialysable macromolecules was by the anterior and posterior infundibulum and oviductal tube compared with the transition zone and the uterus. When status was included, the biosynthetic activity of the anterior and posterior portion of the tract in non-reproductive alligators was not different, whereas that of the posterior region of the reproductive group (vitellogenic, gravid, post-clutch) was significantly lower than that of the anterior region. This finding indicates that regulation of protein synthesis and secretion by the non-reproductive alligator tract is different from that in the tract of the reproductive group. Explant-conditioned media were analysed by one-dimensional and two-dimensional SDS-PAGE and fluorography. Sixteen major proteins in culture media were identified as de novo synthesized, by relative molecular weight, by isoelectric point and by differences in distribution determined for reproductive status and oviductal region. Six proteins were examined by N-terminal amino acid microsequence analysis. On the basis of a 29 amino acid sequence, the major oviductal protein, alligator protein 1 (aP1: M(r) 55,000, basic), found in the infundibulum and tube of vitellogenic alligators, was identical to the major protein isolated from alligator egg albumen. Four proteins (aP4-aP7) were sequenced and shown to be significantly related to immunoglobulin heavy chains from several species. This study demonstrated that a large number of proteins are synthesized de novo and released by the female alligator reproductive tract and that there are biosynthetic activity differences by reproductive status and region. Six proteins have been identified, several of which may be incorporated into alligator egg albumen and some of which appear to be different from proteins found in the egg albumens of other species.

Expression of tissue inhibitor of metalloproteinase-1 protein and messenger ribonucleic acid by the oviduct of cyclic, early-pregnant, and ovariectomized steroid-treated gilts.

It has been suggested that tissue inhibitor of metalloproteinases (TIMP)-1 has a role in reproductive tissues, regulating tissue remodeling or enhancing embryonic development. Oviductal TIMP-1 mRNA levels and protein expression were examined in gilts during the estrous cycle and early pregnancy and in steroid-treated ovariectomized (OVX) gilts by explant culture, two-dimensional SDS-PAGE and fluorography, dot-blot hybridization, immunoblot analysis, RIA, and immunocytochemical studies. TIMP-1 mRNA levels in the oviduct during the estrous cycle were greater (p < 0.02) on Days 2, 15, and 18 than on other days examined, and analysis of oviductal functional segments indicated an effect of day (p < 0.003), an effect of segment (p < 0.007), and a day x segment effect (p < 0.03). The level of TIMP-1 mRNA was greater (p < 0.003) in the isthmus (I) on Day 2 than in the ampulla (A) or infundibulum (INF) or on other days examined (0 and 12). In steroid-treated OVX gilts, an effect of treatment with estradiol valerate (EV) + progesterone (P4) was shown with increased (p < 0.003) TIMP-1 mRNA levels. De novo synthesis of TIMP-1 protein was found throughout the estrous cycle and early pregnancy in all functional segments, but protein expression was greater in the I and greatest on Day 2. In steroid-treated OVX gilts, TIMP-1 protein synthesis was greatest in the I regardless of treatment, but with increased intensity after EV+P4 treatment. TIMP-1 protein was found in oviductal flushings during the estrous cycle and early pregnancy, and in steroid-treated OVX gilts regardless of day, status, or treatment. Differences in TIMP-1 concentrations in oviductal fluid were found by day (p < 0.001), with breed differences detected between the Meishan and standard Western breeds. TIMP-1 protein was immunolocalized primarily to luminal epithelium of the INF, A, and I on all days of the estrous cycle and early pregnancy and to some cells in the stroma and blood vessel walls. Staining intensity correlated with TIMP-1 protein levels in oviductal flushings. The role of TIMP-1 in the oviduct remains to be established.

Oviductal regulation of fertilization and early embryonic development.

During the period of late follicular development and the first four days of the oestrous cycle, the oviduct occupies a central role in the establishment of pregnancy. Oviductal function is regarded as being either 'passive' or biologically active, providing an environment that sustains and enhances fertilization and early cleavage-stage embryonic development. Recent reports have focused on this microenvironment and shown that ovarian steroids induce marked morphological, physiological and biochemical changes. Alterations include changes in the biosynthetic activity and release of macromolecules by the oviductal epithelium which become part of the luminal microenvironment. Furthermore, both regional and temporal differences in activity and protein production occur through hormonal changes during the oestrous cycle and early pregnancy. Studies on identification, characterization and regulation of several proteins synthesized de novo have indicated oocyte-oviduct and embryo-oviduct interactions. However, the identification of oviduct-derived proteins, their regulation and their potential function in vivo needs to be examined. Studies in other species also suggest roles for growth factors in early embryonic development, but little information is available for the pig. We propose that ovarian hormones control changes in synthetic activity, synthesis of some oviduct-derived proteins and the presence of specific factors in the luminal microenvironment which sustain and enhance fertilization and early cleavage-stage embryonic development.

