Stage and cell-specific expression and intracellular localization of the small heat shock protein Hsp27 during oogenesis and spermatogenesis in the Mediterranean fruit fly, Ceratitis capitata

Highlights

• CcHsp27 is expressed during oocyte development in a stage and cell-specific manner.

• CcHsp27 is located in the nuclei of the somatic cells of the late egg chambers.

• CcHsp27 is located in the nuclei of the primary spermatocytes and actin cones.

• CcHsp27 is located in the nuclei of several organs of the male reproductive system.

Abstract

The cell-specific expression and intracellular distribution of the small heat protein Hsp27 was investigated in the ovaries and testes of the Mediterranean fruit fly, Ceratitis capitata (medfly), under both normal and heat shock conditions. For this study, a gfp-hsp27 strain was used to detect the chimeric protein by confocal microscopy. In unstressed ovaries, the protein was expressed throughout egg development in a stage and cell-specific pattern. In germarium, the protein was detected in the cytoplasm of the somatic cells in both unstressed and heat-shocked ovaries. In the early stages of oogenesis of unstressed ovaries, the protein was mainly located in the perinuclear region of the germ cells and in the cytoplasm of the follicle cells, while in later stages (9–10) it was distributed in the cytoplasm of the germ cells. In late stages (12–14), the protein changed localization pattern and was exclusively associated with the nuclei of the somatic cells. In heat shocked ovaries, the protein was mainly located in the nuclei of the somatic cells throughout egg chamber’s development. In unstressed testes, the chimeric protein was detected in the nuclei of primary spermatocytes and in the filamentous structures of spermatid bundles, called actin cones. Interestingly, after a heat shock, the protein presented the same cell-specific localization pattern as in unstressed testes. Furthermore, the protein was also detected in the nuclei of the epithelial cells of the deferent duct, the accessory glands and the ejaculatory bulb. Our data suggest that medfly Hsp27 may have cell-specific functions, especially in the nucleus. Moreover, the association of this protein to actin cones during spermatid individualization, suggests a possible role of the protein in the formation and stabilization of actin cones.

Introduction

One widely conserved mechanism of cellular response to heat shock and other stressors, is the rapid and massive synthesis of heat shock proteins (Hsps) (reviewed by Morimoto et al., 1994). Hsps are molecular chaperones that confer proper protein folding and play important functions in stress response, by preventing the accumulation of damaged or misfolded proteins in cells (reviewed by Hartl and Hayer-Hartl, 2002). Small heat shock proteins (sHsps) are the most diverse in structure and function amongst the various families of stress proteins, varying greatly in amino acid sequence and size (∼12 to 43 kDa) (reviewed by de Jong et al., 1998). Despite these differences, the majority of sHsps form a ubiquitous group of proteins (a-crystalin/sHsp superfamily) sharing two common features: An evolutionary conserved C-terminal α-crystalin domain of about 80–100 amino acid residues, bounded by variable amino and carboxyl-terminal extensions, and the formation of oligomeric complexes up to 800 kDa (reviewed by de Jong et al., 1998, Macrae, 2000, Nakamoto and Vígh, 2007). They form large, dynamic, oligomeric complexes of variable sizes and are able to bind several non-native proteins per complex (Haslbeck et al., 2008). In addition to their roles in protecting cells from stress, they have been reported to be expressed during normal development where they are involved in many cellular functions such as stress tolerance, protein folding, proteasome-mediated degradation of selected proteins, actin polymerization, cytoskeleton stabilization, cell death, development and differentiation, cell cycle and signal transduction (reviewed by Arrigo, 1998, Macrae, 2000, Mounier and Arrigo, 2002, Arrigo, 2005, Garrido et al., 2012). Several studies have shown the involvement of certain sHsps in human diseases such as cardiovascular, neurological and muscular diseases, cancer, ischemia etc., suggesting their potential use as therapeutic agents (reviewed by Boncoraglio et al., 2012, Kampinga and Garrido, 2012, Bakthisaran et al., 2015).

In Drosophila melanogaster, four members of the sHsp family (Hsp27, Hsp26, Hsp23 and Hsp22) have been studied in detail (reviewed by Michaud et al., 1997a, Morrow and Tanguay, 2012). These proteins share the ability to prevent heat-induced protein aggregation and are able to maintain proteins in a refoldable state, even with different efficiencies (Morrow et al., 2006). Hsp27, Hsp26 and Hsp23, are the main sHsps expressed during normal development and are mostly observed in the nervous system and gonads where they are expressed in different cell types (Glaser and Lis, 1990, Marin et al., 1993, Marin et al., 1996, Michaud et al., 1997a). Flies depleted of either Hsp22, Hsp23 or Hsp27 are viable but have a reduced resistance to certain types of stress and a reduced longevity (Seong et al., 2001, Michaud and Tanguay, 2003, Morrow et al., 2004a, Hao et al., 2007, Colinet et al., 2010). Overexpression of these proteins extended Drosophila lifespan and increased stress resistance of the flies (Morrow et al., 2004b, Wang et al., 2004). Drosophila melanogaster Hsp27 (DmHsp27) displays a distinct pattern of expression at specific stages of development in the absence of stress (Arrigo and Tanguay, 1991, Pauli et al., 1992, Arrigo and Landry, 1994). DmHsp27 is synthesized during embryogenesis and its’ synthesis persists, at a lower level, during entire development (Mason et al., 1984, Arrigo et al., 1987, Haass et al., 1990). This protein exhibits distinct patterns of intracellular localization during oogenesis and spermatogenesis (Marin and Tanguay, 1996, Michaud et al., 1997b).

We are interested in investigating the role of the Hsp27 in development and thermal adaptation of the Mediterranean fruit fly, Ceratitis capitata (medfly). In a previous work we isolated and characterized the medfly hsp27 (Cchsp27) gene and we showed that its protein is homologous to DmHsp27 (Kokolakis et al., 2008). The developmental expression pattern of the Cchsp27 gene is similar to the respective pattern of Dmhsp27, although there are some differences in certain developmental stages (Kokolakis et al., 2008). Medfly is a tephritid pest with a worldwide distribution and a history of rapid and devastating invasions to various countries (Harris, 1989, Malacrida et al., 2007). This species is the most thoroughly studied fruit fly pest at genetic and molecular levels. Several genetic tools of Drosophila have been adapted to medfly, allowing scientists to study the regulation and function of medfly genes and to create transgenic strains useful for biological control of this notorious insect pest (reviewed by Wimmer, 2003, SCOLARI et al., 2014). The main goal of the present work was to understand the function of the Hsp27 during oogenesis and spermatogenesis of the medfly. Toward this goal we studied the expression and intracellular localization of the medfly Hsp27 in adult gonads by using a transgenic GFP-CcHsp27 strain and confocal microscopy. Our results show a stage and cell-specific expression of this protein during oogenesis and spermatogenesis, implying specific roles of the protein in these processes.