Regulation of placental development

Abstract

Bovine peri-attachment factor (bPF) mRNA is a novel transcript originally detected in early conceptus and adult kidney. Two known homologs of bPF are murine protein G90, which has been detected in embryonic and adult mouse brain, adult mouse intestine, kidney and testis and a human hypothetical protein detected in placenta. Our first objective was to screen genomic DNA from several species by Southern hybridization for PF homologs. Our second objective was to examine cow, sheep, horse, rat, mouse and porcine conceptus and adult tissues for expression of PF/G90 mRNA. Finally, our third objective was to localize PF within the cell following expression in mammalian cell lines. Southern hybridization revealed genomic PF homologs in the human, dog, cow, pig, rabbit, yeast, and horse. Northern hybridization and/or RT-PCR analysis detected PF in bovine (day 14,17, 21 and 28), ovine (day 13 and 15), porcine (day 10 and 12) and equine (day 14, 16, 20 and 30) conceptus tissue, coinciding with the stage of conceptus development when bPF was previously detected. Analysis of adult sheep tissues detected PF in kidney and lung. Interestingly, no PF could be detected in rat or mouse adult tissues. Screening of day 135 fetal ovine fetal tissues revealed detectable PF mRNA in the lung but not in the heart. Western analysis of a PF fusion protein detected PF in both cytoplasmic and nuclear fractions of stably transfected CHO and Cos-7 cells. The Southern hybridization data indicates a conserved gene for PF exists across several species of different phylogenetic orders. Results of bovine conceptus tissue expands the previous temporal expression window of PF mRNA to day 28, and results with ovine and equine conceptus tissues are similar to those reported for cattle; additionally ovine PF was detected in adult and fetal lung. The predicted nuclear targeting sequence of PF is apparently functional as evidenced by the immunoblot analysis. Given the detection of PF fusion protein in the nucleus and absence of DNA and RNA binding domains, we hypothesize that PF acts as a co-activator or co-repressor to modify transcription within the nucleus. While several proteins have been implicated in transcriptional regulation of PL in in vitro studies, little is known about the transcriptional regulation of oPL in vivo. The purpose of this research was to determine the promoter sequence necessary to give cell specific expression of oPL in BNCs. Ovine PL promoter deletion constructs were made to drive EGFP expression, and stably transfected into ovine fetal fibroblasts for nuclear cloning and embryo transfer. A 47 dGA cell line was chosen for nuclear cloning for advantages in cell culture stability before nuclear transfer and for appropriate growth, quality, and number of blastocysts derived from this cell line. Transgenic tissues were evaluated under fluorescence microscopy, where it was discovered that auto-fluorescence within placental tissue was going to make specific EGFP expression difficult to determine. Unsuccessful attempts were made to detect EGFP by immunohistochemistry and in situ hybridization; however, a positive control could be detected. The exact reason for the inability to detect EGFP and the erratic detection of the neomycin positive control is unknown. Our fixation and immunohistochemistry and in situ hybridization techniques have been used successfully in previous research; therefore, we believe there was most likely a problem with the tissue. With the lack of success in analyzing oPL transcriptional regulation by using transgenic nuclear cloning, we focused on culture of placental cells in vitro and subsequent infection of the cells with adenovirus and lentivirus that carried CMV promoter driven GFP cassettes. Infection of multiple cell types including BNCs was successful with both adenovirus and lentivirus. Ovine growth hormone was previously reported to be expressed in placental tissue for a brief (20 days) period during maximal placental growth and development. Our objectives here were to define the cellular source of oGH in the placenta, and study the impact of exogenous GH during the normal window (35-55 dGA) of placental expression. The results of in situ hybridization indicate that oGH is expressed by uterine epithelium and no tissues of fetal origin. For GH treated ewes, serum GH and IGF-I concentrations were increased approximately 10-fold (P < 0.001) by day 5 of treatment and the increase was maintained throughout the treatment period. Serum progesterone concentrations were unaffected by treatment. Uterine, uterine fluid, placental and fetal weights were unaffected by treatment for both day 55 and 135 groups. Fetal length, liver weight, and liver weight per kg of body weight were not changed by maternal GH treatment. Maternal GH administration did not significantly alter GH (caruncle), or oPL (cotyledon) mRNA concentration as detected by Northern hybridization. In the cotyledon, IGF BP-1 and BP-4 were significantly (P < 0.05) increased, while IGF BP-2 was significantly decreased. The expression of IGF BP-3 was unaffected by treatment. Within the caruncle, IGF BP-1 was decreased, while IGF BP-3 and IGF BP-4 were increased, and IGF BP-2 was unchanged due to GH treatment. In contrast to pigs, these data indicate that maternal serum concentrations of GH and IGF-I have no significant effect on placental and fetal growth. Although we did not impact placental or fetal growth, exogenous GH administration does impact the abundance of mRNA encoding IGF binding proteins within the placenta. Increase in caruncular IGF BP-3 is likely a response to increased maternal serum IGF-I. The increase in IGF BP-4 may also be due to increased IGF-I as IGF BP-4 is a negative regulator of IGF-I action. The decrease in IGF BP-1 is possibly the result of increased maternal insulin in response to GH treatment as insulin decreases IGF BP-1 transcription. While we cannot explain the changes in transcription within the cotyledon, they are important in trying to explain the role of the IGF system at the maternal-fetal interface in growth and differentiation.