, Waltham, MA). Immunohistochemistry Proliferating Palbociclib Phase 3 cell nuclear antigen (PCNA) (PC10; DAKO, Kingsgrove, Australia), activated caspase-3 (Asp175, Cell Signaling), activin-��C (clone-115), and Smad-2 (no. 3122, Cell Signaling) were detected using the DAKO Autostainer universal staining system (DAKO A/S, Glostrup, Denmark). Microwave antigen retrieval for PCNA was performed in 0.01 mol/L citrate buffer (pH 6.0), activin-��C in 0.1 mol/L glycine buffer (pH 4.5), caspase-3, and Smad-2 in 0.01 mol/L sodium citrate buffer (pH 6.0). Sections were treated with CAS blocking reagent (DAKO, Kingsgrove, Australia), antibodies detected with avidin-biotin complex (ABC) and color-reacted with diaminobenzidine (DAKO, Kingsgrove Australia).
Fertility Assessment Male mice (WT, SH, and DH) of varying ages (6 to 30 weeks) were cohabited with 10- to 12-week female C57BL/6 WT mice (1:1). Females were checked daily for the presence of copulatory plugs. Females with positive plugs were separated from males, and the number of pups per plug was recorded. Daily sperm production (DSP) was determined using a frozen left testis from WT, SH, and DH mice by a procedure that has been previously described.24 Sperm Motility Analyses Sperm were collected and capacitated in modified Tyrode��s medium without sodium pyruvate or sodium lactate. Sperm from 23 mice were collected at 14 to 16 weeks (n = 10 WT, 5 SH2, and 9 DH2). Both caudae epididymidis were dissected from the mice and a small slit was made in each before transfer to a 1.
5-ml tube containing 1 ml of pre-equilibrated modified Tyrode��s medium and incubation in 5% CO2 in air at 37��C for 10 minutes to allow the sperm to swim out from the tissue. After incubation the caudae were removed and a 10-��l aliquot of the sperm suspension placed into an 80-��m chamber (Hamilton-Thorne Biosciences, Beverly, MA). Replicates (two per mouse) were analyzed using a Hamilton-Thorne IVOS computer-assisted sperm analyzer (Hamilton-Thorne Biosciences). At least 1000 sperm were analyzed per replicate. Analyses were performed using the recommended settings for mouse sperm. Data were arcsin-transformed and then subjected to general linear analysis, and the difference between means was determined by Tukey��s HSD test (SPSS for Windows; SPSS, Chicago, IL). In Situ Detection of DNA Fragmentation in Testis Sections Apoptosis in testis sections was analyzed by the ApopTag in situ apoptosis detection kit (Chemicon Int., Temecula, CA).25 Testis Stereology Slides were masked Cilengitide before quantitation to facilitate unbiased counting. PCNA-positive cell types were identified based on their location within the tubule, their size, and the shape of the cell nucleus.