**3. Morphology of spermatids in** *A. dumerilii* **during spermiogenesis**

spermatocytes contain a haploid, but duplicated, number of chromosomes (**Figure 2C**). These cells are seen less frequent, since they divide during the second part of meiosis after a very short

connective tissue (c), tunica albuginea (T), and blood vessels (v). B: H-E, C: PAS. Bar = 20 μm.

**Figure 2.** Spermatogenesis in the testis of *Ambystoma dumerilii*. (A) Cephalic region of the testis. Spermatogonia surrounded by connective tissue. Lobules with cysts containing cells in different stages of spermatogenesis. Masson's trichrome. Bar = 30 μm. (B, C) Periphery of the testis in the adjacent region to spermatogonia. Cysts containing primary spermatocytes during the first meiotic prophase, in pachytene, with thick fibrillar chromatin, in diplotene with pairs of chromosomes showing chiasms and during the first meiotic division, secondary spermatocytes and early spermatids. Around the lobules, there is connective tissue. The tunica albuginea with blood vessels surrounds the testis. Spermatogonia (Sg), primary spermatocytes (S1), primary spermatocytes in pachytene (S1p), primary spermatocytes in diplotene (S1d), primary spermatocytes during the first meiotic division (S1md), secondary spermatocytes (S2), spermatids (St),

interphase, rapidly giving rise to two spermatids.

12 Spermatozoa - Facts and Perspectives

The spermatids of *A. dumerilii* initiate the spermiogenesis, occurring during a sequence of morphological changes transforming the spermatids into spermatozoa. Early spermatids are spherical in shape and attain a diameter of 14–17 μm; their nuclei contain light fibrillar chromosomes.

**Figure 4.** Early spermatids in the testis of *Ambystoma dumerilii*. (A) Primary spermatocytes during pachytene and early spermatids with round nucleus. H-E. Bar = 20 μm. (B) Early spermatids with fine fibrillar chromatin. H-E. Bar = 20 μm. (C) Initial elongation of the spermatids and more compact aspect of the nucleus. H-E. Bar = 20 μm. Early spermatids (St1), (St2) and interlobular connective tissue (c).

larger (**Figures 5A**–**C** and **6A**–**C**). The shape of spermatids in spermiogenesis is gradually performing an elongated cell developing head, midpiece, and flagellum. These three parts of the cell are clearly distinguished, additionally to their shape and position in the cell, because their different staining affinity: the head is basophilic; the midpiece is intensely acidophilic; and the flagellum is also acidophilic but less intense than the midpiece. The head of the spermatozoa contains the acrosome and the nucleus, with a narrower cephalic part at the acrosome. All the germinal cells in a cyst maintain the same orientation, with the heads to the same side

Sertoli cells nuclei (Se), connective tissue (c).

**Figure 5.** Spermiogenesis in the testis of *Ambystoma dumerilii*. (A–C) Early spermatids, when they are spherical (1). Various cysts present the evident progressive elongation of the spermatids. The nuclei of Sertoli cells are seen around the cysts. Alcian blue. Bar = 20 μm. Early spermatids (St1). Spermatids in elongation (St2), (St3), (St4), (St5), and (St6).

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The early spermatid nuclei soon are seen as fine granular and progressively come to dense. The early spermatids become progressively elongated and the chromatin shows increasing degree of condensation (**Figure 4A**–**C**). As spermiogenesis proceeds, the nuclei of spermatids become Sequence of Germ Cells Differentiation During Spermiogenesis of the Amphibian Urodele *Ambystoma dumerilii* http://dx.doi.org/10.5772/intechopen.71508 15

**Figure 5.** Spermiogenesis in the testis of *Ambystoma dumerilii*. (A–C) Early spermatids, when they are spherical (1). Various cysts present the evident progressive elongation of the spermatids. The nuclei of Sertoli cells are seen around the cysts. Alcian blue. Bar = 20 μm. Early spermatids (St1). Spermatids in elongation (St2), (St3), (St4), (St5), and (St6). Sertoli cells nuclei (Se), connective tissue (c).

**Figure 4.** Early spermatids in the testis of *Ambystoma dumerilii*. (A) Primary spermatocytes during pachytene and early spermatids with round nucleus. H-E. Bar = 20 μm. (B) Early spermatids with fine fibrillar chromatin. H-E. Bar = 20 μm. (C) Initial elongation of the spermatids and more compact aspect of the nucleus. H-E. Bar = 20 μm. Early spermatids

The early spermatid nuclei soon are seen as fine granular and progressively come to dense. The early spermatids become progressively elongated and the chromatin shows increasing degree of condensation (**Figure 4A**–**C**). As spermiogenesis proceeds, the nuclei of spermatids become

(St1), (St2) and interlobular connective tissue (c).

