Bovine Corpus Luteum Research Articles

Effect of Intraluteal Injection of Endothelin Type A Receptor Antagonist on PGF2.ALPHA.-induced Luteolysis in the Cow

Endothelin-1 (ET-1) is a luteolytic mediator in the bovine corpus luteum (CL), and its action appears to be via endothelin type A receptor (ETR-A). Thus, the aim of the present study was to determine the effect of ETR-A antagonist on PGF2alpha-induced luteolysis in the cow. Cows on days 10-12 of the estrous cycle were subjected to five intraluteal injections of the ETR-A antagonist LU 135252 in saline or only saline at -0.5, 2, 4, 6, and 8 h after PGF2alpha administration (=0 h). Serial luteal biopsies were conducted to determine the expression of mRNA in the luteal tissue. There were no significant differences in the decrease in plasma progesterone (P) concentrations and the mRNA expressions of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase/Delta5, Delta4-isomerase between the ETR-A antagonist-treated group and the control group. However, the start of the decline in CL volume and blood flow area surrounding the CL was delayed for almost two days in the ETR-A antagonist-treated group compared to the control group. The mRNA expression of preproET-1 and endothelin type B receptor increased in both groups, while the ETR-A mRNA remained unchanged. In addition, caspase-3 mRNA expression increased significantly at 24 h in the control group only and its level was higher than that of the ETR-A antagonist-treated group. Thus, the present study suggests that ET-1 regulates structural luteolysis via ETR-A by controlling blood vessel contraction in the CL of the cow.

Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH(2) terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3', which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH(2)-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

Vasculogenesis by Endothelial Progenitor Cells In Vitro

Introduction and Aim: Vasculogenesis is the de novo formation of blood vessels from embryonic precursor cells. The formation of additional vessels is called angiogenesis, the sprouting from pre‐existing vessels by migration, proliferation and differentiation of endothelial cells. Until recently vasculogenesis was considered to be restricted to embryogenesis, however, evidence suggests that progenitor cells exist in the adult, able to participate in neovascularization. In order to study cellular and molecular control mechanisms without animal testing we have established in featuring the three‐dimensional formation of capillary‐like structures from microvascular endothelial cells. The aim of this study was to identify the specific subpopulation of cells that triggers the primary process of vasculogenesis in vitro.Material and Methods: Endothelial cells were isolated from the bovine corpus luteum and examined by phase contrast and transmission electron microscopy. Immunolabelling was done for the most important pro‐angiogenic factor (VEGF) and its endothelium‐specific receptors (VEGF‐R1, ‐R2) as well as for endothelial cell surface markers (CD 31, CD51/61, CD29) and presumptive stem cell marker CD 34. Additionally VEGF and VEGF‐R1, ‐R2 expression was demonstrated by polymerase chain reaction (RT‐PCR).Results: The beginning of vessel formation in vitro was initiated by confluence of endothelial cells. A linear and circular side‐by‐side arrangement of cells and the formation of capillary‐like structures with an internal lumen could be observed. Specific clusters of cells were identified. These cells were arranged in a star‐like formation, i.e., one particular round cell in the centre surrounded by several cells with extensions. Immunolocalization of VEGF, VEGF‐R1, ‐R2, CD 31, CD51/61, CD29 and CD34 was positive. VEGF‐R2 and CD 34 were found to be co‐localized in specific cells exclusively.Conclusions: Results for the first time reveal the specific subpopulation of endothelial cells that trigger the start of vasculogenesis in vitro. These progenitor cells are characterized by a specific morphology, by the presence of endothelial cell markers and by the expression of stem cell marker in vitro models

Phytoestrogens and Their Metabolites Inhibit the Sensitivity of the Bovine Corpus Luteum to Luteotropic Factors

The aim of this study was to examine whether active metabolites of phytoestrogens (equol and para-ethyl-phenol) inhibit sensitivity of bovine corpus luteum (CL) to luteinizing hormone (LH) and to auto/paracrine luteotropic factors (prostaglandin E2-PGE2 and prostaglandin F(2alpha)-PGF(2alpha)), and whether they influence pulsatile progesterone (P4) secretion by the bovine CL. In in vivo experiments, high levels of equol and para-ethyl-phenol were found in plasma and in the CL tissue of heifers and cows fed a soy bean diet (2.5 kg/animal/day), along with lower concentrations of P4 (P < 0.05). Both Prostaglandins (PG) and LH strongly stimulated P4 secretion in cultured pieces of CL that were collected from cows fed a standard diet (P < 0.01). There was no effect of PGs and LH on P4 stimulation in CLs obtained from cows fed a diet rich in soy bean. Finally, we examined whether active metabolites of phytoestrogens participated in regulation of pulsatile P4 secretion and LH-stimulated P4 secretion in vitro using a microdialysis system. Equol and para-ethyl-phenol had no effect on basic and pulsatile P4 secretion in CLs during 240 min of perfusion when compared to the control (P < 0.05). However, they inhibited LH-stimulated P4 secretion (P < 0.05). Phytoestrogens and their metabolites may disrupt CL function by inhibiting PG- and LH-stimulated P4 secretion.

