title: Flutamide induces alterations in the cell-cell junction ultrastructure and reduces the expression of Cx43 at the blood-testis barrier with no disturbance in the rat seminiferous tubule morphology.
abstract: BACKGROUND
  Present study was designed to establish a causal connection between changes in the cell-cell junction protein expression at the blood-testis barrier and alterations in the adult rat testis histology following an anti-androgen flutamide exposure. Particular emphasis was placed on the basal ectoplasmic specialization (ES) in the seminiferous epithelium and expression of gap junction protein, connexin 43 (Cx43).
  
  
  METHODS
  Flutamide (50 mg/kg body weight) was administered to male rats daily from 82 to 88 postnatal day. Testes from 90-day-old control and flutamide-exposed rats were used for all analyses. Testis morphology was analyzed using light and electron microscopy. Gene and protein expressions were analyzed by real-time RT-PCR and Western blotting, respectively, protein distribution by immunohistochemistry, and steroid hormone concentrations by radioimmunoassay.
  
  
  RESULTS
  Seminiferous epithelium of both groups of rats displayed normal histology without any loss of germ cells. In accord, no difference in the apoptosis and proliferation level was found between control and treated groups. As shown by examination of semi-thin and ultrathin sections, cell surface occupied by the basal ES connecting neighboring Sertoli cells and the number of gap and tight junctions coexisting with the basal ES were apparently reduced in flutamide-treated rats. Moreover, the appearance of unconventional circular ES suggests enhanced internalization and degradation of the basal ES. These changes were accompanied by decreased Cx43 and ZO-1 expression (p < 0.01) and a loss of linear distribution of these proteins at the region of the blood-testis barrier. On the other hand, Cx43 expression in the interstitial tissue of flutamide-treated rats increased (p < 0.01), which could be associated with Leydig cell hypertrophy. Concomitantly, both intratesticular testosterone and estradiol concentrations were elevated (p < 0.01), but testosterone to estradiol ratio decreased significantly (p < 0.05) in flutamide-treated rats compared to the controls.
  
  
  CONCLUSIONS
  Short-term treatment with flutamide applied to adult rats exerts its primary effect on the basal ES, coexisting junctional complexes and their constituent proteins Cx43 and ZO-1, without any apparent morphological alterations in the seminiferous epithelium. In the interstitial compartment, however, short-term exposure leads to both histological and functional changes of the Leydig cells.
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Chojnacka
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Hejmej
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Zarzycka
authors.affiliation: Department of Developmental Biology and Morphology of Invertebrates, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Waclaw
authors.initials: W
authors.lastname: Tworzydlo
authors.affiliation: Department of Developmental Biology and Morphology of Invertebrates, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Szczepan
authors.initials: S
authors.lastname: Bilinski
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Laura
authors.initials: L
authors.lastname: Pardyak
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Alicja
authors.initials: A
authors.lastname: Kaminska
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland. barbara.bilinska@uj.edu.pl.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bilinska
mesh_terms: D000726:Androgen Antagonists / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D018031:Connexin 43 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism; D005485:Flutamide / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007365:Intercellular Junctions / Q000187:drug effects* / Q000648:ultrastructure; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012671:Seminiferous Tubules / Q000187:drug effects* / Q000473:pathology; D013737:Testis / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D000726:Androgen Antagonists; D018031:Connexin 43; C580592:Gja1 protein, rat; D012333:RNA, Messenger; D013739:Testosterone; D004958:Estradiol; D005485:Flutamide
keywords: Basal ectoplasmic specialization; Cx43; Flutamide; Rat; Testis; Ultrastructure