Ultrastructure of spermatogenesis in the white clam Chione californiensis (Broderip, 1835) (Mollusca: Pelecypoda).

ABSTRACT Although studies of this species have focused on its reproductive cycle, there is no literature available on the ultrastructure of its germ cells. This study consequently aims to describe the latter cells as well as the shape of the acrosome, a character that may be of help in the taxonomic allocation of species of this family or other bivalve families. Results indicate spermatogonia have a central, spherical nucleus as well as scattered heterochromatin granules throughout the nucleoplasm. Rough endoplasmic reticulum is scarce, but the cytoplasm is rich in mitochondria and disperse glycogen granules. In primary spermatocytes, heterochromatin is more highly condensed and there is a reduction in the amount of cytoplasm and number of mitochondria as compared with spermatogonia. In secondary spermatocytes, heterochromatin is present in peripheral areas of the nucleus, forming occasional projections towards its center. In spermatids chromatin is fully condensed, taking up the entire nucleus. Mitochondria increase in size and migrate to the basal pole, giving rise, along with the centriole, to the spermatozoon neck. In spermatozoa, the nucleus is ovoid, the acrosome is round and the centriole is surrounded by four mitochondria, unlike other bivalves, like Anadara tuberculosa, in which the nucleus is reported to be round, the acrosome is pyramidal and there are five mitochondria.

KEY WORDS: white clam, Chione, spermatogenesis, ultrastructure

INTRODUCTION

The white clam, Chione californiensis (Broderip, 1835) is a bivalve mollusc of the family Veneridae, a group regarded as a potential fishery and aquaculture resource (Baqueiro 1987). One of several species exploited by the traditional fishing industry along the coasts of the Sea of Cortez, it is for the most part a local food item (Baqueiro 1989). The reproductive cycle of several species of the genus Chione in Mexico has been described previously. Garcia-Dominguez et al. (1993), in particular, have described the gonadal cycle of Ch. californiensis.

The ultrastructure of the spermatozoa has been described in many bivalve genera, such as Bamia candida (Pasteels & Harven 1962), Hyriopsis schlegelii (Higashi 1964), Turtonia minuta (Ockelmann 1964), Mytilus edulis and M. perna (Niijima & Dan 1965, Bourcart et al. 1965), Spisula solidissima (Longo & Anderson 1969), Corbicula sandal (Hachiri & Higashi 1970), Crassostrea virginica (Galtsoff & Philpott 1960, Daniels et al. 1973), Bankia spp. (Popham et al. 1974), Lyrodus spp. and Teredo spp. (Popham 1974), Ligumia rostrata (Trimble & Gaudin 1975), Nucula hartvigiana (Popham & Marshall 1977), Anadara trapezia, Anomia descripta, Fulvia tennuicostate, Myodara brevis and Notocorbula vicaria (Popham, 1979).

The ultrastructure of the different germ cells occurring during spermatogenesis and spermiogenesis was described in Anaclara granosa by Suwanrajat and Parnrong (1999) and by Ortiz et al. (2003) in A. tuberculosa. Differences were found as to number of mitochondria and acrosome shape. Our study thus aims to describe the ultrastructure of these cells in Ch. californiensis, a factor that may help significantly to improve the taxonomic allocation of different bivalve species.

MATERIALS AND METHODS

Male specimens were collected in an intertidal coral reef located north of Ensenada de la Paz, a coastal lagoon lying southwest of Bahia de la Paz, B.C.S., Mexico, at 24[degrees]06" to 24[degrees]10"N and 110[degrees]19" to 110[degrees]25"W. Small samples of gonad tissue were removed and fixed in 2.5% glutaraldehyde in seawater with a pH of 8 for 48 h, then rinsed with 2.5% sodium bicarbonate and postfixed with osmium tetroxide for 1 h (Buckland et al. 1986). Dehydration was done in three 10-min shifts with different concentrations of ethanol, followed by two 20-min propylene oxide shifts. Samples were then permeated with 1:1 propylene oxide/epoxy and embedded in Epon 812. Very thin 70-nm sections were obtained and set in mesh copper grids. These were treated with uranyl acetate and lead citrate to enhance contrast and later examined under the transmission electron microscope (Jeol-100SX).

RESULTS

The first germ cells to mature during spermatogenesis are spermatogonia, which have a large central nucleus and, typically, heterochromatin granules of varied size scattered throughout the nucleoplasm. The cytoplasm is rich in mitochondria, whereas rough endoplasmic reticulum, found near the nucleus, is scarce (Fig. 1A).

