Luteal blood flow is a more appropriate indicator for luteal function during the bovine estrous cycle than luteal size

The objective of this study was to assess the reliability of luteal blood flow (LBF) as recorded by color Doppler sonography to monitor luteal function during the estrous cycle of dairy cows and to compare the results with that for the established criterion luteal size (LS) as determined by B-mode sonography. In total, 14 consecutive sonographic examinations were carried out in 10 synchronized lactating Holstein-Friesian cows (Bos taurus) on Days 4, 5, 6, 7, 8, 10, 12, 14, 16, -5, -4, -3, -2, -1 of the estrous cycle (Day 1 = ovulation). Plasma progesterone concentrations in venous blood (P₄) were quantified by enzyme immunoassay. Luteal size was determined by sonographic measurement of the maximal cross-sectional area of the corpus luteum (CL). Luteal blood supply was estimated by calculating the maximum colored area of the CL from power Doppler sonographic images. Luteal size doubled during the luteal growth phase (until Day 7) and remained at this level during the luteal static phase (Day 8 to 16) before decreasing rather slowly during luteal regression (Days -5 to -1). Luteal blood flow doubled during the growth phase, doubled furthermore during the static phase, and decreased rapidly during luteal regression. Thus, LBF values represented highly reliable predictors of luteal status. Luteal blood flow predicted reliably a P₄ > 1.0 ng/mL by reaching only 35% of the maximal values, whereas LS had to exceed 60% of the maximal values to indicate reliably a functional CL. It is concluded that LBF reflected luteal function better than LS specifically during luteal regression.     

Luteal blood flow during the estrous cycle in mares

Transrectal color Doppler ultrasound was used for the noninvasive investigation of luteal blood flow during the estrous cycle in six mares. Color was displayed in Power-Mode, in which the number of color pixels on the ultrasound image is related to the number of moving blood cells. Three pictures with a maximum number of color pixels of the corpus luteum (CL) during an examination period of about 20 min were selected and digitized on a laptop equipped with an external frame grabber card. The intra-class correlation coefficient for the number of color pixels was 0.90. In all estrous cycles similar patterns of changes in (C), in the cross-sectional area of sectional planes of the CL (A), and in plasma progesterone levels (P) occurred. Variance component estimates for the effect of the mare on (C), (A) and (P) were 14, 23 and 4%, for the influence of day of estrous cycle they were 41, 5 and 58% and for the effect of estrous cycle they were 7, 5 and 5%, respectively. There were high positive correlations between cyclic changes in (C) and (P) (r = 0.58; P < 0.0001). The increase in (C) between Days 0 and 5 (Day 0: ovulation) remained at high levels until Day 7 and then decreased until Day 15. There were relationships between (C) and (A) (r = 0.37; P < 0.0001) and between (A) and (P) (r = 0.24; P < 0.05), but correlation coefficients were not as high as between (C) and (P). Differences in (C), (A) and (P) between estrous cycles within mares and between mares were not related to each other (P > 0.05). The results show that transrectal color Doppler sonography is a useful, noninvasive method for examining luteal blood flow in mares, and that there are cyclic changes and individual differences in the vascularization of the CL. The possible influence of luteal perfusion on fertility in mares needs to be investigated in further studies.     

Plasma progesterone concentrations in the mid-luteal phase are dependent on luteal size, but independent of luteal blood flow and gene expression in lactating dairy cows

