ZHENGXuebinWANGJingqianGAOXinmingDUChenHOUCongcongXIEQingpingLIUFengZHUJunquanandLOUBao

Testis Development and Ultrastructural Features During Spermatogenesis in Cultured Small Yellow Croaker,

ZHENG Xuebin1), WANG Jingqian1), GAO Xinming1), DU Chen1), HOU Congcong1),XIE Qingping2), LIU Feng2), ZHU Junquan1),*, and LOU Bao2),*

1),,,315211,2),310021,

The small yellow croakeris an economically important marine fish in Northeast Asia. Currently, its natural resources are threatened by overfishing and environmental pollution. Therefore, research on the reproductive system of the fish is crucial. Here, we studied the testis development and ultrastructural features of spermatogenesis in culturedusing anatomical, histological, and ultrastructural techniques. A pair of testes, consisting of a central sperm duct and radial seminiferous lobules, were observed. The reproduction cycle of testes can be divided into stages I – VI. March to May was confirmed as the breed- ing season for male, while April is the ideal period for artificial breeding. The malecan attain sexual maturity within 1 year. The spermatogenesis ofcomprised spermatogonium, spermatocyte, spermatid, and mature spermatozoon. The morphology of spermatogenic cells changed obviously during spermiogenesis, including nuclear shaping, midpiece and flagel- lum formation. The mature sperms consist of an ellipsoidal head, a short midpiece, and a long flagellum. The anterior of the head with a kidney-shaped nucleus can be distinguished. The midpiece is located posterior to the head and includes four to six spherical mitochon- dria. The flagellum has irregular lateral fins. The testis ofis an unrestricted lobular type, with cystic spermatogenesis, type II spermiogenesis, and type II spermatozoa. These features are highly similar to those of other Sciaenid species. Our findings provide useful insights into the mechanism underlying testis development and spermatogenesis of, which can facilitate the artificial breeding of this species.

testis development; spermatogenesis;; histology; ultrastructure

1 Introduction

Testis development and spermatogenesis are key parts of fish reproductive biology. Elucidating the characteristics and regularity of testis development is important for fish breeding and determining the ideal breeding season (Cui., 2013). Elucidation of the morphological changes of spermatogenic cells during spermatogenesis can provide a theoretical basis for exploring the mechanism of sperma- togenesis in certain species (Tang., 2020). In addition, information on the ultrastructure of fish spermatozoa can provide data for fish phylogeny, as the sperm structure tends to be relatively conserved within a given family or subfa- mily (Quagio-Grassiotto., 2020). Testis development and spermatogenesis in model fish or economically impor- tant fish have been extensively studied. For instance, in ma- rine fish, testis development or spermatogenesis have been reported for species such as(Lin., 1992; You., 2001),(Gwo., 1993),(Brown-Peterson., 2002),(Gusmao-Pompiani., 2005),(Ashida., 2010),(Guan., 2011),(Miao., 2013), and(Ma., 2014).

The small yellow croaker (), be- longing to the order Perciformes and family Sciaenidae, is one of the most important marine fish species in the North- east Asian countries, including China, Korea, and Japan (Liu., 2017). It is widely distributed in the Bohai, Yellow, and East China Seas, and was one of the four most popular marine products in China (Wang., 2015). However, in recent years, the natural resources ofhave de- teriorated significantly due to overfishing and environmen- tal pollution (Liu., 2017; Liu., 2019). Furthermore, these factors have led to considerable changes in the popu- lation structure of, such as early maturation and a reduction in individual size and age (Liu., 2017). Therefore, a series of studies on the reproductive biology ofhave been conducted, including artificial breeding (Chen., 2016), interspecific hybridization (Liu., 2019), sex differentiation (Yang., 2021), and ova- rian and oocyte development (Wu, 1980, 1981). Further- more, Han. (2010) have reported on the reproductive cycle ofin the southern waters of Korea, while Kang. (2013) have reported on the spermatogenesis and functions of the Leydig and Sertoli cells associated with spermatogenesis in. However, the structure of the testes, morphological changes, type of spermatogenesis, and ultrastructural features of mature sperms ofhave not been studied thoroughly.

