Expression dynamics of adipokines during induced ovulation in the bovine follicles and early corpus luteum.

The study aimed to assess the local gene expression of adipokine members, namely vaspin, adiponectin, visfatin, resistin and their associated receptors - heat shock 70 protein 5 (HSPA5), adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) - in bovine follicles during the preovulatory period and early corpus luteum development. Follicles were collected before gonadotropin-releasing hormone (GnRH) treatment (0 h) and at 4, 10, 20, 25 and 60 h after GnRH application through transvaginal ovariectomy (n = 5 samples/group). Relative mRNA expression levels were quantified using real-time reverse transcription polymerase chain reaction (RT-qPCR). Vaspin exhibited high mRNA levels immediately 4 h after GnRH application, followed by a significant decrease. Adiponectin mRNA levels were elevated at 25 h after GnRH treatment. AdipoR2 exhibited late-stage upregulation, displaying increased expression at 20, 25 and 60 h following GnRH application. Visfatin showed upregulation at 20 h post-GnRH application. In conclusion, the observed changes in adipokine family members within preovulatory follicles, following experimentally induced ovulation, may constitute crucial components of the local mechanisms regulating final follicle growth and development.

Comprehensive in silico analysis of prolactin receptor (PRLR) gene nonsynonymous single nucleotide polymorphisms (nsSNPs) reveals multifaceted impact on protein structure, function, and interactions.

This study delves into the functional and structural implications of non-synonymous single nucleotide polymorphisms (nsSNPs) within the Prolactin Receptor (PRLR) gene. Thirteen deleterious nsSNPs were identified through bioinformatics tools, with SIFT predicting 168 out of 395 nsSNPs as detrimental, exhibiting tolerance index (TI) scores ranging from 0 to 0.05. Polyphen2 assigned likelihood scores >0.99 to all 13 nsSNPs, indicating high probability of harm, while Panther scores classified most nsSNPs as 'probably damaging', with specific mutations like W218R scoring 0.74, suggesting a higher impact. Stability analysis using DDG I-Mutant and DDG Mupro consistently predicted decreased stability for all mutations, with CUPSAT indicating mutations like V125G and W218R significantly decreasing stability. Structural analysis through DynaMut predicted destabilization for all mutations except L196I and L292H. MutPred2 highlighted structural alterations for all nsSNPs except L196I, L293V, R315W, and S353N. Domain analysis revealed key mutations within essential functional domains, with five nsSNPs located within Fibronectin type-III domains. Bayesian analysis through ConSurf identified 9 critical residues, with 11 nsSNPs exhibiting notably high conservation. STRING analysis unveiled a complex interaction network, indicating involvement in vital biological processes like lactation. Molecular dynamics (MD) simulations, spanning 100 nanoseconds, elucidated structural dynamics induced by detrimental missense SNPs. Post-translational modification (PTM) analysis identified specific mutations, such as R351, involved in methylation, while S353 was implicated in phosphorylation and glycosylation. These findings offer comprehensive insights into the molecular and phenotypic effects of deleterious nsSNPs in the PRLR gene, crucial for selective breeding.Communicated by Ramaswamy H. Sarma.

Prostaglandins as local regulators of ovarian physiology in ruminants.

Prostaglandins are synthesized from arachidonic acid through the catalytic activities of cyclooxygenase, while the production of different prostaglandin types, prostaglandin F2 alpha (PGF) and prostaglandin E2 (PGE), are regulated by specific prostaglandin synthases (PGFS and PGES). Prostaglandin ligands (PGF and PGE) bind to specific high-affinity receptors and initiate biologically distinct signalling pathways. In the ovaries, prostaglandins are known to be important endocrine regulators of female reproduction, in addition to maintaining local function through autocrine and/or paracrine effect. Many research groups in different animal species have already identified a variety of factors and molecular mechanisms that are responsible for the regulation of prostaglandin functions. In addition, prostaglandins stimulate their intrafollicular and intraluteal production via the pathway of prostaglandin self-regulation in the ovary. Therefore, the objective of the review article is to discuss recent findings about local regulation patterns of prostaglandin ligands PGF and PGE during different physiological stages of ovarian function in domestic ruminants, especially in bovine. In conclusion, the discussed local regulation mechanisms of prostaglandins in the ovary may stimulate further research activities in different methodological approaches, especially during final follicle maturation and ovulation, as well as corpus luteum formation and function.

