Bovine Corpus Luteum Research Articles

Molecular characterization and expression of DERL1 in bovine ovarian follicles and corpora lutea

The endoplasmic reticulum (ER) is a major site of protein synthesis and facilitates the folding and assembly of newly synthesized proteins. Misfolded proteins are retrotranslocated across the ER membrane and destroyed at the proteasome. DERL1 is an important protein involved in the retrotranslocation and degradation of a subset of misfolded proteins from the ER. We characterized a 2617 bp cDNA from bovine granulosa cells that corresponded to bovine DERL1. Two transcripts of 3 and 2.6 kb were detected by Northern blot analysis, and showed variations in expression among tissues. During follicular development, DERL1 expression was greater in day 5 dominant follicles compared to small follicles, ovulatory follicles, or corpus luteum (CL). Within the CL, DERL1 mRNA expression was intermediate in midcycle, and lowest in late cycle as compared to early in the estrous cycle. Western blot analyses demonstrated the presence of DERL1 in the bovine CL at days 5, 11, and 18 of the estrous cycle. Co-immunoprecipitation using luteal tissues showed that DERL1 interacts with class I MHC but not with VIMP or p97 ATPase. The interaction between DERL1 and MHC I suggests that, in the CL, DERL1 may regulate the integrity of MHC I molecules that are transported to the ER membrane. Furthermore, the greater expression of DERL1 mRNA is associated with the active follicular development and early luteal stages, suggesting a role of DERL1 in tissue remodeling events and maintenance of function in reproductive tissues.

Nitric Oxide and Luteal Blood Flow in the Luteolytic Cascade in the Cow

The corpus luteum (CL) of the estrous cycle in the cow is a dynamic organ which has a lifespan of approximately 17-18 days. The main function of the CL is to produce progesterone (P) that requires for achievement and maintenance of pregnancy. If pregnancy does not occur successfully, the CL must regress within a few days to induce the next chance of ovulation. As the CL matures, the steroidogenic cells establish contact with many capillary vessels, consequently the CL is composed of a large number of vascular endothelial cells that can account for up to 50% of all cells. Therefore, blood vessels and blood flow within the CL have an essential role in luteal function. Nitric oxide (NO), a strong vasorelaxant, is now known to play key roles in a variety of physiological process. Indeed, NO has established itself as a polyvalent molecule which plays a decisive role in regulating multiple functions within female reproductive system. In the CL, NO is produced and regulates luteal blood flow, P secretion and apoptosis of luteal cells as well as endothelial cells. This review describes the current investigation for possible roles of NO in the luteolytic cascade within the bovine CL.

Expression and Localization of cFLIP, an Anti-apoptotic Factor, in the Bovine Corpus Luteum

The objective of the present study was to investigate the potential mechanisms regulating cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic factor, in the bovine corpus luteum (CL). Expression of cFLIP mRNA was highest at the developing stage and then decreased significantly during the mid, late and regressed stages (P<0.05). Western blot analysis revealed that expression of the long isoform of cFLIP (cFLIP(L)) protein was high during the early and developing luteal stages, remained steady during the mid and late luteal stages and then decreased significantly (P<0.05) by the regressed stage. However, the expression levels of the short isoform of cFLIP (cFLIP(S)) remained low during the early, developing and mid luteal stages. Immunostaining of cFLIP was strongest in the cytoplasm of luteal and non-luteal cells, including endothelial and immune cells, remained high during the early, developing and mid luteal stages and then decreased significantly (P<0.05) in the late and regressed luteal stages. Immunostaining of cFLIP was observed only in macrophage-like cells in the regressing CL. However, cultured mid luteal cells had a higher percentage of cFLIP-positive cells and a lower percentage of TUNEL-positive cells than luteal cells treated with tumor necrosis factor alpha (TNF)/interferon gamma (IFNG; P<0.01). These results indicate downregulation of cFLIP during structural luteal regression, suggesting that cFLIP plays a survival role in the bovine CL.

