Use of prostheses made of ePTFE in anastomoses of the rat uterine horn

ePTFE prostheses were used in the microsurgical anastomosis of the rat uterine horn. Handling characteristics of this synthetic material during the performance of anastomosis were tested. Patency rates, infections and local tissue reaction were evaluated after thirty and sixty days. Connective tissue infiltration and incomplete endothelialization by normal endometrial elements were observed on the luminal surface of the prosthesis.     

The source of the 5-alpha-pregnanes that occur during gestation in mares.

[1,2,6,7-3H]Progesterone was injected into the uterine artery of umbilical vein of 4 pregnant Ponies to determine whether 5 alpha-pregnane-3,20-dione (DHP), 20 alpha-hydroxy-5 alpha-pregnan-3-one (20 alpha-ol), and 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-ol) are produced by the placenta, fetus, or mare during late gestation. Plasma samples were collected from indwelling catheters in the uterine artery and vein and the umbilical artery and vein at frequent intervals until 6 h after isotope injection. The plasma samples were extracted with organic solvents and the respective pregnanes were isolated by thin-layer chromatography. Mean plasma levels of radioactive DHP in the umbilical vein were greater (P less than 0.05) than those in the umbilical artery but were similar to those in the uterine artery and vein. Plasma levels of radioactive 20 alpha-ol in the umbilical artery and vein were less (P less than 0.05) than those in the uterine artery but similar to those in the uterine vein. Levels of radioactive 3 beta-ol in the umbilical artery were greater (P less than 0.05) than those in uterine artery and vein and similar to those in the umbilical vein. We suggest that DHP was produced by the placenta and a maternal source, that 20 alpha-ol was primarily of maternal origin, and that 3 beta-ol was formed primarily by the fetus.     

Fetal-maternal secretion of prostaglandins in the cow

A study was conducted to measure the blood plasma concentrations of prostaglandin F2 alpha (PGF2 alpha), 13,14-dihydro-15-keto-prostaglandin F (PGFM), 6-keto-prostaglandin F1 alpha (6-keto), prostaglandin uterine artery, uterine vein, umbilical artery and umbilical vein in 24 cows from days 80 to 260 of pregnancy. Blood was collected during surgery and all prostaglandins were measured using specific radioimmunoassay procedures. Results indicate that PGF2 alpha blood levels are higher in the umbilical vessels and uterine vein than in the ovarian vein and uterine artery. PGFM and PGE2 showed a trend towards higher values in the umbilical than in the maternal vessels, but the levels of 6-keto and TBX2 were not different among the vessels studied. No differences across time could be observed in any of the prostaglandins measured, partly due to the great variability in blood levels among animals during the same stage of pregnancy.     

Uterine blood flow, oxygen and glucose uptakes at mid-gestation in the sheep

In early ovine fetal development, the placenta grows more rapidly than the fetus so that at mid-gestation the aggregate weight of placental cotyledons exceeds fetal weight. The purpose of this study was to compare two separate methods of measuring uterine blood flow and glucose and oxygen uptakes in seven mid-gestation ewes, each carrying a single fetus. Uterine blood flow to both uterine horns was measured by microsphere and by tritiated water steady-state diffusion methodology. Calculations of tritiated water blood flows and oxygen and glucose uptakes were based on measurements of arteriovenous concentration differences across each uterine horn. The distribution of blood flow and oxygen uptake between the two uterine horns was strongly correlated with placental mass distribution. The two methods gave comparable results for uterine blood flow (457 +/- 35 vs 476 +/- 35 ml/min), oxygen uptake (457 +/- 35 vs 476 +/- 35 mumol/min), and glucose uptake (63 +/- 8 vs 64 +/- 6 mumol/min). Uterine blood flow was approximately 38% of the late gestation value and 56.1 +/- 1 times higher than umbilical blood flow. Uteroplacental oxygen consumption was about 58% of late gestation measurements and 3.9 +/- 0.5 times higher than fetal oxygen uptake. We confirm that the large placental mass of mid-gestation is associated with high levels of maternal placental blood flow and placental oxidative metabolism.     

