UNICAM P
UN
I
VERSIDADE ESTADUAL DE CAMP
I
NAS
Instituto de Biologia
BRUNO DOMINGOS AZEVEDO SANCHES
EVALUATION OF THE EFFECTS OF 17β-ESTRADIOL INTRAUTERINE
EXPOSURE ON PROSTATE POSTNATAL DEVELOPMENT IN
MONGOLIAN GERBIL
AVALIAÇÃO DOS EFEITOS DA EXPOSIÇÃO INTRAUTERINA AO
17β-ESTRADIOL SOBRE O DESENVOLVIMENTO PROSTÁTICO PÓS-NATAL
NO GERBILO DA MONGÓLIA
Campinas
2017
I
BRUNO DOMINGOS AZEVEDO SANCHES
EVALUATION OF THE EFFECTS OF 17β-ESTRADIOL
INTRAUTERINE EXPOSURE ON PROSTATE POSTNATAL
DEVELOPMENT IN MONGOLIAN GERBIL
AVALIAÇÃO DOS EFEITOS DA EXPOSIÇÃO INTRAUTERINA AO
17β-ESTRADIOL SOBRE O DESENVOLVIMENTO PROSTÁTICO
PÓS-NATAL NO GERBILO DA MONGÓLIA
Dissertation presented to the Institute of Biology of the University of Campinas in partia l fulfillment of the requirements for the degree of Doctor, in the area of
Cell Biology
Dissertação apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutor em Biologia Celular e Estrutural, na Área de Biologia Celular
Supervisor/Orientador: Prof. Dr. Sebastião Roberto Taboga
ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELO ALUNO Bruno Domingos Azevedo Sanches,
E ORIENTADA PELO PROF. DR. Sebastião Roberto Taboga
Campinas 2017
Agência de fomento e nº do processo: FAPESP, 2013/15939-0
Ficha catalográfica
Universidade Estadual de Campinas Biblioteca do Instituto de Biologia Mara Janaina de Oliveira - CRB 8/6972
Informações para Biblioteca Digital
Título em outro idioma: Avaliação dos efeitos da exposição intrauterina ao 17β-estradiol
sobre o desenvolvimento prostático pós-natal no gerbilo da Mongólia Palavras-chave em Inglês: Prostate - Development Female prostate Sex diferences Telocytes Estradiol
Área de concentração: Biologia Celular
Titulação: Doutor em Biologia Celular e Estrutural
Banca examinadora:
Sebastião Roberto Taboga [Orientador] Murilo Vieira Geraldo
Daniela Carvalho dos Santos Cristiane Damas Gil
Silvia Borges Pimentel de Oliveira Data da defesa: 10-11-2017
Programa de Pós-Graduação: Biologia Celular e Estrutural
Sanches, Bruno Domingos Azevedo, 1990-
Sa55e Evaluation of the effects of 17β-estradiol intrauterine exposure on prostate postnatal development in Mongolian gerbil / Bruno Domingos Azevedo Sanches. – Campinas, SP : [s.n.], 2017.
Orientador: Sebastião Roberto Taboga.
Tese (doutorado) – Universidade Estadual de Campinas, Instituto de Biologia.
1. Próstata Desenvolvimento. 2. Próstata feminina. 3. Sexo -Diferenças. 4. Telócitos. 5. Estradiol. I. Taboga, Sebastião Roberto. II. Universidade Estadual de Campinas. Instituto de Biologia. III. Título.
Campinas, 10/11/2017.
COMISSÃO EXAMINADORA
Prof. Dr. Sebastião Roberto Taboga Prof. Dr. Murilo Vieira Geraldo
Profa. Dra. Daniela Carvalho dos Santos
Profa. Dra. Cristiane Damas Gil
Profa. Dra. Silvia Borges Pimentel de Oliveira
Os membros da Comissão Examinadora acima assinaram a Ata de defesa, que se encontra no processo de vida acadêmica do aluno.
AGRADECIMENTOS
Agradeço primeiramente à minha família pelo apoio emocional recebido durante a realização deste trabalho, bem como ao Professor Sebastião Roberto Taboga pelo apoio intelectual e à Universidade Estadual Paulista (UNESP), à Universidade estadual de Campinas (UNICAMP), à Universidade de São Paulo (USP) pelo apoio instrumental e à Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) pelo apoio financeiro recebido.
ABSTRACT
Prostate development in rodent males has been extensively studied, which involves complex epithelial-mesenchymal interactions between the urogenital sinus epithelium (UGE) and the urogenital sinus mesenchyme (UGM). However, studies on the development of the prostate in females are scarce. In parallel, cells called telocytes have been recently described in the prostate, these are supposed to exert several functions in the development and physiology of various organs, but there are no studies on the role of this cellular type in the prostate. The present study employed 3D reconstructions, histochemical, immunohistochemical and immunofluorescence techniques to describe comparatively the postnatal prostate development among Mongolian gerbil males and females, as well as to evaluate the effects of intrauterine exposure to oestradiol on development and, finally, to investigate the presence of telocytes. We observed that the prostate of the females has its branching and differentiation process induced by a single mesenchyme, which presents a ventrolateral disposition, which we denominate: Ventrolateral Mesenchyme (VLM), in addition, the female prostate shows an earlier pre-maturation than that of male, which could be justified by the higher estrogen sensitivity and the reduction in ERα expression along with the earlier expression of epithelial AR. The female prostate was also more sensitive to intrauterine exposure to 17β-estradiol, it was verified a reduction in the number of prostatic branches and morphological changes due to a low dose exposure of this hormone (500ng.BW/D). In addition, our data point to the occurrence of prostate telocytes closely associated with the periductal smooth muscle, which could indicate a possible role in the differentiation process of this muscle and, later, these cells are verified in the interalveolar region and between the prostatic stroma and the periuretral smooth muscle, which may indicate a role of telocytes in the prostatic tissue organization. Finally, c-Kit positive Interstitial Cajal-like cells (ICLCs) were detected in the stroma and we found evidences that telocytes could be progenitor cells of these. In general terms, our data point to a greater sensitivity of the female prostate to the E2, which becomes worrisome due to the presence of prostate in a portion of the women, moreover, our findings indicate a greater complexity of the prostate stroma at the cellular level, evidenced by the presence of telocytes and c-Kit positive ICLCs.
RESUMO
O desenvolvimento da próstata em machos de roedores tem sido extensivamente estudado, tal processo envolve complexas interações epitélio-mesenquimais entre o epitélio do seio urogenital (UGE) e os mesênquimas do seio urogenital (UGM). No entanto, estudos sobre o desenvolvimento da próstata em fêmeas são escassos. Paralelamente, células chamadas de telócitos foram recentemente descritas na próstata, tais são supostas de exercer uma série de funções no desenvolvimento e na fisiologia de vários órgãos, contudo, faltam estudos sobre o papel deste tipo celular na próstata. O presente trabalho fez uso de reconstruções 3D, técnicas histoquímicas, imunohistoquímicas e imunofluorescências para descrever o desenvolvimento pós-natal prostático de modo comparativo entre machos e fêmeas do gerbilo da Mongólia, bem como avaliar os efeitos da exposição intrauterina ao estradiol sobre o desenvolvimento e, por fim, investigar a presença dos telócitos. Nós observamos que a próstata das fêmeas tem seu processo de ramificação e diferenciação induzido por um mesênquima único, que apresenta uma disposição ventrolateral, o qual denominamos: Mesênquima Ventrolateral (VLM), além disso, a próstata nas fêmeas apresenta uma pré-maturação mais precoce do que a dos machos, o que poderia ser justificado pela maior sensibilidade estrogênica e pela redução da presença do ERα juntamente com a presença mais precoce do AR epitelial. A próstata das fêmeas também se mostrou mais sensível a exposição intrauterina ao 17β-estradiol, de modo a apresentarem redução no número de ramificações prostáticas e alterações morfológicas devido a uma dosagem considerada baixa deste hormônio (500ng.BW/D). Além disso, nossos dados apontam para a ocorrência de telócitos na próstata intimamente associados à musculatura lisa periductal, o que poderia indicar um possível papel destas células no processo de diferenciação desta musculatura e, posteriormente, eles também são verificados na região interalveolar e entre o estroma prostático e a musculatura lisa periuretral, o que pode indicar um papel dos telócitos na organização tecidual prostática. Por fim, foram verificadas células intersticiais semelhantes às células de Cajal (ICLCs) c-Kit positivas no estroma e indícios de que os telócitos poderiam ser células progenitoras destas. Em linhas gerais, nossos dados apontam para uma maior sensibilidade da próstata das fêmeas ao E2, o que se torna preocupante em face da presença de próstata em uma parcela das mulheres, como também indicam uma maior complexidade do estroma prostático em nível celular, evidenciada pela presença de telócitos e ICLCs c-Kit positivas.