Changes in the Levels of Progesterone Receptor mRNA and Protein in the Bovine Corpus Luteum During the Estrous Cycle

Progesterone (P4) is synthesized in the luteal cells of many species. The objective of the present study was to determine the expression pattern of P4 receptor (PR) mRNA and the distribution of PR protein in the bovine corpus luteum (CL) during the estrous cycle. The gene expression of PR in the bovine CL throughout the estrous cycle was determined by real-time PCR analysis, and the PR protein expression was evaluated by immunohistochemistry. Messenger RNA of PR was clearly expressed in the CL throughout the estrous cycle. The level of PR mRNA in the CL was highest at the early stage of the estrous cycle and was higher at the mid and late stages than at the regressed stage (P<0.01). In regard to the distribution of PR, the protein was expressed in both small and large luteal cells and in vascular endothelial cells throughout the estrous cycle. These results suggest that P4 has a role in regulating luteal and endothelial cell function in the bovine CL, especially at the early luteal stage as an autocrine/paracrine regulator.

Toxic effects of cypermethrin and methamidophos on bovine corpus luteal cells and progesterone production

This study was planned and executed with the aims to explore corpus luteal primary cell culture as an “animal alternate testing system” in toxicity studies and in vitro toxic effects of cypermethrin (CY) 90% (pyrethroid) and methamidophos (MTP) 73% (organophosphate) on morphology and progesterone secretory activity of bovine corpus luteal cells and tissue. For this purpose, primary cell cultures of bovine corpus luteum (CL) cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal calf serum (FCS). Toxicity evaluation were based on viable CL cell counts, morphological changes in CL cells, ability of CL cells to produce progesterone and histological changes in CL tissue at different hours post exposure to CY and MTP. The changes induced by both the insecticides were time and dose dependant. Viable cell counts and progesterone concentration decreased significantly ( P< 0.05) with the treatment of CY and MTP when compared to control. Corpus luteal cells exposed to CY showed more severe toxic effects as compared to MTP, though the difference was non-significant. Cellular or tissue alterations included degenerative changes in luteal cells, pleomorphic changes, nuclear degeneration and vacuolation, cell shrinkage and rupture, cloudy swelling and hydropic degeneration, less cytoplasmic granulation, cell elongation, hyalinization and cytoplasmic haziness and stripling and necrosis. It was concluded that both the insecticides induce toxic effects in terms of viable counts, morphological and histological changes and progesterone production of bovine CL cells. Cypermethrin exhibited more adverse toxic changes in viable cell counts, progesterone production and histological findings as compared to methamidophos.

The expression of leptin and its receptor during different physiological stages in the bovine ovary

Leptin, the hormonal product of the obese (ob) gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety. In cattle, leptin has also been implicated in the control of ovarian function, but its local production in the ovary and role in the control of ovarian function in autocrine/paracrine manner is unknown. The aims of this study were to document the expression of leptin and its receptor (Ob-R) in detail in bovine corpus luteum (CL) obtained from different stages of the oestrous cycle and during pregnancy-and to determine if the leptin/Ob-R system is expressed clearly in bovine follicles during final growth to preovulatory follicles. Real-time RT-PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, we demonstrated leptin and its receptor transcripts and leptin protein are consistent with in vivo luteinisation of bovine CL and decline coincidental with luteal regression. The highest co-expression of leptin/Ob-R system was observed in TI and GC of the smallest follicles with E2 concentration <0.5 ng/ml followed by significant down regulation in growing follicles with the increase of follicular size and E2 content in the follicular fluid. Furthermore, expression of the leptin/Ob-R system does not show any significant variation in the CL throughout pregnancy. To conclude, our results are the first to demonstrate the possible involvement of locally produced leptin/Ob-R system in the bovine ovary, suggesting roles in the function and/or development of the CL and growth of small follicles in an autocrine/paracrine fashion.

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 hCG on protein synthesis and progesterone production in the bovine luteal cells.

The study was carried out to investigate whether the luteal steroidogenesis in response to tropic stimulus is mediated through de novo synthesis of protein(s). Luteal cell suspension was prepared by collagenase-DNAse treatment of the bovine corpora lutea, weighing 4-6 g. Cells equivalent to 250 micrograms of protein were incubated in a total volume of 550 microliter of medium 199 with or without various test substances. Production of progesterone by the luteal cells was stimulated by hCG in a dose dependent manner. Incorporation of 3H-leucine into the cellular proteins was concomitantly enhanced. Emetine, cycloheximide and puromycin inhibited both basal and hCG stimulated protein synthesis as well as progesterone production in the cells. Thus, luteal steroidogenesis induced by hCG seems to be dependent upon de novo synthesis of a protein(s).

Expression of Lymphangiogenic Factors in Bovine Corpus Luteum During Estrous Cycle, Pregnancy, and Induced Luteolysis.

