Fatty acids goat follicular fluid: effect on oocyte competence

Els acids grassos (AAGG) son una important font d’energia pels oocits en fase de creixement. Per tant, l’estudi del fluid fol·licular (FF) on aquests oocits maduren es d’especial interes. En estudis previs, Romaguera i col. (2011) va concloure que els oocits de fol·licles grans (? 3mm) eren mes competents que els de fol·licles petits. A mes, Catala i col. (2015) van demostrar que hi havia una produccio diferencial d’embrions entre les estacions de l’any, sent significativament mes baixes a la tardor que a l’hivern. La nostra hipotesi va ser que el perfil d’AAGG de cabres adultes i prepubers, aixi com de diferents mides de fol·licle i de diferents estacions de l’any, ens podria donar informacio per poder-ho relacionar amb la competencia oocitaria. El nostre objectiu va ser determinar si es podria fer servir aquest perfil com a marcador molecular per la qualitat oocitaria. En l’experiment 1, varem analitzar el perfil d’AAGG en FF de cabres adultes i prepubers, segons edat, mida del fol·licle i epoca de l’any. Entre d’altres, varem trobar diferencies en el total d’AAGG poliinsaturats (PUFA), la ratio n6:n3 segons edat de la femella i mida de fol·licle i per estacions de l’any. En l’experiment 2, varem avaluar la competencia de l’oocit provinent de femelles prepubers afegint diferents ratios dels acids linoleic (LA: n6 PUFA) i ? -linolenic (ALA: n3 PUFA) en el medi de MIV. Varem observar que la ratio LA:ALA 200:50 µM tenia un efecte perjudicial sobre el desenvolupament embrionari dels oocits quan es comparava amb els grups control i la resta dels tractaments (100:50 i 50:50 µM), quan els oocits eren fecundats in vitro (FIV) (2.63 % vs ? 13 %) pero no quan eren activats partenogeneticament. L’experiment 3 consistia en l’estudi de l’efecte de les diferents ratios de LA:ALA en l’oocit de cabra prepuber avaluant l’activitat i distribucio mitocondrial, la concentracio d’ATP i l’expressio genica relativa. Varem observar que hi havia un canvi si es comparava els oocits en el moment de recol·leccio i despres de 24h de MIV en l’activitat mitocondrial, en la distribucio d’aquests organuls i en la produccio d’ATP. A mes, en el grup de 200:50 µM l’activitat mitocondrial era mes alta que en la resta de grups. Es van analitzar l’expressio relativa de 9 gens que es troben alterats si la cel·lula pateix estres, comprometent la seva viabilitat. D’entre aquests gens, es varen trobar diferencies d’expressio entre les 0 i 24h de maduracio pels gens: GPX1, RPL19 i SOD1, pero no entre els diferents tractaments. En conclusio, es va trobar que el perfil d’AAGG de FF de cabra es similar a la del fluid fol·licular de vaques, ovelles i dona. Les principals diferencies trobades en les cabres es devien principalment a l'edat de la femella. Es va trobar una relacio directa entre la ratio n6:n3 present en el FF segons mida del fol·licle i estacio de l’any amb resultats previs en el nostre laboratori en la produccio in vitro d’embrions, suggerint que aquesta ratio podria ser un bon biomarcador de la competencia dels oocits. L'efecte negatiu de la mes alta ratio de LA:ALA en la competencia dels oocits no estava relacionada, tal com s’havia observat en altres estudis, amb una funcio mitocondrial alterada, ni amb un augment de la produccio d’especies reactives d’oxigen o d’estres del reticle endoplasmatic com a consequencia dels resultats obtinguts en l'expressio relativa dels gens estudiats. Per tant, hem hipotetitzat que l'addicio d'altes concentracions de LA: ALA es pot relacionar amb una alteracio en l'estructura de la membrana plasmatica causada per una incorporacio d'aquests acids grassos en els fosfolipids de la membrana acompanyats amb el consum d'ATP. Fatty acids (AAGG) are an important source of energy for oocyte growth phase. Therefore, the study of follicular fluid (FF) where they mature, is of special interest. In previous studies, Romaguera et al. (2011) concluded that oocytes from large follicles (? 3mm) were more competent than the small follicles. Moreover, Catala et al. (2015) showed that there was a differential production of embryos between seasons, being significantly lower in autumn than in winter. Our hypothesis was that the FA profile of FF from prepubertal and adult goats, as well as from different follicle sizes and different seasons, could give us information of the oocyte competence. Our objective was to determine whether this profile could be used as a molecular marker for the quality oocytes. In the first experiment, we analysed the profile of FA in FF. Among others, we found differences in total AAGG acids (PUFA), and n6: n3 ratio according to age, size of the follicle and seasons. In the second experiment, we evaluate the competence of oocytes from prepubertal females adding different ratios of linoleic acid (LA: n6 PUFA) and ? -linolenic acid (ALA: n3 PUFA) in IVM. We observed ratio LA: ALA 200: 50 µM had a detrimental effect on embryo development of oocytes when compared with control groups and other treatments (100: 50 and 50:50 µM) when oocytes were in vitro fertilized (IVF) (? 2.63% vs 13%) but not when they were activated partenogeneticament. According to the results of the experiment 2, the aim of the experiment 3 was to study the effect of LA:ALA ratios on prepubertal goat oocyte quality by assessing mitochondrial distribution and activity, ATP concentration and relative gene expression. Assessing mitochondrial activity, active mitochondria distribution and ATP concentration in the oocyte, we found that there was a change in this parameters when they were analysed on immature oocytes (collection point) compared to IVM oocytes (after 24 h of maturation). Moreover, the addition of 200:50 µM at IVM modified the mitochondrial activity of these oocytes, being higher compared with the other treatment groups, but no changes were observed in the active mitochondria distribution or ATP concentration. Concerning mRNA relative expression, we analysed 9 genes that are shown to be altered if the cell is under stress, and which development could be compromised: ATF4, DNMT1, GAPDH, GCLC, GPX1/GSH-Px, HSPA5/GPR78, RPL19, SLC2A1/GLUT1, SOD1/CuZnSOD. Among these genes, GPX1, RPL19 and SOD1 showed significant differences when comparing immature and IVM oocytes, but not among groups of treatment. In conclusion, we found that FA profile of goat FF is similar to the follicular fluid found in cows, sheep and woman. The main differences that we found in goats were mainly due to the age of the female. However, we found a direct relationship between n6:n3 PUFA composition in follicular fluid regarding follicular size and season of the year, with previous results in our lab suggesting that this ratio could be a biomarker of oocyte competence. Moreover, we found that adding 200:50 µM LA:ALA had a detrimental effect on blastocyst production of prepubertal goat oocytes produced by IVF but not by parthenogenetic activation. Contrarily to what was previously concluded in another studies found in the literature, the negative effect of the highest LA:ALA ratio on oocyte competence was not related to impaired mitochondrial function, ROS production or ER stress according to the relative expression of the studied genes. Thus, we hypothesized that the effect of the addition of high concentrations of LA:ALA was related to an alteration on the structure of the plasma membrane caused for an incorporation of these fatty acids in the membrane phospholipids accompanied with ATP consumption.