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L. P. Reynolds and S. P. Ford

Summary. The vasoconstrictor activity of the ovarian vascular bed in vitro was investigated during the oestrous cycle and early pregnancy. Gilts were killed during the follicular phase (Days 20 to + 1; N = 5) or luteal phase (Days 11 to 13; N = 4) of the oestrous cycle, or on Day 13 of pregnancy (N = 5). Immediately before death, a sample of vena cava blood was obtained for determination of progesterone and oestrogen (oestrone and oestradiol-17β) concentrations. One ovary was removed, cannulated, perfused in vitro, and subjected to 10-min infusions of saline (vehicle control) and noradrenaline. Vasoconstriction was provoked by electrical stimulation at the end of each infusion. Ovaries from luteal-phase gilts exhibited greater (P < 0·01) vasoconstriction than did ovaries from follicular-phase and pregnant gilts at the end of saline and noradrenaline infusions. The oestrogen to progesterone ratio was less (P < 0·01) for luteal-phase and pregnant than for follicular-phase gilts. Vasoconstriction was negatively correlated (r = −0·99, P < 0·01) with the oestrogen to progesterone ratio in systemic blood of gilts during the oestrous cycle but not during early pregnancy (r = +0·39, P > 0·10), possibly due to an effect of the conceptuses.

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L. P. Reynolds and D. A. Redmer

Summary. Samples of maternal and fetal placental tissues were obtained from cows on Days 100 (N = 4), 150 (N = 5), 200 (N = 6) and 250 (N = 6) of gestation and incubated for 24 h. Conditioned media from caruncular explants were mitogenic for bovine aortic endothelial cells (BAEC) on all days of gestation. Media from intercaruncular endometrium were stimulatory for proliferation of BAEC on Day 100 but inhibitory on Days 150, 200 and 250. Media from cotyledonary and intercotyledonary tissues inhibited proliferation of BAEC on all days. Caruncular-conditioned media stimulated migration of BAEC on Days 150, 200 and 250. Cotyledonary-conditioned media inhibited migration of BAEC on all days. Effects of media from intercaruncular and intercotyledonary tissues on migration of BAEC varied with stage of gestation. Angiogenic activity of media from caruncular (all stages) and intercaruncular (Day 100) tissues appeared to have an M r > 100 000. In cows, therefore, the maternal placentome (caruncle) appears to be the primary source of placental angiogenic activity throughout gestation. The fetal placentome (cotyledon) secretes activity which inhibits two major components of angiogenesis (proliferation and migration of endothelial cells) throughout gestation. Intercaruncular and intercotyledonary tissues may modulate placental angiogenesis throughout gestation. Placental vascular development in the cow is therefore probably controlled by an interaction between stimulatory and inhibitory factors produced by the placenta itself.

Keywords: angiogenic factor; placenta; gestation; cow

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L. P. Reynolds, D. A. Robertson and S. P. Ford

Summary. Non-pregnant heifers (4/group) received intrauterine infusions of vehicle, oestradiol-17β (150 ng), PGE-2 (250 μg), or oestradiol + PGE-2 every 6 h from 12:00 h on Day 13 to 06:00 h on the day of subsequent oestrus or 06:00 h on Day 21 (day of oestrus = Day 0). Ten of 12 heifers receiving vehicle, oestradiol or PGE-2 returned to oestrus by Day 21, whereas none of the heifers receiving oestradiol + PGE-2 returned to oestrus by Day 21. Jugular venous progesterone concentrations of vehicle- and PGE-2-treated heifers declined rapidly after Day 15 and were basal (< 1 ng/ml) by Day 20. For heifers receiving oestradiol infusions, systemic progesterone levels did not decline until after Day 18, but were again basal by Day 20. Heifers treated with oestradiol + PGE-2 maintained elevated systemic progesterone levels until Day 21 after oestrus. In addition, the corpora lutea of the heifers treated with oestradiol + PGE-2 were heavier (P < 0·01) and contained more (P < 0·05) progesterone than did corpora lutea of the heifers in the other 3 groups on Day 21 (3·4 g and 19·52 μg/g and 1·2 g and 1·65 μg/g, respectively). It is concluded that oestradiol-17β and PGE-2, both of which are produced by the bovine conceptus and secreted from the gravid uterus, may act synergistically to maintain luteal function during early pregnancy in the cow.