Expression of Lymphangiogenic Factors in Bovine Corpus Luteum During Estrous Cycle, Pregnancy, and Induced Luteolysis.

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)

EG-VEGF Protein Expression in Superovulated Buffalo Corpus Luteum.

Endocrine gland derived endothelial growth factor (EG-VEGF) also termed prokinectin 1(PK-1), is a recently identified protein, which expression has been described in a variety of tissues, including steroidogenic glands, gastrointestinal tract, and nervous system. EG-VEGF is a multifunctional factor: in bovine corpus luteum (CL) it induces endothelial cell proliferation and permeability. Superovulation has been shown to promote important morphologic and functional changes in buffaloes CL: gonadotropins stimulates progesterone synthesis and induces the expression of angiogenic factors as VEGF and bFGF increasing vascular permeability. Based on these data, we hypothesized that EG-VEGF is present in buffalo CL and that superovulatory treatment influences its expression. To test these hypotheses, we used 12 water buffaloes corpora lutea collected on day 6 post ovulation: six from non-treated (control group) and six from animals submitted to superovulatory treatment. Superovulation was carried out using repeated dosis of follicle-stimulating hormone (FSH). EG-VEGF protein expression was assessed by immunohistochemistry and western blotting (WB). Data were analyzed by Student's t test and differences were considered significant at P < 0.05. A positive immunostaining was detected in the cytoplasm of luteal and endothelial cells, but stroma cells as well as pericytes were not stained for EG-VEGF. Western blots of buffalo CL protein extracts were able to detect one band, at approximately 12 kDa, which correspond to EG-VEGF. In both immunohistochemistry and densitometry, we observed a higher expression of EG-VEGF in CL of superovulated buffaloes in relation to control group (P<0.05). Our data demonstrated that EG-VEGF protein is expressed in buffaloes CL, and that FSH stimulates its expression. Our data agree with previously studies, in which one has demonstrated that hCG stimulates the synthesis of EG-VEGF in human luteinized granulosa cells. In conclusion, the presence of EG-VEGF in CL, mainly in luteal and endothelial cells, points towards a paracrine or even autocrine role of this growth factor in modulating CL function. Moreover, the increased expression observed in superovulated CL may be related to increased vascularization and hormone production, features already described for this model. (poster)

Vasohibin Expression in the Bovine Corpus Luteum: Regulation by VEGF and Prostaglandin F2 alpha.

Vasohibin Expression in the Bovine Corpus Luteum: Regulation by VEGF and Prostaglandin F2 alpha.

Stage-Specific Responses in Gene Expression and Signaling Pathway Activation after Treatment with Prostaglandin F2 Alpha (PGF) and Interferon-Gamma (IFNG) in Bovine Corpus Luteum (CL) and Luteinizing Granulosa Cells.

Stage-Specific Responses in Gene Expression and Signaling Pathway Activation after Treatment with Prostaglandin F2 Alpha (PGF) and Interferon-Gamma (IFNG) in Bovine Corpus Luteum (CL) and Luteinizing Granulosa Cells.

Expression of Adiponectin and Its Receptors in the Bovine Corpus Luteum and T Lymphocytes.

Expression of Adiponectin and Its Receptors in the Bovine Corpus Luteum and T Lymphocytes.

Genetic Over-Expression of Cytokeratin 18 in Bovine Luteal Cells: Effects on Intermediate Filament Formation and FasL-Induced Death.

