Mid-cycle Corpus Luteum Research Articles

Endothelin-1 receptors and biosynthesis in the corpus luteum: Molecular and physiological implications

Endothelin-1 (ET-1), a 21-amino acid peptide was initially identified as a potent vasoconstrictor, ET-1 plays an important role in the female reproductive cycle: its quick ascent during luteal regression, ability to inhibit steroidogenesis in vitro and in vivo, combined with the observation that the luteolytic effects of prostaglandin F2α (PGF2α) were delayed by pretreatment with ET-1 receptors type A (ETA) antagonists suggest that this peptide functions as an important element of the luteolytic cascade. The observation that ETA receptor expression was inversely correlated with steroidogenesis in luteal cells; namely factors which stimulated steroidogenesis inhibited ETA receptor levels is also in accord with the inhibitory role of ET-1 in corpus luteum (CL) function. Contrary to the mature mid cycle CL, the CL of early cycle is refractory to PGF2α-induced luteolysis. PGF2α administered at early luteal phase (day 4 of the cycle) failed to increase luteal ET-1 gene expression or its ETA receptors. In contrast, both genes were markedly induced in mid cycle CL exposed to PGF2α. ET-1 gene is transcribed as prepro ET-1 (ppET-1) and the active form of peptide is derived from the inactive intermediate big ET-1, by endothelin-converting enzyme-1 (ECE-1), therefore alterations in mature ET-1 levels can be achieved by modulating the expression of ppET-1 and/or ECE-1. Analysis using in situ hybridization and enriched luteal cell subpopulations showed that both steroidogenic and endothelial cells of the CL expressed high levels of ECE-1 mRNA. The ppET-1 mRNA, on the other hand, was only expressed by resident endothelial cells, suggesting that luteal parenchymal and endothelial cells cooperate in the biosynthesis of mature bioactive ET-1. A significant, four-fold elevation in ECE-1 expression (mRNA and protein levels) occurred during the transition of the CL from early to mid luteal phase. This increase was accompanied by a significant rise in ET-1 peptide. Surprisingly however, ppET-1 mRNA levels remained similar during early and mid luteal phase. Collectively, these studies demonstrate that: (a) the various components of ET-1 system (ET-1/ECE-1/ETA) are dynamically and independently regulated during bovine luteal life span. (b) The CL becomes PGF2α-responsive only when both ppET-1 and ECE-1 genes are expressed at a level which enable an uninterrupted ET-1 biosynthesis.

Ultrasonographic appearance of tissue is a better indicator of CL function than CL diameter measurement in dairy cows

In this study, the ovaries of 99 randomly selected Friesian cows were examined by ultrasonography measuring the diameter and evaluating the appearance of corpora lutea (CLs) in order to assess the most reliable method for their functional classification. Concurrently, blood samples were taken and analyzed for plasma progesterone (P4) concentration. On the basis of the ultrasonographic measurement of the diameter of the CL, three groups were established: (A) CL not detected ( n=30), (B) CL Ø < 20 mm ( n=22), and (C) CL Ø ≥ 20 mm ( n=47). On the basis of the ultrasonographic appearance, three different groups were established: (A) CL not detected ( n=30), (B) evolving CL ( n=25), and (C) mid-cycle CL ( n=44). On the basis of the P4 values, CLs were functionally classified in the following three groups: (A) CL not detected when plasma P4 was lower than 1 ng/ml ( n=27), (B) evolving CL when plasma P4 was between 1 and 4 ng/ml inclusive ( n=29), and (C) mid-cycle CL when plasma P4 was more than 4 ng/ml ( n=43). The degree of agreement between plasma P4 concentrations and either ultrasonographic classification (diameter or appearance) was highly significant ( P<0.001). However, the results of the present study suggest that for the evaluation of functional classification of the CL in cows ultrasonographic appearance is more reliable than the evaluation of the diameter.

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.

Fas-Fas ligand system mediates luteal cell death in bovine corpus luteum.

Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells when bound to the Fas ligand (Fas L). The present study was undertaken to identify the presence of a Fas-Fas L system in bovine corpus luteum (CL) and to evaluate the regulation of Fas-mediated luteal cell death by leukocyte-derived cytokines. The reverse transcription-polymerase chain reaction showed higher levels of Fas mRNA expression in CL in the regressed luteal stage (Days 19-21) than in the other stages (P < 0.05). Bovine luteal cells from midcycle CL (Days 8-12) were exposed for 24 h to interferon gamma (IFN; 50 ng/ml) and/or tumor necrosis factor alpha (TNF; 50 ng/ml). After 24 h of culture, the expression of Fas mRNA was detected in the cultured cells and was increased by IFN. Moreover, TNF augmented the stimulatory action of IFN, whereas TNF alone did not affect the expression of Fas mRNA. The effects of IFN and TNF on Fas-mediated cell death were also examined. Cells were exposed to IFN and/or TNF for 24 h and were further treated with IFN and/or TNF in the presence or absence of Fas L (100 ng/ml) for 24 h. Treatments of the cells with IFN alone and in combination with TNF resulted in killing of 30% and 50% of the cells (P < 0.05), respectively, whereas TNF alone did not have a cytotoxic effect on the cells. On the other hand, Fas L killed 60% of the cells treated with IFN (P < 0.01) and 85% of the cells treated with the combination of TNF and IFN (P < 0.01), respectively, whereas Fas L showed no effect on the viability of the luteal cells treated with or without TNF. Furthermore, shrunken nuclei and apoptotic bodies were observed in the cells treated with Fas L in the presence of TNF and IFN. The overall results suggest that a Fas-Fas L system is present in bovine CL and that leukocyte-derived TNF and IFN play important roles in Fas-mediated luteal cell death.

The lipoxygenase pathways are involved in LH-stimulated progesterone production in bovine corpus luteum

We have examined the effects of endogenous lipoxygenase products on basal progesterone (P4) production by cultured bovine mid-luteal cells. The involvement of lipoxygenase products in the stimulatory effect of LH on luteal cAMP accumulation and P4 production was also examined. Bovine luteal cells from mid-cycle corpora lutea (CL) were exposed for 16 h to a lipoxygenase inhibitor (nordihydroguaiaretic acid: NDGA; 0.33–33 μM). For the last 4 h of incubation, the cells were exposed to LH and/or three different lipoxygenase products, 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE). NDGA inhibited P4 production by the cells in a dose-dependent manner ( P < 0.05). NDGA-reduced P4 production was reversed by the addition of 12-HETE, but not 5- or 15-HETE, whereas 5-, 12- and 15-HETE alone showed no significant effect on P4 production in the intact cells. Furthermore, NDGA (33 μM) blocked the stimulatory action of LH on P4 production ( P < 0.05), without changing cAMP accumulation ( P > 0.1). When the cells were exposed to 5-, 12- or 15-HETE with LH and NDGA, only 15-HETE maintained the stimulatory effect of LH on P4 production in the cells ( P < 0.05). These results suggest that endogenous lipoxygenase products play important roles in P4 production by bovine CL, i.e. basal P4 production is supported by 12-HETE, and LH-stimulated P4 production is partially mediated via the activation of lipoxygenase and subsequent 15-HETE formation downstream of the LH-activated cAMP-PKA-phosphorylation pathway.

Differential effects of prostaglandin F2alpha on in vitro luteinized bovine granulosa cells

Prostaglandins have been implicated in various aspects of ovarian function including ovulation and luteolysis. In this study, the expression and regulation of inducible prostagland in G/H synthase (PGHS-2) and PGF(2alpha) receptors were investigated in bovine granulosa cells at various stages of differentiation. Firstly, the induction of PGF(2alpha) receptor mRNA and PGHS-2 mRNA in preovulatory granulosa cells was evaluated. Granulosa cells were collected from preovulatory follicles and cultured for 1, 4, 7 or 10 days. Cells were treated with hCG (10 iu) or with increasing doses of forskolin (0-10 micromol l(-1)) for 24 h. Forskolin increased steady-state concentrations of mRNA for PGHS-2 (> 20-fold) and PGF(2alpha) receptor (> 1000-fold) in a dose-dependent fashion. Use of selective protein kinase A inhibitor (H89) reduced both hCG- and forskolin-induced expression of PGF(2alpha) receptor mRNA and PGHS-2 mRNA. The hypothesis that luteinized granulosa cells would acquire PGF(2alpha) responsiveness similar to responses to PGF(2alpha) observed in vivo was also evaluated. Treatment with PGF(2alpha) (100 nmol l(-1)) reduced forskolin-induced expression of PGF(2alpha) receptor mRNA on days 4, 7 and 10, but not on day 1 of culture (n = 3). Treatment with PGF(2alpha) did not change forskolin-induced expression of PGHS-2 mRNA on or before day 4 of culture. In contrast, PGF(2alpha) significantly increased PGHS-2 mRNA expression in granulosa cells primed with forskolin for 7 or 10 days. In conclusion, expression of PGHS-2 and PGF(2alpha) receptor mRNA is protein kinase A-dependent in preovulatory bovine granulosa cells. Granulosa cells become PGF(2alpha)-responsive soon after expression of PGF(2alpha) receptor, whereas further differentiation is required before PGF(2alpha) induces PGHS-2 mRNA upregulation. These results demonstrate that at least two key transitions are required in PGF(2alpha)-induced luteal regression in the mid-cycle corpus luteum.