LISTA DE ABREVIAÇÕES
α-SMA (Alpha-smooth muscle actin) AR (Androgen receptor)
amp (anterior mesenchymal pad) Bmps (bone morphogenetic proteins) BSA (Bovine serum albumin)
CD31 (Cluster of differentiation 31) CD34 (Cluster of differentiation 34) DAB (diaminobenzidine)
DAPI (4',6-diamidino-2-phenylindole) DHT (dihydrotestosterone)
DMEM (Dubelcco’s modified eagle medium) dmp (dorsal mesenchymal pad)
EDCs (endocrine-disrupting chemicals) EE (Ethinylestradiol)
ERα (Estrogen receptor α) ERβ (Estrogen receptor β) E2 (17β-Estradiol)
H&E (hematoxylin-eosin) FBS (Fetal bovine serum)
FGF-10 (Fibroblast growth factor 10) FITC (Fluorescein isothiocyanate) G-spot (Gräfenberg spot)
HBSS (Hanks’ balanced salt solution) ICCs (Interstitial Cajal cells)
ICLCs (Interstitial Cajal-like cells) IF (Immunofluorescence)
Igf (insulin-like growth factor) IgG (Immunoglobulin G)
MIS (Müllerian inhibiting substance) pam (paraurethral mesenchyme)
PB (Prostatic budding)
PBS (Phosphate-buffered saline)
PCNA (proliferating cell nuclear antigen) PCOS (polycystic ovary syndrome)
PDGFR (Platelet-derived growth factor receptor) PI3K (Phosphatidylinositol-4,5-bisphosphate 3-kinase) PM (periurethral mesenchyme)
PSA (prostate specific antigen) PSAP (prostate acid phosphatase) PSM (Periurethral smooth muscle) SCF (Stem cell factor)
Scr (Proto-oncogene tyrosine-protein kinase sarcoma) SD (standard deviation)
SK3 (Small conductance calcium-activated potassium channel 3) sml (smooth muscle layer)
TCs (telocytes)
TGF-β1 (Transforming growth factor β 1) UGE (Urogenital sinus epithelium)
UGM (Urogenital sinus mesenchyme) VLM (Ventrolateral mesenchyme) VMP (Ventral mesenchymal pad)
SUMÁRIO
1. INTRODUÇÃO ...11
1.1. Aspectos gerais do desenvolvimento prostático...11
1.2. Vias de sinalização parácrinas...16
1.3. Efeitos da exposição estrogênica...17
1.4. Os telócitos prostáticos...18 1.5. Perguntas científicas...21 1.6. Objetivos...21 2. PRODUÇÕES CIENTÍFICAS ...22 2.1. Artigo 1………...22 2.1.1.Introduction………...24
2.1.2.Material and Methods………...27
2.1.3.Results………...30
2.1.4.Discussion………...33
2.1.5.Conclusion...37
2.1.6.Figures and Legends...38
2.1.7.References...46
2.2. Artigo 2………....50
2.2.1.Introduction………...53
2.2.2.Material and Methods………...55
2.2.3.Results………...60
2.2.4.Discussion……….…..65
2.2.5.Conclusion……….….69
2.2.6.Figures and Legends………...70
2.2.7.References……….…..83
2.3. Artigo 3………....89
2.3.1.Introduction………...91
2.3.2.Material and Methods………...94
2.3.3.Results………...96
2.3.4.Discussion……….…..99
2.3.5.Figures and legends...103
2.3.6.References...125 3. DISCUSSÃO ...129 4. CONCLUSÃO ...137 5. REFERÊNCIAS GERAIS ...139 6. ANEXOS...151 6.1. Outras publicações...151 6.1.1. Artigo 4...153 6.1.2 Capítulo 1...181 6.1.3 Artigo 5...203 6.2. Documentos...230
6.2.1. Certificado do Comitê de Ética...230
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1. INTRODUÇÃO
1.1. Aspectos gerais do desenvolvimento prostático
A próstata é uma glândula reprodutiva acessória presente nos mamíferos placentários, que porta uma arquitetura túbulo alveolar cuja principal função é a de produzir uma secreção glicoprotéica alcalina essencial para a capacitação dos espermatozoides e para a sobrevivência dos mesmos via neutralização do pH ácido do trato reprodutivo feminino (Thomson, 2008). A próstata é formada por uma porção epitelial composta um epitélio secretor cilíndrico pseudoestratificado que forma os alvéolos prostáticos (ou ácinos prostáticos), a unidade secretora da glândula, e por ductos que conectam a porção secretora e a uretra, além de um estroma formado especialmente por fibroblastos, que são células especializadas na produção de fibras conjuntivas, células musculares, que envolvem os alvéolos sendo essenciais para a contração dos mesmos, bem como células nervosas, que também atuam na contração dos alvéolos e liberação de secreções, entre outras. Os alvéolos e ductos prostáticos estão envolvidos pelo estroma prostático (Fig. 1), tecido de origem mesenquimal que nutre e dá sustentação a próstata, além de participar ativamente de sua funcionalidade (Thomson, 2008; Prins e Putz, 2008; Sanches et al., 2014).
Em linhas gerais, a próstata é uma glândula de grande importância para o sucesso reprodutivo, o que se evidencia pela sua presença em diversas espécies de mamíferos. Em homens, a próstata a se apresenta como uma estrutura compacta, dividida em zonas, virtualmente, três: zona periférica, central e de transição (Timms, 2008). Diferentemente do que é verificado em roedores e outros mamíferos nos quais a próstata é dividida em lobos. Em ratos, por exemplo, a próstata é dividida nos lobos ventral, lateral e dorsal, além da glândula coaguladora (Timms, 2008). Em gerbilos da Mongólia (Meriones unguiculatus), embora exista uma pequena diferença em termos de classificação dos lobos (que são: lobo ventral, dorso-lateral, dorsal e glândula coaguladora) (Rochel et al., 2007), a morfologia e os aspectos fisiológicos desta glândula são muito semelhantes aos de outros roedores.
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Fig. 1. Seção da próstata do gerbilo da Mongólia corada em HE, na qual se evidenciam os
principais componentes histológicos prostáticos.
O desenvolvimento da próstata em machos é um processo consideravelmente conservado durante a evolução dos mamíferos (Timms, 2008) que inicialmente envolve interações epiteliais-mesenquimais entre o epitélio do seio urogenital (UGE) e o mesênquima seio urogenital (UGM). As interações epitélio-mesenquimais são trocas de fatores de crescimento e diferenciação que se dão inicialmente com fatores mesenquimais que atuam sobre o epitélio, que responde secretando fatores que possuem atuação sobre o mesênquima (Fig. 2). Tal processo é essencial para a formação de vários órgãos (Spemann e Schotte, 1932), entre os quais os pulmões, rins, glândulas mamárias e a próstata (Botchkarev e Kishimoto, 2003; Shannon e Hyatt, 2004; Ribatti e Santoiemma, 2014).
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Fig. 2. (A) Desenho esquemático da próstata em desenvolvimento no qual se evidenciam
interações epitélio-mesenquimais entre as ramificações prostáticas e o VMP. (B) Secção da próstata em desenvolvimento no 3° dia pós-natal corada pela técnica da HE. VMP (Pé mesenquimal ventral), PSM (Musculatura lisa periuretral), UE (Epitélio uretral), Linha tracejada (Brotamento prostático se ramificando no VMP), barra (200 µm).