The objective of the present study was to investigate if plasma progesterone (pP(4)) concentrations are dependent on luteal size, blood flow, or gene expression in luteal tissue. To induce cycles with high and low pP(4) concentrations, respectively, 20 lactating dairy cows received either a single treatment with 25 mg prostaglandin F(2α) (PGF(2α)) on Day 4 Hour 12 (PG1; n=8), or two treatments (25 mg PGF(2α) each) on Day 4 Hours 0 and 12 (PG2; n=12) of the estrous cycle (Day 1, Hour 0=ovulation). In four cows, ovulation occurred between 4 and 6d after the second PGF(2α) treatment; these cows and one lame cow were excluded from the study. In the 15 remaining cows with physiological interovulatory intervals, pP(4), area (LTA) and volume (LTV) of luteal tissue, as well as absolute (LBF) and relative (rLBF) luteal blood flow were determined on Day 9, and relative luteal P(4) (rLP(4)) as well as luteal mRNA expression of important receptors, angiogenic, vasoactive, and steroidogenic factors were quantified on Day 11 (±1) during two successive estrous cycles. Furthermore, rLP(4) was multiplied by LTV to produce a semiquantitative assessment of absolute luteal P(4) (LP(4)). There was no effect (P>0.05) of treatment (one or two PGF(2α) treatments), neither on pP(4) concentrations nor on any other parameter in the present study. Nevertheless, there was a lower LP(4) (P=0.01), LTA (P=0.03), and LTV (P=0.02), as well as tendencies of lower pP(4) (P=0.06) and LBF (P=0.09) at first compared with second diestrus. Plasma P(4) was related with LP(4) (r=0.43, P=0.04), LTA (r=0.65, P=0.0001), and LTV (r=0.43, P=0.02), but not with rLBF (r=-0.18, P=0.34). Furthermore, there was no significant correlation between gene expression of important steroidogenic factors and P(4) concentrations in luteal tissue. Results indicate that plasma P(4) concentrations in the mid-luteal phase were dependent on luteal size, but independent of blood flow and gene expression per luteal tissue unit.     

Luteal blood flow increases during the first three weeks of pregnancy in lactating dairy cows

The objectives of this experiment were to characterize luteal blood flow in pregnant and non-pregnant cows and to determine its value for early pregnancy diagnosis. Lactating dairy cows (n = 54), 5.2 ± 0.2 y old (mean ± SEM), average parity 2.4 ± 0.2, and ≥ 6 wk postpartum at the start of the study, were used. The corpus luteum (CL) was examined with transrectal color Doppler ultrasonography (10.0-MHz linear-array transducer) on Days 3, 6, 9, 11, 13, 15, 18, and 21 of the estrus cycle (estrus = Day 0). Artificially inseminated cows (n = 40) were retrospectively classified as pregnant (embryonic heartbeat on Day 25; n = 18), nonpregnant (interestrus interval 15 to 21 d, n = 18), or having an apparent early embryonic loss (interestrus interval >25 d, n = 4). There was a group by time interaction (P < 0.001) for luteal blood flow from Days 3 to 18; it was approximately 1.10 ± 0.08 cm² (mean ± SEM) on Day 3, and increased to approximately 2.00 ± 0.08 cm² on Day 13 (similar among groups). Thereafter, luteal blood flow was numerically (albeit not significantly) greater in pregnant cows, remained constant in those with apparent embryonic loss, and declined (not significantly) between Days 15 and 18 in nonpregnant cows. Luteal blood flow was greater in pregnant than in nonpregnant (P < 0.05) and nonbred cows (P < 0.05, n = 14) on Day 15 (2.50 ± 0.16, 2.01 ± 0.16, and 2.00 ± 0.18 cm², respectively) and on Day 18 (2.40 ± 0.19, 1.45 ± 0.19, and 0.95 ± 0.21 cm²). In cows with apparent early embryonic loss, luteal blood flow was 2.00 ± 0.34 and 2.05 ± 0.39 cm² on Days 15 and 18, which was less (not significantly) than in pregnant cows, but greater (P < 0.05) than in nonbred cows on Day 18. Although mean luteal blood flow was significantly greater in pregnant than nonpregnant (and nonbred) cows on Days 15 and 18, due to substantial variation among cows, it was not an appropriate diagnostic tool for pregnancy status.     

Low plasma progesterone concentrations are accompanied by reduced luteal blood flow and increased size of the dominant follicle in dairy cows

