Cell Localization of ACTH, Dopamine, and GnRH Receptors and PPARγ in Bovine Corpora Lutea during Diestrus

Corpora lutea (CL) are transient organs, essentials for the pregnancy to be successful, that results from the ovulatory follicle rupture. If pregnancy fails to occur, the CL undergo luteolysis, a dynamic regression process that ends with their complete functional and structural demise. The life span of CL is characterized by luteal development, maintenance, and regression regulated by complex interactions between luteotropic and luteolytic mediators. The life-span of bovine CL is regulated by a multifactorial system that includes various hormonal regulators that up to date are not yet well studied, therefore the aim of the present study was to evaluate the immunohistochemical presence of receptors for adrenocorticotropic hormone (melanocortin-2 receptor, MC2R), dopamine (DR1-5), gonadotropin-releasing hormone (GnRHR), and peroxisome proliferators-activated receptor γ(PPARγ) in early, mid, late and regressive CL during diestrous cycle of bovine. Materials, Methods & Results: Ovaries from 24 clinically healthy cyclic cows were collected from licensed abattoirs. The animals had undergone a general veterinary assessment of their reproductive tract and ovaries before slaughter to exclude any apparent abnormalities and early pregnancy. Corpora lutea were separated by blunt dissection from the surrounding ovarian tissues and classifi ed into four groups, covering the entire diestrous cycle length: early, mid, late and regressive. Tissue specimens were immediately processed for immunohistochemical investigation. The slides were incubated with the following primary antibodies: rabbit polyclonal anti-GnRHR, rabbit polyclonal anti-MC2R, rabbit polyclonal anti-DRD1-DR5, and mouse monoclonal anti-PPARγ. The slides were incubated with the specific biotinylated secondary antibody exposed to avidin-biotin complex and the peroxidase activity sites were visualized using the DAB kit as chromogen. Tissue sections in which the primary antibody was omitted or substituted by the specifi c IgG were used as negative controls of non-specific staining. The intensity of receptor immunostaining in CL was assessed and comparated microdensitometrically. The data of the densitometric analysis were examined by Levene's test and one-way ANOVA followed by Student-Newman-Keuls t-test. Differences were considered significant at P < 0.01. GnRHR, PPARγ, and DR2 immunostained the cytoplasm of luteal cells in early, mid and late stages, whereas they did not react in regressive CL. The immunostaining of MC2R was strong in the cytoplasm and nucleus of luteal cells during early, mid and late CL, and decreased (P < 0.01) in regressive ones. PPARγ immunostained the cytoplasm of luteal cells in early, mid and late stages, whereas they did not react in regressive CL. DR2 immunosignals were only found in early, mid and late stages, whereas DR5 was evidenced strongly in the cytoplasm of early and mid CL, and weakly (P < 0.01) in late and regressive ones. DR1, -3, and -4 were immunonegative in all CL types. Discussion: The present study showed that in bovine CL GnRHR, MC2R, PPARγ, DR2 and DR5 are variously present throughout the different luteal phases of the diestrous cycle. These preliminary data confi rm the multifactorial complexity of the bovine CL life span regulation, in particular the fine tuning of the mediators that balance the CL luteotropic and luteolytic condition.