Male reproductive system and spermatogenesis of Limodromus assimilis (Paykull 1790)

Abstract

Based on advanced light and electron microscopy, we describe the male reproductive system and sperm development of Limodromus assimilis. The genital tract consists of pairs of uni-follicular testes, spermatic ducts with diverticula regions, seminal vesicles, accessory glands, an unpaired ejaculatory duct and an aedeagus containing an internal sac equipped with sclerotic scales. Based on their morphology, we draw conclusions about their functions. After spermatogenesis within the follicle, the spermatozoa become released from the sperm cysts. The single spermatozoa move into the diverticula of the vasa deferentia I. Here, they become attached to central rods (spermatostyles), forming secondary conjugates (spermiozeugmata). The coordinated flagella movement of the conjugates possibly improves sperm velocity. Using super-resolution microscopy, we identified highly condensed reticulate chromatin in the lancet-shaped spermatozoa heads and the mitochondrial derivates of the flagella, likely formed by genomic and mitochondrial DNA, respectively. The results show, for the first time, sperm bundle formation in a Platynini species mainly corresponding to that found in Pterostichini species.

Fig 1. Male imago of L. assimilis.

The wide tarsen (arrow) allows one to distinguish males from females.

Fig 2. Organs of the male reproductive system.

(A) The different colours in the scheme (B) where the left testis is shown untwisted indicate the main functional parts.

Fig 3. Spermatogenesis stages in the testis.

(A) Testis section showing different stages of sperm development after haematoxylin-eosin (HE) staining. (a₁) The young part of a follicle surrounded by the epithilial wall (ew) contains sperm cysts with spermatogonia (sg) inside. The spermatogonia enclosed by sperm cyst membranes (cm) undergo several mitoses. (a₂) Spermatogonia during meiosis. Metaphase I (MI) and Anaphase I (AI) cells are marked. (a₃) Early spermatids with globular nuclei in the head and short flagella (fl). The sperm heads are disposed to the cyst membrane (labelled by the blue line). (a₄) Advanced spermatid stage. The mitochondrial chromatin becomes stretched along the elongated flagella. (a_5-1) Final stage of spermatogenesis with fully elongated spermatozoa. Within a cyst (sperm package) spermatozoa are parallel to each other. A fixed flagellar movement is visible, in which the mitochondrial derivates (md) appear darkly stained by HE. (a_5-2) Sperm package extracted from the posterior region of the testis. (B) Dissipation of sperm packages (arrow) by dissolution of the sperm cyst membranes in the posterior part of the testis. (C) Mature spermatozoon stained by the DNA specific dye DAPI. SIM reveals the chromatin structure of the mitochondrial derivates (md) (c1, c2) and of the nucleus (n) in lateral (c3) and plan (c4) view.

Fig 4. Sperm bundle formation in vasa deferentia I (A-E), their transport in vas deferens II (F) and seminal vesicle (F, G), and the structures of spermiozeugmata (J-L).

(A) Part of vas deferens I with diverticula, each containing a spermiozeugma (true colour bright field microscopy). (B) Part of vas deferens I with differently contracted diverticula (black arrows). The direction of the sperm bundle transport is marked by the yellow arrow (DIC). (C, D) Sections of vas deferens I stained with HE show diverticula with a cuboidal epithelium surrounded by a muscular layer (mu) (C) and a spermiozeugma located within a diverticulum consisting of a central rod and attached spermatozoa (D). (E) Oblique section of vas deferens I. AF-AB staining shows blue- and magenta-coloured sperm. (F) Longitudinal section of a part of vas deferens II without diverticula (HE stained) showing a columnar epithelium and a thin muscular layer (mu). (G) Longitudinal section of seminal vesicle (HE stained) filled with spermiozeugma. The squamous epithelium is marked. (H) Longitudinal section of the seminal vesicle. AB-PAS staining shows spermatozoa in magenta and central rods blue-red coloured. (I) Longitudinal section of the seminal vesicle. AF-AB staining shows magenta coloured spermiozeugmata. (J-L) Spermiozeugma isolated from the seminal vesicle. (J) Many spermatozoa are attached to the apical part of the central rod (arrow) of a spermiozeugma (darkfield microscopy). (K) Lateral and plan view of central rods. The apical parts are marked by arrows, the tail piece (inset) by an asterisk (DIC). (L) Anterior part of a spermiozeugma shown in DIC (left) and DNA specific (DAPI) fluorescence (right). The DNA labelling of the nuclei allows to quantify the number of spermatozoa within a sperm bundle.

Fig 5. Longitudinal sections of accessory glands stained with HE (A–C), AB-PAS (D) and AF-AB (E).

(A) Posterior part of an accessory gland filled with numerous spermiozeugmata. (B) Circular (ci mu) and longitudinal (lo mu) muscular layers are visible in the surface section. (C) The columnar gland wall epithelium is surrounded by circular and longitudinal muscle layers. (D) The secretion (asterisk) in the glandular lumen shows a mixed colour of blue and magenta indicating acid and neutral glycoproteins, polysaccharides, glyco- and phospholipids. (E) The blue colour of the secretion (asterisk) indicates that the acid glycoproteins yield carboxyl groups.

Fig 6. (A) Aedeagus (lateral view) shown by light (A, B) and its internal sac structures by electron microscopy (C–E).

Aedeagus with internal sac invaginated and (B) evaginated. Both parameres are also shown separately in (A). (C) Basal region of the internal sac with flat scales. (D) Median region of the internal sac with more or less taper scales. (E) Apical region of the internal sac with spicule-like scales.