Expression of Locally Produced Adipokines and Their Receptors during Different Physiological and Reproductive Stages in the Bovine Corpus Luteum.

This study aimed to determine the gene expression of different local novel adipokines, such as vaspin, adiponectin, visfatin, and resistin, and their known receptors, namely, heat shock 70 protein 5, adiponectin receptor 1, and adiponectin receptor 2, in the bovine corpus luteum (CL) during different phases of the estrous cycle (on days 1-2, 3-4, 5-7, 8-12, 13-18, >18) and pregnancy (at months 1-2, 3-4, 5-7, >7). The mRNA expression was measured by reverse transcription polymerase chain reaction (RT-qPCR). The mRNA expression levels were normalized to the geometric mean of all three constantly expressed reference genes (cyclophilin A, ubiquitin, ubiquitin C). Our findings suggest that adipokines are expressed and present in all investigated groups, and are specifically up- or downregulated during the estrus cycle and during pregnancy. Vaspin and adiponectin levels were upregulated in the middle and late cycle stages. Resistin was abundant during the CL regression stage and in the first months of pregnancy. The specific expression of adipokine receptors indicates their involvement in the local mechanisms that regulate CL function. Further investigations are required to elucidate the regulative mechanisms underlying the different local effects of adipokines on the ovarian physiology of cows.

Local Expression Dynamics of Various Adipokines during Induced Luteal Regression (Luteolysis) in the Bovine Corpus Luteum.

The study aimed to evaluate the mRNA expression levels of various local novel adipokines, including vaspin, adiponectin, visfatin, and resistin, along with their associated receptors, heat shock 70 protein 5, adiponectin receptor 1, and adiponectin receptor 2, in the corpus luteum (CL) during luteal regression, also known as luteolysis, in dairy cows. We selected Fleckvieh cows in the mid-luteal phase (days 8-12, control group) and administered cloprostenol (PGF analog) to experimentally induce luteolysis. We collected CL samples at different time points following PGF application: before treatment (days 8-12, control group) and at 0.5, 2, 4, 12, 24, 48, and 64 h post-treatment (n = 5) per group. The mRNA expression was measured via real-time reverse transcription polymerase chain reaction (RT-qPCR). Vaspin was characterized by high mRNA levels at the beginning of the regression stage, followed by a significant decrease 48 h and 64 h after PGF treatment. Adiponectin mRNA levels were elevated 48 h after PGF. Resistin showed upregulation 4 h post PGF application. In summary, the alterations observed in the adipokine family within experimentally induced regressing CL tissue potentially play an integral role in the local regulatory processes governing the sequence of events culminating in functional luteolysis and subsequent structural changes in the bovine ovary.

Regulatory changes of local produced prostaglandins in corpus luteum after experimentally induced luteolysis in the cow.

The objective of the study was to evaluate the expression patterns of prostaglandin F2alpha (PGF), prostaglandin E2 (PGE), PGF receptor (FP), PGE receptors (EP2 and EP4), prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandin synthases (PGFS and PGES) in corpora lutea (CL) during experimentally induced luteolysis in cow. The Fleckvieh cows in the mid-luteal phase (days 8-12, control group) were injected with cloprostenol (PGF analogue), and CL were collected by transvaginal ovariectomy before (days 8-12, control group) and at 0.5, 2, 4, 12, 24, 48 and 64 h after PGF application (n = 5 per group). The mRNA expression was determined by RT-qPCR, the hormone concentrations by enzyme immunoassay and localization by immunohistochemistry. PTGS2 gene expression increased significantly 2 h after PGF application, followed by continuous and significant downregulation afterwards. The PGF tissue concentration increased significantly just after PGF injection and again during structural luteolysis (after 12 h), whereas PGE concentration significantly decreased during structural luteolysis. The FP receptor mRNA decreased significantly at 2 h and again at 12 h after PGF. In contrast, EP4 receptor mRNA increased significantly just after the PGF application (0.5 h). The immunostaining of PGES and PTGS2 on day 15-17 shows numerous positive luteal cells, followed by lower activity afterwards on day 18 (luteolysis). In conclusion, the changes of examined prostaglandin family members in CL tissue after PGF application may be key components of the local mechanisms regulating the cascade of actions leading to functional and subsequent structural luteolysis in the bovine ovary.

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis.

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.

Prostaglandins in Superovulation Induced Bovine Follicles During the Preovulatory Period and Early Corpus Luteum.