Durante o período pré-natal, cordões epiteliais sólidos (brotamentos prostáticos) derivados do UGE invadem o UGM, ao mesmo tempo em que o UGM origina três compartimentos distintos: o mesênquima periuretral uma camada de musculatura lisa chamada de musculatura lisa periuretral (PSM), e mesênquimas periféricos do seio urogenital. A continuidade do desenvolvimento dos brotamentos prostáticos (PB) é induzida por estes mesênquimas periféricos do seio urogenital (Cunha, 1973; Thomson e marcador, 2006; Sanches et al, 2014). Tais mesênquimas induzem a ramificação do PBs de uma maneira lobo-específica. O mais estudado desses mesênquimas é Pé Mesenquimal Ventral (VMP), que é responsável pelo desenvolvimento do lobo ventral da próstata (Timms et al., 1995), ao passo que outros mesênquimas específicos induzem o desenvolvimento dos lobos restantes nos roedores (Timms e Hofkamp, 2011). Além do processo de ramificação, a canalização e a diferenciação morfofuncional dos alvéolos também ocorre nesses mesênquimas, que darão origem ao estroma prostático. Em ratos, a diferenciação morfofuncional da glândula ocorre até o final do primeiro mês pós-natal (Fig.3). (Prins e Putz, 2008).
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Fig. 3. Desenhos esquemáticos do desenvolvimento prostático (lobo ventral) e seções da
próstata coradas em HE da fase respectiva do desenvolvimento. (A) Fase de brotamento, na qual projeções do epitélio do seio urogenital (UGE) invadem o mesênquima do seio urogenital (UGM). (B) Fase das ramificações, os brotamentos prostáticos se ramificam no VMP. (C) Fase de canalização/lumenização das ramificações prostáticas e progressiva diferenciação do VMP em estroma prostático. (D) Diferenciação morfofuncional dos alvéolos. VMP (Pé mesenquimal ventral), Seta larga (Sentido de crescimento em direção ao VMP adotado pelos brotamentos prostáticos), PSM (Musculatura lisa periuretral), UE (Epitélio uretral), Linha tracejada preto (Brotamento prostático), Linha tracejada branca (Brotamento prostático se ramificando no VMP), Cabeças-de-seta (Canalização dos ductos prostáticos), barra (200 µm).
Em mulheres e fêmeas de várias espécies também tem sido verificada a existência de próstatas, tais seriam menores do que as observadas em homens e machos de outras espécies. Estudam indicam que pelo menos metade das mulheres possuam próstata (Zaviačič, 1999; Dietrich et al., 2011), ela está localizada na porção distal da uretra e possui secreções semelhantes às verificadas na próstata dos homens, tal teria uma possível função na lubrificação vaginal e na capacitação dos espermatozóides (Santos e Taboga, 2006; Wimpissinger et al., 2007). O desenvolvimento da próstata das fêmeas ainda não foi bem estudado, há evidências de que a maturação da glândula nas fêmeas ocorre antes do que na próstata de machos (Custodio et al., 2004, 2010), uma das razões para a escassez de dados sobre o desenvolvimento da próstata das fêmeas é a baixa ocorrência em próstata nos roedores de laboratórios mais utilizados, como os camundongos e os ratos. No gerbilo da Mongólia (Fig. 4), diferentemente de outros roedores de laboratório, a presença de próstata funcional nas fêmeas está acima de 80% (Santos e Taboga, 2006), nesta espécie a próstata de fêmeas adultas expressam AR e ERα na região periductal, além de apresentarem secreções prostáticas, bem como, grande sensibilidade a flutuações hormonais (Fochi et al., 2013). Assim, o gerbilo da Mongólia é um modelo experimental importante para se investigar as patologias prostáticas em ambos os sexos (Santos e Taboga, 2006).
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Fig. 4. Imagem do roedor de laboratório utilizado nos experimentos: o gerbilo da Mongólia
(Meriones unguiculatus), que é filogeneticamente aparentado ao camundongo.
1.2. Vias de sinalização parácrinas empregadas no desenvolvimento prostático
As interações epitélio-mesenquimais na próstata em ambos os sexos são moduladas pela testosterona e pelos estrógenos que atuam por meio de seus receptores (respectivamente o Receptor de Andrógeno – AR e os receptores de estrogénio 1 e 2 - ERα e ERβ) sobre diversas vias de fatores parácrinos e justácrinos implicadas no desenvolvimento da próstata (Prins e Putz, 2008; Timms e Hofkamp, 2011). Por exemplo, o fator de crescimento de fibroblastos 10 (FGF-10) é regulado pelo estradiol e pela testosterona e é um fator essencial para o desenvolvimento da próstata (Donjacour et al, 2003; Bryant et al, 2014).
O padrão de expressão de FGF-10 em ratos é inicialmente amplo no VMP, mas, posteriormente, condensa-se em torno dos ductos prostáticos em crescimento. O mesênquima em torno das extremidades dos ductos apresenta a expressão mais intensa para o FGF-10 durante a morfogênese das ramificações nos primeiros dias após o nascimento e tal regride antes do fim da primeira semana pós-natal (Prins e Putz, 2008). Ao mesmo tempo, o AR é
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inicialmente expresso no UGM e, com a progressão do desenvolvimento, passa a ser expresso também no epitélio prostático em camundongos (Donjacour e Cunha, 1993) em ratos (Prins e Birch, 1995) e também em homens (Wen et al., 2015). A presença do AR epitelial é também observada na próstata de mulheres adultas (Dietrich et al., 2011). Essa marcação está associada com a atividade secretora da glândula (Donjacour e Cunha, 1993).
Durante o desenvolvimento prostático, a musculatura lisa periuretral além de atuar como uma barreira física que impede a comunicação entre os brotamentos e os mesênquimas condensados indutivos, como o VMP, também é fonte de fatores inibidores de proliferação da próstata, tais como o fator de transformação do crescimento beta 1 (TGF-β1) (Prins e Putz, 2008). Este fator reduz a proliferação epitelial prostática, regula negativamente a expressão do FGF-10, e está implicado na diferenciação das células da musculatura lisa periductal (Niu et al., 2011). No entanto, faltam dados comparativos entre machos e fêmeas acerca do padrão de marcação para esses fatores ao longo do desenvolvimento, bem como uma descrição morfológica das diferenças e similaridades existentes entre o desenvolvimento prostático em machos e fêmeas. No artigo 1, utilizou-se reconstruções tridimensionais (3D), técnicas histoquímicas e ensaios de imunohistoquímica para comparar o desenvolvimento pós-natal da próstata em gerbilos da Mongólia de ambos os sexos. Para se averiguar os possíveis mecanismos moleculares intrínsecos, avaliou-se a relação entre o AR, ERα, FGF-10 e a proliferação celular entre os sexos em dois períodos distintos do desenvolvimento pós-natal.
1.3. Os efeitos da exposição estrogênica sobre o desenvolvimento prostático
Além da falta de dados comparativos entre os sexos sobre os padrões de marcação do AR, ERα, ERβ, FGF-10 e outros fatores implicados no desenvolvimento da próstata ao longo do desenvolvimento prostático pós-natal, são igualmente escassos dados sobre o efeito de hormônios exógenos e de desreguladores endócrinos (ED), fatores capazes de mimetizar a ação hormonal no organismo, sobre tais fatores moleculares e diferenças de susceptibilidade a alterações morfofisiológicas decorrentes de manipulações hormonais entre os sexos. Hormônios presentes no ambiente e EDs apresentam grande potencial para provocar alterações no sistema endócrino de várias espécies, incluindo nos humanos (Diamanti-Kandarakis et al, 2009; Combalbert e Hernandez-Raquet, 2010; Rebuli e Patisaul, 2015; Perez et al, 2016). A presença de esteroides em formas ativas no ambiente também pode gerar preocupação devido
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à elevada afinidade destes compostos pelos receptores de esteróides presentes nos animais e a presença de poucos estudos sobre o assunto, bem como sobre o efeito desses hormônios em baixas concentrações (Combalbert e Hernandez-Raquet, 2010; Anderson et al, 2012; Manickum e John, 2013; Chen e Chou, 2016).
