Effects Of α-enolase On Granulosa Cells In Follicles From Zi Geese And Its Mechanisms

Zi goose is one of the better laying performance goose in the world, and theresearch on the regulatory mechanism of egg laying has great theoretic and actualsignificance. Granulosa cells are very important in follicles, which change inquantity, morphology and function could affect the follicle growth status. Ourprevious results showed that the relative expression levels of α-enolase (ENO1) inthe ovaries of laying geese increased compared with those of the pre-laying geese(P<0.05), which suggested that ENO1possibly plays an important role in follicularcells growth. ENO1is a glycolytic enzyme, which catalyzes the ATP-generatingconversion of2-phosphoglycerate (PGA) to phosphoenolpyruvate (PEP).Based on the characteristics of the birds follicles, in this study, histologicallocalization and expression of ENO1in different grades of follicles during the layingperiod firstly were analyzed, and then overexpression and interference vectorstargeting to ENO1were successfully constructed and transfected into folliculargranulosa cells in F1follicles from Zi geese. The effects of ENO1on glycolysis,steroid hormone secretion, reproductive hormone receptor expression, proliferationand apoptosis of granulosa cells were measured by flow cytometry, real timeRT-PCR and Western blot, which provided a better understanding of the function ofENO1in poultry reproductive physiology.(1)H-E staining and immunohistochemical were used to obtain morphologicalcharacteristics of different grades of follicular granulosa cells and ENO1gene andprotein expression in different grades of follicles, and then reproductive hormonesreceptor including follicle stimulating hormone receptor (FSHR), luteinizinghormone receptor (LHR), estrogen receptor alpha (ERα), estrogen receptor beta(ERβ), growth hormone receptor (GHR), insulin-like growth factor binding protein-1(IGFBP-1) mRNA and two gene related to apoptosis including Bcl-2mRNA and Caspase-3mRNA expression in different grades of follicles were analyzed, whichsupplied the basic data for the study of birds egg production mechanism.(2)Overexpression and interference vectors targeting to ENO1weresuccessfully constructed, and the infectious droplets obtained could reach1.1×1011pfu/mL of overexpression recombinant adenovirus. Overexpression and interferenceexpression vectors were transfected into granulosa cells and can be used for thefollowing experiments.(3)ENO1gene interference results:①reduced cells uptake capacity to glucose,decreased pyruvic acid, lactic acid concentration in the supernatant fluid and cellularATP content and pyruvate kinase (PK) mRNA expression, which suggested ENO1gene interference slowed the glycolysis process;②improved the secretion ofestrogen, progesterone and testosterone of granulosa cells, inhibited the productionof inhibin, and reduced the expression of ERα, ERβ, GHR and IGFBP-1mRNAexpression, which showed ENO1could regulate the reactivity of granulosa cells toreproductive hormone and cell growth and development by adjusting their hormonesecretion and reproductive hormone receptor expression;③inhibited the cellproliferation, increased apoptosis and the proportion of granulosa cell in G2/M phase,which suggested that ENO1gene interference could make the granulosa cells blockin G2/M phase, promote cell apoptosis, and inhibit the proliferation.(4)ENO1gene overexpression results:①reduced the glucose levels, improvedpyruvic acid, lactic acid content in the supernatant fluid, cellular ATP content andpyruvate kinase mRNA expression in granulosa cells, which suggested that ENO1gene overexpression speeded up the process of glycolysis, and increased theproportion of energy source supply from glycolysis.②enhanced activin, inhibin andinhibited testosterone concentrations; while promoted the reproductive hormonereceptor ERα, ERβ, GHR and IGFBP-1mRNA expression, which showed ENO1up-regulated activin and inhibin, down-regulated testosterone, and reproductivehormone receptor of granulusa cells to regulate cell growth and development;③decreased cell apoptosis, and made the proportion of granulosa cell in G0/G1phasedecrease, the proportion of granulosa cell in S and G2/M phase increase, whichsuggested that ENO1overexpression could induce DNA synthesis, and increase the proportion of granulosa cells in proliferating phase, and then inhibit cell apoptosis,and promote the proliferation.In conclusion, ENO1could regulate glycolysis, steroid hormone secretion ability,reproductive hormone receptor expression, apoptosis and cell cycle of granulosacells and finally affects goose follicular development.