Angiogenic activity of bovine corpora lutea at several stages of luteal development

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-2 alpha 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-2 alpha alone had no effect on production of endothelial cell mitogen or migration-stimulating factor(s) from luteal incubations; however, the ability of LH to enhance secretion of the migration-stimulating factor(s) was blocked by PGF-2 alpha. 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.     

Luteal function of induced corpora lutea in the bitch

Nineteen anestrous bitches with a mean of 22 kg body weight and ranging from 2 to 4 years of age were induced to exhibit estrus and ovulate using PMSG and HCG. Twelve days after the first day of estrus, bitches were assigned to four treatment groups. Group (A) consisted of six bitches, Group (B) of five bitches and Groups (C) and (D) of four bitches each. At this time, bitches in Groups (A), (B) and (C) were laparotomized and those assigned to Groups (A) and (B) were bilaterally hysterectomized leaving the cervix and oviducts intact. Although bitches in Group (C) were laparotomized, they were not hysterectomized. Group (D) bitches were not subjected to any surgical procedures. Homologous uterine extract was prepared from each bitch in Group (A) and administered intramuscularly beginning on day 25 (day 0 = first day of estrus) and continued every other day for 61 days post-estrus. Bitches in Group (B) were similarly injected with equal volumes of 0.9% saline. Blood samples, obtained prior to laparotomy and every other day for 85 days thereafter, were assayed for plasma progesterone concentrations using radioimmunoassay. One bitch in each of Groups (A) and (D) did not form luteal tissue following treatment with PMSG and HCG although both bitches exhibited estrus following treatment. All other bitches showed an increase in progesterone levels (4 to 19 ng/ml) between the first day of estrus and 10 days post-estrus. Thereafter, progesterone levels progressively declined in all groups with levels below 1 ng/ml between 38 to 40 days post-estrus. Results of this study suggested that CL formed in the bitch following PMSG and HCG treatment have a reduced function compared to non-induced CL of a normal, non-fertile estrous cycle. Such premature CL regression appears to be independent of the presence or absence of the uterus.     

Inhibition of estrus and corpora lutea function with Norgestomet

Forty-five nonpregnant, nonlactating, Angus and Brangus cows were utilized to determine how long a Norgestomet ear implant would inhibit estrus when administered at various stages of an estrous cycle. All cows completed a nontreated estrous cycle to ensure normal cyclicity. At the second observed estrus (estrus = Day 1), cows were randomly allotted to be treated at metestrus (Day 3 or Day 4, n = 15); at diestrus (Day 9 or Day 10, n = 14); or at proestrus (Day 15 or Day 16, n = 16). All cows received a 2-ml intramuscular injection of 3 mg of Norgestomet accompanied by a 6-mg Norgestomet ear implant, which remained in situ for 21 days, or until individual cows were observed in estrus. Estrus was inhibited for a mean (+/- SEM) of 18.7 +/- 0.7, 19.9 +/- 0.8, and 17.0 +/- 0.8 days, respectively, when cows were treated at metestrus, diestrus, and proestrus (metestrus and diestrus vs proestrus; P < 0.05). Estrus was inhibited for an entire 21-day implantation period in 27, 50, and 38% of cows treated at metestrus, diestrus, and proestrus, respectively (P > 0.10). Norgestomet inhibited estrus in all cows for 11, 17, and 11 days after implantation when treatment was initiated at metestrus, diestrus, and proestrus, respectively (P > 0.10). These data indicate that a 6-mg Norgestomet ear implant effectively inhibits estrus in all cows for a maximum of 11 days, with some cows exhibiting estrus by Day 12 with the Norgestomet implant in situ.     