ウシ黄体細胞における apoptosis 抑制因子の検索

ウシ黄体細胞における apoptosis 抑制因子の検索

Is nitric oxide luteolytic or antiluteolytic?

Nitric oxide (NO) has been reported to be luteolytic based on treatment of cows in vivo with an inhibitor of nitric oxide synthase (NOS-produces NO), which delayed the decline in progesterone by two to three days [Jaroszewki J, Hansel, W. Intraluteal administration of a nitric oxide synthase blocker stimulates progesterone, oxytocin secretion and prolongs the life span of the bovine corpus luteum. Proc Soc Exptl Biol Med 2000;224:50–5; Skarzynski D, Jaroszewki J, Bah, M, et al. Administration of nitric oxide synthase inhibitor counteracts prostaglandin F 2α-induced luteolysis in cattle. Biol Reprod 2003;68:1674–81]. The objective of this experiment was to determine the effect of a long acting NO donor or a NOS inhibitor infused chronically into the interstitial tissue of the ovarian vascular pedicle adjacent to the ovary with a corpus luteum on secretion of progesterone during the ovine estrous cycle. Ewes were treated either with Vehicle ( N = 5); Diethylenetriamine (DETA-control for DETA-NONOate; N = 5); ( Z)-1-[2-(2-aminoethyl)- N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONOate-long acting NO donor; N = 6); or l-nitro-arginine methyl ester ( l-NAME-NOS inhibitor; N = 6) every 6 h from 24:00 h (0 h) on day 8 through 18:00 h on day 18 of the estrous cycle. Jugular venous blood was collected every 6 h for analysis for progesterone and corpora lutea were collected at 18:00 h on day 18 and weighed. Weights of corpora lutea were heavier ( P ≤ 0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes, which did not differ amongst each other ( P ≥ 0.05). Profiles of progesterone in jugular venous blood on days 8–18 differed ( P ≤ 0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes did not differ ( P ≥ 0.05) amongst each other. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis.

Analysis of gene expression in non-regressed and regressed bovine corpus luteum tissue using a customized ovarian cDNA array

The lifespan of the bovine corpus luteum (CL) is an important factor in the control of normal ovarian cyclicity and the establishment and maintenance of pregnancy. There is increasing evidence that CL lifespan is regulated by alternative expression of genes that promote or inhibit luteolysis. To gain further insights into these events a 434 character ovarian cDNA array comprising genes attributed to key aspects of CL function including more than 100 anonymous expressed sequence tags (ESTs) was constructed and screened with α 33P dATP labeled RNA isolated from non-regressed ( n = 6) and regressed ( n = 6) CL tissue. Significance analysis of microarrays (SAM) identified 15 genes that changed expression 1.7-fold or more with a false discovery rate of <5%. The differentially expressed genes encoded enzymes involved in steroid biosynthesis and oxygen radical metabolism and proteins involved in extracellular matrix remodeling, apoptosis and cell structure. Results for five of the differentially expressed genes including matrix gla protein and collagen α1(I) (extracellular matrix), glutathione- S-transferase αI (oxygen metabolism), clusterin (apoptosis) and scavenger receptor BI (steroid biosynthesis) were confirmed by Northern blot analysis and found to be significantly different ( P < 0.01) between non-regressed and regressed CL tissue. Collectively this study identified genes with recognized roles in CL regression, genes with potential roles in this process and genes whose function have yet to be defined in this event.

Immunohistochemical Localization of Relaxin‐Like Factor/Insulin‐Like Peptide‐3 in the Bovine Corpus Luteum

Relaxin-like factor/insulin-like peptide (INSL)-3 is highly expressed in the bovine corpus luteum throughout the estrous cycle and pregnancy. Demonstration of translation of the relaxin-like factor message was previously shown for the follicle but not the corpus luteum. In this study, relaxin-like factor mRNA was highly expressed in the corpus luteum on days 7 and 14 of pregnancy. Tissues were fixed in 10% neutral buffered formalin, and utilizing two different antibodies to relaxin-like factor, W3 rabbit anti-bovine and 2-8F mouse anti-bovine, relaxin-like factor was localized in fibroblast-like cells. Staining was also observed in the Leydig cells of bovine testicular tissue. No staining was observed in small and large steroidogenic luteal cells, indicating a nonsteroidogenic source of luteal relaxin-like factor. Definitive cell type identification is currently being determined via electron microscopy.

