Abstract
ABSTRACT
Progesterone governs the establishment and maintenance of pregnancy in ruminants, acting through the nuclear progesterone receptor (PGR) and, potentially, through membrane progesterone receptors (mPRs) of the PAQR family. The cellular distribution of mPRs in ruminant reproductive tissues, however, remains undefined. We generated and validated rabbit polyclonal antibodies against the intracellular C-terminal peptides of bovine PAQR6 (mPRδ) and PAQR8 (mPRβ), and used them, with quantitative PCR of the uterus and conceptus, to map these receptors in the ovine uterus and conceptus and in the bovine ovary. Both antisera showed high ELISA titer against their immunizing peptides, negligible preimmune reactivity, and complete loss of tissue signal after peptide pre-absorption. By qPCR,
PAQR6
,
PAQR8
and
PGR
transcript levels did not change significantly across the peri-implantation period in either the conceptus or the endometrium; in contrast, the progesterone-responsive gene ZBTB16 increased markedly in the conceptus (up to ∼45-fold by day 21, p < 0.05 at days 17, 19 and 21), whereas FKBP5 showed a numerical increase without reaching statistical significance. Both genes remained relatively stable in the endometrium, indicating conceptus-specific changes in progesterone-responsive gene expression. At the protein level, PAQR6 and PAQR8 localized to the luminal and glandular epithelium of the endometrium across the cycle and early pregnancy, and to follicular (granulosa) and luteal cells of the ovary. Dual immunofluorescence in the ovine endometrium showed that both PAQR6 and PAQR8 are enriched at the apical surface of the luminal epithelium and are spatially distinct from the basolateral adherens-junction marker E-cadherin. These results provide a protein-level map of PAQR6 and PAQR8 in the ruminant reproductive tract, identify the apical epithelial surface as a site of receptor enrichment, and show that the endometrial epithelium retains membrane progesterone receptors while progesterone-responsive gene expression changes dynamically in the conceptus during the peri-implantation period. The validated ruminant-reactive antibodies provide a resource for future functional studies.