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Micron
j our na l ho me p age: www.elsev ier .com/ locate /micron
ytoarchitectonical dynamic of Sertoli cells in Melanorivulus punctatusCyprinodontiformes: Rivulidae)
ônica Cassel ∗, Débora Fabiane Neves da Silva, Adelina Ferreiranstituto de Biociências, Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, n◦ 2367, Bairro Boa Esperanc a, Cuiabá 78060-900, MT, Brazil
r t i c l e i n f o
rticle history:eceived 25 July 2012eceived in revised form6 November 2012ccepted 17 November 2012
The Sertoli cell contributes to spermatogenesis acting in the differentiation of germ cells and being theonly somatic cells present in the germinal compartment. So that spermatogenesis is primarily dependentof Sertoli–Sertoli and Sertoli–germ cell interactions once Sertoli cells provide critical factors necessaryfor a successful differentiation of germ cells to sperm. In teleost fish the cytoplasmic extensions of Sertolicells support the cysts that remain closed until spermiogenesis. The number of Sertoli cells determines thetesticular size, the number of testicular germ cells and the production capacity of spermatozoa. Our objec-tive was to describe the morphology and the cytoarchitectonical dynamic of Sertoli cells in Melanorivuluspunctatus, which were collected in the municipality of Chapada dos Guimarães, Mato Grosso, Brazil. Thegonads were extracted and prepared according to histological routine for light microscopy and transmis-sion electron microscopy. Sertoli cells have cytoplasmic extensions which provide the conformation ofcysts in the interior of the lobes. These cells possess a polymorphic nucleus with a well-defined nuclearenvelope and a prominent and eccentric nucleoli. Each cyst is sustained for more than one Sertoli celland the cysts seem to share the Sertoli cells with each other regardless the stage of development of germcells within these cysts. This disposal promotes a reticulated arrangement of Sertoli cells. The Sertoli cellslining the ducts assume rectangular shape with rounded nucleus. Thus, the morphological characteris-
tics of Sertoli cells observed did not differ from what has been described for other teleosts. Despite thesimilarity in the morphology of these cells, we observed that its disposal in the extension of the gonadseems to differ from what is described for fish. The arrangement by which the cytoplasmic extensionsof Sertoli cells connect the ends of lobes prevents the proliferation of spermatogonia on the lobe sidewalls and are only observed in the end of the lobes, which ensures the testicular characteristic–lobularrestricted in Atherinomorpha.
. Introduction
The testicular germinal compartment of vertebrates consists ofwo distinct populations of cells – Sertoli cells and spermatogenicells (Grier, 1993; Pudney, 1993; Nagahama, 1994). The Sertoli cells the only somatic cells in this compartment (Sofikitis et al., 2008)nd their morphology and function are highly conserved in all ver-ebrates (Gubbay et al., 1990), playing a key role in differentiationnd development of the gonad (Griswold, 1993; Sharpe et al., 2003).
Sertoli cells support a relatively fixed number of germ cells,hich remains constant for each species but varies among differ-
nt species (Russell and Peterson, 1984; Franc a and Russell, 1998;ofikitis et al., 2008). The number of Sertoli cells determines the tes-icular size, the number of testicular germ cells and the production
capacity of spermatozoa (Orth et al., 1988; Hess et al., 1993). Con-trary to what is reported to mammals, where the Sertoli cellsproliferate only during fetal and pubertal periods (Hess and Franc a,2007), in teleost the Sertoli cells are able to proliferate as the germcells also proliferate and differentiate (Miura, 1999; Schulz et al.,2000; Koulish et al., 2002; Vilela et al., 2003). During the process ofspermiation the Sertoli cells hypertrophy and modify themselvesin duct cells or degenerate, being absorbed by the cells of the duct(Lo Nostro et al., 2003).
In teleost fish, germ cells mature into cysts where synchronousclones are surrounded by cytoplasmic extensions of Sertoli cells(Callard, 1991; Grier, 1993). Spermatogenesis is primarily depend-ent of Sertoli–Sertoli and Sertoli–germ cell interactions (Pudney,1993; Loir et al., 1995; Schulz and Miura, 2002; Walker and Cheng,2005). Sertoli cells provide critical factors necessary for a success-
ful differentiation of germ cells to sperm. These factors can be:physical support, either through junctional complexes or barrier,known as “blood-testis” barrier which provides a specialized andprotected environment (Pudney, 1993; Walker and Cheng, 2005);
nd biochemical stimuli, in the forms of growth factors and nutri-nts (Setchell, 1986; Griswold, 1988; Maddocks and Setchell, 1988;ruk and Cheng, 2004).The testicular cystic structure in which the Sertoli cells feed only
ne type of germ line at a time is an interesting model for studyinghe regulation of spermatogenesis (Chaves-Pozo et al., 2005). Weescribe the morphology and the cytoarchitectonical dynamic ofertoli cells in the gonads of Melanorivulus punctatus.
