P-209 Implication of delta opioid receptor in the in vitro maturation of oocytes and its effects on subsequent fertilization and embryo development in mice
Authors: Totorikaguena L , Aguirregoitia N , Olabarrieta E 
Published: June 2023

Abstract

Study question
Could opiods modulate in vitro maturation of oocyte and improve fertilization rates?

Summary answer
Delta opioid receptor was present in mice oocytes, changing its expression pattern depending on the maturation stage and helping oocytes to reach blastocyst stage.

What is known already
The molecular mechanisms responsible for oocyte maturation are not completely understood and different molecules have been reported to be implicated as modulators of this process. The endogenous opioid peptides could be interesting candidates because they are involved in the regulation of reproductive physiology at multiple sites. When those receptors are activated, the adenylyl cyclase is inhibited, thus reducing cyclic AMP and blocking PKA. Moreover, opioids are also able to activate proteins involved in maturation. There is evidence suggesting a role for opioids during oocyte maturation because the signaling exerted by opioids is similar to that necessary for the meiosis resumption.

Study design, size, duration
Immature cumulus–oocyte complexes (COCs) from female 8 to 10 week old WT (C57BL/6xCBA) were retrieved after EGF stimulation. Mouse in vitro maturation culture was performed. In vitro grown oocytes, their corresponding cumulus (CC) and granulosa cells (GC) were classified at germinal vesicle stage (GV), at metaphase 1 (MI) and at metaphase 2 (MII).

Participants/materials, setting, methods
The presence of DOR was analysed in granulosa cells and oocytes at each stage of maturation by qRT-PCR and immunocytochemistry. AKT and MAPK signalling pathway (ERK 1/2) was studied by immunocytochemistry. Similarly, COCs were matured in vitro in the absence and presence of DPDPE and naltrindole at various concentrations. After IVM, in vitro fertilisation (IVF) was performed and development was observed until the blastocyst stage was reached, analysing fertilisation and embryo production percentages (p &lt; 0.05).

Main results and the role of chance
Our findings showed that OPRD1 was present in mice oocytes and granulosa cells, changing its expression pattern depending on the maturation stage. The evaluation of RT-PCR examination and immunohistochemical (IHC) staining revealed the presence of the transcript for Oprd1 gene in the different stages of the mouse oocyte's maturation, as well as in the granulosa cells at the moment of the extraction from the ovary. The obtained transcript was compared with the transcript for Oprd1 of mouse cerebral cortex as a positive control. The expected 482 bp fragment was detected in the germinal vesicle stage and in granulosa cells, whereas in MI and MII stage the signal was lower. Immunofluorescence analysis revealed that OPRD1 protein was present in mouse oocytes but, its localization differed at the different stages of the maturation process. Furthermore, the selective delta opioid agonist DPDPE, modulating PI3K/Akt and MAPK pathways, helped oocytes to reach blastocyst stage. Finally, we examined that the observed effect of DPDPE during oocyte maturation in the in vitro fertilization and subsequent embryo development could be blocked by the opioid antagonist naltrindole.

Limitations, reasons for caution
Although we have obtained higher rates of blastocysts, the quality of these embryos still needs to be analysed and transferred to recipient females to evaluate the offspring.

Wider implications of the findings
Our study has clinical implications for the improvement of oocyte in vitro maturation techniques (IVM). OPRD1 could be a possible therapeutic target for in vitro maturation culture medium, as it could improve the blastocyst rates obtained in the actual reproduction assisted techniques.

Trial registration number
not applicable