The endocrine disrupting effect of hypoxia on pituitary cells

Abstract

Hypoxia is an endocrine disruptor which can affect the synthesis and balance of sex hormones and thereby impairs reproduction of fish. However, the precise mechanisms of endocrine disruption remain unclear. Moreover, effects of hypoxia on reproduction in higher vertebrates such as mammals remain largely unknown. Gonadotropin releasing hormone (GnRH) is known to play a pivotal role in vertebrate reproduction. Previous studies have suggested that suppressed expression of GnRH and/or GnRH receptor (GnRHR) along the Hypothalamus-Pituitary-Gonad (HPG) axis could be an important mechanism contributing to reproductive impairments in hypoxic fish. Given that the HPG axis and genes controlling steroidogenesis as well as the sex hormones are highly conserved across vertebrate groups, the endocrine-disrupting effects of hypoxia found in fish may also occur in mammals including human. The aim of the present study is to determine whether hypoxia would affect the expression of GnRHR gene (one of the key genes along the HPG axis) in mammalian pituitary cells. A mouse pituitary cell line, LbetaT2, was used as an in vitro model to test the hypothesis that hypoxia can affect the expression of GnRHR in pituitary, thereby affecting reproduction in mammals. LbetaT2 cells were incubated for 24, 36, or 48 hours in a hypoxic chamber with 2% O2 and an identical set of cells were incubated under normoxic condition to serve as controls. The expression level of GnRHR mRNA was subsequently determined by SYBR Green-based real-time RT-PCR. Furthermore, the effect of hypoxia on transcriptional activity of GnRHR gene in the LbetaT2 pituitary cells was determined. A 1.2 kb fragment of mouse GnRHR promoter was cloned into pGL3-Basic vector and transfected into the LbetaT2 cells. The transfected cells were put into hypoxic chamber with 2% O2 and incubated for 24, 36, or 48 hours. The promoter activity determined from hypoxic cells was compared with that determined from normoxic cells. The effects of hypoxia on GnRHR gene expression and promoter activity in the LbetaT2 pituitary cells will be discussed in this presentation. Our current study represents the first important attempt to decipher the effects of hypoxia (an endocrine disruptor) on mammalian reproduction.