To investigate the influence of low plasma progesterone (P₄) concentrations on luteal and ovarian follicular development as well as endometrial gene expression in the concomitant and subsequent estrous cycle, 20 lactating dairy (Holstein Friesian and Brown Swiss x Holstein Friesian) cows received either a single treatment with 25 mg prostaglandin F_2α (PGF_2α) on Day 4 Hour 12 (PG1; n = 8), or two treatments (25 mg PGF_2α each) on Day 4 Hours 0 and 12 (PG2; n = 12) of the estrous cycle (Day 1, Hour 0 = ovulation). In four cows, ovulation occurred between 4 and 6 d after the second PGF_2α treatment; these cows and one lame cow were excluded. In the 15 remaining cows with physiological interovulatory intervals (18 to 24 d), P₄, luteal size (LS) and blood flow (LBF), as well as follicular size (FS) and blood flow (FBF), were determined daily until Day 4, immediately prior to (0 h) and 12 h after each PGF_2α treatment, and then every 2 d, from Day 5 to 8 d after the subsequent ovulation. Because P₄ did not differ (P > 0.05) between PG1 and PG2, cows were regrouped according to their mean P₄ concentration from Days 7 to 15, either P₄ <2 ng/mL (P₄L; n = 7) or P₄ >2 ng/mL (P₄H; n = 8). In the treatment cycle, LS was smaller in P₄L than P₄H on Days 13 (P = 0.01) and 15 (P = 0.03), and LBF was lower in P₄L than P₄H on Day 15 (P = 0.02). The dominant follicle of the first follicular wave was larger in P₄L than P₄H on Days 13 (P = 0.03), 15 (P = 0.03), and 17 (P = 0.01). In the subsequent cycle, there were no significant differences between P₄L and P₄H for P₄, FS, LS, and LBF; however, FBF was lower (P = 0.01) in P₄L than P₄H on Day 7. In Group P₄L, endometrial expressions of estrogen receptor α and oxytocin receptor were lower (P = 0.05 and P = 0.03, respectively) at the estrus that preceded treatment compared to the post-treatment estrus. In summary, low P₄ during diestrus was associated with smaller LS, reduced LBF, and larger FS in the treatment cycle, but not in the subsequent cycle.     

Local changes in blood flow within the early and midcycle corpus luteum after prostaglandin F(2 alpha) injection in the cow

One of the postulated main luteolytic actions of prostaglandin (PG) F(2 alpha) is to decrease ovarian blood flow. However, before Day 5 of the normal cycle, the corpus luteum (CL) is refractory to the luteolytic action of PGF(2 alpha). Therefore, we aimed to determine in detail the real-time changes in intraluteal blood flow after PGF(2 alpha) injection at the early and middle stages of the estrous cycle in the cow. Normally cycling cows at Day 4 (early CL, n = 5) or Days 10--12 (mid CL, n = 5) of the estrous cycle (estrus = Day 0) were examined by transrectal color and pulsed Doppler ultrasonography to determine the blood flow area, the time-averaged maximum velocity (TAMXV), and the volume of the CL after an i.m. injection of a PGF(2 alpha) analogue. Ultrasonographic examinations were carried out just before PG injection (0 h) and then at 0.5, 1, 2, 4, 8, 12, 24, and 48 h after the injection. Blood samples were collected at each of these times for progesterone (P) determination. The ratio of the colored area to a sectional plane at the maximum diameter of the CL was used as a quantitative index of the changes in blood flow within the luteal tissue. Blood flow within the midcycle CL initially increased (P < 0.05) at 0.5-2 h, decreased at 4 h to the same levels observed at 0 h, and then further decreased to a lower level from 8 h (P < 0.05) to 48 h (P < 0.001). Plasma P concentrations decreased (P < 0.05) from 4.7 +/- 0.5 ng/ml (0 h) to 0.6 +/- 0.2 ng/ml (24 h). The TAMXV and CL volume decreased at 8 h (P < 0.05) and further decreased (P < 0.001) from 12 to 24 h after PG injection, indicating structural luteolysis. These changes were not detected in the early CL, in which luteolysis did not occur. In the early CL, the blood flow gradually increased in parallel with the CL volume, plasma P concentration, and TAMXV from Day 4 to Day 6. The present results indicate that PGF(2 alpha) induces an acute blood flow increase followed by a decrease in the midcycle CL but not in the early CL. This transitory increase may trigger the luteolytic cascade. The lack of intraluteal vascular response to PG injection in the early CL appears to be directly correlated with the ability to be resistant to PG.     

Change in morphology of corpora lutea, central luteal cavities and steroid secretion patterns of postpartum suckled beef cows after melengestrol acetate with or without prostaglandin F2alpha