Differential effects of prostaglandin F2alpha on in vitro luteinized bovine granulosa cells.

Prostaglandins have been implicated in various aspects of ovarian function including ovulation and luteolysis. In this study, the expression and regulation of inducible prostagland in G/H synthase (PGHS-2) and PGF(2alpha) receptors were investigated in bovine granulosa cells at various stages of differentiation. Firstly, the induction of PGF(2alpha) receptor mRNA and PGHS-2 mRNA in preovulatory granulosa cells was evaluated. Granulosa cells were collected from preovulatory follicles and cultured for 1, 4, 7 or 10 days. Cells were treated with hCG (10 iu) or with increasing doses of forskolin (0-10 micromol l(-1)) for 24 h. Forskolin increased steady-state concentrations of mRNA for PGHS-2 (> 20-fold) and PGF(2alpha) receptor (> 1000-fold) in a dose-dependent fashion. Use of selective protein kinase A inhibitor (H89) reduced both hCG- and forskolin-induced expression of PGF(2alpha) receptor mRNA and PGHS-2 mRNA. The hypothesis that luteinized granulosa cells would acquire PGF(2alpha) responsiveness similar to responses to PGF(2alpha) observed in vivo was also evaluated. Treatment with PGF(2alpha) (100 nmol l(-1)) reduced forskolin-induced expression of PGF(2alpha) receptor mRNA on days 4, 7 and 10, but not on day 1 of culture (n = 3). Treatment with PGF(2alpha) did not change forskolin-induced expression of PGHS-2 mRNA on or before day 4 of culture. In contrast, PGF(2alpha) significantly increased PGHS-2 mRNA expression in granulosa cells primed with forskolin for 7 or 10 days. In conclusion, expression of PGHS-2 and PGF(2alpha) receptor mRNA is protein kinase A-dependent in preovulatory bovine granulosa cells. Granulosa cells become PGF(2alpha)-responsive soon after expression of PGF(2alpha) receptor, whereas further differentiation is required before PGF(2alpha) induces PGHS-2 mRNA upregulation. These results demonstrate that at least two key transitions are required in PGF(2alpha)-induced luteal regression in the mid-cycle corpus luteum.

Expression of insulin-like growth factor binding protein (IGFBP)-3, and the effects of IGFBP-2 and -3 in the bovine corpus luteum

The present study was conducted to gain insight into the insulin-like growth factor (IGF) system in the bovine corpus luteum (CL). Specific aims were to measure the levels of IGF binding protein-3 (IGFBP-3) and RNA encoding IGFBP-3 in the CL throughout diestrus, and to investigate the effects of IGFBP-2 and -3 on IGF-I-stimulated progesterone (P4) production and IGF-I-receptor binding. Bovine CL were collected from a local abattoir and classified according to stage of diestrus based on anatomical characteristics. Corpora lutea from early, mid and late diestrus were each analyzed for the presence of IGFBP-3 by ligand blot analysis, and for RNA encoding IGFBP-3 by Northern blot analysis. Dissociated cells from mid-cycle CL were treated with IGF-I, IGFBP-2 or -3, or a combination of IGF-I and IGFBP-2 or -3. The effect of IGFBP-2 and IGFBP-3 on [ 125I] IGF-I binding to its receptor on CL plasma membranes also was investigated. IGFBP-3 protein and RNA expression were higher in early CL, compared to mid or late CL ( p < 0.05). IGF-I stimulated P4 production in a dose-dependant manner ( p < 0.05). IGFBP-2 and -3 blocked the stimulatory effect of IGF-I on P4 production ( p < 0.05). Both IGFBP-2 and -3 inhibited [ 125I]-IGF-I binding to its receptor in a dose-dependant manner. These results demonstrate that IGFBP-3 protein and RNA are expressed predominantly during early diestrus in the bovine CL. Moreover, both IGFBP-2 and -3 can modulate IGF-I actions in the CL by interfering with binding of IGF-I to its receptor.