Molecular cloning and characterization of an estrogen-dependent porcine oviductal secretory glycoprotein.

A family of estrogen-dependent porcine oviductal secretory glycoproteins (POSPs) that exhibit structural similarities are synthesized and secreted into the oviductal lumen at proestrus, estrus, and metestrus. The objectives of this study were to clone the POSP cDNA, obtain the full-length cDNA and protein sequence, examine tissue specificity and species distribution, characterize its regulation, and establish its identity by comparison to other known protein, RNA, or DNA sequences. A full-length cDNA of 2022 base pairs was obtained with an open reading frame of 1581 nucleotides, coding for a deduced protein of 527 amino acids (57 970 M(r)). The deduced protein contained three potential N-glycosylation sites, a consensus heparin-binding site, and potential O-glycosylation sites. Amino acid analysis of POSP-E3 confirmed the presence of a 21-amino acid signal sequence. Northern blot analysis revealed an oviduct-specific mRNA species of 2.25 kb in the infundibulum (INF), ampulla (A), and isthmus (I). An mRNA of similar size was detected in the oviduct of the sheep, cow, and rabbit, and one of slightly greater size (2.8 kb) in the mouse and hamster oviduct but not in the horse or alligator oviduct. Dot blot analysis indicated that steady-state levels of POSP mRNA were significantly greater (p = 0.0001) in the A than in the INF or I regardless of day of the estrous cycle and were greater on Day 0 (estrus; p = 0.0001) regardless of location. Further, steady-state mRNA levels were significantly increased (p = 0.02) on Days 0 and 1, declining rapidly to Day 2 through Day 15 of the estrous cycle. Steady-state POSP mRNA levels were significantly greater (p < 0.003) in ovariectomized gilts treated with estradiol valerate than those treated with other steroid regimens, vehicle, or no treatment (Control), consistent with estrogen control of mRNA expression. The POSP protein exhibited significant identity to oviductal glycoproteins from the baboon, cow, hamster, human, mouse, and sheep, to several mammalian nonoviductal glycoproteins; and to several chitinases. POSP joins a growing subfamily of the chitinase gene family that lacks chitinase enzymatic activity.

Purification and characterization of a uterine retinol-binding protein in the bitch.

A major canine endometrial secreted protein (cP6, 23,000-M(r)) was purified by ion-exchange and gel-filtration chromatography and characterized by two-dimensional gel electrophoresis. Anti-[human retinol-binding protein (hRBP)] serum identified cP6 on immunoblot analysis and immunoprecipitated cP6 from culture medium. This major protein was also shown to bind [3H]retinol. N-terminal and internal amino acid sequences were determined and compared with previously identified protein, RNA, or DNA sequences. N-terminal analysis revealed that cP6 had high identity and similarity to serum retinol-binding proteins (RBPs), while internal sequence analysis showed a strong similarity to rat androgen-dependent epididymal protein and beta-lactoglobulins. Amino acid analysis, however, showed significant differences between these proteins and cP6 in both total amino acid content and certain selected amino acids. Immunohistochemical analysis showed staining for RBP only in the uterine luminal epithelium. These studies suggest that bitch endometrium secretes a family of proteins (cP6), some of which bind [3H]retinol, are immunologically related to the RBP family, and have N-terminal and internal sequences with a high similarity to RBP, beta-lactoglobulins and other members of the lipocalin family. This family of proteins may be important in early development for supplying retinol or derivatives to the developing embryo.

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.

Immunogold localization of porcine oviductal secretory proteins within the zona pellucida, perivitelline space, and plasma membrane of oviductal and uterine oocytes and early embryos.

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.

Identification and immunolocalization of proteins synthesized by dog endometrium and membranes.

