An immunohistochemical analysis of the effects of androgen receptor knock out on gubernacular differentiation in the mouse.

AIM: Cryptorchidism affects 2%-4% of newborn boys and causes infertility and cancer. While normal androgen function is required for successful inguinoscrotal descent, its exact role on gubernacular morphology remains unidentified. We aimed to decipher the effect of androgen blockade on the gubernaculum and surrounding structures. METHODS: Genetically modified mice with androgen receptor knock out (ARKO) were sectioned at ages E17, D0, and D2 for immunohistochemical analysis and D4 for morphological analysis (with ethical approval; A644). Mutants and control littermates were labeled with Ki67, Desmin, and Pax7 to identify the degree of gubernaculuar eversion and the composition of the growth center in the gubernaculum, using light or confocal microscopy. RESULTS: Androgen blockade prevented gubernacular eversion in all animals aged between E17 and D2 when compared to wild types. Furthermore, a growth center was visible, as indicated by a 'swirl' of immature fibroblasts, in D2 animals but was absent in ARKO mice. Moreover, the gubernacular cord was seen to increase in ARKO mice when compared to wild types and increased in size with age. There were no labeling differences in the antibodies tested for gubernacular differentiation. CONCLUSION: Gubernacular eversion in rodents prior to inguinoscrotal migration was androgen dependent, as well as maintenance of gubernacular cord length. This study shows that androgen blockade causes cryptorchidism in mice by preventing gubernacular eversion and possibly by preventing shortening of the gubernacular cord. Altering the morphology of the gubernaculum in response to androgen clearly contributes to the clinical problem of cryptorchidism.

Molecular signals governing cremaster muscle development: clues for cryptorchidism.

BACKGROUND/AIM: Cryptorchidism affects 2-4% of newborn boys. Testicular descent requires the gubernaculum to differentiate into cremaster muscle (CM) during androgen-mediated inguino-scrotal descent, but the cellular mechanisms regulating this remodeling remain elusive. ß-Catenin, a marker of canonical Wnt signaling, promotes myogenic genes and cellular adhesion. We aimed to determine if androgen receptor (AR) blockade altered ß-catenin and its downstream myogenic proteins within the CM. METHOD: Gubernacula from male rats (n=12) and rats treated with anti-androgen, flutamide (n=12) at E19, D0, D2 were processed for immunohistochemistry. Antibodies against ß-catenin, embryonic myosin, and myogenin were visualized by confocal microscopy. RESULTS: At E19, ß-catenin immuno-reactivity (IR) localized to the CM membrane. By D2, cytoplasmic ß-catenin-IR was noted with overall ß-catenin-IR decreasing. Myogenic proteins resided primarily in cells containing ß-catenin on their plasma membrane. Embryonic myosin-IR was high at E19 and then decreased by D2, while myogenin-IR increased. AR blockade increased cytoplasmic ß-catenin at D2 and reduced levels of both myogenic proteins. CONCLUSION: Myogenic proteins are present in CM cells containing ß-catenin. AR blockade did not alter cellular adhesion via ß-catenin. In contrast, blocking AR prevented ß-catenin entering the nucleus and impaired CM myogenesis. Mutations in this pathway may result in idiopathic cryptorchidism.

Gonocyte transformation to spermatogonial stem cells occurs earlier in patients with undervirilisation syndromes.

AIM: Fertility post-orchidopexy is dependent on transformation of neonatal gonocytes (G) into adult dark spermatogonia at about 3 months, the same time as gonadotrophins stimulate androgen secretion. We examined how androgen blockade affects transformation of gonocytes to spermatogonial stem cells (SSC) during this period in patients with undervirilisation syndromes. METHODS: Patients with undervirilisation syndromes (n=30, 1.5 weeks-16 years) underwent review of medical records, pathology reports, and H&E slides of testes (ethics HREC32164). Fluorescent immunohistochemistry against anti-Mullerian hormone (AMH, Sertoli cells), mouse VASA homologue (MVH, germ cells) and DAPI (nuclei) allowed the number of MVH-positive gonocytes/spermatogonial stem cells per seminiferous tubular cross-section (G/T or SSC/T) to be counted. RESULTS: Gonocytes (MVH-positive cells in the tubular lumen) were present in 15/16 patients under 2 years old. SSC (MVH-positive cells on the tubule basement membrane) were present in 25/30 patients. With increasing age, the mean number of SSC/T decreased from ~4 to 0, and G/T decreased from ~1.5 to 0. SSC were present in CAIS and PAIS patients at 1.5 and 3.5 weeks old, respectively. CONCLUSIONS: Gonocytes transform into SSC earlier than expected in patients with undervirilisation syndromes. Lack of androgens may stimulate non-androgenic regulators to trigger transformation. Understanding how gonocytes transform may enable optimization of spermatogonial development to preserve fertility post-orchidopexy.