14 Spermatozoa - Facts and Perspectives

larger (**Figures 5A**–**C** and **6A**–**C**). The shape of spermatids in spermiogenesis is gradually performing an elongated cell developing head, midpiece, and flagellum. These three parts of the cell are clearly distinguished, additionally to their shape and position in the cell, because their different staining affinity: the head is basophilic; the midpiece is intensely acidophilic; and the flagellum is also acidophilic but less intense than the midpiece. The head of the spermatozoa contains the acrosome and the nucleus, with a narrower cephalic part at the acrosome. All the germinal cells in a cyst maintain the same orientation, with the heads to the same side

**Figure 6.** Spermiogenesis in the testis of *Ambystoma dumerilii*. Details of the progressive elongation of the spermatids. (A–C) Cysts contain spermatids during the progressive elongation. Progressive elongation of spermatids (St2), (St3), and (St5). Sertoli cells nuclei (Se). (A, B) Alcian blue. (C) H-E. Bar = 10 μm.

(**Figure 7A**–**C**). As maturation advances the spermatozoa have a swirl arrangement inside the cyst, keeping their heads oriented in the same direction (**Figure 8A**–**C**). The large of the spermatozoa may attain 460 μm [1, 6].

widely in Urodeles as examples are: *Hynobius boulengeri* (197 μm); *Salamandrella keyserlingii* (212 μm) [21]; *Lissotriton italicus* (360 μm) [22]; *Desmognathus aeneus* (388 μm) [23]; *Ambystoma mexicanum* (444 μm) [6]; *Eurycea bislineata* (459 μm), *E. lucifuga* (523 μm), *Pleurodeles dorsalis* (536 μm), *P. dunni* (626 μm) [23]; *Triturus helveticus* (650 μm) [4]; and *Aneides aeneus* (770 μm) [23]. The biological significance of the differences in the lengths of

H-E. Bar = 10 μm. Spermatids (St7), head (h), midpiece (m), flagella (f), and Sertoli cells nuclei (Se).

**Figure 7.** Spermiogenesis in the testis of *Ambystoma dumerilii*. (A–C) The elongation of the late spermatids advances into the cysts. The development of the head, the midpiece, and the flagella is observed by the different staining affinity. The cells conserve the same position, with the heads directed to the same side. (A) H-E. Bar = 20 μm. (B) H-E. Bar = 20 μm. (C)

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spermatozoa is unknown.

The total length of spermatozoa of Urodeles is usually longer than those of other amphibians and other vertebrates. The shortest spermatozoa were reported for *Hynobius nebulosus* with a length of 156 μm, whereas the longest, as we documented before, with a length of 840 μm, was observed in *Necturus maculosus* [4, 5]. The lengths of spermatozoa differ Sequence of Germ Cells Differentiation During Spermiogenesis of the Amphibian Urodele *Ambystoma dumerilii* http://dx.doi.org/10.5772/intechopen.71508 17

**Figure 7.** Spermiogenesis in the testis of *Ambystoma dumerilii*. (A–C) The elongation of the late spermatids advances into the cysts. The development of the head, the midpiece, and the flagella is observed by the different staining affinity. The cells conserve the same position, with the heads directed to the same side. (A) H-E. Bar = 20 μm. (B) H-E. Bar = 20 μm. (C) H-E. Bar = 10 μm. Spermatids (St7), head (h), midpiece (m), flagella (f), and Sertoli cells nuclei (Se).

(**Figure 7A**–**C**). As maturation advances the spermatozoa have a swirl arrangement inside the cyst, keeping their heads oriented in the same direction (**Figure 8A**–**C**). The large of the sper-

**Figure 6.** Spermiogenesis in the testis of *Ambystoma dumerilii*. Details of the progressive elongation of the spermatids. (A–C) Cysts contain spermatids during the progressive elongation. Progressive elongation of spermatids (St2), (St3), and

The total length of spermatozoa of Urodeles is usually longer than those of other amphibians and other vertebrates. The shortest spermatozoa were reported for *Hynobius nebulosus* with a length of 156 μm, whereas the longest, as we documented before, with a length of 840 μm, was observed in *Necturus maculosus* [4, 5]. The lengths of spermatozoa differ

matozoa may attain 460 μm [1, 6].

