Ovine uterine artery hydrogen sulfide biosynthesis in vivo: effects of ovarian cycle and pregnancy†

Abstract

Uterine vasodilation dramatically increases during the follicular phase of the estrous cycle and pregnancy, which are estrogen-dominant physiological states. Uterine vasodilation is believed to be mainly controlled by local uterine artery (UA) production of vasodilators and angiogenic factors. The extremely potent vasodilator and proangiogenic hydrogen sulfide (H2S) is synthesized via metabolizing L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). This study was designed to determine if UA H2S production increases with augmented expression and/or activity of CBS and/or CTH during the ovarian cycle and pregnancy in sheep. Uterine arteries from intact nonpregnant (NP) luteal and follicular phase and late (130-135 days, term ≈ 145 days) pregnant (P) ewes were collected; endothelium-enriched proteins (UAendo) and endothelium-denuded smooth muscle (UAvsm) were mechanically prepared for accessing CBS and CTH proteins by immunoblotting; their cellular localization was determined by semi-quantitative immunofluorescence microscopy. H2S production was measured by the methylene blue assay. Immunoblotting revealed that CBS but not CTH protein was greater in P > > > NP follicular > luteal UAendo and UAvsm (P < 0.001). H2S production was greater in P > > > NP UAendo and UAvsm (P < 0.01). Pregnancy-augmented UAendo and UAvsm H2S production was inhibited by the specific CBS but not CTH inhibitor. CBS and CTH proteins were localized to both endothelium and smooth muscle; however, only CBS protein was significantly greater in P vs NP UA endothelium and smooth muscle. Thus, ovine UA H2S production is significantly augmented via selectively upregulating endothelium and smooth muscle CBS during the follicular phase and pregnancy in vivo.

Keywords: cystathionine β-synthase; hydrogen sulfide; in vivo; ovarian cycle; pregnancy; sheep; uterine artery.

Figure 1.

Uterine artery (UA) endothelial (endo) CBS/CTH expression in nonpregnant (luteal and follicular) and late pregnant ewes. CBS and CTH proteins in mechanically purified UA endothelium samples were determined by immunoblotting. Data (means ± SEM) are from 2–6 ewes/group. Bars with different letters differ significantly among the groups (P < 0.05).

Figure 2.

Uterine artery (UA) endothelial (endo) H₂S production in nonpregnant and late pregnant ewes. Uterine artery endothelium (UAendo) protein lysates from nonpregnant luteal or pregnant ewes were pooled and subjected to the methylene blue assay for measuring H₂S production in the presence or absence of the specific inhibitors of CBS (CHH), CTH (BCA), or their combination. Data (means ± SEM) are presented as fold of NP luteal without inhibitors and are pooled from 3–5 ewes per group. Bars with different letters differ significantly among the groups (P < 0.05). * P < 0.01.

Figure 3.

Uterine artery (UA) vascular smooth muscle (vsm) CBS/CTH expression in nonpregnant (luteal and follicular) and late pregnant ewes. CBS and CTH proteins were determined by immunoblotting. Data (means ± SEM) are from 3–6 ewes/group. Bars with different letters differ significantly among the groups (P < 0.05).

Figure 4.

Uterine artery (UA) vascular smooth muscle (vsm) H₂S production in nonpregnant and late pregnant ewes. Protein lysates from nonpregnant luteal or pregnant or UAvsm were pooled and subjected to the methylene blue assay for measuring H₂S production in the presence or absence of the specific inhibitors of CBS (CHH), CTH (BCA), or their combination. Data (means ± SEM) are presented as fold of NP luteal without inhibitors and are pooled from 3–5 ewes per group. Bars with different letters differ significantly among the groups (P < 0.05). * P < 0.05.

Figure 5.

Uterine artery (UA) CBS and CTH protein localization and expression in endothelium and smooth muscle of nonpregnant (NP) luteal and late pregnant ewes. (A) UA sections were labeled with primary antibodies against CBS or CTH, followed by secondary Alex⁴⁸⁸ (Green)-labeled secondary antibody. Endothelial cells were labeled with marker PECAM/CD31 followed by Alex⁵⁵⁵ (Red)-labeled secondary antibody and cell nuclei were stained with DAPI (blue); IgG control bottom right insert panel. Representative outlines of borders between UA intima (int) and media (SM) were indicated and UA lumen (lum) and tunic intima (int) were denoted in the 2^nd CD31/DAPI panel. (B) Fluorescence images were captured for analyzing relative green fluorescence intensity (RFI) to quantify CBS or CTH proteins. Data (means ± SEM) are from 3–4 different ewes/group. Bars with different letters differ significantly among the groups (P < 0.05). Scale bar = 25 μm.