Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the presence of numerous fluid-filled cysts, extensive fibrosis, and the progressive decline in kidney function. Transforming growth factor-β1 (TGF-β1), an important mediator for renal fibrosis and chronic kidney disease, is overexpressed by cystic cells compared with normal kidney cells; however, its role in PKD pathogenesis remains undefined. To investigate the effect of TGF-β1 on cyst growth, fibrosis, and disease progression, we overexpressed active TGF-β1 specifically in collecting ducts (CDs) of phenotypic normal ( Pkd1 RC/+ ) and Pkd1 RC/RC mice. In normal mice, CD-specific TGF-β1 overexpression caused tubule dilations by 5 wk of age that were accompanied by increased levels of phosphorylated SMAD3, α-smooth muscle actin, vimentin, and periostin; however, it did not induce overt cyst formation by 20 wk. In Pkd1 RC/RC mice, CD overexpression of TGF-β1 increased cyst epithelial cell proliferation. However, extensive fibrosis limited cyst enlargement and caused contraction of the kidneys, leading to a loss of renal function and a shortened lifespan of the mice. These data demonstrate that TGF-β1-induced fibrosis constrains cyst growth and kidney enlargement and accelerates the decline of renal function, supporting the hypothesis that a combined therapy that inhibits renal cyst growth and fibrosis will be required to effectively treat ADPKD.

The epididymis exhibits a less restrictive physical blood–tissue barrier than the testis and, while numerous immunosuppressive factors have been identified in the latter, no mechanisms for epididymal immunotolerance have been identified to date. Therefore, data are currently insufficient to explain how the immune system tolerates the extremely large load of novel antigens expressed on sperm, which become present in the male body after puberty, i.e., long after central tolerance was established. This study tested the hypothesis that transforming growth factor beta (TGFβ) signaling in dendritic cells (DCs) is required for immunotolerance to sperm located in the epididymis, and that male mice lacking TGFβ signaling in DCs would develop severe epididymal inflammation. To test this, we employed adult Tgfbr2ΔDC males, which exhibit a significant reduction of Tgfbr2 expression and TGFβ signaling in DCs, as reported previously. Results show that Tgfbr2ΔDC males exhibit sperm-specific immune response and severe epididymal leukocytosis. This phenotype is consistent with epididymal loss of immunotolerance to sperm and suggests that TGFβ signaling in DCs is a factor required for a non-inflammatory steady state in the epididymis, and therefore for male tract homeostasis and function.

The goal of this study was to test for expression of HCO3 (-) exchangers SLC26A3 and SLC26A6 in primary cultures of porcine vas deferens epithelial cells (1°PVD) and native porcine vas deferens. Quantitative RT-PCR revealed that mRNA coding for SLC26A6 was six times more abundant than mRNA coding for SLC26A3 in 1°PVD cells. Western blot analyses combined with surface biotinylation of 1°PVD demonstrated SLC26A3 and SLC26A6 immunoreactivities in whole-cell lysates and apical surfaces of monolayers. Laser scanning confocal microscopy (LSCM) of the 1°PVD cell monolayers demonstrated that SLC26A3 immunoreactivity was primarily in the apical region but present throughout the basal-apical cellular axis, whereas SLC26A6 immunoreactivity was present in the apical region and sometimes accumulated in the nuclear region. LSCM also demonstrated SLC26A3 and SLC26A6 immunoreactivities present along the entire apical lining of the native porcine vas deferens epithelium and in basal cells. The patterns and apparent abundance of SLC26A3 and SLC26A6 immunoreactivities in the proximal vas deferens were not different from the corresponding immunoreactivities in the distal region. There is no evidence of preferential expression of SLC26A3 or SLC26A6 in any portion of the vas deferens, as has been proposed for epithelia that secrete HCO3 (-) in other duct systems. Thus, vas deferens epithelia express transporters throughout the duct that can contribute to rapid alkalinization of the luminal contents as it has been demonstrated in vivo.