The expression of angiopoietin-tie system members in CL during oestrous cycle and induced luteolysis in cow

Local mechanisms of angiogenesis and angiolysis in the corpus luteum (CL) directly relate to the luteal function. Recently, several studies have shown that members of the angiopoietin-tie system regulate remodelling of microvasculature in the ovary. The aim of this study was to determine the mRNA profiles of the angiopoietins ANPT-1 and ANPT-2 and the receptors Tie1 and Tie2 during different stages of bovine corpora lutea (CL) function. The CL were classified into 4 stages of oestrous cycle: early: Day 2–5, mid: Day 8–12, late: Day 15–17 and regressing: Day >18. For the experimental luteolysis cows during mid luteal stage (Days 8–12) were injected with PGF2α analogue (Cloprostenol) and CL were collected by transvaginal ovariectomy before and at 2, 4, 12, 24, 48 and 64h after PGF2α injection. The expression of ANPT-1, ANPT-2, Tie1 and Tie2 mRNA in the bovine CL during the oestrous cycle and PGF2α induced luteolysis (real-time changes before and after injection) was investigated by RT-PCR. The levels of ANPT-1 mRNA in early and regressing CL were lower (p<0.05) than those in mid and late CL, whereas ANPT-2 mRNA expression did not change during the oestrous cycle. Consequently, the ANPT-2/ANPT-1 ratio (an index of instability of blood vessels) was high in the early and regressing CL. The Tie1 mRNA expression did not change, while Tie2 mRNA expression decreased as the CL aged. During PGF2α induced luteolysis, ANPT-2 mRNA expression was acutely and temporally increased at 2h after PGF2α injection. The expression of ANPT-1 mRNA was decreased from 4h after PGF2α injection and remained at low levels, which results in gradual increase in the ANPT-2/ANPT-1 ratio. It is likely that the decrease of ANPT-1 is a basic mechanism of vascular remodelling in CL. Tie1 mRNA decreased at 64h while Tie2 mRNA declined at 48h after PGF2α. These data indicate that the ANPT-Tie system is involved in the local mechanisms regulating angiogenesis and angiolysis in the developing and regressing CL in cow.

Role of tumor necrosis factor-α and nitric oxide in luteolysis in cattle

Although prostaglandin (PG) F 2α is known to be a principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intra-ovarian factors. Tumor necrosis factor-α (TNFα) and its specific receptors are present in the bovine CL with the highest expressions at luteolysis. TNFα in combination with interferon-γ reduced progesterone (P4) secretion, increased PGF 2α and leukotriene C 4 (LTC 4) production, and induced apoptosis of the luteal cells in vitro. Low concentrations of TNFα caused luteolysis, which resulted in a decreased level of P4, and increased levels of PGF 2α, LTC 4 and nitrite/nitrate (stable metabolites of nitric oxide-NO) in the blood. Inhibition of local NO production counteracts spontaneous and PGF 2α-induced luteolysis. Therefore, NO is a likely candidate for the molecule that mediates PGF 2α and TNFα actions during luteolysis. Both PGF 2α and TNFα increase NO concentrations in blood, and stimulate NO synthase expression on protein level in the bovine CL cells. NO stimulates PGF 2α and LTC 4 secretion, inhibits P4 production and reduces the number of viable luteal cells. TNFα and NO induce apoptotic death of the CL by modulating expression of bcl-2 family genes and by stimulating expression and activity of caspase-3. The above findings indicate that TNFα and NO play crucial roles in functional and structural luteolysis in cattle.

Cooperative Expression of Monocyte Chemoattractant Protein 1 Within the Bovine Corpus Luteum: Evidence of Immune Cell-Endothelial Cell Interactions in a Coculture System1

Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF 2alpha together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF 2alpha for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P < 0.05). Basal secretion of EDN1 by EC was substantial (approximately 2 ng/ml), but was not affected by coculture with PBMC (P > 0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P < 0.05), but again, EDN1 secretion was unchanged (P > 0.05). Interestingly, PGF 2alpha did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P > 0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF 2alpha (P > 0.05), but MLC produced less progesterone in the presence of ActPBMC (P < 0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF 2alpha. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.