. Materials and methods
Ten males of M. punctatus were collected in the reservoir ofarque Estadual da Quineira (15◦27′58′′S, 55◦44′46′′W), munic-pality of Chapada dos Guimarães, Mato Grosso, Brazil, andxed in a 2% glutaraldehyde and 4% paraformaldehyde solution
n Sorensen buffer (0.1 M, pH 7.2) for 24 h. The gonads werextracted, dehydrated in ethanol and embedded in HistoresinTechnovit 7100). Sections were made with a glass knife to 3 �mhick, and stained with iron hematoxylin/eosin and toluidinelue/borax.
Some fragments of the same gonads were prepared for transmis-ion electron microscopy. The fragments were post-fixed for 2 h inhe dark in 1% osmium tetroxide in Sorensen’s phosphate buffer0.1 M, pH 7.2), contrasted with aqueous 5% uranyl acetate for 2 h,ehydrated in acetone and embedded in araldite. After selectionsemi-thin cuts), the samples were sectioned by an ultramicro-ome equipped with a diamond knife, contrasted with saturatedranyl acetate in ethanol 0.2% (1:1) and lead citrate in NaOH (1 N)nd documented in a transmission electron microscope (PhilipsM-100).
. Results
Sertoli cells can be seen extending their cytoplasmic extensionsnd giving the structure of cysts, which promote the support oferm cells (Fig. 2A–C), and after retraction of the extensions lininghe ducts where the sperm are released (Fig. 2D–E). Sertoli cellsssociated with germ cells do not exhibit a unique form, adjus-ing itself to the shape of the cyst or the cysts adjacent (Fig. 2A–C).hese cells have polymorphic nucleus with well defined nuclearnvelope and prominent and eccentric nucleoli (Fig. 1A). The cyto-lasm, as observed in electron microscopy, shows a great numberf membranous organelles, with vesicles and mitochondria clearlyvident (Fig. 1C–D). Each cyst is sustained for more than one Ser-oli cell, which are located at the periphery and connect by cellunctions to other extensions of the Sertoli cells of the same cystr cysts adjacent (Fig. 1B). There are links between the Sertoliells and germ cells, however this connection apparently is bro-en when the cells reach the spermiogenic stage (Fig. 1A–D). Theysts seem to share the Sertoli cells with each other regardless thetage of development of germ cells within these cysts (Fig. 2E).his sharing between cysts promotes a reticulated arrangementf Sertoli cells (Fig. 2A and E). After opening the cysts, sperm areeleased in a duct lined by Sertoli cells (Fig. 2D–E). Sertoli cells lin-ng the ducts did not differ in composition from the cytoplasm,ut are characterized by a rectangular shape with rounded nucleusFig. 2D–E).
. Discussion
The morphological characteristics of Sertoli cells observed did
ot differ from what has been described for other teleosts (Grier,981; Nóbrega et al., 2009; Magalhães et al., 2011). Likewise, theresence of cell junctions between the Sertoli cells is very dis-ussed in the reproduction work already performed with fish,
5 (2013) 115–118
they may be occlusion, for the formation of “blood-testis” barrier,desmosomes and interdigitations (Pudney, 1993; Lo Nostro et al.,2003; Walker and Cheng, 2005; Vicentini et al., 2010). Schulz et al.(2010) reported that, during spermiogenesis adherens junctionsare formed between the germ cells and Sertoli cells; these junc-tions will disintegrate later, together with the cytoplasmic bridgesbetween spermatids, in the process of spermiation. Thus, bothSertoli–Sertoli and Sertoli–germ cells bindings seem to occur in M.punctatus.
The Sertoli cells proliferation is an event commonly referred inthe literature for fishes (Koulish et al., 2002; Chaves-Pozo et al.,2005; Schulz et al., 2005, 2012). This proliferation occurs by thegrowth of the individual and subsequent growth of the gonad(Schulz et al., 2005), or due to cysts expansion accompanying thespermatogenesis (Nicholls and Graham, 1972; Leal et al., 2009),which may explain the presence of more than one Sertoli cell bycyst (Grier and Lo Nostro, 2000; Koulish et al., 2002).
