Mid Corpus Luteum Research Articles

Effect of hypoxia on progesterone production by cultured bovine early and mid luteal cells.

A major role of the corpus luteum (CL) is to produce progesterone (P4). The CL has immature vasculature shortly after ovulation, suggesting it exists under hypoxic conditions. To elucidate the mechanism involved in regulation of luteal cell function during CL development, we compared the effect of hypoxia on P4 production by cultured bovine early and mid luteal cells. Luteal cells obtained from early and mid CL were incubated under different O2 concentrations (20% and 3%) with or without hCG (1 U/ml) for 6 h and 24 h. After 6 h of culture in the presence of hCG, P4 production was not affected by hypoxia whereas decrease in its production by mid luteal cells was observed. After 24 h of culture, P4 production was significantly decreased by hypoxia in both stages of luteal cells regardless of the use of hCG. At 6 h of culture, hypoxia increased mRNA expression of hydroxyl-Δ-5-steroid dehydrogenase, 3β- and steroid Δ-isomerase 1 (HSD3B1) in early luteal cells, and decreased mRNA expression of cytochrome P450 cholesterol side chain cleavage (CYP11A1) enzyme in mid luteal cells. At 24 h of culture, mRNA expressions of steroidogenic acute regulatory protein (STAR), CYP11A1, and HSD3B1 were not affected by hypoxia in both stages of luteal cells. The overall results suggest that early luteal cells maintain P4 production under hypoxic conditions, and hypoxia-induced HSD3B1 may support this P4 production in the bovine early CL.

The Dynamics of microRNA Transcriptome in Bovine Corpus Luteum during Its Formation, Function, and Regression.

The formation, function, and subsequent regression of the ovarian corpus luteum (CL) are dynamic processes that enable ovary cyclical activity. Studies in whole ovary tissue have found microRNAs (miRNAs) to by critical for ovary function. However, relatively little is known about the role of miRNAs in the bovine CL. Utilizing small RNA next-generation sequencing we profiled miRNA transcriptome in bovine CL during the entire physiological estrous cycle, by sampling the CL on days: d 1–2, d 3–4, and d 5–7 (early CL, eCL), d 8–12 (mid CL, mCL), d 13–16 (late CL, lCL), and d > 18 (regressed CL, rCL). We characterized patterns of miRNAs abundance and identified 42 miRNAs that were consistent significantly different expressed (DE) in the eCL relative to their expression at each of the analyzed stages (mCL, lCL, and rCL). Out of these, bta-miR-210-3p, −2898, −96, −7-5p, −183-5p, −182, and −202 showed drastic up-regulation with a fold-change of ≥2.0 and adjusted P < 0.01 in the eCL, while bta-miR-146a was downregulated at lCL and rCL vs. the eCL. Another 24, 11, and 21 miRNAs were significantly DE only between individual comparisons, eCL vs. the mCL, lCL, and rCL, respectively. Irrespective of cycle stage two miRNAs, bta-miR-21-5p and bta-miR-143 were identified as the most abundant miRNAs species and show opposing expression abundance. Whilst bta-miR-21-5p peaked in number of reads in the eCL and was significantly downregulated in the mCL and lCL, bta-miR-143 reached its peak in the rCL and is significantly downregulated in the eCL. MiRNAs with significant DE in at least one cycle stage (CL class) were further grouped into eight distinct clusters by the self-organizing tree algorithm (SOTA). Half of the clusters contain miRNAs with low-expression, whilst the other half contain miRNAs with high-expression levels during eCL. Prediction analysis for significantly DE miRNAs resulted in target genes involved with CL formation, functionalization and CL regression. This study is the most comprehensive profiling of miRNA transcriptome in bovine CL covering the entire estrous cycle and provides a compact database for further functional validation and biomarker identification relevant for CL viability and fertility.

Expression and functional role of Bone Morphogenetic Proteins (BMPs) in cyclical corpus luteum in buffalo (Bubalus bubalis)