Effect of side of insemination on transuterine transport of spermatozoa in superovulated dairy cattle

Eleven superovulating Friesian lactating cows were inseminated deep into one uterine horn with one unit of frozen semen, containing 2.5 million total spermatozoa, with more than 40% postthaw progressive motility and with 14% morphologically abnormal spermatozoa. Semen was deposited into the right or left uterine horns of alternate cows. There was no difference in the proportions of fertilized recovered ova from ipsilateral horns between right and left inseminations (P>0.05). The fertilization rate in the contralateral horns was higher (P<0.01) for right uterine horn insemination (50%) than for left uterine horn insemination (15.6%).     

Progesterone and placentation increase secreted phosphoprotein one (SPP1 or osteopontin) in uterine glands and stroma for histotrophic and hematotrophic support of ovine pregnancy

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.     

Poly(styrenesulfonate)/poly(allylamine) multilayers: a route to favor endothelial cell growth on expanded poly(tetrafluoroethylene) vascular grafts

Small-diameter synthetic vascular grafts of expanded poly(tetrafluoroethylene) (ePTFE) polymer concern one of the most common alternatives for the replacement of diseased vessels. However, high failure rates arise especially due to the lack of endothelial cells (ECs). EC seeding was developed to build a monolayer on the luminal surface. Because ECs show little or no adhesion on synthetic prostheses, it is necessary to promote their retention. On ePTFE surfaces we successfully deposited polyelectrolyte multilayer films (PMFs) consisting of poly(ethylenimine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) to obtain PEI-(PSS-PAH)3 films. EC adhesion and spreading on modified ePTFE were assessed by scanning electron and confocal microscopies. Cell viability was evaluated by Alamar Blue assay. After 7 days of culture, the ePTFE modified with PMF exhibited improvements of EC viability as compared to that of the controls (nonmodified ePTFE) or even ePTFE coated by a PAH monolayer (p < 0.05). Moreover, the spreading of ECs was largely enhanced compared to that of the same controls, resulting in a healthy confluent cell monolayer formation. Positive staining for the von Willebrand factor confirmed the EC phenotype. Promoting EC attachment and function on ePTFE modified with PMFs could become in the future a promising treatment for synthetic small-diameter vascular grafts.     

Fetal-maternal secretion of prostaglandins in the cow

A study was conducted to measure the blood plasma concentrations of prostaglandin F₂α (PGF₂α), 13,14-dihydro-15-keto-prostaglandin F (PGFM), 6-keto-prostaglandin F₁α (6-keto), prostaglandin E₂ (PGE₂), and thromboxane B₂ (TBX₂) in the ovarian vein, uterine artery, uterine vein, umbilical artery and umbilical vein in 24 cows from days 80 to 260 of pregnancy. Blood was collected during surgery and all prostaglandins were measured using specific radioimmunoassay procedures. Results indicate that PGF₂α blood levels are higher in the umbilical vessels and uterine vein than in the ovarian vein and uterine artery. PGFM and PGE₂ showed a trend towards higher values in the umbilical than in the maternal vessels, but the levels of 6-keto and TBX₂ were not different among the vessels studied. No differences across time couls be observed in any of the prostaglandins measured, partly due to the great variability in blood levels among animals during the same stage of pregnancy.     

Effect of the bovine viral diarrhea (BVD) virus on preimplantation bovine embryos: A preliminary study

