Oxytocin induces PGE2 release from bovine cervical mucosa in vivo.

Oxytocin receptor (OTR) concentrations in bovine cervical mucosa rise steeply a few days before estrus to high concentrations and fall rapidly after estrus. To study the physiological role of these OTR, the effect of OT on the release of PGE, from the cervical mucosa of periestrous cows in vivo was determined by inserting bags made of dialysis tubing containing isooncotic saline solution in the endocervix for two 2-h periods, a fresh bag for each period. During the first period no treatment was given, during the second period OT (100 IU) or saline was injected i.m. PGE2 content in the second bag was significantly greater in OT-treated cows than in saline-treated cows. In a second experiment cervical resistance to stretch, achieved by distention of a balloon inside the cervical canal, was measured in periestrous cows before and 10 h after i.m. injection of OT, or endocervical application of 2.5mg PGE1 in a jelly, or the inactive jelly. A significant reduction in the resistance was achieved with both OT and PGE1; in the doses given the effect of PGE1 was longer lasting than that of OT.

Multiple isoforms of porcine aromatase are encoded by three distinct genes.

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal enzyme in the estrogen biosynthetic pathway, is synthesized by the ovary, endometrium, placenta, and peri-implantation embryos in the pig and other mammals, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activity, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the present study examined if these aromatase isoforms represent products of multiple genes or of a single gene via complex splicing mechanisms. Porcine genomic DNA from a single animal was used as a template in the polymerase chain reaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragments revealed the presence of only clones corresponding to the three known aromatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) library for aromatase gene by PCR yielded a single clone approximately 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3, 4-9, and 10 sequences indicated that the BAC genomic clone contains the entirety of the coding exons as well as the proximal promoter region. Sequence analysis of the fragment generated with exon 4 primers determined that this BAC clone contains only the type II gene. The presence and relative orientation of the untranslated 5'- exons 1A and 1B, previously demonstrated for the type III isoform were evaluated in the BAC clone and genomic DNA by PCR. The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous to each other and separated by only three bp. A diagnostic procedure for typing aromatase isoforms was developed, based on the presence of specific restriction sites within isoform-specific exons. The use of this protocol confirmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometrium as a function of pregnancy stage. The presence of distinct genes encoding each of the aromatase isoform predicts important differences in the mechanisms underlying the molecular evolution and regulation of porcine aromatase, unique from those of other mammals, and suggests a critical role for P450 aromatase steroidal products in uterine functions related to pregnancy events.