The role of growth factors in the modulation of ovarian function is an interesting area of research in reproductive biology. Recently, we have shown the expression and role of IGF, EGF, VEGF and FGF in the follicle and CL. Here, we report the presence of Bone Morphogenetic Proteins (BMPs) and their functional receptors in the corpus luteum (CL) of buffalo. The bubaline CL was classified into four stages according to the morphology and progesterone (P4) concentration. The qPCR, immunoblot and immunohistochemistry studies revealed that BMP2 and BMP Receptors (BMPR1A, BMPR1B and BMPR2) were significantly upregulated during the mid stage whereas BMP4 and BMP7 were upregulated during the early stage of CL (P<0.05). Studies on primary luteal cell culture (LCC) using mid CL showed a significant time and concentration dependent effect of BMP4 and BMP7 (P<0.05). At 100ngml−1, the BMPs maximally stimulated the transcripts of StAR, CYP11A1 and 3βHSD that paralleled with P4 accretion in the media (P<0.05). Further, the BMP4 as well as BMP7 upregulated the transcripts of PCNA and downregulated CASPASE3 in the LCC at the same concentration (P<0.05). Though the combined effect of BMP4 and 7 was significantly higher (P<0.05) than that of individual one, it was not additive. In conclusion, the expression of BMPs and their receptors were dependent on the stages of CL in the buffalo. Treatment of LCC with BMPs in vitro confirmed the presence of functional receptors that stimulated the P4 production and luteal cell survival. Moreover, the results support the concept that the upregulation of P4 and its biosynthetic pathway enzymes such as CYP11A1, StAR and 3βHSD in the CL is likely due to the autocrine and /or paracrine effects of BMP4 and BMP7 under physiological milieu.

Status of autophagy, lysosome activity and apoptosis during corpus luteum regression in cattle.

Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3α, LC3β, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL.

The luteotrophic function of galectin-1 by binding to the glycans on vascular endothelial growth factor receptor-2 in bovine luteal cells.

The corpus luteum (CL) is a temporary endocrine gland producing a large amount of progesterone, which is essential for the establishment and maintenance of pregnancy. Galectin-1 is a β-galactose-binding protein that can modify functions of membrane glycoproteins and is expressed in the CL of mice and women. However, the physiological role of galectin-1 in the CL is unclear. In the present study, we investigated the expression and localization of galectin-1 in the bovine CL and the effect of galectin-1 on cultured luteal steroidogenic cells (LSCs) with special reference to its binding to the glycans on vascular endothelial growth factor receptor-2 (VEGFR-2). Galectin-1 protein was highly expressed at the mid and late luteal stages in the membrane fraction of bovine CL tissue and was localized to the surface of LSCs in a carbohydrate-dependent manner. Galectin-1 increased the viability in cultured LSCs. However, the viability of LSCs was decreased by addition of β-lactose, a competitive carbohydrate inhibitor of galectin-1 binding activity. VEGFR-2 protein, like galectin-1, is also highly expressed in the mid CL, and it was modified by multi-antennary glycans, which can be recognized by galectin-1. An overlay assay using biotinylated galectin-1 revealed that galectin-1 directly binds to asparagine-linked glycans (N-glycans) on VEGFR-2. Enhancement of LSC viability by galectin-1 was suppressed by a selective inhibitor of VEGFR-2. The overall findings suggest that galectin-1 plays a role as a survival factor in the bovine CL, possibly by binding to N-glycans on VEGFR-2.

Expression and localization of cFLIP anti-apoptotic protein in the porcine corpus luteum and corpora albicans during the estrous cycle and pregnancy.

We determined expression and localization of the anti-apoptotic cellular FLICE inhibitory protein (cFLIP) in the porcine corpora lutea (CL) and corpus albicans (CA) during estrous and pregnancy. The CL and CA were collected at different stages of estrous to determine cFLIP immunolocalization, and mRNA and protein expression. The mRNA expression of the short cFLIP isoform (cFLIPS) was higher at the early and mid CL stages, and lower by the late CL stage (P < 0.01); mRNA expression of the long cFLIP isoform (cFLIPL) was higher at the mid CL stage, and lower at the early and late CL stages (P < 0.01). Levels of cFLIPS and cFLIPL were steady and high during the early and mid CL stages, and had significantly decreased (P < 0.01) by the late stage. The cFLIP protein was highly expressed in the early and mid CL stages of estrous, but weakly ex-pressed in the late stage. Expression of cFLIPS showed no significant difference between preovulatory corpus albicans (CA1) and corpus albicans (CA2) coexistent with the CL from the previous estrus, but cFLIPL mRNA expression was higher during CA1 than CA2. The expression of cFLIPS showed no significant difference between CA1 and CA2, but cFLIPL was not detected. The cFLIP protein was weak-ly expressed in the CA. Expression of cFLIPS and cFLIPL mRNA and proteins was observed in the CL, and the cFLIP protein was highly expressed during pregnancy. We propose that cFLIPS/L acts as a survival factor, and performs an anti-apoptotic function in the porcine CL.

Opposing roles of leptin and ghrelin in the equine corpus luteum regulation: an in vitro study.