Despite the similarity in the morphology of these cells, weobserved that its disposal in the extension of the gonad seems todiffer from what is described for fish (i.e. Grier, 1981). For this, wemust consider the existing gonad classification for the groups offish. A classification of testicular types in fish was proposed byGrier (1993) and Grier and Uribe Aranzábal (2009) based on theshape of the gonad and the organization of the germinal compart-ment. Under this proposal, the gonads can be classified into tubularand lobular, and the lobular type can be subdivided in restrictedand unrestricted. According to this classification, M. punctatus wasdescribed as the lobular restricted type, in which the germinalcompartment is fingerlike and ends in a blind bottom, where sper-matogonia are restricted (Cassel et al., 2013). Restricted testicles“demonstrates a progression of stages of germ cell maturationtoward the lobules (Parenti et al., 2010). According to Grier (1993)this testicular type is very common among Neoteleostei, especiallyin Atherinomorpha and Percomorpha.
The evolutionary process supported by the transition from atubular testis to a lobular one may have occurred as: (a) a simplechange in the formation of basement membrane which supportsthe germinal epithelium, and (b) the restriction of spermatogoniato the end of the lobes by the establishment of distal epithelioidcords of Sertoli cells (Brown-Peterson et al., 2002; Sàbat et al.,2009; Guerrero-Estévez and Moreno-Mendoza, 2012). The term“epithelioid” was first applied in reference to the cords of spermato-gonia and Sertoli cells, which grow from the final part of the lobesduring the regression stage in the reproductive cycle of Rachycen-tron canadum (Rachycentridae) (Brown-Peterson et al., 2002). Thus,there is a change in the disposal of Sertoli cells in Atherinomorpha,as noted by Parenti and Grier (2004), these cells extend their cyto-plasmic extensions through the lobes, and the lumen of the lobe isabsent. The authors also state that this arrangement by which thecytoplasmic extensions of Sertoli cells connect the ends of lobesprevents the proliferation of spermatogonia on the lobe side wallsand are only observed in the end of the lobes, which ensures thetesticular characteristic–lobular restricted (Grier, 1993) in Atheri-nomorpha.
It is described for Sertoli cells in Poeciliidae, Cyprinodonti-dae, Oryziatidae and Goodeidae its hypertrophy and modificationin columnar cells, forming an efferent duct (Grier, 1981; Sàbatet al., 2009), a feature also observed in M. punctatus. The testicularchanges that accompanied the evolution in Atherinomorpha arehypothesized to impose mechanisms that prevent the repopula-tion of spermatogonia along the lobule walls during the regressionstage and when regressed (Parenti and Grier, 2004). It can be said
that Atherinomorpha has a “testicular regression” with functionalmaturation, i.e. a maturation independent of the stage of the repro-ductive cycle and which is induced in order that the lobe is notinvaded by spermatogonia (Parenti and Grier, 2004).
M. Cassel et al. / Micron 45 (2013) 115–118 117
Fig. 1. Ultrastructure of Sertoli cell: present polymorphic nuclei (N) with well defined nuclear envelope and prominent and eccentric nucleoli (arrows) (A); connect withextensions of adjacent Sertoli through cellular junctions (circle) (B); cytoplasm with large set of membranous organelles, with vesicles (*) and mitochondria (m) clearlyevident (C–D). Links between Sertoli cells and germ cells, and its break when in spermiogenesis (A–D).
Fig. 2. Distribution and arrangement of the Sertoli cells: they are located at the periphery of the cysts and involve germ cells through cytoplasmic extensions (arrows) (Cand E), and more than a single cell (*) covers the wall of the cyst (B–C); cysts seem to share Sertoli cells (A and E); and with the opening of the cyst (white arrow), there are acolumnar repositioning of cells (circle) that will compose the wall of the duct (D–E), these Sertoli cells assume a rectangular format (circle) with rounded nucleus (D–E).
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Vilela, D.A.R., Silva, S.G.B., Peixoto, M.T.D., Godinho, H.P., Franc a, L.R., 2003. Sper-
18 M. Cassel et al. / Mi
. Conclusion
In M. punctatus the Sertoli cells had no difference in structurehen compared with the other teleost. These cells are located in
he periphery of the cysts, which share the Sertoli cells to eachther regardless of the stage of development of germ cells. Duringhe spermiogenesis occurs the opening of the cysts and Sertoli cellstart to present a rectangular shape with rounded nucleus and formhe ducts where the sperm are released. The reticulated disposal ofertoli cells when sharing cysts, observed in M. puctatus, indicates
change in the arrangement of these cells in Atherinomorpha.
cknowledgement
We thank Irani Quagio-Grassiotto (UNESP-Botucatu) for thepace provided for the histological preparations for light and elec-ron microscopy.
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