Table 1.
Sources, sampling periods and numbers of sharks.
Fig 1.
The immature pole of the subadult testis.
(A) Low magnification of the germinal ridge (GR) showing numerous cell-lined spaces of various shapes and sizes medially in its dense stroma. Insets: a full transverse section (upper) of the elongated subadult testis for orientation of the GR, and approximate locations of the unorganized (blue arrowhead, blue arrow) and organized (lower, black arrowhead) testicular parenchyma. (B) The cell-linings of the spaces comprise either exclusively oblong somatic cells or the latter interspersed with a few large spermatogonia (asterisks), that otherwise are separately clustered together with oblong somatic cells (small black arrows) in between the spaces. Inset: clusters of spermatogonia (parenthesis) are also found distolaterally a short distance from the core of the GR underneath the testis capsule. (C) Another variation of the somatic cell-lined space with what clearly indicates a developing collecting duct (cd). Inset: The latter is distinctly different from an adjacent thick-walled intratesticular duct typically seen at the immature pole. Scr, somatic cell rings. Bar: A = 200 μm; B, C = 20 μm.
Fig 2.
Other modes of the assembly of cysts and their fusion at the immature pole of the subadult testis.
(A, B) Sometimes the clusters of primary spermatogonia reveal dark (green open arrowheads) and light speckled (blue open arrowheads) primary spermatogonial subtypes. Cysts may additionally arise from: (1) the rearrangement of aggregating oblong somatic cells (open arrows) around an increasingly discernible lumen, with the former now the new Sertoli cells (black underlined) on either side of the first two or three secondary spermatogonia, and (2) Sertoli cells that extend in a bridge-like manner towards an adjacent small cyst’s Sertoli cells giving rise to a larger cyst with a polar organization (square). (C, D) Adjoining newly formed and developing cysts, that are typically Sertoli cell-dominant, may initially share a common cyst wall (red filled arrowheads) that eventually becomes gradually disassembled due to retraction of the two or so Sertoli nuclei back into cyst periphery, culminating in cyst fusion and the “prompt” formation of a larger but asymmetrical cyst. (E) A developing cyst displaying a polar organization with bridge-like arranged Sertoli cells still distinguishable (black underlined). In contrast, Sertoli nuclei (black filled arrowheads) are less easily discerned among the opposite pole’s predominantly secondary spermatogonia, due in part, also to cross-sectioned profiles of elongated Sertoli nuclei that appear round. (F) An advanced developed ellipsoidal spermatogonial cyst showing randomly distributed Sertoli nuclei in an unevenly thick cyst epithelium. Collecting duct (cd), spermatogonia (open arrowheads, their complete numbers are shown in A, C, D). Bar = 20 μm.
Fig 3.
Developing spermatogonial cysts in the maturing testis (Bouins fixed tissues).
(A). The appearance of spaces in the nest of primary spermatogonia (open arrowheads). Note the continuous lumen with that of presumptive collecting duct (cd). Bouins fixation accentuates visualization of the spermatogonium’s cell membrane (open arrowheads) around its voluminous cytoplasm, neither of which is discernible around the Sertoli nuclei (filled arrowheads). (B). An unevenly thick epithelium in a recently formed cyst may be due to a germ cell-deficient area at one end of the cyst, and thus a cyst with polar asymmetry. Adjacent are rarely seen metaphase figures (green asterisk) amongst somatic cells from which the collecting duct is derived. (C, D). These polar asymmetries in cysts become more overt during subsequent cyst enlargement due to the appearance of initially one or two and eventually densely packed slender or spindle-like basophilic cells (black arrows) at end of the cyst. Scrutiny of adjacent cysts reveals the appearance of a second slender or elongated dark Sertoli cell type (yellow arrow). Bouins fixation commonly accentuates the discrimination of the M-phase spermatogonia’s mitotic spindle and metaphase figures (blue asterisk) and consequently the extent of their sizes. Bar = 20 μm.
Fig 4.
Phenomena at the most common type of cyst—Duct transition area (parentheses) in larger spermatogonial cysts in the maturing and adult testis.
