Methotrexate induces germ cell apoptosis and impairs spermatogenesis in a rat
Authors: Arnold G Coran
Ofer Nativ
Daniel Bejar
Jorge G Mogilner
Omri Nativ
Igor Sukhotnik
Alex Roitburt
Journal: Pediatric Surgery International (2013)
Discipline: CLINICAL MEDICINE
Keywords: Methotrexate; Germ cell apoptosis; Spermatogenesis; Rat; Testes
Affiliations: The Bruce Rappaport Faculty of Medicine, Laboratory of Intestinal Adaptation and Recovery, Technion-Israel Institute of Technology, Haifa, Israel. 

Abstract:  Purpose: The primary toxic effects of methotrexate (MTX) are myelosuppression and/or intestinal mucositis. The objective of the present study is to investigate the effect of MTX on germ cell apoptosis and spermatogenesis in a rat. Methods: Male Sprague-Dawley rats were divided into three experimental groups: control rats treated with vehicle; MTX-2 rats treated with one dose (20 μg/kg) of MTX given IP and killed on the second day; and MTX rats treated with IP MTX (20 μg/kg) and killed on day 4. Johnsen's criteria and the number of germinal cell layers in the testes were used to categorize the spermatogenesis. TUNEL assay was used to determine germ cell apoptosis. Western blotting was used to determine Bax and Bcl-2 protein levels. Statistical analysis was performed using the non-parametric Kruskal-Wallis ANOVA test, with p less than 0.05 considered statistically significant. Results: On day 2, MTX-treated animals demonstrated minimal changes in the histological parameters of spermatogenesis, but germ cell apoptosis increased significantly (threefold increase, p = 0.002) compared to control rats. On day 4, MTX-treated rats demonstrated a trend toward a decrease in germ cell apoptosis, compared to day 2, and showed histological signs of impaired spermatogenesis (decreased number of germ cell layers and Johnsen's criteria). A significant increase in cell apoptosis in MTX-treated rats was correlated with higher Bax/Bcl-2 protein levels. Conclusions: MTX induced germ cell apoptosis and impaired spermatogenesis in rat testes. 

Retracted: No

PubPeer comments:
 This paper is one of at least four papers by the same research group that appear to share the same ERK2 blot to represent different experiments (marked in red). Two of these papers also seem to share another blot (marked with blue boxes). The four papers are: * Ben-Lulu S et al., PLOS ONE (2012), DOI: 10.1371 journal.pone.0045221 * Sukhotnik I et al., Pediatr Surg Int (2013), DOI: 10.1007 s00383-012-3197-0 * Koppelman T et al., BMC Gastroenterology (2012), DOI: 10.1186 1471-230X-12-41 * Koppelman T et al., British Journal of Nutrition (2013), DOI: 10.1017 S0007114512000542 Here is a collage of the four figures, one from each of these papers. ![file] , 
 Figure 1 in this paper also appears to show two panels, shown in cyan and orange, that look similar to panels in an older paper by the same authors, i.e. Sukhotnik et al., Pediatr Surg Int (2008), DOI 10.1007 s00383-007-2042-3 - https: pubpeer.com publications A704F9F94F8290B7A3FD478A2A6A56 Of note, the 2008 Sham panel reflects a sham-operated animal, while the 2013 CONTR animal was only injected with saline. These are very different experiments, presumably done years apart, so it is surprising to see such similar photos. ![file] ,