The ruminant conceptus undergoes a period of elongation that is required for
maternal recognition of pregnancy, prior to attaching to the endometrium. The
purpose of these studies was to investigate the role of proline-rich 15
(PRR15) in the sheep conceptus by examining mRNA
expression, protein localization, and the effect of PRR15 mRNA
degradation. Conceptuses were collected on Days 11, 13, 15, 16, 17, 21, and 30
after mating. Quantitative RT-PCR showed expression of PRR15
mRNA corresponded with the process of trophoblast elongation, with peak
expression occurring on Days 15 and 16. A recombinant ovine PRR15 was generated
and used to create polyclonal antibodies against PRR15. Immunohistochemistry of
a Day 15 conceptus indicated that PRR15 was localized predominantly in the
nucleus of the trophectoderm and extraembryonic primitive endoderm. To test
whether PRR15 was required during early conceptus development,
RNA interference was employed. Blastocysts collected on Day 8 after mating were
infected with a lentivirus expressing a short-hairpin RNA (shRNA) that targeted
PRR15 mRNA for degradation, an shRNA containing a
three-nucleotide mismatch to PRR15 mRNA, or a lentivirus
expressing no shRNA. After infection, blastocysts were transferred into
recipient ewes and collected back on Day 15 of gestation. Although the majority
of the control and mismatched shRNA-treated conceptuses elongated and survived
to Day 15, none of the embryos treated with the lentivirus expressing shRNA
against PRR15 mRNA elongated, and most died. In conclusion,
expression of PRR15 mRNA occurred during a narrow window of
conceptus development, and degradation of PRR15 mRNA led to
conceptus demise or abnormal development