Elsevier

Experimental Cell Research

Volume 314, Issue 1, 1 January 2008, Pages 213-226
Experimental Cell Research

Research Article
The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

https://doi.org/10.1016/j.yexcr.2007.09.022Get rights and content

Abstract

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood–testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150Glued, EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.

Introduction

Sertoli cells play a pivotal role in spermatogenesis [1], [2]. They belong to a unique type of polarized epithelial cells and are the only somatic cells in the seminiferous epithelium of testis. Sertoli cells interact with each other through tight junctions at the base of the seminiferous tubule to form the blood–testis barrier [3], [4], [5]. Their cell bodies extend into the lumen both laterally and apically in form of numerous processes to enwrap germ cells of different developing stages through Sertoli–germ cell junctions [3], [4], [5]. Sertoli cells support germ cell proliferation and differentiation by providing nutrients and regulatory factors [1], [3], [4], [5]. Their morphologies appear to change in the cycle of the seminiferous epithelium in accordance with germ cell development [3], [4], [5]. Nevertheless, due to their structural complexities and extensive associations with germ cells, direct visualization of their shapes in situ is very difficult. So far, only the Sertoli cell morphologies at stage V of the seminiferous cycle have been documented in rat and monkey through 3-D reconstruction of electron microscopic (EM) images [6], [7].

Cytoskeletons are critical for structure and function of Sertoli cells. Microfilaments are essential for formation of cell–cell junctions between Sertoli–Sertoli cells or Sertoli–germ cells. Sertoli cells also form an actin-rich structure, the apical ectoplasmic specialization, at the interface with spermatids [5], [8], [9]. Disruption of microfilaments with Cytochalasin D results in malorientation of spermatids and separation of elongating spermatids from Sertoli cells under osmotic stress [10]. Like in other polarized epithelial cells, microtubule (MT) arrays are generally parallel to the long axis of Sertoli cells, with their minus ends directed toward apical surfaces [11], [12]. Their distribution patterns, however, change periodically during spermatogenesis [13], [14], [15]. Disassembly of MTs with drugs blocks seminiferous tubule fluid secretion by Sertoli cells, induces sloughing of apical fragments of Sertoli cells along with germ cells [16], [17], [18], [19], and attenuates apical movement of elongate spermatids [20]. Nevertheless, since the drugs affect both Sertoli and germ cells, it is difficult to distinguish effects of MT disassembly in Sertoli cells from those in germ cells. Moreover, how the ordered MT arrays contribute to Sertoli cell structure and function is also not known.

The plus ends of MTs are normally associated with a group of proteins, namely plus end-tracking proteins, among which are EB1, dynactin, APC, CLIP-170, and CLASP1/2 [21], [22]. These proteins stabilize MTs and anchor MTs to various cellular structures including cell cortex [21], [22]. EB1, a protein initially identified through interaction with APC [23], appears to be a central player due to its ability to bind many plus end-tracking proteins [21], [22]. It also exhibits centrosome localization and is critical for MT anchoring at the centrosome [24], [25]. EB1 is well conserved and has been diversified into a family during evolution. There are at least three members in EB1 family in Drosophila, whereas four members, EB1–EB3 and EBF3, have been reported in human, along with other highly related proteins such as RP1, RP2, and RP3 [26], [27].

In this study, we investigated whether MT organizations contribute to Sertoli cell morphology and function. We showed that EB1 was highly expressed and located along MT bundles in Sertoli cells. We also developed a microinjection method for efficient and stable expression of exogenous proteins in testis by lentivirus, which has been shown to specifically infect Sertoli cells [28]. We found that overexpressing a dominant-negative mutant of EB1 [24] disrupted MT organization in Sertoli cells and altered Sertoli cell shapes in vivo, resulting in defects in seminiferous tubule morphology and spermatogenesis. Therefore, an intact MT array is important for function-related morphological changes of Sertoli cells during the cycle of seminiferous epithelium.

Section snippets

Source of tissues

All experiments were carried out according to local and national guidelines for the care and use of laboratory animals. C57BL/6 mice obtained from Shanghai Laboratory Animal Center, Chinese Academy of Sciences, were used as source of tissues.

Cell culture

HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% calf serum (Sijiqing Company, Hangzhou, China) in an atmosphere containing 5% CO2. Primary culture of murine Sertoli cells was performed as described [29]

EB1 is highly expressed in Sertoli cells in murine testis

To investigate expression patterns of MT plus end proteins in testis, we performed immunoblotting using testis tissues from different developmental stages. Compared to epididymis and brain, EB1 showed high-level expression in testis (Fig. 1A). CLIP-170 and p150Glued, however, were expressed in comparable levels in these tissues (Fig. 1A). Existence of a larger isoform of CLIP-170 in testis after 28 days postpartum (dpp) is due to an extra exon in mRNA [33]. Similarly, the presence of lower

EB1 exhibits high-level expression and distinct localizations in Sertoli cells

We find that the high-level expression of EB1 in murine Sertoli cells is not a common feature of plus end-tracking proteins including CLIP-170 and the dynactin subunit p150Glued (Fig. 1, [2], Fig. 2; data not shown), implying a role of EB1 in MT organization and dynamics in this type of cells.

In squashed samples of isolated seminiferous tubules, EB1 mainly exhibited dense, punctate localization along tightly packed MTs in Sertoli cell processes (Fig. 1, [2], Fig. 2). This is unlikely an

Acknowledgments

We thank Dr. Tianjin Liu for technical assistance and Dr. Yixian Zheng (Carnegie Institution of Washington, USA) for critical comments on this manuscript. We are also grateful to Dr. Qiwei Zhai (Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for kindly providing the lentivirus system and Dr. Shoichiro Tsukita (Department of Cell Biology, Kyoto University, Japan) for EB1 cDNA. This work was supported by the National Science

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