ABSTRACT
The aim of the study was to find the most stable reference genes from: ACTB, GAPDH, RPL30, CYC, RPL17, RPS7 and YWHAZ in the feline endometrium. Three free software packages, geNorm, NormFinder and BestKeeper were used. In geNorm analysis, the most stable gene was RPS7 (at a primer concentration 1000 nM) or YWHAZ (500 and 250 nM). According to NormFinder and BestKeeper, ACTB (at all examined primer concentrations) followed by RPS7 and CYC were the most stable genes. Based on geNorm results at least two genes from among RPS7, RPL30, ACTB or YWHAZ should be chosen for Real Time-PCR result normalization.
Subject(s)
Cats/genetics , Endometrium/metabolism , Genes/genetics , Genetic Markers/genetics , Models, Animal , Real-Time Polymerase Chain Reaction/veterinary , Animals , DNA Primers/genetics , Female , Real-Time Polymerase Chain Reaction/standards , Software , Statistics, NonparametricABSTRACT
Progesterone (P4) derivatives which are commonly used to block the cyclicity of domestic cats disturb the endocrine balance in the endometrium. The aims of this study were (i) to examine whether lipopolysaccharide (LPS) is responsible for enhancement of tumor necrosis factor-α (TNFα) secretion by the feline endometrial epithelial and stromal cells in vitro, (ii) to know whether immunolocalization of TNFα/TNFR1 and TNFR2 differs in cats at estrus or diestrus, receiving medroxyprogesterone acetate and suffering from pyometra, and (iii) to determine if TNFα-challenged prostaglandin secretion is stopped by prostaglandin synthases inhibitors. A total of 37 domestic adult cats in estrus or diestrus, receiving octane medroxyprogesterone or having clinical symptoms of pyometra, were enrolled in this study. The results obtained showed a distinct increase in LPS-challenged TNFα secretion in endometrial epithelial, but not stromal cells. TNFα augmented PG secretion was blocked by phospholipase A2 (PLA2) and cyclooxygeanase-2 (COX-2), but not by mitogen-activated protein kinase (MAPK) inhibitor. TNFα/TNFR1 and 2 protein expressions were limited mostly to the surface and glandular epithelium. TNFα/TNFRs protein was upregulated in the inflammatory uterus and hence may be involved in development of pathologic changes in the endometrial glands in cats receiving exogenous P4 as a hormonal contraceptive.
Subject(s)
Diestrus/metabolism , Endometrium/drug effects , Endometrium/metabolism , Estrus/metabolism , Lipopolysaccharides/pharmacology , Medroxyprogesterone Acetate/pharmacology , Prostaglandins/metabolism , Pyometra/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Cats , Diestrus/drug effects , Estrus/drug effects , FemaleABSTRACT
In the present study, the question was addressed whether the feline placenta can synthesize prostaglandin F2α (PGF2α). The PGFS protein was elevated, particularly at 2.5-3 weeks of pregnancy compared to 7-8 (P < 0.05) and 8.5-9 weeks (P < 0.001). Transcripts for PGFS were significantly upregulated at 2.5-3 weeks of pregnancy and then gradually declined towards the end of gestation (P < 0.001). Transcripts for PTGS2 were only upregulated in placentas from queens close to term (P < 0.001) compared with earlier phases. Staining of PTGS2 showed distinct positive signals in placentas obtained during the last week before labor, particularly in the strongly invading trophoblast surrounding blood vessels, and also in decidual cells. Shortly after implantation, signals for PGFS were localized in the trophoblast cells. Near term, PGFS staining was seen mainly in decidual cells. Both placental PGF2α and plasma PGFM were elevated towards the end of pregnancy (P < 0.001) compared with earlier weeks of pregnancy. The content of PGF2α in extracted placenta mirrored the PGFM level in plasma of pregnant females. During late gestation there is a significant increase in PGFM levels in maternal blood and of PGF2α levels in placental tissue concomitant with an upregulation of placental PTGS2.
Subject(s)
Dinoprost/metabolism , Gene Expression Regulation, Developmental , Placenta/metabolism , Animals , Cats , Dinoprost/analogs & derivatives , Dinoprost/blood , Female , Gene Expression Profiling , Immunohistochemistry , Pregnancy , Pregnancy, Animal , Progesterone/metabolism , Time Factors , Trophoblasts/metabolismABSTRACT
BACKGROUND: Until recently, the corpus luteum (CL) was considered to be the main source of progesterone (P4) during pregnancy in the domestic cat (Felis catus). However, other possible sources of P4 have not been ruled out. Although feline placental homogenates were found to be capable of synthesizing P4, expression of the respective steroidogenic enzymes has not been investigated at the molecular level. Therefore, in the present study, expression of the two major factors involved in the synthesis of P4 - 3beta-hydroxysteroid dehydrogenase (3betaHSD) and steroidogenic acute regulatory protein (StAR) - was investigated in the feline CL and placenta during the course of pseudopregnancy and pregnancy. METHODS: The mRNA levels of StAR and 3betaHSD were determined using Real Time PCR and their localizations were determined by immunohistochemistry. Placental P4 concentrations, after ethyl extraction, were measured by EIA. RESULTS: Luteal 3betaHSD and StAR mRNA levels were strongly time-dependent, peaking during mid-pregnancy. The placental 3betaHSD mRNA level was significantly upregulated towards the end of pregnancy. In the CL, 3betaHSD and StAR protein were localized in the luteal cells whereas in the placenta they were localized to the maternal decidual cells. Placental P4 concentrations were low in early pregnant queens, but increased along with gestational age. CONCLUSIONS: These results confirm that the placenta is an additional source of P4 in pregnant queens and can thereby be considered as an important endocrine organ supporting feline pregnancy.