Five crossbred-Holstein cows, approximately three to seven years of age, were superovulated using pregnant mare's serum gonadotropin (PMSG) and prostaglandin F_2α (Prostin F_2α). At the induced estrus, each cow was artificially inseminated with frozen semen. Seven days after insemination, the lumen of the right uterine horn of each cow was inoculated with BVD virus in Eagle's minimum essential tissue culture medium, and the lumen of the left horn was infused with tissue culture medium only. Three days later, each cow was subjected to midventral laparotomy under general anesthesia and embryos were collected. A total of 22 embryos were recovered; 12 were from infected uterine horns and ten were from non-infected uterine horns. All embryos from the non-infected uterine horns were in the late blastocyst stage without the zona pellucida. Of the embryos collected from the infected uterine horns, eight of 12 (66.6%) still possessed zona pellucida and appeared in a degenerative state. The remaining four embryos were morphologically similar to those from the non-infected uterine horns. Electron microscopic examination of the degenerated embryos from the infected uterine horns demonstrated the presence of a structure which morphologically resembled the BVD virus. The results of this preliminary study indicate that the BVD virus within the uterine horns may interfere with normal development of preimplantation bovine embryos. Therefore, it is proposed that the BVD virus could adversely affect early stages of gestation in the cow, resulting in infertility.     

Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels

Cyclic changes in estrogen receptor expression in the uterine tissue of 60 female dogs were evaluated, using an immunohistochemical technique on formalin-fixed paraffin-embedded sections. The expression of estrogen receptors in the uterine horns, body and cervix was quantified by means of an immunohistochemical score. A negative correlation was found between staining scores in the uterine horns and serum progesterone levels. Generally, staining scores in the uterine horns were highest during proestrus, declined during estrus and were lowest during early metestrus. During anestrus high staining scores for estrogen receptors were observed, indicating sensitivity for estrogens in a sexual quiescence stage. Compared with the uterine horns, high staining scores were found in the uterine body and cervix during estrus and metestrus. No positive staining for estrogen receptors was detected in 1 pregnant uterus. Fluctuations in estrogen receptors were more pronounced in endometrial stroma cells than in epithelial cells of the uterine horns. The importance of stromal cells in the sexual cyclicity of the canine uterus should not be underestimated when studying uterine endocrinology and pathology.     

Fetal and uteroplacental heat production in sheep

To separate heat production of the fetus from that of the placenta, endometrium, and uterine muscle, we measured total uterine heat production first with the fetus intact and then after the umbilical cord was snared and the fetus killed. Heat production was measured with the Fick principle using thermistors chronically implanted in a maternal artery and major uterine vein and a flowmeter placed on the common internal iliac artery. In nine ewes, carrying lambs weighing 4.46 +/- 0.42 (SE) kg, total uterine heat production fell from 10.6 to 2.9 W after fetal death. Uterine blood flow fell progressively to 90% of control levels during the first hour after death. The caloric equivalent for O2 averaged 4.1 cal/ml O2 for the uterus, 2.2 for the uteroplacenta, and 4.6 for the fetus per se. It was not possible to explain these results using a simple model of maternal-fetal heat transfer. Rather, it was necessary to assume an additional pathway for heat transfer between small uterine veins on the surface of the uterus and cooler structures in the maternal abdomen, presumably the ventral abdominal wall.     

Unilateral luteotropic effect of uterine venous effluent of a gravid uterine horn in sheep.

The involvement of the main uterine vein in the unilateral maintenance of CL was studied in bilaterally ovulating unilaterally pregnant ewes. Ewes were mated at estrus (Day 0) and bilaterally ovulating ewes were randomized into three groups at surgery on Day 5. In all ewes, the uterine horns were separated through the intercornual area and one was ligated and transected near the internal bifurcation to produce a nongravid horn. One group served as controls (five ewes). In the other two groups the main uterine vein on one side was surgically anastomosed (end to side) to the corresponding vein of the opposite side (gravid side to nongravid side in one group--three ewes, and nongravid side to gravid side in the other--three ewes). Necropsies were done on Day 20. Mean CL weight was less, (P less than .01) on the nongravid side in control ewes than on the gravid side in control ewes or for either side in the other two groups. There were no significant differences among mean weights of CL on the gravid side in control ewes and either side in the other two groups. The CL regressed when the ipsilateral uterine vein contained blood from only the nongravid horn whereas the CL was maintained when the ipsilateral uterine vein contained venous blood from a gravid horn, whether or not it also contained blood from a nongravid horn. Results indicate that the uterine venous effluent from a gravid uterine horn in sheep has a luteotropic effect on the ipsilateral CL.     

