Testicular structure and germ cells morphology in salamanders

Abstract

Testes of salamanders or urodeles are paired elongated organs that are attached to the dorsal wall of the body by a mesorchium. The testes are composed of one or several lobes. Each lobe is morphologically and functionally a similar testicular unit. The lobes of the testis are joined by cords covered by a single peritoneal epithelium and subjacent connective tissue. The cords contain spermatogonia. Spermatogonia associate with Sertoli cells to form spermatocysts or cysts. The spermatogenic cells in a cyst undergo their development through spermatogenesis synchronously. The distribution of cysts displays the cephalo-caudal gradient in respect to the stage of spermatogenesis. The formation of cysts at cephalic end of the testis causes their migration along the lobules to the caudal end. Consequently, the disposition in cephalo-caudal regions of spermatogenesis can be observed in longitudinal sections of the testis. The germ cells are spermatogonia, diploid cells with mitotic activity; primary and second spermatocytes characterized by meiotic divisions that develop haploid spermatids; during spermiogenesis the spermatids differentiate to spermatozoa. During spermiation the cysts open and spermatozoa leave the testicular lobules. After spermiation occurs the development of Leydig cells into glandular tissue. This glandular tissue regressed at the end of the reproductive cycle.

Keywords: cephalo-caudal spermatogenesis; cyst; lobular testis; salamanders; spermatophores.

Figure 1.

A. Testicular lobes of Taricha granulosa. The testicular lobes (TL) are joined by an interlobar cord (IC). The cephalic region of the lobe contains spermatogonia (1) and spermatocytes (2) and the caudal region contain spermatozoa (3). Hematoxylin-eosin. Bar = 200 μm. Courtesy of Dr. Harry J. Grier.

Figure 2.

Cephalo-caudal disposition of the spermatogenic stages in longitudinal sections of the testes of Ambystoma dumerilii. (A,B). Three testicular regions of spermatogenesis advance progressively along the cephalo-caudal axis. (1) Starting at the cephalic region, the testis contains numerous lobules with early stages of spermatogenesis, which include spermatogonia, primary and secondary spermatocytes. (2) Subsequently, middle stages of spermatogenesis with spermatids. (3) Followed by late stages of spermatogenesis with spermatozoa. The testicular lobules enclose several cysts (cy) with germinal cells in synchronous stages of spermatogenesis. Alcian blue. Bar = 200 μm. Bar = 50 μm. (C,D). Sections of the peripheral cephalic region of the testis with cysts of proliferating spermatogonia. Primary spermatogonia (1Sg) and secondary spermatogonia (Sg2) are seen. The secondary spermatogonia form clusters surrounding a central lumen (L), structures that originate the testicular lobules. The clusters of secondary spermatogonia are delimited by loose connective tissue (ct). One secondary spermatogonium shows mitotic activity (*). Alcian blue. Bar = 20 μm.

Figure 3.

Cephalic region of the testis of Ambystoma dumerilii with primary and secondary spermatogonia. (A). A primary spermatogonium (1Sg) and several secondary spermatogonia (Sg2) surrounded by Sertoli cells (Se). Sertoli cells have elongated nuclei with granular chromatin and bordered by connective tissue (ct). Alcian blue. Bar = 10 μm. (B,C,D). The proliferation of spermatogonia is evident by chromosomes during mitotic phases, as metaphase (*) in Figs. B and C and anaphase (*) in Fig. D. The fibers of the spindle are observed in mitotic phases in Figs. C and D. The clusters of secondary spermatogonia (2Sg) have a central lumen (L). The spermatogonia are surrounded by Sertoli cells (Se). The connective tissue contains blood vessels (v). Hematoxylin-eosin. Bar = 20 μm. Alcian blue. Bar = 10 μm.

Figure 4.

Spermatogenesis in the testis of Ambystoma dumerilii with cysts of primary and secondary spermatocytes. (A,B). Primary spermatocytes in several stages of the prophase I of meiosis are seen, as: zygotene (z1Sc) with fibrillar chromosomes; pachytene (p1Sc) also with fibrillar chromosomes and the cell diameter increses; diplotene (d1Sc), the chromosomes have characteristic chiasmata; first meiotic division (*1Sc) with dense chromosomes in the spindle. Secondary spermatocytes (*2Sc) in the second meiotic division also contain dense chromosomes in the spindle. Compare the different size diameter of the primary and secondary spermatocytes in division, being smaller the secondary spermatocytes. Cysts with spermatids (St), near the secondary spermatocytes. Elongated nuclei of Sertoli cells (Se) surround the different cysts. Hematoxylin-eosin. Bar = 20 μm.

