John Krisinger

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.

Calbindin-D9k mRNA is tightly regulated during the estrous cycle in the rat uterus ☆

Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein with a molecular weight of 9000. CaBP-9k is mainly expressed in intestine, uterus and placenta, with intestinal levels controlled by vitamin D and uterine levels controlled by estrogens. CaBP-9k mRNA levels were measured in rat uterus throughout the estrous cycle. On the morning of proestrus, estrus and diestrus animals were sacrificed. Serum 17 beta-estradiol concentrations were determined using a radioimmunoassay. Whole uterus was used for preparation of total RNA. Northern blot analysis was performed to quantify CaBP-9k and beta-actin mRNA. CaBP-9k levels were highest at proestrus, dropped 10-fold at estrus and were not detectable at diestrus. beta-Actin levels did not change significantly throughout the estrous cycle. Peak 17 beta-estradiol concentrations coincided with maximum CaBP-9k mRNA expression at proestrus. Despite minimal concentrations of 17 beta-estradiol at estrus, CaBP-9k mRNA was still present at 10% of the proestrus level. At diestrus, CaBP-9k mRNA was not detectable despite increasing 17 beta-estradiol. It is concluded that CaBP-9k is subject to 17 beta-estradiol regulation during the estrous cycle. Correlation between CaBP-9k mRNA and 17 beta-estradiol levels indicates a lag period for CaBP-9k induction in diestrus following a rise in steroid hormone levels.

Expression of calbindin-D9k in the early pregnant rat uterus: Effects of RU 486 and correlation to estrogen receptor mRNA

Calbindin-D9k (CaBP-9k) is a calcium binding protein expressed at high levels in the rat uterus. The CaBP-9k gene carries an estrogen response element which is involved in the steroid hormone regulation of the gene during the estrous cycle and gestation. The present study was aimed at determining expression of the gene during the first half of pregnancy and to assess the role of progesterone (P4) and the estrogen receptor (ER). Expression of CaBP-9k mRNA was determined by Northern blot analysis during the first 10 days of pregnancy. On pregnancy day 1 (P1), CaBP-9k mRNA levels were relatively high. On P2, 3 and 5 CaBP-9k mRNA decreased to the detection limit using 10 micrograms total RNA probed with a random primed cDNA. On P10, CaBP-9k transcripts began to reappear at levels of about 30% of P1. Expression of beta-actin mRNA displayed a continuous increase during this period with a rapid rise of 240% between P2 and P3. The typical increase of P4 accompanied by moderate changes of estradiol (E2) was determined in serum of experimental groups. When RU 486 at 10 mg/kg was administered as a single s.c. injection on P3, the CaBP-9k down-regulation was rapidly interrupted and mRNA expression became extremely high. The effect was seen maximally at 24 h post injection and was maintained at 48 and 72 h. Expression of beta-actin mRNA was increased only moderately at 24 h and was unchanged at 48 and 72 h. Serum P4 remained unaffected by the treatment and E2 displayed a slight increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of the full-length cDNA encoding the human calbindin-D9k

The full‐length cDNA encoding the human calbindin‐D9k (CaBP‐9k) has been cloned using reverse transcription/PCR methodology with rat‐ and bovine‐derived primers and intestinal RNA. A core product, and both a 5′and 3′ product encompassing the full‐length cDNA were obtained. The clones include coding region for 79 amino acids, 57 nucleotides 5′‐ and 159 nucleotides 3′‐non‐coding region, and a poly(A) tail. The deduced protein sequence is homologous Lo other mammalian CaBPs. Northern analysis revealed the mRNA in human duodenum to be about 600 nucleotides in length, Expression levels in adult human tissue were substantially lower than in child, rat or porcine intestine.

Calbindin-D9k gene expression during the perinatal period in the rat: correlation to estrogen receptor expression in uterus

Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein mainly expressed in duodenum, placenta and uterus. The gene encoding the rat CaBP-9k is subject to tissue specific induction by 1,25 dihydroxyvitamin D3 (intestine) and estradiol (E2) (uterus). Control of placental expression remains unknown. The expression of CaBP-9k mRNA during the perinatal period was studied (pregnancy day 21 (P21)-lactation day 4 (L4)). In uterus, maximal expression levels were found at P21 and maintained until L1. With the transition to L2, the CaBP-9k mRNA concentration dropped drastically below the detection limit as quantitated by Northern blot analysis. Measurements of E2 and progesterone (P) levels showed a gradual decrease at late pregnancy (P21; birth). Post partum E2 levels continued to decline and P concentrations increased slightly. Uterine estrogen receptor (ER) mRNA levels determined by cDNA/PCR analysis revealed close correlation between expression of ER and CaBP-9k mRNAs. ER mRNA levels were maximal at P22 and declined at parturition and with onset of lactation. At L2 and L3 ER mRNA levels were minimal and had decreased 5-fold compared to late pregnancy. CaBP-9k protein concentrations fluctuated only slightly dependent on the stage of the estrous cycle: estrus > proestrus > diestrus. During the perinatal period CaBP-9k concentration was overall lower than in non-pregnant uterus and revealed only a moderate increase at birth and decrease in early lactation. Similar to the uterine levels, placental CaBP-9k mRNA was highest at P21 and remained high until birth. Fetal duodenal CaBP-9k rose sharply just prior to birth and plateaued in the early postpartal period.(ABSTRACT TRUNCATED AT 250 WORDS)

