The effect of monosaccharide sugars and pyruvate on the differentiation and metabolism of sheep granulosa cells in vitro

B K Campbell,R J Scaramuzzi,N R Kendall,V Onions,L Guo

doi:10.1530/rep-10-0146

Abstract

The objective of this study was to investigate the effect of three monosaccharides or pyruvate on the ability of gonadotrophins to induce cellular proliferation and differentiation of cultured sheep granulosa cells. Lactate production and levels of mRNA expression for the glucose transporters SLC2A1, SLC2A4, SLC2A5 and SLC2A8 were also determined. No energy source in the culture media reduced cell number (50%) and oestradiol (E(2)) production. Dose and type of monosaccharide had a highly significant (P<0.001) effect on FSH-induced differentiation of the granulosa cells, and there was a highly significant interaction (P<0.001). Glucose supported higher levels of E(2) production than fructose, which was in turn higher than galactose (P<0.001). In contrast, pyruvate at low doses supported similar levels of E(2) production as glucose, but higher doses were markedly inhibitory to E(2) production (P<0.001). Cells responded positively to insulin (P<0.001) in the presence of all three monosaccharides. Glucose and the high doses of fructose resulted in the accumulation of lactate (P<0.001), but pyruvate, galactose and the low dose of fructose resulted in low lactate production. SLC2A5 expression was not detected and SLC2A8 expression was not affected, but SLC2A1 and SLC2A4 expression was depressed (P<0.05) by culture in the presence of fructose and glucose. These data show that glucose, metabolised under anoxic conditions to lactate, is the preferred energy substrate to support the gonadotrophin-induced differentiation of ovine granulosa cells in vitro, and that fructose and pyruvate, but not galactose, are alternative energy substrates despite marked differences in the way these substrates are metabolised.

Highlights

In mammalian species, ovulation rate is determined by the number of follicles selected from the ovulatory wave of follicles, which in turn is controlled primarily by the levels and actions of key reproductive hormones (Webb & Campbell 2007)
Glucose (5.5G0.07 log pg/ml) resulted in significantly higher levels of E2 production than fructose (5.0G0.07 log pg/ml; P!0.001), which in turn was higher than galactose (4.8G0.08 log pg/ml; P!0.01)
There was no significant difference in E2 production at 192 h between low (2.1 mM) and high (16.7 mM) doses of each monosaccharide (Fig. 1b), the high dose of galactose resulted in a significant depression (P!0.01) relative to both fructose and glucose (Fig. 1b)

Summary

Introduction

Ovulation rate is determined by the number of follicles selected from the ovulatory wave of follicles, which in turn is controlled primarily by the levels and actions of key reproductive hormones (Webb & Campbell 2007). Extreme energy restriction can lead to suppressed development of ovulatory follicles and anoestrus through inhibition of GNRH pulsatility (Jolly et al 1995, McShane et al 1994, Katz & Vollenhoven 2000). Increasing energy supply over a maintenance level leads to an increase in fecundity by an effect on follicle development and ovulation, which appears to act through a variety of mechanisms at both central and local levels (Scaramuzzi et al 2006). Glucose may act directly on the ovary. Glucose infused in combination with insulin into the ovarian artery of ewes with ovarian autotransplants directly suppressed androstenedione and oestradiol (E2) production from the dominant follicle without affecting peripheral gonadotrophin concentrations (Downing et al 1999)

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