Animals

Two established mouse lines formed the basis of this work: Inha KO, lacking the Inha mature subunit coding sequence (31) and Inhba KO (BA 192), which lacks the Inhba mature subunit coding sequence (33). Both were maintained on a C57/Bl6 background. In addition, we utilised the Inha x OG2 (bearing Oct4-GFP) mouse strain described previously (19). All three lines were maintained by heterozygote breeding, and genotypes were determined by commercial vendor using real-time PCR (Transnetyx, TN, USA). Testes were collected from E13.5, E15.5 and PND 0 testes of Inha WT (wildtype) and KO littermates, E12.5, E13.5, E14.5 and E15.5 Inhba WT and KO littermates, and E15.5 WT and E17.5 WT and KO pups from the Inha x OG2 strain. For timed matings, the presence of a vaginal plug was used to determine embryonic day (E) 0.5 of pregnancy. Embryonic age was determined at the time of collection from fore- and hind-limb development and male gonads identified by the presence of testis cords. Natural birth, occurring on E19.5 – E20.5, was recorded as postnatal day 0 (PND 0). Testes were dissected free from mesonephros or epididymis at the time of collection. Mice were housed at the Monash Medical Centre Animal Facility in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1997), with a 12hr light/12hr dark cycle with food and water available ad libitum. This study was approved by the Monash University Animal Ethics Committee.