Previous studies on cyclic and pregnant bitches on dioestrus days 3-10 indicated synthesis de novo of at least ten protein complexes, two of which are major protein groups (cP5, M(r) 54,600; cP6, M(r) 23,000). Protein expression differed by day but not by status, suggesting that the embryo does not affect uterine protein synthesis. Ovariectomized bitches treated with various steroid regimens showed induction of cP5 and cP6 only after oestrogen priming followed by progesterone. Recently, N-terminal amino acid microsequencing has identified cP6 as a member of the retinol-binding protein (RBP) family. The present study was designed (a) to identify and characterize proteins synthesized de novo in explant culture from cyclic (dioestrus days 10-16) and early pregnant (dioestrus days 10-26) dogs, (b) to examine distribution of proteins by endometrium preimplantation (PI), and between (BI) and at (I) implantation sites; (c) to characterize proteins synthesized by embryonal membranes and (d) to localize RBP and CUPED (a cat oestrogen-dependent uterine glycoprotein) immunocytochemically in the uterus and embryonal membranes. Proteins synthesized by endometrium were examined by incorporation of [3H]leucine or [35S]methionine on two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by fluorography or autoradiography. Nine proteins, characterized by electrophoresis and previously found between dioestrus days 3-10, appeared to be expressed through day 16 of cyclic and day 26 of pregnant dioestrus, although many decreased and cP5 was lost after day 13. In BI and I sites, RBP decreased in number of isoelectric variants as gestation progressed, while the major M(r) form (23,000) was reduced to 21,500. There was no change in M(r) and isoelectric variants of RBP from cyclic dogs, suggesting differences in RBP gene regulation. Proteins synthesized by embryonal membranes appeared to be serum proteins. Immunolocalization of RBP confirmed that oestrogen priming followed by progesterone was required for induction. In pregnant bitches, staining was present in all luminal epithelia on dioestrus day 10, but by day 17 only specific epithelium stained. As pregnancy progressed to day 24 of dioestrus, staining was localized to specific epithelial cells of the deep spongy zone and yolk sac. In ovariectomized bitches, CUPED was confirmed to be oestrogen dependent with staining in both luminal and glandular epithelia.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (M(r) 40,000 and M(r) 42,000), present with all treatments, were intensified with P4. A high-M(r) basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro synthesis of oviductal secretory proteins by estrogen-treated ovariectomized gilts.

The objective of this study was to identify, characterize, and examine oviductal secretory proteins (OSP) synthesized de novo by whole oviduct (WO), ampulla (A), and isthmic (I) tissue from ovariectomized (OVX), corn oil (CO)-, estrogen (E)-, progesterone (P)-, and E + P-treated gilts. Oviducts were collected from OVX gilts after CO, E, P, or E + P treatment for 11 consecutive days and tissue was incubated with 3H-leucine (3H-leu). Rates of 3H-leu incorporation into nondialyzable macromolecules by WO explants were greater (P less than 0.01) with E- compared to CO-, P-, or E + P-treated gilts and greater (P less than 0.05) by A explants with E- compared to CO-, P-, or E + P-treated gilts. An effect of location was noted, with A having a greater (P less than 0.01) rate of incorporation than WO or I. Conditioned culture medium was analyzed by one (1D)- and two-dimensional (2D) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. Analyses by 1D-SDS-PAGE revealed three major E-dependent bands (335,000, 100,000, and 80,000 M(r)) in WO and A, and one (335,000 M(r)) in the I. A 20,000 M(r) band found in A was inhibited by E, while a 60,000 M(r) band found in the A was induced by P. Analyses by 2D-SDS-PAGE resolved major E-dependent bands 2 (100,000 M(r)) and 3 (80,000 M(r)) into basic and acidic 100,000 M(r) proteins and a 75,000-85,000 M(r) protein (pI less than 4), respectively, found in WO and A, but not in I. A basic 20,000 M(r) protein and an acidic 45,000 M(r) complex, both found in A, were inhibited by E. Gel filtration of culture medium revealed a high M(r) fraction (greater than 2 x 10(6)) that was induced by E and was 6.8-fold greater in medium from A than from I. This study clearly demonstrates that 1) WO and A tissue from E-treated gilts de novo synthesize and secrete three major proteins (basic 100,000, acidic 100,000, and 75,000-85,000 M(r)); 2) these E-dependent proteins are not found in I or with other treatment; 3) several protein complexes synthesized by A are inhibited by E treatment; and 4) a high M(r) fraction, produced primarily in the A, is induced or amplified by E.

