Differential gene expression in anterior pituitary glands from anestrous and cycling postpartum beef cows.
ABSTRACT Oligonucleotide microarrays (GeneChip Bovine Genome Arrays, Affymetrix Inc., Santa Clara, CA) were used to evaluate gene expression profiles in anterior pituitary glands collected from 4 anestrous and 4 cycling postpartum primiparous beef cows to provide insight into genes associated with transition from an anestrous to a cycling status. Tissues were collected 40 to 61 d after calving from anestrous cows and from cyclic cows between d 7 and 13 of the estrous cycle (luteal phase) from d 54 to 77 after calving. Hybridization signals were normalized across arrays, and genes with mean differences in expression that were at least 1.5-fold apart and with a minimum difference in mean signal intensity of 10 were compared. Based on these criteria, 47 transcripts were increased (P < 0.025) and 31 transcripts were decreased (P < 0.025) in pituitary gland tissue from cycling compared with anestrous cows. Few transcripts identified in this analysis were associated previously with reproductive function. To provide greater detail on the influence that stage of the estrous cycle (i.e., collection during the luteal phase) might have on the differences detected in gene expression, quantitative real-time PCR was used to compare gene expression in anterior pituitaries of anestrous cows with an additional independent set of anterior pituitary glands collected at 4 different stages of the estrous cycle: 0.5 to 2 d (n = 9), 5 to 6.5 d (n = 5), 11.4 to 13.7 d (n = 5), and 17.9 to 19 d (n = 6) after the onset of estrus. Gastrin-releasing peptide, the gene that exhibited the largest fold increase in expression in the microarray experiment, and IGFBP3 mRNA were expressed at greater (P < 0.004) amounts in samples from the different stages of the estrous cycle than in samples from anestrous cows. In addition, expression of IGFBP3 mRNA was proportional to serum progesterone concentrations throughout the estrous cycle (P < 0.05). Expression of versican mRNA was decreased (P = 0.03) in samples from the different stages of the estrous cycle compared with anestrous cow samples. Results identified numerous genes that may be involved in the transition from anestrous to cycling status, providing novel insight into mechanisms regulating reproductive function.
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ABSTRACT: The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHβ, but reduces FSHβ mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo.Endocrinology 03/2012; 153(3):1421-34. · 4.72 Impact Factor