Gametogenesis and fertilization in Plasmodium yoelii nigeriensis : a transmission electron microscope study

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Abstract
The ultrastructure of the micro- and macro-gametocytes of Plasmodium yoelii nigeriensis and the nuclear and cytoplasmic changes during gametogenesis and fertilization were examined with the electron microscope. Osmiophilic bodies, dispersed in the cytoplasm, served to distinguish the gametocytes from other erythrocytic stages and were thought to play a part in the gametocytes' escape from the host cells by attachment to the parasite's plasmalemma causing dissolution of the overlying erythrocyte cytoplasm. Macrogametocytes were distinguished from the microgametocytes by their greater density of ribosomes, more elaborate endoplasmic reticulum, which contained electron dense material, more numerous mitochondria and smaller nucleus. In microgametogenesis nuclear division was endomitotic and the genome was segregated on three successive spindle formations. Microtubule organizing centres developed adjacent to the nuclear envelope and gave rise to orthogonal tetrads of kinetosomes which were found at opposite poles of the first nuclear spindle. Axoneme assembly from the kinetosome followed the usual pattern by the addition of sub-units terminally on to the A subfibres, and, with a slight lag, on to the B subfibres. The kinetosomes were closely linked to centriolar plaques in pores of the nuclear envelope, at the spindle poles. The attachment of the kinetosomes and their axonemes to the spindle poles provided the mechanism by which each haploid set of chromosomes was eventually endowed with a single axoneme. At the time of the final nuclear segregation the kinetosome and a newly formed juxta-kinetosomal sphere and granule became surrounded by a basket work of irregular tubules which lay close to a bud of the nucleus containing a spindle pole and the now highly condensed chromatin. During exflagellation the juxta-kinetosomal sphere and granule, together with the kinetosome and axoneme were forced through the perikinetosomal basket perpendicularly towards the surface and distended the plasmalemma. In the final stages of gamete formation, the gamete slid off tangentially to the surface and the nuclear bud also passed through the perikinetosomal basket, became separated from the main body of nucleoplasm and was incorporated as the nucleus of the gamete. The free microgamete contained a single axoneme with its kinetosome and distal juxta-kinetosomal sphere and granule. The condensed nucleus was intertwined with the axoneme. After emergence from the erythrocyte there were contrastingly few changes in the macrogamete. The absence of intranuclear spindles and maturation bodies provided evidence that meiosis did not occur at this stage. At fertilization one pole of the microgamete was closely applied to the surface of the macrogamete, and amorphous material on the outer surfaces became confluent. This allowed contact and fusion of the plasmalemmas. The naked axoneme and the nucleus of the microgamete passed into the cytoplasm of the macrogamete. Decondensation of the microgamete chromatin may have occurred before fusion of the two nuclei. A chromosome number of about ten was estimated for the microgametes from numbers of kinetochores and microtubules in the intranuclear spindles. This accords with the estimate for the sporogonic stages. The perikinetosomal basket and juxta-kinetosomal sphere and granule have not been described previously in malaria parasites. The former may have a skeletal function in directing the axoneme and nuclear bud towards the surface. The latter, in their position at the distal end of the microgamete may assist in the penetration of the macrogamete by mediating the fusion of the plasmalemmas of the two gametes, though we have not been able to determine that the kinetosomal end is that one which contacts the macrogamete.