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

Abstract

The expression of type I GNRH receptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidenced GNRHR-I and GNRH-I in luteal cells at early (day 4 pseudopregnancy)-, mid (day 9)-, and late (day 13)-luteal stages. Real-time RT-PCR and western 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 F 2a (PGF 2a ) secretion and cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE 2 at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP 3 , 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 PGF 2a and NOS activity at days 9 and 13, whereas co-incubation with NOS inhibitor (N-nitro-L-arginine methyl 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 directly CL progesterone production via PGF 2a at mid-and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP 3 , DAG, PKC, COX-2, and NOS.

Gonadotropin-Releasing Hormone 1 Directly Affects Corpora Lutea Lifespan in Mediterranean Buffalo (Bubalus bubalis) During Diestrus: Presence and In Vitro Effects on Enzymatic and Hormonal Activities1

“…Apparently, both mRNA and protein abundances did not change at different luteal stages. Positive staining for GNRHR was clearly evident in the cytoplasm of luteal cells during all stages of pseudopregnancy [9]. In the present study, GNRHR immunopresence and gene expression were evident during all luteal stages.…”

“…GNRHR was measurable in varying amounts in CL at different stages; in particular, it was detectable in stage III luteal tissues [19]. Recently, the presence of GNRHR at both transcript and protein levels was detected in CL of pseudopregnant rabbits [9]. Apparently, both mRNA and protein abundances did not change at different luteal stages.…”

“…The pituitary tissue of a female buffalo was included as a positive control to verify whether the antibody anti-GNRHR recognizes the receptor in this species. The intensity of immunostaining for all antibodies was assessed and compared microdensitometrically as previously described [9]. The image analysis system (IAAS 2000 image analyzer; Delta Sistemi) was calibrated by taking the background developed in sections incubated with antiserum as ''zero'' and a conventional value of maximum intensity of staining as ''100.''…”

“…In particular, GNRHR is constitutively expressed in rabbit corpora lutea (CL) independently of luteal stage, whereas GNRH directly downregulates CL progesterone production via PGF2a at mid and late luteal stages of pseudopregnancy [9].…”

Immunopresence and functional activity of prostaglandin-endoperoxide synthases and nitric oxide synthases in bovine corpora lutea during diestrus

“…In fact, PGF2a has been identified as the main luteolysing factor of uterine origin and PGE2 as luteotrophic factor [15]. In many species, the CL themselves synthesize PGF2a and PGE2 [6,17]. The critical step in PG biosynthesis is the enzymatic conversion of arachidonic acid into PGH2 by prostaglandin-endoperoxide synthase 1 (PTGS1) or PTGS2 [19].…”