Cryptorchidism in the Orl Rat Is Associated with Muscle Patterning Defects in the Fetal Gubernaculum and Altered Hormonal Signaling1

Abstract

Cryptorchidism, or undescended testis, is a common male genital anomaly of unclear etiology. Hormonal stimulation of the developing fetal gubernaculum by testicular androgens and insulin-like 3 (INSL3) is required for testicular descent. In studies of the orl fetal rat, one of several reported strains with inherited cryptorchidism, we studied hormone levels, gene expression in intact and hormone-stimulated gubernaculum, and imaging of the developing cremaster muscle facilitated by a tissue clearing protocol to further characterize development of the orl gubernaculum. Abnormal localization of the inverted gubernaculum was visible soon after birth. In the orl fetus, testicular testosterone, gubernacular androgen-responsive transcript levels, and muscle-specific gene expression were reduced. However, the in vitro transcriptional response of the orl gubernaculum to androgen was largely comparable to wild type (wt). In contrast, increases in serum INSL3, gubernacular INSL3-responsive transcript levels, expression of the INSL3 receptor, Rxfp2, and the response of the orl gubernaculum to INSL3 in vitro all suggest enhanced activation of INSL3/RXFP2 signaling in the orl rat. However, DNA sequence analysis did not identify functional variants in orl Insl3. Finally, combined analysis of the present and previous studies of the orl transcriptome confirmed altered expression of muscle and cellular motility genes, and whole mount imaging revealed aberrant muscle pattern formation in the orl fetal gubernaculum. The nature and prevalence of developmental muscle defects in the orl gubernaculum are consistent with the cryptorchid phenotype in this strain. These data suggest impaired androgen and enhanced INSL3 signaling in the orl fetus accompanied by defective cremaster muscle development.

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

“…2 We observed that prenatal muscle development is abnormal in the GCC of the cryptorchid LE/orl rat, and although inversion occurs, the GCC is subsequently diverted into the superficial inguinal pouch postnatally. 12 We were, thus, motivated to perform additional prenatal studies to define the cellular properties of the rat GCC that set the stage for normal inversion.…”

“…Care was provided, timed pregnancies generated and euthanasia performed as described previously; 12 the morning of the day of sperm detection was designated embryonic day 0 (E0).…”

“…compound (VW R) and stored at −70°C as described previously. 12 For organ culture, individual E17 GCCs were placed into 6-well tissue culture dishes coated with poly-L lysine (PLL) 0.01% (70–150 kDa, Sigma) and mouse laminin 8ug/ml (EMD Millipore), and immobilized in Matrigel™ Membrane Matrix 8.5mg/ml (Corning) with warming at 37°C for 10 minutes. We maintained organ cultures in growth medium (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), 10% fetal calf serum (FCS) and penicillin-streptomycin (pen-strep); all from ThermoFisher Scientific) in a humidified incubator at 37°C with 5% CO 2 .…”

“…12 We created image sets, overlapping as needed, to include the entire organ to its point of attachment at the abdominal wall. Z-stack sagittal images were acquired and deconvolved using AutoQuant ® software (Media Cybernetics).…”

“…6,10 Moreover, altered expression of muscle-specific genes and focally defective muscle development occur in the fetal GCC of the congenitally cryptorchid LE/orl rat. 11,12 …”