Retinol improves development of bovine oocytes compromised by heat stress during maturation.

The objectives of this study were to evaluate: 1) effects of a physiologically relevant elevated temperature on in vitro development of maturing oocytes, 2) effects of retinol on in vitro development of maturing oocytes, and 3) effects of retinol to improve development of oocytes compromised by an elevated temperature. Bovine oocytes were matured for 24 h at 38.5 or 41.0 degrees C (first 12 h) in 0 or 5 microM retinol. After insemination, cleavage and blastocyst development were assessed on d 3 and 8, respectively. Temperature, retinol, and their interaction were included in the statistical model. Culture of oocytes at 41.0 degrees C decreased the proportion of 8- to 16-cell embryos and increased that of 2-cell embryos. In addition, culture at 41.0 degrees C decreased the ability of oocytes to develop to the blastocyst stage. Blastocysts derived from oocytes cultured at 41.0 degrees C had fewer total nuclei. In 3 of the 7 experimental replicates, effects of 41.0 degrees C to reduce blastocyst development were minimal (difference in the development of the control vs. heat stress group was <20%). To provide a more precise test of our hypothesis (retinol administration may improve development of oocytes compromised by heat stress), data were analyzed, including only those replicates (n = 4) in which heat stress reduced development to blastocyst >20%. When this was done, a significant temperature x retinol interaction was noted. The addition of retinol to the maturation medium prevented heat-induced reductions in development of oocytes to blastocyst stage. Results indicate that retinol may protect oocytes from some of the deleterious effects of heat stress.

Age-related changes in porcine corticosteroid-binding globulin (pCBG) as determined by an enzyme-linked immunosorbent assay.

The objectives of this study were to develop an assay for the direct measure of porcine corticosteroid-binding globulin (pCBG) and to confirm age-related changes in plasma pCBG concentration. Isolation and purification of pCBG from plasma was performed by affinity chromatography and HPLC-DEAE anion exchange techniques. Analysis by SDS-PAGE revealed two polypeptides (54 and 59 kDa) having similar amino acid homology (>50%) to previously reported sequences of seven mammalian species for the first 33 amino acids. Porcine CBG (20 ng/well) was immobilized to microtiter plates and standards or samples added along with rabbit antiserum developed against the purified pCBG. Goat anti-rabbit IgG-alkaline phosphatase conjugate was added followed by p-NPP substrate. The resultant color development was read at 405 nm. Intra- and interassay coefficients of variation (n=26) of a pooled sample were 10 and 15%, respectively. Age-related changes (P<0.001) in plasma pCBG concentration (n=203) from day 3 through 168 of age confirmed that, in the pig, changes seen in the percent distribution of cortisol among protein bound and free forms around day 28 of age are associated with an increase in CBG concentration.

Partial nucleotide sequence of the porcine corticosteroid-binding globulin (CBG) cDNA and specification of CBG expression sites in postnatal pigs.

The purpose of this study was to develop a porcine CBG cDNA probe in order to examine the porcine CBG mRNA expression in major tissues from the postnatal pig. The reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted to develop the porcine CBG cDNA probe using total RNA extracted from liver of 40-day-old pig. The RT-PCR product was subcloned into the pGEM vector (Promega, Madison, WI) and subjected to restriction enzyme treatments and DNA sequencing. Northern blot analysis was conducted using total RNA extracted from samples (approximately 200 mg) of liver, lung, kidney, and whole adrenal tissue that were collected from pigs on day 3 (n = 2) or day 40 (n = 2) postpartum. A 500 bp partial porcine CBG cDNA encoded 166 amino acids and had 83, 78, and 77% homology to a 494-nucleotide sequence of CBG from sheep, human, and rabbit, respectively. The deduced peptide sequence of the partial porcine CBG showed 77, 62, 60, and 51% homology to sheep, human, rabbit, and rat CBG sequences, respectively. An approximately 1.53 kb CBG mRNA was detected only in the liver tissue. In conclusion, the development of a partial CBG cDNA for swine makes it possible to study the ontogeny and the regulation of CBG synthesis at the molecular level and, based on tissues examined in this study, the liver appears to be the primary source of CBG biosynthesis in the postnatal pig.

