An ultrastructural study of nuage–mitochondria complexes in spermatogonia of the sea urchin, Anthocidaris crassispina, was carried out. Release of mitochondrial contents into the cytoplasm was observed. The mitochondrial derivatives persisted as cristae-containing globules of friable material that subsequently contacted and integrated with nuage. The present ultrastructural findings agree with the results of other researchers who proposed that germ plasm substance probably produced by the nucleus is supplemented by the mitochondrial genome.
The electron-dense germ line materials containing ribonucleoproteins, referred to as ‘nuage’ or ‘germ plasm’, are the most distinct marker of germ line cells for a wide range of organisms ( Eddy 1975; Ikenishi 1998). This cytoplasmic substance has been found both in oogenesis and spermatogenesis. In the oogenesis of some taxons, the components of germ plasm are synthesized during oocyte development and then asymmetrically segregated to the posterior egg pole as ‘oosome’, ‘pole plasm’ or ‘germ plasm’ ( Ding & Lipshitz 1993; Klag & Bilinski 1993; Ikenishi 1998). However, in other studies, the patches of germ plasm disappear after the start of oocyte maturation ( Holland 1988). There are also reports showing continuity of maternal germ line granules throughout the life cycle of Caenorhabditis, Drosophila and Xenopus ( Mahowald 1977; Strome & Wood 1982; Ikenishi 1998). For spermatogenesis, nuage has been observed at the early stages of sperm development (spermatogonia and first spermatocytes) but then it undergoes dissolution after the start of meiosis ( Oke & Suarez-Quian 1992; Reunov & Rice 1993). It is not often that nuage is observed until the spermatid stage ( Werner et al. 1994 ). In taxons where the germ plasm granules disappear during maturation of female and male gametes, the primordial germ cells with reappeared nuage can segregate from somatic cells in relatively late stages of development. Therefore, it seems theoretically possible that the cytoplasmic determinants of the germ line may persist during early embryogenesis, but only at the molecular level, and not be visible by electron microscopy ( Holland 1988; Timmermans & Taverne 1989).
Sea urchins, A. crassispina, were collected from subtidal water at Kat O, Hong Kong, in June 1998. Upon arrival at the laboratory, specimens were dissected for spermatogenesis study. Gonads of three male individuals were dissected, cut into small pieces and fixed for 2 h in primary fixative (containing 1% tannic acid and 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 8.55% sucrose, pH 7.5). Fixed tissues were washed (in decreasing concentrations of sucrose-buffer solutions and buffer), postfixed in 2% buffered OsO4 for 2 h, rinsed in buffer and distilled water, dehydrated in an ethanol series and acetone, infiltrated and embedded in Spurr’s resin. Ultrathin sections were stained with 2% alcoholic uranyl acetate and aqueous lead citrate before being examined with a JEOL 100SX transmission electron microscope at 80 kV.
The gonad particles from three individuals were collected. Three blocks from each individual were sectioned for transmission electron microscopy (TEM) study. The sections were mounted on slot grids coated with formvar film stabilized with carbon. One section from each block was considered. Twenty spermatogonia containing nuage–mitochondria complexes were investigated on each section. From a total of 180 spermatogonia, 1620 mitochondria and mitochondrial derivatives were counted. Percentages were calculated. The result was analyzed statistically using the Student’s t-test.
Nuage-like material in spermatogonia of the sea urchin, A. crassispina, was observed as cytoplasmic conglomerates of electron-dense granulated substances enclosed among the mitochondria ( Au et al. 1998 ). Normally mitochondria are ovoid in shape, have quite prominent cristae and are filled with a friable electron-dense matrix. Mitochondria surrounding nuage could be observed releasing the electron-dense content ( Fig. 1A). Globules of the mitochondrial matrix were found to be devoid of any outside membrane and had mitochondria-like cristae inside ( Fig. 1B). The cristae containing globules of mitochondrial matrix were often attached to nuage aggregates ( Fig. 1C) and probably assimilated with it ( Fig. 1D). Some of the mitochondria surrounding the germ plasm substance had no cristae and appeared ‘empty’, probably after releasing their matrix into the cytoplasm ( Fig. 1D).