As concentrações do estradiol (E2) presentes no esgoto e no solo variam muito entre os países (Combalbert e Hernandez-Raquet, 2010) e entre os diferentes sistemas de tratamento (Silva et al., 2012). Apesar dos avanços na remoção de hormônios, mesmo em países desenvolvidos se atesta a presença de E2 ativo na água tratada (Anderson et al., 2012). Mais preocupante ainda é a presença de E2 no solo devido à atividade pecuária, já que os dados a este respeito são escassos (Combalbert e Hernandez-Raquet, 2010). Dentre os esteroides, a presença de E2 ativo no ambiente se torna particularmente preocupante devido aos dados que apontam para grandes alterações no desenvolvimento de vários órgãos, tais como as glândulas mamárias (Fenton et al, 2011.), testículos e próstata (Säntti et al., 1994; Delbès et al., 2006; Prins et al., 2007, Saffarini et al., 2015) em decorrência da exposição estrogênica intrauterina. Na próstata especificamente a estrogenização intrauterina afeta a morfogênese dos brotamentos, causando hipomorfia em machos (Prins, 1992; Prins et al, 2006; Saffarini et al, 2015), bem como aumenta a susceptibilidade da próstata a tumorigênese com a senescência (Prins et al, 2007; Perez et al, 2012; Perez et al., 2016). Além disso, tal exposição também está associada com mudanças na fase de ramificação da próstata (PU et al., 2004; Saffarini et al., 2015). Em face disso, o artigo 2 utiliza técnicas de imunohistoquímica e reconstruções tridimensionais para avaliar os efeitos da exposição estrogênica intrauterina em diferentes dosagens sobre o desenvolvimento pós-natal da próstata do gerbilo em ambos os sexos.
1.4. Os telócitos prostáticos
Outro aspecto pouco investigado é o papel dos telócitos (Fig. 5), células estromais categorizadas recentemente (Popescu and Faussone-Pellegrini, 2010) e também presentes na próstata (Corradi et al., 2013), bem como o possível efeito da manipulação hormonal sobre tais células. Além da próstata, tais estão presentes também em diversos outros órgãos, como no coração, útero, glândulas mamárias, pulmões, entre outros (Kostin, 2010; Carmona et al., 2011, Creţoiu et al., 2012; Nicolescu et al, 2011; Nicolescu e Popescu, 2012; Rusu et al, 2012; Zheng et al., 2012; Ullah et al., 2014). Em termos morfológicos, os telócitos apresentam o corpo
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celular reduzido, em formato fusiforme, além de possuírem longas projeções citoplasmáticas, os telopódios, divididas entre dilatações, os podomos, e seções fibrilares, os podômeros (Popescu and Faussone-Pellegrini, 2010; Corradi et al. 2013). Aos telócitos foram propostas diversas funções que variam de órgão para órgão, como o papel organizacional do estroma por meio da modulação da comunicação intercelular (Rusu et al., 2012), na regeneração no músculo cardíaco (Kostin, 2010), na atuação na função imune no duodeno (Carmona et al., 2011), na contratibilidade do útero (Creţoiu et al., 2012) e na comunicação elétrica intercelular (Edelstein and Smythies, 2014), entre outras. Contudo, a função dos telócitos na próstata segue ainda pouco conhecida.
Fig. 5. Imagem de microscopia de luz de telócitos. (A) Secções histológicas da próstata de
gerbilo da Mongólia coradas em HE, na qual pode se observar um prolongamento de um telócito com seu aspecto monoliforme na região periductal. (B) Telócitos prostáticos em cultura de células após 96h de cultivo, na qual se verifica a morfologia alongada de tais células. Ep (Epitélio prostático); Setas (Prolongamentos dos telócitos); Barra longa (10 µm); Barra curta (200µm).
A conceituação original dos telócitos abrange parte das então chamadas células intersticiais semelhantes às células de Cajal (ICLCs) (Popescu and Faussone-Pellegrini, 2010) que são células que foram detectadas em vários órgãos além do trato gastrointestinal, e que são similares morfologicamente às células intersticiais de Cajal (ICCs), as quais foram descritas pela primeira vez por Ramon y Cajal (1911) e existiriam exclusivamente nos órgãos do trato gastrointestinal. Às ICCs foi atribuída a função de marca-passo da contração da musculatura lisa dos órgãos do trato gastrointestinal; sendo, portanto, células importantes na regulação do peristaltismo (Keith, 1915; Thuneberg, 1982). Tanto as ICLCs quanto as ICCs possuem o c-Kit como principal marcador (Shafik et al., 2009), porém há evidências de que tais células
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constituem um grupo heterogêneo de células, e uma parcela delas não expressa o c-Kit, portando marcação apenas para o CD34.
Mais recentemente, as ICLCs CD34 positivas ou CD34 e c-Kit simultaneamente positivas foram categorizadas como um grupo distinto de células ao qual foi dado o nome de telócitos (Popescu and Faussone-Pellegrini, 2010). No entanto, verificam-se também na próstata de ICLCs c-Kit positivas, que não estão incluídas na definição de telócitos, desse modo, a própria natureza dos telócitos como células distintas e a relação entre os telócitos e estas outras ICLCs permanece um assunto aberto. O CD34 é um marcador típico de células progenitoras, desse modo foi proposto que as células ICLCs CD34 positivas, posteriormente chamadas de telócitos, seriam células progenitoras das ICCs ou das ICLCs c-Kit positivas e a sobreposição destes fatores ocorreria em células em estágio intermediário de diferenciação (Wang et al., 2009).
Mas, independentemente de os telócitos serem ou não células progenitoras das ICLCs c-Kit positivas, isto não invalidaria as evidências de que os telócitos possam exercer, como supracitado, diversos outros papéis no desenvolvimento e na manutenção do estroma em vários órgãos (Kostin, 2010; Carmona et al., 2011; Popescu et al., 2011; Creţoiu et al., 2012; Corradi et al., 2013; Edelstein and Smythies, 2014; Ullah et al., 2014; Chi et al., 2015). Porém, o papel dos telócitos ou ICLCs CD34 positivas na próstata se mantém elusivo, bem como faltam informações sobre o desenvolvimento dessas células no tecido prostático. Em face disso, o
Artigo 3 teve por objetivo avaliar a presença de telócitos ao longo do desenvolvimento
prostático e a relação com as ICLCs c-Kit positivas por meio de ensaios de imunofluorescência para o CD34 e para o c-Kit, bem como a relação entre os telócitos e o desenvolvimento prostático, especialmente no que tange a organização tecidual e o desenvolvimento da musculatura lisa periductal/perialveolar.
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1.5. Perguntas científicas
O presente estudo visa responder quatro perguntas científicas:
1) Haveria diferenças no desenvolvimento prostático pós-natal entre fêmeas e machos do
gerbilo da Mongólia?
2) A exposição intrauterina ao estradiol poderia causar diferentes efeitos entre a próstata
de machos e fêmeas do gerbilo da Mongólia?
3) Os telócitos prostáticos exerceriam um papel no processo de organogênese prostática? 4) Seriam os telócitos progenitores de ICLCs c-Kit positivas na próstata?
1.6. Objetivos
Tivemos por objetivos descrever comparativamente o desenvolvimento pós-natal da próstata em machos e fêmeas de gerbilo da Mongólia em termos morfológicos, avaliar os efeitos da exposição estrogênica intrauterina sobre o desenvolvimento prostático, especialmente sobre a morfologia e sobre os seguintes fatores implicados no desenvolvimento prostático: AR, ERα, ERβ e TGF-β1. Por fim, objetivamos avaliar a presença de telócitos ao longo do desenvolvimento pós-natal da próstata, uma possível função para estas células e a relação das mesmas com as ICLCs c-Kit positivas.