The goal of this study was to determine whether transforming growth factor-β1 (TGF-β1) affects epithelial cells lining the vas deferens, an organ that is universally affected in cystic fibrosis male patients. In PVD9902 cells, which are derived from porcine vas deferens epithelium, TGF-β1 exposure significantly reduced short-circuit current (Isc) stimulated by forskolin or a cell membrane-permeant cAMP analog, 8-pCPT-cAMP, suggesting that TGF-β1 affects targets of the cAMP signaling pathway. Electrophysiological results indicated that TGF-β1 reduces the magnitude of current inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers. Real-time RT-PCR revealed that TGF-β1 downregulates the abundance of mRNA coding for CFTR, while biotinylation and Western blot showed that TGF-β1 reduces both total CFTR and apical cell surface CFTR abundance. These results suggest that TGF-β1 causes a reduction in CFTR expression, which limits CFTR-mediated anion secretion. TGF-β1-associated attenuation of anion secretion was abrogated by SB431542, a TGF-β1 receptor I inhibitor. Signaling pathway studies showed that the effect of TGF-β1 on Isc was reduced by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). TGF-β1 exposure also increased the amount of phospho-p38 MAPK substantially. In addition, anisomycin, a p38 MAPK activator, mimicked the effect of TGF-β1, which further suggests that TGF-β1 affects PVD9902 cells through a p38 MAPK pathway. These observations suggest that TGF-β1, via TGF-β1 receptor I and p38 MAPK signaling, reduces CFTR expression to impair CFTR-mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and ultimately would compromise male fertility.

This study is designed to identify effects of transforming growth factor β1 (TGF‐β1) on ion transport across vas deferens epithelia. In modified Ussing chambers, PVD9902 cells (derived from porcine vas deferens) that were exposed to TGF‐β1 exhibited attenuated forskolin‐stimulated anion secretion and reduced effects of DASU‐02, a CFTR channel blocker. RT‐PCR and western blots revealed that TGF‐β1 down‐regulates mRNA expression and protein expression of CFTR. Biotinylation assays suggested that TGF‐β1 also reduced the abundance of CFTR in the apical membrane. The effect of TGF‐β1 on anion secretion was abrogated by a TGF‐β1 receptor I inhibitor and was reduced by an inhibitor of p38 MAPK, suggesting that TGF‐β1 interacts with its cognate receptor on vas deferens epithelial cells to activate p38 MAPK signaling. This conclusion was further supported by the observation that TGF‐β1 enhances phospho‐p38 MAPK abundance. Additionally, anisomycin, a p38 MAPK activator, mimicked the effect of TGF‐β1 to reduce forskolin‐stimulated anion secretion. Men harboring profound CFTR mutations exhibit infertility and reproductive duct anomalies, whereas mild mutations have variable presentations. Our observations suggest that TGF‐β1 reduces CFTR‐mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and would compromise male fertility. [NIH HD058398 and RR017686]

Epithelial cells lining the male excurrent duct contribute to male fertility by employing a number of physiological mechanisms that generate a luminal microenvironment conducive to spermatozoa maturation and storage. Among these mechanisms, male duct epithelia establish intercellular tight junctions that constitute a barrier to paracellular diffusion of water, solutes, large molecules, and cells. Mechanisms regulating the male duct epithelial barrier remain unidentified. Transforming growth factor beta (TGFB) is a regulatory cytokine present in high concentrations in human semen. This study examined whether TGFB has any effects on epithelial function exhibited by primary cultures of porcine vas deferens epithelia. TGFB1 exposure caused a 70%-99% decrease in basal transepithelial electrical resistance (R-TE, a sensitive indicator of barrier integrity), while a significant decrease in anion secretory response to forskolin was detected at the highest levels of TGFB1 exposure employed. SB431542, a selective TGFB receptor I (TGFBR1) inhibitor, prevented decreases in barrier function. Results also demonstrated that TGFB1 exposure modifies the distribution pattern of tight junction proteins occludin and claudin 7. TGFBR1 is localized at the apical border of the native porcine vas deferens epithelium. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) 11 (also known as p38-MAPK) did not alter the effect of TGFB1 on R-TE significantly. These data suggest that epithelia lining the vas deferens are subject to disruptions in the physical barrier if active TGFB becomes bioavailable in the luminal fluid, which might be expected to compromise fertility.