Metabolic hormones have been associated with reproductive function modulation. Thus, the aim of this study was: (i) to characterize the immunolocalization, mRNA and protein levels of leptin (LEP), Ghrelin (GHR) and respective receptors LEPR and Ghr-R1A, throughout luteal phase; and (ii) to evaluate the role of LEP and GHR on progesterone (P4), prostaglandin (PG) E2 and PGF2α, nitric oxide (nitrite), tumor necrosis factor-α (TNF); macrophage migration inhibitory factor (MIF) secretion, and on angiogenic activity (BAEC proliferation), in equine corpus luteum (CL) from early and mid-luteal stages. LEPR expression was decreased in late CL, while GHR/Ghr-R1A system was increased in the same stage. Regarding secretory activity, GHR decreased P4 in early CL, but increased PGF2α, nitrite and TNF in mid CL. Conversely, LEP increased P4, PGE2, angiogenic activity, MIF, TNF and nitrite during early CL, in a dose-dependent manner. The in vitro effect of LEP on secretory activity was reverted by GHR, when both factors acted together. The present results evidence the presence of LEP and GHR systems in the equine CL. Moreover, we suggest that LEP and GHR play opposing roles in equine CL regulation, with LEP supporting luteal establishment and GHR promoting luteal regression. Finally, a dose-dependent luteotrophic effect of LEP was demonstrated.

Proliferation of Luteal Steroidogenic Cells in Cattle

The rapid growth of the corpus luteum (CL) after ovulation is believed to be mainly due to an increase in the size of luteal cells (hypertrophy) rather than an increase in their number. However, the relationship between luteal growth and the proliferation of luteal steroidogenic cells (LSCs) is not fully understood. One goal of the present study was to determine whether LSCs proliferate during CL growth. A second goal was to determine whether luteinizing hormone (LH), which is known have roles in the proliferation and differentiation of follicular cells, also affects the proliferation of LSCs. Ki-67 (a cell proliferation marker) was expressed during the early, developing and mid luteal stages and some Ki-67-positive cells co-expressed HSD3B (a steroidogenic marker). DNA content in LSCs isolated from the developing CL increased much more rapidly (indicating rapid growth) than did DNA content in LSCs isolated from the mid CL. The cell cycle-progressive genes CCND2 (cyclin D2) and CCNE1 (cyclin E1) mRNA were expressed more strongly in the small luteal cells than in the large luteal cells. LH decreased the rate of increase of DNA in LSCs isolated from the mid luteal stage but not in LSCs from the developing stage. LH suppressed CCND2 expression in LSCs from the mid luteal stage but not from the developing luteal stage. Furthermore, LH receptor (LHCGR) mRNA expression was higher at the mid luteal stage than at the developing luteal stage. The overall results suggest that the growth of the bovine CL is due to not only hypertrophy of LSCs but also an increase in their number, and that the proliferative ability of luteal steroidogenic cells decreases between the developing and mid luteal stages.

Local Auto-Amplification System for Prostaglandin F2alpha in Bovine Corpus Luteum.

It has been considered that regression of the corpus luteum (CL) is induced by positive feedback loop between uterine prostaglandin F2a (PGF) and luteal oxytocin (OT) in ruminants. However, a previous study showed that an injection of PGF together with an OT receptor antagonist also induced luteolysis with an increase of plasma PGF level. Furthermore, administration of PGF in vivo dramatically increased the PGF production of ovine CL tissue. These findings suggest that PGF stimulates intraluteal PGF production, and that the possible auto-amplification system for PGF production plays an important role in luteolysis. However, it is not clear whether PGF directly stimulates intraluteal PGF production and which PGF synthetic enzymes are involved in PGF-stimulated PGF biosynthesis. In the present study, we examined the effects of PGF 1) on PGF and PGE production in bovine luteal tissue, and 2) on PGF biosynthetic enzymes such as cyclooxygenase (COX)-2, PGF synthase (PGFS) and carbonyl reductase (CBR)-1 in bovine mid luteal cells. Luteal tissues were collected at the early (Day 2-3), developing (Days 5-6), mid (Days 8-12) and late (Days 15-17) luteal stages. These tissues were exposed to PGF (1 μM) or PGF together with a COX inhibitor (indomethacin: 10 μM) for 2 h, and then washed with PBS 6 times to remove the treated PGF2. The tissues were cultured further for 2 h. PGF stimulated intraluteal PGF production in all stages but did not affect PGE2 production. The stimulatory effect of PGF was greatest at the late luteal stage. Indomethacin suppressed the PGF-stimulated PGF production in luteal tissue, indicating that the increased PGF in the medium was luteal origin. The protein expression of COX-2, PGFS and CBR-1 were determined by western blotting in cultured bovine luteal cells obtained from the mid CL. Cells were treated for 4 or 24 h with PGF (1 μM). PGF significantly increased PGFS and CBR-1 expression (P<0.05), and tended to increase COX-2 protein level (P=0.06) at 24 h post-treatment. The overall results strongly suggest the presence of a local auto-amplification system for PGF that is mediated by PGFS, CBR-1 and COX-2 activation.

