Expression of type I GNRH receptor and in vivo and in vitro GNRH-I effects in corpora lutea of pseudopregnant rabbits

The expression of type IGNRHreceptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidencedGNRHR-I andGNRH-I in luteal cells at early (day 4 pseudopregnancy)-,mid (day 9)-, and late (day 13)- luteal stages.Real-timeRT-PCRandwestern blotting revealed GNRHR-I mRNA and protein at the three luteal stages. Buserelin in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24 h respectively. In in vitro cultured CL, buserelin reduced progesterone secretion, increased prostaglandin F2a (PGF2a) secretion and cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE2 at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP3, 2-amino-ethoxydiphenylborate), and diacylglycerol (DAG, 1-hexadecyl-2-acetyl glycerol) or inhibitors for phospholipase C (PLC, compound 48/80), and protein kinase C (PKC, staurosporine) counteracted the buserelin effects. Buserelin co-incubated with COX inhibitor (acetylsalicylic acid) increased progesterone and decreased PGF2a and NOS activity at days 9 and 13, whereas co-incubation withNOS inhibitor (N-nitro-L-argininemethyl ester) increased progesterone at the same luteal stages. These results suggest that GNRHR-I is constitutively expressed in rabbit CL independently of luteal stage, whereas GNRH-I down-regulates directlyCLprogesterone productionvia PGF2a at mid- and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP3, DAG, PKC, COX-2, andNOS.