Expression of SUMO associated proteins in the mouse endometrium is regulated by ovarian hormones throughout the estrous cycle

Abstract

The modification of proteins by small ubiquitin-like modifier (SUMO), known as SUMOylation, regulates biological function by changing protein transcription and translation. During the estrous cycle the endometrium undergoes continual change to processes including cell proliferation, secretion and exfoliation and these changes are regulated by the levels of ovarian hormones. Increasing the expression of SUMO family members has previously been shown to promote proliferation and invasion of endometrial cancer cells. However, limited research has been carried out into the expression and function of SUMO in the mammalian endometrium. In the present study, the level and localization of SUMO-associated proteins throughout the natural estrous cycle in the mouse uterus was determined using immunohistochemical staining and western blot analysis. The association between the spatiotemporal expression of these SUMO modified proteins and SENPs in endometrium and the concentration of ovarian hormones during estrous cycle was revealed. The present study clarified the role of SUMOylation in maintenance of normal estrous cycling and may have important significance in the study of hormone-dependent endometrial diseases.

Keywords: endometrium; estrous cycle; sentrin specific protease; small ubiquitin-like modifier; small ubiquitin-like modifier-conjugating enzyme UBC9.

Figure 1.

PAP staining of vaginal exfoliated cells and serum hormone levels during different stages of the mouse estrous cycle. (A) Images of Pap-stained vaginal exfoliated cells: Proestrus-anterior keratinocyte cells (n=7); estrus-non-nucleated keratinized squamous cells (n=3); metestrus I-reduced non-nucleated keratinized squamous cells and appearance of leucocytes (n=6); metestrus II-predominance of leucocytes (n=4); diestrus-predominantly nuclear epithelial cells, occasional leucocytes and keratinocytes (n=15). (B) Serum FSH, LH, E2 and Pr levels in female mice at different stages of the estrous cycle. E2, estradiol; FSH, follicle stimulating hormone; LH, luteinizing hormone; Pr, progesterone; ★, anterior keratinocyte cell; ←, non-nucleated keratinized squamous cell; #, nuclear epithelial cell; *, leucocytes; P, proestrus; E, estrus; MI, metestrus I; MII, metestrus II; D, diestrus.

Figure 2.

Hematoxylin & eosin staining of mouse endometrial sections at different stages of the estrous cycle. SCEp, simple columnar epithelium; LPM, lamina propria mucosa; UG, uterine gland.

Figure 3.

Immunohistochemical staining showing mouse uterine expression of SUMO-related proteins at different stages of the estrous cycle.

Figure 4.

Western blot analysis of SUMO-related proteins in the mouse uterus at different stages of the estrous cycle. (A) Representative western blot of conjugated and free SUMO1 at different stages of the estrous cycle. (B) Normalization of SUMO1 conjugate expression to GAPDH. (C) Representative western blot of SUMO2/3 conjugates at different stages of the estrous cycle. (D) Normalization of SUMO2/3 conjugate expression to GAPDH. (E) Representative western blots of uterine expression of UBC9 and SENP1, 2, 3 and 5 at different stages of the estrous cycle. GAPDH was used for normalization of the levels of (F) SENP1 and SENP2, (G) SENP3 and SENP5 and (H) UBC9. Data are presented as the mean ± standard error (n=3). *P<0.05, **P<0.01, ***P<0.05 vs. proestrus. SUMO, small ubiquitin-like modifier; SENP, sentrin specific protease; UBC9, small ubiquitin-like modifier-conjugating enzyme UBC9.