Prepubertal heifers versus cows—The differences in the follicular environment

Abstract

The oocyte quality is to a large extent influenced by the sexual maturity of the donor female. Although this phenomenon has already been broadly described in domestic animals, the underlying mechanisms are poorly understood. Published data focus on oocyte ultrastructure, fertilization abnormalities, and blastocyst developmental rate. The goal of the present experiment was to characterize the follicular environment (oocyte, cumulus [CC] and granulosa (GC) cells as well as follicular fluid [FF]) in ovarian follicles of prepubertal heifers and cows. Each experimental replicate included the following set of traits within individual follicles: lipid droplets (LDs) number in oocytes, expression of seven genes involved in energy metabolism (fatty acids [FAs] metabolism—ELOVL2, ELOVL5, SCD, FADS2, glucose transport—GLUT1, GLUT3, GLUT8) in CC and GC as well as FA composition and glucose concentration in FF. According to our results, cow oocytes were larger in diameter and contained more LD than those from prepubertal heifers, both before and after IVM. The LD number was also higher in cow oocytes after IVM, when compared to immature oocytes. The FF from cow follicles had elevated glucose content similarly to the majority of the analyzed FA. Transcript analysis revealed differences for five out of seven analyzed genes (ELOVL, FADS2, SCD, GLUT3, GLUT8) in CC and GC cells. However after considering the female category, the only difference was noticed for the mRNA of SCD gene, which was more abundant in cow GC. This finding may indicate distinct roles of CC and GC in follicular energy metabolism. In conclusions, we suggest that distinct properties of follicular environment in prepubertal heifers and cows may be responsible for differences in the quality of oocytes from the two categories of donors. We hypothesize that suboptimal environment in heifer follicles (glucose and FA lower content in FF) determines reduced quality of their oocytes (lower diameter and LD number) and limited maturation potential. Besides, energy demands of heifer oocytes may be restricted due to a low LD number, exerting a negative effect on the development of the future embryo. The advantages of cow gametes (e.g., higher LD number and diameter) attributed to oocytes of superior quality may support the statement that cows donate oocytes of better quality than heifers.

Introduction

The main source of experimental material dedicated to in vitro production of bovine embryos are oocytes collected from ovaries of the slaughterhouse origin, thus the reproductive history of the oocyte donors is unknown. According to numerous authors, the sexual maturity of the donor exerts a direct impact on the quality of oocytes and embryos. This parameter is understood as the ability of the oocyte to resume meiosis, be fertilized, and develop into a viable embryo [1]. Kauffold et al. [2] pointed to lower developmental competence of in vitro-produced embryos obtained from juvenile calves in comparison with the adult cows. The reported differences included embryo yield at the 4-cell stage, morula, and blastocyst stage. Concomitantly Rizos et al. [3] pointed to the advantage of cow oocytes over those of heifers in relation to embryo production efficiency, under both in vivo and in vitro conditions. Experiments involving wider spectrum of basic procedures revealed properties distinctive for calf oocytes, such as ultrastructure (cortical granules distribution, reduced number of mitochondria), enzyme activity, reduced metabolism of glutamine, pyruvate, and glucose as well as transcript and protein expression patterns. Differential properties included delayed or incomplete maturation, fertilization disturbances, and polyspermy rate which were elevated for calf oocytes (reviewed by [2]). Follicular environment of cows and heifers differs also with regard to the follicular fluid (FF) composition for example: protein distribution and LH and estradiol concentration [4]. A more recent study reports differences in the content of fatty acids (FAs) and amino acids [5]. The differences in the FF composition have also been noted for heifers and lactating cows what reflects the divergence in the metabolic activity of the follicular cells. The activity of follicular cells affects energy metabolism of the cumulus-oocyte complex (COC) and influences oocyte quality. Since oocyte quality is the key factor determining blastocyst yield in vitro [6], a search for mechanisms underlying the differences in the follicular environment between cow and heifer oocytes is fully justified.

Energy metabolism is crucial for oocyte quality. Fatty acids and carbohydrates (e.g., glucose) are well-recognized energy sources, but FAs are far more rich in energy [7]. These energy substrates may originate either from internal reservoir preserved in the COCs or from FF, from which the oocyte may incorporate FA [8], [9]. In addition, oocytes also display some lipogenic and lipolytic capacity [10], [11]. Moreover, FA esterification and storage in lipid droplets (LDs) may protect the oocyte against FA-induced lipotoxicity [12]. Several studies reported a significant impact of high-FA supplementation of the IVM medium on oocyte and the nascent embryo quality [13], [14], [15], [16], indicating the importance of lipid metabolism within the COC for the final oocyte quality.

Gene expression analysis in cumulus (CCs) or granulosa (GCs) cells has been recently recognized as a noninvasive approach to evaluate oocyte quality. Owing to the importance of energy production, the analysis of lipids or glucose metabolism pathways active within the follicular cells seems to be justified in this particular context. Among many genes controlling cellular energy pathways, FADS2, SCD (stearoyl-CoA desaturase), ELOVLs, and GLUTs genes were chosen for the purpose of this experiment. The product of FADS2 (FA desaturase) catalyzes the initial step in the enzymatic cascade of the synthesis of polyunsaturated fatty acids (PUFAs). Stearoyl-CoA desaturase is a microsomal, rate-limiting enzyme involved in the desaturation of stearic acid to oleic acid. The variation in SCD enzyme activity in mammals is likely to affect a variety of key physiological variables, including cellular differentiation, insulin sensitivity, metabolic rate, adiposity, atherosclerosis, cancer, and obesity [17]. ELONGASE enzymes (ELOVL3, 5, and 8) are involved in the elongation of long-chain PUFAs. Particularly, ELOVL2 is involved in the elongation of C20 and C22 PUFA to produce C24:4n-6 and C24:5n-3 PUFAs [18]. It was reported in mouse cell lines that overexpression of ELOVL2 gene promotes accumulation of LDs, together with enhanced FA uptake [19]. GLUT genes control glucose intake. Paczkowski et al. [20] observed that the inhibition of FA oxidation during IVM of mice resulted in increase of mRNA content of GLUT1 gene both in oocytes and CCs.

Following the newest findings in the field, the aim of this study was to investigate whether the follicular environment of prepubertal heifers and adult cows differs with regard to the selected features related to energy metabolism within the follicle. The analyzed set of parameters included the LD content within the oocyte, gene expression analysis of selected seven genes involved in FA metabolism, glucose levels in cumulus and GCs, and FA content and glucose concentration in FF. The investigated set of factors allowed for characterization of the selected aspects of energy metabolism within the ovarian follicle: FF as a source of energy subtracts (FA, glucose), CCs as energy transport pathways (expression of genes involved in FA metabolism), and the oocyte as the FA (LD) reservoir.