Changes in quantities of high-mobility-group protein 1 in oviduct cellular fractions after oestrogen stimulation.

Antiserum against chick oviduct high-mobility-group protein 1 (HMG 1) has been induced in the rabbit. With this antiserum, immunobiochemical techniques have been used to probe the quantitative change of HMG 1 in the cellular fractions of chick oviduct before or after oestrogen stimulation. HMG 1 is detectable in the cytosol, microsomal and nuclear fraction of the chick oviduct cell. After administration of oestrogen to young chicks in vivo for 5 days, the quantity of HMG 1 is increased 4-fold in the cytosol, 3.5-fold in the microsomal fraction and 1.6-fold in the nuclear fraction. The finding of large amounts of HMG 1 in cytoplasm of oviduct cell is not likely due to its leakage from the nucleus. We anticipate that HMG 1 is synthesized in the cytoplasm and then transported into the nucleus. The synthesis and transportation of HMG proteins is probably regulated by oestrogen.     

Co-culture,oviduct secretion and the function of oviduct-specific glycoproteins.

Co-cultures of embryos with somatic cells, usually in the form of monolayers, or conditioned medium from these somatic cells, results in development past the early stage blocks and the formation of hatched blastocysts. Optimum rates of development are not achieved, however, and the task is to investigate components of the oviduct that are obligatory or facilitative for embryo development. Glycine and alanine are amino acids present in much higher concentrations in oviduct fluid than in serum or culture media. Glycoproteins specifically produced by the oviduct around oestrus bind to embryos and aid development but are absent from most culture media. These glycoproteins are induced by oestrogen in vivo but not in vitro. It is our contention that co-cultures of mammalian embryos should include appropriate concentrations of amino acids and a source of embryotrophic glycoproteins as an additive or by including stromal cells in addition to epithelial cells.     

Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunohistochemical localization of taurine in the rat ovary, oviduct, and uterus.

The distribution of the amino acid taurine in the female reproductive organs has not been previously analyzed in detail. The aim of this study was to determine taurine localization in the rat ovary, oviduct, and uterus by immunohistochemical methods. Taurine was localized in the ovarian surface epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In primary and antral follicles, taurine was found mainly in theca cells and oocytes, whereas the zona pellucida, antrum, and most granulosa cells were unstained. However, taurine immunoreactivity in theca cells and oocytes decreased during follicular atresia. During corpora lutea development, the number of immunopositive theca lutein cells increased as these cells invaded the granulosa-derived region. Therefore, most luteal cells from the mature corpora lutea were stained. In the regressing corpora lutea, however, taurine staining in luteal cells decreased. In the fimbriae, infundibulum, and uterotubal junction, taurine was localized in most epithelial cells. In the ampullar and isthmic segments, taurine was found in the cilia of most ciliated cells and in the apical cytoplasm of some non-ciliated cells. In the uterus, most epithelial cells were immunopositive during diestrus and metestrus, whereas most of them were immunonegative during estrus and proestrus. Moreover, taurine immunoreactivity in the oviduct and uterus decreased with pregnancy. (J Histochem Cytochem 49:1133-1142, 2001)     

Hyperactivated sperm progress in the mouse oviduct.

Sperm from naturally mated mice were observed and videotaped moving within mouse oviducts. The typical pattern of sperm progress involved intermittently breaking free and swimming a short distance, then reattaching to the epithelium. The proportion of sperm that swam freely (were not attached to the epithelium) was calculated and analyzed for effects of oviductal region, ovulation status, and sperm location relative to the lumen. A significantly higher proportion of sperm were free in the ampulla than in the isthmus (26.3% +/- 0.8% vs. 11.8% +/- 1.0%; p less than 0.0001) and in post-ovulatory than pre-ovulatory (16.2% +/- 2.0% vs. 10.6% +/- 1.6%; p less than 0.05) oviducts. Flagellar curvature ratio values showed that free sperm (0.716 +/- 0.024) had more sharply curved tails than stuck sperm (0.782 +/- 0.013). While this difference is significant (p = 0.01), the effect of attachment status interacted significantly (p less than 0.05) with the oviductal region such that there was a greater difference in the isthmus than in the ampulla. Only sperm using the more curved tail beats of hyperactivation were seen to break free from the epithelium and to progress along the oviduct. These results indicate that hyperactivation plays a role in moving sperm out of the isthmic reservoir and to the site of fertilization.     