(A) A tangential plane of sectioning that does not include the lumen (upper) reveal slender to spindle-like basophilic cells with pointed ends (yellow arrows) among the peripherally located spermatogonia (open arrowheads). Inset: Bouins fixation enhances the distinction between the cyst’s original Sertoli nuclei (black filled arrowheads) and those of a second dark Sertoli cell (yellow arrows). (B) The same phenomena are observed in mature spermatogonial cysts. Different planes of sectioning also reveal a triangular shape of the second dark Sertoli cell’s nucleus. Inset: enhanced exposure of the boxed area to highlight a dark but bulkier looking Sertoli nucleus with an irregular profile (yellow arrowhead). cd, collecting duct. Bar = 20 μm.
Fig 5.
Comparable internally organized spermatogonial cyst stages in the diametric testis that are conventionally associated with frequent Sertoli cell mitotic figures seen at the luminal border.
(A) Internal reorganization in the Greenland shark cysts is never complete as shown by the, at times, irregularly distributed Sertoli nuclei (encircled). Mitotic figures in such cysts are those of spermatogonia, including the odd dividing one of other spermatogonia ensconced (blue arrows) among the large bulky Sertoli nuclei. Note the next spermatogonial cyst stage’s eccentric peripheral aggregation of Sertoli nuclei (parentheses) in an odd-shaped cyst. (B, C). Positive control sections of the corresponding cyst stages in the blue shark testis showing periluminally located Sertoli cell mitotic figures (black arrows) and spermatogonial mitotic figures on the cyst periphery. The direction (red arrow) of the wave of spermatogonial mitotic activity as discerned from the increasingly dispersed chromatin in the neighboring spermatogonial nuclei. Bar = 20 μm.
Fig 6.
Meiotic divisions and spermiogenesis in the testis of the spermatogenically active mature Greenland shark.
Both meiotic divisions (A) appear to proceed normally. However, in some spermatid cysts, spermiogenesis is notably arrested at right at its initiation (B) or is delayed leading to asynchronous development. (C). Inset: Appearance of a scattering of elongated dark cells with pointed ends (black filled arrows) among spermatids at the site of attachment of the collecting duct (cd). (D) Spermiogenesis and spermiation are eventually completed in most cysts as evident from the spent cysts (square) and spermatozoa in the collecting ducts (red arrow). Inset: a slightly tangential section of an older spent cyst with a polarized appearance. A double row of strongly basophilic elongated nuclei with pointed ends (black filled arrows) are observed at one end of the cyst. Sertoli nuclei (black filled arrowheads); primary spermatocytes (psc); secondary spermatocytes (ssc), spermatids (spt). Bar = 20 μm.
Fig 7.
Testicular degeneration in the spermatogenically inactive male (Bouins-fixed).
A prominent feature is spermatogenic arrest very early in spermiogenesis (A) that elicits heightened Sertoli phagocytic activity (yellow arrowheads) to systematically clear germinal compartment (asterisks) of the developmentally arrested, pyknotic remains of the spermatids. This a cumulative process, as evident from a zone at the mature pole of persisting phagocytically cleared cysts (B) containing only randomly distributed Sertoli nuclei (yellow arrowheads). Insets A, B: These testes also reveal an increasing incidence of exclusively single pyknotic germ cell deaths (open arrowheads) in successive spermatogonial generations. Note concave Sertoli nuclei (yellow arrowheads), each tightly apposed to a pyknotic body. Bar = 30 μm.
Fig 8.
Testicular degeneration in subadult testes (formalin-fixed) manifests as arrested spermatogenesis at the primary spermatocyte stage.
(A) Meiotic arrest manifests as the gradual fragmentation of the spermatocytes into pyknotic bodies (1), which the Sertoli cells (arrowheads) gradually clear via phagocytosis (2→3). However, two of the subadult testes reveal two to three well-delineated patches of leukocyte infiltration (blue asterisk) around a subgroup of these degenerating cysts followed by their invasion (red asterisk). (B). Only the testes of these two subadults reveal the source of infiltrating leukocytes, which is a compact mass of eosinophilic, granule-laden immune cells under the testis capsule. Note the discreet migration of a few of these eosinophilic immune cells in the direction (large arrow) of the numerous intratesticular ducts and degenerating cysts. Inset: the occasional sight of cytoplasmic shedding adjacent to involuting ducts. Bar: A = 20 μm; B = 40 μm.