Gross Morphology of the Male Reproductive Tract
The male internal reproductive tract in T. klimeschi is composed of two units. Each unit is formed by a bilobed testis comprising about 200 cysts, a seminal vesicle which becomes thicker as the maturation of sperm (Fig. 1B) and inserted in the testis depression, two accessory glands (curled gland and strand shaped gland) and vas deferens (Fig. 1A). The strand shaped accessory gland connected to the seminal vesicle while the curly accessory gland surrounded the vas deferens (Fig. 1A). Two units fuse at their ends flowing into an ejaculatory duct (Fig. 1A).
The cysts are full of testes (Fig. 1C), within which spermatogenesis occurs. The cyst cell with a big nucleus (Fig. 2A) constitutes a group of germ cells surrounded by an epithelium. The number of spermatids per cyst is 350 to 512. Most mature sperms are stored in the seminal vesicle.
Spermatogenesis
During spermatogenesis, spermatogonia undergo mitosis to produce spermatocytes that undergo meiosis to produce spermatids which to be spermatozoa eventually via spermiogenesis.
TEM allowed us to determine that the spermatogonia with a diameter of 5.67 ± 0.58 µm are irregular cells and have an irregular and large nucleus (3.45 ± 1.04 µm), homogeneously distributed granular chromatin and irregularly distributed high electron-dense heterochromatin lump (Figs. 2A, B). The cytoplasm contains abundant glycogen granules and mitochondria, as well as Golgi, a large lysosome and smooth endoplasmic reticulum.
The spermatocytes (Fig. 2C), originate from mitosis of spermatogonia, have a diameter of 4.69 ± 0.43 µm. The cells are characterized by their round shaped nucleus of 2.40 ± 0.71 µm in diameter and a low electron-dense band comprised by smooth endoplasmic reticulum around the nucleus. Large lysosome, large mitochondria with well-defined crests, Golgi and abundant glycogen granules can be observed in their cytoplasm.
The spermatids (Fig. 2D) produced by meiotic division of the spermatocytes are 4.88 ± 0.28 µm in diameter with a round nucleus of 3.31 ± 0.22 µm in diameter. Nuclear chromatin exhibits different degrees of compactness and electron density and it is possible to observe the presence of synaptonemal complexes in some nuclei. The cytoplasm contains electron-lucent Golgi and many mitochondria.
Spermiogenesis
There are many changes in spermiogenesis. Nuclear chromatin begins to unhomogeneously concentrate and electron density is enhanced (Figs. 3B, C, 4G, H). At a more mature stage, nuclear chromatin will present two distinct regions (Figs. 4E, F), one is homogeneously compact and another is fibrillar. The posterior end of the nucleus forms a concavity where the axoneme occurring and elongate (Figs. 4A, G). Nearly round preacrosomal vesicle with 452.70 ± 22.82 nm in diameter is visible and close to the nucleus (Fig. 4B) and Golgi apparatus (Fig. 4C) and then became flat (Fig. 4D) as the elongation of nucleus.
At the same period, the spherical-shaped mitochondria that are found dispersed in the cytoplasm begin to migrate toward the basal pole of the nucleus (Fig. 3B). Then the globular mitochondria aggregate (Fig. 3C) and fuse into thin lines (Fig. 3D), which in turns become a coarse state, and then become thicker irregular but chimeric two mitochondrial derivatives (Figs. 3E, F). These two mitochondrial derivatives become slender as the cell’s elongating (Fig. 4G, H). A pair of vertically arranged centrioles are separated and located at the ends of the mitochondrial derivatives (Figs. 4H, I). Shortly after, the centriolar adjunct appeared on both side of the origin of axoneme (Fig. 4L). During spermatogenesis, the sperm components are surrounded by microtubules (Figs. 4E, F) which are visible in spermatozoa (Fig. 6F).
Spermatozoa
The spermatozoa of T. klimeschi are slender (Fig. 5A), measuring about 75 µm in length and 0.5 µm in wide and composed of an acrosomal complex, a nucleus (Figs. 6A, D), an axoneme, two mitochondrial derivatives and two accessory bodies (Fig. 6F).
The 3-layred acrosome complex (Figs. 6A, B) made up of a dense extra acrosomal granular layer with low electron density material, a cup-like acrosomal vesicle and a rod-like conical perforatorium, measuring about 1.2 µm in length. The acrosome complex lies on a slightly concave nuclear face and is separated from nucleus by a thick basal lamina (Fig. 6A).
Close to the acrosome complex is the nucleus (Figs. 6A, B) with circular section, about 6 µm in length and 0.4 µm in diameter indicated by longitudinal section. The nucleus is not homogeneous and has two different states of aggregation (Fig. 6C), there are obvious boundary between thick filamentous nuclear condensation and filamentous nuclear condensation.
Longitudinal section showing (Fig. 6D) that the first to insert into the nucleus is the large mitochondrial derivative, followed by the small mitochondrial derivative, axoneme and two accessory bodies. The cross section indicates that the axoneme (Figs. 6F, G) has a diameter of 277.89 ± 10.58 nm with 9 + 9 + 2 microtubules, the large mitochondrial derivatives with dark crystals accounted for about 60% of its circular cross section is always thinner than the axoneme and will become thinner along the axoneme until disappearing completely (Figs. 6F, H). The dark crystals accounted for about 40% of the drop shape cross section of the small mitochondrial derivative (Fig. 6F). The longitudinal section indicates that mitochondrial derivatives are comb-like (Figs. 5C, 6E). The axoneme and mitochondrial derivatives of T. klimeschi are not embracing each other, but rather run more or less parallel to each other (Fig. 5B).
Two accessory bodies are different (Fig. 6F). The completely crystalline crescent accessory body is smaller than a quarter of axoneme and close to the small mitochondrial derivative. The other consists of a small crystal that was mostly triangular and sometimes crescent and a big compact “puff”-like expansion.
At the end of the sperm, the first to disappear is the two accessory bodies, followed by small mitochondrial derivatives, large mitochondrial derivatives and finally is the disintegration and disappearance of axoneme (Fig. 6H).
In this study, bi- and multi-flagellate spermatids and spermatozoa were observed at a low rate (Figs. 6I, J).
The spermatozoa of T. klimeschi individually stored in the seminal vesicle, its long flagella folded into several turns and the whole sperm is wrapped in a film (Figs. 5D, E). Compare to slender spermatozoon, this state spermatozoon is more like a tadpole. The head of the “tadpole” is a circle surrounded by the middle of spermatozoon, in some circle form an “S” shape, and the tail is juxtaposed by the two ends of the spermatozoon.