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W. A. Ricke, D. A. Redmer and L. P. Reynolds

Corpora lutea were obtained from gilts on days 2, 4, 8,12,15 or 18 after oestrus. Luteal fresh masses and DNA contents increased linearly (P < 0.01) from day 2 to day 12 and day 2 to day 15, respectively. Changes in the ratio of protein:DNA were greatest between days 2 and 4 and days 15 and 18, whereas changes in DNA content were relatively small during the same intervals. Thus, a major component of changes in the size of the corpus luteum during the early and late periods of the luteal phase was cellular hypertrophy. Proliferation of luteal cells in vivo (nuclear incorporation of 5-bromo-2-deoxyuridine, a thymidine analogue) was greatest on day 2 and decreased exponentially (P < 0.01) throughout the oestrous cycle. Results from co-localization of 5-bromo-2-deoxyuridine and factor VIII (von Willebrand factor), a marker of endothelial cells, or 5-bromo-2-deoxyuridine and 3β-hydroxysteroid dehydrogenase, a marker of steroidogenic cells, indicated that some of the luteal steroidogenic cells proliferated early in luteal development. However, during early and mid-cycle, most of the luteal cell proliferation occurred in the endothelial cells. Thus, during growth of the pig corpus luteum, which is extremely rapid, most of the proliferating luteal cells are vascular endothelial cells. This observation is consistent with the high vascularity and blood flow of the mature corpus luteum and implies a critical role for angiogenesis in luteal development in the pig, as has been proposed for several other mammalian species.

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D. S. Millaway, D. A. Redmer, J. D. Kirsch, R. V. Anthony and L. P. Reynolds

Summary. In Study 1, explants of caruncular and intercaruncular endometrium and fetal membrane were collected from ewes (5-6/day) on Days 11–13, 16–18 and 21–23 after mating and Days 10–12 after oestrus, and incubated for 24 h. Explant-conditioned media were evaluated for their effects on endothelial cell proliferation. Both caruncular and intercaruncular endometrium secreted factor(s) which stimulated endothelial cell proliferation, and which appeared to be > 100 × 103 M r and heat-labile. In Study 2, conditioned media from explant incubations of caruncular and intercaruncular endometrium, cotyledon and intercotyledonary fetal membrane obtained from ewes (6-7/day) on Days 40,65,90,115 and 140 after mating were evaluated for their effects on endothelial cell proliferation. Caruncular and intercaruncular endometrium and intercotyledonary fetal membrane secreted factor(s) which inhibited endothelial cell proliferation. Media from cotyledonary explants tended to stimulate endothelial cell proliferation on Day 115. Conditioned media from cotyledonary explants obtained from 3 additional ewes at Day 120 of gestation stimulated endothelial cell proliferation, and this activity also appeared to be > 100 × 103 M r. Placental angiogenesis in ewes therefore appears to be modulated by both maternal and fetal placental tissues via stimulatory and inhibitory factors.

Keywords: angiogenesis; endothelial proliferation; placenta; ewe; gestation

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D. A. Redmer, A. T. Grazul, J. D. Kirsch and L. P. Reynolds

Summary. Samples from corpus haemorrhagicum, mid-cycle corpus luteum (CL) and late-cycle CL were tested for their abilities to stimulate neovascularization of chorioallantoic membranes (CAM) of developing chicks. Responses were graded from 0 to 4 (4 being the greatest response). Luteal tissue implants from each stage of the oestrous cycle stimulated growth of CAM blood vessels, and vascular responses increased with age of CL. Implants from late-cycle CL were typically graded 3 or 4. Luteal tissues from several stages of development were also incubated for 6 h in serum-free medium containing no hormone, LH, PGF-2α or both hormones. Media conditioned by luteal tissues were assayed for progesterone and tested for their ability to stimulate mitogenesis and migration of bovine aortic endothelial cells in vitro. All media conditioned by luteal tissues stimulated mitogenesis and migration of endothelial cells, but media from late-cycle CL exhibited the greatest activity. Luteinizing hormone significantly increased in-vitro secretion of a factor(s) that stimulated migration of endothelial cells. PGF-2α alone had no effect on production of endothelial cell mitogen or migrationstimulating factor(s) from luteal incubations; however, the ability of LH to enhance secretion of the migration-stimulating factor(s) was blocked by PGF-2α. This study demonstrates that angiogenic activity of bovine luteal tissues increases with age of the CL and in-vitro secretion of angiogenic factor is responsive to hormones known to regulate luteal function.

