European Journal of Histochemistry 2010; volume 54:e43

Immunoreactivity of Thymosyn β4 (Tβ4) is a 43-aminoacid , first isolated in human blood platelets;2 it is Correspondence: Dr. Sonia Nemolato, Diparti - beta 4 in human foetal and probably an important intracellular modulator mento di Citomorfologia, Divisione di Anatomia adult genitourinary tract of polymerization3 due to its capacity to Patologica, University of Cagliari, via Ospedale interact with monomeric actin.1 As an extracel- 54, 09124 Cagliari, Italy. 1 2 1 Tel. +39.070.6092370 - Fax +39.070.657882. S. Nemolato, T. Cabras, M.U. Fanari, lular factor, Tβ4 has been documented to be E-mail: [email protected] F. Cau,1 D. Fanni,1 C. Gerosa,1 involved in hormonal activities and in modu- B. Manconi,2 I. Messana,2 M. Castagnola,3 1,4 lating the immune response. It has also been Key words: thymosin β4, genitourinary tract, 1 G. Faa found to have anti-inflammatory efficacy5 and development. 1Dipartimento di Citomorfologia, to be implicated in cardiac protection,6 angio- Divisione di Anatomia Patologica, genesis,7 wound healing,8 blood coagulation,2 Contributions: SN conceived and designed the 1,4 experiments, acquisition of data, performed the University of Cagliari, Cagliari, Italy; hair growth and apoptosis. experiments, analyzed the data, wrote the manu- 2 Recently, a role for Tβ4 has been described Dipartimento di Scienze Applicate ai script; TC designed the experiments, analyzed in developing foetal salivary glands,9 in coro- Biosistemi, Università di Cagliari, Cagliari, the data, wrote the manuscript; MF, FC designed 10 Italy; 3Istituto di Biochimica e di nary vasculogenesis, and during the develop- and performed the experiments, contributed Biochimica Clinica, Università Cattolica ment of different neural cell types in rat reagents/materials/analysis tools; DF conceived 11 Roma, Italy brains. In mouse embryonic kidneys, Tβ4 and designed the experiments, acquisition of activity has been shown to increase during data, performed the experiments, analyzed the development, remaining at high levels during data, wrote the manuscript; BM, conceived and active nephrogenesis.12 In rat foetal kidneys, designed the experiments, analyzed the data, wrote the manuscript; IM conceived and designed Tβ4 has been identified in differentiating Abstract the experiments, performed the experiments, glomeruli, while in adult rats it has been analyzed the data, wrote the manuscript, critical mainly found in collecting ducts.12,13 High lev- Thymosin beta 4 (Tβ4) is a member of the revision of the manuscript and approval of the beta- family, a family of els of Tβ4 mRNA were detected in normal article; MC, GF conceived, designed and per- playing essential roles in many cellular func- human embryonic kidneys and in renal formed the experiments, analyzed the data, wrote 14 the manuscript, critical revision of the manu- tions. Our recent studies suggested Tβ4 plays a tumors. Recent studies have also provided script and approval of the article. All authors read key role in the development of human salivary new information concerning a role for Tβ4 in glands and the gastrointestinal tract. The aim human glomerulosclerosis and in and approved the final manuscript. 15 of this study was to analyse the presence of T fibrosis in adults. Tβ4 also plays an important β4 Acknowledgements: the authors thank Mr. in the human adult and foetal genitourinary role in the development of the reproductive Iganzio Ferru for his secretarial assistance, and tract. Immunolocalization of Tβ4 was studied system. Its secretion has been shown to be Mrs. Sandra Serra and Mrs. Simonetta Paderi for in autoptic samples of kidney, bladder, uterus, modulated by and their technical support. They also would like to 16-18 , and prostate obtained from four in mice and pigs. Increased levels of Tβ4 in acknowledge the financial support of the “Fon - human foetuses and four adults. Presence of the rat ovary have been correlated with the dazione Banco di Sardegna”, Cagliari, Sardinia, the peptide was observed in cells of different process of luteinization16 under the influence Italy. origin: in surface epithelium, in gland epithe- of human chorionic gonadotropin, which stim- lial cells and in the interstitial cells. T was 17 Conflict of interest: the authors report no conflict. β4 ulates Tβ4 gene expression. The Tβ4 gene is mainly found in adult and foetal bladder in the downregulated at transcriptional level by transitional epithelial cells; in the adult Received for publication: 16 July 2010. androgen activity in cell cul- Accepted for publication: 20 September 2010 endometrium, glands and stromal cells were 19 ture. Presence of Tβ4 has been reported dur- immunoreactive for the peptide; Tβ4 was ing the formation of actin-based pseudopodia This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0). mainly localized in the glands of foetal prostate in tumoral cells, facilitating cell migration and while, in the adults a weak T reactivity was β4 prostate cancer progression.20 Our research restricted to the stroma. In adult and foetal kid- ©Copyright S. Nemolato et al., 2010 has also shown high Tβ4 presence in tumour Licensee PAGEPress, Italy ney, Tβ4 reactivity was restricted to ducts and infiltrating mast cells.21 Overall, our research European Journal of Histochemistry 2010; 54:e43 tubules with completely spared glomeruli; a doi:10.4081/ejh.2010.e43 suggests that Tβ4 has an important role in the weak positivity was observed in adult and development of different embryonic and adult foetal oocytes; immunoreactivity was mainly localized in the interstitial cells of foetal and organs. Due to controversy in the scientific community regarding the role of Tβ4 in health Materials and Methods adult testis. In this study, we confirm that Tβ4 could play a relevant role during human devel- and disease, this study was aimed at verifying opment, even in the genitourinary tract, and if similarities exist between Tβ4 presence dur- Four human foetuses, two males and two reveal that immunoreactivity for this peptide ing development and in adult life, and at dis- females, with gestational age ranging from 14 may change during postnatal and adult life. cussing the putative role played by Tβ4 in both to 27 weeks, and four adults, two males and situations. We also found it necessary to bet- two females 35, 66, 68 and 72 year-old respec-