Change in morphology of the corpus luteum (CL) and patterns of progesterone and estradiol secretion after treatment with melengestrol acetate (MGA) were monitored in postpartum beef cows. Twenty Angus cows were randomly assigned to MGA or MGA + prostaglandin F(2alpha) (PGF) treatments. All cows were fed 0.5 mg of MGA per cow per day for 14 d. The MGA-treated cows (n = 10) were allowed to return to estrus spontaneously at the second estrus after withdrawal of MGA from the feed. The MGA + PGF-treated cows (n = 10) received an injection containing 25 mg of PGF(2alpha) 17 d after the last feeding of MGA. Cycle 1 was defined as the first luteal phase after MGA feeding and Cycle 2 represented the subsequent cycle or luteal phase after PGF. Blood sampling and transrectal ultrasonography of the ovaries was done daily through the completion of 2 estrous cycles upon removal of MGA from the feed. Blood samples were analyzed for plasma progesterone and estradiol concentrations. Area of CL and fluid-filled cavities within each CL were determined by ultrasonography. Concentrations of progesterone and area of CL were similar between cycles and treatments. Estradiol concentrations were higher (P < 0.05) in Cycle 2 than in Cycle 1. Fluid-filled cavities were larger (P < 0.001) in Cycle 1 than in Cycle 2 for both mid-luteal (Days 5 to 9) and late-luteal (Days 10 to 14) phases. Multiple CL (2 or more during 1 cycle) were observed in 5 cows. Progesterone concentrations and total area of luteal tissue did not change with respect to treatment or cycle, but CL morphology was altered in the first cycle after MGA treatment. Of the 19 cows that ovulated after withdrawal of MGA, 3 experienced a short luteal phase. These data characterize changes that occur among cows that are fed melengestrol acetate during the postpartum period and enhance observations from prior studies regarding MGA use.     

Comparison of ovarian palpation, milk progesterone and plasma progesterone in the cow

Holstein cows were examined twice weekly, beginning at 13 to 18 days after parturition, using palpation per rectum and milk progesterone (P4) assay to determine the functional status of the corpus luteum (CL). These results were then compared with P4 concentrations in the plasma to determine which test was the more accurate in detecting functional luteal tissue, or if both tests were needed for optimal accuracy. The tests were found to be comparable except when the plasma P4 concentration was <1. 0 ng/ml. At this level, errors due to palpation occurred more frequently than those due to milk P4 concentrations since the still palpable CL of pregnancy could be mistaken for a functional CL at 14 to 21 days after parturition. However, at all the other concentrations including when plasma P4 was <1.0 ng/ml and the cows were more than 21 days post partum, neither the milk P4 assay nor palpation per rectum could be considered the better indicator of luteal status. Therefore, we conclude that the combined use of both tests does not afford better assessment of luteal function than either test alone.     

Impact of angiogenic and innate immune systems on the corpus luteum function during its formation and maintenance in ruminants

The corpus luteum (CL) is formed from an ovulated follicle, and grows rapidly to secrete progesterone (P4) thereby supporting implantation and maintenance of pregnancy. It is now evident that angiogenesis is necessary to form the structure of the developing CL as well as to acquire the steroidogenic capacity to secrete large amounts of P4. It is of interest that the increases in CL size, plasma P4 concentration and luteal blood flow are occurring in parallel during the first seven days after ovulation. Angiogenic factors, such as vascular endothelial growth factor-A (VEFGA) and basic fibroblast growth factor (FGF2), play a central role in promoting cell proliferation and angiogenesis in the developing CL. Angiopoietins regulate the stability of blood vessels, which directly affects angiogenesis or angiolysis via angiogenic factors. Vasohibin-1 is a novel negative feedback regulator, which inhibits VEGF-based vasculogenesis. It became evident that the immune cells, i.e., macrophages, eosinophils and neutrophils are recruited into the CL – using the innate immune system – just after ovulation which is accompanied by bleeding. The immune cells support active angiogenesis and thus the growth of the CL. In cows, the lymphatic system, but not blood vascular system, is reconstituted during early pregnancy, and embryonic trophoblast-derived interferon tau could play a crucial role in inducing lymphangiogenesis. This novel phenomenon may support a maternal recognition of pregnancy in shifting the local systems in such a way that they ensure a long-term supply of P4 over the period of pregnancy. Overall, the current findings support the concept that several major components involved in the regulation of the CL development and maintenance overlap in stimulating steroidogenesis, angiogenesis, vascular function and the innate immune system.     

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.     

