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The expression of Calbindin-D9k (CaBP-9k) in the pig uterus and placenta was measured by Northern blot analysis and reverse transcription polymerase chain reaction (PCR), respectively. Progesterone (P4) administration to ovariectomized pigs decreased CaBP-9k mRNA levels. Expression of endometrial CaBP-9k mRNA was high on pregnancy Days 10-12 and below the detection limit on Days 15 and 18. On Day 60, expression could be detected at low levels. In myometrium and placenta, CaBP-9k mRNA expression was not detectable by Northern analysis using total RNA. Reverse-transcribed RNA from both tissues demonstrated the presence of CaBP-9k transcripts by means of PCR. The partial CaBP-9k gene was amplified by PCR and cloned to determine the sequence of intron A. In contrast to the rat CaBP-9k gene, the pig gene does not contain a functional estrogen response element (ERE) within this region. A similar ERE-like sequence located at the identical location was examined by gel retardation analysis and failed to bind the estradiol receptor. A similar disruption of this ERE-like sequence has been described in the human CaBP-9k gene, which is not expressed at any level in placenta, myometrium, or endometrium. It is concluded that the pig CaBP-9k gene is regulated in these reproductive tissues in a manner distinct from that in rat and human tissues. The regulation is probably due to a regulatory region outside of intron A, which in the rat gene contains the key cis element for uterine expression of the CaBP-9k gene.

The Calbindin-D9k (CaBP-9k) is a cytosolic calcium binding protein expressed in the mammalian intestine, placenta, and uterus. The protein is probably involved in calcium transport across the intestinal and placental epithelia. In uterus, a function in controlling myometrial activity involving intracellular calcium has been postulated. The amino acid sequence of the porcine CaBP-9k has been determined from intestine. The cDNAs for the bovine, murine, and rat CaBP-9k have been cloned. The objective of this study was the cloning of the porcine cDNA encoding the CaBP-9k. We performed the anchored polymerase chain reaction (PCR) technique using rat and bovine cDNA sequence-derived primers for amplification of intestinal cDNA. Both 5' and 3' amplification products were cloned and sequenced. The sequences revealed the full-length cDNA encoding the porcine CaBP-9k, coding region for 79 amino acids, 57 nucleotides 5' and 149 nucleotides 3' noncoding region. The inferred amino acid sequence is identical to the published amino acid sequence, except for one residue. The porcine CaBP-9k cDNA is 82.8% and 69.1% homologous with the bovine and rat sequences, respectively. Both bovine and porcine cDNAs contain a stretch of approximately 50 nucleotides not found in the rat sequence. Northern analysis showed a 600 nucleotide transcript in intestine, kidney, and uterus.

The effects of luteinizing hormone-releasing hormone (LHRH) and its putative intracellular mediators on progesterone (P) and prostaglandin E2 (PGE2) formation were studied in rat granulosa cells. A calcium ionophore (A23187), 12-0-tetradecanoylphorbol-13-acetate (TPA), and melittin (a phospholipase A2-stimulator) were used to later intracellular calcium, protein kinase C, and arachidonic acid levels, respectively. During a 5-h incubation, LHRH increased basal P levels but failed to affect the formation of P induced by cholera toxin (CT). On the other hand, both basal and CT-stimulated PGE2 formation were increased by LHRH. Treatment of the cells with A23187 or TPA attenuated the formation of P induced by CT or FSH. By contrast, A23187 or TPA significantly augmented CT- or FSH-stimulated PGE2 formation. Interestingly, the effects of A23187 and TPA on PGE2 were synergistic, whether or not FSH or CT was present during the incubation. This synergy was not observed with regard to P formation. Melittin also increased basal P and PGE2 levels, and enhanced the stimulation of PGE2 by A23187 or TPA. However, in the combined presence of A23187 and TPA, melittin failed to further enhance the high levels of PGE2 accumulated. These findings further support a role for the intracellular calcium, protein kinase C, and arachidonic acid metabolic pathways in the multiple actions of LHRH in the ovary.