The baboon expresses the calbindin-D9k gene in intestine but not in uterus and placenta: implication for conservation of the gene in primates.

Expression of the Calbindin-D9k (CaBP-9k) gene was studied in the baboon. Northern blot analysis using a human CaBP-9k cDNA probe detected expression in duodenum but not in uterus and placenta. Reverse transcription/polymerase chain reaction (RT/PCR) confirmed this expression pattern and indicated a high degree of identity between the baboon and human CaBP-9k mRNAs. PCR was employed to amplify the intron A region of the baboon CaBP-9k gene using human-derived primers and baboon genomic DNA. The baboon intron was closely related to the human CaBP-9k intron A, including the presence a complete Alu-repetitive element. Most significantly, a 13 nucleotide long element at the 5' end of the baboon intron matched exactly the human sequence. This element represents a nonfunctional variation of an estrogen response element found at the same location in the rat CaBP-9k gene. The rat element functions as an enhancer and mediates uterine and possibly placental CaBP-9k expression in the rat and probably most other mammals. The finding of a modified ERE in baboon as in human suggests that during primate evolution the expression of this mammalian-specific gene has been eliminated in uterus and placenta. This scenario raises the question of the role of CaBP-9k in these reproductive tissues.

The human gonadotropin-releasing hormone receptor gene: complete structure including multiple promoters, transcription initiation sites, and polyadenylation signals.

The interaction of gonadotropin-releasing hormone and its receptor is a critical event in the endocrine regulation of reproduction. We have recently cloned the gene encoding for the human gonadotropin-releasing hormone receptor (hGnRHR). Partial sequence analysis revealed a structural organization consisting of three exons and two introns. Exon II contains only 219 bp and the remainder of the approximately 5 kb transcript is distributed between exons I and III. The complete coding region for the hGnRHR represented only 987 bp leaving an extensive 5' and 3' non-translated region and potentially additional exons unaccounted for. This report provides the complete sequence of exon I and III and demonstrates that further exons are unlikely to be contained within this gene. Sequencing of the 5' end of the gene revealed the presence of five consensus TATA sequences distributed within a 700 nucleotide region. Primer extension analysis detected multiple transcription initiation sites associated with this cluster of TATA sequences. Transcription of this region up to the most 5' initiation site was demonstrated by the reverse transcription-polymerase chain reaction (RT-PCR) method. The 5' non-translated region stretches between 703 and 1393 bp, depending on which initiation site is used. Several consensus cis-acting regulatory sequences were identified within the 5' end. These include, among others, sites for PEA-3, AP-1, and Pit-1. In addition, cAMP response element (CRE)-like and glucocorticoid/progesterone response element (GRE/PRE)-like sequences were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells.

The present study investigated the expression and regulation of GnRH and GnRH receptor (GnRHR) messenger RNAs (mRNAs) in human granulosa-luteal cells using reverse transcription-polymerase chain reaction (RT-PCR). Granulosa-luteal cells were aspirated from preovulatory follicles obtained from women undergoing in vitro fertilization. Two sets of primers derived from human hypothalamic GnRHR complementary DNA (cDNA) were used to amplify cDNAs from granulosa-luteal cells. PCR products corresponding to the expected sizes of GnRH were obtained from granulosa-luteal cells as well as the brain, but not from skeletal muscle cDNA. The authenticity of the PCR products was confirmed by Southern blot hybridization with internal oligonucleotide probes and by subsequent cloning and sequencing. Similarly, using four sets of primers specific for the human pituitary GnRHR cDNA, PCR products with the expected sizes were detected from both brain and granulosa-luteal cells, but not from skeletal muscle. PCR products were subsequently confirmed by Southern blot hybridization using an internal oligonucleotide probe or a cDNA probe which was obtained from screening a human pituitary cDNA library. Cloning and sequencing of the PCR product in the 3'-untranslated region revealed identical sequence with the reported human pituitary GnRHR cDNA sequence. RNA samples obtained from cells immediately after dissociation or after 2, 5, and 8 days of culture were analyzed by RT-PCR, and in all cases, both GnRH and GnRHR mRNA were detected. To investigate how gene expression of GnRH and GnRHR is regulated, we examined the effect of GnRH and hCG on GnRH and GnRHR mRNA levels in cultured human granulosa-luteal cells. Treatment with different concentrations of GnRH induced biphasic responses. Both GnRH and GnRHR mRNA were significantly increased by 1 nM, but slightly decreased by 1 microM GnRH; 1 nM GnRH also significantly inhibited progesterone production, whereas higher doses had no effect. Treatment with hCG (1 IU/ml) decreased GnRHR mRNA levels without altering the expression of the GnRH gene. These results demonstrate for the first time that 1) both GnRH and GnRHR mRNAs are expressed in human granulosa-luteal cells; 2) GnRH mRNA levels are autoregulated by GnRH; and 3) GnRHR gene expression is up-regulated by GnRH, but down-regulated by hCG. These findings provide strong evidence that GnRH is an autocrine regulator in the human ovary.

The human gonadotropin-releasing hormone (GnRH) receptor gene: cloning, genomic organization and chromosomal assignment.

The cDNA encoding the gonadotropin-releasing hormone (GnRH) receptor has recently been cloned and characterized in several species, including human. To determine the structure of the gene encoding the human GnRH receptor, we have screened a human genomic library and isolated seven positive clones, using cDNA probes derived from a human pituitary cDNA library. The isolated genomic clone contains the entire protein coding region of the GnRH receptor which is distributed between three exons and spans over 18.9 kb. Sequence analysis and restriction endonuclease mapping revealed the presence of two introns of 4.2 and 5.0 kb, respectively, both located within the open reading frame, designating the human GnRH receptor gene to the intron-containing class of the G-protein coupled receptor superfamily. Genomic Southern blot analysis indicated the presence of a single copy of the gene encoding for the GnRH receptor within the human genome. Using DNA from human-hamster somatic hybrid cell lines, the GnRH receptor gene was assigned to human chromosome 4, by means of PCR. The present study represents the first report on the GnRH receptor gene and its partial characterization should facilitate further investigation of the mechanisms by which expression of this gene is regulated.