Electron microscopy of cultured angiogenic endothelial cells

Angiogenesis is a multi-step process involving migration, proliferation, and a specific spatial arrangement of endothelial cells. On the basis of a model of cultured microvascular endothelial cells derived from the bovine corpus luteum, all stages of in vitro angiogenesis as well as intussusceptive remodeling were characterized by scanning and transmission electron microscopy. To preserve the delicate three-dimensional cellular structures for electron microscopy, modified processing techniques for both transmission and scanning electron microscopy including micro-corrosion casting of cultured cells were established. The detailed results on morphological alterations and cellular interactions confirmed and complemented earlier studies of in vitro angiogenesis. Electron microscopy proved to be an efficient tool for detection and supervision of all major endothelial differentiation processes resembling in vivo conditions that are generally considered important in a realistic in vitro model of angiogenesis: occurrence of function-related cellular junctions; development of specific surface features indicating cellular polarity; production of extracellular matrix material; mechanisms leading to the formation of an internal lumen; specific spatial arrangement of endothelial cells within capillary-like networks; detachment of apoptotic cells as well as intussusception of specific cells within the course of vascular remodeling. The abundance of quickly available information provided by electron microscopic approaches may be useful for subsequent, e.g., biochemical or molecular, studies and thus delivers important controls for further experimental designs.

143 PRESENCE OF PROSTAGLANDIN F2α RECEPTOR IN IN VITRO-DERIVED MORULA AND BLASTOCYST STAGE BOVINE EMBRYOS

Culture of in vitro and in vivo-derived embryos in medium containing prostaglandin F2� (PGF) decreased embryonic development to blastocyst stage and reduced hatching rates (Scenna et al. Prostaglandins 73, 215-226). Moreover, administration of an inhibitor of PGF synthesis at the time of embryo transfer in bovine recipients improved pregnancy rates (Schrick et al. 2001 Theriogenology 59, 335 abstr.). These findings indicate a direct negative effect of PGF on embryonic development. However, to our knowledge, no evidence of PGF receptor expression in morula or blastocyst stage bovine embryos is available in the literature. Therefore, the objective of the current study was to determine the presence of PGF receptor mRNA using real-time RT-PCR and protein expression by Western blotting in morula or blastocyst stage in vitro bovine embryos. Briefly, isolated total RNA from compact morula or blastocyst stage embryos and from bovine tongue epithelium (positive control for PGF receptor mRNA) were reverse-transcribed into cDNA. A volume from the RT reaction equivalent to 10 embryos per tube was utilized to determine transcripts for PGF receptor and Histone H2A (standard PCR control). Polymerase chain reaction was performed, and identity of PCR fragments was confirmed by ethidium-bromide-stained 2% agarose gel electrophoresis and by DNA sequencing. To determine protein expression, morula and blastocyst stage embryos were lysed in lysis buffer (10% SDS, 1 m Tris pH 7.5, 1 m NaF, 1 m DTT, 0.1 m EGTA with protease inhibitors) and stored at -20�C. Crude proteins isolated from bovine corpora lutea (positive control for PGF receptor protein), embryo samples, and prestained standards were separated by 12% SDS-PAGE under reducing conditions. Proteins were electrotransferred onto a PVDF membrane. Nonspecific binding sites in the PVDF membrane were blocked with 10% nonfat dry milk, and the blot was washed and incubated for 1 h at room temperature with a 1:1000 dilution of the primary antibody (rabbit polyclonal antibody against PGF receptor protein). Subsequently, the blot was washed and incubated for 1 h at room temperature with 1:1000 dilution of mouse anti-rabbit IgG conjugated with horseradish peroxidase. Finally, the blot was washed and revealed by chemiluminescence in a CCD camera. Results indicated that transcripts as well as the protein for PGF receptor were present in early stage bovine embryos. Identification of PGF receptor in morula and blastocyst stage bovine embryos may, in part, explain the increase in pregnancy rates after administration of a PGF synthesis inhibitor at the time of embryo transfer, which opens up the possibility to develop new strategies to prevent detrimental effects of PGF during early embryonic development.

Effects of Selective Protein Kinase C Isozymes in Prostaglandin2α-Induced Ca2+ Signaling and Luteinizing Hormone-Induced Progesterone Accumulation in the Mid-Phase Bovine Corpus Luteum1

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.

Matrix metalloproteinase‐2 (MMP‐2) expression and regulation by tumor necrosis factor alpha (TNFα) 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.

Developmental Sensitivity of the Bovine Corpus Luteum to Prostaglandin F2α (PGF2α) and Endothelin-1 (ET-1): Is ET-1 a Mediator of the Luteolytic Actions of PGF2α or a Tonic Inhibitor of Progesterone Secretion?1

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.

