Biomedical subjects

J D Godkin

Publications and source records attributed to J D Godkin.

At least 37 records · Page 2Linked to original sources

PubMed1992

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.

Amino Acid Sequence↗

PubMed1991

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry.

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.

Animals↗

PubMed1991

Characterization of protein production by ovine placental membranes: identification of a placental retinol-binding protein.

Ovine chorion, allantois, and amnion from days 23, 26, 28, 35, 45, 53, 62, and 72 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 to characterize in vitro synthesis and release of proteins. Proteins synthesized and released into medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Patterns of protein production by these isolated membranes remained relatively unchanged from days 23-72 with the exception of the products of yolk sac, which regress by day 35 of pregnancy. In general, chorion was the source of a number of basic-to-neutral proteins; allantois and amnion were the sources of more acidic proteins; and yolk sac was the source of serum-like proteins. A major low mol wt acidic protein, D4, was produced by all membranes and present in fetal membrane fluids. Protein D4 consisted of three isoelectric variants (isoelectric point 5.3-6.1) with identical mol wts (23,000 +/- 800) and was shown to react immunologically with antibovine placental retinol-binding protein (RBP) serum. Hence, protein D4 is a putative ovine placental RBP. The present study is the first to characterize and compare protein production by isolated ovine chorionic, allantoic, amniotic, and yolk sac membranes from day 23 through midpregnancy and identify the major low mol wt acidic protein produced by each membrane as a placental RBP.

Allantois↗

PubMed1991

Phagocytosis of protein coated colloidal-gold-agarose-gelatin microbeads by cultured uterine glandular epithelial and stromal cells.

A procedure for fixing and immunostaining whole cells from primary cultures of ovine and bovine uterine gland fragments was used to identify keratin in intermediate filaments of epithelial cells to distinguish them from stromal cells. Colloidal gold encapsulated agarose-gelatin microbeads were coated with different proteins and used to investigate uptake by epithelial and stromal cells in culture. Microbeads were taken up by stromal cells and by epithelial cells on the outskirts of colonies. These cells formed ridges where they contacted and grew above stromal cells. Electron microscopy demonstrated that the microbeads had been internalized and appeared to be nontoxic. Individual cells could harbor more than 90 microbeads within their cytoplasm for at least seven to ten days with no apparent harm. Some cells with microbeads were seen to divide.

Animals↗

PubMed1991

Immunohistochemical localization of bovine placental retinol-binding protein.

An immunogold staining method was used in combination with epipolarization microscopic detection to demonstrate the presence of bovine placental retinol-binding protein in bovine extraembryonic membranes. Amnion, chorion and allantois were fixed in Bouin fixation fluid and embedded in polyethylene glycol 1500. Sections (5 mm) were cut and transferred onto Digene silanated slides and immunostained using rabbit antiserum raised against bovine placental retinol-binding protein followed by goat anti-rabbit IgG labeled with 1 nm gold. Gold particles after silver enhancement were viewed and photographed under epipolarization microscopy. Epithelial cells of all three membranes (i.e. amniotic ectoderm, chorionic trophectoderm, and allantoic endoderm) were immunoreactive, while mesodermal cells, collagen, and blood cells were not. These data, together with our previous observation that these three placental membranes synthesize and secrete retinol-binding protein, indicate that epithelial cells lining the amnion, chorion and allantois are the major sources of this protein. The presence of retinol-binding protein in placental membranes and their fluids may be indicative of an important role for retinol in placental differentiation and development.

Allantois↗

PubMed1990

N-glycosylated and unglycosylated forms of caprine trophoblast protein-1 are secreted by preimplantation goat conceptuses.

Goat conceptuses secrete caprine trophoblast protein-1 (cTP-1) which is related antigenically to the abundant embryonic interferon-alpha II of sheep and cattle. Antiserum to ovine and bovine TP-1s immunoprecipitated three molecular weight classes (23,000, 21,000 and 17,000, each with two isotypes) of cTP-1 from goat conceptus culture medium. Cultures which contained tunicamycin resulted in a shift in the Mr = 23,000 and Mr = 21,000 forms to a Mr of 17,000. The Mr = 23,000 and 21,000 forms, but not the Mr = 17,000 form, bound to Concanaval in A-Sepharose and were eluted under conditions selective for glycoproteins bearing complex-type oligosaccharide(s). Thus cTP-1 is a mixture of glycosylated and unglycosylated polypeptides.

Animals↗

PubMed1990

Immunolocalization and endocytosis of the uterine secretory protein, uteroferrin, in pre-implantation pig trophectoderm on day 11 of pregnancy.