Influence of supplemental, dietary vitamin A on retinol-binding protein concentrations in the plasma of preruminant calves.

Transport of retinol (vitamin A alcohol) from retinoid stores in the liver to target tissues is accomplished exclusively by a specific plasma protein, retinol-binding protein. Within individuals, retinol-binding protein concentrations in plasma are regulated and remain constant except in extremes of vitamin A nutriture or in disease. In the present study, retinol-binding protein concentrations in plasma from preruminant calves supplemented with 0, 1700 (i.e., current NRC requirement), 34,000, or 68,000 IU of vitamin A daily from birth to 27 d of age (n = 6/treatment) were quantified. Retinol-binding protein concentrations at birth averaged 21 microg/ml (n = 24) or approximately 50% of concentrations in dairy heifers and cows. Plasma retinol and retinol-binding protein concentrations were correlated positively, corroborating the role of vitamin A nutriture in the regulation of retinol-binding protein secretion from the liver. In this regard, dietary vitamin A influenced positively retinol and retinol-binding protein concentrations and, as a consequence, the degree of saturation of retinol-binding protein with retinol. At 27 d of age, calves fed > or = 34,000 IU of vitamin A had substantially higher retinol and retinol-binding protein concentrations than did calves fed < or = 1700 IU of vitamin A, indicating that dietary vitamin A effects positively vitamin A status. The data also suggest that the current NRC requirement may not be sufficient to assure vitamin A adequacy in preruminant calves. Percent saturation of retionol-binding protein with retinol in all calves was < 35%, much lower than anticipated and suggests that the retinol requirement of vitamin A-responsive tissues exceeded vitamin A availability.

Retinol administration to superovulated ewes improves in vitro embryonic viability.

Retinol and its metabolites, all-trans retinoic acid and 9-cis retinoid acid, are regulators of cellular growth, differentiation, and development and have been implicated in reproductive processes including folliculogenesis and embryonic survival. Three experiments were conducted to identify effects of retinoid treatment of superovulated ewes upon subsequent in vitro embryonic development. Ewes were treated with all-trans retinol (ROH), all-trans retinoic acid (RA), 9-cis retinoic acid (CIS), or vehicle (Control) on the first and last day of FSH treatment. Embryos were recovered at the morula stage, cultured in vitro for 96 h, and observed for blastocyst formation. Embryos from ROH-treated animals had a higher (p < 0.01) incidence of blastocyst formation than RA-, CIS-, or vehicle-treated animals (72% vs. 27%, 33% and 32%, respectively). In experiment 2, ewes were given ROH or vehicle and treated as above. ROH treatment resulted in an increased percentage of embryos forming blastocysts (70% vs. 22%, p < 0.05). In experiment 3, ewes were treated with ROH or vehicle, and embryos were collected at the 1- to 4-cell stage and cultured for 7 days. ROH treatment resulted in increased blastocyst formation (79% vs. 5%, p < 0.05). The majority of embryos (60% vs. 6%; p < 0.01)) from vehicle-treated animals failed to develop beyond the 8-cell stage in comparison with those from ROH animals. ROH treatment of superovulated ewes increased embryonic viability and positively impacted embryonic development.

Steroid regulation of retinol-binding protein in the ovine oviduct.

Two studies were conducted to identify retinol-binding protein (RBP) expression in the ovine oviduct and to determine the role of ovarian steroids in its regulation. Ewes were salpingectomized on Days 1, 5, or 10 of their respective estrous cycles, and oviducts were homogenized for RNA analysis, fixed for immunocytochemistry (ICC), or cultured for 24 h for protein analysis. ICC localized RBP to the epithelium of all oviducts. RBP synthesis was demonstrated by immunoprecipitation of radiolabeled RBP from the medium of oviductal explant cultures. Explant culture medium from oviducts harvested on Day 1 contained significantly more RBP than medium from oviducts collected on Days 5 or 10. Slot-blot analysis demonstrated that steady-state RBP mRNA levels were significantly higher on Day 1 than Day 5 or 10. In the second experiment, ovariectomized ewes were treated with estradiol-17beta (E2), progesterone (P4), E2+P4 (E2+P4), or vehicle control, and oviducts were analyzed as above. P4 alone or in combination with E2 significantly reduced steady-state RBP mRNA levels compared to those in E2-treated animals. Oviductal explants from E2- and E2+P4-treated animals released 3- to 5-fold more RBP into the medium than control and P4 treatments as determined by ELISA. RBP synthesis of metabolically labeled RBP was increased by E2 and E2+P4 treatments. This study demonstrates that P4 applied on an estradiol background negatively regulates RBP gene expression in the oviduct whereas estradiol appears to stimulate RBP synthesis and secretion.