Equine Luteal Function Regulation May Depend on the Interaction Between Cytokines and Vascular Endothelial Growth Factor: An In Vitro Study1

We hypothesized that cytokines influence luteal angiogenesis in mares, while angiogenic factors themselves can also regulate luteal secretory capacity. Therefore, the purpose of this study was to evaluate the role of cytokines--tumor necrosis factor alpha (TNF), interferon gamma (IFNG) and Fas ligand (FASL)--on in vitro modulation of angiogenic activity and mRNA level of vascular endothelial growth factor A (VEGF), its receptor VEGFR2, thrombospondin 1 (TSP1), and its receptor CD36 in equine corpus luteum (CL) throughout the luteal phase. After treatment, VEGF protein expression was determined in midluteal phase (mid) CL cells. The role of VEGF on regulation of luteal secretory capacity was assessed by progesterone (P(4)) and prostaglandin E(2) (PGE(2)) production and by mRNA levels for steroidogenic enzymes 3-beta-hydroxysteroid dehydrogenase (3betaHSD) and PGE synthase (PGES). In early CL cells, TNF increased angiogenic activity (bovine aortic endothelial cell viability) and VEGF and VEGFR2 mRNA levels and decreased CD36 (real-time PCR relative quantification). In mid-CL cells, TNF increased VEGF mRNA and protein expression (Western blot analysis) and reduced CD36 mRNA levels, while FASL and TNF+IFNG+FASL decreased VEGF protein expression. In late CL cells, TNF and TNF+IFNG+FASL reduced VEGFR2 mRNA, but TNF+IFNG+FASL increased TSP1 and CD36 mRNA. VEGF treatment increased mRNA levels of 3betaHSD and PGES and secretion of P(4) and PGE(2). In conclusion, these findings suggest a novel auto/paracrine action of cytokines, specifically TNF, on the up-regulation of VEGF for angiogenesis stimulation in equine early CL, while at luteolysis, cytokines down-regulated angiogenesis. Additionally, VEGF stimulated P(4) and PGE(2) production, which may be crucial for CL establishment.

Seasonal Changes in Luteal Progesterone Concentration and mRNA Expressions of Progesterone Synthesis-related Proteins in the Corpus Luteum of Mares

Although circulating progesterone (P₄) levels tend to change with the season, little is known about the seasonal changes of P₄ synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (~N32°), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P₄ concentration, was used to evaluate the seasonal changes in P₄ synthesis. The luteal P₄ concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) were lowest during early winter and increased during late winter. These results suggest that P₄ synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P₄ synthesis-related proteins in mares.

High Number of Neutrophils Exists in the Developing Corpus Luteum and Promotes Angiogenesis and Progesterone Secretion in the Cow.