Unilateral uteroovarian relationship in pregnant cattle and role of uterine vein

The hypothesis of a unilateral uteroovarian relationship between location of embryos and maintenance of corpora lutea (CL) was tested in superovulated cattle. Ovulations were induced in both ovaries and a uterine horn was isolated subsequent to insemination to produce unilateral pregnancy. The mean weight of CL on day 24 was greater (P<.05) on the gravid side (2580 mg) than on the nongravid side (1200 mg; n=5), supporting the hypothesis. Involvement of the main uterine vein in the unilateral pathway between a gravid horn and the adjacent ovary was demonstrated in an experiment which utilized separation of uterine horns, anastomosis of uterine veins between sides, and insertion of embryos into one side by embryo transfer. Mean weight of CL on day 24 was less (P<.05) when the gravid horn was contralateral to the CL (950 mg; n=3), than when the gravid horn was ipsilateral to the CL (4760 mg) or when the gravid horn was contralateral to the CL and the uterine vein from the contralateral side was anastomosed to the vein on the ipsilateral side (3580 mg; n=3).     

A simple method for removing the luminal epithelium of the mouse uterus for biochemical studies.

The luminal epithelium of the mouse uterus was removed simply and rapidly by opening the uterine horns longitudinally and 'vortexing' them in buffer with glass balls. This procedure, which requires no specialized apparatus, was developed for use with uteri in different hormonal states, and 2 uterine horns/tube, 5 glass balls and vortexing for 2 min are suggested for routine use. Under these conditions, 79--100% of the cells were removed, yielding epithelial fractions of 65--90% purity. The suspension of cell constituents recovered contained intact nuclei which could be further purified. Extensive histological examination demonstrated that the other uterine tissues were minimally damaged.     

Concentrations of spermatozoa in the vagina of heifers after deposition of semen in the uterine horns, uterine body or cervix

In Exp. I, virgin Holstein heifers (N = 18) were induced into oestrus with PGF-2 alpha. Animals which stood to be mounted were paired for insemination approximately 8 h later with 56.1 x 10(6) spermatozoa from a single bull. Semen was deposited in the uterine body of one female. Each matched female was inseminated by deposition of one-half of the inseminate into the right uterine horn and one-half into the left uterine horn approximately 7.0 cm anterior to the internal cervical os. In Exp. II, additional heifers (N = 18) were induced into oestrus and inseminated by deposition into the uterine horns or cervix (2.0 cm anterior to the external cervical os). A 1.0 ml aspirate of vaginal mucus was collected at hourly intervals for 8 h after insemination. Concentration of spermatozoa was determined by haemocytometry. In Exp. I, cumulative percentage spermatozoa recovered in an 8 h collection period were similar (P greater than 0.10) for insemination into the uterine horns (17.9 +/- 2.9%) and uterine body (18.5 +/- 4.5%). In Exp. II, cumulative % sperm recovery from the vagina was greater (P less than 0.10) for cervical deposition (59.1 +/- 14.1%) than for that into the uterine horns (30.9 +/- 7.8%). In Exp. II, the insemination treatment x hour of sample interaction was significant (P less than 0.08). Recovery of spermatozoa from the vagina was greatest (P less than 0.05) within 3 h after cervical insemination (31.4 +/- 9.9% compared to 9.4 +/- 2.5% for uterine horn deposition). Percentage recovery of spermatozoa from the remaining hourly collections were similar (P greater than 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)     

The position effect in mice on day 14

Previous publications have produced conflicting results concerning the effect and nature of uterine horn position on embryonic weight and development in the uterus bicornis. For this investigation we used 503 embryos from 41 litters of NMRI - mice on gestational day 14. The relative position of each embryo was determined by measuring the distance from the center of each placenta to the cervix in relation to the total length of the uterine horn. After cessation of shrinkage due to fixation the umbilical cord and remaining membranes were removed under the dissecting microscope and the embryos were weighed to the nearest 0.1 mg. Analysis of potential embryonic positions along the uterine horns indicated that 10% at the extreme ovarian and cervical ends were almost devoid of implantations. A mixed nested analysis of variance was used as the appropriate model to test a position on weight effect. Not only was a position effect found but, in contrast to other studies, it is also quantitatively represented. Embryos were lighter both at the ovarian and cervical ends, the mean weight difference being in the range of less than 10% of the average embryo weight.     