The aim of this study was to characterize the regulation pattern of prostaglandin family members namely prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES) in the bovine follicles during preovulatory period and early corpus luteum (CL). Ovaries containing preovulatory follicles or CL were collected by transvaginal ovariectomy (n = 5 cows/group), and the follicles were classified: (I) before GnRH treatment; (II) 4 h after GnRH; (III) 10 h after GnRH; (IV) 20 h after GnRH; (V) 25 h after GnRH, and (VI) 60 h after GnRH (early CL). In these samples, the concentrations of progesterone (P4), estradiol (E2), PTGF and PTGE were investigated in the follicular fluid (FF) by validated EIA. Relative mRNA abundance of genes encoding for prostaglandin receptors (PTGFR, PTGER2, PTGER4), COX-2, PTGFS and PTGES were quantified by RT-qPCR. The localization of COX-2 and PTGES were investigated by established immunohistochemistry in fixed follicular and CL tissue samples. The high E2 concentration in the FF of the follicle group before GnRH treatment (495.8 ng/ml) and during luteinizing hormone (LH) surge (4 h after GnRH, 574.36 ng/ml), is followed by a significant (P<0.05) downregulation afterwards with the lowest level during ovulation (25 h after GnRH, 53.11 ng/ml). In contrast the concentration of P4 was very low before LH surge (50.64 mg/ml) followed by a significant upregulation (P < 0.05) during ovulation (537.18 ng/ml). The mRNA expression of COX-2 increased significantely (P < 0.05) 4 h after GnRH and again 20 h after GnRH, followed by a significant decrease (P < 0.05) after ovulation (early CL). The mRNA of PTGFS in follicles before GnRH was high followed by a continuous and significant downregulation (P < 0.05) afterwards. In contrast, PTGES mRNA abundance increased significantely (P < 0.05) in follicles 20 h after GnRH treatment and remained high afterwards. The mRNA abundance of PTGFR, PTGER2, and PTGER4 in follicles before GnRH was high, followed by a continuous and significant down regulation afterwards and significant increase (P < 0.05) only after ovulation (early CL). The low concentration of PTGF (0.04 ng/ml) and PTGE (0.15 ng/ml) in FF before GnRH, increased continuously in follicle groups before ovulation and displayed a further significant and dramatic increase (P < 0.05) around ovulation (101.01 ng/ml, respectively, 484.21 ng/ml). Immunohistochemically, the granulosa cells showed an intensive signal for COX-2 and PTGES in follicles during preovulation and in granulosa-luteal cells of the early CL. In conclusion, our results indicate that the examined bovine prostaglandin family members are involved in the local mechanisms regulating final follicle maturation and ovulation during the folliculo-luteal transition and CL formation.

Changes in the expression of prostaglandin family members in bovine corpus luteum during the estrous cycle and pregnancy.

The aim of this study was to characterize certain prostaglandin family members in the bovine corpus luteum (CL) during the estrous cycle and pregnancy. The CL tissue was assigned to the stages 1-2, 3-4, 5-7, 8-12, 13-16 and >18 days (after regression) of the estrous cycle and 1-2, 3-4, 6-7, and >8 months of pregnancy. In these samples, we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, and PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES). The expression of messenger RNA (mRNA) was measured by reverse transcription quantitative polymerase chain reaction, hormones by enzyme immunoassay, and localization by immunohistochemistry. The mRNA expression of COX-2, PTGFS, and PTGES in CL during the early-luteal phase was high followed by a continuous and significant downregulation afterward, as well as during all phases of pregnancy. The concentration of PTGF in CL tissue was high during the early-luteal phase, decreased significantly in the mid-luteal phase, and increased again afterward. In contrast, the concentration of PTGE increased significantly during the late-luteal phase followed by a decrease during regression. The PTGE level increased again during late pregnancy. Immunohistochemically, the large granulose-luteal cells show strong staining for COX-2 and PTGES during the early-luteal stage followed by lower activity afterward. During pregnancy, most of the luteal cells were only weakly positive or negative. In conclusion, our results indicate that the examined prostaglandin family members are involved in the local mechanisms that regulate luteal function, specifically during CL formation, function, and regression and during pregnancy in the cow.

Aflatoxin M1 contamination of raw cow's milk in five regions of Kosovo during 2016.