After ovulation, the bovine corpus luteum (CL) rapidly develops within a few days together with angiogenesis and progesterone (P) production. Ovulation and CL formation closely resemble an inflammatory response due to the influx of immune cells. It is well known that polymorphonuclear neutrophils (PMNs) play a role in host defense, inflammation, tissue remodeling and wound healing, and they secrete chemoattractants stimulating angiogenesis. We therefore hypothesized that PMNs and its chemoattractant interleukin-8 (IL-8) exist in the early CL just after ovulation and may play a physiological role in the CL development. PMNs were detected in CL tissue by Pas-staining, and IL-8 was measured in supernatant of the CL tissue culture by ELISA: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). Extremely low levels of PMNs and IL-8 were observed in the mid and late luteal phase, but increased during luteal regression. In immunohistochemical analysis, IL-8 protein was stained mainly in endothelial cells and immune cells such as PMNs and mononuclear cells in the CL. In transmigration assay of PMNs isolated from days 2, 5 and 10 of the estrous cycle, the different phase of estrous cycle did not affect the levels of PMN migration. On the other hand, the PMN migration was higher by the supernatant derived from the early CL as well as the recombinant IL-8 compared with that of the mid CL, and the level of PMN migration was inhibited by neutralizing with an anti-IL-8 antibody. These findings indicated that IL-8 is one of major chemoattractants to migrate PMNs within the early CL. We further investigated the role of PMNs and IL-8 in luteal function such as angiogenesis in luteal endothelial cells and P secretion from the early luteal cells. To assess the ability to stimulate angiogenesis, the peripheral PMNs from the early luteal phase were activated in culture with N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10 and 100 nM) or without fMLP as control at 37°C for 1 h. Supernatant of activated PMNs was harvested and used for the assessment of capillary tube formation. In in vitro matrigel assay, the supernatant of activated-PMNs as well as IL-8 (1-100 ng/ml) stimulated sprouting of capillary-like structures of luteal endothelial cells. Also, IL-8 (10 and 100 ng/ml) increased the proliferation of luteal endothelial cells. In co-culture model with early luteal cells (2x105 cells/well) and PMNs (1x105 cells/well), PMNs stimulated P secretion either with or without direct contact, but this was not accompanied with the increase of mRNA for steroidogenic acute regulatory protein (StAR) in luteal cells. On the other hand, IL-8 (1-100 ng/ml) did not affect P secretion and StAR mRNA. In conclusion, neutrophils are novel regulators of CL development after ovulation, and IL-8 secreted from the early CL especially endothelial and immune cells is a major chemoattractant for the migration of PMNs into the CL in the cow. Supported grants by Global COE and JSPS programs. (poster)

Evidence that polymorphonuclear neutrophils infiltrate into the developing corpus luteum and promote angiogenesis with interleukin-8 in the cow

BackgroundAfter ovulation in the cow, the corpus luteum (CL) rapidly develops within a few days with angiogenesis and progesterone production. CL formation resembles an inflammatory response due to the influx of immune cells. Neutrophils play a role in host defense and inflammation, and secrete chemoattractants to stimulate angiogenesis. We therefore hypothesized that neutrophils infiltrate in the developing CL from just after ovulation and may play a role in angiogenesis of the CL.Methods and ResultsPolymorphonuclear neutrophils (PMN) were detected in CL tissue by Pas-staining, and interleukin-8 (IL-8, a neutrophil-specific chemoattractant) was measured in supernatant of the CL tissue culture: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). PMNs and IL-8 were low levels in the mid and late luteal phases, but IL-8 was increased during luteal regression. The PMN migration in vitro was stimulated by the supernatant from the early CL but not from the mid CL, and this activity was inhibited by neutralizing with an anti-IL-8 antibody, indicating the major role of IL-8 in inducing active PMN migration in the early CL. Moreover, IL-8 stimulated proliferation of CL-derived endothelial cells (LECs), and both the supernatant of activated PMNs and IL-8 stimulated formation of capillary-like structures of LECs.ConclusionPMNs migrate into the early CL partially due to its major chemoattractant IL-8 produced at high levels in the CL, and PMNs is a potential regulator of angiogenesis together with IL-8 in developing CL in the cow.

Prostaglandin F2.ALPHA. Differentially Affects mRNA Expression Relating to Angiogenesis, Vasoactivation and Prostaglandins in the Early and Mid Corpus Luteum in the Cow

Administration of prostaglandin (PG) F(2alpha) in cattle during the mid-luteal phase (Days 8-12 of the estrous cycle) drastically reduces the plasma progesterone concentrations and the volume of the corpus luteum (CL). However, PGF(2alpha) does not induce luteolysis during the early luteal phase (up to Day 5 of the estrous cycle). To characterize the possible distinct difference in acute response to a luteolytic dose of PGF(2alpha) administration, we determined various mRNA expressions in the early and mid CL relating to angiogenesis, vasoactivation and PG-related factors at 30 min after PGF(2alpha) injection in cyclic cows. The experiments were conducted on Day 4 (early CL) and Days 10-12 (mid CL). Cows were either injected with 500 microg PGF(2alpha) analogue or saline as the control (early CL control, n=5; early CL PGF(2alpha) treated, n=5; mid CL control, n=5; mid CL PGF(2alpha) treated, n=7). Thirty min after injection of PGF(2alpha) or saline, the cows were ovariectomized transvaginally, and the CL tissues were collected from regions designated as the periphery and center of the CL. Administration of PGF(2alpha) up-regulated the mRNA expressions of angiogenic-related factors such as vascular endothelial growth factors, vasohibin, fibroblast growth factor 2 and insulin-like growth factor-II in the early CL, whereas PGF(2alpha) down-regulated these mRNA expressions in the mid CL. In the vasoactive factors, PGF(2alpha) stimulated the mRNA expressions of endothelin-1, angiotensin converting enzyme, endothelial nitric oxide synthase (NOS) and inducible NOS in the periphery area of the mid CL, but not in the early CL. However, PGF(2alpha) drastically down-regulated PGF(2alpha) receptor mRNA expression in both regions of the early and mid CL. The results indicated a clear difference in the acute action of PGF(2alpha) depending not only on the luteal phase (immature vs. mature) but also the region (periphery vs. center) within the CL at 30 min after PGF(2alpha) injection in the cow.

