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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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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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R. K. Christenson, J. J. Ford and D. A. Redmer

Summary. The crossbred gilts studied were aged 80 days (prepubertal), 180 days (prepubertal or postpubertal) and 260 days (postpubertal or pregnant). Estimates of metabolic clearance rate (MCR) of oestradiol and progesterone were consistently less (21 and 27%) in plasma than in blood, and these differences were not influenced by age of gilt. The MCR (1/day per kg body weight) for oestradiol and progesterone in plasma was greater (P < 0·05) for 80-day-old prepubertal gilts than for older gilts. The MCR values of oestradiol and progesterone were similar in 180-day-old and 260-day-old gilts independent of reproductive state. Production rate (PR) of oestradiol and progesterone increased with age (80–180 days), and age and reproductive state differences were much more pronounced for PR of progesterone than of oestradiol. These results support the hypothesis that a reduction in the MCR and an increase in PR of oestradiol and progesterone in the gilt are associated with the process of pubertal development, and changes in gonadal steroid concentrations appear not to alter the MCR of oestradiol and progesterone.

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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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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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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 M r < 10 000, < 30 000 and < 100 000, but was retained in fractions of M r > 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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M Carvalho, L Mateus, F Afonso, S Van Harten, L Alfaro Cardoso, D A Redmer and G Ferreira-Dias

The objective of this study was to evaluate the effects of two different levels of food restriction on testicular angiogenic activity, microvascularization, tissue growth, and regression, using the rabbit as a study model. The rabbits (Oryctolagus cuniculus cuniculus) were randomly assigned to a control group (A, n=5), fed ad libitum, and to groups B (n=5) and C (n=5), with two different levels of food restriction. Food restriction was responsible for a 21.2% decrease in body weight in group B and 34.7% in group C. Testis explants were cultured for 24 h and conditioned media were tested for their ability to stimulate mitogenesis of bovine aortic endothelial cells (BAEC). There was an increase in testicular microvascular area and mitogenesis of BAEC in group C rabbits. Despite no change in testicular DNA concentration among groups, food restriction decreased both RNA and protein compared with control. No treatment differences in the percentage of seminiferous tubules filled with all stages of spermatogenesis (spermatogonia, spermatocytes, and spermatids) and spermatozoa, as well as the area occupied by seminiferous tubules, were observed. Nevertheless, serum testosterone was markedly less in group C compared with groups A and B. These results suggest that angiogenesis may play a role in overcoming testicular nutritional impairment in rabbits subjected to food restriction.