Formation and function of GnRH-induced subnormal corpora lutea in cyclic ewes

Of 19 dioestrous ewes given 50 micrograms GnRH on Day 10 of the oestrous cycle, 15 (79%) formed corpora haemorrhagica within 2 days after injection of GnRH. After excision of the Day 10 spontaneous CL, the GnRH-induced CL were short lived when compared to spontaneous CL in saline-treated ewes (3.1 +/- 0.4 vs 17.3 +/- 0.3 days, respectively). Hysterectomy of ewes bearing the GnRH-induced CL prevented regression of the short-lived CL, thus extending functional lifespan greater than or equal to 38 days. Serum concentrations of progesterone produced by the GnRH-induced CL in hysterectomized ewes were less than those observed during a comparable interval (Days 7-14) in saline-treated, non-hysterectomized ewes (2.24 +/- 0.1 vs 3.67 +/- 0.15 ng/ml, respectively; P less than or equal to 0.001). When GnRH was given before (5 h before) or during (5 h after) PGF-2 alpha-induced regression of the Day 10 spontaneous CL, the GnRH-induced CL which formed were also short-lived. In contrast, when GnRH was given following (36 h after) PGF-2 alpha-induced regression of the Day 10 spontaneous CL, the CL which formed were not different in lifespan or production of progesterone from spontaneous CL. Efforts to enhance function of the GnRH-induced subnormal CL by treating ewes with the synthetic progestagen, norgestomet, to suppress follicular development after CL formation, were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloprostenol induced luteal regression in the beef cow. II. Histological evaluation of corpora lutea and correlation with luteal progesterone

Forty-two cycling, multiparous beef cows (percentage-Brahman) were injected twice at 11-d intervals with 500mug Cloprostenol (a prostaglandin F(2alpha) analog) to induce luteolysis. Cows were randomly assigned for ovariectomy at 12 hr intervals from 0 to 72 hr post-injection. Corpora lutea were excised and one-half of the corpus luteum was stored in phosphate-buffered formalin until mounting and staining with hematoxylin and eosin. The other half of the CL was snap-frozen for determination of progesterone content and concentration. Luteal cell density increased following Cloprostenol injection and was significantly correlated with a shift from predominantly healthy Type I cells to predominantly degenerating Types III and V cells. Cell mitosis tended to decrease by 12 hr and was lower by 24 hr post-injection. Cell pyknosis increased by 24 hr post-injection and was correlated with the decrease in percentage of healthy luteal cells. No pattern was detectable in cell karyorrhexis. Histological regression of the CL was inversely correlated with CL progesterone content. Therefore, we conclude that a reduction in cell mitosis is the earliest morphological sign of degeneration of the CL and that the CL follows a well-defined sequence of regression which is accompanied by a decrease in progesterone content.     

Ultrasonic morphology of corpora lutea and central luteal cavities during the estrous cycle and early pregnancy in heifers

Ultrasonography was used once daily to quantify corpora lutea, central luteal cavities, and luteinized tissue during interovulatory intervals (n = 66) and during Days 0 to 60 of pregnancy (n = 14) in nulliparous Holstein heifers (ovulation = Day 0). The corpus luteum of the estrous cycle was detectable by ultrasonography in most heifers from the day of ovulation (mean, Day 0.5) and extending into the regressive phase beyond the next ovulation (mean, Day 1.4 +/-0.2 after the next ovulation). During pregnancy, the corpus luteum was detected until Day 60 (end of study). Maximal central luteal cavity area detected on Days 0 to 20 was used retrospectively to group luteal glands into four cavity categories: no, small, medium, and large. These categories corresponded to approximate cavity diameters of <2 mm, 2 to 5 mm, 6 to 10 mm, and >10 mm, respectively. The incidence of each cavity category was similar between interovulatory intervals and pregnancies (combined incidence, 17 80 , 8 80 , 33 80 , and 22 80 for no, small, medium, and large cavities, respectively; total with cavities, 63 80 , 79%). Mean day of first detection of a central cavity was earliest for large cavities during interovulatory intervals (means, Days 4.7, 4.4, and 3.0 for small, medium, and large cavities, respectively; P<0.04) and during pregnancies (means, Days 5.5, 4.2, and 3.3, respectively; NS). However, the day that the cavities reached maximum size (range of means, Days 5.5 to 7.0) did not differ among categories. Mean day of last detection of the central cavity was significantly different among cavity categories during interovulatory intervals (means, Days 9.3, 11.1, and 17.4 for small, medium, and large cavities, respectively) and pregnancies (means, Days 7.0, 8.8, and 20.2, respectively). Time of loss of central cavities was similar between nonbred and pregnant heifers, and there was no significant difference among cavity categories in the length of the interovulatory interval (mean, 20.1 d). Luteal tissue area was not significantly different among cavity categories during interovulatory intervals. There were no indications that cavities were functionally important. Luteal tissue area increased linearly in pregnant heifers on Days 21 to 60 (mean slope, 2.6 mm(2)/day).     