Transforming growth factor beta and the endometrium.

During the oestrous or menstrual cycle and throughout much of pregnancy, the uterine endometrium undergoes rapid, as well as progressive, morphological and functional modification. During the preimplantation stage of pregnancy, the endometrium provides an environment that sustains embryonic development, and then participates in the nidation process. Later, the endometrium contributes the maternal component of the fetomaternal placenta. For a successful pregnancy, the placenta must orchestrate and regulate opposing forces. Trophoblast invasion must be limited to protect the uterus from destruction, while the allogenic fetus must be guarded from maternal immunological attack. Because of their powerful effects on the cellular and molecular processes associated with cellular proliferation and differentiation, angiogenesis and immunomodulation, the transforming growth factor beta (TGF-beta) polypeptides have been identified as potential modulators of many endometrial functions. Here, we examine the literature concerning cell-specific and temporal patterns of TGF-beta expression in the uterine endometrium during the oestrous cycle and pregnancy and evaluate the influence of ovarian steroids on TGF-beta expression in a range of species. Studies of the function of TGF-beta in the endometrium and at the fetomaternal interface are reviewed and discussed.

Immunolocalization of retinol-binding protein and profiles of its mRNA as related to testicular development in the beef bull.

A study was conducted to identify the cell types that express retinol-binding protein (RBP) in the bovine testis and to compare relative steady-state levels of RBP mRNA expression at different times of testicular development. At the ages of 10 (n = 3), 20 (n = 8), and 34 (n = 7) wk, Angus bulls were bled three times at 1.5-hr intervals, then surgically castrated. Blood samples were analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T), by radioimmunoassay and the degree of seminiferous tubule development was evaluated histologically in sections of fixed tissue samples stained with hematoxylin and eosin. Immunolocalization of RBP was based on the biotin-strepavidin-horseradish peroxidase method. Testis weight and concentrations of LH and T increased with age (P < 0.05), but those of FSH did not change (P > 0.05) between 10 and 34 wk. Seminiferous tubules at 10 wk contained immature Sertoli cells and gonocytes whereas, at 20 wk, spermatogonia and few spermatocytes were detected. At 34 wk, Sertoli cells appeared differentiated and spermatids were observed. RBP was immunolocalized in Sertoli, Leydig, and peritubular cells at the ages of 10, 20, and 34 wk. Furthermore, no differences in staining between Sertoli cells from tubules with or without germ cells were detected. Northern hybridization of testicular RNA with an RBP cDNA probe revealed the presence of a 1.4-Kb mRNA, which was similar to previous RBP transcripts found in other bovine tissues. Quantitative slot blot analysis revealed that steady-state RBP mRNA levels were 50% higher at 10 wk (P < 0.05) than at 20 and 34 wk of age.

Developmental gene expression of procollagen III in bovine extraembryonic membranes during early pregnancy.

A major secretory protein produced by bovine chorioallantoic membranes, in vitro, was previously identified as the carboxyl-propeptide of alpha-1 type III collagen. In the present study, the protein and gene expression of procollagen III by bovine chorioallantois between days 17 and 45 of pregnancy was investigated. In addition, differential usage of multiple transcription termination sites by chorioallantois was examined. Two-dimensional PAGE of proteins synthesized and released by whole conceptuses or isolated chorioallantoic membranes into culture medium demonstrated that the C-terminal of procollagen III was not detectable before day 21 of pregnancy and concentrations increased thereafter. Developmental gene expression was determined by Northern blot analysis using a probe (A) that preceded all five polyadenylation sites of the previously sequenced clone 9.22. Procollagen III mRNA expression was undetectable at day 17, low on day 20, and increased through day 36. Two major transcripts of 5.9 and 4.9 kb were identified, the latter of which was expressed more prominently. A second probe (B), which terminated between poly-A sites 2 and 3, was designed to identify transcripts that terminated at poly-A site 1 or 2. This probe bound to the 5.9-kb mRNA only. Two additional procollagen III cDNA clones were isolated from our bovine conceptus cDNA library and sequenced. One, designated 9.29, terminated at poly-A site 5. The other, designated 11.7, terminated at poly-A site 2, indicating that the bovine conceptus uses these stop sites in procollagen III transcription. Results from this study demonstrate that procollagen III gene and protein expression coincide with the development of the allantois, which progressively fuses with the chorion forming the chorioallantois placenta. In addition, multiple termination sites are used in procollagen III transcription.

