Embryo implantation is closely associated with dynamic expression of proprotein convertase 5/6 in the rabbit uterus

Abstract

Background: Proprotein convertase 5/6 (PC5/6) is critical for embryo implantation in women, regulating both uterine epithelial receptivity and stromal cell decidualization. PC5/6 is likewise essential for implantation in mice, but involved only in decidualization. An alternative animal model is required to address the function of PC5/6 in the uterine epithelium. This study aimed to establish whether PC5/6 is associated with embryo implantation in rabbits.

Methods: Virgin New-Zealand white rabbits aged 3-4 moths were mated with males of the same strain, or pseudo-pregnancy induced. After mating, uterine tissues were collected over a 10 day (d) period (n = 3 per time point) for RNA, protein and histological analyses to determine the temporal and spatial uterine expression pattern of PC5/6 during the initial stages of pregnancy or induced pseudo-pregnancy.

Results: PC5/6 mRNA was up-regulated just prior to embryo attachment on d6, and the elevated expression was maintained throughout implantation on d6.5-10. Western analysis revealed a preferential up-regulation of PC5/6 in the implantation sites. Immunohistochemical analysis identified that both the amount and cellular localization of PC5/6 changed with increasing pregnancy stages. Before embryo attachment, PC5/6 was low and localised in the luminal and glandular epithelium. It increased on d6.5 in the basal glands and mucosal folds, and then strongly intensified on d7-10 in the multinucleated luminal symplasma and decidual cells at the site of embryo implantation. In contrast, the pseudo-pregnant uterus displayed relatively low and static PC6 mRNA expression throughout the 10 days, with no obvious changes in either PC5/6 level or cellular localization.

Conclusions: These findings demonstrate that embryo implantation in the rabbit is closely associated with dynamic expression of uterine PC5/6, and that the rabbit may be an appropriate model to investigate the function of PC5/6 in the uterine epithelium during embryo attachment.

Figure 1

RT-PCR confirmation of PC5/6 mRNA expression in the rabbit uterus. (A) Agarose gel electrophoresis of RT-PCR products of PC5/6 (220 bp) and 18S (187 bp) respectively. Two negative controls are: -RT, no reverse transcription enzyme in RT reaction; -RNA, no RNA in RT reaction. +RT+RNA, RT reaction contained RNA and reverse transcription enzyme. M, DNA marker. (B) Comparison between sequences of rabbit PC5/6 mRNA (Rabbit-PC5/6) and RT-PCR amplified uterine PC5/6 product (PCR-PC5/6).

Figure 2

Real-time RT-PCR analysis of PC5/6 mRNA expression in the rabbit uterus between d0 and d10 in pregnant and pseudo-pregnant animals. For pregnant uterus from d6.5 onwards, implantation and inter-implantation sites were collected separately. Data are mean + SEM, n = 3 animals, * P < 0.001 (inter-implantation site vs implantation site and pseudo-pregnant uterus on the same day).

Figure 3

Western blot analysis of PC5/6 protein in the pregnant rabbit uterus. Total protein extracts from inter-implantation (Inter) and implantation (Imp) sites on pregnant d7, 8 and 10 were analysed for PC5/6 (upper panel) and β-actin (lower panel) respectively.

Figure 4

Immunolocalization of PC5/6 protein in the rabbit uterus during pregnancy d0-6.5. Representative microphotographs are shown for pregnant d0-1 (A, B and C), d5 (D, E and F) and d6.5 (G, H and I). For each time point, panels depict an overview (A, D and G), endometrial mucosal folds (B, E and H) and basal glands (D, F, I). Arrowhead in G demonstrates increased basal glandular staining at the time of embryo attachment. Insets in A, D and G are negative controls. Scale bar = 25 μm.

Figure 5

Immunolocalization of PC5/6 protein in the rabbit uterus on pregnant d7-10 and pseudo-pregnant d8. Representative microphotographs are shown for pregnant uterus on d7 (A, B and C), d8 (D, E and F) and d10 (G, H and I), and pseudo-pregnant uterus on d8 (J, K and L). For pregnant uterus, panels depict an overview (A, D, G), attachment region (B, E and H) and non-attachment region (D, F and I) for each time point. Arrows in A, D and G highlight the embryonic tissues. Arrowheads in H show decidual cells; the insert shows a higher magnification. For pseudo-pregnant uterus, an overview (J), mucosal folds (K) and deep basal glands (I) are shown. Inserts in A, D, G and J are negative controls. Scale bar = 25 μm.