Λουτράδης Δημήτριος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Δρακάκης Πέτρος, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ντόμαλη Αικατερίνη, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Μελέτη έκφρασης των υποδοχέων της CRH, CRH-R1 και CRH-R2 σε έμβρυα ποντικιού στα στάδια δύο, τεσσάρων και οκτώ κυττάρων και βλαστοκύστης
Study on the expression of CRH receptors, CRH-R1 and CRH-R2 in mice embryos, at the stages of two, four and eight cells and blastocyst
CRH (Corticotropin Releasing Hormone) is a 41-amino acid neuropeptide, synthesised in the hypothalamus, regulating the hypothalamus-pituitary-adrenal axis and its expression and biological functions are mediated by its membrane receptors, CRH-R1 (α, β, γ, c-h) and CRH-R2 (α, β, γ). CRH receptors are also activated by other endogenous agonists, such as urocortin (UCN). CRH is a very important regulator of stress in mammals. Although initially reported to be expressed in the CNS where it inhibits the inflammatory reaction, several studies have shown that CRH is as well expressed in several peripheral tissues mediating the stress effect and stimulating local inflammation. CRH is involved in human pathophysiology since pathogenesis is considered a disturbance of homeostasis, and therefore a stress condition.
CRH and its receptors are expressed in several sites of the female reproductive system, including the ovaries, endometrial glands, decidualized stroma, trophoblast, syncytiotrophoblast and placental decidua. CRH is secreted at inflammatory sites, acting as proinflammatory factor. Reproductive CRH has been shown to serve as an autocrine and paracrine modulator and to participate in ovulation, decidualization, embryo implantation and maintenance of human pregnancy. CRH produced by the deciduas and the trophoblast induces the secretion of FasL (pre- apoptotic cytokine). During the implantation of the blastocyst, the endometrium has inflammatory response characteristics, nevertheless, the embryo is not rejected as a semi-allograft it is. During this procedure the role of Fas/FasL pathway is in great importance. Due to the Fas/FasL pathway the excess of T cells is removed, through an inflammatory reaction, leading to the acceptance of the embryo.
The purpose of this study was to investigate the expression of genes encoding the CRH-R1 and CRH-R2 in the preimplantation developmental stages of 2-, 4-, and 8- cells and blastocyst in mice. For the collection of embryos, female mice were allowed to mate with males of the same age and strain overnight. The females were then sacrificed, their oviducts were removed and zygotes were collected and cultured. Two-, four- and eifght-cell embryos and blastocysts were collected after specific hours. Subsequently, total RNA was extracted from the embryos and reverse transcripted to cDNA. The reverse transcription reaction was followed by the technique of Real-Time PCR. The amplified products were electrophoresed on a 3% agarose gel containing ethidium bromide.
According to the results, only CRH-R1 is significantly expressed at the four- and eight-cell stage embryo as well as the blastocysts. The detection of CRH-R1 in blastocysts is consistent with findings of previous studies. However, the detection of CRH-R1 in the other two earlier stages, is a novel findng that is confirmed only by one previous study, in which a different technique was used. Thus, further studies are needed to clarify the presence of the receptor 1 in those earlier stages. These findings suggest that CRH plays an important role in blastocyst implantation and that it may also affect earlier developmental stages. Therefore, understanding the mechanisms by which CRH affects preimpantation development, may potentially lead to ways to treat stress as an ifertility factor.
CRH-R1 receptor, CRH-R2 receptor, Preimplantation stages, Blastocyst, Mice embryo