Porcine leptin receptor: Molecular structure and expression in the ovary

The porcine leptin receptor complementary DNA was cloned and sequenced and the leptin receptor gene expression evaluated in the porcine ovary. An open reading frame of 3498 nt cDNA was amplified from pig liver mRNA by RT-PCR. Sequence homology with the extracellular, transmembrane, and cytoplasmic domains of human, mouse, rat, sheep, and cow leptin receptors varied between 45% and 90%. Leptin receptor mRNA was present in porcine kidney, liver, spleen, lung, brain, testis, uterus, ovary, corpus luteum (CL), theca, and granulosa cells. The abundance of leptin receptor transcripts and protein varied during luteinization of granulosa cells in vitro and in the CL during the pig luteal phase. In the postovulatory CL, both mRNA and protein were low but detectable, maximal expression was observed in the midcycle CL, and lowest abundance occurred in regressed CL. Leptin receptor mRNA was present in granulosa cells at isolation and increased in abundance as the cells luteinized over 96 hr in culture. Leptin receptor protein was detectable after 12 hr of in vitro luteinization. We conclude that leptin receptor is expressed in granulosa and luteal cells, and varies during pig ovarian cell differentiation. Mol. Reprod. Dev. 56:465–474, 2000. © 2000 Wiley-Liss, Inc.

Administration of Prostaglandin F2α During the Early Bovine Luteal Phase Does Not Alter the Expression of ET-1 and of Its Type A Receptor: A Possible Cause for Corpus Luteum Refractoriness

Luteal regression is initiated by prostaglandin F(2 alpha) (PGF(2 alpha)). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF(2 alpha). Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF(2 alpha)-induced luteolysis. Therefore, in this study, we compared the effects of PGF(2 alpha) administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF(2 alpha) receptors (PGF(2 alpha)-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF(2 alpha) analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF(2 alpha) analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF(2 alpha) injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450(scc)) was only affected when PGF(2 alpha) was administered during midcycle. Nevertheless, PGF(2 alpha) elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF(2 alpha)-R was transiently affected. Such effects probably result from PGF(2 alpha) acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF(2 alpha), possibly the endothelium, could yet be nonresponsive during the early luteal phase.

Different actions of noradrenaline and nitric oxide on the output of prostaglandins and progesterone in cultured bovine luteal cells

The effects of noradrenaline (NA) and nitric oxide (NO) on prostaglandins (PGs) and progesterone (P4) secretion during the development of the bovine corpus luteum (CL) were investigated. Bovine luteal cells of early and mid-cycle CL were cultured for 20 to 24 h in medium containing 10% calf serum, washed, and treated with NA or nitrergic agents for an additional 16 h in a serum-free medium. NA (10 −5 M) stimulated P4 from early and mid-cycle CL by 238% and 154% ( P < 0.01), respectively. Moreover, although NA induced a twofold increase in PGE 2 secretion ( P < 0.01) in both examined periods, the effect of NA on PGF 2α secretion was approximately 1.5 times higher ( P < 0.05) in early than in mid-cycle CL. Two NO synthase inhibitors, l-NAME and l-NOARG (both 10 −4 M), stimulated P4 secretion only in mid-luteal cells ( P < 0.01), although they did not affect the cells from early CL. Although a NO donor, S-NAP (10 −4 M) inhibited P4 secretion from mid-cycle luteal cells ( P < 0.05), it strongly stimulated PGE 2 in both examined phases ( P < 0.001). On the other hand, the output of PGF 2α was stimulated by S-NAP only in the cells of the mid-cycle CL ( P < 0.01). The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL functions. Whereas NA may play a supporting role in luteal development, NO may participate in the functional regression of the bovine CL by inhibiting steroidogenesis.

