Discovery of LH-regulated genes in the primate corpus luteum

Molecular Human Reproduction, Mar 2004

Circulating LH is essential for the development and function of the primate corpus luteum (CL) during the menstrual cycle. However, the cellular and molecular processes whereby LH controls luteal structure and function are poorly understood. Therefore, studies were initiated to identify gene products that are regulated by gonadotrophin in the monkey CL. Rhesus monkeys either were untreated (controls, CTRL; n=3) or received the GnRH antagonist Antide (ANT; 3 mg/kg body weight, n=3) to inhibit pituitary LH secretion on day 6 of the luteal phase in spontaneous menstrual cycles. The CL was removed 24 h later. RNA was extracted and converted to cDNA. The CTRL and ANT cDNA were differentially labelled with fluorescent dyes (Cy3-CTRL and Cy5-ANT) and hybridized onto microarrays containing 11 600 human cDNA. The selected cDNA were analysed further via semi-quantitative RT–PCR (a) to validate the microarray results and (b) to determine if their expression varies in the CL (n=3/stage) between the mid (day 6–8), late (day 14–16), or very late (day 18–19, menses) luteal phase of the natural cycle. After normalization of the fluorescence data, 206 cDNA (1.8% of the total) exhibited ≥2-fold change in expression after ANT. Of the 25 cDNA exhibiting a ≥6-fold change, 6 were up-regulated and 19 were down-regulated. Twenty-two of these 25 cDNA were validated by RT–PCR as differentially expressed in the ANT group, relative to the CTRL group, and 11 of 25 changed (P<0.05) correspondingly in the late-to-very late luteal phase. Thus, we have identified gene products that are regulated by gonadotrophin in the primate CL that may be important in luteal regression during the menstrual cycle.

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Discovery of LH-regulated genes in the primate corpus luteum

J.Xu 1 2 3 R.L.Stouffer 1 2 3 5 R.P.Searles 0 2 J.D.Hennebold 1 2 4 0 OHSU Gene Microarray Shared Resource, Oregon National Primate Research Center , Beaverton, OR 97006 , USA 1 Division of Reproductive Sciences 2 Beaverton , OR 97006 , USA 3 Department of Environmental & Biomolecular Systems, OGI School of Science & Engineering 4 Department of Obstetrics/Gynecology, Oregon Health & Science University , Portland, OR 97239 , USA 5 Department of Physiology/Pharmacology, Oregon Health & Science University Circulating LH is essential for the development and function of the primate corpus luteum (CL) during the menstrual cycle. However, the cellular and molecular processes whereby LH controls luteal structure and function are poorly understood. Therefore, studies were initiated to identify gene products that are regulated by gonadotrophin in the monkey CL. Rhesus monkeys either were untreated (controls, CTRL; n 5 3) or received the GnRH antagonist Antide (ANT; 3 mg/kg body weight, n 5 3) to inhibit pituitary LH secretion on day 6 of the luteal phase in spontaneous menstrual cycles. The CL was removed 24 h later. RNA was extracted and converted to cDNA. The CTRL and ANT cDNA were differentially labelled with fluorescent dyes (Cy3-CTRL and Cy5-ANT) and hybridized onto microarrays containing 11 600 human cDNA. The selected cDNA were analysed further via semi-quantitative RT - PCR (a) to validate the microarray results and (b) to determine if their expression varies in the CL (n 5 3/stage) between the mid (day 6 - 8), late (day 14 - 16), or very late (day 18 - 19, menses) luteal phase of the natural cycle. After normalization of the fluorescence data, 206 cDNA (1.8% of the total) exhibited $ 2-fold change in expression after ANT. Of the 25 cDNA exhibiting a $ 6-fold change, 6 were up-regulated and 19 were down-regulated. Twenty-two of these 25 cDNA were validated by RT - PCR as differentially expressed in the ANT group, relative to the CTRL group, and 11 of 25 changed (P < 0.05) correspondingly in the late-to-very late luteal phase. Thus, we have identified gene products that are regulated by gonadotrophin in the primate CL that may be important in luteal regression during the menstrual cycle. - Introduction The development and maintenance of the functional corpus luteum (CL) in primates during the menstrual cycle requires the actions of the pituitary-derived gonadotrophin, LH (Zeleznik and Benyo, 1994; Niswender et al., 2000; Stouffer, 2003). Several lines of investigation indicate a critical role for LH in supporting the proper structure and function of the primate CL. The inhibition of LH synthesis and secretion (by lesioning the arcuate nucleus in the hypothalamus or administering a GnRH antagonist; Hutchison and Zeleznik, 1984; Collins et al., 1986; Dubourdieu et al., 1991; Duffy et al., 1999) or action (by administering a neutralizing LH antibody; Asch et al., 1981; Groff et al., 1984) reduces progesterone production to a level that results in premature luteal regression as evidenced by menstruation. Replacement of LH in such protocols (Hutchison and Zeleznik, 1984), notably with an escalating dose-regimen (Duffy et al., 1999), leads to the maintenance of luteal function as determined by the continued progesterone production, as well as normal luteal phase length. From these studies, it is clear that LH is an essential luteotropin required for the primate CL during the menstrual cycle. Nevertheless, the intracellular processes and mechanisms whereby LH acts to develop and maintain primate CL structure and function are not well understood. Previous investigations identified individual LH-regulated genes in the primate CL of the menstrual cycle that are involved in steroid synthesis or action, such as steroidogenic enzymes (Ravindranath et al., 1992a), steroidogenic acute regulatory protein (STAR; Devoto et al., 2004) and estrogen receptor (ERb; Duffy et al., 2000), or tissue remodelling, e.g. members of the matrix metalloproteinase family (Young and Stouffer, 2004) and angiogenic factors (Ravindranath et al., 1992b). Differential mRNA display was also used to identify novel genes that are regulated by LH in the ovulatory, luteinizing follicle of the rat (Espey and Richards, 2002) and the CL of the non-human primate (Yadav et al., 2004). However, relatively little information exists regarding the specific genes that are under the regulatory control of gonadotrophin in the primate CL. The present study was initiated, therefore, to systematically investigate those genes that are acutely dependent on gonadotrophin for expression in the monkey CL, following its development in the natural menstrual cycle (Xu et al., 2003b). Using spotted (human cDNA) microarrays, the mRNA of 11 600 genes were compared in CL from control and GnRH antagonist-treated monkeys. Twenty-five cDNA exhibiting a $ 6-fold change among groups were further analysed by semi-quantitative RT PCR. In addition, since there is evidence for less L (...truncated)


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J. Xu, R.L. Stouffer, R.P. Searles, J.D. Hennebold. Discovery of LH-regulated genes in the primate corpus luteum, Molecular Human Reproduction, 2004, pp. 151-159, 11/3, DOI: 10.1093/molehr/gah157