Stage-Specific Gene Expression During Fish Spermatogenesis as Determined by Laser-Capture Microdissection and Quantitative-PCR in Sea Bass (Dicentrarchus labrax) Gonads

BIOLOGY OF REPRODUCTION 79, 738–747 (2008) Published online before print 9 July 2008. DOI 10.1095/biolreprod.108.069708 Stage-Specific Gene Expression During Fish Spermatogenesis as Determined by Laser-Capture Microdissection and Quantitative-PCR in Sea Bass (Dicentrarchus labrax) Gonads1 Jordi Viñas and Francesc Piferrer2 Institut de Ciències del Mar, Consejo Superior de Investigaciones Cientı´ficas (CSIC), 08003 Barcelona, Spain The role of genes implicated in the regulation of spermatogenesis and their patterns of expression is still poorly understood. In this study, we took advantage of the cystic arrangement of the teleost testis to set up a laser capture microdissection procedure to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids, or spermatozoa. We then used quantitative PCR to determine the stage-specific expression patterns of the germ cell marker vasa; gonadal aromatase (cyp19a); estrogen receptors (ers) alpha, beta1, and beta2 (era, erb1, and erb2, respectively); 11beta-hydroxylase (cyp11b1); androgen receptor beta (arb); insulinlike growth factor 1 (igf1); and sox17. vasa had the highest mRNA levels, followed by genes involved in androgen metabolism (cyp11b1 and arb). Most genes associated with estrogen metabolism (cyp19a, era, and erb1) had a lower expression, whereas igf1 and sox17 exhibited the lowest mRNA levels. Comparison of changes in mRNA levels revealed five patterns of gene expression, in general with progressively lower expression seen as spermatogenesis advanced. igf1 and sox17 were exclusively expressed in spermatogonia-containing cysts, suggesting effects during the proliferative stage. Genes involved in androgen synthesis (cyp11b1) and action (arb) peaked during the early stages of spermatogenesis and then sharply decreased. In contrast, genes associated with estrogen action, particularly erb2 and era, showed a more gradual decrease. Together, these results demonstrate the usefulness of fish models and suggest that whereas androgens are required at high levels and may exert their major actions at the initial stages of spermatogenesis, estrogens are also essential, albeit required at lower levels, and with a more generalized influence. 11b-hydroxylase, androgen receptor, aromatase, estradiol receptor, estrogen receptors, gene expression patterns, Igf1, laser capture microdissection, sea bass, Sox17, spermatogenesis, steroid hormones, testis, vasa INTRODUCTION Spermatogenesis is a tightly regulated and complex process involving several cell-cell interactions perfectly 1 This work was supported by a Genome Spain-Genome Canada grant (‘‘Pleurogene’’) to F.P. J.V. was supported by a postdoctoral contract from the Pleurogene project and an I3P postdoctoral grant from the Spanish Ministry of Education and Science. 2 Correspondence: Francesc Piferrer, Institut de Ciències del Mar, Consejo Superior de Investigaciones Cientı́ficas (CSIC), Passeig Marı́tim, 37-49, 08003 Barcelona, Spain. FAX: 34 932309555; e-mail: Received: 8 April 2008. First decision: 17 May 2008. Accepted: 18 June 2008. Ó 2008 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 738 ordered in space and time. It begins with rapid proliferation of spermatogonia through mitotic divisions, leading to primary spermatocytes, which through a first meiotic division become secondary spermatocytes. A secondary meiotic division without DNA replication generates haploid spermatids that finally are transformed into mature spermatozoa. The basic features and the regulatory mechanisms of spermatogenesis are, in essence, well conserved throughout vertebrates [1, 2]. Spermatogenesis is triggered by the release of gonadotropin hormones by the pituitary gland that stimulate androgen production in the testis [1, 3, 4]. The hormonal control of spermatogenesis is accomplished in a stage-specific and cellspecific manner by a vast number of factors [5–9]. However, due to the complexity of the testis structure, it is difficult to quantify the expression of genes in specific cell types. Gene expression has been studied by whole-tissue approaches, including in situ hybridization [10, 11]. However, this methodology is not quantitative. Alternatively, serial analysis of gene expression (SAGE) allows the quantification of mRNA levels of a large number of genes [12, 13]. However, SAGE cannot determine with enough precision the cellular localization of expressed genes. Cell-specific gene expression can be studied by in vitro culture of isolated specific cell types [14, 15]. The major drawback of this approach is that it is time consuming and, further, obtaining a sufficient amount of viable cell populations from the same stage of development in enough purity and quantity is not always straightforward. The development of laser capture microdissection (LCM) in the late 1990s [16, 17] made possible the isolation of specific cell populations from sections of complex tissues and, in some cases, to obtain individual cells. Laser capture microdissection was initially conceived for the study of gene expression in cancer cells [16], but was quickly adopted to study gene expression in several tissues and different experimental situations [18]. Thus, LCM coupled with reverse transcriptase-quantitative real-time polymerase chain reaction (RTqPCR) is a powerful method to accurately quantify gene expression in particular cell types, and its specificity has made this technique an ideal tool for studying gene expression during spermatogenesis [19, 20]. In mammals, for example, LCM has been used to quantify the stage-specific expression of selected genes during rat [7] and mouse [9] spermatogenesis, and the identification of differentially expressed genes between primary spermatocytes and round human spermatids [21]. Nevertheless, the intricate association between Sertoli cells and germ cells typical of mammals during the spermatogenic wave still makes difficult the isolation of staged germ cells. In teleost fish, the largest group of vertebrates, two major testis structures are present, both clearly differentiated from the characteristic tubular organization of the mammalian testis. One, the anastomosing tubular testis, is only present in basal teleosts. The other, the lobular testis, is more widespread and is present in ABSTRACT GENE EXPRESSION DURING FISH SPERMATOGENESIS MATERIALS AND METHODS Animals and Tissue Dissection Testes were obtained from twelve 330-day-old sea bass males with an average body weight of 89.85 6 6.13 g (mean 6 SEM) that were maintained under natural conditions of photoperiod and temperature in our experimental aquarium facilities, and were fed with a commercial pelleted diet. Fish were anesthetized with an overdose of MS-222 (Sigma, St. Louis, MO) and killed by decapitation. Gonads were excised under RNase-free conditions. One of the two testes was processed for conventional histological analysis to reliably identify ge (...truncated)