In vivo regulation of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase: increased enzyme protein concentration and catalytic efficiency in human leukemia and lymphoma.

The activity of microsomal HMG-CoA reductase in freshly isolated leukocytes from patients with a variety of hematologic malignancies was significantly increased (up to 20-fold) when compared to enzyme activity in leukocytes from normal subjects (average 10.3 +/- 0.8 pmol/min per mg). Increased enzyme activity was not due to nonspecific leukocyte stimulation or to the presence of a malignancy, since normal enzyme activity was observed in subjects with either viral illnesses or solid tumors. Increased HMG-CoA reductase activity accompanying hematologic malignancy could also not be attributed to alterations in enzyme-substrate kinetic parameters (Km), or to alterations in the phosphorylation state or thiol-disulfide status of the enzyme, nor was it correlated with differences in serum lipid or lipoprotein concentrations. The increase (3.6-fold) in HMG-CoA reductase activity in leukocytes from patients with preleukemia was due entirely to a rise in enzyme catalytic efficiency (specific activity), whereas the increase (4.3-fold) observed in leukocytes from patients with overt leukemia or non-Hodgkin's lymphoma was due to a concomitant increase in both enzyme catalytic efficiency (2.5-fold) and enzyme protein concentration (1.6-fold). Similar increases in HMG-CoA reductase activity and catalytic efficiency were also noted for both transformed, nonmalignant, and malignant cultured leukocytes, suggesting that increased enzyme catalytic efficiency is not a nonspecific consequence of physiological changes occurring in response to the malignancy but may be an integral aspect of the malignant phenotype. HMG-CoA reductase protein concentrations, however, were not elevated in either transformed, nonmalignant, or malignant cultured leukocytes, suggesting that increases in enzyme protein levels may be secondary to other physiological changes that occur during the development of overt leukemia. Taken together, these observations suggest that an increase in the activity of HMG-CoA reductase, the rate-controlling enzyme in cholesterol synthesis, is a common occurrence in human hematologic malignancies and that a biphasic elevation of enzyme activity may exist in malignant leukocytes, such that changes in catalytic activity may occur early in tumorigenesis and may be followed by secondary changes in enzyme levels.

In vitro synthesis of oviductal proteins associated with estrus and 17 beta-estradiol-treated ovariectomized ewes.

The objectives of this study were: 1) to identify and characterize ewe oviductal secretory proteins (OSP) de novo synthesized during the estrous cycle; 2) to examine the effect of estrogen (E) and progesterone (P) on synthesis of OSP; and 3) to determine synthesis of OSP by ampulla (A) and isthmus (I). Oviducts were collected from cyclic ewes on days 1, 8, and 16 and ovariectomized (OVX) ewes after treatment with corn oil (CO), E, P, and E + P. Tissue was incubated in the presence of 3H-leucine or 3H-glucosamine. Rates of 3H-leucine incorporation into nondialyzable macromolecules by explants was greater (P less than 0.05) on day 1 than days 8 and 16, greater (P = 0.01) in OVX E-treated ewes, greater in A than I regardless of treatment and greater (P less than 0.05) in A than I with E. Culture medium was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and fluorography. Two major basic proteins (Mr 90-92,000) and three minor proteins were only present on day 1 of estrus. E treatment demonstrated these same proteins which were not found with CO or P treatment. Two-dimensional analysis of medium from A and I cultured separately indicated that A synthesized the two major basic and three minor E-dependent proteins. Of three protein complexes present regardless of treatment or cycle day, two were dominant in the A and one was dominant in the I suggesting a synthetic gradient. Tunicamycin did not inhibit incorporation of 3H-glucosamine into E-dependent proteins. Gel filtration revealed a high Mr fraction (2 x 10(6] 2.5-fold greater on day 1 than day 8 and 2.2-fold greater with E than CO treatment. This study clearly demonstrates: 1) that the oviduct from estrus and E-treated ewes synthesizes two major basic and three minor glycoproteins not detectable on other cycle days or with OVX or P treatment; 2) the E-dependent glycoproteins are synthesized by the A; and 3) a high Mr fraction is induced or amplified by E.

Identification and characterization of de novo-synthesized porcine oviductal secretory proteins.

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.