Testosterone upregulates anion secretion across porcine vas deferens epithelia in vitro

Abstract

Testosterone induces and maintains prostaglandin endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase 2) expression in vas deferens epithelial cells, but it remains unknown whether this has a physiological role in the context of male reproductive biology. Prostaglandins induce concentration-dependent increases in anion secretion in porcine vas deferens epithelial cell (1 degrees PVD) monolayers, where bicarbonate contributes to cAMP-stimulated anion secretion. Moreover, bradykinin induces anion secretion across 1 degrees PVD monolayers that is indomethacin sensitive, and both PTGS2 and PTGS1 are expressed in this model system. Therefore, it was hypothesized that testosterone modulates anion secretion across vas deferens epithelia via PTGS-dependent pathways and prostaglandin synthesis. Porcine vas deferens epithelial cells were isolated and cultured as monolayers on permeable supports until assayed in modified Ussing chambers. RNA and protein were isolated concurrently for semiquantitative expression analysis. Testosterone upregulated basal and bradykinin-induced short-circuit current across 1 degrees PVD monolayers, indicative of anion secretion. Testosterone also induced greater transepithelial electrical resistance. Increases in anion secretion were associated with preferential upregulation of PTGS2 at the mRNA and protein levels. In addition, testosterone induced greater basal and bradykinin-induced anion secretion across vas deferens epithelial cells isolated from the distal segment of the duct. Taken together, these results suggest that testosterone upregulates epithelial responsiveness to acute modulations of anion secretion (likely bicarbonate secretion), which ultimately modifies the environment to which sperm are exposed.

FIG. 1.

Testosterone cypionate upregulates basal I_SC and basal R_TE in primary cultures of 1°PVDs. Basal I_SC (A) and basal R_TE (B) are greater in monolayers exposed to testosterone (100 μM). n = 17 in each condition. *Indicates significant difference from basal medium (P < 0.05).

FIG. 2.

Testosterone upregulates BK-induced anion secretion across 1°PVDs. A) Typical 1°PVD responses to LBK (1 nM) derived from paired monolayers cultured in the absence or presence of TC supplementation (100 μM). Summarized observations (n = 16–17) reveal that TC supplementation upregulates LBK responses as measured by ΔI_SC-MAX (B), net ion flux over 900 sec (C), and ΔR_TE-MAX (D). *Indicates significant difference from basal medium (P < 0.05).

FIG. 3.

Testosterone upregulates basal I_SC in 1°PVDs cultured in conditions that included PR-free medium, CS-FBS, or typical FBS. Basal I_SC (A) is greater in the PR-free + CS-FBS + TC compared with the PR-free + CS-FBS or PR-free + FBS. Basal R_TE (B), although slightly greater in PR-free + CS-FBS + TC, was not significantly different from that of the two other conditions. *Indicates significant difference (P < 0.05, n = 7).

FIG. 4.

Testosterone upregulates BK-induced anion secretion across 1°PVDs cultured in conditions optimized to reduce the effect of other steroids. Summarized observations (n = 7) reveal that TC upregulates LBK responses as measured by ΔI_SC-MAX (A), net ion flux over 900 sec (B), and ΔR_TE-MAX (C). *Indicates significant difference from PR-free + CS-FBS (P < 0.05).

FIG. 5.

Testosterone upregulates PTGS2 mRNA abundance in 1°PVDs. A) Cells cultured in TC-supplemented medium exhibit PTGS2 upregulation compared with paired cells cultured in basal medium (n = 10 and 12 reactions for PTGS1 and PTGS2, respectively; RNA derived from five and six animals, respectively). B) More prominent PTGS2 upregulation was detected in 1°PVDs cultured in PR-free + CS-FBS + TC compared with 1°PVDs cultured in PR-free + CS-FBS (n = 10 and 12 reactions for PTGS1 and PTGS2, respectively; RNA derived from five and six animals, respectively). *Indicates significant difference from PR-free + CS-FBS (P < 0.05).

FIG. 6.

Testosterone upregulates apparent PTGS2 protein abundance in 1°PVDs. A) Immunoblots exhibited a band of ∼75 kDa that is consistent with porcine PTGS2 and of greater density in protein samples derived from TC-supplemented 1°PVDs. After normalization to ACTIN, PTGS2 expression was 2-fold greater in TC-supplemented monolayers assayed in two-cell culture systems: regular medium (n = 5) and optimized medium (n = 6). B) PTGS1 immunoreactivity, which in porcine kidney and white blood cells exhibits the same mobility as in 1°PVDs, is unchanged by testosterone (n = 6). Arrowheads indicate bands of expected mobility.

FIG. 7.

Testosterone induces the greatest basal I_SC in the distal segment of porcine vas deferens. A) Basal I_SC reaches the greatest levels in TC-supplemented distal 1°PVD monolayers. In addition, distal 1°PVDs cultured in basal medium exhibit lower basal I_SC than that of proximal 1°PVDs cultured in basal medium. B) Monolayers presented similar levels of basal R_TE across experimental groups and treatments. Summarized results (n = 6) of paired cell isolates derived from the same vas deferens for each animal. *Indicates significant difference compared with proximal basal medium (P < 0.05). #Indicates significant difference compared with proximal TC-supplemented medium (P < 0.05) or compared with all other groups (P < 0.05). Proximal, Basal represents proximal basal condition; Proximal, TC represents proximal TC-supplemented condition; Distal, Basal represents distal basal condition; and Distal, TC represents distal TC-supplemented condition.

FIG. 8.

Testosterone induces greater upregulation of BK-induced anion secretion across distal 1°PVDs. Summarized results (n = 6) reveal that BK-induced ΔI_SC-MAX is greatest in distal TC-supplemented 1°PVDs (A). Net ion flux over 900 sec is greater in distal TC-supplemented 1°PVDs (B). *Indicates significant difference (P < 0.05) from proximal TC-supplemented 1°PVDs (Student t-test) and/or from the three other experimental groups (ANOVA). Proximal, Basal represents proximal basal condition; Proximal, TC represents proximal TC-supplemented condition; Distal, Basal represents distal basal condition; and Distal, TC represents distal TC-supplemented condition.