ter investigate the presence of Tβ4 in the tively, were selected for this study. In all cases, human urinary and reproductive tract of foe- no pathological changes were evident in the Introduction tuses and adults in order to add new scientific organs examined in this study. We obtained data for a more complete description of the tissue samples at autopsy from each subject Beta-thymosins are N-terminally acetylated several, and not completely known, functions including the following segments of the male peptides of 5 kDa molecular mass, which are of this versatile peptide. and female genitourinary tract: kidney, blad- composed of 40-44 amino acid residues.1 der, uterus, ovary, testicle and prostate. Tissue

[European Journal of Histochemistry 2010; 54:e43] [page 193] Original paper samples were fixed in 10% formalin, routinely in the foetal or adult prostatic gland epithelium processed and paraffin-embedded. Immuno - (Figure 4a,b). We found a diffuse immunore- histochemistry was performed on 5 μm-thick activity of Tβ4 in the stroma of adult prostate sections, using the labelled streptavidin-biotin (Figure 4b), but no reactivity was detected in complex system (LSAB2, Dako) in a Dako the foetal prostate stroma (Figure 4a). A fine Autostainer (DakoCytomation, Carpintera, CA, granular positivity was present in the foetal USA). Heat-induced antigen retrieval was car- ovary (Figure 5a), while immunoreactivity of ried out by steaming unstained sections in the peptide was homogeneously weak in adult Target Retrieval Solution (Dako TRS pH 6.1) for 30 min. Tissue sections were incubated (30 Table 1. Immunoreactivity of Tβ4 in differ- min at room temperature) with the monoclon- ent organs and cells of the human geni- al anti-thymosin β4 antibody (Bachem, tourinary tract. Bubendorf, Switzer land). Sections of a reac- Foetus Aduld tive human adult lymph node with activated Kidney 22 macrophages were used as positive controls. Ducts Diffuse Diffuse As a negative control the same procedure was Glomeruli Negative Negative applied omitting the primary antibody. Bladder Transitional epithelium Focal Diffuse Ethics statements Stroma Negative Focal The study protocol and written consent forms Endometrium Glands Negative Diffuse were approved by the Ethics Human Studies Stromal cells Focal Diffuse Committee of University Medical Centre of Prostate Figure 2. Bladder. (a) Foetuses: the developing Cagliari (according to the instructions of the Glands Negative Focal transitional epithelium shows a weak and dif- Declaration of Helsinki). Full written consent Stroma Negative Diffuse fuse cytoplasmic immuoreactivity of Tβ4 forms were obtained from the parents of the Isolated cells Focal Diffuse (arrow). The surrounding stroma shows a mild diffuse immunoreactivity of the peptide. Scale newborns and all rules were respected. For the Ovary bar: 50 µm. (b). Adults: perinuclear coarse gran- specimens from adults, we obtained written Oocyte Focal Diffuse ules, immunoreactive for Tβ4, are detected in consent from their next of kin. Testicle the cytoplasm of transitional epithelium Spermatic ducts Negative Focal (arrow). The stroma shows a focal positivity of Interstitial cells Focal Diffuse the peptide (arrowhead). Scale bar: 50 µm. Results

