Investigation of the molecular mechanism of luteolysis in dogs

Hormonal regulation of the luteal stage of the estrous cycle between pregnant and non-pregnant dogs is similar except for the production of prostaglandin F2 alpha (PGF2α) before parturition. In pregnant dogs, PGF2α that lysis corpus luteum (CL) is produced by uteroplacental tissues before parturition. However, production of PGF2α is not reported in non-pregnant dogs and the CL regress itself slowly in those animals. This slow regression occurs despite of the presence of several luteotropic factors that supports the CL function during the luteal stage in non-pregnant dogs. Therefore, these aspects of luteolysis in non-pregnant dogs have not been fully understood. The aim of present thesis was to investigate molecular mechanisms of luteolysis in non-pregnant dogs. For this purpose, PGF2α were administrated subcutaneously on the 30th to 35th days of dioestrus when CL is sensitive to PGF2α to induce luteolysis in non-pregnant dogs. Non-pregnant dogs were assigned to control group (n=4, no PGF2α injection); single PGF2α injection group (1PGF, n=4); twice PGF2α injection group (2PGF, n=4); and thrice PGF2α injection group (3PGF, n=4). Because of the side effects of high dose PGF2α in dogs, a low dose of PGF2α (20 µg/kg) administiration was preferred. The CL samples were obtained with ovariohysterectomy (OHE) 1 hour after the last PGF2α injection in the treatment groups (1st, 9th, 25th hours) and control group (no treatment) in order to investigate the molecular changes during the luteolysis. Total RNA was extracted from the CL samples and converted to complementary DNA (cDNA) with the reverse transcriptase reaction. mRNA expression levels of genes that are known to play a role in luteolytic mechanism (Steroidogenic acute regulatory protein (StAR), Cholesterol side-chain cleavage enzyme (P450scc/CYP11A1), Luteinizing hormone receptor (LH-R), Monocyte chemoattractant protein-1 (MCP-1), Interleukin-8 (IL-8), Nuclear receptor subfamily 4 group A member 1 (NR4A1/Nurr77), Cyclooxygenase 1-2 (COX-1-COX-2), Prostaglandin transporter (PGT), Prostaglandin F2α receptor (PGFR), BAX, Caspase-3, Fas-Ligand (Fas-L)) were evaluated by Real-Time Polymerase Chain Reaction (Real-Time PCR). Cellular localization of NR4A1 and MCP-1 mRNA levels, cellular localization and density of StAR protein level were determined by respectively in situ hybridization (ISH) and immunohistochemistry (IHC) methods in luteal tissue. After PGF2α induction, mRNA expression level of NR4A1 was higher in treatment groups and it was localized in luteal and endothelial cells according to ISH. mRNA levels of PGFR, PGT, and COX-2 were significantly reduced with the increase of the number of PGF2α injections. Cyclooxygenase 1 expression was similar among the groups. mRNA levels of MCP-1 and IL-8 genes, which are related to immune response, significantly increased after the first injection (1PGF). In situ hybridization showed MCP-1 were localized in the luteal tissue between the luteal and endothelial cells after PGF2α induction. The expression level of Fas-L mRNA increased only in the 3PGF group. mRNA expression levels of LH-R, StAR, and CYP11A1 genes which are responsible for progesterone (P4) synthesis, decreased after PGF2α injections. The reduction of StAR protein density depending on the increased number of PGF2α injections was also observed under the microscope with IHC. The expression of apoptosis-related genes, BAX and Caspase-3, was similar among the groups. Serum P4 levels sharply reduced after PGF2α injections in the treatment groups but were still higher than the basal level at the end of the study. According to the results (serum P4, mRNA and protein (StAR) expression levels of studied genes), it may be suggested that repeated injections of PGF2α on the 30th to 35th days of diestrous in non-pregnant dogs induces functional luteolysis in canine CL. During his induced luteolysis model, the mechanism related to PGF2α production, impact, and transport decreased at mRNA level in CL, and similarly, the mechanisms responsible for the production and continuation of P4 decreased after repeated PGF2α applications. Furthermore, the reduction of LH-R with PGF2α application shows that exogenous PGF2α application prevents luteotropic effect in canine CL. Moreover, the increase in the expression levels of immune response genes after PGF2α injection indicates that PGF2α induces acute inflammation in canine CL. Under these experimental conditions, the unresponsiveness of apoptosis-related genes to PGF2α injections can be interpreted as that PGF2α induces functional luteolysis but does not start apoptosis. In conclusion, it was indicated that exogenously repeated PGF2α applications could induce luteolytic mechanisms in the CL in non-pregnant dogs. Furthermore it can also be concluded that some aspects of molecular regulation of luteolysis in canine CL in non-pregnant dogs are similar to other domestic animals.