Sertoli Cells: What's Androgen Got to Do with It

Testicular development postnatally is important for understanding male fertility. During testicular development Sertoli cells, Leydig cells and germ cells proliferate and mature. Sertoli cell proliferation begins a few days before birth and continues until puberty. Proliferation of Sertoli cells occurs in two distinct intervals during the neonatal and the pre pubertal periods. Sertoli cells cease proliferating peripubertally. Sertoli cells are intimately associated with germ cell development, where their number in the testes is correlated with the amount of sperm produced. Sertoli cells provide structural support for the developing germ cells through the development of the blood testes barrier. The blood testes barrier is made up of multiple junctional complexes; tight junctions, adhesion junctions and gap junctions along with a cytoskeleton frame work. Androgens may play a role in the proliferation and differentiation of Sertoli cells. Few studies have explored the requirements for testosterone during Sertoli cell proliferation. Not only may androgens play a role in Sertoli cell proliferation but they may regulate junctional complex protein expression. To our knowledge, no studies have analyzed the effects of androgen receptor inactivation on the first interval of Sertoli cell proliferation in the boar. Methods: Boars were randomly divided between treatment with a daily oral dose of flutamide (Sigma, CA) suspended in canola oil at 10 mg/kg body weight or the canola oil vehicle alone. Two groups of five pairs of littermate boars were treated beginning at one week of age and treatment ended at 6.5 weeks of age. Tissues were collected from 6.5 and 11 week old animals. A third group of five pairs of littermate boars were treated beginning at one week of age and treatment ended at 2, 3 or 4 weeks of age. Three of these litters provided an additional littermate pair that was hemicastrated at 8 days of age. Blood was collected weekly from the jugular vein during treatment and again before tissue collection. Testes were collected, weighed and a slice of testis removed from the equator was fixed overnight in 4% paraformaldehyde in PBS at 4°C. The fixed tissues were then washed in PBS and dehydrated in 30%, 50% and 70% ethanol, and embedded in paraffin. Sertoli cell nuclei in 25 microm testes sections were immunolabeled for GATA-4 and Sertoli cell numbers were determined in 17 microm thick counting frames randomly selected by the CAST Grid software. 5 microm testis sections were immunolabeled for junctional complex proteins; zona occulden-1, occludin, claudin-11, N-Cadherin, connexin 43 and vimentin. Hormones were measured in serum using radioimmunoassays. Results: Approximately 80% more Sertoli cells were present in flutamide-treated boars at 6.5 weeks of age compared with their vehicle-treated littermates (P = 0.003). Testis weight was increased by approximately 50% in these flutamide-treated boars compared with the vehicle-treated littermates (P = 0.013). Animals that were hemicastrated at 2 weeks of age also had significantly more Sertoli cells per testis then their vehicle littermates (P = 0.0263). Plasma testosterone concentration was significantly increased in flutamide-treated boars during the treatment period of 1 to 6.5 weeks (P = 0.0234) compared with vehicle-treated littermates. Overall, plasma estradiol concentration was increased in flutamide-treated boars compared with vehicle-treated littermates (P = 0.0017). Plasma FSH and LH concentrations were increased at week 4 (P = 0.0013 and P = 0.0015) and week 5 (P = 0.0024 and P = 0.027). Zona Occluden -1 labeling intensity was significantly lower at 2 weeks (P= 0.013) in the hemicastrated vehicle-treated group of boars compared with intact littermates. Zona Occluden -1 protein expression was significantly different at 6.5 weeks of age between the vehicle-treated and flutamide-treated boars (P= 0.04). Occludin, claudin-11, CDH2, CX43 and vimentin protein expression did not differ between flutamide-treated and vehicle-treated boars at any age. Further understanding of androgen regulation of Sertoli cell proliferation in multiple species will contribute to a better understanding of testicular development. Understanding the biological mechanism of testicular development, Sertoli cell proliferation and development will provide a strong platform for more applied work in male fertility.