Uteroferrin is a progesterone-induced, iron-binding glycoprotein secreted by the glandular epithelium of the pig endometrium. Evidence is presented that maternal uteroferrin is present in trophectoderm of preimplantation pig blastocysts on day 11 of pregnancy. Although [35S]-methionine was not incorporated into uteroferrin during in vitro culture of blastocysts, solubilized tissue extracts from 10-20-mm-diameter blastocysts contained uteroferrin by western blotting with monospecific antiserum to uteroferrin. Uteroferrin was detected in the apical and basolateral cytoplasm of trophectoderm by immunocytochemistry of paraffin-embedded blastocysts. Immunostaining was excluded from cells of the endoderm and the inner cell mass. Furthermore, blastocysts internalized fluorescein-labeled uteroferrin from medium during in vitro culture in a temperature-dependent manner. Fluorescent label was located in apical and basolateral cytoplasm in a punctate distribution, and clustered in the supranuclear region of trophectoderm. Addition of a threefold excess of unlabeled uteroferrin to culture medium did not inhibit uptake. These results suggest that the pre-implantation pig blastocyst actively endocytoses uteroferrin from glandular secretions in utero. Uptake was restricted to trophectoderm.

Acid Phosphatase↗

PubMed1990

Immunocytochemical localization of BP, a conceptus secretory protein, in trophectoderm during pig trophoblast elongation.

Tissue sections of periimplantation pig conceptuses (days 9-15 of pregnancy) were incubated with antiserum to the basic protein (BP), a major secretory protein of filamentous pig blastocysts. Bound antibody was detected by the peroxidase-antiperoxidase method. BP was restricted to trophectoderm in conceptuses which had made the transition from a spherical to an ovoid shape having a diameter of greater than 5 mm (day 11). Tubular (day 11, 10-20 mm) and filamentous (day 11-15) conceptus trophectoderm contained BP. These results suggest that BP synthesis commences at the time of rapid trophoblast growth.

Animals↗

PubMed1990

Purification and characterization of bovine placental retinol-binding protein.

A major low mol wt acidic protein, 3B3, produced from cultures of day 29-90 bovine allantoic membranes (in the presence of [3H]leucine or [35S]methionine) and from day 29-60 allantoic fluid, has been purified. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical mol wt (23,200 +/- 900) when analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequence analysis of 3B3 isolated from allantoic fluid on the first 43 amino acids showed that 3B3 had 93% and 91% homology with rabbit and human plasma retinol-binding protein (RBP), respectively. The UV absorption spectrum and the fluorescence excitation and emission spectra of purified 3B3 from both sources indicated the presence of bound retinol. Rabbit antiserum was raised against placental RBP (3B3) isolated from allantoic membrane culture medium. Placental RBP was immunoprecipitated from radiolabeled allantois and chorion culture medium and was detected in allantoic membrane culture medium and allantoic fluid by Western blotting. These results suggested that bovine placental membranes secrete RBP into allantoic fluid and that placental RBP may play important roles in vitamin A metabolism in the developing embryo.

Allantois↗

PubMed1990

Ultrastructural morphometric analysis of the uterine epithelium during early pregnancy in the sheep.

Stereological techniques were used to quantify ultrastructural changes in the caruncular epithelium during the pre- (Day 13), peri- (Day 16) and post- (Days 19 and 22) attachment periods of placentation. Tissues from Day-13 non-pregnant ewes were used as controls. Uteri for stereological evaluation were perfused via the uterine artery with 3% glutaraldehyde and separated into proximal, middle and distal regions. Tissues from caruncular areas were processed for electron microscopy. Volume fractions (Vv) of nuclei, mitochondria, lipid and cytoplasmic granules were estimated by point-counting volumetry. Surface areas per unit tissue volume (Sv) of mitochondrial membranes and cristae, Golgi, plasmalemma, endoplasmic reticulum and nuclear membranes were estimated by line-intersection counting. The only significant difference between pregnant and non-pregnant uterine epithelium at Day 13, a time before attachment, was a lower Sv of smooth endoplasmic reticulum (SER) in tissue from pregnant ewes. This value returned to control (non-pregnant Day 13) levels at Day 16, and was again significantly reduced at Days 19 and 22. The Vv of lipid decreased significantly at Day 16 and remained at reduced levels thereafter. These changes may reflect the effects of conceptus products on lipid storage and mobilization. The Sv of rough endoplasmic reticulum (RER) significantly increased on Day 16 of gestation, and remained elevated on Day 19. These results may reflect increased synthesis of protein for export at these times. In general, several of the values measured which may be indicative of cellular metabolism were reduced at Day 22 of pregnancy, perhaps suggesting diminished metabolism by the uterine epithelium after attachment of the trophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Isolation, characterization and immunocytochemical localization of bovine trophoblast protein-1.

