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.

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.

Obtaining Reliable RT-qPCR Results in Molecular Diagnostics-MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery.

In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of "key steps" in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine.