A histological study of testis development and ultrastructural features of spermatogenesis in cultured Acrossocheilus fasciatus

Tissue and Cell 48 (2016) 49–62

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A histological study of testis development and ultrastructural features of spermatogenesis in cultured Acrossocheilus fasciatus Su-Yan Fu a , Jian-Hu Jiang a,c , Wan-Xi Yang b , Jun-Quan Zhu a,∗ a b c

The Key Laboratory of Applied Marine Biotechnology by the Ministry of Education, College of Ocean, Ningbo University, Ningbo 315211, China The Sperm Laboratory, College of Life Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou, Zhejiang 310058, China Zhejiang Institute of Freshwater Fisheries, Huzhou, Zhejiang 313001, China

a r t i c l e

i n f o

Article history: Received 11 August 2015 Received in revised form 15 October 2015 Accepted 15 October 2015 Available online 26 November 2015 Keywords: Acrossocheilus fasciatus Histology Spermatogenesis Testis development Ultrastructure

a b s t r a c t Testis development and ultrastructural features of spermatogenesis in Acrossocheilus fasciatus (Cypriniformes, Barbinae), a commercial stream fish, were studied using light and electron microscopy. The reproduction cycle in A. fasciatus testes is classified into six successive stages from Stage I to Stage VI. Based on an analysis of previous results, May to July can be confirmed as the best breeding season for A. fasciatus males. During this time, the A. fasciatus testes are in Stage V and the sperm in males is most abundant. In the first reproductive cycle, sexually mature male testes return to Stage III in October, subsequently overwintering at this stage. In the lobular-type testes of A. fasciatus, cystic type spermatogenesis occurs with restricted spermatogonia. All spermatogenic cells at different stages are distributed along the seminiferous lobules, which contain spermatogonia, spermatocytes, spermatids and spermatozoa. At the end of spermatogenesis, spermatogenic cysts open to release spermatozoa into the lobule lumen. Ultrastructural observation of A. fasciatus spermiogenesis reveals that electron-dense substances appear at the different stages of germ cells, from primary spermatogonia to secondary spermatocytes. We have termed these dense substances as “nuage” when free in the cytoplasm or adjacent to the nuclear envelope, while those close to the mitochondria are called inter-mitochondrial cement. The spermatozoa in A. fasciatus can be classified as type I due to the presence of nuclear rotation. Although the nuclear chromatin in the head of sperm was highly condensed, no acrosome was formed. The cytoplasmic canal, a common ultrastructural feature of Teleostei spermatozoa, was also present in the midpiece. In addition, numerous fused mitochondria were observed. The distal centriole and proximal centriole constituting the centriolar complex were oriented incompletely perpendicular to each other. The flagellum showed a typical 9 + 2 arrangement pattern. Conversely, our study on A. fasciatus yielded no information concerning the lateral fins although an enlarged saclike area was present at the end of some flagella. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Acrossocheilus fasciatus (Cypriniformes, Barbinae), commonly known as the freshwater grouper, is widely distributed between the mountains and streams in Jiangsu, Zhejiang, and Taiwan, among other regions (Dong et al., 1991). This is a newly developed aquaculture species and is a commercial stream fish of high economic value. In addition, this species also has an ornamental value. Existing reports on A. fasciatus concentrate on its classification, distribution, reproductive biology, artificial reproduction technology, embryonic development, and farming (Yao et al., 2013). In recent years, the artificial breeding of A. fasciatus has made rapid

∗ Corresponding author. E-mail address: [email protected] (J.-Q. Zhu). http://dx.doi.org/10.1016/j.tice.2015.10.005 0040-8166/© 2015 Elsevier Ltd. All rights reserved.

progress, thus promoting the development of aquatic breeding (Zhang and Jiang, 2010). Recently, increasing attention is also being paid to the reproduction and growth of cultured A. fasciatus. Jiang et al. (2012) reported on the development of embryonic, larval, and juvenile fish of cultured A. fasciatus. Moreover, Guo et al. (2008) carried on detailed research concerning the annual variation of gonad development in A. fasciatus living in Puxi River in the Huangshan Mountains. However, few reports are available on gonad development and the reproduction period of cultured A. fasciatus. One of these reports, by Yao et al. (2013), described the histological features and annual variation of ovary development of cultured A. fasciatus by biological investigation and examination of histological sections. Concurrently, other researchers also described ovary development by histological observation in A. fasciatus cultured in Xinchang County, Zhejiang Province (Jiang et al., 2013). Nevertheless, to date, no information is available

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concerning testis development and spermatogenesis in cultured A. fasciatus. Typically, testis development and spermatogenesis is an important part of the study of fish propagation. The male gonad structure has been described in many marine fish species. As recently as a few years ago, several researchers, including Billard (1986), Grier (1993), and Parenti and Grier (2004), have discussed and classified the male gonads of fish. The ultrastructure of teleost spermiogenesis has been reviewed by Mattei (1969), Yasuzumi (1971), and Billard (1986). In particular, spermiogenesis has been well documented in Liza aurata (Brusl˜e, 1981), Oreochromis niloticus (Lou and Takahashi, 1989), and Sorubim lima (Quagio-Grassiotto and Carvalho, 2000). As a model organism, the cellular organization of the mature testes, stages of spermiogenesis, and the reproductive cycle in the male gonads of Danio rerio (Cypriniformes, Cyprinidae) were described by Rupik et al. (2011) and Husznoa and Klag (2012). At present, only a few reports exist concerning the testis development and spermatogenesis of freshwater Barbinae fishes that belong to the group of commercial fishes. Tang and Liu (1998) investigated the gonad development of Spinibarbus sinenisis and pointed out that it possesses ampullar-type testes. Xie and He (1988) studied the gonad development and annual variation of Tor brevifilis and classified the reproductive cycle into six successive stages from Stage I to Stage VI. Moreover, this species begins to overwinter when its testes are in Stage IV. Lin et al. (2003) stated that the testes of Spinibarbus caldwelli have a lobular-type structure, where all the germ cells, except primary spermatogonia and Sertoli cells, together constitute many spermatogenic cysts within the lobule. According to the characteristics of cell differentiation at each stage, whole germ cells in the process of spermatogenesis are divided into six different types: primary spermatogonia, secondary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa. Through experimental observation, Wen et al. (2005) classified Spinibarbus hollamdi testis development into several periods, namely the primordial germ cell period, proliferating period, early growth period, late growth period, mature period and removal period. It was thus found that the male fish (three years of age) developed to sexual maturity, with a breeding period from April to September. In the current study on cultured A. fasciatus, testis development during the reproductive cycle and the ultrastructural features of spermatogenesis at light and electron microscope levels are described for the first time. Thus, one aim of the current study is to investigate the testicular structure and annual changes in testis development in the first reproductive cycle using gross anatomy and histological sectioning techniques. Another aim is to observe the ultrastructure of germ cells during spermatogenesis using both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is well known understanding the characteristic and law of A. fasciatus testis development and spermatogenesis can provide further theoretical guidance for aquaculture.

2. Materials and methods 2.1. Fish samples Experimental A. fasciatus were collected from the second largest aquaculture farm in Xinchang County, Zhejiang Province. The area of the rearing ponds made of cement was 50–80 m2 . Clean groundwater and water from reservoirs was used for fish farming, which was conducted in a temperature of 5–26 ◦ C throughout the year. The breeding way of water-flowing type was conducted, and the breeding density was 50–200 fish/m2 . The dissolved oxygen in the water (pH 6.7–7.2) was maintained above 4 mg/L. Fish were provided with compound feed containing 35% crude protein. During

the breeding, the rearing pond was cleared regularly to have a clean and tidy environment. The fish fry were bred in early July every year from 2008 to 2011. A total of 20 A. fasciatus fish were sampled randomly every month from November 2010 to October 2011 among the generations of 2008, 2009, and 2010 in the rearing ponds. In addition, A. fasciatus samples were taken from the 2011 generation from August 2011 to October 2011, and 20 fish were sampled monthly. Among 780 samples taken, 365 were male. Sampled fish were dissected after being anesthetized. The testes were subsequently removed from the abdomens and photographed. At the same time, measurements were taken for the body length, body weight, and the weight of the testes, among other measurements. 2.2. Tissue processing 2.2.1. Light microscopy (LM) Some excised testis samples were fixed in Bouin’s solution for LM and later embedded in paraffin. The histological sections after HE staining were then examined using an Olympus BX51 light microscope and photographed with Image-Pro Plus 6.0 image analysis software. The developmental stage of the testes was determined as described by Liu (1993) and Lou (1999). 2.2.2. Electron microscopy (TEM and SEM) For electron microscopy, the testes were cut into small fragments and fixed for 2 h in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and stored at 4 ◦ C. Thereafter, the material was post-fixed for 1–2 h in 1% osmium tetroxide in the same buffer, dehydrated in a graded ethanol series (30%, 50%, 70%, and 90%) and subsequently transferred to ascending concentrations of acetone (90% and 100%). Finally the material was embedded in Epon 812 (Luft, 1961) and acetone (1:1) for 1 h, followed by araldite resin and acetone (2:1) for 1.0–1.5 h, and lastly, in pure araldite resin for 1 h. Ultrathin sections were cut with a LKB-II ultramicrotome, stained with uranyl acetate for 40 min and counterstained with lead citrate for one minute, for later examination using a JEM-1200EX transmission electron microscope. The samples were also observed and photographed using a JSM 6300 scanning electron microscope. 3. Results 3.1. Testicular structure and morphology A. fasciatus testes proved to be paired, elongated organs of varying color depending on the developmental stage. In general, the left and right testes were the same in size, although differences were occasionally observed. Notable alterations in appearance, volume, and color were used to define the different reproductive classes (Fig. 1A–F). The testes were located symmetrically in the dorsal region of the coelomic cavity, connected to the ventral surface of the swim bladder at the cranial end and to the kidneys at the caudal end. Ultimately, the left and right testes merged into a Y-shape at the caudal end to form a short spermatic duct, allowing communication with the external environment via the cloacal orifice. Histologically, the testicular parenchyma was enclosed by a connective tissue capsule that is comparable to the tunica albuginea of mammals. The tunica albuginea found here invaginated into the organ interior forming numerous septa that subdivided the testis into anastomosed seminiferous tubules. The septa contained masses of muscle cells, individual fibrocytes (Fig. 2A), testicular vessels (Fig. 2b and c), and Leydig cells. The tubules were internally lined by germinal epithelium composed of germ cells and Sertoli cells. Cytoplasmic processes of the Sertoli cells surrounded the germ cells, forming cysts. The tubules were filled with cysts, thus, forming sites of spermatogenesis in this fish.

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Fig. 1. Anatomical images of testes at different development stages in A. fasciatus. (A) Stage I testis. The testis is a transparent filament in shape and is less than 1 mm in diameter. (B) Stage II testis. The testis is a translucent narrow strip and is approximately 1.0–1.7 mm in diameter. (C) Stage III testis. The flat-tape shaped testis is pale red owing to the development of blood vessels. (D) Stage IV testis. The testis is weak pink and more regular tape shape. Its size is larger compared to the previous stage. Surface vessels are well-developed. The midpiece is slightly thick, but both ends gradually become thinner. (E) Stage V testis. The testis is strip-shaped and milky in the reproduction period with the largest volume. White semen flows out when pressure is applied on the belly. (F) Stage VI testis. The testis is a single fold in this regressing stage with a notable reduction in size. Surface vessels are well-developed. Scale bars: A and B = 1 mm; C–F = 4 mm.

Analysis of the paraffin-embedded sections showed that A. fasciatus testes could be classified as a typical lobular-type, with a lobular cavity at the center (Fig. 2b, B). The seminiferous lobules in the peripheral section of the testes were arranged radially and regularly distributed. However, the seminiferous lobules in the central section were arranged irregularly in a crisscross pattern (Fig. 2a and b). Spermatogenic cysts were abundant within each seminiferous lobule. These spermatogenic cysts of different sizes, occurring in various developmental stages, were distributed along the entire length of the lobular lumen. Spermatogenesis occurred inside the cysts. Within a single cyst, germ cells developed synchronously, while germ cells in different cysts were not necessarily in the same period. Many cysts could be found in a single lobule, each containing germ cells at different stages, that is, spermatogonia, spermatocytes, spermatids, and spermatozoa (Fig. 2C). Nevertheless, during the mature period, spermatozoa were not enclosed within cysts but were found at the center of the lobule’s lumen (Fig. 2f, F). A spermatic duct lined by simple epithelium was located in the central portion of each testis and extended until the urogenital papilla, located caudally to the anal opening. As spermatogenic cells proliferated and grew continuously, the size of spermatogenic cysts increased significantly, but the spermatogenic cyst wall gradually became thinner. At the end of spermatogenesis, spermatogenic cysts opened to release spermatozoa into the lobule lumen, and were subsequently excreted from the body via spermatic ducts. 3.2. The cycle of testis development According to the histological characteristics observed from the paraffin-embedded sections, and the anatomical features from different developmental periods, the reproductive cycle in A. fasciatus

testes was classified into six successive stages: (1) Stage I, (2) Stage II, (3) Stage III, (4) Stage IV, (5) Stage V, and (6) Stage VI. Each period had its salient features.

3.2.1. Stage I testes In this stage, the testes resembled a very transparent filament of less than 1 mm in diameter. We could not distinguish the gender by the naked eye, but easily identified male and female fish by observation of gonadal tissue slices (Fig. 1A). The sections showed that the testes were divided into many seminiferous lobules by the surrounding connective tissue, which is lobular interstitium consisting of interstitial cells, fibroblasts, and blood capillaries, among other substances. At this point, the lobular cavity could not yet be observed at the center of the seminiferous lobules. Spermatogonia and a small number of primary spermatocytes could be very clearly seen in the seminiferous lobules. The spermatogonia, which appeared near the elongated Sertoli cells, were ovoid or spherical in shape, and approximately 7–9 ␮m in diameter. These were the largest germ cell type with a large central nucleus (6–8 ␮m), de-condensed chromatin, and a prominent nucleolus. The basophilic nucleolus was usually situated at a neutral position or on the lateral side. Spermatogonia divided by mitosis and differentiated until they became primary spermatocytes that subsequently entered meiotic prophase. In primary spermatocytes, several stages were not observed, including leptotene, zygotene, pachytene, and diplotene, although the synaptonemal complexes appeared. Primary spermatocytes in the shape of a circle or ellipse were smaller than spermatogonia. The cell diameter was about 5–6 ␮m and the cytoplasm was lightly stained (Fig. 2a, A). In the first reproductive cycle, this stage of testis development was usually

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Fig. 2. Histological images of testes at different development stages in A. fasciatus. (a) Stage I testis. Seminiferous lobules are shown but the lobular cavity is not yet evident; (A) Enlarged image of Fig. a. Spermatogonia, primary spermatocytes and fibroblasts can be observed. (b) Stage II testis. The lobular cavity is evident; (B) Enlarged image of Fig. b. Spermatogonium and primary spermatocytes can be observed. (c) Stage III testis. Seminiferous lobules and developed surface vessels are shown; (C) Enlarged image of Fig. c. Spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatids can be observed. Spermatogenic cysts (asterisks) show an independent distribution pattern. (d) Stage IV testis. Some spermatozoa are present in the lobular cavity; (D) Enlarged image of Fig. d. Primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa can be observed. (e) Stage V testis. The lobular cavity is filled with spermatozoa; (E) Enlarged image of Fig. e. A large number of spermatozoa can be observed. (f) Stage VI testis. The majority of spermatozoa have been discharged from the lobular cavity; (F) Enlarged image of Fig. f. Some spermatozoa remain in the lobular cavity. SL, seminiferous lobule; LL, lobular cavity; FI, fibroblast; BV, blood vessel; SG, spermatagonia; PS, primary spermatocyte; SS, secondary spermatocyte; ST, spermatid; SP, spermatozoon. Scale bars: a, b and c = 30 ␮m; A, B, C and D = 10 ␮m; d, e and f = 50 ␮m; E, F = 15 ␮m.

observed in 4-month-old males (2010 generation) and occurred only once in the life of A. fasciatus (Table 1).

and similarly, occurred only once in the life of A. fasciatus (Table 1).

3.2.2. Stage II testes The testes at this stage were translucent narrow strips with a diameter of approximately 1.0–1.7 mm (Fig. 1B). Observation of the tissue sections revealed that the lobular cavity appeared in the middle of part of the seminiferous lobules. Spermatogonia and primary spermatocytes in seminiferous lobules dramatically increased in number and the arrangement between them was much denser, thus, morphologically, this stage appeared similar to stage I (Fig. 2b, B). In the first reproductive cycle, this stage of testis development was usually observed in 5-month-old males (2010 generation),

3.2.3. Stage III testes The testes were a similar shape to flat-tape and were pale red owing to the blood vessels (Fig. 1C). Remarkably, these vessels were distributed in the interlobular stroma. Observation of microscope sections allowed us to see that the lobular cavity had expanded in this developmental stage. During this stage, spermatogenic cells were mainly composed of spermatogonia, primary spermatocytes, secondary spermatocytes, and a small amount of sperm cells. Various spermatogenic cells at different developmental stages were usually gathered forming several spermatogenic cysts. Although the spermatogenic cyst wall was thin, it could

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Table 1 Testis development of A. fasciatus of 2010 generation in the first sexual cycle. Months of age

Date

Average body length (cm)

Average body weight (g)

Stage of testis development

4 5

2010.11 2010.12

3.20 ± 0.23 3.34 ± 0.31

0.89 ± 0.11 1.00 ± 0.13

I II

6

2011.01

3.46 ± 0.25

1.02 ± 0.18

III

7 8

2011.02 2011.03

3.54 ± 0.29 4.02 ± 0.34

1.07 ± 0.14 1.40 ± 0.17

III III

9 10 11 12 13

2011.04 2011.05 2011.06 2011.07 2011.08

4.70 ± 0.26 5.44 ± 0.41 6.07 ± 0.43 7.17 ± 0.47 7.53 ± 0.46

2.15 ± 0.12 3.29 ± 0.41 4.50 ± 0.33 7.05 ± 0.76 8.02 ± 0.98

IV V V V IV

14 15

2011.09 2011.10

8.38 ± 0.54 9.33 ± 0.39

10.30 ± 1.54 13.63 ± 2.75

IV III

still easily be observed. The primary spermatocyte developed into secondary spermatocytes following the first meiotic division. Compared to primary spermatocytes, secondary spermatocytes were of a smaller size (3–4 ␮m in diameter). The second most noticeable trend was that the nuclear basophilia increased significantly (Fig. 2c, C). In the first reproductive cycle, testes of males of 6–8 months of age (2010 generation) were all in this stage. After maturation and regression, sexually mature male testes returned to this stage in October, and subsequently, this species overwintered in this stage (Table 1). 3.2.4. Stage IV testes As the males matured, the testes remarkably enlarged compared to those of previous stage, with well-developed surface vessels. The testes also became light pink in color, and were of a more regular, tapered shape than that observed previously. At this stage, the midpiece of the testes was slightly thick, although both ends gradually became thinner (Fig. 1D). Longitudinal sectioning of the testes showed that spermatogenic cells, which were located in the central section, developed faster than those in the peripheral section. We found that the spermatogenic cells located in central section of the testis were mainly spermatids, with a small number of spermatozoa. On the other hand, the spermatogenic cells located in the peripheral section were mostly secondary spermatocytes and spermatids. Secondary spermatocytes developed into spermatids via the second meiotic division. Spermatids were the smallest germ cells inside the cysts, with a diameter of about 2–3 ␮m. As the testicular development proceeded, the nuclear basophilia strengthened further so these spermatids were deeply stained. In late Stage IV, some parts of the spermatogenic cysts ruptured, releasing sperm into the lobular cavity (Fig. 2d, D). In the first reproductive cycle, this stage of testis development appeared in 9-month-old males (2010 generation). Sexually mature male testes again underwent development to this stage in April of the following year (Table 1). 3.2.5. Stage V testes In terms of overall appearance, the testes were strip-shaped and milky in color, and had the largest volume in this stage (Fig. 1E). If light pressure was applied to the fish belly at this point, a significant quantity of white semen flowed out. Thus this stage was also known as the reproduction period. According to observation of histological sections, the lobular cavity was filled with sperm with a diameter of about 1.5 ␮m. After HE staining, the sperm nuclei were dark blue (Fig. 2e, E). In the first reproductive cycle,

Note A few male testes were still in Stage I A few male testes were still in Stage II A few male testes had developed to Stage IV

Semen still could be squeezed out in part of the male fish

testes of males of 10–12 months of age (2010 generation) were all in this stage. Thus the males all reached sexual maturity for the first time at this point. From May to July of the following year, the testes of sexually mature males once again developed to this stage (Table 1). 3.2.6. Stage VI testes After spermatozoa were released into the tubule lumen, a notable reduction in size of the regressing testes was subsequently observed. The testes varied in shape from a thick strip in stage V to a single fold in this stage, on which surface vessels were well-developed (Fig. 1F). Subsequent observation found that the majority of sperm in the lobular cavity had emptied, and only part of lobular cavity still contained a small amount of sperm (Fig. 2f, F). In the first reproductive cycle, this stage of testis development appeared in males in 13–14 months of age (2010 generation). Sexually mature male testes again underwent development to this stage from August to September of the following year (Table 1). 3.3. Spermatogenesis and ultrastructure of germ cells Spermatogenesis occurred in the seminiferous lobules of the testis in A. fasciatus. According to the characteristics observed by LM and TEM, the process of germ cell development was classified into four successive stages: (1) spermatogonia, (2) spermatocytes, (3) spermatids, and (4) spermatozoa. 3.3.1. Spermatogonia stage Spermatogonial cells observed in the testis of A. fasciatus comprised two types: primary (type A) and secondary (type B) spermatogonia distributed randomly along the lobular wall. 3.3.1.1. Primary (type A) spermatogonia. Primary (type A) spermatogonia (SGA) were the largest germ cell type in the testis of A. fasciatus. SGA were located in the basal portion of the seminiferous tubules, and appeared ovoid or spherical in shape. The SGA nuclei were approximately 4–5 ␮m in diameter (Fig. 3A), with a regular nuclear envelope outline. In the cytoplasm, several spherical or elongated mitochondria were randomly distributed around the nucleus that contained a few electron-lucent chromatin. Some electron-dense substances, here referred to as nuage, were free in the cytoplasm, either adjacent to the nuclear envelope or close to the mitochondria (Fig. 3A–C).

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Fig. 3. Primary spermatogonia (SGA) in A. fasciatus testis. (A) TEM micrograph showing the nucleus (N) containing sparse electron-lucent chromatin. Electron-dense substances (arrows) also known as nuage are adjacent to the nuclear envelope (arrowheads). Spherical or elongated mitochondria (M) are randomly distributed around the nucleus (N). (B, C) The cysts with SGA in the same phase of development. Nuclei (N) with nucleoli (Nu), mitochondria and nuage (arrows) can be observed. N, nucleus; NU, nucleolus; M, mitochondria; Scale bars: A–C = 0.5 ␮m.

3.3.1.2. Secondary (type B) spermatogonia. Secondary (type B) spermatogonia (SGB), developed from the primary spermatogonia and were surrounded by some of the Sertoli cell cytoplasmic processes. Compared to the SGA, these cells were smaller in size with nuclei of approximately 3.5–4.0 ␮m in diameter (Fig. 4A). SGB were oval-shaped with a notable nucleolus within nucleus, although a few of the cells undergoing mitosis were elongated (Fig. 4B and C). In terms of the ultrastructure, the spherical nucleus had decondensed chromatin. More mitochondria were observed, but the number of nuage was apparently reduced compared to the SGA. The

electron-dense inter-mitochondrial cement was at a short distance from the nuclear envelope or associated with mitochondria (Fig. 4A–C). 3.3.2. Spermatocyte stage Two types of spermatocytes were observed in the testes of A. fasciatus: primary and secondary spermatocytes. However, secondary spermatocytes were rarely observed, because this stage lasted very short time and had no spectacular ultrastructure. Some cysts contained a few intact secondary spermatocytes, but in most cases,

Fig. 4. Secondary spermatogonia (SGB) in A. fasciatus testis. (A) TEM micrograph showing the nucleolus (Nu) within the nucleus (N) containing sparse electron-lucent chromatin. Some inter-mitochondrial (M) dense substances (arrow) are close to the circular nuclear envelope (arrowheads). Some cytoplasmic processes of the Sertoli cells (ST) surround the cell. (B, C) Elongated cells with notable nucleoli (Nu) within nuclei (N) undergoing mitosis. Mitochondria (M), inter-mitochondrial dense substances (arrows), nuage accumulation (asterisks) and centriole (CT) are seen. N, nucleus; NU, nucleolus; M, mitochondria; ST, Sertoli cells; CT, centriole. Scale bars: A = 0.5 ␮m; B and C = 0.5 ␮m.

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Fig. 5. TEM micrographs of primary spermatocytes in A. fasciatus. (A). The nucleus has a regularly circular outline (arrowhead) containing electron-dense areas (asterisk). An inter-mitochondrial (M) dense substance (arrow) is notable. (B). Notice a small number of synaptonemal complexes (arrows) within nucleus (N) containing relatively higher electron-dense chromatin compared with those spermatogonia. M, mitochondria; N, nucleus. Scale bars: A and B = 0.5 ␮m.

they were observed as dividing cells in metaphase of the second meiotic division. 3.3.2.1. Primary spermatocyte. Primary spermatocytes were formed by SGB via the last mitotic division, which entered meiotic prophase. These germ cells differed from spermatogonia in their nuclear size, density, and characteristics of chromatin condensation and distribution. The primary spermatocytes also had oval-shaped nuclei with regular outlines, but were smaller than spermatogonia. The nuclei measured about 3.0–3.5 ␮m in diameter. Compared to spermatogonia, primary spermatocytes had relatively more electron-dense chromatin in their nuclei, with numerous short synaptonemal complexes (Fig. 5B). A small number of mitochondria were distributed in the cytoplasm, and some dense inter-mitochondrial cement could be observed (Fig. 5A). There was an absence of nuage material (Fig. 5A and B). 3.3.2.2. Secondary spermatocyte. Primary spermatocytes developed into secondary spermatocytes following the first meiotic division. However, secondary spermatocytes had smaller cytoplasmic volume, measured about 3–4 ␮m in width and had variably shaped nuclei with a clear nuclear envelope. The electron density in the nucleus was much higher than that of primary spermatocyte nuclei. In the cytoplasm, some electron-dense substances were distributed around mitochondria. Sertoli cells rested on the basement membrane that contributed to forming spermatogenic cysts. Secondary spermatocytes were rarely encountered due to their short lifespan, since they quickly entered the second meiotic division to form spermatids (Fig. 6). 3.3.3. Spermatid stage Spermatids were seen at different stages of cell differentiation. Three maturational stages of spermatids were identified in A. fasciatus during the course of spermatogenesis, namely early, intermediate, and late spermatids (Figs. 7–9). These maturational stages were identified based on the condensation and distribution of chromatin material in the nucleus, development of the midpiece and flagellum, and exocytosis of residual cytoplasm for spermatozoa formation. 3.3.3.1. Early spermatids. Spermatids developed from secondary spermatocytes following the second meiotic division, and were the smallest germ cells inside the cysts. Early spermatids were generally ovoid or spheroidal in shape. The nucleus had a circular outline measuring about 3.0 ␮m in diameter, and contained translucent areas adjacent to electron-dense areas. Spherical or slightly elongated mitochondria were symmetrically distributed around the

Fig. 6. TEM micrograph of a secondary spermatocyte in A. fasciatus. A dividing cell during the metaphase has an irregular shape. The electron density in the nucleus (N) is much higher than that of primary spermatocyte nuclei. Some of electrondense substances (arrows) are distributed around mitochondria (M). Sertoli cells (ST) resting on the basement membrane are evident. N, nucleus; M, mitochondria; ST, Sertoli cells. Scale bars = 0.5 ␮m.

nucleus in the cytoplasm. Some spermatids showed flagella growing out of the side of the nuclei (Fig. 7B). Well-developed Sertoli cells with significant nucleoli were clearly distributed along the periphery of spermatids (Fig. 7A and B). 3.3.3.2. Intermediate spermatids. Intermediate spermatids were in the second stage of spermiogenesis, and possessed regularly spherical nuclei (measuring about 3.0 ␮m in diameter) containing filamentous clusters of chromatin that were more highly condensed than that of the previous stage. However, the chromatin was not completely condensed forming chromatin clusters interspersed with electron-lucent areas (Fig. 8C). In this stage, the nucleus was capped with a thin cytoplasm containing lamellar configurations that would be eventually eliminated, while the excess cytoplasm was rejected as the residual body (Figs. 8A and B and 11A). Several vesicles were present, either isolated or interconnected with each other (Fig. 8A). 3.3.3.3. Late spermatids. During the third stage of spermiogenesis, the morphology of the late spermatids gradually changed due to the lateral insertion of flagellum. The diameter of nucleus was

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Fig. 7. TEM micrographs of early spermatids in A. fasciatus. (A) The nucleus has a circular outline (arrowheads) containing translucent areas, which is present near the electron-dense areas (asterisk). Spherical or slightly elongated mitochondria (M) are symmetrically distributed around the nucleus (N). Sertoli cells with significant nucleoli are distributed along the periphery of spermatids. (B) The nucleus remains regularly spherical and contains filamentous clusters of more highly condensed chromatin. The flagellum (F) growing out of the side of the nucleus (N) is visible. F, flagellum; N, nucleus; M, mitochondria; ST, Sertoli cells. Scale bars: A and B = 0.5 ␮m.

Fig. 8. TEM micrographs of intermediate spermatids in A. fasciatus. (A, B) Sections of the spermatids showing the pattern of nuclear chromatin condensation (asterisk), the distribution of mitochondria (M) and vesicles (V). The nucleus (N) is capped with a thin cytoplasm containing lamellar configurations (arrow). Residual body (RB) and flagellum (F) can be observed. (C) A white arrow indicates the electron-lucent areas surrounded by more compact chromatin (asterisk). The spermatid is limited with Sertoli cells (ST). N, nucleus; M, mitochondria; V, vesicles; F, flagellum; RB, residual body; ST, Sertoli cells. Scale bars: A and C = 1 ␮m; B = 0.5 ␮m.

about 2.5 ␮m. As the condensation continued, numerous clusters of highly compacted chromatin material were packed within the nucleus. In addition, the nuclear envelope with an irregular outline was surrounded by heterogeneous cytoplasm containing ovoid mitochondria that moved to a position just posterior to the nucleus of the spermatid. With the spermatid development proceeding, the flagellum surrounded by a cytoplasmic channel grew from the distal centriole, which was oriented towards the nucleus. In the late spermatid stage, a shallow depression called nuclear fossa formed in one hemisphere of the nuclei. The distal centriole was located outside of the nuclear fossa, while the proximal centriole was nearer to the depression (Fig. 9). 3.3.4. Spermatozoa stage Mature spermatozoa were mostly found in the lumen of the cysts, which comprised the head (nucleus), midpiece (mitochondrion) and a single tail (flagellum), as is the case for other teleosts (Figs. 10A–D and 12A–E). In this species, no acrosome was formed. The mature spermatozoon was characterized by a spherical head (measuring about 1.5–1.8 ␮m in diameter) with an extremely reduced volume of cytoplasm. The round-shaped nucleus, about 1.4–1.6 ␮m in diameter, contained completely condensed

chromatin. The midpiece was short and situated laterally in relation to the flagellum (Fig. 12D and E). Some large mitochondria forming a collar encircled the insertion site of the flagellum (Figs. 11A and 12D and E). These mitochondria were separated from the flagellum by the cytoplasmic channel, which was a space between the cytoplasmic sheath and the initial region of the flagellum (Figs. 10A and 11A). The proximal and distal centrioles appeared as electron-dense substances were inserted into the initial section of the flagellum (Fig. 10A and B). The tail length measured about 25–30 ␮m (Fig. 12C). The flagellum with visible axoneme had a small amount of cytoplasm enveloped by a plasma membrane (Fig. 10A). The structure of the axoneme revealed a typical 9 + 2 arrangement pattern (9 peripheral doublets and a pair of single central microtubules) (Fig. 11A and B). An enlarged saclike area appeared at the end of some flagella (Fig. 10C). 4. Discussion 4.1. Testicular structure According to the distribution and development characteristics of germ cells in testes, teleost testicular structure was classified as

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Fig. 9. TEM micrograph of a late spermatid in A. fasciatus. Notice numerous clusters of highly compacted chromatin material (asterisk) packed within the nucleus (N) and the nuclear envelope (arrowheads) with irregular outline surrounded by heterogeneous cytoplasm containing ovoid mitochondria (M). The flagellum (F) surrounded by the cytoplasmic channel (CC) grows from the distal centriole (DC) which is connected with the proximal centriole at the anterior. CC, cytoplasmic channel; DC, distal centriole; PC, proximal centriole; F, flagellum; N, nucleus; M, mitochondria. Scale bar = 0.2 ␮m.

anastomosing tubular- and lobular-types (Billard, 1986). These two categories have significant differences. In lobular-type testes, spermatagonia are evenly distributed along the edge of seminiferous lobules. As the testicular development proceeds, spermatagonia,

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spermatocytes, and spermtids gather to form spermatogenic cysts, while spermatozoa gradually move to the center of the lobule cavity. Since spermatogonia are located at the terminal end of the lobule, this type of testis has also been called the restricted-type structure (Grier and Uribe-Aranzabal, 2009). In tubular-type testes, on the other hand, the spermatogonia are distributed through the tube walls; thus, it is also known as the unrestricted-type structure (Grier, 1993; Parenti and Grier, 2004; Grier and Uribe-Aranzabal, 2009). The results from histological study of the testicular structure show that A. fasciatus testes can be classified as lobular-type, which is similar to most teleosts. However, Rupik et al. (2011) reported that the gonads of D. rerio, were rather of the tubular anastomosing type with an unrestricted distribution of spermatogonia based on light and electron microscopy observations. In the seminiferous lobules of A. fasciatus testes, spermatogenic cells did not migrate as spermatogenesis proceeded, but rather, developed in situ. When these spermatogenic cells developed into mature spermatozoa, spermatogenic cysts ruptured and spermatozoa were subsequently released into the central cavity. This phenomenon also occurs in Carassius auratus (Guan et al., 1990), Bostrichthys sinensis (Jiang et al., 2004), Tridentiger trigonocephalus (Zhang et al., 2009), and Alcolapia grahami (Papah et al., 2013). According to the arrangement of seminiferous lobules in the testes, the lobular-type structure could be subdivided into ampullar- or radiant-types (Su, 1995). Our studies in A. fasciatus indicated that the seminiferous lobules in the peripheral section of the testes were arranged radially and regularly. However, the seminiferous lobules in the central section were arranged irregularly in a crisscross pattern. Therefore, the seminiferous lobules of A. fasciatus testes were neither of the ampullar nor radiant types. Similar reports have been made concerning the species S. caldwelli belonging to the Barbinae subfamily as well (Lin et al., 2003). Further research is necessary to establish whether this lobe arrangement is particular to the Barbinae subfamily. 4.2. The features of testis development In the current study of the testis development of A. fasciatus belonging to the 2010 generation in the first sexual cycle,

Fig. 10. Transmission electron micrographs of spermatozoa of A. fasciatus. (A) Longitudinal section through the spermatozoa head (N) with group of mitochondria (M) and proximal part of flagellum (F) surrounded by cytoplasmic channel (CC) and centriolar complex (PC and DC). The nucleus (N) with completely condensed chromatin is about 1.5 ␮m in diameter. (B) The proximal and distal centrioles lie laterally outside of the nucleus. (C) There is an enlarged saclike area at the end of some flagella. (D) Relatively more mature flagella without residual cytoplasm. CC, cytoplasmic channel; DC, distal centriole; PC, proximal centriole; F, flagellum; N, nucleus; M, mitochondria. Scale bars: A = 0.25 ␮m; B and D = 0.5 ␮m; C = 0.2 ␮m.

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Fig. 11. Transmission electron micrographs of midpiece and flagellum cross section. (A) Midpiece cross section revealing the central location of the flagellum (F) within the cytoplasmic channel (CC). Some large mitochondria (M) forming a ring encircled the insertion site of the flagellum (F). The residual body (RB) surrounded by spermatozoa laterally can be observed. (B) Flagellum cross section revealing the typical 9 + 2 arrangement pattern (9 peripheral doublets and a pair of single central microtubules). CC, cytoplasmic channel; F, flagellum; N, nucleus; M, mitochondria; RB, residual body. Scale bar: A = 0.25 ␮m; B = 0.2 ␮m.

Fig. 12. Scanning electron micrographs of spermatozoa of A. fasciatus. (A, B) Spermatozoa in the lumen of a lobule. The head (H) and flagellum (F) are shown. (C) A single spermatozoon. The head is bulbous (about 1.5–1.8 ␮m in diameter). The tail measures about 25–30 ␮m in length. (D, E) Spermatozoa showing the head (H), midpiece (MP) and flagellum (F). The midpiece (MP) is situated laterally in relation to the flagellum (F). The mitochondrial collar can also be observed. SP, spermatozoa; H, head; F, flagellum; MP, midpiece. Scale bars: A = 5 ␮m; B = 1 ␮m; C = 2 ␮m; D = 0.2 ␮m; E = 0.3 ␮m.

we found that males of 10 months of age reached sexual maturity. Among other species belonging to the same subfamily, it has previously been reported that 2-year-old T. brevifilis (Xie et al., 1999) males developed to sexual maturity, while most males of Spinibarbus sinensis (Cai et al., 2003), Spinibarbus hollandi (Wen et al., 2005), and Onychostoma simus (Chen et al., 2008) developed to sexual maturity at 3 years of age. In conclusion, the males of A. fasciatus reached sexual maturity earlier than other fishes belonging to the same subfamily. In addition, research on Pimelodidae reported by Batlouni et al. (2006) revealed that 6-month-old

Pseudoplatystoma fasciatum males had free spermatozoa. However, researchers performed hormone induction only when fish were 1 year and 4 months old, while females of similar age were raised in the same conditions and also entered into their first spawning period (Romagosa et al., 2003c). In the reproductive cycle of teleosts, female gonad development is susceptible to various factors, while males are rarely conditioned by the surrounding environment (Wu et al., 2009). As a result, the varying age of sexual maturity among Barbinae males can be mainly attributed to interspecific genetic differences. In addition, the length and weight in

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the youngest sexually mature A. fasciatus male individual was only 5.44 ± 0.41 cm and 3.29 ± 0.41 g respectively. This is far lower than that observed in T. brevifilis (Xie et al., 1999) and S. hollandi (Wen et al., 2005) where the youngest sexually mature male individuals had lengths of 13.7 cm and 33.6 cm and weights of 44.0 g and 990.0 g respectively. During A. fasciatus breeding, we found that the artificial settings for reproduction were often occupied by larger males. As the males that first reach sexual maturity are often smaller, it still needs to be established whether they can participate in normal reproduction. It is well known that the criteria for classification of reproductive stages vary between different species. Five reproductive stages were detected during the annual cycle of gonad development in P. fasciatum: early maturation, middle maturation, late maturation, regression, and recrudescence (Batlouni et al., 2006). This method was comparable to the traditional criteria of classification that was initially utilized in Perciforme fish species, such as Centropomus undecimalis (Grier and Taylor, 1998), Cynoscion nebulosus (Brown-Peterson and Warren, 2001), and Rachycentron canadum (Brown-Peterson et al., 2002). Similarly, the reproduction cycle in A. fasciatus testes is classified into six successive stages: (1) Stage I, (2) Stage II, (3) Stage III, (4) Stage IV, (5) Stage V, and (6) Stage VI. However, after sexual maturation, A. fasciatus testes undergo annual changes and experience only Stages III, IV, V, and VI. In October, the postpartum male testes reverted to Stage III, and this species subsequently overwinters in this stage. For fish from the same subfamily, such as T. brevifilis (Xie et al., 2002) and Acrossocheilus hemispinus (Liu, 2010), the postpartum male testes also reverted to Stage III, but these species begin to overwinter when their testes are in Stage IV. In A. fasciatus, however, the testes only enter Stage IV of development in April the following year and thus have much shorter duration than that of T. brevifilis (Xie et al., 2002) and A. hemispinus (Liu, 2010). From May to July, the testes of A. fasciatus are in Stage V. In this stage, sperm in males is the most abundant, and if light pressure is applied to the belly of the fish at this point, a significant amount of thick white semen flows out. In August and September, some semen of thinner density can still be squeezed from some males. At this point, the testes have degenerated to Stage VI. Therefore, May to July can be confirmed as the best breeding season of A. fasciatus males. Except phases and continuity, another important characteristic in the testis development of A. fasciatus is the asynchronism, which can be summarized in four aspects. Firstly, asynchronism between individuals in testis development can be observed since some fish from the same batch lagged behind or were ahead of others in testis development. Secondly, bilateral testes in a single individual may be asynchronous during development since the sizes of the left and right testes were inconsistent, with the right testis of some males further developed than the left. Similar findings have been reported for B. sinensis (Jiang, 2002) and Leiocassis crassilabris (Chen, 2007). Thirdly, asynchronism of dorsoventral testis development in the same individual was observed. The testis development of the ventral testis was usually faster than that of the dorsal. This phenomenon also has been observed in the development of the testes in Ctenopharyngodon idellus (Lin et al., 1981). We hypothesize that these findings may be due to differences in the internal environment. Lastly, asynchronism was observed in front-back testis development in the same individual. Before A. fasciatus started their breeding, the back-end testis developed ahead of the front-end testis. That is to say, the back-end testis started to release sperm first. After large quantities of sperm were released, the front-back testis developed to the regressed class earlier than the back-end testis. This adaptive characteristic may have arisen in the A. fasciatus reproductive season during long-term race multiplication to ensure that the largest number of sperm is released.

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4.3. Spermatogenesis and spermatogenic cell ultrastructure Based on the developmental features of germ cells, spermatogenesis in teleosts can be separated into two distinct categories: synchronous and asynchronous spermatogenesis. Synchronous spermatogenesis occurs in most teleosts, and is also known as cystic type spermatogenesis. During this type of spermatogenesis, the developing germ cells are enclosed within cysts. In each cyst, germ cells are connected by cytoplasmic bridges, which cause synchronous differentiation (Andrade et al., 2001). At the end of spermatogenesis, the cytoplasmic bridges break off and the mature spermatozoa are released into the lumen of the lobules. On the other hand, in asynchronous spermatogenesis, also known as semi-cystic type spermatogenesis, spermatogenesis occurs partially outside the cyst (Andrade et al., 2001; Mattei et al., 1993). This type of spermatogenesis exists in other species, such as Bryconops affinis (Characidae) (Andrade et al., 2001). In contrast to the synchronous spermatogenesis, the cysts open before the end of spermatogenesis, and then complete development in the lobule lumen. Cytoplasmic bridges break down when the germ cells are released. In some species the cysts open at various stages of germ cell differentiation. In such cases, mixed germ cell types are found in the lumen of the lobules. Asynchronous spermatogenesis, first named by Mattei et al. (1993), is unusual for the spermatogenesis of fishes. Our study indicates that A. fasciatus has cystic type spermatogenesis with synchronous differentiation of germ cells. All of the stages of spermatogenesis proceed within cysts. Only completely differentiated sperm are released into the lobule lumen. As observed by Schulz et al. (2010), in the model organism D. rerio, several distinct types of germ cells constituting consecutive stages of spermatogenesis were present: spermatogonia A and B, primary and secondary spermatocytes, spermatids, and spermatozoa. In addition, four stages of spermiogenesis can be distinguished in D. rerio, which differ from each other in the condensation of chromatin, migration of centrioles, formation of flagella, migration of mitochondria to the midpiece, and elimination of excess cytoplasm (Rupik et al., 2011). Using similar criteria, we also divided A. fasciatus spermiogenesis into four stages. However, in some species such as L. aurata (Teleostei, Mugilidae), spermiogenesis is divided into five stages based on cytological features (Brusl˜e, 1981). Using two African catfish species (Clarias ngamensis and Clarias gariepinus), Mokae et al. (2013) subdivided the African catfish spermatogenesis into five histological stages from Stage I to Stage V. Based on the feature of each stage, they could determine whether the species were mature and ready for spawning, breeding, and reproduction at the time of sampling. During the testis development in large number of teleosts, a specific electron-dense substance appears in the cytoplasm of partial germ cells. When free in the cytoplasm, these substances may be either adjacent to the nuclear envelope or close to the mitochondria, and are respectively referred to as nuage or inter-mitochondrial cement (Hamaguchi, 1993; Stoumboudi and ˜ et al., 2002; Fishelson, 2003; Jun et al., 2006; Abraham, 1996; Munoz Chung, 2008; Chung et al., 2010; Papah et al., 2013). Hamaguchi (1993) found that these dense substances only occurred in primary spermatogonia in the Japanese rice fish, also known as the medaka (Oryzias latipes), and presumed that their presence was associated with synthetic activities of this cell type, although their precise role was still unknown. A similar report was made concerning Lake Magadi tilapia (A. grahami) where inter-mitochondrial dense substances were observed in secondary spermatogonia (Papah et al., 2013). The presence of nuage or inter-mitochondrial cement was associated with mitochondria in primary spermatogonia, secondary spermatogonia, and metaphase spermatocytes during the first meiotic division that may have resulted from a cytoplasm–nuclear exchange, as reported by Billard (1984). Chung

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(2008) stated that inter-mitochondrial cement appeared in the cytoplasm of the primary and secondary spermatocytes during spermatogenesis of Boleophthalmus pectinirostris. However, ultrastructural observation of A. fasciatus spermatogenesis in our study revealed that electron-dense substances appeared in different stages of germ cells from primary spermatogonia to secondary spermatocytes. Some reports on the structure and function of these electron-dense substances have been made in related articles. For instance, Toury et al. (1977) reported that the isolated inter-mitochondrial cement contains 5S, 18S and 28S RNAs, transfer RNA, proteins, cytochromes, and lipids. In particular, RNA and protein are the major constituents of the cement. The majority of proteins are destined to be incorporated into the mitochondria in clumps. In addition, it was recently reported that these electrondense substances have further been regarded as the early indicators of spermatogonia formation, although they disappear in later stages of germ cell development (Koulish et al., 2002; Fishelson et al., 2006). In Teleostei with external fertilization, three basic spermatozoon types occur during spermiogenesis: type I, II, and III spermatozoa, which are identified based on the presence or absence of nuclear rotation and the orientation of flagellum with respect to the nucleus (Mattei, 1970, 1991; Jamieson, 1991; Lahnsteiner and Patzner, 1997; Shahin, 2006, 2007; Quagio-Grassiotto and Oliveira, 2008; Schulz et al., 2010). In type I spermatozoa, the flagellar axis lies perpendicularly to the nucleus due to nuclear rotation. As reported by Lo Nostro et al. (2003), Synbranchus marmoratus possessed a biflagellated sperm, and could belong to the type I spermatozoa due to the position of the flagellum in relation to the nucleus and the shape of the head. In type II spermatozoa, by contrast, there is no nuclear rotation so the flagellum remains parallel to the base of the nucleus. An extraordinary spermatozoon type exists as an intermediate between the first two types, in which nuclear rotation is incomplete or partial and the flagellar axis thus is eccentrically oriented with respect to the nucleus. This phenomenon can be seen in some fish species, such as Cichlid, O. niloticus (Lou and Takahashi, 1989); Cyprinid, Cyprinus carpio (Billard, 1986); and Characid, Paracheirodon innesi (Jamieson, 1991). Type III spermatozoa, unique to the family Pimelodidae, is a new type of spermatozoon and has only been recently described. In contrast to all other Teleostei, the nucleus does not rotate during spermiogenesis and the flagellar axis develops centrally to the nucleus (Quagio-Grassiotto and Carvalho, 2000; Quagio-Grassiotto and Oliveira, 2008). According to the current study, the flagellar axis lies parallel to the nucleus in A. fasciatus spermatozoa during early spermiogenesis. After spermiogenesis is complete, the flagellar axis is perpendicular to the nucleus in the final spermatozoon under the action of nuclear rotation. Hence, the spermatozoa in A. fasciatus can be classified as type I. Generally speaking, type I spermatozoa have small rounded or ovoid nuclei but no acrosome. In this study, the spermatozoa of A. fasciatus lacked an acrosome. Similarly, the rounded heads of A. graham (Papah et al., 2013) spermatozoa have no evidence of an acrosome or acrosomal vesicle. Based on the general characteristics of the spermatozoa of Melanorivulus punctatus, Cassel et al. (2014) have classified this species as an aquasperm that is devoid of an acrosome vesicle. This phenomenon is similar to that of other teleost fishes (Yamamoto and Colak, 1974; Poirier and Nicholson, 1982; Romagosa et al., 1999; Lo Nostro et al., 2003; Chung, 2008). Another distinguishing feature of bony fish spermatozoa is the presence of nuclear fossa and centriolar complex. The nuclear fossa, containing the centriolar complex, is a depression of varying depth that forms at the point where the nuclear envelope invaginates into the base of the nucleus. Gwo et al. (2006) reported that there is no proximal centriole in Spratelloides gracilis. However, earlier research found that the proximal centriole of Engraulis

japonicus (Engraulididae) is indistinct (Hara and Okiyama, 1998). Kim et al. (2013) suggested that the shape of the nucleus is involved in varying based on the depth of the depression (shallow, moderate, and deep), which is the position of the centriolar complex in the nuclear fossa. In M. punctatus (Cassel et al., 2014), the nuclear fossa is moderately deep and contains a part of the centriolar complex. In Teleostei, the position of the centriolar complex is related to the shape of the nuclear fossa (Quagio-Grassiotto and Oliveira, 2008). Without doubt, the perpendicular position of the centrioles, as noted, is the most common (Quagio-Grassiotto et al., 2003; Gusmão-Pompiani et al., 2005; Greven and Schmahl, 2006; Fishelson et al., 2007; Chung, 2008; Vázquez et al., 2012). However, we found that the two centrioles of A. fasciatus are incompletely perpendicular. However, the proximal centriole in Lagodon rhomboids is inclined at 30◦ to the distal centriole (Gwo et al., 2005). In the sperm of the gobiid fish, B. pectinirostris (Gobiidae family), the nucleus is asymmetrical with a considerable basal fossa containing the proximal centriole (Chung, 2008). The nuclear fossa of Pagellus bogaraveo (Maricchiolo et al., 2010) spermatozoa appears deep and bell-shaped in a sagittal longitudinal section. It appears smaller at the apical end and enlarges posteriorly. The anterior region of the nuclear fossa is occupied by the proximal centriole, whereas the distal centriole (serving as the basal body of the flagellum) is located in the posterior region. Spadella et al. (2012) reported that a deep nuclear fossa at the posterior region of the nucleus contains the centrioles. In a longitudinal section, the shape of the nuclear fossa is an arc with irregular walls. The proximal centriole is immediately anterior and perpendicular to the distal one. Interestingly, in Epinephelus bruneus spermiogenesis, the two centrioles are located outside of nuclear fossa and are perpendicular to each other. However, after nuclear rotation is complete, the proximal centriole is located inside the nuclear fossa while the distal centriole is located outside the nuclear fossa (Kim et al., 2013). It is known that the cytoplasmic canal is a common ultrastructural feature of spermatozoa in Teleostei, and A. fasciatus is no exception. Some large mitochondria forming a collar encircled the site of insertion of the flagellum. These mitochondria were separated from the flagellum by a cytoplasmic channel of varying length. Some findings concerning the cytoplasmic canal indicate that the Percichthyidae family, for example, have a long cytoplasmic canal (Jamieson, 1991), while other species, except the Carangidae family, have a medium cytoplasmic canal (Maricchiolo et al., 2002). In contrast, fish of the Sciaenidae family have a short cytoplasmic canal. However, in the sperm of blue sprat, S. gracilis, Gwo et al. (2006) observed that the mitochondrial collar encircled the flagellum but no cytoplasmic canal was observed separating the flagellum from the midpiece. In addition, some reports show that mitochondria and vesicles are present in the midpiece of teleost spermatozoa (Jamieson, 1991; Mattei, 1991). Nevertheless, vesicles are not observed within the midpiece of Astroblepus spermatozoa (Spadella et al., 2012). To date, the physiological function of the cytoplasmic canal has not been reported in detail, but according to its architectural features we presume that it plays a role in movement of the flagellum. A classic flagellum in most groups of fishes is comprised of an axoneme with two central microtubules and nine pairs of peripheral microtubules surrounded by the plasma membrane. However, spermatozoa in Synbranchus marmoratus have been revealed to be biflagellate cells utilizing both centrioles as basal bodies for the development of each flagellum (Lo Nostro et al., 2003). In particular, lateral fins have also been reported as an ultrastructural feature of the flagellum in many fish spermatozoa (Mattei, 1970; Zaki et al., 2005; Chung, 2008; Jamieson, 2009; Kim et al., 2013; Cassel et al., 2014). Based on previous research, flagellar lateral fins are present in most externally fertilizing spermatozoa, except for fish of the Ostariophysi superorder (Characiform, Cypriniform, Siluriform)

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(Jamieson, 1989). However, in most cases of internally fertilizing spermatozoa, the flagellar lateral fins are absent. Lahnsteiner and Patzner (1995, 2007) found only one unpaired fin in Diplodus sargus. However a study concerning spermatozoon lateral fins in B. pectinirostris (Chung, 2008) agreed with other reports that the spermatozoon has two flagellar lateral fins. Similarly, bilateral fins are present on the flagellum of M. punctatus spermatozoa, although the final part of the flagellum does not display these fins (Cassel et al., 2014). However, as demonstrated by SEM observation by Kim et al. (2013), Epinephelus bruneus spermatozoa have six segmenttype lateral fins along the length of the flagellum. Therefore, along the cross-section of flagellum, fish lateral fins show one or two pairs of variously shaped fins. Conversely, our study on A. fasciatus provided no information concerning spermatozoon lateral fins. Lateral fins on spermatozoa vary in number among species, and even within a species, but these variations are not associated with any other structural changes in spermatozoa (Thiaw et al., 1986). Mattei (1988) reported that lateral fins can make flagellar movement more efficient not depending on the environment in which the spermatozoa move, the number of flagella, and the type of spermatozoa. Acknowledgements The authors are grateful to the College of Ocean at Ningbo University for the electron microscopy assistance. We would also like to acknowledge Z. Sheng and L.-L. Long for their suggestions on writing. This work was supported by the second largest aquaculture farm in Xinchang County, Zhejiang Province foe the assistance in fish sample collection. This project was financially supported by the National Natural Science Foundation of China (nos. 31272642 and 41276151), the Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo Science and Technology Plan Projects (no. 2013C910015), and the K.-C. Wong Magna Fund in Ningbo University. References Andrade, R.F., Bazzoli, N., Rizzo, E., Sato, Y., 2001. Continuous gametogenesis in the neotropical freshwater teleost Bryconops affinis (Pisces: Characidae). Tissue Cell 33, 524–532. Batlouni, S.R., Romagosa, E., Borella, M.I., 2006. The reproductive cycle of male catfish Pseudoplatystoma fasciatum (Teleostei, Pimelodidae) revealed by changes of the germinal epithelium—an approach addressed to aquaculture. Anim. Reprod. Sci. 96, 116–132. Billard, R., 1984. Ultrastructural changes in the spermatogonia and spermatocytes of Poecilia reticulata during spermatogenesis. Cell Tissue Res. 237, 129–226. Billard, R., 1986. Spermiogenesis and spermatology of some teleost fish species. Reprod. Nutr. Dev. 26, 877–920. Brown-Peterson, N.J., Grier, H.J., Overstreet, R.M., 2002. Annual changes in germinal epithelium determine male reproductive classes of the cobia. J. Fish Biol. 60, 178–202. Brown-Peterson, N.J., Warren, J.W., 2001. The reproductive biology of spotted sea trout, Cynoscion nebulosus, along the Mississipi Gulf Coast. Gulf Mex. Sci., 61–63. Brusl˜e, S., 1981. Ultrastructure of spermiogenesis in Liza aurata Risso, 1810 (Teleostei: Mugilidae). Cell Tissue Res. 233, 415–424. Cai, Y.Z., He, C.R., Cai, Y.Q., Jiang, J., 2003. The preliminary studies on the biology of Seinibarbus sinensis. Freshw. Fisheries 33 (3), 16–19. Cassel, M., Ferreira, A., Mehanna, M., 2014. Ultrastructural features of spermatogenesis in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae). Micron 62, 1–6. Chen, X.J., Zhou, J., Li, M.J., 2008. A Look at the biological characteristics and reproductive technique of Onychostoma simus. Jiangsu Agric. Sci. 41 (6), 222–224. Chen, Y.Q., 2007. A Study on Gonadal Histology and Ultrastructure of Leiocassis crassilabris. Southwest University, Chongqing, pp. 15–21. Chung, E.Y., 2008. Ultrastructure of germ cells, the Leydig cells, and Sertoli cells during spermatogenesis in Boleophthalmus pectinirostris (Teleostei, Perciformes, Gobiidae). Tissue Cell 40, 195–205. Chung, E.Y., Yang, Y.C., Kang, H.W., Choi, K.H., Jun, J.C., Lee, K.Y., 2010. Ultrastructure of germ cells and the functions of Leydig cells and Sertoli cells associated with spermatogenesis in Pampus argenteus (Teleostei: Perciformes: Stromateidae). Zool. Stud. 49, 39–50.

61

Dong, Y.M., Zhu, G.Y., Huang, M.H., 1991. Freshwater Fishes. Fauna Zhejiang. Zhejiang Science and Technology Press, Hangzhou, pp. 104–105. Fishelson, L., 2003. Comparison of testes structure, spermatogenesis, and spermatocytogenesis in young, aging, and hybrid cichlid fish (Cichlidae, Teleostei). J. Morphol. 256, 285–300. Fishelson, L., Delarea, Y., Gon, O., 2006. Testis structure, spermatogenesis, spermatocytogenesis, and sperm structure in cardinal fish (Apogonidae, Perciformes). Anat. Embryol. 211, 31–46. Fishelson, L., Gon, O., Holdengreber, V., Delarea, Y., 2007. Comparative spermatogenesis, spermatocytogenesis, and spermatozeugmata formation in males of viviparous species of clinid fishes (Teleostei: Clinidae, Blennioidei). Anat. Rec. 290, 311–323. Greven, H., Schmahl, G., 2006. A note on the spermatozoon ultrastructure of the four eyed fish Anableps anableps (Atherinomorpha, Cyprinodontiformes). Z. Fischk. 8, 83–88. Grier, H.J., 1993. Comparative organization of Sertoli cell including the Sertoli cell barrier. In: Russell, L.D., Griswold, M.D. (Eds.), The Sertoli Cell. Cache River Press, Clearwater, FL, pp. 704–730. Grier, H.J., Taylor, R.G., 1998. Testicular maturation and regression in the common snook. J. Fish Biol. 53, 521–542. Grier, H.J., Uribe-Aranzabal, M.C., 2009. The testis and spermatogenesis in teleost. In: Jamieson, B.G.M. (Ed.), Reproductive Biology and Phylogeny of Fishes (Agnathans and Neotelestomi). Phylogeny-Reproductive System-Viviparity-Spermatozoa. Science Publishers Inc., Enfield, pp. 119–142. Guan, T.L., Huang, D.Q., Huang, G.P., 1990. Cellular organization of the testis, spermatogenesis and spermiogenesis in goldfish (Carassaius auratus). Acta Hydrobiol. Sin. 14 (3), 233–238. Guo, L.L., Yan, Y.Z., Xi, Y.L., 2008. Development and annual change of the gonads of Acrossocheilus fasciatus in Puxi River of Huangshan Mountain. Freshw. Fisheries 38 (6), 8–13. Gusmão-Pompiani, P., Oliveira, C., Quagio-Grassiotto, I., 2005. Spermatozoa ultrastructure in Sciaenidae and Polynemidae (Teleostei: Perciformes) with some consideration on Percoidei spermatozoa ultrastructure. Tissue Cell 37, 177–191. Gwo, J.C., Chiu, J.Y., Lin, C.Y., Su, Y., Yu, S.L., 2005. Spermatozoal ultrastructure of four Sparidae fishes: Acanthopagrus berda, Acanthopagrus australis, Lagodon rhomboids and Archosargus probatocephus. Tissue Cell 37, 109–115. Gwo, J.C., Lin, C.Y., Yang, W.L., Chou, Y.C., 2006. Ultrastructure of the sperm of blue sprat, Spratelloides gracilis; Teleostei, Clupeiformes, Clupeidae. Tissue Cell 38, 285–291. Hamaguchi, S., 1993. Alterations in the morphology of nuages in spermatogonia of the fish, Oryzias latipes, treated with puromycin or actinomycin. Reprod. Nutr. Dev. 33, 137–141. Hara, M., Okiyama, M., 1998. An ultrastructural review of the spermatozoa of Japanese fishes. Bull. Ocean Res. Inst. Univ. Tokyo 33, 1–138. Husznoa, J., Klag, J., 2012. The reproductive cycle in the male gonads of Danio rerio (Teleostei, Cyprinidae). Stereological analysis. Micron 43, 666–672. Jamieson, B.G.M., 1989. Complex spematozoon of the live-bearing half-beak, Hemirhamphodon pogonognathus (Bleeker): ultastructural description (Euteleostei, Atherinomorpha, Beloniformes). Gamete Res. 24, 247–259. Jamieson, B.G.M., 1991. Fish Evolution and Systematics: Evidence from Spermatozoa. Cambridge University Press, Cambridge, pp. 1–319. Jamieson, B.G.M., 2009. Reproductive Biology and Phylogeny of Fishes (Agnathans and Bony Fishes), Part A. Science Publishers, Enfield, NH, USA, pp. 1–788. Jiang, H.X., 2002. Studies on the Testicular Cycle Development and Spermatogenesis of Bostrichthys sinensis. Fujian Normal University, Fujian, pp. 18–19. Jiang, H.X., You, Y.L., Lin, D.J., Su, M., 2004. Observation on the testicular morpha and structure of Chinese ocellated gudgeon, Bostrichthys sinensis. J. Fujian Agric. Forestry Univ. (Nat. Sci. Ed.) 33 (1), 89–93. Jiang, J.H., Dai, H.P., Zhu, J.Q., Zhang, Y.M., Lei, S.Y., 2013. Histological observation on the ovary development of cultivated Acrossocheilus fasciatus. Oceanol. Limnol. Sin. 44 (2), 348–354. Jiang, J.H., Zhang, D.M., Zhu, J.Q., Lei, S.Y., Zhang, Y.M., 2012. The development of embryo, larva, and juvenile of Acrossocheilus fasciatus. Oceanol. Limnol. Sin. 43 (2), 280–287. Jun, J.C., Chung, E.Y., Yang, Y.C., 2006. Ultrastructure of germ cells, cyst epithelialcells and interstitial cells during spermatogenesis of the stone flounder, Kareius bicoloratus. Kor. J. Ichthyol. 18, 311–318. Kim, S.H., Lee, C.H., Song, Y.B., Hur, S.W., Kim, H.B., Lee, Y.D., 2013. Ultrastructure of late spermatids and spermatozoa during spermiogenesis in longtooth grouper Epinephelus bruneus from Jeju, Korea. Tissue Cell 45, 261–268. Koulish, S.C., Kramer, R.C., Grier, H.J., 2002. Organization of the male gonad in aprotogynous fish, Thalassoma bifasciatum (Teleostei: Labridae). J. Morphol. 254, 292–311. Lahnsteiner, F., Patzner, R.A., 1995. Fine structure of spermatozoa of two marine teleost fishes, the red mullet, Mullus barbatus (Mullidae) and the white sea bream, Diplodus sargus (Sparidae). J. Submicrosc. Cytol. Pathol. 27, 259–266. Lahnsteiner, F., Patzner, R.A., 1997. Fine structure of spermatozoa of four littoral teleosts, Symphodus ocellatus, Coris julis, Thalassoma pavo and Chromis chromis. J. Submicr. Cytol. Pathol. 29, 477–485. Lahnsteiner, F., Patzner, R.A., 2007. Sperm morphology and ultrastructure in fish. In: Alavi, S.M.H., Cosson, J., Coward, K., Rafiee, G. (Eds.), Fish Spermatology. Alpha Science International Ltd, Oxford, UK, pp. 1–61.

62

S.-Y. Fu et al. / Tissue and Cell 48 (2016) 49–62

Lin, D.J., You, Y.L., Su, M., 2003. Studies on testicular histology and spermatogenesis of teleost, Spinibarbus caldwelli (nichols). Acta Hydrobiol. Sin. 27 (6), 563–571. Lin, G.H., Weng, S.C., Zhang, F.W., 1981. Studies on the development of testes in the first sexcycle of the Chinese ide, (Ctenopharyngodon idellus). Oceanol. Limnol. Sin. 12 (6), 559–565. Liu, J., 1993. China Farmed Fish Reproduction Physiology. China Agriculture Press, Beijing, pp. 29–41. Liu, L.L., 2010. The Study on Gonad Development and Artificial Reproduction of Acrossocheilus hemispinus. Fujian Normal University, Fujian, pp. 17–40. Lo Nostro, F.L., Grier, H., Meijide, F.J., Guerrero, G.A., 2003. Ultrastructure of the testis in Synbranchus marmoratus (Teleostei, Synbranchidae): the germinal compartment. Tissue Cell 35, 121–132. Lou, Y.D., 1999. Histology and Embryology. China Agriculture Press, Beijing, pp. 126–137. Lou, Y.H., Takahashi, H., 1989. Spermiogenesis in the Nile tilapia Oreochromis niloticus with notes on a unique pattern of nuclear chromatin condensation. J. Morphol. 200, 321–330. Luft, J.H., 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409. Mattei, X., 1969. Contribution à l’étude de la spermiogenèse et des spermatozoïdes de Poissons par la méthode de la micrsocopie électronique. In: Thèse Doctoral d’Etat, Sciences, Montpellier., pp. 148. Mattei, X., 1970. Spermiogenèse comparée des poisons. In: Baccetti, B. (Ed.), Comparative Spermatology. Academic Press, New York, pp. 57–70. Mattei, X., 1988. The flagellar apparatus of spermatozoa in fish. Ultrastructure and evolution. Biol. Cell 63, 151–158. Mattei, X., 1991. Spermatozoon ultrastructure and its systematic implication in fishes. Can. J. Zool. 69, 3038–3055. Maricchiolo, G., Genovese, L., Guerrera, M.C., Micale, V., Muglia, U., 2010. Fine structure of spermatozoa in the blackspot sea bream Pagellus bogaraveo (Brünnich, 1768) with some considerations about the centriolar complex. Tissue Cell 42, 88–96. Maricchiolo, G., Genovese, L., Laurà, R., Micale, V., Muglia, U., 2002. The ultrastructure of amberjack (Seriola dumerilli) sperm. Eur. J. Morphol. 40, 289–292. Mattei, X., Slau, Y., Thiaw, O.T., Thiam, D., 1993. Peculiarities in the organization of testis of Ophidion sp. (Pisces: Teleostei). Evidence for two types of spermatogenesisin in teleost fish. J. Fish Biol. 43, 931–937. Mokae, L.M., Smitb, N.J., Wagenaara, G.M., 2013. Comparative histomorphological assessment of the testes of two Clarias species from the Okavango Delta Panhandle, Botswana. Tissue Cell 45, 7–20. ˜ Munoz, M., Sàbat, M., Mallol, S., Casadevall, M., 2002. Gonadal structure and gametogenesis of Trigla lyra (Pisces: Triglidae). Zool. Stud. 41, 412–420. Papah, M.B., Kisiaa, S.M., Ojooa, R.O., Makanyaa, A.N., Wood, C.M., Kavembe, G.D., Maina, J.N., Johannsson, O.E., Bergman, H.L., Laurent, P., Chevalier, C., Bianchini, A., Bianchini, L.F., Onyango, D.W., 2013. Morphological evaluation of spermatogenesis in Lake Magadi tilapia (Alcolapia grahami): A fish living on the edge. Tissue Cell 45, 371–382. Parenti, L.R., Grier, H.J., 2004. Evolution and phylogeny of gonad morphology in bony fishes. Intergr. Comp. Biol. 44, 333–348. Poirier, G.R., Nicholson, N., 1982. Fine structure of the testicular spermatozoa from the channel catfish, Ictalurus punctatus. J. Ultrastruct. Res. 80, 104–110. Quagio-Grassiotto, I., Antoneli, F.N., Oliveira, C., 2003. Spermiogenesis and spermultrastructure in Cichla intermedia with some considerations about Labroidei spermatozoa (Teleostei, Perciformes, Cichlidae). Tissue Cell 35, 441–446. Quagio-Grassiotto, I., Carvalho, E.D., 2000. Ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermiogenesis. J. Submicrosc. Cytol. Pathol. 32, 629–633. Quagio-Grassiotto, I., Oliveira, C., 2008. Sperm ultrastructure and a new type ofspermiogenesis in two species of Pimelodidae, with a comparative review of sperm ultrastructure in siluriformes (Teleostei: Ostariophysi). J. Comp. Zool. 247, 55–66.

Romagosa, E., Narahara, M.Y., Borella, M.I., Parreira, S.F., Fenerich-Verani, N., 1999. Ultrastructure of the germ cells in the testis of matrinx Brycon cephalus (Teleostei, Characidae). Tissue Cell 31, 540–544. Romagosa, E., Paiva, P., Andrade-Talmelli, E.F., Godinho, H.M., 2003c. Biologia reprodutiva de fèmeas de cachara, Pseudoplatystoma fasciatum (Teleostei, Siluriformes, Pimelodidae), mantidas em cativeiro. B. Inst. Pesca 29 (2), 151–159. Rupik, W., Huszno, J., Klag, J., 2011. Cellular organisation of the mature testes and stages of spermiogenesis in Danio rerio (Cyprinidae; Teleostei)—structural and ultrastructural studies. Micron 42, 833–839. Schulz, R.W., de Francs, L.R., Lareyre, J.J., Le Gac, F., Chiarini-Garcia, H., Nobrega, R.H., Miura, T., 2010. Spermatogenesis in fish. Gen. Comp. Endocrinol. 165, 390–411. Shahin, A.A.B., 2006. Semi-cystic spermatogenesis and biflagellate spermatozoon ultrastructure in the Nile electric fish Malapterurus electricus (Teleostei; Siluriformes: Malapteruridae). Acta Zool. (Stockholm) 87, 215–227. Shahin, A.A.B., 2007. A novel of spermiogenesis in the Nile catfish Chrysichthys auratus (Siluriformes: Bagridae) in Egypt, with description of spermatozoon ultrastructure. Zool. Res. 28, 193–206. Spadella, M.A., Oliveira, C., Ortega, H., Quagio-Grassiotto, I., Burns, J.R., 2012. Male and female reproductive morphology in the inseminating genus Astroblepus (Ostariophysi: Siluriformes: Astroblepidae). Zoologischer Anzeiger 251, 38–48. Stoumboudi, M.T.H., Abraham, M., 1996. The spermatogenic process in Barbus longiceps, Capoeta damascina and their natural sterile hybrid (Teleostei, Cyprinidae). J. Fish Biol. 49, 458–468. Su, J.X., 1995. Ichthyography and Marine Fish Culture. China Agriculture Press, Beijing, pp. 90–93. Tang, H.Y., Liu, J.H., 1998. Observation on the gonadal development of Barbodes (Spinibarbus) sinensis (bleeker). J. Southwest Agric. Univ. 20 (1), 90–94. Thiaw, O.T., Mattei, X., Romand, R., Marchand, B., 1986. Reinvestigation of spermatic flagella structure: the teleostean Cyprinodontidae. J. Ultrastruct. Mol. Struct. Res. 97, 109–118. Toury, R., Clérot, J.C., André, J., 1977. Les groupements mitochondriaux des poissons téléostéens cyprinidés. IV. Analyse biochimique des constituents du “ciment” intermitochondrial isolé(1) . Biol. Cellulaire 30, 225–232. ˜ R.H., Meijide, F.J., Guerrero, G.A., 2012. Spermatogenesis Vázquez, G.R., Da Cuna, and changes in testicular structure during the reproductive cycle in Cichlasoma dimerus (Teleostei, Perciformes). Acta Zool. 93, 338–350. Wen, C.Y., Xu, J., Zou, P.Z., Chen, J.R., Zhong, L.M., Luo, Q.H., Wu, Y.X., 2005. A study on the testis development of cultured Spiniparbus hollamdi Oshima. Freshw. Fisheries 35 (3), 41–43. Wu, H.F., Sheng, J.Q., Hong, Y.J., Wang, J.H., Liu, T.X., Gui, J.F., 2009. Gonadal development in natural wildness triploid mutant Pingxiang red-transparent crucian carp, Carassius auratus L. Acta Hydrobiol. Sin. 33 (6), 1185–1192. Xie, N.Y., He, X.F., 1988. The development and annual change in the Gonads of Tor brevifilis (Peters). Life Sci. Res. 2 (2), 140–146. Xie, N.Y., He, X.F., Yang, Q.F., 1999. Cytochemical in vestigations on oogenesis of the freshwater crab Sinopotamoin yangtsekiense. Chin. J. Zool. 15 (2), 1–5. Xie, N.Y., Yang, Q.F., He, X.F., 2002. The embryonic and larval development of Tor brevifilis brevifilis. J. Fisheries China 26 (2), 115–121. Yamamoto, K., Colak, A., 1974. Ultrastructure of the Japanese eel spermatozoon. Annot. Zool. Jpn. 47, 48–54. Yao, Z.L., Mi, G.Q., Lian, Q.P., Lv, Y.P., Xu, S.J., Lu, J., Wang, Y.C., 2013. Histological development and annual change in ovary of cultured Acrossocheilus fasciatus. Fisheries Sci. 32 (1), 31–35. Yasuzumi, F., 1971. Electron microscope study of the fish spermiogenesis. J. Nara. Med. Assoc. 22, 343–345. Zaki, M.I., Negm, R.K., El-Agamy, A., Awad, G.S., 2005. Ultrastructure of male germ cells and character of spermatozoa in Boops boops (family Sparidae) in Alexandria Coast, Egypt. Egypt J. Aquat. Res. 31, 293–313. Zhang, L.Z., Chen, L.H., Zhuang, P., Feng, G.P., Xu, B., Zhao, Y., 2009. Histological studies on gonad of Tridentiger trigonocephalus (Gill) of the Yangtze Estuary in reproductive season. J. Fisheries China 33 (2), 245–252. Zhang, Y.M., Jiang, J.H., 2010. A study on the artificial propagation of Acrossocheilus fasciatus. J. Zhejiang Ocean Univ. (Nat. Sci.) 29 (3), 211–214.