Minimal essential medium alpha (α-MEM) was purchased from Invitrogen (USA), fetal bovine serum (FBS) from American Type Culture Collection (ATCC; USA), insulin-transferrin-selenium (ITS) from VWR (USA), and androstenedione from Sigma (USA). The sensitive estradiol enzyme-linked immunosorbent assay (ELISA) kit was purchased from DRG International (USA).

This study was performed in accordance with recommendations in Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal handling was done in accordance with the protocols approved by the University of Illinois at Urbana-Champaign's Institutional Animal Care and Use Committee (protocol No. 16036). Seven naturally farrowed domestic female Yorkshire piglets aged 3 weeks (n = 3) and 12 weeks (n = 4) were obtained from the University of Illinois swine herd and used in this study.

To measure estradiol-synthetic capacity, Pp, mLN, and ovary tissues were dissected from individual piglets euthanized by intracardiac administration of sodium pentobarbital (78 mg/kg body weight, Fatal Plus; Vortech Pharmaceuticals, USA), weighed, and cultured overnight in steroid-free media (α-MEM supplemented with 1% ITS [10 ng/mL insulin, 5.5 ng/mL transferrin, 5.5 ng/mL selenium], 5% FBS, and penicillin-streptomycin). Ovary tissue was then washed with fresh media to remove any steroid that might have been released to the culture media. Tissues were further cultured in the absence (vehicle) or presence of androstenedione (200 nM) [24]. Media were collected at 24, 48, and 72 h to measure estradiol content.

Estradiol concentrations were measured in blood collected via cardiac puncture, as well as in culture media and tissues (mLNs and gonads). Culture media estradiol content was measured directly, whereas, for blood and tissue samples, total lipids were extracted, following a standard procedure [25], and stored at −80℃ until needed for hormone assay. The concentrations of estradiol in the extracted lipids were measured by using ELISA (EIA-4399; DRG International, Germany) as previously reported [24]. Final estradiol concentrations were expressed by weight per unit volume or by weight of tissue, sera, or culture media (pg/mL or pg/mg). The samples were run in triplicate and had intra- and inter-assay coefficients of variability below 10%. The detection range was 0.1 to 30 pg/mL.

For gene expression analysis, mLNs, Pps, and ovaries were collected. Total RNA was extracted, by using TrizolVR solution (Ambion, USA), from porcine tissues (Pps, mLNs, and gonads) and then purified with an RNEasy Kit (Qiagen, USA). Concentration and quality of total RNA were analyzed by using a Nanodrop spectrophotometer (Thermo Scientific, USA) and the samples were then stored at −80℃ until use. Complementary DNA was generated by using M-MLV Reverse Transcriptase (Thermo Scientific) and random primers. The following primers were used for reverse transcription polymerase chain reaction (RT-PCR): 17β-Hsd 5′-AGC CAG AAT ATG TGG CAC CC-3′/5′-CAA CAA GTC CTG ATG GGG CT-3′, Cyp19a1 5′-GGA AAT CCA GAC TGT TGT TG-3′/5′-GCT GGA AGT ACC TGT AAG GA-3′, StAR 5′-GAC TTT GTC GGC TGT-3′/5′-ATC CCT TGA GGT CAA TGC TC-3′, 3β-Hsd 5′-CGT CCT GAC ACA CAA CTC CAA-3′/5′-CCA CGT TGC CGA CGT AGA-3′, CYP17a1 5′-TCC GAG AGG TGC TTC GAT TC-3′/5′-GGC GCT CCT TGA TCT TCA CT-3′, Rpl19 5′-CCT GAA GGT CAA AGG GAA TGT G-3′/5′-GTC TGC CTT CAG CTT GTG GAT-3′. PCR products were run on 2% agarose gels containing ethidium bromide and photographed under UV light. The RT-PCR products were quantified by determining band intensities measured with 1Dscan EX (Scanalytics, USA); Rpl19 was used as the internal control.

Collected tissues were fixed in 4% paraformaldehyde, embedded in paraffin wax, sectioned at 6 µm thickness, and stained with hematoxylin and eosin. Immunofluorescent labeling of CYP19 and PNAd were performed as previously described [24] by using monoclonal anti-CYP19 (MCA2077S; Bio-Rad Laboratories, USA) or purified rat anti-mouse PNAd carbohydrate epitope (MECA-79; BD Biosciences, USA). After primary antibody incubation overnight, slides were incubated with a secondary biotinylated horse anti-mouse IgG (Vectastain ABC kit; Vector Labs, USA) or rabbit anti-rat IgG (Vectastain ABC kit) and then treated with avidin-biotin complex solution (Vectastain Elite ABC kit; Vector Labs). For color development, 3,′-diaminobenzidine (DAB; Vector Labs) was applied. Slides were then counter-stained with hematoxylin, mounted, and imaged by using an Olympus BX51 microscope (Olympus, Japan).

Datasets were first tested for normality and homogeneity of variance and were transformed before statistical analysis. All figures depict non-transformed data. Statistical analyses were performed by applying two-tailed Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's honest significant difference.

In porcine species, the ileum contains multiple Pps. At three weeks of age, Pps are visibly identifiable by their unique luminal form, which appears as whitish round segments. These segments are follicles that contain densely populated lymphocytes (panel A in Fig. 1; insets). In pigs at this age, mLNs are easily identifiable as they are morphologically distinctive, but the follicles in this organ are less clearly defined than those in Pps (panel B in Fig. 1). The estradiol concentrations of serum, mLN, and ovary were 0.27 pg/mg, 0.66 pg/mg, and 1.84 pg/mg, respectively (panel C in Fig. 1). To see if lymphoid tissues synthesize estradiol, Pps-containing and Pp-free ileal tissue samples were cultured for 72 h, and the estradiol concentrations in the culture media were measured. During the culture period, estradiol concentrations continued to rise in the culture media containing Pps, but not in the media without Pps (panel D in Fig. 1). When the estradiol-synthetic capacity of the Pps was compared with those of mLN and ovary in 12-week-old pigs, the mLN and Pp samples exhibited a robust estradiol-synthetic capacity (Fig. 2). Notably, the estradiol-synthetic capacity of mLN (12.36 pg/mg at 72 h) was comparable to that of ovary (12.45 pg/mg at 72 h). Addition of androstenedione to the tissue cultures increased estradiol production slightly, but not significantly, in all examined tissues.

De novo estradiol synthesis in tissue requires the expression of StAR, 3β-Hsd, Cyp17a1, Cyp19a1, and 17β-Hsd genes (panel A in Fig. 3). To determine whether mLNs and Pps synthesize estradiol de novo, mRNA expression levels of steroidogenic genes were examined by performing RT-PCR (panel B in Fig. 3). Both Pps and mLNs expressed mRNAs for all of the steroidogenic genes examined, with minor differences in expression levels. Specifically, 3β-Hsd mRNA expression in both Pps and mLNs were significantly lower than the ovarian expression level (p < 0.01). The expression levels of 17β-Hsd in Pps and mLNs were 2-fold higher than that in ovary (p < 0.01). Pps and mLNs showed lower Cyp19a1 mRNA expression than that in ovary (panel B in Fig. 3). Importantly, immunohistochemical results showed that ileal tissue containing Pps of 12-week-old piglets expresses aromatase protein (CYP19) in endothelial cells of high endothelial venules (HEVs) (Fig. 4).