Mitogenic factors of corpora lutea

The mammalian corpus luteum (CL), which plays a central role in the reproductive process because of its production of hormones such as progesterone, appears to be an exceptionally dynamic organ. Its rate of growth and development are extremely rapid and, even when the CL is functionally mature, its rate of cell turnover remains relatively high. Associated with this high rate of cell turnover, the mature CL receives the greatest blood supply per unit tissue of any organ, and also exhibits a relatively high metabolic rate. Although numerous growth factors have been identified in luteal tissue, their role in growth and differentiation of this dynamic organ remains unclear. Recently, while attempting to identify mitogenic factors of ovine and bovine CL, we have found that they produce several mitogens during the estrous cycle as well as pregnancy. The majority of these luteal-derived mitogenic factors are heparin-binding, and although some may represent previously identified factors, several appear to be novel heparin-binding growth factors. Isolation and purification of mitogenic factors produced by the CL will enable us to determine their roles in luteal growth, development and differentiated function, which will contribute to our understanding not only of the regulation of fertility but also of tissue growth and development in general.     

Effects of human chorionic gonadotropin on luteal blood flow and progesterone secretion in cows and in vitro-microdialyzed corpora lutea

To check human chorionic gonadotropin (hCG) effects on luteal blood flow (LBF) and progesterone (P₄) synthesis, six cows received either 3000 IU hCG or saline (NaCl) on Day 7 (Day 1 = ovulation) during two estrous cycles. Plasma P₄ and LBF were measured before (0 h) and up to 48 h after treatment. Luteal blood flow increased by 51% (P < 0.05) at 1 h after hCG administration and returned to baseline levels thereafter. Plasma P₄ levels were increased from pretreatment levels by 30% at 1 h (P = 0.05) and 81% at 48 h (P = 0.02) after hCG treatment. In contrast, NaCl did not cause changes in LBF and P₄ (P > 0.05). Additionally, central and peripheral parts of 14 abattoir-derived corpora lutea of the mid-luteal phase (Day 8 to 12) were perfused with Ringer solution in an in vitro microdialysis system, supplemented with 50 or 150 IU/mL hCG for 1 h. Application of 50 IU/mL hCG showed no influence on P₄ response (P > 0.05) in both central and peripheral parts, whereas 150 IU/mL hCG resulted in an increase of P₄ synthesis (P = 0.002) in the central parts only. In vivo, hCG provoked an immediate and long-term rise in P₄ but only a temporary elevation of LBF. Luteal blood flow itself does not seem to be the exclusive cause for an increase in P₄, because the in vitro data clearly showed direct effects of hCG on P₄ secretion. Interestingly, different P₄ secretion patterns could be found between central and peripheral parts of the corpus luteum in both control and hCG perfused corpora lutea.     

Regression of the corpora lutea in sheep in response to cloprostenol is not affected by loss of luteal oxytocin after hysterectomy

Concentrations of oxytocin in corpora lutea were reduced from 1706 to less than 15 ng/g wet wt after hysterectomy in sheep during the oestrous cycle. Hysterectomy also blocked the appearance of raised levels of oxytocin in ovarian and jugular venous plasma caused by cloprostenol. Administration of cloprostenol to hysterectomized ewes resulted in luteal regression, which occurred as rapidly as in intact animals. Therefore oxytocin in the corpus luteum during the oestrous cycle is unlikely to be involved in intraluteal events mediating prostaglandin-induced luteolysis.     

Cytokeratin expression in bovine corpora lutea

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corporalutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed zonation such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.     