The Fas-Fas ligand (FasL) system is an integral component of luteal regression, yet the intrinsic mechanism(s) sensitizing luteal cells to FasL-induced cell death are unknown. Susceptibility to FasL in some cell types is influenced by cytokeratin-containing intermediate filaments, and within these cells the filaments impart resistance to cell death. In previous work we have observed that steroidogenic cells of the bovine corpus luteum (CL) contain cytokeratin 18 (CK18) filaments and thus might be resistant to FasL-induced cell death. Here, we tested this possibility by infecting primary cultures of bovine luteal cells with a full-length cDNA construct for hCK18 in a GFP-containing adenovirus vector with cytomegalovirus promoter. The cells were obtained from bovine CL in the early (Day 5) and late stages (Days 16-18) post-ovulation and prepared for primary cell culture. Cells were cultured overnight and exposed to culture medium (Control), GFP-containing adenovirus vector (Ad.GFP transduced), or CK18-adenovirus vector, also containing GFP (Ad.CK18 transduced) at an optimized MOI for 1 hr. Subsequently, the cells were incubated an additional 16-24 hr in virus-free media, and then exposed to either vehicle or a cytokine cocktail containing FasL (50ng/mL FasL) for 24 hr to induce cell death. The cells were fixed and prepared for detection of GFP, immunocytochemical detection of CK18, and an assessment of cell death. Overall infection efficiency averaged 75% in cultures from early stage CL; whereas only 40% efficiency was achieved for cultures from late stage CL. Approximately 45% of the cells from early stage CL expressed CK18 filaments prior to transduction; whereas >75% of the cells expressed CK18 following treatment with Ad.CK18. In cells from late stage CL, endogenous expression of CK18 filaments was relatively low (~16% of all cells), but increased to ~40% of the cells following treatment with Ad.CK18. Intriguingly, virtually all transduced cells from early and late stage CL contained abundant CK18 aggregates rather than filamentous structures. These aggregates were not observed in Control or GFP-transduced cells nor did they affect the viability of the cells in CK18-transduced cultures. FasL-induced cell death was similar among Control, Ad.GFP-, and Ad.CK18-transduced cultures (14%, 18%, and 16% death, respectively) regardless of CL stage, indicating aggregation of CK18 in transduced cultures did not prevent FasL-induced death. Aggregation of CK18 also did not influence progesterone secretion by cells of early CL before or after FasL treatment (62 versus 60 ng/ml, respectively), but potentially enhanced progesterone secretion by cells of late stage CL (82 versus 181 ng/ml for control and FasL-treated cultures, respectively). Knowing that intermediate filaments consist of heterodimeric proteins (e.g., CK18 paired with CK8), the aggregation of CK18 following transduction might have resulted from a lack of other available cytokeratins. In conclusion, genetic over-expression of CK18 in bovine luteal cells results in CK18 aggregation rather than filament formation, which fails to impair FasL-induced cell death but may influence progesterone secretion. This project was supported by National Research Initiative Competitive Grant no. 2007-35203-18074 from the USDA Cooperative State Research, Education, and Extension Service. (poster)

Disruption of Cytokeratin 18-Containing Intermediate Filaments in Bovine Luteal Cells: Effects of Acrylamide on Progesterone Secretion, Fas Expression, and FasL-Induced Apoptosis.

One proposed mechanism of cell death during regression of the bovine corpus luteum (CL) is Fas-Fas Ligand (FasL)-induced apoptosis. Fas is a protein receptor found on the surface of luteal cells that binds with FasL and induces cell death through a series of signaling pathways involving caspase enzymes and cytokeratin-containing intermediate filaments. Others have shown in non-ovarian cell types that cytokeratin 18 (CK18)-containing intermediate filaments impair FasL-induced apoptosis, yet these filaments are also directly cleaved by caspases during apoptotic signaling. The purpose of the present study was to determine whether disruption of CK18-containing intermediate filaments in bovine luteal cells increases the cell surface expression of Fas and enhances FasL-induced apoptosis. Early and late stage CL (Days 5 and 16-18 postovulation, respectively) were collected and prepared for primary cell culture following collagenase dissociation. The cultures were exposed initially to culture medium (control) or acrylamide (5mM) for 4 hours to disrupt CK18-containing intermediate filaments. Subsequently, the culture medium in both groups was exchanged and the cells were then exposed to vehicle (control) or a cytokine cocktail containing FasL (50ng/mL) for 24 hours to induce cell death. Immunocytochemical staining revealed that acrylamide effectively disrupted CK18-containing intermediate filaments, resulting in perinuclear aggregation. In contrast, acrylamide had no effect on microtubule organization, progesterone secretion (177 versus 197 ng/5x105cells in control and acrylamide cultures, respectively), or cell viability (19% versus 14% cell death in control and acrylamide treated cultures, respectively) regardless of the stage of CL. Flow cytometric analysis revealed that Fas expression on the surface of cultured luteal cells from early and late stage CL was relatively high (an average of 84% and 61% of luteal cells from early and late stages, respectively). Pretreatment of the cultures with acrylamide, however, did not substantially enhance Fas expression (86% and 63% of luteal cells from early and late stage CL, respectively). Similarly, acrylamide did not augment FasL-induced cell death (an average of 42% versus 40% cell death for control and acrylamide-treated cultures, respectively), regardless of stage of CL. Consistent with the relatively high expression of Fas on cells from early stage CL, the incidence of FasL-induced death was greater in cultures from early stage CL than late stage CL (52% versus 29% cell death, respectively). The results indicate that while acrylamide effectively disrupts CK18-containing intermediate filaments in bovine luteal cells, its transient actions fail to increase Fas expression on the cell surface or to promote FasL-induced apoptosis. This project was supported by National Research Initiative Competitive Grant no. 2007-35203-18074 from the USDA Cooperative State Research, Education, and Extension Service. (poster)

Changes in the Vasculature of Bovine Corpus Luteum During the Estrous Cycle and Prostaglandin F2alpha-Induced Luteolysis.