[FIGURE 1 OMITTED]

Primary spermatocytes are formed by mitosis of the spermatogonia. The nucleus has a larger amount of condensed heterochromatin, but there is less cytoplasm and fewer mitochondria than in spermatogonia (Fig. 1B). Primary spermatocytes give rise by meiosis to secondary spermatocytes. Heterochromatin is characteristically more condensed in the latter, migrating to peripheral areas of the nucleus (Fig. 1C).

Spermatids arise from secondary spermatocytes, which have become significantly smaller. There is less cytoplasm and it forms a halo around the nucleus. Chromatin takes up all of the latter and it is fully condensed. During this stage, mitochondria increase in size and migrate to the basal pole of the nucleus (Fig. 1D).

Lastly, spermatids undergo a complex change during spermiogenesis giving rise to spermatozoa, which display three regions: head, middle piece and tail. The head is formed by an elongate nucleus and a round acrosoma (Fig. 2A). The neck or middle piece contains four mitochondria with well-developed cristae arrayed in a circle (Fig. 2B). Central to the four mitochondria lies the centriole, which gives rise to the flagellum. The latter exhibits the typical axoneme structure of nine paired peripheral microtubules and two central ones.

[FIGURE 2 OMITTED]

DISCUSSION

During the first two stages of spermatogenesis involving maturation of the spermatogonia, in the course of formation of the primary spermatocytes, which undergo mitotic multiplication to give rise to secondary spermatocytes and during meiosis of the secondary spermatocytes in the course of which spermatids arise, germ cells contain a large central nucleus with heterochromatin granules of varied size and there are few organelles. In spermatids, chromatin is fully condensed and a reduced cytoplasm forms a halo about the nucleus, whereas mitochondria increase in size and migrate to the basal pole of the nucleus. These characters are similar to those reported by Suwanrajat and Parnrong (1999) in Anadara granosa and Ortiz et al. (2003) in A. tuberculosa.

During spermiogenesis the name given to the last stage of spermatogenesis spermatids are fully transformed to give rise to spermatozoa. Existing characters confirm the primitive nature of the latter, as in several other bivalves with external fertilization (Franzen 1955).

The acrosome of molluscs varies widely as to size, composition, morphology and position in spermatozoa. Arising usually in a vesicle derived from the Golgi body (Popham 1979), its function is to dissolve the various egg layers so that fusion of spermatozoon and ovum can occur (Colwin & Colwin 1967, Franklin 1970, Longo 1973). Chione californiensis has a round acrosome, unlike A. tuberculosa where the latter is pyramidal (Ortiz et al. 2003) and A. granosa in which it is triangular (Suwanrajat & Parnrong 1999).

The spermatozoon nucleus displays great morphological diversity in mollusc species. In spermatozoa of a primitive type the nucleus may be spherical, ovoid or conical (Popham 1979). Franzen (1955, 1970) mentions this structure is used as a systematic character.

Mitochondria are usually spherical in shape and their array as spherical masses on the proximal end of the spermatozoon head is also typical of species with external fertilization. Suwanrajat and Parnrong (1999) say the number of mitochondria may be species specific. Five mitochondria are present in A. tuberculosa (Ortiz et al. 2003) and four in Ch. californiensis, confirming the fact that this may be a species specific character.

Axoneme structure of the flagellum varies little in bivalve molluscs. It is comprised of a central pair of microtubules, which is surrounded by nine peripheral pairs (Ortiz et al. 2003). This concurs with our observations in Ch. californiensis.

We may conclude from this that the spermatozoa of Chione californiensis exhibit as species specific characters an ovoid nucleus, a round acrosome and a centriole surrounded by four mitochondria.

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ESPERANZA ORTIZ-ORDONEZ, (1,3) * ESTHER URIA GALICIA, (1,3) ANGELICA SILVA-OLIVARES, (2) VICTOR TSUTSUMI (2) AND MINEKO SHIBAYAMA (2)

(1) Dept. de Morfologia.Escuela Nacional de Ciencias Biologicas, I.P.N. Prol. Carpio y Plan de Ayala S/N Mexico, D.F. 11340; (2) Dept. Patologia Experimental.Centro de Investigacion y de Estudios Avanzados, I.P.N. Mexico; (3) Becario COFAA

* Corresponding author. E-mail: [e-mail]