Nearly all male cystic fibrosis (CF) patients exhibit tissue abnormalities in the reproductive tract, a condition that renders them azoospermic and infertile. Two swine CF models have been reported recently that include respiratory and digestive manifestations that are comparable to human CF. The goal of this study was to determine the phenotypic changes that may be present in the vas deferens of these swine CF models. Tracts from CFTR-/- and CFTR Delta F508/Delta F508 neonates revealed partial or total vas deferens and/or epididymis atresia at birth, while wild-type littermates were normal. Histopathological analysis revealed a range of tissue abnormalities and disruptions in tubular organization. Vas deferens epithelial cells were isolated and electrophysiological results support that CFTR-/- monolayers can exhibit Na+ reabsorption but reveal no anion secretion following exposure to cAMP-generating compounds, suggesting that CFTR-dependent Cl- and/or HCO3- transport is completely impaired. SLC26A3 and SLC26A6 immunoreactivities were detected in all experimental groups, indicating that these two chloride-bicarbonate exchangers were present, but were either unable to function or their activity is electroneutral. In addition, no signs of increased mucus synthesis and/or secretion were present in the male excurrent ducts of these CF models. Results demonstrate a causal link between CFTR mutations and duct abnormalities that are manifested at birth.

The objectives were to develop a transplantation assay for equine testicular cells using busulfan-treated prepubertal immunocompetent rats as recipients, and to determine if putative equine spermatogonial stem cells (SSCs) could be enriched by flow cytometric cell sorting (based on light scattering properties), thereby improving engraftment efficiency. Four weeks after transplantation of frozen/thawed PKH26-labeled equine testicular cells, 0.029 ± 0.045% (mean ± SD) of viable donor cells transplanted had engrafted. Donor cells were present in seminiferous tubules of all recipient rats forming chains, pairs, mesh structures, or clusters (with two to >30 cells/structure). Cells were localized to the basal compartment by the basement membrane. Although equine cells proliferated within rat seminiferous tubules, no donor-derived spermatogenesis was evident. Furthermore, there was no histologic evidence of acute cellular rejection. No fluorescent cells were present in control testes. When equine testicular cells were sorted based on light scattering properties, the percentage of transplanted donor cells that engrafted was higher after injection of cells from the small, low complexity fraction (II; 0.169 ± 0.099%) than from either the large, high complexity fraction (I; 0.046 ± 0.051%) or unsorted cells (0.009 ± 0.007%; P < 0.05). Seminiferous tubules of busulfan-treated prepubertal immunocompetent rats provided a suitable niche for engraftment and proliferation, but not differentiation, of equine testicular cells. Sorting equine testicular cells based on light scattering properties resulted in a 19-fold improvement in colonization efficiency by cells with high forward scatter and low side scatter, which may represent putative equine SSCs.

Data are accumulating to demonstrate that pH regulation in the male reproductive tract has a vital role in modulating sperm cell fertilizing capacity, and therefore male fertility. Bicarbonate uptake by sperm cells is required for the achievement of motility levels required for fertilization. Vas deferens epithelial cells can carry out measurable bicarbonate secretion, but the available literature to date reports that the vas deferens luminal content is typically acidic. This study aimed to determine pH in the boar vas deferens lumen and whether modulatory mechanisms exist for regulation of pH in this compartment of the male reproductive tract. A fiberoptic pH probe was used to assess pH in the vas deferens of anesthetized adult boars. The mean pH, derived from multiple measurements at variable positions along the vas deferens lumen, was 7.39 +/- 0.09. Furthermore, administration of xylazine, an alpha-2 adrenergic receptor agonist rapidly (< 10 min) alkalinized the vas deferens lumen in most cases. Because the duct was transected proximal to the site of measurements, the observations rule out the possibility that alkalinization resulted from secretion in more proximal portions of the duct. These results indicate that the boar vas deferens lumen can be alkaline, and they suggest that porcine vas deferens epithelia increase net bicarbonate secretion in vivo after systemic alpha-2 adrenergic stimulation. This secretory response greatly changes the luminal environment to which sperm cells are exposed, which will initiate or enhance motility, and is expected to modulate male fertility.