In this study, we evaluated the testis development and spermatogenesis ofby using anatomical, histo- logical and ultrastructural (transmission electron micros- copy, TEM; and scanning electron microscopy, SEM) tech- niques. We aimed to gain information on the reproductive biology ofand explore the mechanism of sper- matogenesis into provide basic data for im- proving artificial breeding of the species.

2 Materials and Methods

2.1 Experimental Fish

Fishwere cultured in an indoor cement pool of the Xi- xuan Island, Marine Fisheries Research Institute of Zheji- ang Province, Zhoushan, China. The water temperature was controlled at 8 – 11℃ during November – February and 16 – 18℃ during March – April. In other months, the water temperature was maintained at the sea-water temperature of the East China Sea. A total of 30 fish were sampled randomly from the generations of 2017 every one or two months from August, 2017 to August, 2018. The fish were starved and temporarily cultured in sink for 24 h prior to sampling. All experiments were conducted in accordance with the principles and procedures approved by the Ani- mal Care and Use Committee of Ningbo University.

2.2 Sampling and Tissue Processing

2.2.1 Anatomical and histological analysis

Sampled fish were anaesthetized by ethyl 3-aminoben- zoate methanesulfonate salt (50 mg L?1, Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the testes were immedi- ately removed from the abdominal cavity and photograph- ed. The testes were then cut into small sections and fixed in Bouin’s solution for 24 h. Next, the tissue samples were dehydrated with alcohol gradient and embedded in paraf- fin. Subsequently, they were sliced into 6-μm-thick sections and stained with hematoxylin-eosin (HE). Finally, the his- tological structure of testis was observed using a micro- scope (Nikon Ds-Ri2P).

2.2.2 Ultrastructural analysis by transmission electron microscopy and scanning electron microscopy

The ultrastructure of germ cells was observed by TEM (Hitatchi H-7650, Japan) during the complete process of spermatogenesis, including the stages of spermatogonia, spermatocytes, spermatids, and mature spermatozoa deve- lopment. For TEM analysis, testes were cut into small sec- tions and fixed in 2.5% glutaraldehyde for 2 h, osmium te- troxide for 2 h, then dehydrated with alcohol, permeated by ethoxyline resin (Epon-812, Sigma-Aldrich), and embed- ded into araldite resin. The ultrathin microtome (LKB-II, LKB-Produkter AC Bromma. Sweden) was used to slice the sections. Then the sliced sections were subsequently counterstained with uranyl acetate and lead citrate. Semen was extracted from matureand fixed in 2.5% glutaraldehyde for 24 h, dehydrated with alcohol, dried at the critical point, coated with gold, and observed by the SEM (Hitachi S-3400P, Japan).

2.3 Statistical Analysis

All data were expressed as the mean ± standard devia- tion (SD). The gonadosomatic index () was calculated as Eq. (1)

whereandare the gonad weight and body weight respectively. The processing and the measurement of germ cell images were performed by Photoshop (CS5, Adobe).

3 Results

3.1 Morphological Features of the Testis

We constructed a model (Fig.1) oftestis based on our morphological and histological findings of the development of the testes (Figs.2 – 4). A pair of testes with similar size were located symmetrically in the dorsal region of the coelomic cavity and connected to the ventral surface of the swim bladder. The testes were posteriorly elongated, uniting into a Y-like shape in the terminal region and finally opening to the cloaca (Fig.1A). Histologically, the testes consisted of the outermost peritoneum, a longi- tudinal blood vessel, a longitudinal sperm duct, several se- miniferous lobules, and the interlobular connective tissues (Figs.1A, B). A blood vessel, located in the inner surface of the ventral side of testis, was visible by naked eye. The sperm duct was connected to the blood vessel, which was located near the centre of the testis. The interlobular connective tissues, which contained the Leydig cells, blood/ lymphatic vessels, fibrocytes, and connective tissue cells, were comparable to the tunica albuginea of mammals. The interlobular connective tissues were invaginated into the testis interior and formed numerous septa. These septa sub- divided the testis into several anastomosed seminiferous lobules. The seminiferous lobules in the testis were ar- ranged radially around the sperm duct, with regular distri- bution, including numerous spermatogenic cysts. Many syn- chronously developing spermatogenic cells were observ- ed in the same cyst, while the development was not neces- sarily synchronous in different cysts. Upon full develop- ment of spermatogenic cells, the cysts opened to release the mature sperm into the lobular lumen and subsequently into the sperm duct. Finally, the sperm was excreted from the coelomic cavitythe cloaca (Fig.1B).

Fig.1 Diagram of testis morphology and structure in Lari- michthys polyactis. A, the outline of the L. polyactis tes- tis. B, the cross-section features of the L. polyactis testis. a, testis; b, sperm duct; c, blood vessel; d, cloacal orifice; e, seminiferous lobule; f, peritoneum; g, tunica albuginea; h, spermatogenic cyst.

3.2 Cycle of Testis Development

Testis development ofwas divided into six different stages according to the criteria set by Liu (1993) and Lou (1996). The morphological and histological cha- racteristics are presented in the following sections.

3.2.1 Stage I

In this stage, the testes were transparent and appeared like a filament, while it was impossible to differentiate the gender with the naked eyes (Fig.2A). Theof this stage ranged around 0.01% – 0.04%, with an average of 0.03% (= 4). The histological results showed that abundant con- nective tissues occupy the majority of the testis interior, and interspersed with irregularly distributed seminiferous lobules. A small number of spermatogonia that dispersed in the seminiferous lobules were the only germ cell type de- tected in this stage. The spermatogonia were the largest germ cells present in the testes, showing an ovoid or spherical outline and containing a large nucleus with a prominent nucleolus (Figs.3A1 – A3). In the first reproductive cycle, 36.4% of the 4-month-old males were in this stage, while the rest had progressed to stage II (Table 1).

Fig.2 Anatomical features of different developmental stages of the testes in Larimichthys polyactis. A – F, morphology of the testes at stages I – VI. G, morphology of repeat developmental stage II testis.

Table 1 Testis development and the changes of body length, weight and GSI of male L. polyactis during 4 – 16 months

Note: II’ means repeat development stage II.

3.2.2 Stage II

The testes were translucent and had a fine rod-like shape (Fig.2B). The range ofat this stage is 0.04% – 0.11% with an average of 0.07% (= 24). The histological analy- sis showed a noticeable sperm duct, with a big central blood vessel near the sperm duct (Fig.3B1). The connective tis- sues extended to form several thin septa dividing the tes- tes into many seminiferous lobules. Meanwhile, the sperma- togonia in the seminiferous lobules generally proliferated and formed numerous spermatogenic cysts. Thus, a large number of spermatogonia located closely against each other were detected in this stage (Figs.3B2, B3). In the first re- productive cycle, 63.6% of the 4-month-old and all the 6-month-old males were in this stage (Table 1).

3.2.3 Stage III

The testes were translucent and showed flat, tape-like shape. A red blood vessel was observed on the inner sur- face of the ventral side of the testis by naked eyes (Fig.2C). Theof this stage ranged 0.20% – 1.03% with an aver- age of 0.59% (= 13). Histological findings revealed many spermatogenic cysts containing spermatogonia, similar to those observed in the stage II testes. However, they had dif- ferentiated into spermatocyte cysts in this stage, while a small number of cysts had already differentiated into spermatid cysts. Consequently, the germ cells in the seminiferous lo- bules included a large number of spermatogonia and sper- matocytes, as well as a small number of spermatids. How- ever, no mature sperms were observed in the testes at this stage. The spermatocytes, including the primary and second- ary spermatocytes, showed a smaller cell size and a more basophilic nucleus compared to the spermatogonia, while the spermatids were smaller and more basophilic than the spermatocytes (Figs.3C2, C3). In the first reproductive cycle, 83.6% of the 8-month-old males were in this stage (Table 1).

Fig.3 Histological characteristics of the Larimichthys polyactis testis at stages I – IV. (A1) Stage I testis. Blood vessel and several connective tissues were observed, the sperm duct was not evident in this stage. (A2, A3) Enlarged images of A1, the connective tissues divide the testis into several seminiferous lobules, and a few of spermatogonia are dispersed inside. (B1) Stage II testis. Blood vessel and sperm duct were evident and the number of seminiferous lobules significantly in- creased in this stage. (B2, B3) Enlarged images of B1, the spermatogonia proliferated and attached to each other closely in the spermatogenic cysts. (C1) Stage III testis. Blood vessel and sperm duct were evident in this stage. (C2, C3) En- larged images of C1, several spermatogenic cysts with spermatocytes and a small number of spermatogenic cysts with spermatid was detected in the seminiferous lobules. (D1) Early stage IV testis. The mature sperm could be detected in the lobular lumen and sperm duct. (D2, D3) Enlarged images of D1, several spermatocytes and spermatids were observed in the spermatogenic cysts, and the mature sperms were mainly distributed in the lobular lumen and sperm duct. (D4) Late stage IV testis. More mature sperms were detected in the lobular lumen and sperm duct. (D5, D6) Enlarged images of D4, several spermatids in different stages were observed in the spermatogenic cysts. BV, blood vessel; SG, spermatogonia; SD, sperm duct; SC, spermatocyte; ST, spermatid; SP, spermatozoa; ST1, early spermatid; ST3, late spermatid.

3.2.4 Stage IV

The testes in this stage were markedly enlarged com- pared to those in stage III, and a blood vessel was observed on the surface of the testes. The testes were in ivory color and displayed a flat, tendon-like shape (Fig.2D). Theof this stage ranged from 1.03% – 4.66% with an average of 3.12% (= 13). Histological findings revealed that all types of spermatogenic cells could be detected in the semi- niferous lobules, including spermatogonia, spermatocytes, spermatids, and mature sperms (Fig.3D). A small number of spermatogonia were observed, occasionally in the bound- ary of seminiferous lobules. The spermatocytes and sper- matids were the main cell types present in this stage. Many spermatids in different stages were distributed in the semi- niferous lobules, and could be divided into early, middle, and late phases according to the cell size and basophils. In addition, some of the spermatids were developed into ma- ture sperm and the spermatogenic cysts opened to release them into the lobular lumen. Subsequently, the sperm pass- ed into the sperm duct, resulting in a slightly expanded sperm duct (Figs.3D1 – D6). In the early stage IV testes, the testis volume was slightly lower and the spermatogonia were more frequently observed in the lobules (Figs.3D1 – D3), while in later stage IV testes, the testes were enlarged and the number of spermatids and mature sperms were signi- ficantly higher (Figs.3D4 – D6). In the first reproductive cy- cle, 16.7% of the 8-month-old and 85.7% of the 10-month- old males were in this stage (Table 1).

3.2.5 Stage V

In this stage, the volume of the testes was the highest. The testes were ivory in color and showed a long cystiform shape. In addition, a large amount of semen exuded from the cloaca when slight pressure was applied to the fish ab- domen (Fig.2E). Theof this stage ranged from 1.27% – 5.81%, with an average of 2.75% (= 30). Histological findings revealed abundant mature sperm distributed in the seminiferous lobules and enlarged sperm duct. Simultane- ously, the ratio of other germ cell types, such as the sperma- togonia, spermatocytes, and spermatids, was significantly lower due to the final process of spermiogenesis. In the ear- lier stage V testes, the testis volume was the highest, as less mature sperm were released from the coelomic cavity. Nu- merous spermatids were in the final process of spermio- genesis to produce more mature sperm (Figs.4A1 – A3). As the reproduction progressed, some mature sperm were spent, and the lobules and sperm duct were filled with the remain- ing mature sperm. Only a few spermatocytes and sperma- tids remained in the peripheral regions of the seminiferous lobules (Figs.4A4 – A6). The mature sperms were the small- est germ cells in the testes, which were delineated by a high- ly compacted and basophilic nucleus (Fig.4A6). In the first reproductive cycle, 14.3% of the 10-month-old, 100% of the 12-month-old, and 56.5% of the 13-month-old males were in this stage (Table 1).

