130 EXPRESSION AND LOCALIZATION OF μ OPIOID RECEPTOR IN PORCINE PRE-IMPLANTATION EMBRYOS

Abstract

Embryonic stem cells can become any tissue in the body, excluding a placenta. Growth factors, hormones, and neurotransmitters have been implicated in the regulation of their fate. Because various neural precursors express functional neurotransmitter receptors, as G-protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. Moreover, a high level of endogenous opioids linked to G-protein-coupled receptor above all μ opioid receptors (MOR) has been shown to interfere with normal calcium metabolism and with the activity of the mitogen-activated protein kinase (MAPK). Thus it is very important to understand the possible influence of opioid activities in the regulation of stem cell fate. In this study we investigated the presence of MOR on porcine in vitro-produced embryos at one-cell, 4-cell, morula, and blastocyst stages by immunostaining. The COC were collected by aspiration, cultured in NCSU-37 medium supplemented with hormones for 20 to 22 h, and then in maturation medium without hormones for 24 h. After this time, COC were inseminated with frozen-thawed epididymal spermatozoa at the concentration of 10 × 5 sperm cells mL^-1 for 3 h. After removal of cumulus cells, putative zygotes were cultured in IVC Pyr-Lac medium for the first 2 days and in IVC Glu medium until Day 6 (the day of IVF was defined as Day 0). Embryos at different stages were collected at 12, 36, 120, and 144 h post fertilization, and kept in 4% (v/v) paraformaldehyde until examination. All samples were washed and incubated for 30 min in PBS-1%BSA. Controls were incubated in PBS-1% BSA for 90 min, whereas embryos were incubated with a 1 : 2500 dilution of the primary rabbit antibody against the third extracellular loop of MOR. Prior to examination, all samples were washed in PBS and incubated with a FITC-conjugated anti rabbit IgG-secondary antibody diluted 1:200 in Evans Blue/PBS1x. Samples were visualized by laser scanning confocal microscope (Nikon). The immunofluorescence localize, by intense brilliant green, the presence of MOR on blastomers of all stage embryos examined, whereas the embryos of negative control did not show any fluorescent region or spotted coloring. Our results support specific implication of the opioid receptors in developmental process of porcine embryos. Their presence suggests a possible role of MOR in embryonic development. Thus it can be speculated that there is a role for MOR in controlling key events of the stem cell life. However, these primary results must be confirmed by the demonstration of protein expression (by Western blot) of MOR in the embryos and deeply studied to understand the exact functional role of MOR in them at this level.

JSPS short-term scholarship.