The immunostaining for Tβ4 appeared homogeneous or granular and was always restricted to the cytoplasm of positive cells. No nuclear reactivity was observed in this study. No significant differences were found in the immunohistochemical pattern for Tβ4 among the four foetuses analyzed, as well as the four adults observed in this study (Table 1). The immunoreactivity for Tβ4 in foetal kidneys par- alleled the immunoreactivity in the adult. A weak diffuse cytoplasmic positivity was detect- ed in developmental and mature ducts while the glomeruli were constantly negative (Figure 1a,b). In the bladder, a weak immunoreactivity of the peptide was observed in the immature transitional epithelium, which changed to a coarse granular reactivity in the mature epithelium of the adult bladder (Figure 2a,b). The surrounding stroma showed mild diffuse immunoreactivity of T in the foetus and focal Figure 3. Endometrium. (a) Foetuses: no β4 immunoreactivity of Tβ4 is observed in devel- in adults (Figure 2b). No immunoreactivity for oping endometrial glands (arrow). Only scat- Tβ4 was observed in foetal endometrial glands. Figure 1. Kidney. (a) Foetuses: a diffuse tered stromal cells show a weak cytoplasmic Only scattered stromal cells showed a weak cytoplasmic immunoreactivity of Tβ4 is immunoreactivity (arrowhead). Scale bar: cytoplasmic immunoreactivity (Figure 3a). A present in developing ducts (arrow). The 100µm. Inset: scattered stromal cells with a immature glomuruli are negative (arrow- cytoplasmic immunoreactivity (arrows). Scale mild, granular cytoplasmic immunoreactivity head). Scale bar: 25 µm. (b) Adults: pri- bar: 25 µm. (b) Adults: a mild granular cyto- of Tβ4 was present in the endometrial glands mary and secondary ducts show a diffuse plasmic immunoreactivity of Tβ4 is observed in of adults, while the absence of any significant cytoplasmic immunoreactivity of the endometrial glands (arrow) while the sur- immunoreactivity was found in the stroma Tβ4(arrow). The glomeruli are negative rounding stroma has no significant immunore- (arrowhead). Scale bar: 25 µm. activity of the peptide. Scale bar: 25 µm. (Figure 3b). No reactivity for Tβ4 was detected

