Regional Variation in Androgen Receptor Expression and Biomechanical Properties May Contribute to Cryptorchidism Susceptibility in the LE/orl Rat.

Background: The process of testicular descent requires androgen and insulin-like 3, hormones secreted by fetal Leydig cells. Knowledge concerning distinct and common functions of these hormones in regulating development of the fetal gubernaculum remains limited and/or conflicting. The current studies were designed to better define characteristics of androgen receptor (AR) expression, function and regulation, as well as the biomechanical properties of normal and cryptorchid gubernaculum during fetal development. Methods: We studied fetal gubernacula from Long Evans outbred (LE/wt) rats and an inbred (LE/orl) strain with an inherited form of cryptorchidism associated with an AR signaling defect. Gubernacular cells or whole organs obtained from LE/wt and LE/orl fetal gubernacula underwent AR immunostaining and quantitative image analysis. The effects of dihydrotestosterone (DHT) on AR expression, muscle fiber morphology, hyaluronan (HA) levels and glycosaminoglycan (GAG) content were measured in LE/wt gubernacula. Finally, the spatial mechanics of freshly harvested LE/wt and LE/orl fetal gubernacula were compared using micropipette aspiration. Results: AR is expressed in the nucleus of mesenchymal core, tip and cord cells of the embryonic (E) day 17 and 21 fetal gubernaculum, and is enhanced by DHT in primary cultures of gubernacular mesenchymal cells. Enhanced AR expression at the tip was observed in LE/wt but not LE/orl gubernacula. In in vitro studies of whole mount fetal gubernaculum, DHT did not alter muscle fiber morphology, HA content or GAG production. Progressive swelling with reduced cellular density of the LE/wt gubernaculum at E19-21 was associated with increased central stiffness in LE/wt but not in LE/orl fetuses. Conclusions: These data confirm nuclear AR expression in gubernacular mesenchyme with distal enhancement at the tip/cord region in LE/wt but not LE/orl rat fetuses. DHT enhanced cellular AR expression but had no major effects on muscle morphology or matrix composition in the rat fetal gubernaculum in vitro. Regional increased stiffness and decreased cell density between E19 and E21 were observed in LE/wt but not LE/orl fetal gubernacula. Developmental differences in cell-specific AR expression in LE/orl fetal gubernacula may contribute to the dysmorphism and aberrant function that underlies cryptorchidism susceptibility in this strain.

Fetal Rat Gubernaculum Mesenchymal Cells Adopt Myogenic and Myofibroblast-Like Phenotypes.

PURPOSE: Gubernaculum-cremaster complex development is hormonally regulated and abnormal in a cryptorchid rat model. Using cell tracking techniques and imaging we studied myogenic phenotypes and fates in the fetal rat gubernaculum-cremaster complex. MATERIALS AND METHODS: Embryonic day 17 gubernaculum-cremaster complexes were labeled with CellTracker™ or the DNA synthesis marker EdU (5-ethynyl-2'-deoxyuridine), or immobilized in Matrigel® and grown in culture. Embryonic day 17 to 21 gubernaculum-cremaster complex sections and cells were imaged using wide field and deconvolution immunofluorescence microscopy, and muscle and/or myofibroblast specific antibodies. Deconvolved image stacks were used to create a 3-dimensional model of embryonic day 21 gubernaculum-cremaster complex muscle. RESULTS: PAX7 (paired box 7) positive and myogenin positive muscle precursors were visible in a desmin-rich myogenic zone between muscle layers that elongated and became thicker during development. Gubernaculum-cremaster complex inner mesenchymal cells expressed desmin and αSMA (α smooth muscle actin) at lower levels than in the myogenic zone. After pulse labeling with CellTracker or EdU mesenchymal cells became incorporated into differentiated muscle. Conversely, mesenchymal cells migrated beyond Matrigel immobilized gubernaculum-cremaster complexes, expressed PAX7 and fused to form striated myotubes. Mesenchymal gubernaculum-cremaster complex cell lines proliferated more than 40 passages and showed contractile behavior but did not form striated muscle. Our 3-dimensional gubernaculum-cremaster complex model had 2 orthogonal ventral layers and an arcing inner layer of muscle. CONCLUSIONS: Our data suggest that mesenchymal cells in the peripheral myogenic zone of the fetal gubernaculum-cremaster complex contribute to formation of a distinctively patterned cremaster muscle. Nonmyogenic, desmin and αSMA positive gubernaculum-cremaster complex mesenchymal cells proliferate and have a myofibroblast-like phenotype in culture. Intrinsic mechanical properties of these divergent cell types may facilitate perinatal inversion of the gubernaculum-cremaster complex.

Phenotype specific association of the TGFBR3 locus with nonsyndromic cryptorchidism.

PURPOSE: Based on a genome-wide association study of testicular dysgenesis syndrome showing a possible association with TGFBR3, we analyzed data from a larger, phenotypically restricted cryptorchidism population for potential replication of this signal. MATERIALS AND METHODS: We excluded samples based on strict quality control criteria, leaving 844 cases and 2,718 controls of European ancestry that were analyzed in 2 separate groups based on genotyping platform (ie Illumina® HumanHap550, version 1 or 3, or Human610-Quad, version 1 BeadChip in group 1 and Human OmniExpress 12, version 1 BeadChip platform in group 2). Analyses included genotype imputation at the TGFBR3 locus, association analysis of imputed data with correction for population substructure, subsequent meta-analysis of data for groups 1 and 2, and selective genotyping of independent cases (330) and controls (324) for replication. We also measured Tgfbr3 mRNA levels and performed TGFBR3/betaglycan immunostaining in rat fetal gubernaculum. RESULTS: We identified suggestive (p ≤ 1× 10(-4)) association of markers in/near TGFBR3, including rs9661103 (OR 1.40; 95% CI 1.20, 1.64; p = 2.71 × 10(-5)) and rs10782968 (OR 1.58; 95% CI 1.26, 1.98; p = 9.36 × 10(-5)) in groups 1 and 2, respectively. In subgroup analyses we observed strongest association of rs17576372 (OR 1.42; 95% CI 1.24, 1.60; p = 1.67 × 10(-4)) with proximal and rs11165059 (OR 1.32; 95% CI 1.15, 1.38; p = 9.42 × 10(-4)) with distal testis position, signals in strong linkage disequilibrium with rs9661103 and rs10782968, respectively. Association of the prior genome-wide association study signal (rs12082710) was marginal (OR 1.13; 95% CI 0.99, 1.28; p = 0.09 for group 1), and we were unable to replicate signals in our independent cohort. Tgfbr3/betaglycan was differentially expressed in wild-type and cryptorchid rat fetal gubernaculum. CONCLUSIONS: These data suggest complex or phenotype specific association of cryptorchidism with TGFBR3 and the gubernaculum as a potential target of TGFß signaling.