Localization and subcellular distribution of prolyl oligopeptidase in the mouse placenta

Abstract

Prolyl oligopeptidase (POP) is a serine endopeptidase which selectively digests a -Pro-X-peptide bond.Our previous study showed that POP mRNA was strongly expressed in the spongiotrophoblast of the mouse placenta at E17.5, suggesting its importance in development. To gain more insight into POP's role during gestation, we investigated its expression using different developmental stages of placenta. As a result of in situ hybridization, we found that localization of POP mRNA changed at E12.5. POP mRNA was strongly expressed in the spongiotrophoblast and labyrinth at E10.5 and E11.5 but thereafter only in the spongiotrophoblast. Immunohistochemistry revealed that POP was present in the parietal trophoblast giant cell, the spongiotrophoblast cell, and the labyrinth at E11.5 but the strong expression in the labyrinth was maintained only in the canal-associated and sinusoidal trophoblast giant cells at E16.5 and E18.5. To determine subcellular distribution of the POP protein, we fractionated the placental extract into cytoplasmic, membrane, and nuclear subfractions. By Western blot analysis, POP was detected in the cytoplasmic and membrane fractions but not in the nuclear fraction at E11.5 and E16.5. Interestingly, the cytoplasmic POP exhibited higher enzymatic activity than the membrane-associated type. These data suggest that the cytoplasmic and membrane-associated POP have distinct roles in different types of placental cells.

A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

“…These expression patterns of EGFP mRNA were different from that of the endogenous POP mRNA which was expressed at higher levels in the ovary and placenta (Fig. 1, Matsubara et al, 2010Matsubara et al, , 2011. This indicated that the 914-bp promoter was insufficient to recapitulate the tissue distribution of POP mRNA.…”

“…Moreover, we have shown that the mouse POP mRNA was localized to granulosa cells in the ovary (Matsubara et al, 2010) and to spermatids in the testis (Kimura et al, 2002). In the placenta, POP mRNA changed its localization at embryonic day 12.5 (E12.5) from the junctional and labyrinthine zones to the junctional zone alone (Matsubara et al, 2011). These indicate that the POP gene transcription is highly regulated in a tissue-specific manner.…”

“…Placentas were isolated from wild type and transgenic pregnant mice as previously described (Matsubara et al, 2010(Matsubara et al, , 2011. We washed the tissues with phosphate buffered saline, and the EGFP fluorescence was analyzed using a Leica MZ FLIII Fluorescence Stereo Microscope (Leica Microsystems, Tokyo, Japan) equipped with a cooled charged-coupled device camera (KEYENCE VB-7010, KEYENCE, Osaka, Japan).…”

“…For example, POP mRNA was detected at a high level in the porcine heart and muscle (Rennex et al, 1991), the human skeletal muscle (Goossens et al, 1997;Shirasawa et al, 1994), the rat brain and thymus (Kimura and Takahashi, 2000), and the mouse ovary and placenta (Matsubara et al, 2010(Matsubara et al, , 2011), compared to other tissues. Moreover, we have shown that the mouse POP mRNA was localized to granulosa cells in the ovary (Matsubara et al, 2010) and to spermatids in the testis (Kimura et al, 2002).…”

A testis-specific serine protease, Prss41/Tessp-1, is necessary for the progression of meiosis during murine in vitro spermatogenesis

“…The germ cells were further fractionated into nuclear, membrane, and cytoplasmic subfractions as previously described [8].…”