The role of trophoblast interferons in the maintenance of early pregnancy in ruminants.

PROBLEM: Are the effects of ruminant trophoblast interferon-tau (IFN-tau) on uterine prostaglandin (PG) secretion a specific action of this cytokine and what are the effects of IFN-tau on expression of uterine genes not generally associated with pregnancy maintenance? METHODS: The effects of IFN-tau and IFN-alpha on bovine uterine explant and epithelial cell production of PGF2 alpha and PGE2 were determined in the presence and absence of oxytocin (OT). The effects of intrauterine administration of IFN-tau were determined on uterine expression of retinol-binding protein (RBP) and transforming growth factor-beta (TGF-beta) isoforms. RESULTS: IFN-tau attenuated uterine endometrial secretion of PGF2 alpha and PGE2 in vitro and diminish PG stimulation by OT. IFN-tau and IFN-alpha were observed to be equipotent. Intrauterine infusion of IFN-tau resulted in a significant decrease in steady-state RBP mRNA levels and expression of TGF-beta 1, 2, and 3 mRNA levels were lowest in IFN-tau treated animals. CONCLUSION: Negative regulation of gene expression may be a general strategy in IFN activity. This may explain the similar activities of IFN-tau and IFN-alpha on a broad variety of cell types, including ruminant uterine endometrium.

Bovine endometrial retinol-binding protein secretion, messenger ribonucleic acid expression, and cellular localization during the estrous cycle and early pregnancy.

Retinol-binding protein (RBP) mRNA was localized to luminal and glandular epithelial cells of bovine endometrium by in situ hybridization. Relative levels of endometrial RBP mRNA expression during the estrous cycle and early pregnancy were determined by quantitative slot blot analysis and contrasted with uterine luminal concentrations of RBP. Expression of mRNA was moderate at Day 1 after estrus, declined at Day 5, reached lowest levels by Day 10, rose significantly through Days 15-17, and peaked at Day 20. RBP mRNA expression in pregnant animals was similar to that in cyclic animals on Day 15, doubled between Days 17 and 20, remained constant through Day 22, and rose slightly thereafter. Luminal RBP concentrations of cyclic cows, as determined by ELISA, decreased from Day 1 through Day 10, rose dramatically on Day 15, then declined through Day 20. Concentrations of RBP in uterine flushes from pregnant animals were similar to those of cyclic cows on Day 15 but remained relatively constant through Day 17. It is concluded that 1) RBP synthesis occurs in the luminal and glandular epithelial cells, 2) RBP transcription and secretion are correlated with each other, and 3) ovarian steroids, possibly in conjunction with uterine concentrations of their receptors, modulate uterine RBP expression.

Isolation and identification of porcine embryonic basic protein as a fragment of pregnancy-associated glycoprotein-2.

Between days 11 and 12 of gestation, the porcine conceptus undergoes a metamorphosis from a spherical blastocyst to an elongate thread-like form. During this process, the conceptus secretes a variety of products. One of these products is protein previously referred to as porcine embryonic basic protein (BP). This protein has been shown to be a major secreted product between days 13 and 18. In this study, we report a simple two-step procedure to isolate BP from day 15 porcine conceptus conditioned medium, utilized ion-exchange chromatography and reverse-phase HPLC. Purified BP was subjected to Edman degradation amino-terminal sequencing and a 25 amino acid residue sequence was obtained. Comparing the N-terminal sequence of BP to sequences in the GenBank database determined that BP shared amino acid homology with porcine pregnancy-associated glycoprotein-2 (PAG-2). The region of identity corresponded to an internal site of PAG-2, suggesting BP was a proteolytic fragment of PAG-2. The purified protein was confirmed to be BP by Western blot using a previously characterized anti-BP antiserum. Also, the BP was immunolocalized with the trophectoderm of day 11 blastocysts. Staining intensity was diminished in spherical blastocysts compared to elongated blastocysts. Although the function of PAG-2 and its cleavage product BP are unknown, the large quantity produced by the porcine conceptus and its sequence conservation across species may indicate a necessary role in early pregnancy.