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

Levels of prostaglandin F (PGF) in bovine endometrium uterine venous, ovarian arterial and jugular plasma during the estrous cycle (38789).

Prostaglandins F (PGF) were measured in uterine vein, ovarian artery, and jugular vein plasma and in the endometrial tissues at various times during the bovine estrous cycle, and were compared to peripheral plasma progesterone levels. Four groups of heifers at days 1-5, 10-14, 15-17 and 20-0 of the estrous cycle were studied. Low levels of PGF (48 plus or minus 12 ng/g dry tissue were measured in the endometrium on days 1-14 of the cycle. Higher values (131 plus or minus 9.0) were found at days 15 until the day of estrus (p less than 0.001). Similarly, very low levels of PGF were observed in the uterine vein plasma at days 1-14 (0.162) plus or minus 0.044) ng/mlM plus or minus S.E.), whereas on days 15 until the day of estrus the levels ranged from 1.5 to 3.0 ng/ml. The increases in uterine vein PGF on day 15 occurred even while peripheral plasma progesterone levels were still high. However, PGF was not elevated in either the ovarian artery or the jugular vein at any time during the cycle, even when uterine vein levels were greatly elevated. No differences in PGF content were detected in endometrial tissue from uterine horns adjacent or opposite to the functional corpus luteum.     

Dietary melatonin supplementation alters uteroplacental amino acid flux during intrauterine growth restriction in ewes

Dietary melatonin supplementation during mid- to late-gestation increased umbilical artery blood flow and caused disproportionate fetal growth. This melatonin-induced increase in umbilical artery blood flow may alter nutrient availability to the fetus, which may lead to alterations in fetal size. The objectives of the current experiment were to determine amino acid (AA) and glucose concentrations as well as AA and glucose flux across the uteroplacenta using a mid- to late-gestation model of intrauterine growth restriction supplemented with dietary melatonin as a 2 × 2 factorial design. At day 50 of gestation, 32 ewes were supplemented with 5 mg of melatonin (MEL) or no melatonin (CON) and were allocated to receive 100% (adequate; ADQ) or 60% (restricted; RES) of nutrient requirements. On day 130 of gestation, uterine and umbilical blood flows were determined via Doppler ultrasonography during a non-survival surgery. Blood samples were collected under general anesthesia from the maternal saphenous artery, gravid uterine vein, umbilical artery, and umbilical vein for AA analysis and glucose. Total α-AA concentrations in maternal artery and gravid uterine vein were decreased (P < 0.05) in RES v. ADQ fed ewes. Maternal arterial − venous difference in total α-AA was increased (P ⩽ 0.01) in RES v. ADQ fed ewes, while total uterine α-AA flux was not different (P > 0.40) across all treatment groups. Fetal venous − arterial difference in total α-AA as well as uteroplacental flux of total α-AA were decreased (P < 0.05) in CON-RES v. CON-ADQ, and similar (P > 0.20) in MEL-RES v. CON-ADQ. Maternal concentrations and uterine flux of branched-chain AA (BCAA) were not different across all treatment groups; however, fetal uptake of BCAA was decreased (P < 0.05) in CON-RES v. CON-ADQ, and similar (P > 0.20) in MEL-RES v. CON-ADQ. Uterine uptake of glucose was not different (P ⩾ 0.08) across all treatment groups, while uteroplacental uptake of glucose was increased (P ⩽ 0.05) in RES v. ADQ ewes. In conclusion, maternal nutrient restriction increased maternal arterial − venous difference in total α-AA, while total uterine α-AA flux was unaffected by maternal nutrient restriction. Melatonin supplementation did not impact maternal serum concentrations or uterine flux of glucose or AA; however, melatonin did improve fetal BCAA uptake during maternal nutrient restriction.     

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.