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2. PRODUÇÕES CIENTÍFICAS
▪ 2.1. ARTIGO 1
The Androgen Receptor and Estrogen Receptor Alpha immunolabeling pattern is related
to Sex Dimorphism in the Gerbil Prostate Development
Bruno D A Sanches
1, Juliana S Maldarine
2, Bruno C Zani
2, Manoel F Biancardi
1,2,
Fernanda C A Santos
3, Rejane M Góes
1,2, Patricia S L Vilamaior
2, Sebastião R
Taboga
1,21Department of Structural and Functional Biology, State University of Campinas, Av. Bertrand
Russel s/n, Campinas, São Paulo, Brazil
2Univ. Estadual Paulista – UNESP, Department of Biology, Laboratory of Microscopy and
Microanalysis, Rua Cristóvão Colombo, São José do Rio Preto, São Paulo, Brazil
3Department of Morphology, Federal University of Goias, Samambaia II, Goiania, Goias,
Brazil
*Corresponding author: Dr. Sebastião R. Taboga, Department of Biology, Laboratory of Microscopy and Microanalysis, São Paulo State University. Rua Cristóvão Colombo 2265, Jardim Nazareth, São José do Rio Preto, São Paulo, Brazil. CEP 15054-000, Brazil. E-mail: taboga@ibilce.unesp.br
This dissertation text is adapted from an article published as:
Sanches BDA, Maldarine JS, Zani BC, Biancardi MF, Santos FCA, Góes RM, Vilamaior PSL. and Taboga SR. 2016. The expression of the androgen receptor and estrogen receptor 1
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ABSTRACT
The development of the prostate gland in females has not yet been clearly elucidated, and the sexual dimorphism associated with such gland development in general is far from being understood. In the present study, we used tri-dimensional (3D) reconstructions and histochemical and immunohistochemical techniques to describe the sexual dimorphism and its causes in the early postnatal development of the prostate in male and female Mongolian gerbils (Meriones unguiculatus). We observed that the female prostate was smaller, had fewer branches throughout the development, and underwent differentiation earlier than that in males. Also, the presence of estrogen receptor alpha (ERα) and fibroblast growth factor 10 (FGF-10) was decreased in the periductal region, and the presence of the androgen receptor (AR) was increased in the epithelium. All together, these changes decreased proliferation and branching and led to an earlier pre-maturation of the female prostate. These new data shed light on the underlying mechanisms involved with the sexual dimorphism in the development of the prostate.
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2.1.1. INTRODUCTION
The initial development of the prostate involves complex epithelial-mesenchymal interactions that occur between the urogenital sinus epithelium (UGE) and the urogenital sinus mesenchyme (UGM) (Cunha, 1973; Timms, 2008; Sanches et al, 2014.). Solid projections from the UGE, or prostatic buds (PB), invade the UGM, which in turn differentiates into three separate compartments: the periurethral mesenchyme, located in contact with the UGM; a layer of periductal smooth muscle layer (SM) that surrounds the UGM; and peripheral mesenchymes external to the periurethral smooth muscle layer (PSM) (Thomson and Marker, 2006; Sanchez et al, 2015.). In males, the PB invades the periurethral mesenchyme (PM) and reaches the peripheral mesenchymes before the closing of the PSM. Once inside these mesenchymes, which are lobe-specific, the PB branch and their distal ends dilate. After that, the PB undergo canalization and differentiate into prostatic alveoli. The proximal portions of the PB give rise to the conductive portion of the gland, and the peripheral mesenchymes differentiate into the prostatic stroma (Prins and Putz, 2008). The epithelial-mesenchymal interactions in the prostate involve testosterone and estradiol, which modulate several paracrine and juxtacrine factors (Prins and Putz, 2008; Timms and Hofkamp, 2011) by means of the androgen receptor (AR) and the estrogen receptors (ER-alpha and -beta). The fibroblast growth factor 10 (FGF-10) is regulated by these hormones and is an essential factor for the prostate development (Donjacour et al, 2003; Bryant et al, 2014).
The immunolabeling pattern of FGF-10 in rats is initially broad in VMP, but subsequently condenses around the elongating ducts showing the strongest presence at the distal tips during the branching morphogenesis in the first days after birth and recedes before the end the first postnatal week (Prins and Putz, 2008), At the same time, the AR, that is primarily expressed in the mesenchyme, also becomes expressed in the epithelium, which is observed in mice (Donjacour and Cunha , 1993) in rats (Prins and Birch, 1995) and in the developing prostate of men (Wen et al., 2015). Epithelial AR prostate is also observed in adult women (Dietrich et al., 2011). Such marking is associated with the epithelial secretory activity of the gland (Donjacour and Cunha, 1993).
As previously shown (Huang et al., 2005), the immunolabeling pattern of FGF-10 in the mesenchymal pad is broad during early stages of ductal budding and elongation and
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subsequently condenses tightly around the elongating ducts with strongest presence at the distal tips during active branching morphogenesis
There is an extensive literature on the development of the prostate in males (Prins and Birch, 1995; Thomson et al, 2002; Timms and Hofkamp, 2011; Sanches et al, 2014); however, studies in females are still scarce, mainly because female rodents used in research do not have a functional prostate (Thomson et al., 2002). In view of this practical limitation, the female Mongolian gerbil, which has a large functional prostate, constitutes a promising experimental model for research on the female gland. Moreover, breeding and handling of this strain are an easy task in the laboratory setting (Santos and Taboga, 2006; Sanches et al, 2014, 2015; Jesus et al, 2015).
Morphological and physiological data indicate that the maturation of the prostate gland in gerbils occurs earlier in females than that in males (Custodio et al., 2004, 2010). Researchers believe that the maturation depends on a specific inductive mesenchyme, the ventrolateral mesenchyme (VLM), to which the PB migrate still in the prenatal period (Sanches et al., 2016). Nevertheless, studies evaluating the sexual dimorphism in the development of the prostate and the underlying mechanisms are still necessary.
The presence of a prostate in female mammalians and Mongolian gerbils is an obscure and controversial subject, but morphophysiological evidence confirms that the paraurethral glands, or the Skene's glands, are, in fact, the prostate gland in females (Tepper et al, 1984; Pollen and Dreilinger; 1984; Zaviačič, 1999; Santos and Taboga, 2006; Sanches et al., 2016). Nonetheless, the smooth muscle hypothesis suggests that lower levels of testosterone in females could lead to a premature closure of the SM, preventing the communication between the PB and the inductive mesenchyme, which would justify the absence of a functional prostate gland in female rats (Thomson et al., 2002).
The evaluation of sexual dimorphism in the development of the Mongolian gerbil prostate is based on similarities observed between the female gerbil and woman prostates (Santos and Taboga, 2006; Custodio et al, 2010). The similarities consist in the fact that the prostate in women and in gerbil females comprise a cluster of alveoli and ducts that are concentrated on both sides of the urethra. Moreover, the female prostate in gerbils, as in humans, expresses epithelial AR (Santos and Taboga, 2006), stromal ERα (Dietrich et al., 2011), PSA and PSAP (Custos et al., 2004). The difficulties and ethical issues associated with the study of the woman prostate (Zaviačič, 1999; Zaviačič et al, 2000) make the gerbil an
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excellent model to examine prostatic pathologies that resemble those in humans (Santos and Taboga, 2006). Also, the investigation of the underlying molecular mechanisms could help us to understand the sexual variations in the development of prostatic pathologies that result from the exposure to endocrine disruptors and steroids (Santos and Taboga, 2006; Perez et al, 2011, 2012), or from lifestyle habits (Jesus et al, 2015).
In the present study, we used tri-dimensional (3D) reconstructions, histochemical techniques and immunohistochemical assays to compare the postnatal early development of the prostate in male and female Mongolian gerbils. To understand the intrinsic molecular mechanisms, we evaluated the relationship between the AR, ERα, FGF-10 and cell proliferation.
