Implications of the RhoA/Rho associated kinase pathway and leptin in primary uterine inertia in the dog

Abstract

The underlying functional and molecular changes in canine primary uterine inertia (PUI) are still not clarified. Leptin (Lep) and obesity negatively affect uterine contractility in women, partly mediated by the RhoA/Rho associated kinase pathway, affecting myometrial calcium sensitization. We hypothesized that increased uterine Lep/Lep receptor (LepR) or decreased RhoA/Rho associated kinase expression contributes to PUI in dogs, independent of obesity. Dogs presented for dystocia were grouped into PUI (n = 11) or obstructive dystocia (OD, still showing strong labor contractions; n = 7). Interplacental full-thickness uterine biopsies were collected during Cesarean section for relative gene expression (RGE) of RhoA, its effector kinases (ROCK1, ROCK2), Lep and LepR by qPCR. Protein and/or mRNA expression and localization was evaluated by immunohistochemistry and in situ hybridization. RGE was compared between groups by one-way ANOVA using body weight as covariate with statistical significance at P < 0.05. Uterine ROCK1 and ROCK2 gene expression was significantly higher in PUI than OD, while RhoA and Lep did not differ. LepR RGE was below the detection limit in five PUI and all OD dogs. Litter size had no influence. Lep, LepR, RhoA, ROCK1, ROCK2 protein and/or mRNA were localized in the myometrium and endometrium. Uterine protein expression appeared similar between groups. LepR mRNA signals appeared stronger in PUI than OD. In conclusion, lasting, strong labor contractions in OD likely resulted in downregulation of uterine ROCK1 and ROCK2, contrasting the higher expression in PUI dogs with insufficient contractions. The Lep-LepR system may affect uterine contractility in non-obese PUI dogs in a paracrine-autocrine manner.

Keywords: Canine; Contractility; Dystocia; Parturition; Uterus.

Fig. 1.

Comparison of relative gene expression (RGE) of A) RhoA, B) ROCK1, C) ROCK2, and D) Leptin in inter-placental uterine tissue samples of dogs diagnosed with primary uterine inertia (PUI) or obstructive dystocia (OD). Bars represent the observed (RhoA, ROCK1, ROCK2) or geometric mean (Leptin), and whiskers the standard deviation or the deviation factor, respectively. Different superscripts denote significant difference between groups at P < 0.05.

Fig. 2.

Comparison of relative gene expression (RGE) of A) RhoA, B) ROCK1, C) ROCK2, and D) Leptin in inter-placental uterine tissue samples of dogs diagnosed with primary uterine inertia (PUI) carrying small (PUI-S), average (PUI-N) or large (PUI-L) litters relative to the breed average litter size. Bars represent the observed (RhoA, ROCK2) or geometric (ROCK1, Leptin) mean, and whiskers the standard deviation or the deviation factor, respectively. There was no significant difference in gene expression among groups.

Fig. 3.

Correlation of inter-placental relative gene expression (RGE) of ROCK1 and ROCK2 in the whole study population (n = 18; r = 0.835, P < 0.0001).

Fig. 4.

Immunohistochemical detection of RhoA, ROCK1, and ROCK2 in uterine interplacental tissue in the PUI and OD groups. For RhoA (A–C), strong signals are visible in the smooth muscle cells of the myometrium (A; representative image from the PUI group). Both the circular (solid arrowheads) and longitudinal (open arrowheads) layer of the myometrium stained (B; representative image from the OD group). In the endometrium (C), the surface epithelium (thin black arrows), superficial (open arrow) and deep (solid arrows) uterine glands and stromal immune cells (small white arrowheads) showed positive immunoreactivity. Blood vessels (asterisks) also stained (B, C). No signals are visible in the isotype control for RhoA (lower inset in B). For ROCK1 (D–F), positive staining is seen in myocytes (D; representative image from the PUI group) of both the circular (solid arrowheads) and longitudinal (open arrowheads) uterine muscle layers (E; representative image from the OD group). The luminal epithelium (thin black arrows), superficial (not shown) and deep (solid arrows) uterine glands, and stromal immune cells (small white arrowheads) stained positive for ROCK1 (F). Blood vessels (asterisks) also stained (D–F). The isotype control is devoid of signals (lower inset in E). ROCK2 (G–I) signals are visible in smooth muscle cells (G; representative image from the OD group) of both myometrial layers, i.e., circular (solid arrowheads) and longitudinal (open arrowheads) (H; representative image from the PUI group), and strong immunostaining is also present in blood vessels (asterisks) (H, I). In the endometrium (I), no or occasional weak ROCK2 signals are present in the luminal epithelium (thin black arrows) and deep uterine glands (solid arrows). There is no staining in the isotype control (lower inset in H).

Fig. 5.

Localization of RhoA, ROCK1 and ROCK2 mRNA in uterine interplacental tissue by in situ hybridization. For RhoA, positive signals are visible in myocytes (A) of the circular (solid arrowheads) and longitudinal (open arrowheads) myometrial layers (B, C). The same expression pattern is visible for ROCK1 (D–F) and ROCK2 (G, H). In the endometrium, positive signals are seen in the luminal epithelium (thin black arrows), superficial (open arrows) and deep (solid arrows) uterine glands for RhoA (C), ROCK1 (F) and ROCK2 (I). There was variable positive staining in blood vessels (asterisks) for RhoA and ROCKs. Negative controls for RhoA, ROCK1 and ROCK2 have no signals (lower insets in B, E, H, respectively).

Fig. 6.

Immunohistochemical detection of Lep, and LepR mRNA detection by in situ hybridization in uterine interplacental tissues in the PUI and OD groups. Positive immunostaining for Lep (A–C) is visible in the myocytes (A; representative image from the PUI group) of the circular (solid arrowheads) and longitudinal (open arrowheads) myometrial layers (B; representative image from the OD group). Weak positive signals for Lep are visible in the uterine luminal epithelium (thin black arrow), and in the superficial (open arrow) and deep glands (solid arrows) of the endometrium; stromal immune cells (small white arrowheads) also stained (C). Blood vessels showed positive immunosignals for Lep (asterisks). The isotype control is devoid of signals (lower inset in B). LepR mRNA (D–F) was localized in myocytes of the circular (solid arrowheads) and longitudinal (open arrowheads) myometrial layers. LepR mRNA signals were also found in the luminal (not shown) and glandular epithelial cells (deep glands shown with solid arrows in F). No signals are seen in the negative control (lower inset in F).