Fig.4 Histological characteristics of stages V and VI and repeat development stage II testis of Larimichthys polyactis. (A1) Early stage V testis. Many spermatids and mature sperms are in the seminiferous lobules and sperm duct. (A2, A3) En- larged images of A1, a large number of spermatids, in the final process of spermiogenesis, are distributed in the semini- ferous lobules. (A4) Late stage V testis. The seminiferous lobules and sperm duct are filled with numerous mature sperms. (A5, A6) Enlarged images of A4, numerous mature sperms are distributed in the lobular lumen, a few spermatocytes and spermatids are located at the boundary of seminiferous lobules. (B1) Stage VI testis. The majority of the mature sperms are released, only a few residual sperms remain in the sperm duct and lobular lumen. (B2, B3) Enlarged images of B1, a few spermatocytes and spermatids are in the spermatogenic cysts, and the spermatogonia occasionally occurred in the connec- tive tissues. (B4) The end of stage VI testis. The mature sperms are totally spent. (B5, B6) Enlarged images of B4, the con- nective tissues left by spermatogenic cysts are cancellated in the testis (arrow) with the Sertoli cells dispersed in it. Only a small part of spermatogonia are located on the boundary of germinal epithelium, and the residual sperm or other germ cells are spent or absorbed. (C1) Repeat development stage II testis. The testis renew and show similar features to stage II testis in the first cycle. (C2, C3) Enlarged images of C1, the spermatogonia proliferate and attach to each other closely in the spermatogenic cysts. BV, blood vessel; SG, spermatogonia; SD, sperm duct; SE, Sertoli cell; ST, spermatid; ST1, early spermatid; ST2, middle spermatid; ST3, late spermatid; SP, spermatozoa.

3.2.6 Stage VI

After the process of spermiation, the volume of the tes- tes significantly reduced and shrunk into an oblate, tape- like shape, while the blood vessel was still prominent on the inner surface of the testes (Fig.2F). Theof this stage ranged from 0.12% – 1.01%, with an average of 0.82% (= 9). Histological findings indicated that a majority of the mature sperm had been released. Only a small number of residual sperms remained dispersed in the sperm duct and lobular lumen. In addition, a small number of spermatogo- nia and spermatocytes were located in the connective tis- sue near the border of the seminiferous lobules (Figs.4B1 – B3). At the end of this stage, all mature sperm and other germ cells were spent or degenerated, except for a small number of spermatogonia present only at the boundary of the germinal epithelium. In addition, a lot of cancellated connective tissues formed by the remaining spermatogenic cysts were observed in the testes, and some Sertoli cells were dispersed in the connective tissues (Figs.4B4 – B6). In the first reproductive cycle, 43.5% of the 13-month-old males were in this stage (Table 1).

3.2.7 Repeat development stage II

The testes were translucent and again displayed a flat, tape-like structure. However, the volume of the testes was higher than that during the primary stage II (Fig.2G). Theof this stage ranged from 0.17% – 0.34%, with an ave- rage of 0.27% (= 10). Histology revealed that the distri- bution and constitution of the spermatogenic cells were si- milar to those of stage II testes in the first cycle. There were a large number of spermatogenic cysts, containing many spermatogonia grouped closely with each other in the seminiferous lobules. More connective tissues were ob- served in the testes, which resulted in a thicker lobular wall than those observed during the first stage II (Figs.4C1 – C3). In the second reproductive cycle, all the 16-month-old maleswere in this stage (Table 1).

3.3 Spermatogenesis and Ultrastructure of the Germ Cells

The spermatogenesis ofcan be divided into four stages, including spermatogonia, spermatocyte, sper- matid, and mature sperm.

3.3.1 Spermatogonia stage

The spermatogonia stage can be divided into primary and secondary spermatogonia.

3.3.1.1 Primary spermatogonia

The primary spermatogonia were the largest germ cells in the testes, measuring 13.23 μm ± 1.09 μm in cell diame- ter and 8.84 μm ± 0.36 μm in nuclear diameter (= 20). They usually occur individually and separately in the boundary of seminiferous lobules (Fig.5a). The primary spermato- gonia were round or oval, with an oval nucleus. The nu- cleolus was large. They are located eccentrically within the nucleus with a high electron density. The chromatin was fine, fiber-shaped, and evenly dispersed, with a low electron density. In the cytoplasm, several nuages were detected, which had a high electron density and were mainly distri-buted in the perinuclear area; the mitochondria were round and in small size (Fig.5A).