Ovine endometrial expression of transforming growth factor beta isoforms during the peri-implantation period.

During the estrous cycle and early pregnancy, the uterus undergoes a variety o morphological changes. Because of their powerful effects on angiogenesis, on extracellular matrix modification, and on cellular proliferation and differentiation, the transforming growth factor beta (TGF beta) family of polypeptides may be involved in the regulation of pregnancy-related endometrial modification. In this study, endometrial stead-state mRNA expression of the three isoforms, TGF beta 1, beta 2, and beta 3, was quantified, and the proteins were localized during the later part of the estrous cycle (Day 13 and 16) and during early pregnancy (Days 13 through 30) in sheep. TGF beta 1 mRNA was expressed as a single transcript with steady-state mRNA expression levels 2-fold higher on Day 16 of the estrous cycle than on Day 13 of the estrous cycle (p < or = 0.002) or Days 13 and 16 (p < or = 0.004) and 0.008, respectively) of gestation. During pregnancy, levels increased progressively to peak on Day 27 (2.5-fold above Day 16 of pregnancy; p < or = 0.0001) and then leveled off at Day 30. Immunocytochemical localization of TGF beta 1 demonstrated protein in glandular protein in glandular and luminal epithelium at all days examined. TGF beta 2 mRNA was expressed as five distinct transcripts, and mRNA expression levels were lowest in Day 16 pregnant endometrium. Thereafter the expression level increased steadily through Day 30 (p < or = 0.0003). TGF beta 2 protein was localized in epithelium, diffusely within the endometrial stroma, and in leukocyte-like cells within the stroma. TGF beta 3 was expressed as one major transcripts and two minor transcripts. As with TGF beta 1, there was a dramatic difference in TGF beta 3 levels between Day 16 of pregnancy and Day 16 of the estrous cycle (3.8-fold, p < or = 0.0001). In pregnant ewes, endometrial TGF beta 3 levels increased 1.9-fold (p = 0.13) between Days 16 and 23 and remained relatively constant through Day 30. Immunohistochemistry localized TGF beta 3 protein most prominantly in the subepithelial stroma of the caruncule from Day 16 of the cycle in endometrial tissue. The observed changes in mRNA and protein expression patterns of TGF beta s within the ovine endometrium suggest that TGF beta s play a role in restructuring and modifying endometrium for a subsequent estrous cycle and/or pregnancy.

Characterization of protein production by caprine placental membranes: identification and immunolocalization of retinol-binding protein.

Caprine chorion, allantois and amnion from days 23, 28, 35, 39 and 45, and yolk sac from day 23 of pregnancy were isolated by dissection and cultured for 24 h in modified minimum essential medium in the presence of [35S] methionine. De novo-synthesized proteins released into the culture medium were analyzed by two-dimensional PAGE and fluorography. Patterns of protein production by these isolated extraembryonic membranes remained relatively unchanged from days 23 to 45 of pregnancy. Electrophoretic profiles of proteins synthesized by allantois and amnion were identical but distinct from that produced by chorion. Yolk sac was the major source of serum-like proteins. An acidic (pI 5.3-6.3) 22 kDa protein, which consisted of four isoelectric variants, was produced by all extraembryonic membranes and demonstrated to immunoreact with antiserum produced against bovine placental retinol-binding protein (RBP). Limited N-terminal sequence analysis of one major isoform indicated that the protein had complete homology with bovine RBP over the first 15 amino acids. Immunoreactive RBP was localized in epithelial cells lining the chorion, allantois and amnion. In this study, we have characterized and compared protein production by isolated extraembryonic membranes through days 23 to 45 of pregnancy and identified the 22 kDa protein as caprine RBP of placental origin.

