Immunolocalization, gene expression, and enzymatic activity of cyclooxygenases, prostaglandin e2‐9‐ketoreductase, and nitric oxide synthases in mediterranean buffalo (bubalus bubalis) corpora lutea during diestrus

Abstract

Immunopresence, gene expression, and enzymatic activity of cyclooxygenase 1 (COX1), COX2, PGE2-9-ketoreductase (PGE2-9-K), endothelial (eNOS), and inducible nitric oxide synthases (iNOS), and hormone in vitro production were examined in early, mid, late, and regressive buffalo corpora lutea (CL). COX1 immunosignals were detected in the cytoplasm of small luteal cells, COX2 in large luteal cells, and PGE2-9-K in all luteal cells. COX2 and PGE2-9-K immunosignals were greater in late CL. Immunopresence of both NOS types were evidenced in the nuclei and cytoplasm of all luteal cells, as well as in the nuclei of endothelial cells, during all stages studied. The eNOS and iNOS immunosignals increased during the early stage. COX1 transcripts were lower in late and regressive CL, COX2 in late, PGE2-9-K higher in regressive, and iNOS higher in early and lower in regressive CL. COX1 enzymatic activity was lower in regressive CL, COX2 increased in mid and late stages, and PGE2-9-K was higher in late CL. Endothelial NOS activity was higher during mid and late stages and lower in regressive, whereas iNOS was greater in late and lower in early. Progesterone in vitro release was higher in mid and lower in late phase, while PGF2α synthesis was higher in late CL and lower in regressive, and PGE2 was higher during regressive stage. These results support the idea that COX, NOS, and PGE2-9-K regulate buffalo CL life span. In particular, regressive CL seems involved in the development of the contralateral early CL, through the production of the luteotrophic PGE2.

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

“…2). In previous studies on buffalo CL [10,11], it was shown that PTGS1 enzymatic activity was higher in late stage and lower in regressive CL, while PTGS2 increased from early to late CL and, differently from the present data, became higher in regressive CL. It is interesting to note that regressive CL is accompanied by general decline (p < 0.01) of PTGS and NOS enzymatic activities (Fig.…”

“…CLs were separated by blunt dissection from the surrounding ovarian tissues and classified into four groups, covering the entire cycle length, as described previously [1,3]: early (Days 4-6), mid (8)(9)(10)(11)(12), late (14)(15)(16), and regressive (19)(20)(21). CLs were cut in half longitudinally for the following immunohistochemical investigation and enzymatic activity assays.…”

“…Tissue specimens were immediately processed for immunohistochemical investigation according to procedures previously described [11]. The slides were incubated with the following primary polyclonal antibodies (Abcam, Cambridge): rabbit anti-nNOS (1:250), rabbit anti-eNOS (1:10), rabbit anti-iNOS (1:200), rabbit anti--PTGS1 (1:50), and goat polyclonal anti-PTGS2 (1:50).…”

Lectin-binding sites in the seminal vesicles of entire and castrated horses

“…All the animals did not present reproductive abnormalities. The specimens were immediately fixed and processed for lectin histochemistry using procedures previously described (Parillo & Diverio 2009b;Parillo et al 2012Parillo et al , 2013Zerani et al 2012, in press). The sections were dipped in 0.3% H 2 O 2 /methanol for 1 hour to inhibit endogenous peroxidase activity, washed with PBS, then incubated in a moist chamber for 1 hour at room temperature with a solution of horseradish peroxidase (HRP) conjugated lectins (Sigma-Aldrich, St Louis, MO, USA) in 0.1 M PBS, pH 7.2, containing 0.1 mM CaCl 2 , MgCl 2 and MnCl 2 .…”

Immunolocalisation of nitric oxide synthase isoforms in the ductuli efferentes and epididym

“…The slides were incubated with the following primary rabbit polyclonal antibodies (Abcam, Cambridge): anti-nNOS (1:250), anti-eNOS (1:10) and anti-iNOS (1:200) [15,17]. Then, the slides were incubated with biotinylated goat antirabbit secondary antibody (Santa Cruz Biotechnology, CA, USA), exposed to avidin-biotin complex (ABC kit, Vector Laboratories) and the peroxidase activity sites were visualised using the DAB kit (Vector Laboratories) as chromogen [4,20].…”