Bovine trophoblast protein-1 (bTP-1) was isolated to 90% purity from culture medium of Day 18-20 conceptuses incubated in vitro (in the presence of L-[3H]leucine) by a combination of Sephacryl S-200 gel filtration chromatography and O-(diethylaminoethyl) (DEAE) anion-exchange high-performance liquid chromatography (DEAE-HPLC). The radiolabeled protein had an Mr of 21,200 +/- 800 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had three isoelectric variants (pI 5.7-6.5) by two-dimensional SDS-PAGE. DEAE-HPLC-enriched bTP-1 cross-reacted with anti-o TP-1 serum on Western blots. A monospecific antiserum against bTP-1 was produced by excising the bTP-1 polypeptide band from preparative SDS-PAGE gels. Antiserum reacted with a single polypeptide with an Mr of 21,200 as determined by Western blotting of SDS-PAGE-separated conceptus medium proteins and by immunoprecipitation from L-[35S]methionine-labeled medium proteins followed by SDS-PAGE and autoradiography. Bovine trophoblast protein-1 was localized by immunocytochemistry in the cytoplasm of both mono- and binuclear trophectoderm cells of Day 20 bovine conceptuses, indicating that it is a product of the trophoblast.

Animals↗

PubMed1989

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy.

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Characterization of bovine conceptus proteins produced during the peri- and postattachment periods of early pregnancy.

Bovine conceptuses removed from the uterus during the peri- and postattachment periods of placentation (Days 17-24 and 26-38, respectively) were cultured in a modified minimum essential medium in the presence of L-[3H]leucine to characterize in vitro synthesis of proteins released into the medium. Patterns of protein production were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography of dried gels. Four groups of low molecular weight acidic proteins (LMWAP) were observed to be synthesized during the peri- and postattachment periods. The number and relative concentrations of these changed with development. One group (A) consisted of three major and two or more minor isoelectric species (pI approximately equal to 5.8-6.8); these were the major synthesized proteins observed from Days 17-22. The major polypeptides of Group A were present at all time points examined through Day 38 and, in several preparations, appeared as doublets (Mr approximately equal to 22,000 and 24,000) through Day 29 but not thereafter. Group A polypeptides from Day 19 and 36 conceptus cultures were demonstrated by immunoblot analysis to cross-react with antiserum produced against ovine trophoblast protein-1 (oTP-1). A second group of proteins (A1) and a single protein (B) in the 20,000-24,000 Mr range were observed between Days 17 and 22. These were acidic relative to Group A and were not detected after Day 22. A fourth group (C) of LMWAP (Mr approximately equal to 14,000-18,000) was first observed around Day 21 and appeared to increase relative to Group A through Day 29. One protein from this group, C3, was the predominant LMWAP at Day 38.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Characterization of protein production by bovine chorionic and allantoic membranes.

Bovine allantoic (A) and chorionic (C) membranes from Days 29, 32, 36, and 40 of pregnancy were isolated by dissection and cultured in a modified minimum essential medium in the presence of L-[35S]methionine to characterize in vitro synthesis and release of proteins. Membranes were also cultured in the presence of the glycosylation inhibitor tunicamycin. Proteins synthesized and released into the medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography of dried gels. Stained gels were used to analyze protein from allantoic fluids. Percent incorporation of the radiolabeled amino acid into nondialyzable protein was higher for A than for C (A = 8.0 +/- 1.2 vs. C = 6.4 +/- 0.5 per 200 mg tissue) but not significantly different. C released significantly more total protein (nonradioactive) into the medium (57.0 +/- 3 vs. 9.6 +/- 0.6 micrograms/ml). Of the 25 proteins analyzed, 19 appeared to be produced primarily by one membrane or the other. In general, C was the source of a number of basic-to-neutral glycosylated proteins and A was the source of a number of more acidic glycosylated proteins. Many but not all proteins synthesized by A were present in allantoic fluid. The present study is the first to characterize protein production by isolated chorionic and allantoic membranes and to demonstrate that both tissues contribute to the production of secretory conceptus proteins.

Allantois↗

PubMed1988

Purification, characterization, and immunocytochemical localization of the major basic protein of pig blastocysts.

The major basic protein (BP) synthesized and secreted by elongating pig blastocysts was purified from medium of Day 14-17 conceptus cultures. Sequential ion-exchange and gel-filtration chromatographies resulted in isolation of BP as a single polypeptide of Mr = 43,100 or 42,800 under denaturing or native conditions, respectively. BP was found to be a glycoprotein by incorporation of [3H] glucosamine and susceptibility to N-glycopeptidase F. Two BP polypeptides were produced by N-glycopeptidase F (Mr = 39,800 and 36,300). Antiserum to BP immunoprecipitated radiolabeled BP from blastocyst culture medium. BP was not detected in medium from 1-2 mm diameter spherical (Day 10) blastocysts but was found in medium from 3-5 mm spherical (Day 10) and filamentous (less than 50 cm, Day 12) conceptuses, suggesting that BP synthesis and secretion began at the initiation of trophoblast expansion. With immunocytochemical procedures, BP was located in the apical cytoplasm of trophectoderm cells of Day 11 expanding (5-7 and 10-20 mm) blastocysts. These results suggest that trophoblast epithelium secrete BP apically toward the uterine lumen and that BP may play a role in maternal-fetal interactions during the peri-implantation period.

