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Open AcceResearchStructural alterations in the seminiferous tubules of rats treated with immunosuppressor tacrolimusBreno H Caneguim1, Paulo S Cerri2, Lus C Spolidrio3, Sandra M Miraglia1 and Estela Sasso-Cerri*2

Address: 1Department of Morphology and Genetics, Federal University of So Paulo (UNIFESP), So Paulo, Brazil, 2Department of Morphology, Laboratory of Histology and Embryology, Dental School So Paulo State University (UNESP), Araraquara, Brazil and 3Department of Physiology and Pathology, Dental School So Paulo State University (UNESP), Araraquara, Brazil

Email: Breno H Caneguim - [email protected]; Paulo S Cerri - [email protected]; Lus C Spolidrio - [email protected]; Sandra M Miraglia - [email protected]; Estela Sasso-Cerri* - [email protected]

* Corresponding author

AbstractBackground: Tacrolimus (FK-506) is an immunosuppressant that binds to a specific immunophilin,resulting in the suppression of the cellular immune response during transplant rejection. Except forsome alterations in the spermatozoa, testicular morphological alterations have not been describedin rats treated with tacrolimus. In the present study, we purpose to evaluate if the treatment withtacrolimus at long term of follow-up interferes in the integrity of the seminiferous tubules.

Methods: Rats aging 42-day-old received daily subcutaneous injections of 1 mg/kg/day oftacrolimus during 30 (T-30) and 60 (T-60) days; the rats from control groups (C-30 and C-60)received saline solution. The left testes were fixed in 4% formaldehyde and embedded in glycolmethacrylate for morphological and morphometric analyses while right testes were fixed in Bouin'sliquid and embedded in paraffin for detection of cell death by the TUNEL method. The epithelialand total tubular areas as well as the stages of the seminiferous epithelium and the number ofspermatocytes, spermatids and Sertoli cells (SC) per tubule were obtained.

Results: In the treated groups, seminiferous tubules irregularly outlined showed disarrangedcellular layers and loss of germ cells probably due to cell death, which was revealed by TUNELmethod. In addition to germ cells, structural alterations in the SC and folding of the peritubulartissue were usually observed. The morphometric results revealed significant decrease in thenumber of SC, spermatocytes, spermatids and significant reduction in the epithelial and totaltubular areas.

Conclusion: Tacrolimus induces significant histopathological disorders in the seminiferoustubules, resulting in spermatogenic damage and reduction in the number of Sertoli cells. A carefulevaluation of the peritubular components will be necessary to clarify if these alterations are relatedto the effect of FK-506 on the peritubular tissue.

Published: 25 February 2009

Reproductive Biology and Endocrinology 2009, 7:19 doi:10.1186/1477-7827-7-19

Received: 29 September 2008Accepted: 25 February 2009

This article is available from: http://www.rbej.com/content/7/1/19

2009 Caneguim et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundThe immunosuppressive therapy in organ transplantrecipients has included the use of calcineurin inhibitorssuch as tacrolimus (FK-506) and cyclosporine [1].Although the mechanism of action of tacrolimus is similarto that of cyclosporine, tacrolimus is 10 to 100 times morepotent and has been referred as useful therapeutic optionfor the transplant recipients. Tacrolimus is a macrolideimmunosuppressant isolated from fermentation broth ofStreptomyces tsukubaensis [2]. This drug binds to a specificimmunophilin, called FKBP12, forming a complex in thelymphocytes that inhibits calcineurin, a Ca2+- and cal-modulin-dependent serine/threonine phosphatase. Cal-cineurin plays a critical role in interleukin (IL)-2 promoterinduction after T-cell activation [3]. Thus, the tacrolimus-FKBP12 complex prevents dephosphorylation of the cyto-plasmic subunit of nuclear factor of activated T-cell (NF-ATc) by calcineurin, resulting in the suppression of IL-2transcription and inhibition of the cellular immuneresponse during transplant rejection [1]. High levels oftacrolimus in the blood are observed 2 hours after oraladministration [3,4]. This drug (98%) circulates in theblood, bound to plasma proteins, such as albumin, lipo-proteins and globulins [4,5] and accumulates in severaltissues such as lung, spleen, kidney, heart, pancreas, mus-cle and liver [1,3].

