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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.34 n.2 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000200021 

Targeting and fusion proteins during mammalian
spermiogenesis

JOÃO RAMALHO-SANTOS2 AND RICARDO D. MORENO1,*

1Unit of Reproduction and Developmental Biology, Pontifical Catholic University of Chile, Santiago, Chile and

2Center for Neuroscience and Cell Biology of Coimbra, Department of Zoology, University of Coimbra, 3000 Coimbra, Portugal

Corresponding Author: Dr Ricardo D. Moreno, Physiology Department, Faculty of Biological Sciences, Pontifical Catholic University of Chile. Alameda 340, Santiago, Chile. Tel: (562) 686 2885. Fax: (562) 222 5515. Email:rmoreno@genes.bio.puc.cl

Received: May 20, 2001. Accepted: July 10, 2001

ABSTRACT

Regulated exocytosis is controlled by internal and external signals. The molecular machinery controlling the sorting from the newly synthesized vesicles from the Golgi apparatus to the plasma membrane play a key role in the regulation of both the number and spatial location of the vesicles. In this context the mammalian acrosome is a unique vesicle since it is the only secretory vesicle attached to the nucleus. In this work we have studied the membrane trafficking between the Golgi apparatus and the acrosome during mammalian spermiogenesis. During bovine spermiogenesis, Golgi antigens (mannosidase II) were detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgi-acrosome flow may be relatively unselective, with Golgi residents retrieved before spermiation is complete. Surprisingly, rab7, a protein involved in lysosome/endosome trafficking was also found associated with the acrosomal vesicle during mouse spermiogenesis. Our results suggest that the acrosome biogenesis is associated with membrane flow from both the Golgi apparatus and the endosome/lysosome system in mammalian spermatids.

Key Terms: acrosome, spermatid, Golgi, fertilization, spermiogenesis.

INTRODUCTION

Although biogenesis is an important aspect of mammalian spermiogenesis, the nature of the acrosome itself is still debated. The acidic characteristic of the compartment, as well as its rich hydrolase content, suggests similarities with lysosomes (Hartree, 1975; Moreno et al, 2000a). However, some typical lysosomal markers are not present on the acrosome (Martinez et al 1996a), which raises the possibility that it might more accurately be described as a modified secretory granule. Nevertheless, the acrosome is a complex organelle that includes several structurally and biochemically distinct regions (Olson et al, 1998), and its growth and shaping relies on intense membrane trafficking, possibly involving both clathrin-coated and COPI-coated vesicles (Griffiths et al, 1981; Martinez et al, 1996b; Moreno et al 2000b; Olson et al, 1998).

During mammalian spermiogenesis several distinct stages of spermatid differentiation also reflect differences in the nascent acrosome (Lebond & Clermon, 1952). The initial Golgi-phase is marked by fusion of Golgi-derived dense pro-acrosomal granules (which contain some, but not all, acrosome components) into a single acrosomal granule that attaches to the cell nucleus. During the cap-phase, fusion of many (presumably Golgi-derived) vesicles takes place around the granule, forming the acrosomal vesicle (Susi et al, 1971). This initially rounded vesicle spreads over the nucleus concomitantly with Golgi apparatus migration to the opposite cell pole. In both these stages the spermatid nucleus maintains a round morphology. During the acrosome phase the spermatid begins to elongate as the manchette microtubules start to grow from the nuclear ring. The acrosome reaches its final shape at the end of the maturation phase, and most of the cytoplasm and organelles are discarded in the cytoplasmic droplet/residual body (Susi et al, 1971).

We initiated this study by addressing the puzzling fact that some Golgi-specific antigens seem to be found in the acrosome at several stages of spermiogenesis (Igdoura et al, 1999; Moreno et al, 2000a, b; Ramalho-Santos et al, 2001). In parallel, we have set out to characterize components of the membrane trafficking machinery present during acrosome biogenesis in an attempt to identify molecular players potentially active in the recognition/docking/fusion of the membrane vesicles that will ultimately form the organelle. Important regulators of membrane trafficking are the members of the rab family of small GTPases (Martinez & Goud, 1998; Pfeffer, 1999) . These proteins play a crucial role in determining that transport vesicles will fuse with their appropriate targets (Pfeffer, 1999; Somsel Rodman & Wandinger-Ness, 2000). We describe here that rab7 is found in association with the acrosome during the early stages of spermiogenesis and may also play a role in the biogenesis of this organelle.

MATERIALS AND METHODS

Chemicals

All chemicals were obtained from Sigma Chemical Co (St. Louis, MO), unless otherwise stated.

Antibodies

Antibodies directed against rab1A, rab2, rab3A, rab5, rab6, rab7, rab8, rab11 and rabaptin-5 were from Santa Cruz Biotechnology (Santa Cruz, CA). Additional probes against rab5 were obtained from CalBiochem (La Jolla, CA) and CytoSignal (Irvine, CA); against rab6 from CalBiochem; and against rab7 from CytoSignal. A monoclonal antibody against the mouse protein sp56, related to the guinea pig acrosomal matrix protein AM67 (Foster et al, 1997) was obtained from QED Bioscience (San Diego, CA).

