title: Optimization of a filter-lysis protocol to purify rat testicular homogenates for automated spermatid counting.
abstract: Quantifying testicular homogenization-resistant spermatid heads (HRSH) is a  powerful indicator of spermatogenesis. These counts have traditionally been  performed manually using a hemocytometer, but this method can be time  consuming and biased. We aimed to develop a protocol to reduce debris for the  application of automated counting, which would allow for efficient and  unbiased quantification of rat HRSH. We developed a filter-lysis protocol that  effectively removes debris from rat testicular homogenates. After filtering  and lysing the homogenates, we found no statistical differences between manual  (classic and filter-lysis) and automated (filter-lysis) counts using 1-way  analysis of variance with Bonferroni's multiple comparison test. In addition,  Pearson's correlation coefficients were calculated to compare the counting  methods, and there was a strong correlation between the classic manual counts  and the filter-lysis manual (r = 0.85, P = .002) and the  filter-lysis automated (r = 0.89, P = .0005) counts. We also  tested the utility of the automated method in a low-dose exposure model known  to decrease HRSH. Adult Fischer 344 rats exposed to 0.33% 2,5-hexanedione in  the drinking water for 12 weeks demonstrated decreased body (P = .02)  and testes (P = .002) weights. In addition, there was a significant  reduction in the number of HRSH per testis (P = .002) when compared  to controls. A filterlysis protocol was optimized to purify rat testicular  homogenates for automated HRSH counts. Automated counting systems yield  unbiased data and can be applied to detect changes in the testis after  low-dose toxicant exposure.
authors.affiliation: Department of Pathology and Laboratory Medicine, Brown University, Box G-E5, Providence, RI 02912, USA.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Pacheco
authors.forename: Linnea M
authors.initials: LM
authors.lastname: Anderson
authors.forename: Kim
authors.initials: K
authors.lastname: Boekelheide
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D057205:Automation, Laboratory; D002457:Cell Extracts; D003902:Detergents / Q000494:pharmacology; D005374:Filtration; D006588:Hexanones / Q000633:toxicity; D008297:Male; D015588:Observer Variation; D009929:Organ Size / Q000187:drug effects; D051381:Rats; D011916:Rats, Inbred F344; D015203:Reproducibility of Results; D013076:Sperm Count / Q000379:methods*; D013077:Sperm Head / Q000187:drug effects / Q000473:pathology*; D013091:Spermatogenesis / Q000187:drug effects; D013501:Surface-Active Agents / Q000494:pharmacology; D013737:Testis / Q000187:drug effects / Q000473:pathology*; D013997:Time Factors
chemical_list: D002457:Cell Extracts; D003902:Detergents; D006588:Hexanones; D013501:Surface-Active Agents; C011269:2,5-hexanedione