Effects of porcine oocytes on the expression levels of transcripts encoding glycolytic enzymes in granulosa cells

Abstract

Oocytes play critical roles in regulating the expression of transcripts encoding the glycolytic enzymes phosphofructokinase, platelet (PFKP) and lactate dehydrogenase A (LDHA) in granulosa cells in mice, but whether this is the case in pigs or other mammals has not been adequately investigated. Therefore, the aim of this study was to determine whether porcine oocytes regulate the expression levels of these transcripts in granulosa cells in vitro. Porcine cumulus cells expressed higher levels of PFKP and LDHA transcripts than mural granulosa cells (MGCs). However, co-culturing with oocytes had no significant effect on the isolated cumulus cells. While murine oocytes promoted the expression of both Pfkp and Ldha transcripts by murine MGCs, porcine oocytes promoted the expression of only Pfkp, but not Ldha transcripts by murine MGCs. Neither murine nor porcine oocytes affected PFKP and LDHA expression by porcine MGCs. Moreover, in the presence of porcine follicular fluid, porcine oocytes maintained the expression of PFKP, but not LDHA by porcine cumulus cells. Therefore, porcine oocytes are capable of regulating the expression of PFKP but not LDHA in granulosa cells in coordination with unknown factor(s) present in the follicular fluid.

Age-associated changes in granulosa cells and follicular fluid in cows

“…It was also reported that low oxygen stimuli in mice enhanced the expression of glycolytic genes in GCs, as well as the survival rate and development of follicles [ 28 ]. Furthermore, oocytes themselves activate glycolytic genes through the secretion of cytokines in mice and pigs [ 29 , 30 , 31 ]. Contrary to the activation of glycolysis, hypoxic conditions decreased mitochondrial functions and quantity, as determined by mitochondrial mass and DNA copy number [ 27 ].…”

Effects of oocyte‐derived paracrine factors on release of extracellular vesicles by murine mural granulosa cells in vitro

“…Real‐time PCR analysis was conducted as reported previously (Matsuno et al., 2016). In brief, total RNA was extracted from MGCs using a ReliaPrep TM Cell Miniprep System (Promega KK), and reverse transcribed using a ReverTraAce qPCR Master Mix with gDNA Remover (Toyobo, Osaka, Japan).…”

Effects of exosome-like vesicles on cumulus expansion in pigs <i>in vitro</i>

“…Porcine COCs and MGCs were collected from antral follicles 2–5 mm in diameter as previously reported [ 23 , 24 , 25 ].…”

“…Real-time PCR analysis was conducted as previously reported [ 25 ]. Briefly, total RNA was extracted from COCs using a ReliaPrep TM Cell Miniprep System (Promega) and reverse-transcribed using a ReverTraAce qPCR Master Mix with gDNA Remover (Toyobo, Osaka, Japan) according to the manufacturer′s protocols.…”

“…To avoid false-positive signals, dissociation-curve analyses were performed at the end of the analyses, and the PCR products were subjected to agarose gel electrophoresis to confirm the single amplification and sizes of the products. The PCR primers were used according to previous reports [ 25 , 30 , 31 ]. The ranges of amplification efficiencies were 92–97% and 88–90% for primers used to amplify cDNA from pigs and mice, respectively.…”