16 Spermatozoa - Facts and Perspectives

(St5). Sertoli cells nuclei (Se). (A, B) Alcian blue. (C) H-E. Bar = 10 μm.

widely in Urodeles as examples are: *Hynobius boulengeri* (197 μm); *Salamandrella keyserlingii* (212 μm) [21]; *Lissotriton italicus* (360 μm) [22]; *Desmognathus aeneus* (388 μm) [23]; *Ambystoma mexicanum* (444 μm) [6]; *Eurycea bislineata* (459 μm), *E. lucifuga* (523 μm), *Pleurodeles dorsalis* (536 μm), *P. dunni* (626 μm) [23]; *Triturus helveticus* (650 μm) [4]; and *Aneides aeneus* (770 μm) [23]. The biological significance of the differences in the lengths of spermatozoa is unknown.

**Figure 8.** Spermatozoa in the testis of *Ambystoma dumerilii*. (A–C) The cellular maximum elongation is observed in the spermatozoa, and additionally, the spermatozoa screw in where the head is in the interior part and the flagella in the exterior part of this roll, maintaining the cystic condition. (A) Alcian blue. Bar = 20 μm. (B) H-E. Bar = 20 μm. (C) H-E. Bar = 10 μm. Spermatozoa (Z), head (h), midpiece (m), and flagella (f).

#### **4. Spermiation**

The region of spermiation is observed at the caudal end of the testis, where the density of cysts with spermatozoa decreases, there are abundant empty cysts containing remnants of Sertoli cells, and few cysts containing spermatozoa, compared with the region before spermiation where there are abundant cysts with spermatozoa (**Figure 9A** and **B**).

Upon the conclusion of the spermiation, when the cysts open and the spermatozoa leave the testis, Sertoli cells remain inside the lobule and undergo morphological changes during their degeneration until they disappear [9, 19, 24]. During the emptying of the cysts, some spermatozoa remain in some of the cysts which show abnormal morphology (**Figure 9C**); these spermatozoa

**Figure 9.** Spermiation in the testis of *Ambystoma dumerilii*. (A, B) Portion of the testis where is seen the limit between the regions before and during spermiation. The region before spermiation contains lobules with abundant cysts with spermatozoa. The region during spermiation contains few cysts with spermatozoa, compared with these seen in the other region and there are also empty cysts, without spermatozoa, containing only the residue of the Sertoli cells. A portion of an intratesticular duct containing spermatozoa is seen. Masson's trichrome. Bar = 0.1 mm. H-E. Bar = 20 μm. (C) During spermiation, abnormal spermatozoa, showing irregular morphology, may remain into the cysts. H-E. Bar = 20 μm. Testicular regions: Before spermiation (BSp) and during spermiation (DSp), spermatozoa (Z), empty

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are phagocytized by the Sertoli cells [14].

cyst (Ec), intratesticular duct (iD), and abnormal spermatozoa (aZ).

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**Figure 9.** Spermiation in the testis of *Ambystoma dumerilii*. (A, B) Portion of the testis where is seen the limit between the regions before and during spermiation. The region before spermiation contains lobules with abundant cysts with spermatozoa. The region during spermiation contains few cysts with spermatozoa, compared with these seen in the other region and there are also empty cysts, without spermatozoa, containing only the residue of the Sertoli cells. A portion of an intratesticular duct containing spermatozoa is seen. Masson's trichrome. Bar = 0.1 mm. H-E. Bar = 20 μm. (C) During spermiation, abnormal spermatozoa, showing irregular morphology, may remain into the cysts. H-E. Bar = 20 μm. Testicular regions: Before spermiation (BSp) and during spermiation (DSp), spermatozoa (Z), empty cyst (Ec), intratesticular duct (iD), and abnormal spermatozoa (aZ).

**4. Spermiation**

18 Spermatozoa - Facts and Perspectives

The region of spermiation is observed at the caudal end of the testis, where the density of cysts with spermatozoa decreases, there are abundant empty cysts containing remnants of Sertoli cells, and few cysts containing spermatozoa, compared with the region before spermiation

**Figure 8.** Spermatozoa in the testis of *Ambystoma dumerilii*. (A–C) The cellular maximum elongation is observed in the spermatozoa, and additionally, the spermatozoa screw in where the head is in the interior part and the flagella in the exterior part of this roll, maintaining the cystic condition. (A) Alcian blue. Bar = 20 μm. (B) H-E. Bar = 20 μm. (C)

where there are abundant cysts with spermatozoa (**Figure 9A** and **B**).