Distribution of Arteriolovenous Vessels, Capillaries and eNOS Expression in the Bovine Corpus Luteum During the Estrous Cycle: a Possible Implication of Different Sensitivity by Luteal Phase to PGF2.ALPHA. in the Increase of Luteal Blood Flow

We have shown that luteal blood flow increases in the peripheral vasculature of the mature corpus luteum (CL) prior to the onset of luteolysis in response to prostaglandin F(2alpha) (PGF(2alpha)) in the cow, but this phenomenon does not occur in the early CL. We therefore hypothesize that this acute increase of luteal blood flow occurs by vasodilation of large blood vessels due to local release of nitric oxide (NO). In the present study, we characterized the CL vasculature together with endothelial NO synthase (eNOS) expression during the estrous cycle in the cow. Immunohistochemistry was used to quantify the number of arteriolovenous vessels (surrounded with smooth muscle actin-positive smooth muscle cells), capillaries (with von Willebrand Factor-positive endothelial cells) and eNOS protein. The arteriolovenous vessels existed more in the periphery of the matured CL (mid, late and regressing CL) than in the center region. In the early CL, there were as many arteriolovenous vessels in the periphery as in the center, while more capillaries existed in the center than in the periphery of the mid and late CL. Also, eNOS protein existed in the periphery more than in the center of the matured CL. These results indicate that the early CL has a homogeneous vascular and eNOS distribution. In contrast, the matured CL is a heterogeneous organ having a higher vascular and eNOS distribution in the periphery than in the center. In conclusion, the distribution of arteriolovenous vessels and eNOS in the matured CL was higher in the periphery than in the center of the CL. Thus, this suggests that this structural change from the early (homogeneous) to the mid (heterogeneous) luteal phase is related to the difference in the CL response of blood flow increase due to PGF(2alpha), which is only observed in the mature CL.

Effect of Cytokines and Steroid Hormones on Nitric Oxide Generating System in Bovine Luteal Endothelial Cells.

Injection of a luteolytic dose of prostaglandin (PG) F2α induces an acute increase in plasma concentration of nitric oxide (NO) metabolites (nitrite/nitrate). Furthermore, NO suppresses progesterone (P4) production and induces apoptosis in bovine luteal cells. These findings suggest that NO plays important roles in luteolysis. However, the regulation of NO generating system in bovine CL is not fully understood. Although tumor necrosis factor α (TNF) and interferon γ (IFNG) produced by immune cells have been demonstrated stimulate NO production in extragonadal cells, the roles of TNF and IFNG in the regulation of NO production, NO synthase (NOS) expression and NOS activity in the bovine corpus luteum (CL) remain unclear. To elucidate whether NO generating system in luteal endothelial cells (LECs) is regulated by cytokines, the effects of TNF or IFNG on the expression of NOS (inducible NO synthase; iNOS and endothelial NO synthase; eNOS) mRNA, protein and NO production were examined. In addition, the direct effects of estrogen (E2) and P4 on NOS proteins and NOS activity were examined. LECs were obtained from mid CL (Days 8-12 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. Cultured bovine LECs were treated for 24 h with TNF (2.9 nM), IFNG (2.5 nM), E2 (10 nM) or P4 (1 μM). Both TNF and IFNG stimulated iNOS mRNA, protein expression and NO production. Furthermore, E2 stimulatediNOS protein expression, whereas P4 inhibited iNOS protein expression. In contrast to iNOS, eNOS protein expression was not affected by treatments with TNF, IFNG, E2 or P4. By observing the conversion of [3C]L-arginine to [3C]L-citrulline, we found that TNF, IFNG and E2 stimulated the NOS activity in cultured LECs (P<0.05). The overall results demonstrate that TNF, IFNG and E2 accelerate NO production by stimulating iNOS protein expression as well as NOS activity. On the other hand, P4 was able to suppress iNOS expression in LECs. This research was supported by JSPS Grants (No. 19580326 and No. 18380166), and by a research fund from the Livestock Technology Association (LTA), Japan. (poster)

