Porcine intestinal lymphoid tissues synthesize estradiol

Abstract

Estradiol (17β-estradiol) is synthesized primarily in the gonads of both sexes and regulates the development and function of reproductive organs. Recently, we reported that intestinal lymphocyte homeostasis is regulated by estradiol synthesized de novo in the endothelial cells of the high endothelial venules (HEVs) of mesenteric lymph nodes and Peyer's patches in mice. This observation prompted us to hypothesize that HEVs of intestinal lymphoid tissues are sites of estradiol synthesis across species. In this study, we examined whether estradiol is synthesized in the intestinal lymphoid tissues of adolescent piglets. Comparisons of estradiol levels in blood and tissue showed that estradiol concentrations in mesenteric lymph nodes and Peyer's patches were significantly higher than the level in serum. Reverse transcription polymerase chain reaction showed that porcine intestinal lymphoid tissues express mRNAs for steroidogenic enzymes (StAR, 17β-Hsd,3β-Hsd, Cyp17a1, and Cyp19a1), and immunohistochemical results in ilial tissue showed expression of aromatase (CYP19) in Peyer's patch-localized endothelial cells of HEVs. When mesenteric lymph node and Peyer's patch tissues were cultured in vitro, they produced estradiol. Taken together, the results indicate that mesenteric lymph nodes and Peyer's patches are sites of estradiol synthesis in adolescent piglets.

Keywords: aromatase; estradiol; intestines; lymph nodes; swine.

Fig. 1. Histological organization of Peyer's patch (Pp) and mesenteric lymph node (mLN) tissue, estradiol synthesis in dissected ileal Pp, and tissue estradiol concentrations. Tissues were dissected from a 3-week-old piglet. (A) Anatomical and histological view of representative Pp in luminal surface and of ileal tissues without Pp. Pp is indicated by arrows. Lu, lumen; Vi, villi; Mu, muscularis externa. (B) Histological view of mesenteric mLN with lymphoid follicle (LF) surrounded by the dotted line. (C) Concentrations of estradiol in serum, mLN, and ovary (n = 4, respectively). (D) Changes in estradiol concentration in ileal tissues with and without Pp. Tissues were dissected from 3-week old piglets (n = 4), cultured in vitro, and the amount of estradiol released from the tissues was measured until 72 h at 24 h intervals. Scale bars = 3 mm (A), 0.5 mm (insets in A), 0.3 mm (B). Significantly different from the ileum at ^*p < 0.01 or ^**p < 0.005, respectively, obtained via one-way ANOVA and Tukey's post hoc test.

Fig. 2. Estradiol synthesis in dissected ileal Peyer's patch (Pp), mesenteric lymph node (mLN), and ovary tissues showing changes in estradiol concentrations. Tissues were dissected from 12-week-old piglets and cultured in vitro (n = 4) in the absence (Veh) or presence of androstenedione (AN; 200 nM). The amount of estradiol released from the tissues was measured until 72 h at 24 h intervals.

Fig. 3. Expression of steroidogenic enzyme genes in porcine Peyer's patch (Pp) and mesenteric lymph node (mLN) tissues in a 12-week-old pig. (A) Key enzymes responsible for precursor synthesis (StAR, 3β-HSD, and CYP17) and the rate-limiting estradiol-synthetic enzymes (17β-HSD and CYP19) in the steroidogenic pathway. (B) Quantitation of the relative mRNA expression and representative agarose gel images of StAR, 3β-Hsd, Cyp17a1, 17β-Hsd, and Cyp19a1 in ovary, ileal Pp, and mLN tissues of 12-week-old female piglets. Rpl19 was used for internal control. Significantly different from ovary at ^*p < 0.01 or ^**p < 0.005, respectively, based on one-way ANOVA and Tukey's post hoc test results (n = 4). Note that 17β-Hsd expression is higher in Pp and mLN than in ovary, whereas Cyp19a1 expression is lower in these lymphoid tissues than in the ovary.

Fig. 4. Aromatase (CYP19) expression in high endothelial venule (HEV) cells of Peyer's patch (Pp) tissue. Adjacent sections of a Pp of a 12-week-old female piglet's ileum containing Pp were stained with antibodies against PNAd (a HEV marker) or CYP19. Lower panels show enlarged views of boxed areas in the upper panels. HEVs are indicated by asterisks. Arrows indicate CYP19-expressing HEV cells. Scale bars = 100 µm (upper), 50 µm (lower).