Autotransfer of Day 4 embryos from oviduct to oviduct versus oviduct to uterus in the mare

Embryo autotransfer is defined as the collection of an embryo from and the transfer of this embryo into the same animal. The objectives of this study were to: 1) test the hypothesis that oviduct transport of the equine embryo from the oviduct into the uterus is not dependent on a unilateral embryo-corpus luteum interaction, 2) develop an embryo autotransfer technique for the mare and 3) compare the success rates of Day 4 embryos surgically autotransferred from the oviduct ipsilateral to ovulation to either the oviduct (n=10 mares) or the uterine horn (n=10 mares) contralateral to ovulation. Seventy percent (7 10 ) of the Day 4 embryos which were autotransferred to the oviduct contralateral to ovulation were transported through the oviduct and subsequently developed into embryonic vesicles detectable by ultrasonography between 10 and 21 days postovulation. This finding supported the hypothesis that oviductal embryo transport is not dependent upon the ipsilateral corpus luteum. Overall, sixty percent (12 20 ) of the autotransfers were successful. The success rate of uterine-transferred embryos was not significantly less (P>0.3) than that of oviductal-transferred embryos (5 10 vs 7 10 , respectively). Therefore, the Day 4 equine embryos were apparently mature enough to survive in the mare's uterus.     

Cannulation of the equine oviduct and chemical analysis of oviduct fluid

Siliconized rubber tubes were used to cannulate one oviduct in 7 mares, and secretions were collected in a polycarbonate container located externally, in the region of the left paralumbar fossa. Secretion rates were recorded daily during the estrous cycle. Concentrations of calcium, magnesium, sodium, potassium, inorganic phosphorus and glucose were determined in the oviduct fluids secreted throughout the estrous cycle. Secretion rates were greatest during estrus (days 1-9), with a significant decrease (P<.01) noted during nonestrus (days 10-21). Concentrations of all constitutents measured were low during estrus, with a marked elevation in concentration during the nonestrus period. Histologic examination of oviducts following long-term cannulation demonstrated infiltration of lymphocytes, plasma cells, and small areas of degenerated epithelium.     

The chick oviduct in tissue culture. I. Initial characterization of growing primary oviduct tissue cultures

A simple three-enzyme treatment of collagenase, dispase and hyaluronidase on finely minced chick oviduct yields clumps of 50-150 cells. These cells attach to collagen-treated dishes and survive in culture for at least 2 weeks without subculturing. Oviduct cell cultures can also be induced to grow. Estradiol or epidermal growth factor (EGF) induce a 40% increase in cells in 4 days when cultures are grown in serum levels that do not support growth. Serum from estrogen-stimulated chicks promotes rapid cellular proliferation (doubling times of 1-2 days). Sera from estrogen withdrawn chicks, laying hen or horse do not support as rapid proliferation. The oviduct growth-promoting factors in serum from estrogen-stimulated chicks are not steroids or fibroblast growth factors (FGF). Removal of steroids from these sera by charcoal treatment or delipidization does not decrease the rate of growth. The addition of 1-100 nM estradiol does not increase a serum's ability to promote growth. Purified FGF or platelet-derived growth factor (PDGF) do not induce oviduct proliferation. These results were reproduced in oviduct cell cultures started from estrogen-stimulated and withdrawn chicks as well as laying hens. Thus the factors in serum from estrogen-stimulated chicks that promote rapid oviduct growth are induced by estrogen treatments in vivo, but do not seem to be only steroids.     

Relationship between cellular DNA synthesis,PCNA expression and sex steroid hormone receptor status in the developing mouse ovary,uterus and oviduct

The proliferative activities of the different cellular compartments of the developing mouse ovary, uterus, and oviduct were studied by radioautographic assessment of DNA synthesis with [³H]-thymidine labeling and by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). The distributions of estrogen and progesterone receptors (ER and PR) were studied by immunohistochemical staining. The values of the PCNA positive staining indices were a little higher than that of the radioautographic labeling indices. However, linear relations were shown for the two indices. The proliferative activities were high from postnatal day 1–7 and decreased from day 14 in the different cellular compartments of the ovary. The proliferative activities were high on days 1, 3 and decreased from day 7 in the uterus and oviduct. Staining of ER and PR was very weak in the surface epithelium, stroma and large follicles of the ovary. Positive staining for ER occurred from day 14 in the uterine epithelium and from day 7 in oviductal epithelium. Positive staining for PR was observed from day 1 in both the uterine and oviductal epithelium. However, the positivity of both ER and PR occurred from postnatal day 1 in the stromal cells of the uterus and oviduct. These results suggest that the appearance of the steroid receptors differ between the different cellular compartment of the reproductive organs. The proliferative activities have an inverse relation with the expression of the steroid hormone receptors in the female reproductive organs during developmental stages. Therefore, we propose that there is an autonomous proliferation mechanism in the development of the reproductive organs or that the proliferation is moderated by factors other than steroid hormones.     

Oxytocin receptors in rat oviduct.

