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A blue fluorescence with characteristics similar to those of reduced pyridine nucleotide of isolated mitochondria has been demonstrated to be localized in the nebenkerns of cricket, cockroach, and gerris spermatids, and in the mitochondria of intact ascites cells. Further evidence for the association of fluorescence with pyridine nucleotide is its response to oxygen tension. It is possible to oxygenate cells which have already reached anaerobiosis by illumination of photosynthetic algae which are included in the same preparation. The nebenkern of spermatids and the mitochondria of ascites cells show cyclic increase and decrease of fluorescence in response to alternation of darkness (consumption of oxygen by respiratory activity) and light (oxygen evolution by photosynthesis). Repeated microfluorimetric determinations in the same cell, through successive cycles of oxidation-reduction are made possible by suitable improvements in the suspension media.

A systematic study of differentiation of the spermatids in Drosophila melanogaster et virilis and Gelastorrhinus bicolor de Haan was made with the electron microscope. The chromidial bodies with a concentric laminated structure, possibly related to the cytoplasmic membrane-bound apparatus, have been found in the juxtanuclear region of the undifferentiated spermatid. At a particular stage in the evolution of spermatid, namely after the caudal migration of the paired nebenkern derivatives, a conspicuous membrane-bound apparatus appears in the ground substance of the cytoplasm. This apparatus consists of broad, flattened cisternae 5 mμ or less in width, but no fine granules lie between this membrane-bound system and on the surface of the membrane. However, the apparatus is associated with the chromidial body, the Golgi complex, and the nebenkern derivative. The dense body appears in the matrix of the nebenkern derivative in a particular stage of differentiation and grows by accretion of new material to its surface, thus bringing about its progressive enlargement. The origin and fate of the dense body have been discussed. The structure of the insect sperm tail flagellum is fundamentally very similar to that of the mammalian sperms: the middle-piece of the insect sperm is enclosed by the elongated nebenkern derivative; the main-piece by the loosely running fibrous helical sheath; the end-piece is devoid of the helical sheath; the axial filaments consist of two sets, a central set and a peripheral set.

During early spermatid elongation, at which time germ cells of the mutant l(3)pl (lethal-polyploid) of Drosophila hydei are entirely blocked in their differentiation [16], the microtubular system displays numerous abnormalities on the ultrastructural level. Unstable localization of the otherwise normal centriole, fractionation of the nebenkern, and lack of both nuclear and cellular elongation reflect a general disorganization in cellular arrangement. Moreover, in the growing flagellum, the central tubules are missing. The cytoplasmic microtubules are reduced in number and show various structural abnormalities. Less than one-third have a normal cylindrical cross-section. Single tubules or doublets with one or several side projections of varying lengths are observed, the most frequent representing an α-shape. Cross-bridged tubules may form short chains. The similarity of the observed malformations to the effects of antimitotic drugs suggests that the gene l(3)pl acts at the level of microtubular assembly.

The fine structure of mitochondria and the structural changes leading to the formation and evolution of the mitochondrial body or “nebenkern” of the spermatid of insects have been investigated in the grasshopper Laplatacris dispar. Each mitochondrion possesses a double mitochondrial membrane, tubule-like crests and a mitochondrial matrix. In this material it is clearly demonstrated that the crests are folds of the inner mitochondrial membrane that end freely in the mitochondrial matrix. In the early spermatid the localization and the early fusion of some mitochondria in one region of the cytoplasm were observed. This period is followed by the approachment and agglutination of all mitochondria to form the mitochondrial body. The probable mechanisms that may be involved in this agglutination are discussed. This stage is continued by an intense process of remodelling of the mitochondrial body, leading to a differentiation of an inner region made of fused small mitochondrial profiles and an outer one of large profiles filled with abundant mitochondrial matrix. The inner and outer profiles are in ample continuity. An active process of flow of the mitochondrial membranes and of the matrix is postulated. The mitochondrial matrix shows the presence of a fine macromolecular filamentous component. This phenomenon is another indication of the fact that there is a continuity in the mitochondrial cavity and that there are no complete partitions of it. A labyrinthic even space, 240 A wide, occupied by a material of weak electron density separates the fused mitochondrial membranes. The intense remodelling of the mitochondrial body leads to a diminution of the membranes and finally to the division of the “nebenkern” into two bodies. In later stages the “nebenkern” is drawn into an elongated body that still maintains a modified but characteristic mitochondrial structure. The fine structure of other cytoplasmic components of the seminal cell, including the centrioles, Golgi bodies and endoplasmic reticulum, is described. The endoplasmic reticulum is mainly agranular but shows differentiated regions with attached granules (granular endoplasmic reticulum). Both types of reticulum are in direct continuity.