During February and August 2016 (winter and summer season), 192 samples of raw bulk tank milk have been collected from small dairy farms in five different regions of Kosova (Prishtina, Prizren, Peja, Mitrovica, and Gjilan). These samples were analyzed for aflatoxin M1 (AFM1) contamination level by ultra-high performance liquid chromatography coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UHPLC-ESI-MS/MS). The limit of quantification for AFM1 was 0.02 µg/L and recovery at 0.1-0.5 µg/L was 90-95% (coefficient of variation 15-25%). A total of 74 milk samples (38%) were found to be contaminated with AFM1 and 11 samples (5.7%) slightly exceeded the European Union maximum level of 0.05 µg/L. The maximum AFM1 level was at 0.082 µg/L. There was no relevant difference between summer and winter with regard to AFM1 frequency and levels. Although the frequency of AFM1-positive samples varied between regions, from 7% (Prishtina) to 53% (Prizren), only minor regional differences were observed with regard to average and maximum toxin levels. The high percentage of milk samples which were non-compliant with AFM1 maximum levels indicates that efforts to reduce the contamination levels of aflatoxin B1 in cows feed in Kosovo are necessary.

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 localization of members of the thrombospondin family during final follicle maturation and corpus luteum formation and function in the bovine ovary.

The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (< 0.5, 0.5-5, 5-40, 40-180 and >180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16 and >18 of the estrous cycle and of pregnancy (month 1-2, 3-4, 6-7 and > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis.

Induction of endothelin-2 expression by luteinizing hormone and hypoxia: possible role in bovine corpus luteum formation.

The pattern and regulation of endothlin-2 (EDN2) expression and its putative roles in bovine ovaries were investigated. EDN2 mRNA was determined in corpus luteum (CL) and during folliculoluteal transition induced by GnRH in vivo. EDN2 was elevated only in the early CL and was not present in older CL. In the young CL, EDN2 mRNA was identified mainly in luteal cells but not endothelial cells that expressed the EDN1 gene. Similarly, in preovulatory follicles, EDN2 was expressed in the granulosa cells (GCs) and not in the vascular theca interna. LH and hypoxia are two major stimulants of CL formation. Therefore, GCs were cultured with bovine LH, under hypoxic conditions. GCs incubated with bovine LH resulted in increased EDN2 mRNA 42 h later. CoCl2, a hypoxia-mimicking agent, elevated EDN2 in GCs in a dose-dependent manner. Incubation of the human GC line (Simian virus 40 large T antigen) under low oxygen tension (1%) augmented EDN2 6 and 24 h later. In these two cell types, along with EDN2, hypoxia augmented VEGF. EDN2 induced in GCs changes that characterize the developing CL: cell proliferation as well as up-regulation of vascular endothelial growth factor and cyclooxygenase-2 (mRNA and protein levels). Human chorionic gonadotropin also up-regulated these two genes. Small interfering RNA targeting EDN-converting enzyme-1 effectively reduced its mRNA levels. This treatment, expected to lower the mature EDN2 peptide production, inhibited VEGF mRNA levels and GC numbers. Together these data suggest that elevated EDN2 in the early bovine CL, triggered by LH surge and hypoxia, may facilitate CL formation by promoting angiogenesis, cell proliferation, and differentiation.

Effect of prostaglandin F2 alpha on local luteotropic and angiogenic factors during induced functional luteolysis in the bovine corpus luteum.

The essential role of endometrial prostaglandin F2 alpha (PTGF) for induction of the corpus luteum (CL) regression is well documented in the cow. However, the acute effects of PTGF on known local luteotropic factors (oxytocin [OXT] and its receptor, insulin-like growth factor [IGF] 1, and progesterone and its receptor), the principal angiogenic factor vascular endothelial growth factor (VEGF) A and the capillary destabilization factor angiopoietin (ANGPT) 2 were not thoroughly studied in detail. The aim of this study was therefore to evaluate the tissue concentration of these factors during PTGF induced luteolysis. In addition the mRNA expression of progesterone receptor (PGR), OXT receptor (OXTR), IGF1, IGFBP1, ANGPT1, and ANGPT2 was determined at different times after PTGF treatment. Cows (n = 5 per group) in the mid-luteal phase (Days 8-12, control group) were injected with the PTGF analog (cloprostenol), and CL were collected by transvaginal ovariectomy at 0.5, 2, 4, 12, 24, 48, and 64 h after injection. The mRNA expression was analyzed by quantitative real-time PCR, and the protein concentration was evaluated by enzyme immunoassay or radioimmunoassay. Progesterone concentrations, as well as mRNA expression of PGR, in CL tissue were significantly down-regulated by 12 h after PTGF. Tissue OXT peptide and OXTR mRNA decreased significantly after 2 h, followed by a continuous decrease of OXT mRNA. IGF1 and VEGFA protein already decreased after 0.5 h. By contrast, the IGFBP1 mRNA was up-regulated significantly after 2 h to a high plateau. ANGPT2 protein and mRNA significantly increased during the first 2 h, followed by a steep decrease after 4 h. The acute decrease of local luteotropic activity and acute changes of ANGPT2 and VEGFA suggest that modulation of vascular stability may be a key component in the cascade of events leading to functional luteolysis.