[page 194] [European Journal of Histochemistry 2010; 54:e43] Original paper

oocytes (Figure 5b). Stroma was negative in even in the genitourinary tract, and reveal that tionship between Tβ4 presence and the devel- both adult and in foetal (Figure 5a,b). immunoreactivity for this peptide may change opmental status does not coincide with our The interstitial cells of the adult testicle were during postnatal life with marked differences previous data regarding the gastrointestinal 23 strongly positive for Tβ4 while, in the foetus, between foetal and adult organs. Our previous tract. In previous studies, Tβ4 presence pat- the same cells showed a mild immunoreactivi- studies on Tβ4 protein presence in salivary terns were characterized by a higher presence ty for the peptide (Figure 6a,b). Moreover, a glands9 and in the gastrointestinal tract23 first in the foetus and by a marked decrease in the disclosed the uneven distribution of the pep- mild reactivity of Tβ4 was observed in the sper- adult suggesting a major role of Tβ4 during matic ducts of the adult testicle (Figure 6b). tide in different organs of the same system and embryogenesis. This hypothesis was con- within each organ in the different epithelial firmed by our studies of Tβ4 content in human and mesenchymal structures and cells. 9 saliva, which clearly showed high Tβ4 levels Moreover, in recent years, T presence has β4 during gestation followed by the disappear- been reported to increase in different Discussion ance of the peptide in the adult saliva. Our tumors,24-26 mainly in its infiltrative borders study reinforces our hypothesis that T func- and in their metastastic stage (Nemolato et al, β4 This is the first comprehensive study that unpublished data). The uneven distribution of tions are not totally known and stresses the analyzes Tβ4 presence in the human genitouri- different function this peptide could play in Tβ4 among different organs as well as among nary tract, and compares the presence of this different cell types within each organ is con- different human organs, not only during devel- peptide during development with its reactivity firmed by this study also in the genitourinary opment, but also even during adult life. Some in the adult. We convincingly demonstrate that peculiar features related to the patchy Tβ4 dis- tract. Tβ4 was clearly present in all organs Tβ4 presence is particular for every organ and, examined in this study; it was more diffuse in tribution in different organs deserve to be inside each organ, it appears restricted to cer- kidney, adult bladder and in interstitial cells of noted in: i) kidney restriction of Tβ4 reactivity tain structures or to particular cell types. Many the testis. to ducts and tubules with completely spared studies in experimental animals and recently Interestingly, we found differences in this glomeruli (Figure 1a,b); ii) a positivity in the in humans suggest that Tβ4 could play a rele- study between foetal and mature organs. In oocytes of the ovary (Figure 5a,b); iii) vant role during development.9,23 Our data con- general, we found direct associations between immunoreactivity restricted to interstitial cells firms this hypothesis. In this study, we find presence of Tβ4 and the degree of development in the testis (Figure 6a,b). What is the rela- that Tβ4 is present during intrauterine life in all the organs tested. The presence of a rela- tionship between these very different types of

Figure 4. Prostate. (a) Foetuses: no immu - Figure 5. Ovary. (a) Foetuses: scattered Figure 6. Testicle. (a) Foetuses: no immuno - no reacti vity of Tβ4 is observed in the Tβ4 reactive fine granules are present in reactivity for Tβ4 is observed in the imma- prostate glands and in the surrounding the cytoplasm of the oocyte (arrow). A ture spermatic ducts (arrow). A mild cyto- stroma. Scale bar: 50 µm. (b) Adults: no weak diffuse immunoreactivity of the sur- plasmic immunoreactivity of the peptide is significant immuoreactivity of Tβ4 is rounding stroma is detected. Scale bar: 25 observed in isolated interstitial cells (arrow- detected in the prostate glands. The sur- µm. (b) Adults: a weak and homogeneous head). Scale bar: 25 µm. (b) Adults: a granu- rounding stroma shows a diffuse cytoplasmic immunoreactivity for Tβ4 is lar cytoplasmic immunoreactivity of Tβ4 is immunoreactivity for the peptide (arrow- detected in the oocyte of the primary fol- detected in the interstitial cells of the testicle head). Scale bar: 50 µm. Inset: at higher licle (arrow). A weak diffuse immunopos- (arrow). A weak immunoreactivity is obser - magnification, no significant immunoreac- itivity in the surrounding stroma is ved in the cells of the spermatic ducts (arrow- tivity for Tβ4 is observed in the prostate observed. Scale bar: 25 µm. head). Scale bar: 50 µm Inset: cells of the gland. Scale bar: 25 µm. spermatic ducts with a weak immunoreactiv- ity (arrows). Scale bar: 25 µm.

[European Journal of Histochemistry 2010; 54:e43] [page 195] Original paper cells? For the time being, we have no clear (EPDCs) as cardiac stem cells. Anat Rec A modulation of thymosin genes in the answer. The immunoreactivity of oocytes of Discov Mol Cell Evol Biol 2004;276:43-57. immature rat ovary by . Mol

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