The uterine expression of SEC63 gene is up-regulated at implantation sites in association with the decidualization during the early pregnancy in mice

Abstract

Background: Sec63 is a key component of the protein translocation machinery in the mammalian endoplasmic reticulum (ER), and involved in the post-translation processing of secretory proteins. The aim of this study was to determine the expression pattern of SEC63 gene in mouse uterus during the early pregnancy.

Methods: Real-time quantitative PCR and Western blot analyses were used to evaluate the alteration in levels of uterine SEC63 gene expression during the peri-implantation period in mice. Further, both in situ hybridization and immunohistochemical analyses were performed to examine the spatial localization of SEC63 gene expression in mouse uterine tissues. The presence of Sec63 protein in human uterine tissue was also detected by immunohistochemical analysis. Statistical analysis was carried out using Tukey test.

Results: Uterine SEC63 gene expression was up-regulated and predominantly localized in mouse decidual cells during days 5-8 of pregnancy. More interestingly, Sec63 protein was also detected in human decidua of 10-week pregnancy, whereas was not observed in human endometrial tissues both at proliferative and secretory phases of menstrual cycle.

Conclusion: The pattern of SEC63 gene expression is consistent with a possible role for SEC63 in decidualization.

Figure 1

The tissue distribution of Sec63 mRNA in the mouse. The tissue distribution of Sec63 mRNA in the mouse was analyzed by RT-PCR by using Gapdh mRNA as the control. Sec63 mRNA was detected in all of the tested tissues. (B: brain; Lu: lung; H: heart; Li: liver; Sp: spleen; K: kidney; M: muscle; SI: small intestine; Sk: skin; T: testis; E: epididymis; O: ovary; U: uterus.)

Figure 2

Uterine expression level of Sec63 mRNA during the early pregnancy. Real-time quantitative PCR analysis of the expression level of Sec63 mRNA in mouse uterine tissues of day 4 (4), day 5 (5) and day 7 (7) pregnancy (panel A), as well as the inter-implantation sites (nI), implantation sites (I) and embryo (Em) of day 7 pregnancy (panel B), by using the β-Actin as the housekeeping gene. In panel A and panel B, the data were normalized relative to that of day 4 and nI (reference groups) respectively.

Figure 3

Uterine expression level of SEC63 protein during the early pregnancy. Western blot analysis of SEC63 expressed in mouse uterine tissues during the early pregnancy using the goat anti-sera specific for both mouse and human Sec63p (1:50) as the primary antibody. Total protein (100 μg) was exacted from uteri of day 1 (D1) and day 4 (D4) pregnant mice, and from inter-implantation sites (i-I) and implantation sites (I) on day 5 (D5) and day 6 (D6). On day 7 (D7), embryos (e) were also separated from the implantation sites. Panel A shows the SEC63 signal detected by goat anti-sera specific for both mouse and human Sec63p. Panel B shows the equal of loading samples stained by the rabbit anti-β-actin antibody.

Figure 4

Determination of uterine Sec63 mRNA in the mouse by in situ hybridization analysis. In situ hybridization of Sec63 mRNA was performed using digoxigenin-labeled anti-sense probes generated against Sec63 cDNA in the mouse uterus on day 1(A) and day 4 (B) of pregnancy; at the implantation site of day 5(C), day 6 (D) and day 8 (E); at the inter-implantation site of day 6 (H); in the uterus of day 5 pseudopregnant mouse (G); delayed implantation (I) before and (J) after activation. (F) is the same as (E), but sense sequence was used for substitution as control. L: lumen of uterus; G: gland; SC: stromal cells; DC: decidual cells; EM: embryo. Scale bars represents 50 μm.

Figure 5

Examination of uterine SEC63 protein in the mouse by Immunohistochemical staining. SEC63p immunohistochemical staining of mouse uterine tissues was performed by using goat anti-sera specific for both mouse and human Sec63p as the primary antibody. The uterine sections were stained with Sec63 for day 1 (A) and day 4 (B) pregnant uterus; the implantation site of day 5 (C), day 6 (D) and day 8 (F); day 5 pseudopregnant uterus (G); and delayed implantation before (H) and 24 hr after activation (I). Panel E is the negative control for panel B, in which normal goat serum is substituted for the primary antibody. L: lumen of uterus; G: gland; SC: stromal cells; DC: decidual cells; EM: embryo. Scale bars represents 10 μm.

Figure 6

The present of SEC63 in human tissues detected by IHC analysis. Immunohistochemical staining for SEC63 in human decidua of 10-week pregnancy (A), human oviduct of 10-week ectopic pregnancy (C), human endometrium at proliferative phase (D) and human endometrium at secretory phase (E) by using goat anti-sera specific for both mouse and human Sec63p as the primary antibody. Panel B and F are the negative controls respectively for panel A and C, in which normal goat serum is substituted for the primary antibody. Scale bars represents 50 μm.