Changes in the Vasculature of Bovine Corpus Luteum During the Estrous Cycle and Prostaglandin F2alpha-Induced Luteolysis.

Loss of Regulatory T Lymphocytes in the Regressing Bovine Corpus Luteum.

Loss of Regulatory T Lymphocytes in the Regressing Bovine Corpus Luteum.

The expression of the nuclear receptors NR5A1 and NR5A2 and transcription factor GATA6 correlates with steroidogenic gene expression in the bovine corpus luteum

The corpus luteum (CL) is the major site of progesterone (P4) production during the luteal phase of the estrous cycle in cattle. To better understand the molecular mechanisms underlying P4 production, we compared the mRNA and protein expression profiles of key components of the steroidogenic pathway (StAR, CYP11A, and 3beta-HSD) during the bovine CL luteal phase with that of several transcription factors (NR5A1, NR5A2, GATA4, GATA6) known for their roles in the control of steroidogenic gene expression. In the bovine CL, StAR, CYP11A, and 3beta-HSD mRNA and protein levels remained constant at the mid and late luteal phases but markedly declined at the regressed luteal stage. NR5A1 and NR5A2 exhibited a similar pattern with a significant decrease in expression at the regressed luteal stage. Both GATA4 and GATA6 mRNA and proteins could be detected in bovine CL; GATA6 levels, however, were generally higher. Although GATA4 expression did not change during the luteal phase, GATA6 showed a marked decrease at the regressed luteal stage, like NR5A1, NR5A2, and the other steroidogenic markers. Thus, we suggest that NR5A1, NR5A2, and GATA6, but not GATA4, contribute to the transcriptional regulation of steroidogenic gene expression, and hence P4 production, in the bovine CL. Furthermore, we have demonstrated the association of NR5A1 and NR5A2 with the bovine StAR promoter in the mid-luteal CL using chromatin immunoprecipitation, suggesting that these factors have definitive roles in the regulation of StAR gene transcription in vivo.

Expression and Localization of Gap Junctional Connexins 26 and 43 in Bovine Periovulatory Follicles and in Corpus Luteum During Different Functional Stages of Oestrous Cycle and Pregnancy

The aim of this study was to characterize the regulation of connexins (Cx26 and Cx43) in the bovine ovary (experiment 1-3). Experiment 1: ovaries containing preovulatory follicles or corpora lutea (CL) were collected at 0, 4, 10, 20, 25 (follicles) and 60 h (CL) relative to injection of GnRH. Experiment 2: CL were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous cycle and of early and late pregnancy (<4 and >4 months). Experiment 3: induced luteolysis, cows on days 8-12 were injected with PGF2alpha analogue (Cloprostenol), and CL were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2alpha injection. Real-time RT-PCR was applied to investigate mRNA expression and immunofluorescence was utilized for protein localization. Cx26 mRNA increased rapidly 4 h after GnRH injection (during LH surge) and decreased afterwards during the whole experimental period. Cx43 mRNA expression decreased continuously after GnRH application. Cx26 mRNA in CL increased significantly in the second part of oestrous cycle and after regression. In contrast, the highest mRNA expression for Cx43 in CL was detected during the early luteal phase. After induced luteolysis the mRNA expression of Cx26 increased significantly at 24 h. As shown by immunofluorescence, Cx26 was predominantly localized in the connective tissue and blood vessels of bovine CL, whereas Cx43 was present in the luteal cells and blood vessels. This resulted in a strong increase of Cx26 expression during the late luteal phase and after luteal regression. Subsequently, Cx43 expression was distinctly decreased after luteal regression. These data suggest that Cx26 and Cx43 are involved in the local cellular mechanisms participating in tissue remodelling during the critical time around periovulation as well as during CL formation (angiogenesis), function and regression in the bovine ovary.