Prostaglandin F2α Stimulates Endothelial Nitric Oxide Synthase Depending on the Existence of Bovine Granulosa Cells: Analysis by Co‐culture System of Endothelial Cells, Smooth Muscle Cells and Granulosa Cells

Prostaglandin F(2 alpha) (PGF(2 alpha)) induces luteolysis in the mid but not in the early luteal phase; despite this, both the early and the mid corpus luteum (CL) have PGF(2 alpha) receptor (FPr). We previously indicated that the luteal blood flow surrounding the CL drastically increases prior to a decrease of progesterone (P) in the cows, suggesting that an acute increase of luteal blood flow may be an early sign of luteolysis in response to PGF(2 alpha) and that this may be induced by a vasorelaxant nitric oxide (NO). The aim of this study was to investigate the luteal stage-dependent and the site-restricted effect of PGF(2 alpha) and NO on the mRNA expressions and P secretion. To mimic the local luteal region both of peripheral and central areas of the CL, we utilized co-cultures using bovine aorta endothelial cells (EC), smooth muscle cells (SMC) and luteinizing granulosa cells (GC) or fully-luteinized GC. PGF(2 alpha) stimulated the expression of endothelial NO synthase (eNOS) mRNA at 0.5 h in mix-cultures of EC and SMC with fully-luteinized GC but not with luteinizing GC. The expression of eNOS mRNA in EC was increased by PGF(2 alpha) at 1 h only when EC was cultured together with fully-luteinized GC but not with luteinizing GC. In all co-cultures, PGF(2 alpha) did not affect the mRNA expression of FPr. Treatment of NO donor inhibited P secretion at 0.5 h. In conclusion, the present study suggests that the coexistence of the mature luteal cells (fully-luteinized GC) with EC/SMC may be crucial for acquiring functional NO synthesis induced by PGF(2 alpha).

LOCALIZATION AND CHANGES OF ENOS IN THE CORPUS LUTEUM AFTER ADMINISTRATION OF PGF2ALPHA DURING EARLY AND MID LUTEAL PHASE IN THE COW

We have indicated that luteal blood flow (BF) temporarily but drastically increases in the peripheral vasculature of mature corpus luteum (CL) at 0.5–2 hours after prostaglandin F2alpha (PGF) administration to induce luteolysis in the cow. However, this phenomenon has not been seen in early CL. We hypothesized that this acute increase of luteal BF occurs due to vasodilation induced by the local action of nitric oxide (NO) on the resistance vessels that possess smooth muscle layer, and might be one of the first signs to start the cascade of luteolysis. Firstly, we aimed to determine the site-dependent expression of endothelial nitric oxide synthase (eNOS) protein together with distribution of resistance vessels and capillaries. The CLs were obtained from local abattoir (early; Days2–5 n=5, mid; Days8–12 n=5), and were compared in immunohistochemical localization of eNOS with distribution of resistance vessels and capillaries. The number of the resistance vessels and capillaries were determined by immunohistochemistry for smooth muscle actin (SMA), and expressed as the number of vessels per 40,000 square micro meter. Also, the expression of eNOS protein was confirmed as the percentage of eNOS positive areas per 40,000 square micro meter. To examine the sitedependent effect, the area in the CL was divided into center area (without connective tissue) and periphery (1mm from the border of the luteal tissue and ovarian parenchyma) of the CL. The number of the resistance vessels was high only in periphery of the mid CL (early; 1.7±0.5 in periphery vs. N.D. in center, mid; 6.7±1.5 in periphery vs. 0.3±0.3 in center). The number of capillaries was at the same level between periphery and center in the early and mid CL respectively (early; 11.3±1.7 in periphery vs. 14.3±0.9 in center, mid; 7.0±1.2 in periphery vs. 7.7±1.5 in center). However, the number of capillaries decreased from early to mid stage. The eNOS protein in early CL was localized evenly in periphery and center. (periphery; 1.4±0.2, center; 1.3±0.2). In the mid CL, eNOS protein was localized mainly in periphery of the CL (periphery;1.4±0.1, center; 0.1±0.01). Secondly, to define the effect of PGF on the eNOS expression, PGF or saline was treated to cows (n=4–5, each treated group) during early (Day4) or mid luteal phase (Day10–12). At 30 min after treatment, we observed the luteal BF using the color Doppler ultrasound, and confirmed the luteal BF increase in the mid CL of the PGF treated group only. Cows were then ovariectomized, the CL was stained immunohistochemically for eNOS, and its mRNA expression was determined with real-time PCR. PGF increased both eNOS protein and mRNA expression only in the periphery of the mid CL (P<0.01), but not in the center of the mid CL and both area of the early CL. These results indicate that capillaries and eNOS in the early CL are distributed homogeneously between periphery and center, but in the mid CL, the resistance vessels and eNOS are evolved only in periphery. In conclusion, PGF stimulates eNOS expression together with luteal BF increase in the resistance vessels in the periphery of the matured CL. (poster)