Keywords: angiogenesis; corpus luteum; cow; oestrous cycle

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L. P. Reynolds, D. S. Millaway, J. D. Kirsch, J. E. Infeld and D. A. Redmer

Summary. In Exp. 1, maternal (caruncle) and fetal (cotyledon) portions of the placenta as well as uterine endometrium were obtained from cows at mid-gestation and evaluated for angiogenic activity by placing tissue samples on chick chorioallantoic membranes (CAM). Only caruncular tissues exhibited angiogenic activity in the CAM assay. In Exp. 2, lyophilized homogenates of caruncular tissues obtained from cows at mid-gestation were evaluated for angiogenic activity on CAM and for their ability to stimulate mitosis of bovine aortic endothelial cells in vitro. Homogenates of caruncular tissues again were angiogenic on the CAM and also were mitogenic for endothelial cells. In Exp. 3, maternal (caruncle and endometrium) and fetal (cotyledon and fetal membrane) portions of the placenta were obtained from cows at mid-gestation and fine minces (explants) of each were cultured for 24 h. Explant-conditioned media were then tested for angiogenic activity by their abilities to stimulate mitosis and migration of bovine aortic endothelial cells in vitro. Conditioned media from caruncular explants, but not from explants of other tissues, exhibited both mitogenic and migration-stimulating activities. When pools of caruncular explant-conditioned media were fractionated by ultrafiltration, mitogenic activity was not present in fractions of Mr < 10 000, < 30 000 and < 100 000, but was retained in fractions of Mr > 10 000, > 30 000 and > 100 000. Mitogenic activity was not observed in any fractions subjected to heat treatment. On the basis of these data, we conclude that, during mid-gestation, angiogenic activity of bovine placenta is associated primarily with the maternal caruncular portion of the placenta which, therefore, may direct growth of the placental microvasculature. This angiogenic activity seems to have a molecular weight of > 100 000 and be heat labile.

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L. P. Reynolds, D. S. Millaway, J. D. Kirsch, J. E. Infeld and D. A. Redmer

Summary. Weight of placental tissues of cows increased exponentially from Day 100 to Day 250 of gestation, but at much slower relative and absolute rates than fetal weight. In addition, growth rate of fetal placental tissues was less than that of maternal placental tissues. Concentrations of DNA, RNA and protein, however, increased in fetal placental but not in maternal placental tissues. Fetal placental tissues therefore exhibited hyperplasia, which probably contributes to increased functional capacity of the placenta during late gestation. The rate of O2 uptake in vitro was greatest for maternal placental tissues, suggesting that the maternal portion of the placenta accounts for most of the large rate of placental O2 utilization in vivo. Compared with other placental tissues, rate of secretion of macromolecules by intercaruncular endometrium was high, but decreased from Day 100 to 250, suggesting that uterine glandular secretory activity may decrease as gestation advances. Rate of secretion of macromolecules also was high for intercotyledonary tissues and increased with day of gestation, suggesting a role for secretory products of chorioallantois in gravid uterine function.

Keywords: placenta; growth; metabolism; cow; gestation

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D. A. Redmer, Y. Dai, J. Li, D. S. Charnock-Jones, S. K. Smith, L. P. Reynolds and R. M. Moor

The corpus luteum undergoes tremendous growth, development and regression each oestrous or menstrual cycle. These changes are reflected by equally impressive growth and regression of the luteal vasculature. We have previously shown that angiogenic factors from corpora lutea are primarily heparin binding and that one of these factors is similar to vascular endothelial growth factor (VEGF). In an effort to identify this factor, and to define its role in luteal vascular development, the cDNA for the coding region of ovine VEGF was sequenced and a sensitive RNase protection assay was developed to quantitate mRNA encoding VEGF in luteal tissues from ewes in the early (days 2–4), mid- (day 8) and late (days 14–15) stages of the oestrous cycle. In addition, an N-terminal peptide was synthesized from the translated ovine cDNA sequence for VEGF and an antiserum was raised against this peptide for use in western immunoblotting procedures. Nested reverse transcriptase (RT)-PCR of RNA from ovine corpora lutea resulted in three products that correspond in size to the alternatively spliced variants of VEGF VEGF120, VEGF164, and VEGF188) predicted from other species. The RNase protection assay revealed that the proportion of mRNA encoding VEGF was 2- to 3-fold greater on days 2–4 than on day 8 or days 14–15. Densitometric analysis of gels from the RNase protection assay showed that VEGF120 represented approximately one third of the total mRNA encoding VEGF in the corpus luteum and that this proportion did not vary with stage of the oestrous cycle. SDS-PAGE and western immunoblot analysis of a homogenate from corpora lutea showed a single 18 kDa protein. These data demonstrate that VEGF is expressed in luteal tissue throughout the ovine oestrous cycle and that expression of mRNA encoding VEGF is upregulated during the period of rapid luteal development, when luteal vascular growth is at its maximum.

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Alan J Conley, Erin L Legacki, C Jo Corbin, Scott Stanley, Carl R Dahlen and Lawrence P Reynolds

Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3β-hydroxysteroid dehydrogenase (3βHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3βHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3βHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3βHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.