Sensitivity of bovine corpora lutea to prostaglandin F2alpha is dependent on progesterone, oxytocin, and prostaglandins.

Prostaglandin (PG) F2alpha that is released from the uterus is essential for spontaneous luteolysis in cattle. Although PGF2alpha and its analogues are extensively used to synchronize the estrous cycle by inducing luteolysis, corpora lutea (CL) at the early stage of the estrous cycle are resistant to the luteolytic effect of PGF2alpha. We examined the sensitivity of bovine CL to PGF2alpha treatment in vitro and determined whether the changes in the response of CL to PGF2alpha are dependent on progesterone (P4), oxytocin (OT), and PGs produced locally. Bovine luteal cells from early (Days 4-5 of the estrous cycle) and mid-cycle CL (Days 8-12 of the estrous cycle) were preexposed for 12 h to a P4 antagonist (onapristone: OP; 10(-4) M), an OT antagonist (atosiban: AT; 10(-6) M), or indomethacin (INDO; 10(-4) M) before stimulation with PGF2alpha. Although OP reduced P4 secretion (p < 0.001) only in early CL, it reduced OT secretion in the cells of both phases examined (p < 0.001). OP also reduced PGF2alpha and PGE2 secretion (p < 0.01) from early CL. However, it stimulated PGF2alpha secretion in mid-cycle luteal cells (p < 0.001). AT reduced P4 secretion in early and mid-cycle CL (p < 0.05). Moreover, PGF2alpha secretion was inhibited (p < 0.05) by AT in early CL. The OT secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2alpha. PGF2alpha had no influence on OT secretion, although [Ca2+]i increased (p < 0.05) in the early CL. However, the effect of PGF2alpha was augmented (p < 0.01) in cells after pretreatment with OP, AT, and INDO in comparison with the controls. In mid-cycle luteal cells, PGF2alpha induced 2-fold increases in OT secretion and [Ca2+]i. However, in contrast to results in early CL, these increases were magnified only by preexposure of the cells to AT (p < 0.05). These results indicate that luteal P4, OT, and PGs are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and may be responsible for the resistance of the early bovine CL to the exogenous PGF2alpha action.

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

We evaluated the agreement between ultrasonographic characteristcs of the corpus luteum (CL) and plasma progesterone (P4) concentration in dairy cows. In Phase 1 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 susequent 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.

Angiotensin II interacts with prostaglandin F2alpha and endothelin-1 as a local luteolytic factor in the bovine corpus luteum in vitro.

Recent findings suggest that the ovarian renin-angiotensin system may regulate ovarian function through the paracrine/autocrine actions of angiotensin II (Ang II). In this study, we have examined and characterized the local effects of Ang II as a luteolytic factor and its interaction with prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1) in the bovine corpus luteum (CL) of the mid-luteal phase, by using an in vitro microdialysis system (MDS). Ang II was detected in the MDS perfusate (4 pg/ml), and infusion of PGF2alpha (10(-6) M) for 2 h increased the Ang II release by 50-100% during the following experimental period, in addition to its stimulation of ET-1 release. Two 2-h infusions of Ang II (10(-7)-10(-5) M) separated by a 2-h interval induced a dose- and time-dependent decrease of progesterone (P4) release by 41-66%. When the luteal explants were pre-perfused with PGF2alpha (10(-6) M) for 2 h, two consecutive perfusions of Ang II (10(-6) M) at a 2-h interval rapidly reduced the P4 release (by 50%). This reduction occurred 6 h earlier than those of infusions of PGF2alpha or Ang II alone. The simultaneous infusion of either 1) Ang II (10(-6) M) with PGF2alpha (10(-6) M), 2) ET-1 (10(-7) M) with PGF2alpha, or 3) Ang II + ET-1 with PGF2alpha (10(-6) M) for 2 h also induced a rapid and pronounced (60%) decrease in P4 release. Perfusion with the Ang II antagonist blocked the P4-suppressing activity of Ang II alone or PGF2alpha + Ang II infusion. Ang II stimulated the release of ET-1 and oxytocin during infusion but inhibited them after infusion. These results show that Ang II is released in the bovine midcycle CL in vitro, and this peptide, either alone or together with PGF2alpha, can suppress the release of P4. As PGF2alpha directly stimulated Ang II release, Ang II may influence the critical period for starting the cascade of functional luteolysis in vivo and might lead to structural luteolysis with ET-1 as a major vasoconstrictor. The overall results suggest that Ang II may have an important role at luteolysis in the bovine CL.