Transforming growth factor 1 (TGFB1) is associated with pulmonary disease progression in cystic fibrosis (CF) patients although the impact of TGFB1 on the male reproductive duct is unknown. Cystic fibrosis, the most common recessive genetic disease in Caucasians, is almost universally (>98%) associated with congenital bilateral absence of the vas deferens (CBAVD) resulting in male infertility although pulmonary and pancreatic manifestations, which affect slightly smaller proportions of the population (~85%), are more likely to lead to mortality. Male CF patients may with present infertility or subfertility without CBAVD. In addition, a small number of CBAVD cases occur that are not accompanied by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and various cases of idiopathic human male infertility or subfertility are reported that are not of testicular-based etiology. This study aimed to determine whether TGFB1 can alter human and porcine vas deferens epithelial cell physiology and to investigate the signal transduction pathway through which TGFB1 may induce changes in vas deferens epithelial cell function. Primary porcine (1°PVD) and human (1°HVD) vas deferens epithelial cell monolayers were exposed to either vehicle or TGFB1, for 24 and/or 72 hours, and then assessed in modified Ussing chambers. Vehicle-treated 1°PVD presented basal transepithelial resistance (RTE, a sensitive measure of epithelial barrier function) of 4800 Ohms/cm2, while paired 1°PVD exposed to TGFB1 (100 ng/ml) for 24 and 72 hours had 470 and 45 Ohms/cm2, respectively. 1°HVD exposed to TGFB1 (5 ng/ml) for 72 hours also revealed significant reductions in RTE. The ability of both 1°HVD and 1°PVD to respond with increases in short-circuit current (ISC, a sensitive measure of anion secretion) upon forskolin exposure (2 μM) was also impaired in TGFB1-exposed monolayers at 72 hours. To determine if protein kinases modulate the TGFB1 effect, 1°PVD monolayers were exposed to TGFB1 (5 ng/ml) for 24 hours, either in the absence or presence of selective inhibitors of mitogen-activated protein kinases 1 and 3 (MAPK1/3, previously known as ERK1/2), MAPK8 (previously known as JNK-1), MAPK11 (previously known as p38-MAPK) and phosphoinositide-3-kinase (PIK3). Outcomes suggest that SP600125, a selective MAPK8-inhibitor (10 μM), and SKF86002 (10 μM), a selective MAPK11-inhibitor (10 μM), are able to reduce the decreases in RTE induced by TGFB1. These data suggest that both human and porcine vas deferens epithelial cells respond functionally to activation of the TGFB1 receptor in vitro with a significant reduction in epithelial barrier function. Thus, TGFB1 signaling seems to lead to a loss of cell-cell association that could lead to a change in tissue architecture that may ultimately result in destruction of the duct. (Supported by NIH HD058398 and RR017686) (platform)

The goal of this study was to detect bicarbonate exchangers SLC26A3 and SLC26A6 in intact vas deferens epithelia. Bicarbonate uptake by sperm cells while still in the male reproductive tract is required for the development of fertilizing capacity. The expression of SLC26A3 and SLC26A6 at the mRNA level has been reported in the porcine vas deferens (PVD) PVD9902 cell line, but it remains unknown whether SLC26A3 and SLC26A6 are expressed in intact PVD epithelium. Laser scanning confocal microscopy (LSCM) was employed to investigate SLC26A3 and SLC26A6 immunoreactivities in intact PVD and primary PVD cultured (1oPVD) cells. qRT‐PCR was used to determine the mRNA copy number of SLC26A3 and SLC26A6 in total RNA isolated from 1oPVD cells. LSCM demonstrated that SLC26A3 and SLC26A6 immunoreactivities are located at the apical membrane of principal cells in the proximal and distal segments of PVD. In contrast to tissue sections 1oPVD cells (cultured for 14 days) demonstrated diffuse immunolabelling close to apical area. 1oPVD cells expressed 391±70 copies of SLC26A3 and 2324±265 copies of SLC26A6 mRNA per 500 ng of total RNA. The study was supported by the NIH (RO1HD058398) and (RR017686 ‐ Imaging and Molecular Cores).