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.

The expression of apelin and its receptor APJ during different physiological stages in the bovine ovary.

Recent studies implicate that apelin and its receptor APJ may have important role for the modulation of angiogenesis. The aim of this study was to further characterise the regulation of apelin/APJ system in bovine ovary. Experiment 1: corpora lutea (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 gravidity (month <3, 3-5, 6-7 and >8). Experiment 2: Follicles during maturation were divided into granulosa cells (GC) and theca interna (TI) and were examined separately. Classification of follicles occurred by follicle size and oestradiol-17beta (E2) concentration in the follicular fluid (FF) (<0.5 ng/ml, 0.5-5 ng/ml; 5-40 ng/ml; 40-180 ng/ml; >180 ng/ml). Real-time RT-PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, the expression level of apelin during the oestrous cycle was significantly higher compared to the one during pregnancy. Apelin mRNA levels were always high during the cycle with a tendency of decrease after CL regression. The APJ mRNA in the CL was significantly up regulated on days 5-7 and 8-12 followed by a decrease on days 13-16, and further on days >18. The expression of APJ does not show any significant regulation in the CL throughout pregnancy. The expression of apelin and APJ was not statistically regulated in GC, but was significantly up regulated in follicles with an E2 concentration of more than 5 ng/ml and showed an increase according to growth and maturation of follicles. In conclusion, our data suggest that apelin/APJ system is involved in the mechanism regulating angiogenesis during follicle maturation as well as during CL formation and function in the bovine ovary.

Expression and possible role of fibroblast growth factor family members in porcine antral follicles during final maturation.

The aim of this study was to investigate the possible participation of fibroblast growth factor (FGF) family members (FGF1, FGF2 and FGF7 and their receptors) in porcine follicles (polyovulatory species) under special consideration for FGF2 during final growth. A classification of follicles was done by size and follicular fluid content of oestradiol-17beta, progesterone and prostaglandin F2alpha. The mRNA expression of examined factors was analysed by real-time PCR. The hormone concentration was estimated by enzyme immunoassay, protein characterisation by western blotting and localisation by immunohistochemistry. Follicle tissue separated in theca interna and granulosa cells was extracted and tested for mRNA of FGF1, FGF2, FGF7 and receptors (FGFR1IIIc, FGFRIIIb and FGFR2IIIc). Additionally, the mRNA expression of FSHR, LHR and aromatase cytochrome P450 for further characterisation of follicles was analysed. Significantly, higher FGF2 protein levels were measured in stroma when compared with total follicle or corpus luteum tissue. This result was confirmed by western blot with two strong bands. Immunological localisation of FGF2 only in stroma (fibroblasts) confirms the protein measurements. The results show a clear difference for FGF2 protein expression during final growth of follicles if monovulatory (bovine) and polyovulatory (porcine) species are compared. FGF2 protein in porcine ovary may be (due to localisation and concentration in stroma) important for support of angiogenesis of more follicles (polyovulatory species) and not of a single follicle like in cows.

Expression and localization of apelin and its receptor APJ in the bovine corpus luteum during the estrous cycle and prostaglandin F2alpha-induced luteolysis.