VEGF and imaging of vessels in rheumatoid arthritis

Angiogenesis is a prominent feature of rheumatoid synovitis. Formation of new blood vessels permits a supply of nutrients and oxygen to the augmented inflammatory cell mass and so contributes to perpetuation of joint disease. Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific growth factor that is upregulated by proinflammatory cytokines and by hypoxia. Serum VEGF concentrations are elevated in rheumatoid arthritis (RA) and correlate with disease activity. Furthermore, serum VEGF measured at first presentation in RA is highly significantly correlated with radiographic progression of disease over the subsequent year. Power Doppler ultrasonography is a sensitive method for demonstrating the presence of blood flow in small vessels and there is a very close relation between the presence or absence of vascular flow signal on power Doppler imaging and the rate of early synovial enhancement on dynamic gadolinium-enhanced magnetic resonance imaging (MRI) of joints with RA. Images obtained by both dynamic enhanced MRI and power Doppler ultrasonography correlate with vascularity of synovial tissue as assessed histologically. In early RA, there is a striking association between joint erosions assessed on high-resolution ultrasonography and vascular signal in power Doppler mode. Collectively, these findings implicate vascular pannus in the erosive phase of disease and strongly suggest that proangiogenic molecules such as VEGF are targets for novel therapies in RA. Animal model data supports this concept. It seems likely that serological and imaging measures of vascularity in RA will become useful tools in the assessment of disease activity and response to therapy.     

Oviductal progesterone concentration and its spatial distribution in cyclic and early pregnant cows

Changes and local distribution of oviductal progesterone (P(4)) concentration during the estrous cycle and early pregnancy in cows were investigated. Intact reproductive tracts were collected from 16 Holstein cows at an abattoir. Samples were classified in to 4 stages (follicular, postovulatory, luteal and early pregnant,< 20 d) based on visual observation of corpus luteum (CL), uterine characteristics and luteal P(4) concentrations. Oviducts were separated from the uterus at the utero-tubal junction and divided into 4 parts: fimbriae, proximal, medial and distal parts. Luteal tissue samples were also collected. Progesterone levels in oviductal and luteal tissues were determined by radioimmunoassay (RIA). Comparatively higher (P < 0.001) P(4) levels were found in stages with a functioning CL ( luteal phase and early pregnancy) than in those with a regressing CL (follicular phase and post ovulation). The oviduct ipsilateral to the CL bearing ovary during the luteal phase and early pregnancy showed higher ( P < 0.001) P(4) concentrations than the contralateral side. Such a difference was not observed during the follicular phase or post ovulation. The ipsilateral oviduct to the functioning CL at early pregnancy showed higher (P <0.05) P(4) levels than at the luteal phase, while no significant difference in luteal P(4) levels between these 2 stages was observed. Neither were any differences in P(4) concentration within the oviduct observed during any phase of the estrous cycle or during early pregnancy. A positive relationship between luteal and oviductal P(4) concentrations was noted. In conclusion, changes in P(4) levels in the oviduct depend on the location and functional stage of the CL. Localized levels of P(4) in the oviduct may be due to local delivery of P(4) from the CL.     

Agreement between ultrasonographic classification of the CL and plasma progesterone concentration in dairy cows

We evaluated the agreement between ultrasonographic characteristics of the corpus luteum (CL) and plasma progesterone (P4) concentration in dairy cows. In Phase I of the study, the ovaries of 8 cows were ultrasonographically examined, and P4 was analyzed daily from estrus (Day 0) to Day 4, then at Day 7 and Day 10, and again daily from Day 17 to the onset of next estrus. In Phase 2, the ovaries of 157 randomly selected Friesian cows were examined once by ultrasonography, and blood samples collected concurrently were analyzed for plasma P4. On the basis of the P4 values, the function of CLs was classified as follows: 1) non-secretory CL when plasma P4 was lower than 1 ng/mL (n=41); 2) evolving CL when plasma P4 was between 1 and 4 ng/mL (n=55); and 3) mid-cycle CL when plasma P4 was more than 4 ng/mL (n=61). On the basis of ultrasonographic examination, 3 additional groups were established (absence of CL, evolving CL, midcycle CL). Ultrasonographic characteristics and size of Day 3 to 4 CLs and their respective plasma P4 concentrations were not distinguishable from those of CLs observed 3 to 4 d before the subsequent estrus. The degree of agreement between the two classification was 72%. The data indicate that the functional classification of CLs is difficult to determine based on ultrasonography alone.     