Some adverse effects associated to tacrolimus treatmenthave been related in the treated patients, such as nephro-toxicity, neurotoxicity, Diabetes mellitus, gastrointestinaldisturbances and hypertension [1,6,7]. The inhibition ofcalcineurin leads to decrease in the intracellular transcrip-tion of insulin by the pancreatic cells. Thus, hyperglicemiaand post-transplant Diabetes mellitus (PTDM) are the mostcommon metabolic diseases caused by calcineurin inhib-itors such as FK-506 [1,8]. The nephrotoxicity has beenassociated to vasoconstriction, reduction in the glomeru-lar filtration [9] and histopathological alterations, such ashyaline arteriolopathy, striped interstitial fibrosis andtubular atrophy [10-12].

Studies focusing the effects of calcineurin inhibitor immu-nosuppressants, mainly tacrolimus, on the reproductivesystem are scarce in the literature. Some testicular effectshave been demonstrated in rats treated with the other cal-cineurin inhibitor immunosuppressant cyclosporine.Among the effects, reduction in the testicular weight [13],decrease in the diameters of the seminiferous tubules [13-16], decrease in the number of spermatocytes at stage VII[17] and desquamation of round spermatids [16,17] havebeen reported. Additionally, testicular ultrastructuralanalyses revealed the presence of damaged spermatids, aswell as disconnection of the head from the tail of sperma-tozoa and abnormal development of flagella [16]. In con-trast to cyclosporine, testicular alterations have not beenfound in tacrolimus-treated rats for 2 weeks [18,19].

Moreover, the plasma levels of testosterone [18,19] andLH [18] were normal after tacrolimus treatment. How-ever, the number and motility of spermatozoa reducedand the number of live fetus and the embryonic implan-tation index reduced after mating of the treated male ratswith untreated female rats [19]. Since these authors haveobserved degenerative germ cells in the lumen of epidi-dymis, it has been suggested that the quantitative andfunctional alterations in the spermatozoa resulted fromthe effect of tacrolimus in the epididymis rather than theseminiferous epithelium. According to the transplantedorgan, the period of therapy in the patients receiving tac-rolimus-based immunosuppression varies from 1 to 5years of follow-up. In the present study, we purpose toevaluate the structural integrity of the seminiferoustubules in rats treated with tacrolimus at long term (30and 60 days) of follow-up.

MethodsAnimals and treatmentTwenty Holtzman male rats (Rattus norvegicus albinus)aging 42 day-old (weighing around 160 g) were main-tained in plastic cages under 12 h light/12 h dark cycle atcontrolled temperature (23 2C), with water and foodprovided ad libitum. The animal care and the experimentalprocedures were conducted following the national law onanimal use. This protocol was approved by the EthicalCommittee for Animal Research of So Paulo Federal Uni-versity, Brazil (UNIFESP/EPM).

The animals were distributed into four groups: two con-trol groups (C-30 and C-60) and two tacrolimus (Prograf

Janssen Cilag, So Jos dos Campos, SP, Brazil) groups(T-30 and T-60). The rats from T-30 and T-60 wereweighed weekly and received daily subcutaneous injec-tions of 1 mg/kg/day of tacrolimus [20] during 30 and 60days, respectively. This dosage provides plasma peak andthrough levels of tacrolimus of approximately 11.2 ng/ml[21-23] and has been reported to be immunosuppressiveand clinically relevant, resulting in a consistent response[20].

The animals from control groups received subcutaneousinjections of saline solution during the same periods.

Histological proceduresThe rats were anaesthetized with 80 mg/Kg of body weightof Ketamine (Francotar; Virbac do Brazil Ind. e Com.Ltda., So Paulo, SP, Brazil). The testes were removed,weighed and the rats were killed by an overdose of Keta-mine. The right testes were fixed in Bouin's liquid, embed-ded in paraffin and the sections were submitted to TUNELmethod for detection of cell death. The left testes werefixed in 4% formaldehyde (prepared from paraformalde-hyde), at pH 7.4 with 0.1 M sodium phosphate, embed-ded in glycol methacrylate (Historesin-Embedding Kit,

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Jung, Germany) and the sections were stained with Gill'shematoxylin and eosin HE [24] for morphometric anal-yses.

Morphometric analysesSeminiferous tubules cross sections were randomly cho-sen in three non-serial sections per animal, totalizing 100tubules/animal. Taking into account that th

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