Low mitochondrial activity within developing earthworm male germ-line cysts revealed by JC-1.

The male germ-line cysts that occur in annelids appear to be a very convenient model for spermatogenesis studies. Germ-line cysts in the studied earthworm are composed of two compartments: (1) germ cells, where each cell is connected via one intercellular bridge to (2) an anuclear central cytoplasmic mass, the cytophore. In the present paper, confocal and transmission electron microscopy were used to follow the changes in the mitochondrial activity and ultrastructure within the cysts during spermatogenesis. JC-1 was used to visualize the populations of mitochondria with a high and low membrane potential. We used the spot detection Imaris software module to obtain the quantitative data. We counted and compared the 'mitochondrial spots' - the smallest detectable signals from mitochondria. It was found that in all of the stages of cyst development, the majority of mitochondria spots showed a green fluorescence, thus indicating a low mitochondrial membrane potential (MMP). Moreover, the number of active mitochondria spots that were visualized by red JC-1 fluorescence (high MMP) drastically decreased as spermatogenesis progressed. As much as 26% of the total number of mitochondrial spots in the spermatogonial cysts showed a high MMP - 19% in the spermatocytes, 24% in the isodiametric spermatids and 3% and 6%, respectively, in the cysts that were holding early and late elongate spermatids. The mitochondria were usually thread-like and had an electron-dense matrix and lamellar cristae. Then, during spermiogenesis, the mitochondria within both the spermatids and the cytophore had a tendency to form aggregates in which the mitochondria were cemented by an electron-dense material.