Progesterone is a suppressor of apoptosis in bovine luteal cells

Progesterone is suggested to be a suppressor of apoptosis in bovine luteal cells. Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells. Furthermore, apoptosis is known to be controlled by the bcl-2 gene/protein family and caspases. This study was undertaken to determine whether intraluteal progesterone (P4) is involved in Fas L-mediated luteal cell death in the bovine corpus luteum (CL) in vitro. Moreover, we studied whether an antagonist of P4 influences gene expression of the bcl-2 family and caspase-3 and the activity of caspase-3 in the bovine CL. Luteal cells obtained from the cows in the midluteal phase of the estrous cycle (Days 8-12 of the cycle) were exposed to a specific P4 antagonist (onapristone [OP], 10(-4) M) with or without 100 ng/ml Fas L. Although Fas L alone did not show a cytotoxic effect, treatment of the cells with OP alone or in combination with Fas L resulted in killing of 30% and 45% of the cells, respectively (P <0.05). DNA fragmentation was observed in the cells treated with Fas L in the presence of OP. The inhibition of P4 action by OP increased the expression of Fas mRNA (P <0.01); however, it did not affect bax or bcl-2 mRNA expression (P >0.05). Moreover, OP stimulated expression of caspase-3 mRNA (P <0.01). The overall results indirectly show that intraluteal P4 suppresses apoptosis in bovine luteal cells through the inhibition of Fas and caspase-3 mRNA expression and inhibition of caspase-3 activation.     

Influence of l-arginine supplementation on reproductive blood flow and embryo recovery rates in mares

Supplementation with l-arginine can increase uterine arterial blood flow and vascular perfusion of the preovulatory follicle in mares. Increased vascular perfusion of the preovulatory follicle has been correlated with successful pregnancy in mares. The objective of this study was to determine if supplemental l-arginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle, and embryo recovery rates in mares. Mares were blocked by age and breed and assigned at random within block to l-arginine supplementation or control groups. Mares were fed l-arginine beginning 17 days before and through the duration of the study. Transrectal Doppler ultrasonography was used to measure ovarian arterial blood flow and vascular perfusion of the preovulatory follicle daily when it reached 35 mm and subsequent CL on Days 2, 4, and 6. Mares, on achieving a follicle of 35 mm or more were bred via artificial insemination and an embryo collection was attempted 7 days after ovulation. Treatment did not affect interovulatory interval (arginine-treated, 18.1 ± 2.6 days; control, 20.7 ± 2.3 days) or embryo recovery rate (arginine-treated, 54%; control, 48%). Mares treated with l-arginine had a larger follicle for the 10 days preceding ovulation than control mares (30.4 ± 1.2 and 26.3 ± 1.3 mm, respectively; P < 0.05) and vascular perfusion of the dominant follicle tended (P = 0.10) to be greater for the 4 days before ovulation. No differences were observed between groups in diameter or vascular perfusion of the CL. Resistance indices, normalized to ovulation, were not significantly different between groups during the follicular or luteal phase. Oral l-arginine supplementation increased the size and tended to increase perfusion of the follicle 1, but had no effect on luteal perfusion or embryo recovery rates in mares.     

Matrix metalloproteinase-2 (MMP-2) expression and regulation by tumor necrosis factor alpha (TNFalpha) in the bovine corpus luteum

Angiogenesis and tissue remodeling events in the corpus luteum (CL) are mediated by matrix metalloproteinases (MMPs). We have recently reported the cloning of bovine membrane-type 1 metalloproteinase (MT1-MMP) and have shown that active MT1-MMP is correlated to MMP-2 activity in the CL during the estrous cycle. Given the important role that MMP-2 plays in neovascularization, we became interested in understanding the role of this enzyme in the CL, a system in which angiogenesis is exquisitely regulated in the course of its lifespan. The aims of the present study were to clone bovine MMP-2 cDNA, to investigate its temporal and spatial expression in three stages of CL during the estrous cycle and to study its regulation by TNFalpha, a key cytokine regulator of CL physiology. Bovine MMP-2 cDNA was isolated from a UNI-ZAP II bovine capillary endothelial cell cDNA library and sequenced. This gene encoded a protein of 662 amino acids. Luteal tissues were collected from non-lactating dairy cows on days 4, 10, and 16 of the estrous cycle. Northern and Western blotting revealed that the levels of MMP-2 mRNA (3.1 kb) and immunoreactive pro-MMP-2 protein (68 kDa) did not differ (P > 0.05) in any age of CL examined. In addition to large luteal cells, MMP2 was localized to endothelial cells in all ages of CL by immunohistochemistry. Studies using in vitro luteal cell cultures showed that MMP-2 mRNA, protein expression and activity was upregulated by TNFalpha in a dose- and time-dependent manner. The present study suggests that MMP-2 is predominantly produced by large luteal cells and endothelial cells, and that it plays an essential role in luteal remodeling and angiogenesis. These data also suggest that cytokines such as TNFalpha may modulate these processes by regulating MMP-2 expression.     

