ИСТИНА

Peculiarities of spermatogenesis and organization of spermatozoa are usually useful for reconstruction of phylogeny in close related groups of invertebrates. It is especially important for reconstruction of phylogeny of such an enigmatic group as brachiopods. In addition, organization of mature spermatozoon may help to understand the reproductive biology of deep sea animals. The spermatogenesis and ultrastructure of sperm are studied in deep sea water discinid brachiopod Pelagodiscus atlanticus. Testes of P. atlanticus are voluminous sacs, which are located along the lateral sides of the body. Each testis has complex shape with large external portion and folded inner portion. Spermatogonia develop around the blood capillaries and contain germ plasm, numerous mitochondria of small diameter, Golgi apparatus, and lipid droplets. Spermatocytes form clusters of four cells, which connect via cytoplasmic bridges. In secondary spermatocytes, Golgi apparatus produces several proacrosomal vesicles, which are located at the posterior pole of cell and then fuse each other and migrate to anterior pole. The orthogonal distal and proximal centrioles are visible in secondary spermatocytes. In early spermatid, the nucleus undergoes changes and condensation of the chromatin. In early spermatids, the acrosomal vesicle is large and consists of peripheral electron dense material and central content of middle electron density. Mitochondria of early spermatids are not such numerous as they are in spermatogonia; mitochondria become bigger and their matrix becomes denser. Early spermatids still contain Golgi apparatus, which is located near the centrioles. Late spermatid looks like spermatozoom (see below), but have larger amount of cytoplasm, which is then eliminated as residual body. Each spermatozoon has a barrel-shaped head. The acrosome vesicle looks like a hat with a central small invagination on its basal surface. A margin of the acrosome vesicle narrows forming a small projection that reminds a brim of the hat. The inner space of the acrosome vesicle is heterogeneous. About 20 bundles of electron-dense material occur around of the brim and each of them gives the narrow bands to the top of the acrosome vesicle. The central part of the acrosomal vesicle, in front of the invagination, contains an electron-dense homogeneous material. Subacrosomal material consists of fibrils. The round nucleus has an anterior broad depression with deeper central invagination that is filled with subacrosomal material. The posterior part of the nucleus has a fossa that partially contains the proximal centriole. The lateral sides of the nucleus is indented laterally to accommodate the mitochondria. The nucleus contains electron-dense chromatin with several small electron-lucent lacunas. The most part of the nucleus is surrounded by 8 mitochondria arranging into two rows. The proximal and distal centrioles are orthogonal in their arrangement and located in the center of the mitochondria ring. Nine satellite projections give rise from the distal centriole. Each of them bifurcates twice and has striated structure. The flagellum has typical 9+2 pattern of microtubules. Organization of mature spermatozoon is studied in several brachiopod species from all three clades: Discinisca and Lingula (Linguliformea), Crania (Craniiformea), Calloria, Kraussina, Terebratulina, and Coptothyris (Rhynchonelliformea) (Sawada, 1973; Chuang, 1983; Afzelius, Ferraguti, 1978; James et al., 1992; Hodgson, Reunov, 1994; Drozdov et al., 2012). Spermatozoa of all brachiopods studied to date, may be considered as spermatozoa of primitive type with some modifications. The presence of such type of mature spermatozoon evidences that deep sea water P. atlanticus has external fertilization and its mature oocytes lack of any thick envelops. The organization of mature spermatozoa is similar in P. atlanticus and Discinisca tenius (Hodgson, Reunov, 1994). In both species, all organelles of spermatozoon are represented in their ancestral condition as it is described in literature (Reunov, 2005). Comparative analysis of all data on organization of brachiopod spermatozoa allows to insist that discinids maintained the most primitive type of the spermatozoon. We suppose that the last common brachiopod ancestor had spermatozoon, which looked like spermatozoon of discinids. This primitive spermatozoon has trapeziform acrosome, small compact nucleus, eight mitochondria around the nucleus, and two orthogonal centrioles (Fig. 1). In comparison with this primitive type, spermatozoa of all other brachiopods underwent some changes. Thus, in lingulids, acrosoma little changes in shape and the number of mitochondria reduces to six (Sawada, 1973; Chuang, 1983). In craniid Novocrania anomala, the acrosome drastically changes in shape and number of mitochondria reduces to four (Afzelius and Ferraguti, 1978). In Rhynchonelliformea, acrosoma is very small, there is a single circular mitochondria, and centrioles are located parallel to each other. There are three main ways of evolution of mature spermatozoon in brachiopods. This conclusion supports the brachiopod phylogeny, which is based on data of morphological and molecular analysis and suggests the presence of three main stems within brachiopods: Linguliformea, Craniiformea, and Rhynchonelliformea. At the same time, our results do not corroborate the idea about presence of “articulate” and “inarticulate” brachiopods as taxonomically valid clades. This study is supported by Russian Foundation for Basic Research (#18-04-00586); the ultrastructural researches were done with support from Russian Science Foundation (#18-14-00082).