Electron microscopic mitochondria count
Four morphological types of mitochondrially related organelles were consistently observed in the spermatogonia of A. crassispina ( Fig. 1A–D). Percentages of these structures were calculated ( Fig. 2) from the 1620 mitochondria counted for all spermatogonia investigated (see Materials and Methods). It was found that 67% of the mitochondria were morphologically intact, 3% were observed in the process of matrix release ( Fig. 1A), 21% identified as cristae containing globules of mitochondrial matrix without an outer membrane ( Fig. 1B,C) and 9% could be described as ‘empty’ mitochondria ( Fig. 1D).
Data obtained demonstrate the release of mitochondrial content into the cytoplasm and the transient existence of particles of this substance as electron-dense globules of friable material with mitochondrial cristae inside. In our opinion these mitochondria-derived globules undergo assimilation with cytoplasmic nuage. Actually, the mitochondria-like globules, as well as the ‘empty’ mitochondria mentioned in the current publication, could quite often be observed in the micrographs of other authors ( Boswell & Mahowald 1985; Williams 1989; Cuoc et al. 1993 ). The coexistance of these structures, however, has never been explained as a sequence of mitochondrial content discharge.
For a long time the concept of nuclear synthesis of nuage has been accepted ( Eddy 1975; Aizenstadt 1984; Brough & Dixon 1989; Cuoc et al. 1993 ). However, some authors proposed that germ plasm components arose in the cytoplasm ( Klag & Bilinski 1993; Werner et al. 1994 ) although the mechanism of this synthesis is not clear. It was demonstrated that injection of mitochondrial large rRNA to ultraviolet- irradiated Drosophila embryos induced restoration of pole cell-forming ability ( Kobayashi & Okada 1989). Mitochondrial large rRNA was found outside mitochondria in the germ plasm of Drosophila ( Ding & Lipshitz 1993; Kobayashi et al. 1993 ) and Xenopus ( Kobayashi et al. 1998 ). Despite the function of mitochondrially encoded 16S large rRNA as a stimulant of pole cell formation being subject to doubt by some authors ( Ding et al. 1994 ), they do not deny that mitochondrial RNA is concentrated in the posterior polar plasm during normal development. Mitochondrial small ribosomal RNA has been reported to be associated with polar granules in Drosophila and Xenopus (see Ikenishi 1998; Kobayashi et al. 1998 ). Recently, extra mitochondrial 12 S and 16 S rRNA has been revealed in blastomeres of 16-cell stage sea urchin embryos ( Ogawa et al. 1999 ). Therefore, it seems quite possible that mitochondrial nucleic acids act as the cytoplasmic source of germ plasm components. Our ultrastructural observations coincide with the idea of Kobayashi et al. (1993 , 1998) who suggested that germ plasm as a product of the nuclear genome can be supplemented by the products of mitochondrial genes.
Calculation of more than 1000 mitochondria and mitochondrial derivatives demonstrated that quite significant amounts of these organelles participate in the process of nuage formation. On rare occasions (3%), mitochondria were observed releasing their matrix: this probably could be explained by the high velocity of this process. Most of the mitochondrial derivatives (21%) were composed of cristae containing globules whereas the empty mitochondria comprised a lesser amount (9%). As has been shown, globules of the mitochondrial matrix are associated with nuage. The destiny of empty mitochondria still is not absolutely clear, although our preliminary data suggest that these structures are degenerating in the cytoplasm and this process proceeds faster than the dissolution of matrix globules. Future exploration will be undertaken to elucidate the empty mitochondria disappearance mechanism.
We are grateful to the Research Committee, City University of Hong Kong for the award of a Strategic Grant (No. 7000602) to study spermatogenesis in sea urchins. We would like to thank Dr D. Shkuratov, Miss P. Y. M. Wan, Mr M. W. L. Chiang and staff in the Electron Microscope Units of the University of Hong Kong and the City University of Hong Kong for technical assistance.