Animals↗

PubMed1988

Interferon-alpha mimics effects of ovine trophoblast protein 1 on prostaglandin and protein secretion by ovine endometrial cells in vitro.

Ovine endometrial cells were isolated from ovariectomized oestrogen and progesterone-treated ewes and maintained in primary culture. In-vitro treatment with human interferon-alpha 2 (Roferon, Hoffman La Roche) (5, 50 IU/ml) or purified ovine trophoblast protein 1 (oTP-1, 30 ng/ml) significantly attenuated PGF2 alpha (25 +/- 17, 29 +/- 17, 28 +/- 9% +/- SEM of control [no in-vitro treatment = 100%] respectively, N = 4 ewes) and PGE (11 +/- 4, 16 +/- 4, 16 +/- 5% of control) release from the cultured cells. Fluorography of two dimensional polyacrylamide gel electrophoretic analyses of proteins secreted by the cells following 35S-methionine incorporation, revealed that synthesis and secretion of the same "pregnancy-related" proteins was stimulated by both interferon-alpha 2 and oTP-1. Thus, interferon-alpha 2 (which has sequence homology with oTP-1) acts on the ovine endometrium, eliciting similar biological responses to those of oTP-1.

Animals↗

PubMed1985

Characterization of proteins produced in vitro by periattachment bovine conceptuses.

Bovine conceptuses from Days 16 (n = 4), 19 (n = 6), 22 (n = 3), and 24 (n = 4), and chorion from Day 69 (estrus/mating = Day 0) were cultured for 24 h in modified minimum essential medium (MEM) in the presence of radioactive L-leucine [( 3H] leucine) to characterize de novo synthesis and release of proteins. Proteins released into MEM were identified by two-dimensional polyacrylamide gel electrophoresis, fluorography, and gel and ion exchange chromatography. Major polypeptides identified in MEM were different from those identified in conceptus and chorionic tissues. Both uptake of [3H] leucine and quality of polypeptides produced de novo and released into MEM were related to stage of conceptus development. Percent retention of [3H] leucine in MEM was lowest (P less than 0.01) in Day 16 cultures (1.2 +/- 4.1%), increased in Days 19 (16.8 +/- 3.7%) and 22 cultures (20.9 +/- 5.8%), and decreased (P less than 0.07) in Day 24 cultures (6.9 +/- 4.1%). Complexity of polypeptides increased after Day 16. Days 16, 19, 22 and 24 conceptus culture MEM was enriched in low-Mr, acidic polypeptides (Mr/isoelectric point ranges: 22K-26K/6.5-5.6, 20K-26K/5.5-5.4, and 16K-20K/5.0-4.5), which were not prominent products of Day 29 and 69 tissues. A high-Mr (Mr +/- SEM; 735K +/- 22K) glycoprotein was produced by all conceptus and chorionic tissues. The transient nature of production of low-Mr polypeptides suggests that they may be required during the periattachment period.

Amino Acids↗

PubMed1985

Protein production by cultures established from Day-14-16 sheep and pig conceptuses.

Primary cell cultures were established from cells derived from dissociated Day-14 and -16 sheep and pig blastocysts. The appearance of cells in culture from both species was similar. Cultures contained a variety of cells with distinct morphologies, some were small and compact and formed clumps and multiple layers while others were large, flat and formed a monolayer. Within 4 h of culturing small floating fluid-filled spheres of cells were observed in the medium; some of these increased in size to greater than 1 cm diameter over 1-2 weeks. In addition, fluid-filled domes of cells arose from the underlying monolayer. Contractile cells became evident after about 8 days and some became organized into large patches of contracting tissue. Two-dimensional polyacrylamide gel electrophoresis and fluorography were performed on proteins released into the medium by confluent monolayers, floating spheres and floating cells that failed to attach during the first 24 h. All cultures produced as major products proteins with electrophoretic mobilities identical to certain fetal plasma proteins. In general, cultures did not produce proteins characteristic of short-term cultures of whole conceptuses harvested at Days 14-16. In cultures established from sheep blastocysts only the cells that failed to attach produced ovine trophoblast protein-1, a major polypeptide produced by the trophectoderm of the sheep conceptus between Days 13 and 21 of pregnancy.

Animals↗