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

We evaluated the agreement between ultrasonographic characteristcs of the corpus luteum (CL) and plasma progesterone (P4) concentration in dairy cows. In Phase 1 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 susequent 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.

Luteinizing hormone receptors in the bovine corpus luteum during the oestrous cycle and pregnancy.

The concentration and affinity of luteinizing hormone (LH) receptors in bovine luteal tissues during the oestrous cycle and pregnancy were investigated by Scatchard analysis of the binding of 125I-labeled human chorionic gonadotropin. Corpora lutea (CL) were classified into five stages of the oestrous cycle and three stages of pregnancy. The concentration of LH receptors sharply increased from the early I stage of the oestrous cycle (Days 2-3; 3.09 fmol mg(-1) protein) to the early II stage (Days 5-6; 9.44 fmol mg(-1) protein) and then remained constant until the late luteal stage (Days 15-17; 8.14-9.56 fmol mg(-1) protein). The LH receptors could not be analysed in the regressed luteal tissue due to the small amounts of binding. There was no significant difference in the concentrations of LH receptors (5.63-9.64 fmol mg(-1) protein) among the three stages of pregnancy. Moreover, the concentrations of the receptors in the CL of pregnancy were comparable to those in the mid-cycle CL. The binding affinity did not change significantly during the oestrous cycle and pregnancy. Based on these results, it is assumed that the luteal function during the entire period of pregnancy might be regulated, at least in part, by LH, which is mediated via its specific receptors, and that the luteal function during pregnancy seems not to be regulated by changes in the binding capacity and affinity of LH receptors. To understand the physiological roles of LH in regulating luteal function in pregnant cows, further studies are required.

Prostaglandin F2alpha regulates distinct physiological changes in early and mid-cycle bovine corpora lutea.

Prostaglandin (PG) F2alpha is the primary luteolysin in most species. A single treatment with PGF2alpha will cause regression of the mid-cycle but not the early-cycle (Days 1-5 after estrus) bovine corpus luteum (CL) despite the presence of similar concentrations of high-affinity PGF2alpha receptors (FP receptors). This study was designed to determine whether PGF2alpha activated similar intracellular processes in early- and mid-cycle CL. Cows received saline or 25 mg PGF2alpha injection (i.m.; n = 6/group) on Day 4 or 11 after onset of the LH surge (induced by GnRH injection), and CL were collected at 4 h after treatment. As expected, CL volumes and luteal weights were not different at 4 h after PGF2alpha treatment. Luteal vitamin C concentration and steady-state concentrations of mRNA for 3beta-hydroxysteroid dehydrogenase and for FP receptor were decreased by 4 h in both Day 4 and 11 CL treated with PGF2alpha (p < 0.05). These results demonstrate clear actions of PGF2alpha in the early CL. In contrast, steady-state concentrations of mRNA encoding PG G/H synthase-2 (PGHS-2) were increased by treatment with PGF2alpha in mid-cycle CL but decreased by PGF2alpha in early-cycle CL (p < 0.05). In addition, treatment of mid-cycle but not early-cycle cows with PGF2alpha decreased luteal and serum progesterone concentrations by 4 h (p < 0.05). In summary, PGF2alpha clearly exerts actions in both early- and mid-cycle bovine CL. The lack of PGF2alpha-induced luteolysis in the early CL may be due to specific changes in gene expression, especially PGHS-2, that may prevent intraluteal PGF2alpha production and possibly other key luteolytic processes.