Figure 5.

Spermatogenesis in Ambystoma dumerilii with morphological details of primary spermatocytes and spermatids. (A). Primary spermatocytes during pachytene (p1Sc). The nuclei contain dense paired chromosomes. Sertoli cell nucleus (Se) and the interstitial tissue (it) are seen. (B). Primary spermatocytes during diplotene (d1Sc). The separation of the paired chromosomes is evident, remaining united in the chiasmata. Other spermatocyte is in metaphase of the first meiotic division (*1Sc). A Sertoli cell nucleus (Se) is seen. (C). Primary spermatocytes during metaphase of the first meiotic division (*1Sc). The division of the primary spermatocytes originates the secondary spermatocytes (2Sc). The secondary spermatocytes contain filamentous chromosomes previous to the second division of meiosis. Compare the smaller size of the secondary spermatocytes with the primary spermatocytes. A primary spermatocyte during pachytene (p1Sc) and the limit of the lobule with interstitial tissue (it) are also seen. (D). Early spermatids (eSt) with round nucleus, and middle spermatids (mSt) with elongation of the nucleus are evident. Sertoli cell nuclei (Se) and interstitial tissue (it). (A-D): Hematoxylin-eosin. Bar = 10 μm.

Figure 6.

Caudal region of the testis of Ambystoma dumerilii (A,C,D) and A. mexicanum (B) with details of morphological changes of the spermatids during spermiogenesis. (A,B,C,D). Lobules with cysts that contain early spermatids (eSt) with round nucleus, and middle spermatids (mSt) with different levels of elongation of the nucleus and late spermatids (lSt) with evident and progressive elongation. Sertoli cell nuclei (Se) surround the cysts, interstitial tissue (it). (A): Masson's trichrome. Bar = 10 μm. (B): Hematoxylin-eosin. Bar = 10 μm. (C,D): Masson's trichrome. Bar = 10 μm.

Figure 7.

Caudal region of the testis of Ambystoma dumerilii with details of morphological changes of the late spermatids during spermiogenesis. (A,B,C,D). Lobules with cysts that contain late spermatids (lSt) with final elongation of the nucleus on the head (h) and development of a thin and large flagella (f). Nuclei of Sertoli cells (Se) and interstitial tissue (it) are seen. Masson's trichrome. (A,B,D): Bar = 10 μm. (C): Bar = 50 μm.

Figure 8.

Caudal region of the testis of Ambystoma mexicanum with spermatozoa. (A). Lobules containing abundant cysts with spermatozoa (z). Interstitial tissue (it) surrounds the lobules. Hematoxylin-eosin. Bar = 50 μm. (B). Detail of cysts with spermatozoa (z) formed by the head (h) and flagellum (f). At the end of spermiogenesis the spermatozoa are swirled into the cysts. Masson's trichrome. Bar = 10 μm. (C). During spermiation, abnormal spermatozoa (az), showing irregular morphology, may remain into the cysts. Nuclei of Sertoli cells (Se) are seen. Alcian blue. Bar = 10 μm.

Figure 9.

Caudal region of the testis of Ambystoma mexicanum during spermiation. (A). There are lobules with spermatozoa (Sz) and, more caudally, there are lobules after spermiation without spermatozoa. Leydig cells (Le) hypertrophy around the lobules. Hematoxylin-eosin. Bar = 50 μm. (B). Detail of the Fig. 9A. During hypertrophy, Leydig cells (Le) become cuboidal, columnar or polyhedral in shape. The Leydig cells surround the lobules, where residual Sertoli cells (Se) and some spermatogonia (Sg) may be seen. Hematoxylin-eosin. Bar = 10 μm.

Figure 10.

Efferent ducts of Ambystoma dumerilii after spermiation. (A,B). Panoramic view of sections with 2 types of efferent ducts: transversal ducts (Td) and Wolffian ducts (Wd). The lumen of the Wolffian ducts contains abundant spermatozoa (z). Near the ducts, the periphery of both testes (T) is seen. Hematoxylin-eosin. Bar = 50 μm. Bar = 40 μm. (C,D). Details of the efferent ducts of Ambystoma dumerilii. Both types of ducts, transversal ducts (Td) and Wolffian ducts (Wd) have columnar epithelium (E). The epithelium of the transversal ducts presents long stereocilia (*) in the apical end. Wolffian ducts contain abundant spermatozoa (z). Connective tissue (ct) surrounds the ducts. Masson's trichrome. Bar = 20 μm. Bar = 10 μm.