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2.1.2. MATERIALS AND METHODS
Animals and Experimental Design
Animals used in this study were provided by the Univ. Estadual Paulista (UNESP), São Jose do Rio Preto, SP, Brazil. They were maintained in polyethylene cages under controlled conditions of light and temperature and were provided with filtered water and rodent food ad libtum. Handling of the animals and experiments were conducted in accordance with UNESP ethical guidelines (Ethical committee CEUA number 116/2015) and the Guide for Care and Use of Laboratory Animals (The National Academies Press, 2011, Washington, DC, USA). We used 42 adult female and 42 adult male Mongolian gerbils (Merionis unguiculatus, Gerbilinae: Criscetidae) between 3 and 6 months of age. They were randomly paired and then subdivided into 14 experimental groups, each one composed of 3 pairs. The groups were distributed in a way that covered the early postnatal development of the female prostate: P1, P3, P5, P7, P14, P30 and P45, where "P" represents the postnatal period and the numbers represent the day in that period. In order to cover the initial prostatic postnatal development, we use the P1 group representing the neonate stage; P3, P14 and P30 groups, the pre-pubertal stage and the P45, the onset of puberty. The puberty is more delayed in the gerbil (Fochi et al., 2013) than in the rat (Putz and Prins, 2008), which can be associated with the generation time longer in gerbils compared to the rats. The animals were later sacrificed by lethal injection containing a mixture of an anesthetic, ketamine (100 mg / kg bw, Dopalen, Vetbrands, Brazil), and a muscle relaxant, xylazine (11 mg / kg bw, Rompun, Bayer , Brazil). Lastly, 6 puppies from 3 different pairs were obtained for each experimental group and their prostatic complexes were dissected and removed after sacrifice.
Light Microscopy
The female prostatic complexes were fixed in paraformaldehyde 4%, during 12 hours, or Metacarn (methanol, chloroform and acetic acid in the ratio 6:3:1) for 24 hours. Then, samples were dehydrated in ethanol series, cleared in xylene, and finally embedded in paraffin (Histosec, Merck Dermstadt, Germany) for 3 hours. Samples fixed in paraformaldehyde 4% were evaluated for the presence of the ERα, proliferating cell nuclear antigen (PCNA), AR, and FGF-10 by immunohistochemistry, and samples fixed in Metacarn were employed for
28
morphological analyses and 3D reconstructions. All prostatic complexes were serially sectioned (5μm) in a rotary microtome and mounted on histological slides. Tissue sections were subjected to hematoxylin-eosin (H&E) staining for general morphological analyses or were used for immunohistochemical assays. The specimens were analyzed with an Olympus BX60 light microscope (Olympus, Japan), and the resulting images were digitalized by using the DP-BSW V3.1 (Olympus) and Image-ProPlus 6.1 for Windows (Media Cybernetics, Silver Spring, MD) software.
Immunohistochemistry
Tissue sections were subjected to immunohistochemistry assays to detect the AR, as described in the protocol applied to the prostate (adapted from Cordeiro et al., 2008), ERα, PCNA and FGF-10. The following antibodies were used at a dilution of 1:100: AR (rabbit polyclonal IgG, N-20, sc-816, Santa Cruz Biotechnology, CA, USA), ERα (rabbit polyclonal IgG, MC 20, sc-542, Santa Cruz Biotechnology), PCNA (monoclonal mouse IgG2a, PC10, SC-56, Santa Cruz Bio- technology) and FGF-10 (goat polyclonal, IgG, C-17, sc-7375, Santa Cruz Biotechnology). PolyK4061 (Dako Dako Envision, North America, Carpinteria, USA) was used as a secondary antibody. Samples were incubated for 45 minutes at 37°C and were counterstained with diaminobenzidine (DAB) and Harris Hematoxylin. Finally, histological sections were examined under an Olympus BX60 Light Microscope (Olympus). Negative controls were obtained by omitting the step of incubating the primary antibody and the other steps are the same.
Three-dimensional Reconstruction
Three-dimensional reconstructions were performed for all experimental groups in order to obtain detailed 3D images of the branching and morphofunctional differentiation of the male and female prostates. Serial histological sections of the prostatic complexes (5 μm) were analyzed and photographed with an Olympus BX60 light microscope (Olympus, Japan). The resulting images were digitalized by using the DP-BSW V3.1 (Olympus, Japan) and the Image-ProPlus 6.1 for Windows (Media Cybernetics, Silver Spring, MD) software. Later, the images were reconstructed by using Reconstruct software (Fiala, 2005).
29
Quantification of immunohistochemical assays and morphometry
For the purposes of quantification of immunohistochemical assays, the total number of cells in 30 random microscopic fields was counted for each experimental group in 3 different animals, according to the methodology described in Fochi et al. (2013); this was applied for PCNA, ERα and AR in order to separate the positively and negatively marked cells in 30 random microscopic fields per animal for each group of 3 different animals. A minimum of 1000 cells were counted per group and then the percentage of immunostaining was calculated as the number of positive cells divided by the total number of cells. FGF-10 labelling was assessed qualitatively. Quantitative results are expressed as mean ± standard deviation (SD).
Statistical analysis
Data were initially studied by analysis of variance (One-way ANOVA) and subsequently by the Tukey test for multiple comparisons, with a significance level of 5% (p ≤ 0.05). Statistical tests were performed using Statistica 7.0 software (StarSoft Inc., Tulsa, OK, USA).
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2.1.3. RESULTS
Branching varies between males and females in terms of inductive mesenchyme, the number of branches, and the time it begins.
Our results showed that branching is distinct in males and females. In males, the inductive mesenchyme associated with branching and the development of the ventral lobe was the ventral mesenchymal pad (VMP) (Figure 1A), whereas in females, this process was associated with the ventrolateral mesenchyme (VLM) (Figure 1D). Buds that arose from the male VMP had a V-shape (Figures 1A and 1C), and buds from the female VLM grew parallel to the urethral axis (Figures 1B and 1D). Branching began earlier in males (P1) (Figure 1A) than that in females (P5) (Figure 1F). We detected a strong cell proliferation in the branches of both sexes, especially in males (Figures 1H and 1G). Furthermore, at birth, the periurethral musculature in females was completely formed, and all the buds reached their inductive mesenchyme during the prenatal period, differently from what was observed in males (Figures 1A and C).
After the canalization, the prostate development occurs earlier in females than that in males.
As the prostate developed, the PB underwent canalization and formed the ducts of the gland in its proximal portion and the alveolar lumen in its distal ends. This process began earlier in females (P3) (Figure 1D) than that in males (P7) (Figure 1G) and was characterized by apoptotic activity (Figures 1G and 1J). In males and females, on P14, the distal ends of the branches generated the alveoli, and a strong canalization was observed in these areas. Accumulation of stromal fibers in the stroma indicated the progressive differentiation of the gland (Figures 1I and 1J). On P30, in both sexes, we identified developing alveoli containing luminal secretion and the SM. In females, however, the simple cuboidal epithelium was highly differentiated and the alveoli were more developed, suggesting that the differentiation happened earlier in females than that in males (Figures 1K and 1L). On P45, we observed highly differentiated alveoli in both sexes (Figures 1M and 1N).
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During the postnatal development, the developing prostate arises from a more posterior region of urethra, proximal to bladder, and extends to a more anterior region.
In both sexes, taking the male ventral prostate as a parameter, the prostate initially developed in a more posterior region of the urethra, proximal to the bladder (Figures 2A and B). Throughout its development, the prostate progressively branched and extended up to the most anterior regions of urethra (Figures 2C to 2N). The single lobe of the female prostate did not develop completely (Figure 2H). It usually had a bilateral conformation (Figures 2J to 2C) and smaller and fewer alveoli (Figures 2M and 2N). Moreover, the female prostate differentiation was faster than that in males (Figure 3).
The number of AR positive cells are higher in males than in females in the developing prostate.
In males, on P7, the AR was highly expressed in the stroma and epithelium (Figure 4A), coinciding with a strong cellular proliferation. On P30, stromal periductal cells showed strong staining, while the immunolabeling in the epithelium was weak (Figure 4C). In females, on P7, the number of AR positive cells was lower than in males and restricted to the stroma (Figure 4B). On P30, we detected its presence in the epithelial cells (Figure 4D). Albeit there is a slight decrease in the in the percentage of stromal AR positive cells throughout the development in both sexes (Fig.4R, male varying from 89±4 to 75,8±3, female from 60,4±4 to 54,6±1 between P7 and P30 respectively).