Fibroblast Growth Factor Receptor (FGFR)-1 and -2 in the Ovine Corpus Luteum throughout the Estrous Cycle

Fibroblast growth factors (FGFs) probably play an important role in development and maintenance of the vasculature of the corpus luteum (CL). The objective of the present study was to evaluate the distribution and levels of fibroblast growth factor receptors (FGFRs) in the ovine CL from the early, mid-and late stages of the estrous cycle. Presence of FGFR-1 and -2 receptors was evaluated in CL by using Western analysis, immunohistochemistry and topical autoradiography. Western analysis demonstrated that the levels of FGFR-1 and -2 were similar in the early and mid-cycle CL but increased (p < 0.05) in the late stage of the estrous cycle. Immunohistochemistry and topical autoradiography demonstrate that both parenchymal (steroidogenic) and nonparenchymal (e.g. endothelial, fibroblastic) cells express FGFR-1 and -2. FGFR-1 was localized to the luteal vasculature throughout the estrous cycle; in the parenchymal cells, it was present during mid-cycle but was barely detectable in the late stage. Conversely, FGFR-2 was present in the parenchymal cells at all stages of the estrous cycle but localized to the larger microvessels only at the late stage. These data demonstrate that FGF receptors are present in the parenchyma as well as the vasculature of the CL which suggests that FGF is involved in the regulation of luteal parenchymal and vascular function.

Hormonal regulation of monocyte chemoattractant protein-1 messenger ribonucleic acid expression in corpora lutea.

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that attracts monocytes and macrophages. It is known that macrophages accumulate in the corpus luteum (CL) during luteal regression in many species. In this study, we investigated the regulation of MCP-1 mRNA in ovine and bovine CL during prostaglandin (PG) F2alpha-induced luteolysis, after LH treatment, or after pharmacologic activation of the protein kinase (PK) A or PKC intracellular effector systems. In experiment 1, ewes on day 11 or 12 of the estrous cycle were infused with saline or PGF2alpha. PGF2alpha increased MCP-1 mRNA at 1 and 4 h after treatment. MCP-1 mRNA returned to basal level at 12 h and increased again at 24 h post treatment. In experiment 2, ewes received saline, PGF2alpha, phorbol 12-myristate 13-acetate (PMA), luteinizing hormone (LH), or forskolin infusion and CL were collected at 0 (untreated), 4, 12, or 24 h after infusion. Similar to experiment 1, PGF2alpha induced MCP-1 mRNA at 4 and 24 h post treatment. PMA increased mRNA for MCP-1 at 4, 12, and 24 h. Treatment with LH or forskolin transiently decreased MCP-1 mRNA expression. In experiment 3, cows were treated with a luteolytic dose (25 mg) of PGF2alpha on day 4 or day 11 of estrous cycle and expression of MCP-1 mRNA was quantified. Steady-state concentrations of mRNA for MCP-1 were induced by PGF2alpha treatment only in mid-cycle CL but not in early CL. In summary, administration of PGF2alpha or activation of PKC induced MCP-1 mRNA expression. Expression of MCP-1 may be important for stimulating immune processes during luteal regression.

Hormonal Regulation of Monocyte Chemoattractant Protein-1 Messenger Ribonucleic Acid Expression in Corpora Lutea

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that attracts monocytes and macrophages. It is known that macrophages accumulate in the corpus luteum (CL) during luteal regression in many species. In this study, we investigated the regulation of MCP-1 mRNA in ovine and bovine CL during prostaglandin (PG) F2α-induced luteolysis, after LH treatment, or after pharmacologic activation of the protein kinase (PK) A or PKC intracellular effector systems. In experiment 1, ewes on day 11 or 12 of the estrous cycle were infused with saline or PGF2α. PGF2α increased MCP-1 mRNA at 1 and 4 h after treatment. MCP-1 mRNA returned to basal level at 12 h and increased again at 24 h post treatment. In experiment 2, ewes received saline, PGF2α, phorbol 12-myristate 13-acetate (PMA), luteinizing hormone (LH), or forskolin infusion and CL were collected at 0 (untreated), 4, 12, or 24 h after infusion. Similar to experiment 1, PGF2α induced MCP-1 mRNA at 4 and 24 h post treatment. PMA increased mRNA for MCP-1 at 4, 12, and 24 h. Treatment with LH or forskolin transiently decreased MCP-1 mRNA expression. In experiment 3, cows were treated with a luteolytic dose (25 mg) of PGF2α on day 4 or day 11 of estrous cycle and expression of MCP-1 mRNA was quantified. Steady-state concentrations of mRNA for MCP-1 were induced by PGF2α treatment only in mid-cycle CL but not in early CL. In summary, administration of PGF2α or activation of PKC induced MCP-1 mRNA expression. Expression of MCP-1 may be important for stimulating immune processes during luteal regression.