Coexpression of preprotachykinin A and B transcripts in the bovine corpus luteum and evidence for functional neurokinin receptor activity in luteal endothelial cells and ovarian macrophages

Nonneuronal cell sources of tachykinins, such as substance P (SP) and neurokinin B (NKB), have been demonstrated in leukocytes, endothelial cells and endocrine cells, and may play a role in corpus luteum (CL) development. For this reason, we analyzed mRNA presence for the two tachykinin precursors together with the neurokinin-1 receptor and the neurokinin-3 receptor (NK-1R and NK-3R, preferred by SP and NKB, respectively) in bovine CL at various stages in the luteal phase. Using the RT-PCR technique, we detected coexpression for the preprotachykinin A gene (PPT-A), which encodes SP and neurokinin A (NKA), and the preprotachykinin B gene (PPT-B) for NKB in the CL at the development, secretion and regression stages. Coexpression was also noted for NK-1R and NK-3R gene transcripts. Cultures of endothelial cells (ECs) derived from bovine CL expressed NK-1R and NK-3R mRNA, as did ovarian macrophages. Agonist treatment induced a stronger intracellular calcium ([Ca 2+] i) increase after activation of NK-1R compared to NK-3R, a result that we verified by calcium imaging. This is the first evidence for functional tachykinin receptor activity in luteal ECs and ovarian macrophages from bovine CL.

Expression pattern of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) system members in bovine corpus luteum endothelial cells during treatment with FGF-2, VEGF or oestradiol.

The development of the corpus luteum (CL) is accompanied by very active angiogenesis. We hypothesize that during this process endothelial cells (EC) are under the control of several angiogenic factors and steroids. The aim of this study was to examine the expression of the angiogenic growth factor systems - fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) - in EC derived from the bovine CL. Endothelial cells were cultured in serum-free medium and treated for 24 h with different concentrations of oestradiol (range from 10(-13) to 10(-5) mol/l), VEGF or FGF-2 (1, 10 and 100 ng/ml, respectively) and compared with untreated controls. Cells were harvested, total RNA extracted and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Treatment with oestradiol or FGF-2 stimulated the expression of FGF-2, but VEGF treatment showed no effect on the FGF-2 expression. FGF-2 or VEGF treatment resulted in an up-regulation of the FGF receptor (FGFR) mRNA. However, no FGF-1 expression was detected in EC. For the VEGF system, treatment with FGF-2, VEGF or oestradiol did not affect VEGF expression. However, the presence of FGF-2 in the medium up-regulated the expression of both VEGF receptors (VEGFR-1 and VEGFR-2), whereas oestradiol or VEGF treatment showed no effect on the expression of these receptors. Our results reveal that functional angiogenic growth factor systems were expressed in vitro in bovine EC derived from the CL. This suggests that the angiogenic FGF and VEGF system members were regulated by FGF or VEGF, but not by oestradiol-17beta.

Involvement of the cytoskeleton in oxytocin secretion by cultured bovine luteal cells.

A number of substances have been implicated in the regulation of oxytocin (OT) secretion from bovine corpus luteum in vivo. However, isolated bovine luteal cells cultured in a monolayer lose the ability to secrete OT in response to stimulatory substances. The present study investigated how cell-to-cell contact and the cytoskeleton affect OT secretion by isolated bovine luteal cells. In experiment 1, bovine midluteal cells (Days 8-12 of the estrous cycle) were stimulated with prostaglandin F2alpha (PGF2alpha; 1 microM), noradrenaline (NA; 10 microM), or growth hormone (GH; 5 nM) in two culture systems: In one system, cell monolayers were incubated in 24-well culture plates, and in the other system, aggregates of cells were incubated in glass tubes in a shaking water bath. The cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes, whereas the cells cultured in glass tubes remained fully rounded during the experimental period and soon formed aggregates of cells. Although PGF2alpha, NA, and GH did not stimulate OT secretion by the monolayer cells, all tested substances stimulated OT secretion by the aggregated cells (P < 0.01). In experiment 2, the monolayer cells were pre-exposed for 1 h to an antimicrofilament agent (cytochalasin B; 1 microM) or two antimicrotubule agents (colchicine or vinblastine; 1 microM) before stimulation with PGF2alpha, NA, or GH. Although PGF2alpha, NA, and GH did not stimulate OT secretion by the monolayer cells in the presence of colchicine or vinblastine, they all stimulated OT secretion in the presence of cytochalasin B (P < 0.001). The overall results show that OT secretion by bovine luteal cells depends on microfilament function and cell shape. Moreover, the aggregate culture system that allows three-dimensional, cell-to-cell contact seems to be a good model for studying OT secretion by isolated bovine luteal cells.

Progesterone Is a Suppressor of Apoptosis in Bovine Luteal Cells1

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.