Effects of treatment with a prostaglandin analogue on developmental dynamics and functionality of induced corpora lutea in goats

The aim of this study was to compare morphological and functional features of spontaneous and induced corpora lutea (CLs) in goats. Fourteen adult and cycling Anglo Nubian goats (Argentina) were randomly allocated to two groups: Group N (n = 7) included goats with natural spontaneous oestrus and Group PG (n = 7) included does in which oestrus was synchronized by the administration of two i.m. cloprostenol doses, 10 days apart. In both groups, oestrous behaviour was checked twice daily (Day of oestrus = Day 0) and daily transrectal ultrasonographies were performed for evaluating CLs and follicles dynamics through the complete subsequent oestrous cycle; the luteal activity was determined directly, in terms of progesterone (P4) secretion, and indirectly, by assessing effects of CL on follicular dynamics. All goats exhibited oestrous behaviour and ovulation without differences in ovulation rate (N: 1.67 ± 0.2, PG: 2.0 ± 0.1). The total luteal tissue area showed linear growth from Day 4 to Day 15 of oestrous cycle in all goats, but the developmental dynamics differed between groups, treated goats had larger area (P < 0.01). Plasma P4 concentrations also increased from Day 0 to Day 15 in all the does; however, from Day 5 to Day 15, treated does had a lower concentrations than the untreated group (P < 0.001). There were differences in the development of follicular waves between groups; assessment of size-distribution showed that treated group had a higher number of small and larger follicles (P < 0.05). The largest follicles recorded in treated goats had a higher maximum diameter both at the first (PG: 7.6 ± 0.8 mm; N: 4.9 ± 0.7 mm, P < 0.05) and second follicular waves (PG: 6.3 ± 1.4 mm; N: 5.0 ± 0.4 mm, P < 0.05) and a longer growth phase during the second wave (PG: 6.5 ± 1.7 days; N: 4.6 ± 0.7 days, P < 0.05), coincident with the period of maximal luteal secretion. In conclusion, synchronization of oestrus and ovulation by the administration of a prostaglandin analogue causes differences in developmental dynamics and functionality of induced corpora lutea when compared to natural spontaneous ovulation.     

Inhibition of luteal function by oxytocin antagonist in goats (Capra hircus).

Early corpus luteum development in nonpregnant and pregnant goats was characterized by a steady increase in peripheral plasma concentrations of progesterone and a high release of prostacyclin (PGI-2) but low release of prostaglandin F-2 alpha (PGF-2 alpha). Jugular administration of oxytocin antagonist (OXA) (0.2 microgram/kg/day) on the day of oestrus and for 3 days thereafter to cyclic and mated goats, significantly (P less than 0.001) inhibited progesterone and prostaglandin secretion and reduced conception rate. Co-administration of PGI-2 (200 micrograms/day) with OXA resulted in a steady increase of progesterone and establishment of pregnancy, but co-administration of PGF-2 alpha (175 micrograms/day) with OXA had no effect. It is suggested that oxytocin is required for early development of the corpus luteum and such effects may be mediated via PGI-2 production.     

Secretion of oxytocin and progesterone by ovine corpora lutea in vitro

The mechanisms involved in the control of oxytocin and progesterone secretion by the ovine corpus luteum have been investigated in vitro using luteal slice incubations. Oxytocin and progesterone were secreted at constant rates from luteal slices for 2 h of incubation (366 +/- 60 pg X mg X h and 18.9 +/- 0.18 ng X mg X h, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p less than 0.02) stimulated in the presence of ovine luteinizing hormone. Incubation of luteal slices in medium containing 100 mM potassium, however, resulted in increased secretion of oxytocin and, to a lesser extent, of progesterone (294 +/- 59% and 142 +/- 15%, respectively, p less than 0.05). Basal oxytocin secretion was reduced during incubation in calcium-free medium, compared to secretion in the presence of calcium (70 +/- 15 and 175 +/- 25 pg X mg X 20 min, respectively, p less than 0.01), whereas progesterone secretion was not altered in the absence of calcium. Secretion of both hormones by luteal slices was stimulated by the addition of the calcium ionophore A23187 (p less than 0.05). Addition of prostaglandin F2 alpha (2.8 microM) had no effect on secretion of either oxytocin or progesterone. We have demonstrated that oxytocin and progesterone can be stimulated, independently, from corpus luteum slices incubated in vitro. The pattern of release is consistent with the proposal that oxytocin, but not progesterone, is associated with and actively released from luteal secretory granules. Our results also indicated that prostaglandin F2 alpha does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.