Tissue & Cell, 2000 32 (1) 88–94 © 2000 Harcourt Publishers Ltd DOI: 10.1054/tice.1999.0090, available online at http://www.idealibrary.com
Tissue&Cell
Chromatin condensation during Scrobicularia plana spermiogenesis: a controlled and comparative enzymatic ultracytochemical study M. Sousa,1 C. Cunha,1 M. Erkan,2 R. Guerra,3 E. Oliveira,1 L. Baldaia4
Abstract. In Scrobicularia plana testis, a nuclear acid phosphatase (ACPase) activity was detected in mid and late spermatids with the improved Gomori-chloride procedure. Lead deposits were first observed in mid spermatids at focal points over condensed chromatin strands, increasing in density as chromatin further condensated. In late spermiogenesis, lead deposits became concentrated between chromatin aggregates, and after total DNA compaction were transfered to the nuclear periphery and then shed into the cytoplasm. The specificity of the nuclear ACPase was tested against different pH values (3.9, 7.2, 7.8, 9.0), substrates (TPP, IDP, TMP, p-NCS, ATP, GTP, AMP, ADP, AMP-PNP) and inhibitors (NaF, levamisole, Zn, vanadate, theophylline). To further specify the nature of this nuclear ACPase, other enzymes were comparatively studied at their optimal pH values and at pH 5.0: nucleoside-diphosphatase, thiamin-pyrophosphatase, inorganic trimetaphosphatase, lysosomal arylsulfatases A and B, ATPase, GTPase, 5′-nucleotidase, adenylate kinase, and adenylate cyclase. Several other controls were introduced to exclude artefactual deposits induced by lead ions and tissue molecules. The results showed that the enzyme has an optimal pH at 5.0, a high specific affinity for β-GP, and is inhibited by NaF, which suggests that it behaves as a type B-ACPase, and all controls demonstrated the specificity of the enzymic activity. Because lead deposits were specifically and temporally associated with spermatid chromatin condensation, when DNA and RNA synthesis, histones, phosphoproteins and RNA molecules strongly decrease, it is possible to suggest that the nuclear ACPase could be associated with DNA processing during chromatin compaction or involved in the hydrolysis of 2′ and 3′ nucleotides resulting from nuclear RNase action during RNA degradation. © 2000 Harcourt Publishers Ltd
Keywords: chromatin condensation, enzyme cytochemistry, Mollusca, spermatogenesis, ultrastructure
Introduction
1 Laboratory of Cell Biology and 4Laboratory of Physiology (CIMAR), Institute of Biomedical Sciences, University of Porto, Portugal 2Faculty of Science, University of Istanbul, Turkey 3Department of Biology, Faculty of Sciences, University of Valparaíso, Chile
Received 30 August 1999 Accepted 30 November 1999 Correspondence to: Mário Sousa, MD, PhD, Laboratory of Cell Biology, Institute of Biomedical Sciences, University of Porto, Lg. Prof. Abel Salazar 2, 4099–003 Porto, Portugal. Fax: +351 2 206 22 32; E-mail:
[email protected]
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During spermiogenesis, remarkable changes occur at the ultrastructural level that are accompanied by several modifications on the protein composition of chromatin. The proteins associated with DNA in bivalve spermatozoa display a great variability, being of four main types: somatic histones, sperm-specific histone H1, protamines, and lysine-rich proteins. At early spermiogenesis, somatic histones are replaced by more basic proteins (arginine rich), and as a consequence the ratio of chromatin positive to negative charges increases, and chromatin becomes
NUCLEAR PHOSPHATASE IN SPERMIOGENESIS
filamentous. During the following spermiogenesis stages, these filaments aggregate into a lamellar structure and finally into a homogenously dense, very compact quasicrystalline DNA arrangement, which is made possible due to the richness in protamines (Casas & Subirana, 1994). The transition from histone to protamine-like proteins or to protamines is unique for spermiogenesis and is found in almost all animals. The histone change involves new protein synthesis, low in lysine but rich in arginine residues, and is not associated with any detectable DNA replication. Since the process is not part of chromosome duplication it may be involved in changes of chromosome function (Bloch & Hew, 1960). Chromatin condensation during spermiogenesis in the bivalve Scrobicularia plana is also characterized by a transition of chromatin fibrils into lamellar structures and then to a compact DNA arrangement (Sousa et al., 1989). During a study of the cytochemical localization of enzymic activities in lysosomes and Golgi complexes at the ultrastructural level in S. plana spermatogenesis, a nuclear acid phosphatase activity was observed. Nuclear acid phosphatase activity is generally attributed to a false localization due to cytoplasmic diffusion or as artefactual nucleation by the nuclear matrix (De Jong, 1982), but in some cells it has been confirmed and related to RNA processing (Kessel & Decker, 1972; Sánchez-Pina et al., 1980). In the present study we show that nuclear acid phosphatase activity in S. plana follow a very specific time and spatial-course pattern during spermatid chromatin condensation. An extensive controlled and comparative ultracytochemical study suggests that this nuclear acid phosphatase activity is specific and related to chromatin compaction.
Materials and methods Specimens of S. plana (Mollusca, Bivalvia, Heterodonta, Veneroida) were collected at the estuary of the river Douro, Porto (Portuguese Atlantic North Coast intertidal zone). All chemicals were of analytical grade and purchased from Sigma (St Louis, MA, USA) or Merck (Germany). The improved Gomori-chloride cytochemical procedure for acid phosphatase (ACPase) was performed (De Jong et al., 1979a; 1979b; De Jong, 1982). Small pieces (