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.

Epithelia lining the male reproductive duct modulate fertility by altering the luminal environment to which sperm are exposed. Although vas deferens epithelial cells reportedly express high levels of cyclooxygenases (Ptgs), and activation of bradykinin (BK) receptors can lead to upregulation of PTGS activity in epididymal epithelia, it remains unknown whether BKs and/or PTGSs have any role in modulating epithelial ion transport across vas deferens epithelia. Porcine and human vas deferens epithelial cell primary cultures and the PVD9902 cell line responded to lysylbradykinin with an increase in short circuit current (I SC; indicating net anion secretion), an effect that was 60%-93% reduced by indomethacin. The BK effect was inhibited by the B2 receptor subtype (BDKRB2) antagonist HOE140, whereas the B1 receptor subtype agonist des-Arg9-BK had no effect. BDKRB2 immunoreactivity was documented in most epithelial cells composing the native epithelium and on Western blots derived from cultured cells. Gene expression analysis revealed that the PTGS2 transcript is 20 times more abundant than its PTGS1 counterpart in cultured porcine vas deferens epithelia and that BDKRB2 mRNA is likewise highly expressed. Subsequent experiments revealed that prostaglandin E2, 1-OH prostaglandin E1 (prostaglandin E receptor 4 [PTGER4] agonist) and butaprost (PTGER2 agonist) increase I SC in a concentration-dependent manner, whereas sulprostone (mixed PTGER1 and PTGER3 agonist) produced no change in I SC. These results demonstrate that autacoids can affect epithelial cells to acutely modulate the luminal environment to which sperm are exposed in the vas deferens by enhancing PTGS activity, leading to the production of prostaglandins that act at PTGER4 and/or PTGER2 to induce or enhance anion secretion.

Epithelial cells lining the rat vas deferens express cyclooxygenase (COX) 2 in a testosterone-dependent fashion although no functional role for this expression pattern has been established. It was hypothesized that testosterone modulates anion secretion across vas deferens epithelia via COX-dependent pathways and prostaglandin (PG) synthesis. Vas deferens epithelial cells were isolated from porcine tissues and cultured as monolayers on permeable supports within paired conditions until assayed in modified Ussing chambers. RNA and protein were isolated concurrently for semi-quantitative expression analysis. Native porcine vas deferens was subjected to immunocytochemistry. Testosterone-treated monolayers exhibited bradykinin (BK)-induced ion transport (measured as short circuit current) 65% greater than paired vehicle-treated monolayers. The non-selective COX inhibitor indomethacin inhibited (80% less) anion secretion stimulated by BK. qRT-PCR revealed a modest testosterone induced upregulation in COX-2 mRNA and this transcript was 20 times more abundant than its counterpart COX-1. Immunocytochemistry performed to label the bradykinin 2 receptor subtype (B2R), and the prostanoid receptors EP2 and EP4 support that these receptors are localized at the apical membrane of porcine vas deferens epithelium. Taken together, these results suggest that testosterone plays a key permissive role for BK-stimulated anion secretion. BK acts at the B2R receptor to increase COX activity and the synthesis of PGs that ultimately bind to EP receptors to increase cAMP levels and initiate and/or intensify anion secretion into the vas deferens luminal environment to which sperm is exposed. [Supported by the Cystic Fibrosis Foundation (SCHULT06PO) and the NIH (RR-17686)].