Calbindin-D9k gene expression during pregnancy and lactation in the rat

Calbindin-D9k (CaBP-9k) is a calcium binding protein expressed in mammalian intestine, uterus and placenta. It is believed to be involved in transepithelial calcium transport in intestine and placenta and regulation of cytosolic calcium concentration in uterus. CaBP-9k mRNA levels were measured by Northern blot analysis in maternal duodenum, uterus, placenta and fetal/neonatal duodenum during pregnancy and lactation. In maternal duodenum a maximal increase occurred at day 15 of lactation (2.3-fold) and 20 days post-lactation levels decrease to 30.3% of non-pregnant controls. In non-pregnant uterus a 10-fold variation of CaBP-9k mRNA levels was observed between individual animals despite a uniform expression of beta-actin. During pregnancy high CaBP-9k expression is found, averaging about 20% of duodenal levels, which abruptly drops below detection during early lactation. At late lactation CaBP-9k mRNA levels are again subject to great variation ranging from no expression to maximal levels found in the non-pregnant uterus. Placental CaBP-9k is maximally expressed at the end of pregnancy (day 20) reaching about 2.5% of duodenal levels. Fetal intestinal CaBP-9k mRNA was detectable in 20 micrograms total RNA at day 18 of pregnancy and rose sharply in early lactation reaching about 50% of adult duodenal levels at day 20 lactation. The profound changes of uterine CaBP-9k mRNA in non-pregnant (cycling), pregnant, and lactating rats indicate a rapid hormonal regulation of gene expression, most likely involving 17 beta-estradiol.

Gonadotropin-releasing hormone (GnRH) and cyclic amp positively regulate inhibin subunit messenger RNA levels in human placental cells

Bioactive and immunodetectable levels of both inhibin and activin are present in the placenta, raising questions as to the regulatory control of their synthesis. This study was designed to determine the effect of cyclic AMP (cAMP) and gonadotropin-releasing hormone (GnRH) on inhibin subunit gene expression in short-term incubations of placental cells. A semi-quantitative polymerase chain reaction (PCR) technique was used after isolation of total RNA and first strand cDNA synthesis from mechanically dispersed trophoblast-enriched cells obtained from human placentae at term. The level of gene expression of inhibin subunits was higher for beta A and alpha-subunits mRNA compared to the beta B-subunit mRNA as determined by PCR in combination with Southern blotting or Northern hybridization. Steady-state levels of beta-actin mRNA did not change throughout the 6-h incubation period and was used as a control of PCR amplification of the respective inhibin subunit gene transcripts following treatments with 8-bromo cAMP or GnRH. 8-bromo cAMP dose-dependently increased all three inhibin subunit gene transcripts with maximal responses seen at 150 microM (alpha-subunit mRNA 2.3-fold, beta A-subunit mRNA 1.8-fold and beta B-subunit mRNA 2.8-fold over control). GnRH (100 nM) significantly increased inhibin alpha and beta B-subunit mRNA levels 2.9-fold and 2.0-fold, respectively (P < 0.01), but not beta A-subunit mRNA. Collectively, the present findings demonstrate that in human term placental cells, gene expression of all inhibin subunits is under the direct influence of cAMP and further support a modulatory role of local GnRH in placental trophoblasts during late pregnancy.

Effect of medium-chain triglycerides on calbindin-D9k expression in the intestine

These studies determined the effect of the saturated fat source in infant formula on the expression of calbindin-D9k (CaBP-9k). Piglets were fed from birth to 8 d with milk or formula containing saturated fatty acids as medium-chain triglycerides (MCT), coconut oil, palm oil (Palm 1), or synthesized triglycerides with 16∶0 directed to thesn-2 position (Palm 2). Levels of intestinal CaBP-9k mRNA were significantly (P<0.01) higher in piglets fed formula with MCT than in piglets fed the other formula or milk; and higher in piglets fed the Palm-1 than in piglets fed Palm-2 formula. This is the first evidence that MCT alter piglet intestinal CaBP-9k mRNA.