Particles from rat oviduct homogenates sedimenting between 1,000 × g for 10 min and 48,000 × g for 30 min bound [³H]oxytocin $\text{in vitro}$. The apparent K_d for oxytocin binding to high affinity sites in particles prepared from estrogen-treated rats was 1.8 × 10^?9 M. About 215 fmoles of oxytocin were bound per mg of particulate protein. Oviducal preparations from untreated rats had about 25% the affinity for oxytocin of preparations from estrogen-treated rats. Oxytocin analogues were bound to oviducal particles in the same rank order as their uterotonic potencies: (desamino)oxytocin > (4-threonine)oxytocin > oxytocin > (8-lysine)vasopressin ? desaminotocinol. No oxytocin binding could be shown with the particulate fractions from rat ovary. The binding of oxytocin to the oviduct and uterus are similar in affinity, number of binding sites, ligand specificity, and the increase in response to estrogen treatment.     

Tubulin synthesis during ciliogenesis in the mouse oviduct.

We reported earlier that tubulin levels increase in the developing mouse oviduct during that period after birth when ciliogenesis is at a maximum (Staprans, I., and Dirksen, E. R. (1974) J. Cell Biol., 62, 164). To determine the degree to which de novo synthesis and tubulin pools contribute to this increase, [³H]leucine-incorporation experiments were performed in vivo and in culture. Soluble, particulate and axonemal fractions, obtained from homogenized oviducts of 3-, 5-, 8- and 12-day-old suckling mice, were electrophoresed on sodium dodecyl sulfate gels and the specific activity of the tubulin band determined. The present work shows that more than 90% of the tubulin in 3-day-old and 75% in 5-day-old mouse oviducts is synthesized de novo. From both the in vivo and in culture experiments we conclude that although tubulin pools are present in mouse oviduct, they are continuously being replenished by newly synthesized protein as there is a rapid outflow from the soluble and particulate to the axonemal fraction into structures such as basal bodies and cilia. This burst of de novo tubulin synthesis corresponds to evidence from electron microscopic autoradiography, where label is present to a greater extent over centriole precursors and basal bodies than over other cell organelles. [³H]leucine incorporation into tubulin was inhibited by cycloheximide, demonstrating that we are dealing with synthesis, while colchicine below 10^?3, M concentration had no effect on tubulin assembly into axonemes.     

Proteins in the luminal fluid from the bovine oviduct.

Oviducal fluid was collected by cannulation from four cows and by irrigation from fifteen slaughtered cows.The proteins in the fluid were examined by polyacrylamide gel electrophoresis at pH 4-5 and pH 8-9, isoelectric focusing on polyacrylamide, immunodiffusion, immunoelectrophoresis, affinity chromatography and gel filtration. The macromolecular components found were mainly serum proteins but small amounts of other proteins were detected in oestrous and dioestrous samples by electrophoresis at pH 8-9 following fractionation of the fluid by gel filtration or affinity chromatography. Small amounts of cathodically migrating proteins were detected directly by electrophoresis at pH 4-5 in dioestrous samples but not in oestrous samples. Determination of glycosidase activities revealed that the levels at oestrus were similar to the levels detected in serum. At dioestrus, the activities of B-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase were elevated.     

Estrogen receptor in hen oviduct chromatin, digested by micrococcal nuclease.

Nuclei from laying hen oviduct were prepared according to Hewish and Burgoyne i.e. in the presence of spermine and spermidine and in the absence of divalent cations and were then moderately digested by micrococcal nuclease. When the resulting chromatin was analysed by ultracentrifugation on a sucrose gradient, a peak of specific estradiol-binding sites was observed, sedimenting slightly faster (13-14 S) than the mononucleosomes (12 S). When the chromatin was centrifuged on a gradient containing heparin (5 microngram/ml) the sedimentation coefficient of the estradiol receptor peak shifted to 7-8 S; it returned to the 13-14 S position in the absence of heparin, when target organ chromatin was also present in the gradient. The preparation of the chromatin is described and the validity of the method to explore receptor localisation is discussed, as is the specificity of the receptor-DNA interaction.     

Immunoadsorption of specific chicken oviduct polysomes. Isolation of ovalbumin, ovomucoid, and lysozyme messenger RNA.

The messenger RNA coding for the egg white proteins ovalbumin, ovomucoid, and lysozyme were isolated by immunoadsorption of polysomes synthesizing these proteins. Monospecific antibodies against ovalbumin, ovomucoid, and lysozyme, raised in rabbits, were reacted with chicken oviduct polysomes. The antibody-polysome complexes were isolated by immunoadsorption onto sheep anti-rabbit antibodies coupled to an insoluble matrix. The specifically bound polysomes were eluted and the mRNA was obtained by poly(U)-Sepharose chromatography. The three specific RNAs were further purified by preparative gel electrophoresis. The purity of the mRNA preparations was demonstrated by analytical gel electrophoresis, the capability to direct the synthesis of specific protein products in a wheat germ cell-free system, and by hybridization to cDNA transcribed from mRNAoa and mRNAomu. Purified mRNAoa was shown to contain less than 0.1% mRNAomu and purified mRNAomu was about 99% pure with respect to mRNAoa. Purified mRNAly was contaminated with mRNAoa to 0.34% and with mRNAomu to 2.9%.