Early gestational expression of transforming growth factor beta isoforms by the ovine placenta.

The ruminant blastocyst metamorphoses from a 1-mm sphere to a 1 x 190-mm thread between Days 12 and 15 of gestation. The transforming growth factor beta (TGF beta) family of polypeptide growth factors are potential regulators of embryonic elongation because of their ability to regulate cellular replication, differentiation, and extracellular matrix formation. In mammalian development three isoforms of TGF beta-- TGF beta 1, beta 2, and beta 3--have been identified. Through the use of isoform-specific probes, ovine embryonic TGF beta 1 and beta 2 transcripts were identified and characterized in the present study. TGF beta 1 was expressed as a single transcript 2.6 kb in length. Levels increased by 14.8-fold from Day 13, when levels were barely detectable, to Day 27, when they were highest (p = 0.0001). Enriched samples of Day 20 chorionic and allantoic membranes demonstrated that the allantoic membrane contained 3.9-fold greater levels (p = 0.001) of TGF beta 1 message than the chorion. TGF beta 2 was expressed as five transcripts 6.2, 5.2, 4.8, 4.0, and 2.7 kb in length. From Day 13 to Day 23, there was a 6.2-fold increase (p = 0.0005) in TGF beta 2 expression; thereafter, expression declined 20% by Day 30 (p = 0.03). Extra-embryonic membrane expression of TGF beta 2 was slightly greater (1.5-fold; p = 0.01) in allantois than in chorion. Expression of TGF beta 3 was not detectable by Northern blotting, and only trace quantities of TGF beta 3 transcript were detected by slot-blot analysis. Antisera specific to TGF beta 1, beta 2 and beta 3 were used to immunolocalize the proteins within tissues. TGF beta 1 and beta 2 were identified in Day 16 trophectoderm and yolk sac. Both were also localized in chorion, allantois, and placental endothelium through Day 30. TGF beta 3 protein could not be detected in any conceptus tissue at any of the stages examined. The increase in expression of TGF beta 1 and beta 2 mRNA coincidental with conceptus elongation and placental development, as well as tissue localization of the protein, suggests their involvement in early gestational development in sheep.

Early gestational expression of retinol-binding protein mRNA by the ovine conceptus and endometrium.

Communication between the mother and the early developing embryo is mediated by a variety of signals secreted by either the uterus or the embryo to elicit a response from the other. These signals include prostaglandins, proteins, and steroids. Recently, retinol-binding protein (RBP) has been described as a product of both the conceptus and endometrium in several species. Utilizing a cDNA clone to bovine RBP, we have described RBP mRNA expression in the endometrium, early conceptus, and extraembryonic membranes of sheep. Endometrial RBP mRNA expression did not differ between samples collected on day 13 of the estrous cycle and early pregnancy. In cyclic animals, RBP mRNA expression decreased two-fold between days 13 and 16, presumably a result of luteal regression and the consequential withdrawal of progesterone. In pregnant animals, endometrial RBP mRNA expression likewise decreased between days 13 and 16 and remained at this reduced level through day 30, despite the presence of a functional corpus luteum. Initiation of embryonic RBP expression appeared to coincide with early stages of blastocyst elongation at day 13. Levels of expression increased dramatically with conceptus development, peaked on day 23, and declined afterwards. Results from restriction enzyme analysis of genomic DNA indicated that RBP was encoded by a single gene per haploid genome. Differences in the temporal and tissue-specific expression of the protein, despite the apparent utilization of a single gene, suggest complex regulation of RBP gene expression.

Localization of bovine trophoblast protein-1 in the cow blastocyst during implantation: an immunological cryoultrastructural study.

At implantation in ruminants the signal for maintenance of progesterone production by the corpus luteum, essential for a successful pregnancy, is an interferon-like protein (bovine, ovine or caprine trophoblast protein-1; e.g. bTP-1) produced by the blastocyst trophoblast. This quantitative immunogold cryoultrastructural study demonstrates that bTP-1 immunoreactivity is found only in the Golgi complex and associated clear vesicles in the uninucleate trophoblast cells between 18 and 23 days post insemination. The characteristic trophoblast binucleate cells (up to 20 per cent by number) show no significant label at any time. There is also some evidence for transient labelling of the fetomaternal microvillar junction at the time of maximum bTP-1 production suggesting a transfer of the molecule by exocytosis. No evidence for any labelling within the uterine epithelium was observed.