Influence of nitric oxide and noradrenaline on prostaglandin F(2)(alpha)-induced oxytocin secretion and intracellular calcium mobilization in cultured bovine luteal cells

Although prostaglandin (PG) F(2alpha) released from the uterus has been shown to cause regression of the bovine corpus luteum (CL), the neuroendocrine, paracrine, and autocrine mechanisms regulating luteolysis and PGF(2alpha) action in the CL are not fully understood. A number of substances produced locally in the CL may be involved in maintaining the equilibrium between luteal development and its regression. The present study was carried out to determine whether noradrenaline (NA) and nitric oxide (NO) regulate the sensitivity of the bovine CL to PGF(2alpha) in vitro and modulate a positive feedback cascade between PGF(2alpha) and luteal oxytocin (OT) in cows. Bovine luteal cells (Days 8-12 of the estrous cycle) cultured in glass tubes were pre-exposed to NA (10(-5) M) or an NO donor (S-nitroso-N:-acetylpenicillamine [S-NAP]; 10(-4) M) before stimulation with PGF(2alpha) (10(-6) M). Noradrenaline significantly stimulated the release of progesterone (P(4)), OT, PGF(2alpha), and PGE(2) (P: < 0.01); however, S-NAP inhibited P(4) and OT secretion (P: < 0.05). Oxytocin secretion and the intracellular level of free Ca(2+) ([Ca(2+)](i)) were measured as indicators of CL sensitivity to PGF(2alpha). Prostaglandin F(2alpha) increased both the amount of OT secretion and [Ca(2+)](i) by approximately two times the amount before (both P: < 0.05). The S-NAP amplified the effect of PGF(2alpha) on [Ca(2+)](i) and OT secretion (both P: < 0.001), whereas NA diminished the stimulatory effects of PGF(2alpha) on [Ca(2+)](i) (P: < 0.05). Moreover, PGF(2alpha) did not exert any additionally effects on OT secretion in NA-pretreated cells. The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL function. While NA stimulates P(4) and OT secretion, NO may inhibit it in bovine CL. Both NA and NO are likely to stimulate the synthesis of luteal PGs and to modulate the action of PGF(2alpha). Noradrenaline may be the factor that is responsible for the limited action of PGF(2alpha) on CL and may be involved in the protection of the CL against premature luteolysis. In contrast, NO augments PGF(2alpha) action on CL and it may be involved in the course of luteolysis.     

Bovine corpus luteum is an extrapituitary site of prolactin production

Prolactin (PRL) is known to be synthesized not only in the anterior pituitary, but also in other organs including the ovary. Among its various functions, PRL is regarded as the most important constituent of the luteotropic complex in rodents and pigs. The purpose of the present study was to determine whether PRL is produced locally in bovine corpus luteum (CL) and to determine its possible roles in CL. In the present study, we examined changes during the luteal phase in (1) the expressions of PRL and PRL receptors (long form: l-PRLR, short form: s-PRLR) in CL and (2) the localization of PRL in CL. We also measured the levels of PRL mRNA in cultured luteal cells and luteal endothelial cells. Furthermore, the effect of PRL on progesterone (P4) and prostaglandin (PG) F2alpha production by cultured bovine luteal cells was examined. Semiquantitative RT-PCR analysis revealed that the mRNAs for PRL and its two receptors, l- and s-PRLR, were expressed in all luteal stages examined. PRL mRNA expression was less in the regressed stage (days 19-21 after ovulation) than in the other stages. Both l-PRLR and s-PRLR mRNA expressions were higher in the late luteal stage (days 15-17) than in the other stages, while the ratio of l-PRLR to s-PRLR was less in the regressed stage than in the other stages. PRL mRNA was also detected in cultured luteal cells and luteal endothelial cells. PRL protein was immunohistochemically detected only in CL of the mid- and regressed stages. It was detected in smooth muscle cells of the intraluteal arterioles and endothelial cells but not in luteal cells and other cell types of CL. Exposure of cultured luteal cells obtained from mid-stage CL (days 8-12) to bovine PRL (100, 200 ng/ml) for 24 hr did not affect P4 and PGF2alpha production by the cells. The present study demonstrates for the first time the expressions of PRL and PRLR mRNA in bovine CL throughout the luteal phase. The overall results strongly suggest that the bovine CL is an extrapituitary site of PRL production.