Isolation of spermatogenic cells

Bovine testes were obtained from a local slaughterhouse (Moreno et al, 2000b). Testicular cells were dissected and transferred to a petri dish filled with TALP-HEPES (modified Tyrode-Lactate medium with pyruvate and albumin: 114 mM NaCl, 3.2 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2, 25 mM NaHCO3, 0.4 mM NaH2PO4, 10 mM sodium lactate, 6.5 IU penicillin, 25 µg/ml gentamicin, 3 mg/ml fatty acid-free bovine serum albumin, 0.2 mM pyruvate, buffered with 10 mM Hepes at pH 7.4) and minced with two fine forceps. The minced tissue was filtered through a fine mesh to remove the tissue debris, and the cell suspension was centrifuged for 5 min at 700 x g. The pellet was resuspended in 12 ml of warm TALP-HEPES and centrifuged again for 5 min at 700 x g.

Immunocytochemistry

For immunocytochemistry, cover slips with bovine spermatogenic cells were placed in PBS containing 2% formaldehyde and fixed for 1 h. Following fixation, the samples were permeabilized for 60 min in PBS containing 1% Triton X-100, and nonspecific reactions were blocked by further incubation in PBS containing 2 mg/ml bovine serum albumin and 100 mM glycine. For labeling, the antibodies were solubilized in this blocking solution and incubated with the cover slips for 1-2 h at the appropriate dilutions. For rab proteins the incubation was carried out overnight at 37°C. After extensive washing in PBS containing 0.1% Triton X-100, the samples were sequentially labeled with either FIC-conjugated (Zymed, San Francisco, CA), or Alexa-488 or Alexa-568 (Molecular Probes) appropriate secondary antibodies for 1 h. Following these incubations, cover slips were mounted in VectaShield mounting medium (Vector Labs, Burlingame, CA) and sealed with nail polish. Samples were examined with a Zeiss Axiophot or a Nikon Eclipse E1000 epifluorescence-equipped microscope operated with Metamorph software.

RESULTS

The acrosome of elongating bovine spermatids contains Golgi proteins

In pachytene spermatocytes giantin is present in a rounded structure formed by at least two crescent-shaped structures (Fig 1A). These structures face each other and surround additional central material with a dotted pattern. Mannosidase II (ManII) also shows a rounded shape formed by two crescent-shaped structures, which are probably the same label by giantin (Fig. 1B). In round spermatids, Man II and Giantin can also be conspicuously found on the acrosome (Fig. 1C). This observation was confirmed with other Golgi markers, including Golgin-97 (data not shown) suggesting that the presence of Golgi proteins on the acrosome might constitute a general motif during spermiogenesis.

FIGURE 1: Localization of Giantin in mammalian spermatids. Indirect immunofluorescence of bovine spermatocytes (A,B) and spermatids at step 5 (C) and step 7 (D) of differentiation showing the presence of ManII. The Golgi apparatus in secondary spermatocytes is composed of two half-circles facing each other through their concave sides (A, B). In spermatids ManII localizes both in the Golgi apparatus (arrowhead) and on the acrosome vesicle (arrowhead). The inserts show the DIC picture.

Rabs proteins are present in the mammalian acrosome

Given recent debates regarding both the possible contribution of the endocytic pathway during acrosomal maturation and the nature of the organelle itself, we also tested several rab proteins known to function in endosomes and lysosomes. In order to investigate if the developing acrosome has some lysosome-characteristics during spermiogenesis, we determined the presence of rab7 an specific protein involved in late endosome/lysosome trafficking. Pachytene spermatocytes displayed only a faint label in the cytoplasm (Figure 2A). In contrast early round spermatids showed bright vesicles throughout the entire cytoplasm (Figure 2B). Interestingly, rab7 was found

associated with the developing acrosome in bovine elongating spermatids, as well as with a string cytoplasmic signal (Figure 2C). Unlike rab5, rab7 is present in late, and not early, endosomes (Bucci et al 2000) and has recently been shown to play a role in lysosome biogenesis (Bucci et al, 2000; Somsel Rodman & Wandinger-Ness, 2000).

FIGURE 2 Distribution of Rab7 during bovine spermiogenesis.
The plate shows the distribution of rab7 as evaluated by immunofluorescence (A, B and C) and the respective DIC pictured (A', B', C'). Rab 7 shows a punctate cytoplasmic distribution in pachytene spermatocytes (A, A') and early round spermatids (B, B'). More differentiated spermatids (step 8) rab7 labels cytoplasmic vesicles and the developing acrosome (arrow).