EXPRESSIONS OF ESTROGEN RECEPTORS ALPHA AND BETA ARE REGULATED BY PROSTAGLANDIN F2ALPHA, TUMOR NECROSIS FACTOR ALPHA, AND INTERFERON GAMMA IN BOVINE LUTEAL CELLS

Estradiol (E) functions in gene regulation by binding to a specific intracellular receptor. Although E receptors α (ERα) and β (ERβ) are known to be present in bovine corpus luteum (CL), the functions of ERs throughout the luteal phase and the mechanisms that control ER expressions in luteal cells are not fully understood. To determine possible functions of ERα and ERβ and to clarify the regulatory mechanisms of ER expressions in the bovine CL, we examined 1) changes in the protein expressions of ERs in CL during the luteal phase, and 2) the effects of prostaglandin (PG) F2α, tumor necrosis factor α (TNFα) or interferon γ (IFNγ) on ER mRNA expressions in cultured bovine luteal cells. Western blot analyses revealed that ERα and ERβ proteins were expressed throughout the luteal phase. ERα protein level was higher at the early luteal stage (Days 2–3 after ovulation) and mid luteal stage (Days 8–12) than at the other luteal stages. ERβ protein level was higher at the developing and mid luteal stages than at the other luteal stages. The ratio of ERβ to ERα was higher in the regressed stage than in the other stages. Luteal cells obtained from mid stage CL (Days 8–12) were incubated with PGF2α (3.5–350 ng/ml), TNFα (0.25–2.5 ng/ml) or IFNγ (0.25–2.5 ng/ml) for 24 h. PGF2α and TNFα inhibited ERα and ERβ mRNA expressions. IFNγ suppressed ERβ mRNA expression but did not affect the expression of ERα mRNA. These data suggest that PGF2α, TNFα, and IFNγ regulate ERα and ERβ expressions in bovine luteal cells. The overall findings lead us to hypothesize that, in cow, ERα plays roles in maintenance of CL function, while ERβ functions in luteolysis. (poster)

Expression of mRNA for Cell Adhesion Molecules in the Bovine Corpus Luteum During the Estrous Cycle and PGF2.ALPHA.-Induced Luteolysis

Cell-to-cell interaction via cell contact-dependent pathway is essentially important for maintenance and regulation of corpus luteum (CL) integrity and its physiological actions. The objective of the present study was to evaluate the mRNA expression of the cell adhesion molecules (CAMs) that are constituent factors of gap junctions [connexin (Cx) 43] and adherence junctions (VE-, E-, N-cadherin) in two types of endothelial cells from the mid CL and in CL tissue during the estrous cycle and PGF(2alpha)-induced luteolysis in the cow. Specific mRNA expression for Cx43 and N-cadherin was detected in cytokeratin-positive (CK+) and cytokeratin-negative (CK-) luteal endothelial cells (EC) and fully luteinized granulosa cells (LGC). E-cadherin mRNA was expressed in CK+EC and LGC, but not in CK-EC. VE-cadherin mRNA was expressed in both CK+ and CK-EC. During the estrous cycle, Cx43 mRNA expression was significantly lower in the regressing CL. VE-cadherin expression also tended to increase in the mid CL and increased significantly in the regressing CL. E-cadherin mRNA expression was higher in the early and late CL than in the mid- and regressing CL. N-cadherin mRNA expression gradually increased from the early to late CL followed by a decrease in the regressing CL. During PGF(2alpha)-induced luteolysis, Cx43 mRNA expression appeared to increase, and VE-cadherin and E-cadherin mRNA significantly increased at 24 h. N-cadherin mRNA expression decreased 2 and 4 h after PGF(2alpha) administration. Collectively, expression of the mRNAs for CAMs was different in the two types of luteal endothelial cells and fully luteinized granulosa cells and changed independently in the CL during the estrous cycle and PGF(2alpha)-induced luteolysis in the cow. The results suggest that CAMs play physiological roles in cell-to-cell communication to regulate both gap and adherence junctions during CL development and regression in the cow.