Expression and cellular localization of retinol-binding protein messenger ribonucleic acid in bovine blastocysts and extraembryonic membranes.

A cDNA clone encoding retinol-binding protein (RBP) was isolated from a bovine conceptus cDNA library by use of an antiserum specific for bovine conceptus RBP (bcRBP). The RBP cDNA clone, designated bcRBP-700, is 732 bp in length and codes for a protein whose predicted amino acid sequence is identical to that of bovine plasma RBP. The size of the RBP mRNA transcript in bovine chorioallantois was approximately 1.4 kb as determined by Northern blot analysis. Expression of the protein and its mRNA in expanding bovine conceptuses (Day 13) and extraembryonic membranes (Day 45) was determined by immunocytochemistry with anti-bcRBP serum and in situ hybridization with 35S-labeled bcRBP-700 cDNA. Strong immunostaining for RBP and hybridization signals for RBP mRNA were observed in trophectoderm of tubular but not spherical Day 13 blastocysts. RBP mRNA was localized in epithelial cells lining the chorion, allantois, and amnion at Day 45 of pregnancy. In addition, RBP mRNA was detected in cotyledons, the sites of chorionic attachment to the uterine endometrium and physiological exchange between the embryo and its mother. Expression of RBP in expanding conceptuses, developing extraembryonic membranes, and sites of fetal-maternal attachment suggests that the extraembryonic membranes regulate retinol transport and availability within the conceptus.

Characterization and immunolocalization of bovine uterine retinol-binding protein.

Endometrial explants obtained from cows between Days 13 and 29 of pregnancy were cultured for 24 h in modified minimum essential medium in the presence of [35S]methionine or [3H]leucine. Proteins synthesized and released into medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Uterine luminal flushings were obtained from cyclic cows (Days 2-20 of estrous cycle) and early pregnant cows (Days 17-22). Endometrial tissues from cows on Days 17 and 29 of pregnancy were prepared for immunocytochemistry. A uterine secretory protein, which consisted of five isoelectric variants (pI 5.3-6.1) of identical molecular mass (23,000 Da), was shown to react immunologically with antiserum raised against bovine placental retinol-binding protein (bpRBP). Limited N-terminal sequence analysis of two major isoforms showed that the protein had nearly complete homology with bovine placental and plasma retinol-binding protein (RBP) over the first 25 amino acids. Through use of bpRBP antiserum, immunoreactive RBP was detected in uterine flushings collected from cows in the late luteal phase of the estrous cycle and early pregnancy by Western blotting, and in medium conditioned by uterine explants prepared at Days 13-29 of pregnancy by immunoprecipitation. Immunoreactive RBP was localized in endometrial surface and glandular epithelium on Days 17 and 29 of pregnancy by immunocytochemistry. These results demonstrate that RBP is a product of bovine uterine tissues. The uterine RBP may play an important role in vitamin A transport between maternal tissues and developing embryos.

Purification and immunolocalization of ovine placental retinol-binding protein.

A retinol-binding protein (RBP), synthesized and secreted by ovine allantois in vitro, was purified from culture medium. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical molecular masses of about 23,000 Da as determined by two-dimensional PAGE under reducing conditions. Thirty-one of the first 34 N-terminal amino acids of the purified protein were sequenced and shown to have complete homology with bovine placental and bovine plasma RBP. The ultraviolet absorption spectrum and fluorescence excitation and emission spectra of the purified ovine placental RBP indicated the presence of bound retinol. Metabolic labeling studies demonstrated that the protein was synthesized by placental membranes. Using antiserum to bovine placental RBP, ovine placental RBP was immunolocalized in trophectoderm of 13-day-old blastocysts and trophectodermal cells of the chorion, endodermal cells lining the allantois, and ectodermal cells lining the amnion of 23-, 45-, and 53-day-old conceptuses. Results from this study suggest that ovine placental membrane epithelia synthesize and secrete RBP. Transport, storage, and metabolism of retinol mediated by placental RBP may be essential for normal embryonic development during pregnancy.