Impact of preovulatory follicle maturity on oocyte metabolism and embryo development

Abstract

Improved oocyte competence for embryo development and pregnancy was observed following ovulation of preovulatory follicles with greater physiological maturity, as indicated by estradiol production, prior to the gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) surge. It was hypothesized that follicular fluid from preovulatory follicles of greater maturity better supports the maturing oocyte's metabolic requirements and improves embryo development. The objective was to determine if differences in preovulatory follicular fluid due to follicle maturity influence oocyte metabolism during in vitro maturation (IVM) and affect embryo development. Bovine preovulatory follicular fluid was collected 18 h after a GnRH-induced LH surge. Serum estradiol concentration at GnRH administration categorized follicles as greater or lesser maturity. Immature bovine oocytes were submitted to 24 h IVM in medium supplemented with 20% follicular fluid from preovulatory follicles of greater or lesser maturity. Embryo development was recorded. Oocyte maturation media and media conditioned by developing embryos were submitted for metabolomics. A randomized block design was utilized to determine differences in embryo development and media metabolites (P ≤ 0.05). Blastocysts from oocytes matured in greater vs. lesser maturity follicular fluid had a more moderate rate of development (P = 0.01). At the conclusion of 24 h IVM, abundance of 66 metabolites differed between greater and lesser follicle maturity treatments. Nine metabolites differed in media conditioned by developing embryos. Metabolome results suggest improved amino acid, purine, and glucose metabolism, followed by a more efficient rate of embryo development, in oocytes matured in greater vs lesser maturity follicular fluid.

Keywords: bovine; cumulus-oocyte complex metabolism; embryo development; follicular fluid; preovulatory follicle.

Fig. 1.

A) Pathways enriched with metabolites differentially abundant in oocyte maturation media supplemented with preovulatory follicular fluid of greater vs lesser physiological maturity. Before maturation is represented by the top bar in each pathway, after maturation by the middle bar, and change in metabolite abundance from before to after maturation (delta) by the bottom bar in each pathway. Actual FDR is written in each bar. B) Schematic of purine and pyrimidine metabolism. C) Schematic of arginine biosynthesis. In (B and C), triangles represent metabolites that were more abundant in the greater follicle maturity treatment. Inverted triangles represent metabolites that were less abundant in the greater follicle maturity treatment.

Fig. 2.

A–I) Bar chats representing the nine differentially abundant metabolites in conditioned media following embryo development of oocytes supplemented with follicular fluid from preovulatory follicles of greater vs lesser maturity during 24 h in vitro maturation.

Fig. 3.

Schematic of preovulatory follicular fluid collection, treatment classification, and in vitro maturation treatment scheme. Preovulatory follicular fluid samples were collected 18.70 ± 0.15 h after gonadotropin releasing hormone administration to induce the preovulatory luteinizing hormone surge (GnRH2). Follicles were classified as greater or lesser physiological maturity based on serum estradiol (E2) at GnRH2. Immature bovine cumulus-oocyte complexes were randomly placed into treatment groups to mature at 38.5°C, 5.5% CO₂, 21.0% O₂ for 24 h. Treatment groups consisted of oocyte maturation media (OMM) supplemented with 20% follicular fluid (FF) of greater physiological maturity (Greater), 20% FF of lesser physiological maturity (Lesser), and 20% fetal bovine serum (FBS, reference for lab). After 24 h in vitro maturation, in vitro fertilization (IVF) was performed. At 16 h post IVF (hpi), putative zygotes were denuded of associated cumulus and spermatozoa. At 73 hpi, cleavage of putative zygotes was recorded. At 193 hpi, blastocyst development was recorded. For each treatment, 60 µL OMM was collected before and after 24 h IVM and after blastocyst assessment for metabolome analysis. GnRH1, gonadotropin-releasing hormone first administration; PGF, prostaglandin F2α.