ERα is verified during the prostate development in both sexes, and its presence decreases as the gland differentiates.
In males, on P7, the ERα is present in the epithelial and stromal compartments (Figure 4E), whereas, on P30, it was (Figure 4G) less verified in the stroma. In females, on P7, the receptor was highly present in the stroma and less verified in the epithelium (Figure 4F). On P30, its presence was slightly decreased in the stroma, and the receptor was not detected in the epithelium (Figure 4H). In both sexes a decrease in the percentage of ERα positive cells is verified (Fig.4Q, male varying from 67,9±7 to 25,8±4, female from 81,7±6 to 44,6±4 between P7 and P30 respectively).
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In both sexes, the FGF-10 is observed in the periductal mesenchyme and its presence diminishes as the epithelium differentiates.
In males, on P7, FGF-10 immunolabeling was limited to the periductal stroma (Figure 4I) , and, on P30, its presence was decreased (Figure 4K). In females, on P7, the immunolabeling of the FGF-10, though lower than that in males, had a similar distribution; ie, it was localized to the periductal region (Figure 4J). On P30, as seen in males, the presence of this growth factor was decreased (Figure 4L). In this stage, we identified functional alveoli in both sexes (Figures 1K and 1L).
The cell proliferation decreases with the morphological and functional differentiation of the prostatic alveoli.
In males, on P7, the PCNA was highly present in the prostatic stroma and epithelium of the branches (Figure 4M). On P30, PCNA immunolabeling was high especially in the basal epithelial cells of the prostatic alveoli and low in the stroma (Figure O). In females, on P7, like in males, the PCNA was highly verified in the stroma (Figure 4N). On P30, its presence decreased and it was restricted to a few epithelial and stromal cells (Figure 4D). In face of the importance of ERα to female prostate postnatal development, and of stromal AR to male prostate development, the more pronounced decrease of ERα presence in females (-45,4%) compared to the smaller decrease of AR presence in males (-15,2%) could justify, partially, the greater decrease of the proliferative stimulus on epithelium in females (Fig.4R, male varying from 92,2±3 to 58,4±12, 36,6% of decrease, and female from 52,2±3 to 19,6±5, 62,4 of decrease, between P7 and P30 respectively in the percentage of PCNA positive cells) and on stroma (Fig.4S, male varying from 86,3±5 to 61,3±17, 28,9% of decrease, and female from 64,3±6 to 17,4±4, 72,9% of decrease, between P7 and P30 respectively in the percentage of PCNA positive cells). The decrease of ERα presence in females can also possibly justify the earlier onset of the gland pre-maturation verified in females in comparison to males.
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2.1.4. DISCUSSION
The development of the prostate depends on epithelial-mesenchymal interactions between the PB and the inductive mesenchymes that surround the PSM. This muscle layer is considered an important modulator of the prostate development (Thomson et al 2002, Thomson and Marker, 2006; Timms and Hofkamp, 2011) because it may prevent these interactions and, consequently, its development. Thomson and collaborators speculated that the rapid differentiation of the smooth muscle layer in female rats, possibly due to a low concentration of testosterone, could halt the development of a functional prostate. They suggested that the PB would recede without reaching the inductive mesenchyme, where the prostatic cell differentiation would take place (Thomson et al., 2002). However, in the female Mongolian gerbil, the presence of a functional prostate is nearly ubiquitous (Santos and Taboga, 2006), indicating that, unlike in other laboratory rodents, the PB are able to reach their respective inductive mesenchymes and undergo prostatic cell differentiation.
Studies in our laboratory indicate that the PB of the female Mongolian gerbil reach the inductive mesenchyme, the VLM still in the prenatal period, leading to the development of the prostate during postnatal period (Sanches et al., 2016 unpublished data). In this study, we have seen that, in males, a dozen of buds reach the VMP, while in females, usually two PB reach their inductive mesenchyme.
The prostatic development in the Mongolian gerbil occurs later than that seen in other laboratory rodents. The first epithelial buddings in mice, rats and Mongolian gerbils appear around the 17th, between the 19th and 20th, and between the 20th and 21st day of intrauterine life, respectively. This difference may be caused by a longer gestation time in gerbils (Sanches et al., 2014). The postnatal development also occurs later in the Mongolian gerbil. In rats, functional alveoli are present as early as the 10th day of the postnatal life (P10) (Prins and Putz, 2008). In our study, we confirmed the presence of functional alveoli in male and female gerbils only on P30.
We have already demonstrated that there is sexual dimorphism in the development of the prostate and that the differentiation of the gland happens earlier in females than that in males. For example, acid phosphatase, an enzyme that indicates the functional differentiation of the prostate, is active earlier in females (Custodio et al., 2004; Santos et al, 2006), and the analysis of postnatal development of the female prostate showed a faster cell differentiation
34
(Sanches et al., 2016). At birth, the buds of males are branching in the VMP, and those of females undergo this process only on P5. The canalization occurs earlier in females (P3) than that in males (P7), and, on P30, the alveoli of females have a more differentiated morphology, ie, the alveoli are lined by a cuboidal epithelium similar to that observed in the adult gland. In this work, the 3D analysis permitted us to understand the spatial arrangement of the prostate during its postnatal development, which is initially restricted to the proximal region of the bladder and later proceeds to its distal region. In structural terms, the gerbil female prostate shows during development a more distal disposition, which is similar to that seen in the prostate gland of women, however, the disposition is dorsolateral in women while is ventrolateral in the gerbil females. Another difference is the presence of other glandular structures associated with urethra in women, especially in the distal portion, which do not express the typical prostate markers constituting distinguished paraurethral glands (Dietrich et al., 2011).
The reason for the occurrence of a functional prostate in the Mongolian female gerbil remains unclear; nevertheless, their hormonal dynamics could be a key element. Serum testosterone levels in female Mongolian gerbils, for example, range from 0.4 to 2.7 ng/mL (Santos et al., 2006), while in female rats, the average concentration does not reach 0.2 ng/mL (Albert, Jonik and Walsh, 1990). Testosterone levels in female gerbils, however, are still much lower than in male gerbils (Santos et al., 2006). Our work shows that, in female, there is a lower presence of the AR on P7 comparatively to male, and that the presence of this receptor can be stronger in the prenatal period, which may explain why the PB reach the VLM in this period. The smooth muscle hypothesis (Thomson et al., 2002) proposes that high levels of testosterone could delay the closure of the periductual smooth muscle layer and permit the branching of the epithelial buds towards the VLM, as observed in males. However, at birth, this layer is already closed in the Mongolian gerbil female, like in other female experimental rodents. We thus believe that the smooth muscle hypothesis is not a plausible explanation for these facts.
Other factors could be implicated in the development of the prostate gland in female gerbils. In addition to its role as a physical barrier that prevents the communication between the PB and their corresponding inductive mesenchyme, the smooth muscle is a source of prostatic proliferation inhibitory factors, such as the transforming growth factor beta 1 (TGF-β1) (Prins and Putz, 2008). This factor reduces prostatic epithelial proliferation, downregulates the expression of the FGF-10, and is implicated in the differentiation of periductal smooth muscle cells (Niu et al., 2011). The TGF-β1, and possibly other similarly acting factors of the same
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superfamily, could explain the V-shaped growth pattern observed in males, in which the PB grow to circumvent the smooth muscle.
In the same way, higher levels of circulating estrogens in the blood stream in the early postnatal period in females could explain the occurrence of a functional prostate in female gerbils (Custodio et al., 2008). Researchers have already shown that the ERα expressed in the prostatic stroma promotes epithelial proliferation in the prostate (Ellem and Risbridger, 2009; Takizawa et al, 2015). In female gerbils, the ERα is highly present in the stroma and might as well induce proliferation in the epithelium, a role played by the AR in male gerbils (Rochel-Maia et al., 2013).