Angiogenesis, changes in blood flow, and extracellular matrix remodeling are the processes associated with the development and demise of the bovine corpus luteum (CL) during the estrous cycle. APJ (putative receptor protein related to angiotensin type 1 receptor) is a G-protein-coupled receptor, and its ligand, apelin, has been identified as a novel regulator of blood pressure and as an angiogenic factor. We hypothesized that the apelin-APJ system is involved in luteal function. This study investigated whether apelin-APJ exists in bovine CL and determined their expression profiles and localization during luteal phase and prostaglandin F(2)(alpha) (PGF(2)(alpha))-induced luteolysis. During the luteal phase, apelin mRNA expression increased from early to late CL and decreased in regressed CL. APJ mRNA expression increased from early to mid-CL and remained elevated in late and regressed CL. Apelin and APJ proteins were immunohistochemically detected only in the smooth muscle cells of intraluteal arterioles during the luteal phase. PGF(2)(alpha) stimulated apelin and APJ mRNA expression at 0.5-2 and 2 h respectively, and then the mRNA expression of apelin-APJ was inhibited from 4 h during PGF(2)(alpha)-induced luteolysis. Additionally, apelin mRNA and protein were stimulated at 1 h after PGF(2)(alpha) injection only in the periphery of mid- but not early CL. The present study indicated that the apelin-APJ was localized in the smooth muscle cells of intraluteal arterioles, and responded to PGF(2)(alpha) at the periphery of mid-CL in the cow. Thus, the apelin-APJ system may be involved in the maturation of CL and the luteolytic cascade as a regulator of intraluteal arterioles in cow.

Effect of the luteinising hormone surge on regulation of vascular endothelial growth factor and extracellular matrix-degrading proteinases and their inhibitors in bovine follicles.

The aim of the present study was to evaluate the pattern of regulation of vascular endothelial growth factor (VEGF)-A (isoforms 121, 165, 189), VEGF receptor tyrosine kinases (VEGF-R1 and VEGF-R2), matrix metalloproteinase (MMP)-1, MMP-2, MMP-14, MMP-19, tissue-specific inhibitor of metalloproteinases (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in time-defined follicle classes before (0 h) and after the application of gonadotrophin-releasing hormone (GnRH). Bovine ovaries containing periovulatory follicles or new corpora lutea (CL; Days 1-2) were collected 0, 4, 10, 20 and 25 h (follicles) or 60 h (CL) after the injection of GnRH. Transcripts of VEGF isoforms (VEGF(121), VEGF(165), VEGF(189)) were upregulated 4 h after GnRH injection (during the luteinising hormone (LH) surge) and decreased thereafter to lowest levels around ovulation. All VEGF isoforms and their receptors were upregulated again after ovulation. The VEGF peptide concentration in follicular fluid decreased 20 h after GnRH injection, followed by an increase in follicles 25 h after GnRH. Expression of MMP-1 mRNA increased rapidly 4 h after GnRH injection and remained high during the entire experimental period. In contrast, MMP-19 mRNA increased significantly only after ovulation. Expression of TIMP-1 mRNA increased 4 h after GnRH and again after ovulation. Expression of tPA mRNA increased 4 h after GnRH and remained high during the entire experimental period, whereas expression of uPA transcripts increased significantly only after ovulation. Both uPAR and PAI-1 mRNA levels increased in follicles 4 h after GnRH and again after ovulation. The amount of MMP-1 protein (immunolocalisation) increased in follicles 10 h after GnRH: additional staining was observed in the granulosa cell layer. In conclusion, the temporal and spatial pattern of regulation of VEGF and extracellular matrix-degrading proteinases during periovulation suggests they are important mediators of the LH-dependent rupture of bovine follicles and for early CL formation (angiogenesis).

Effect of local neutralization of basic fibroblast growth factor or vascular endothelial growth factor by a specific antibody on the development of the corpus luteum in the cow.

Active angiogenesis and progesterone (P) synthesis occur in parallel during development of the corpus luteum (CL). Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are known to stimulate angiogenesis and P synthesis in vitro. The aim of the present study was to investigate the impact of bFGF or VEGF on the CL development in the cow by using a specific antibody against bFGF or VEGF. bFGF antibody, VEGF antibody, or saline as a control (n = 4 cows/treatment) were injected directly into the CL immediately after ovulation (Day 1), and the treatment was continued for 3 times/day over 7 days. Luteal biopsies were applied on Day 8 of the estrous cycle to determine the expression of genes associated with P synthesis and angiogenesis. Intraluteal injections with the bFGF antibody or the VEGF antibody markedly decreased the CL volume, plasma P concentration and StAR mRNA expression. bFGF antibody treatment decreased the mRNA expression of bFGF, FGF receptor-1, VEGF120, and angiopoietin (ANPT)-1, and increased ANPT-2/ANPT-1 ratio. However, VEGF antibody treatment decreased ANPT-2 mRNA expression and ANPT-2/ANPT-1 ratio. These results indicate that local neutralization of bFGF or VEGF changes genes regulating angiogenesis and P synthesis, and remarkably suppresses the CL size and P secretion during the development of CL in the cow, supporting the concept that bFGF and VEGF control the CL formation and function.