GASZ Is Essential for Male Meiosis Figure 2. Gross and histological analysis of postnatal testes. (A) Gross analysis of testes from 7-week-old littermates. (B–M) Histological analysis of testes of Gasz+/2 and Gasz2/2 mice. (M) is a higher power magnification of (L) to show the lack of germ cells attached to the base of the tubule and sloughing germ cells in the lumen. G, spermatogonia; L, leptotene spermatocytes; M, meiotically dividing spermatocytes; Mit, mitotically dividing spermatogonia; P, pachytene spermatocytes, PL, preleptotene spermatocytes; Ser, Sertoli cells; Z, zygotene spermato- cytes; dying spermatocytes with compact chromatin (red arrowhead); and with diffuse chromatin (open arrowhead); sloughing germ cells (black arrowheads). [Scale bars: 100 mm (B–C), 20 mm (D–K).] doi:10.1371/journal.pgen.1000635.g002 Figure 1. Targeting of the Gasz allele and generation of Gasz mutant mice. (A) Schematic representation of the Gasz gene, structure of the targeting vector, and the resultant mutant allele. Genomic DNA fragments used as 59 and 39 homology arms in the targeting vector are indicated by thick lines. Exons 1 and 2 (which encode the Gasz transcriptional start site and the initiation ATG codon) are replaced by a PgkHPRT expression cassette (shaded boxes). The 59 and 39 probes (filled boxes) used for Southern blots are indicated. (S) Sph I, (K) Kpn I. The MC1tk expression cassette was used for negative selection. (B) Southern blot analysis of genomic DNA derived from a litter from Gasz+/2 (+/2) intercrosses. Similar percentages of male and female mice were genotyped as Gasz homozygous (2/2). The 59 probe hybridizes to 7.7 kb (wild-type, WT) and 4.8 kb (mutant, Mut) SphI fragments. (C) RT-PCR analysis of Gasz expression in Gasz+/2 (+/2) and Gasz2/2 (2/2) testes from E18.5 to post-natal day 14 demonstrating Gasz expression in embryonic testes and the lack of Gasz mRNA in testes. (D) Western blot analysis of wild type testis samples from different time-points as well as 6-week-old Gasz WT, +/2, and 2/2 mice using a polyclonal antibody to GASZ (Upper) or an antibody to b-actin as a control for sample loading (Lower). GASZ protein is detected as early as postnatal day 2 and peak abundance is reached after 14 days of age. Absence of the GASZ protein in Gasz2/2 testes confirmed that the Gasz2/2 mutation was null. doi:10.1371/journal.pgen.1000635.g001 the composition of Gasz2/2 testes (Figure 2C) had not changed substantially in composition, whereas Gasz+/2 testes (Figure 2B) had advanced to contain preleptotene and leptotene spermatocytes. At P12, Gasz2/2 testes (Figure 2E) contained predominantly sper- matogonia with only 25% of the tubules containing preleptotene spermatocytes but no leptotene spermatocytes as compared with Gasz+/2 testes where more advanced zygotene spermatocytes were present (Figure 2D). By P14, Gasz2/2 testes displayed early pachytene germ cell loss due to apoptosis (Figure S3), with the most advanced germ cells being zygotene spermatocytes (Figure 2G), while Gasz+/2 testes (Figure 2F) had advanced to the mid-pachytene stage. Thus, histological analysis supports a consistent delay in Gasz2/2 spermatogenesis beginning at meiotic prophase. Gasz2/2 spermatogenesis fails at an identical point in juveniles and young adults. However, by 6 months of age, there are few germ cells in the PLoS Genetics | www.plosgenetics.org 3 September 2009 | Volume 5 | Issue 9 | e1000635