H-E. Bar = 10 μm. Spermatozoa (Z), head (h), midpiece (m), and flagella (f).

Upon the conclusion of the spermiation, when the cysts open and the spermatozoa leave the testis, Sertoli cells remain inside the lobule and undergo morphological changes during their degeneration until they disappear [9, 19, 24]. During the emptying of the cysts, some spermatozoa remain in some of the cysts which show abnormal morphology (**Figure 9C**); these spermatozoa are phagocytized by the Sertoli cells [14].

connective tissues, smooth muscle cells, and serosa; at the periphery, some melanocytes are dispersed (**Figure 10B** and **C**). In the lumen of the deferent ducts, the spermatozoa are in irregular position (**Figure 10C**); the cystic condition maintained all along the spermatogenesis is ended when the cyst is open at the spermiation, in the testicular lobules, before the entrance

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We thank Gerardo Gómez-Ríos and Adriana García-Alarcón for processing some histological specimens and valuable discussions during the early progress of this work; we are grateful to Gabino De la Rosa-Cruz for helpful technical assistance with digital photography; and José

Department of Compared Biology, Sciences Faculty, Laboratory of Biology of Reproduction,

[1] Uribe MC, Gómez Ríos G, Brandon RA. Spermatogenesis in the urodele *Ambystoma* 

[2] Propper CR. Chapter 3. Testicular structure and control of sperm development in amphibians. In: Norris DO, Lopez Kristin H, editors. Amphibians, Hormones and Reproduction

[3] Uribe MC, Mejía-Roa V. Testicular structure and germ cells morphology in salamanders.

[4] Picheral B. Structural, comparative and functional aspects of spermatozoa in Urodeles. In: Fawcett DW, Bedford JM, editors. The Spermatozoon. Baltimore, Maryland: Urban

[5] Scheltinga DM, Jamieson BGM. The mature spermatozoa. In: Sever D, Volume editor. Jamieson BGM, Series editor. Reproductive Biology and Phylogeny of Urodela. Enfield

in Vertebrates. Vol. 2. Oxford, UK: Ac Press, Elsevier; 2011. pp. 39-53

(NH), USA/Plymouth, UK: Science Publishers; 2003. pp. 203-274

Spermatogenesis. 2014;**4**(3):e988090. DOI: 10.4161/21565562.2014.988090

Antonio Hernández Gómez who kindly prepared the excellent digital figures.

Mari Carmen Uribe\* and Sergio Gracia-Fernández

\*Address all correspondence to: mari3uribe3@gmail.com

Universidad Nacional Autónoma de México, Mexico City, Mexico

*dumerilii*. Journal of Morphology. 1994;**222**:287-299

and Schwarzenberg; 1979. pp. 267-287

to the deferent duct system.

**Acknowledgements**

**Author details**

**References**

**Figure 10.** Deferent duct of *Ambystoma dumerilii*. (A) Sections of the large and folded deferent duct containing abundant spermatozoa in the lumen. B: H-E, C: PAS. Bar = 0.1 mm. (B) Detail of the deferent duct lined by cuboidal epithelium, connective tissue, muscle cells, and serosa. Subjacent the serosa, there are melanocytes. B: H-E, C: PAS. Bar = 20 μm. (C) Spermatozoa in the lumen of the deferent duct. B: H-E, C: PAS. Bar = 10 μm. Deferent duct (Dd), spermatozoa (Z), lumen (L), cuboidal epithelium (e), connective tissue (c), muscle cells (mu), serosa (s), and melanocytes (mc).

Throughout spermiation, spermatozoa are progressively released from the cysts to the lobular lumen and then to the efferent duct system [14, 16, 17, 19, 24–26].

Intratesticular ducts (*rete testis*) are embedded in the interlobular connective tissue of the testis (**Figure 9A**). Their lumen is lined with squamous epithelium. The efferent ducts include cephalic mesonephric nephrons, corresponding to the type of mesonephric kidneys of amphibians. The nephronic collecting ducts empty into the vas deferens also called primary urinary duct or Wolffian duct [2]. The Wolffian ducts are the largest of the sperm collecting ducts (**Figure 10A**); their lumen is lined with cuboidal epithelium and subjacent there are connective tissues, smooth muscle cells, and serosa; at the periphery, some melanocytes are dispersed (**Figure 10B** and **C**). In the lumen of the deferent ducts, the spermatozoa are in irregular position (**Figure 10C**); the cystic condition maintained all along the spermatogenesis is ended when the cyst is open at the spermiation, in the testicular lobules, before the entrance to the deferent duct system.