 

DISCUSSION

Acrosome biogenesis is essential for fertilization and for the initiation of development. The synthesis of the acrosomal enzyme acrosin, and probably many other components of the acrosome, starts at the pachytene stage (Moreno et al 2000a). Ultra-structural studies report that acrosin is sorted and packed into an electron-dense granule within the proacrosomal vesicles (Moreno et al 2000a, Ramalho-Santos et al, 2001, Susi et al, 1971). In spermatids, the proacrosomal vesicles fuse to each other and attach to the assembled perinuclear theca, forming the acrosomal vesicle. The acrosomal vesicle contains an electron-dense acrosomal granule that gives rise to the acrosomal matrix of mature spermatozoa. At this stage, the Golgi apparatus is located over the acrosomal vesicle, and many vesicles bud from it and fuses with the acrosomal vesicle membrane (Susi et al, 1971). During these stages of spermatid differentiation (cap and acrosome phase), the acrosomal vesicle spreads radially over one-third and, eventually, over one-half of the lengthening and compacting nucleus (Susi et al, 1971). After completing the production of acrosomal proteins, the Golgi apparatus separates from the acrosomal vesicle and starts to migrate towards the caudal portion of elongating spermatids. Finally, the Golgi stacks fragment is possibly digested by autophagocytocis in the discarded cytoplasmic droplet (Moreno et al, 2000a, Susi et al, 1971).

The Golgi apparatus in young rat spermatids consists of a compact hemispherical mass next to the developing acrosomal vesicle, with three to nine parallel saccules perforated with pores of various dimensions (Susi et al, 1971). Immunofluorescence and immunogold studies carried out with probes against cis- and medial- Golgi proteins, such as Golgin95/GM130, giantin, mannosidase II and beta-COP reveal a horseshoe-shaped Golgi apparatus, with the concave side facing the acrosome, in step 2- 7 spermatids (Martinez et al, 1996b; Moreno et al, 2000a, b; Ramalho-Santos et al, 2001). Moreover, all the aforementioned probes are also found in the acrosomal vesicle in step 2-7 spermatids but not in late elongating spermatids or mature sperm. On the other hand, this fact may represent a completely new mechanism of secretory vesicle formation, where the Golgi stacks fall apart and then fuse with the immature vesicle. This membrane flow may help to remodel the composition of the acrosomal membrane, and also may somehow participate in the shaping of the acrosome. There are a number of lysosomal proteins detected in the acrosomal vesicle, which are also subsequently retrieved throughout differentiation (Igdoura et al, 1999; Ramalho-Santos et al, 2001). The mechanism for the removal of these proteins may involve a specific degradation pathway at late steps of spermiogenesis. In this context it is interesting to note that some enzymes of the ubiquitin-mediated degradation pathway are present within the acrosome of developing spermatids (Sutovsky et al, 2000). However, we believed that coated vesicles with clathrin, and probably also beta-COP-coated vesicles, might participate in the retrieval of mis-sorted proteins from the acrosome (Moreno et al, 2000b). These proteins are present in late elongating spermatids, suggesting a membrane remodeling process of the acrosome at that stage (Ramalho-Santos et al, 2001).

The mammalian acrosome seems to be unique in its category since contains both specific and lysosome-derived enzymes (Hartree, 1975). This property has led to many studies suggesting that the acrosome may be a modified lysosome. Secretory lysosomes are found in many cells, and it seems that they are constantly produced not only in exocytic cells (Martinez et al, 2000). However, some typical lysosomal markers are not present on the acrosome, which raises the possibility that it might more accurately be described as a modified secretory granule (Martinez et al, 1996a). Nevertheless, the acrosome is a complex organelle that includes several structurally and biochemicaly distinct regions, and its growth and shaping relies on intense membrane trafficking, possibly involving both clathrin-coated and COPI-coated vesicles. One of the important regulators of membrane trafficking are the members of the rab family of small GTPases (Pfeffer,1999; White et al, 1999). These proteins play a crucial role in determining that transport vesicles will fuse with their appropriate targets. Our results obtained with SNAREs and rab proteins in this study seem to suggest that the acrosome may be a unique structure with several pathways converging on, and possibly contributing to, its biogenesis. Rab7 has been implicated in lysosome biogenesis and may control the fusion rate between late endosome and lysosomes (Bucci et al, 2000). In addition we have also found rab5, a protein involved in early endosome trafficking, in the acrosome of mouse spermatids (Bucci et al, 1992). In this way it is interesting that beta-COP is also involved in endosome trafficking (Whitney et al, 1995). In summary, our results suggest a contribution of the ensodome/lysosome system to the biogenesis of the mammalian acrosome. The way in which the spermatid regulates this membrane flow during spermiogenesis remains to be solved.

ACKNOWLEDGMENTS

This work was supported by research grants from DIPUC (2001/05E) and CONRAD (MFG-00-56) to R.D.M. J. R.-S. was the recipient of a Praxis XXI postdoctoral fellowship from Fundação para a Ciência e Tecnologia (FCT, Portugal). We wish to thank Professor Jaime Alvarez for his encouraging conversations and enthusiastic way of living life. We also wish to thank Cassia Mendes, Verónica Tapia, Diego, Waldo, Hugo, Diana and Minu for their love and inspiration in our lives.

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