Both the AR (Pu et al, 2007; Memarzadeh et al, 2011) and ERα (Omoto, 2008) upregulate the expression of the FGF-10, an essential growth factor for the branching morphogenesis in the prostate which is secreted by stromal cells (Prins and Putz 2008); thereby, the presence of one or the other steroid receptor in the periductal region can increase the expression of the FGF-10. Such paracrine factor promotes the branching morphogenesis in the prostatic epithelium. In males, during the prenatal period, there is a strong AR presence around the buds (Sanches et al., 2014) that is maintained throughout its development. In this study, we observed that, in the periductal region, on P7, the AR is highly observed in males, whereas the ERα is highly present in females, the presence of these factors could explain why both males and females develop branches. Considering that possibly the importance of stromal ERα to female prostate postnatal development is comparable to the AR to male prostate postnatal development (Custodio et al., 2004), we have detected a higher decrease in ERα presence in females comparatively to the reduction in the immunolabeling of the stromal AR in males, which can be related to the decreased presence of FGF-10 in the perdiductal region in females and, in turn, justify possibly the reduced branching. Theses findings could possibly explain why the female prostate has around20% the size of the male (Santos and Taboga, 2006).
In addition to the relationship with FGF-10, the stromal AR (Cunha, Cooke and Kurita, 2004; Yu et al, 2012.) and ERα (Omoto, 2008; Ellem and Risbridger, 2009; Rochel-Maia et al, 2013) have a notorious role in the induction of the prostatic epithelium proliferation. Indeed, in this work, we have confirmed that the decrease in the presence of these factors paralleled the decrease in cell proliferation during prostatic development. In this context, the presence of the epithelial AR appears to reduce the proliferation (Wu et al., 2007) and promote the epithelial differentiation and secretion (Donjacour and Cunha, 1991; McNamara et al., 2013). One possible mechanism by which the epithelial AR reduces proliferation in the
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prostatic epithelium involves the TGF-β1 (Niu et al., 2011). Thus, in the female prostate, the lower presence of the ERα in the epithelium and stroma and the presence of epithelial AR as early as P30 could explain the faster cell differentiation and earlier pre-maturation of the gland (Custodio et al., 2008).
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2.1.5. CONCLUSION
Taken together, the data presented in this study reinforce the concept that the prostate development, regardless of sex, is strongly related to variations in the presence and distribution of the ERα and AR in the prostatic tissue over time (Prins and Putz, 2008; Meeks and Schaeffer, 2011; Lee et al, 2016). Sex-specific variations found during the development, such as the reduced prostate size and early differentiation in females, are possibly caused by the rapid decrease in the presence of the ERα in the periductal region and by the decrease in the presence of the 10, the main prostatic branching promoter. In females, the decreased FGF-10 presence could explain the decrease of branching, and the early presence of the epithelial AR contributed to the early differentiation by means of antiproliferative stimuli. However, additional studies on inhibitory factors in the prostate development and their spatial distribution in the developing gland are needed to build a more reasonable explanation for the occurrence of a functional prostate in the females of Mongolian gerbil and to better understand prostate development in general.
Conflict of interest
The authors declare no conflict of interest to this work.
Acknowledgments
The authors would like to thank the researchers from the Microscopy Laboratory and Microanalysis: Luiz Roberto Falleiros Junior and Rosana Silistino de Souza (UNESP São José do Rio Preto, São Paulo, Brazil) for their technical support and Rodrigo P. A. Barros, MD, PhD, for assisting with preparation of the manuscript. This study was funded by the São Paulo Research Foundation (FAPESP), grant number 2013/15939-0, 2013/16443-9 and by the Brazilian National Research and Development Council and (CNPq), grant number 301596/2011-5.
Authors’ contribution
BDAS and JSM contributed equally to this work. All authors (BDAS, BCZ, JSM, MFB, FCAS, RMG, PSLV and SRT) contributed to the design and interpretation of the results and to the review of the manuscript. BDAS, BCZ and JSM performed the experiments. BDAS wrote the manuscript and SRT conducted the final review of the manuscript.
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Figure 1. Histology of the prostatic complex development in males and females. Histological
sections of the developing prostatic complex were stained with hematoxylin-eosin to evaluate the early postnatal development of the gland. A, C, E: in males, the PB branch in the VMP. In the inserts, strong cell proliferation can be seen, which is confirmed by the presence of cells in mitosis. B: in females, the PB grow parallel to the axis of the urothelium. D: in females, on P3, the PB undergo canalization in the VLM and mitotic cells are present. F, H: in females, on P5 and P7, the branching morphogenesis takes place simultaneously with the canalization of the gland. G, I: in males, on P7 and P14, the canalization occurs simultaneously with the branching morphogenesis. The presence of stromal fibers is confirmed in the differentiating stroma. J: in females, on P14, the canalization proceeds to the distal end of the branches, which give rise to the lumen of the alveoli. Like in males, collagen fibers accumulate in the stroma, indicating its progressive differentiation. K: in males, on P30, prostatic secretions are displayed in the developing alveoli. The differentiation of the prostatic stroma continues and the SM around the alveoli can be seen. L: in females, on P30, the alveoli show a highly differentiated morphology, the secretory epithelium has a simple cubic configuration, the stroma presents collagen fibers, and the SM is visible. M: in males, on P45, the alveoli are fully differentiated. N: in females, on P45, the differentiated alveoli are increased in volume and produce a large amount of secretion. VMP (ventral mesenchymal pad), PSM (periurethral smooth muscle), PM (periurethral mesenchyme), Arrow (prostate branching), UE (urethral epithelium), insertion (details of prostate development), Arrow head (mitotic cells), VLM (ventrolateral mesenchyme), dashed line (prostatic bud undergoing branching), CN (canalization), CF (collagen fibers), Asterisk (apoptotic cells), Large arrow (prostatic alveoli), PS (prostate stroma), SM (periductal smooth muscle), PE (prostate epithelium).
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Figure 2. Three-dimensional reconstructions of the developing prostatic complex of all
experimental groups. A: in males, on P1, the distal ends of the PB reach the VMP, which is located beyond the PSM that surrounds the urethra, and undergo the branching morphogenesis. These buds grow in a caudal-cranial direction and in a V-shaped pattern. B, D: in females, the distal ends of the PB reach the VLM on P1 and dilate on P3. C, E, G, I: in males, the branching morphogenesis of the gland proceeds between P3 and P14. During this period, branches increase in number and size and occupy the VMP region proximal to the bladder. As the gland develops, branches expand parallel to the axis of the urethra, in a region distal to the bladder. F: in females, on P5, prostate branches grow towards portions of the urethra that are distal to the bladder, like in males. H, J: in females, on P7 and P14, branches increase in number and in size, occupy the VLM region that differentiates into the prostatic stroma, and reach the anterior region of urethra. K, M: in males, on P30 and P45, the ends of the branches gradually differentiate into alveoli, forming an intricate network of ducts and alveoli that reach the vicinity of the urethral opening. L, N: in females, on P30 and P45, the ends of the branches differentiate into alveoli that are similar to those seen in males; however, these alveoli exhibit a more differentiated morphology. VMP (ventral mesenchymal pad), Asterisk (distal ends of PB in the VMP undergoing branching), Arrow (prostate branches), V (growth pattern of prostate buds toward the VMP), VLM (ventrolateral mesenchyme), small thick arrow (prostatic budding on the unbranched VLM), large thick Arrow (direction of expansion of prostate branchings - Posterior - Anterior), arrow head (prostate alveoli), green (urethral epithelium), dark Blue (urethral lumen), cyan (budding / branching / prostate alveoli), bar (200μm).
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Figure 3. Schematic representation of the postnatal prostate development and the presence of
selected factors involved in this. In males, the distal ends of the PB reach the VMP on P1, and, in females, they reach the VLM on P5. In males, the canalization begins on P7 and, in females, on P3. In both sexes, alveoli are present after P30. Dark Blue (VLM / prostatic stroma), light blue (periurethral mesenchyme), magenta (buddings / branchings / epithelium of prostatic alveoli), Red (periurethral smooth muscle), green (urothelium / urethral epithelium).