Fig.5 Histological and ultrastructural features of the primary and secondary spermatogonia in Larimichthys polyactis. (a) Histological image shows the primary spermatogonia individually distributed at the lobule boundary. (A) Ultrastructural image shows a large nucleolus located in the nucleus and several nuages (arrow) and mitochondria distributed in the cy- toplasm. (B1) Ultrastructural image shows several secondary spermatogonia distributed in the same cyst. (B2) A large nu- cleolus in the nucleus and several nuages (arrow) and mitochondria in the cytoplasm were observed. (B3) Several mito- chondria adhere to the nuages (arrow). (b) Histological image shows several secondary spermatogonia distributed in the same cyst. PSG, primary spermatogonia; N, nucleus; Nu, nucleolus; M, mitochondria; SSG, secondary spermatogonia.

3.3.1.2 Secondary spermatogonia

Secondary spermatogonia were similar to primary sper- matogonia; however they were smaller in size, with 8.39 μm ± 0.44 μm in cell diameter and 5.11 μm ± 0.46 μm in nu- cleus diameter (= 20). Two or more secondary spermato- gonia were usually distributed in a cyst (Figs.5B1, b). They were oval, while their nuclei were round or oval, with one or two nucleoli observed in the central or boundary area of the nucleus (Figs.5B1, B2, b). The nuages were detected in the cytoplasm, wherein some adhered to the mitochondria(Figs.5B2, B3).

3.3.2 Spermatocyte stage

The spermatocyte stage can also be divided into primary and secondary spermatocytes.

3.3.2.1 Primary spermatocytes

The most frequently recognized primary spermatocytes were those in prophase I of the first meiotic division, main- ly distinguished by the characteristics of the chromatin. Lep- totene spermatocytes, with 4.02 μm ± 0.15 μm in nuclear dia- meter (= 10), were characterized by relatively electron- dense and minimally condensed chromatin appearing as nu- merous small patches. Furthermore, the nucleolus and nu- age were also detected in this stage (Fig.6A1). Additional- ly, zygotene spermatocytes, measuring 3.93 μm ± 0.11 μm in nuclear diameter (n = 12), were observed. The chroma- tin was more condensed and gathered into numerous short synaptonemal complexes (Fig.6A2). Pachytene spermato- cytes, measuring 3.98 μm ± 0.21 μm in nuclear diameter (n = 14), presented the typical structure of synaptonemal com- plexes, the homologous chromosomes were parallel to each other as a pair of fuzzy shapes (Fig.6A3). Diplotene sper- matocytes, measuring 3.79 μm ± 0.10 μm in nuclear diame- ter (n = 10), were also observed. The majority of synaptone- mal complexes were transformed into thick clumps of more electron-dense chromosomes, a few short synaptonemal complexes remained in the nucleus (Fig.6A4). During the metaphase of the first meiotic division, the metaphasic pri- mary spermatocytes showed an oval nucleus with highly condensed chromatin, distributed in the central space of the primary spermatocytes (Figs.6B, b).

Fig.6 Histological and ultrastructural features of spermatocytes in Larimichthys polyactis. (A1) Leptotene spermatocytes, showing the nuages (triangular arrow), nucleolus, and small patch-shaped chromatin (arrow). (A2) Zygotene spermato- cytes, showing the condensation of chromatin and the primary synaptonemal complexes (arrow). (A3) Pachytene sperma- tocytes, showing the homologous chromosomes pairing with each other and presenting the typical structure of synap- tonemal complexes (arrow). (A4) Diplotene spermatocytes, showing the disassembled chromosomes and a few of remain- ing synaptonemal complexes (arrow). (b) Histological image shows the metaphase I of primary spermatocytes. (B) Ultra- structure image shows many secondary spermatocytes closely attached to each other in the cyst, as well as some primary spermatocytes in the same cyst, in metaphase of meiosis I. (C) Ultrastructural image shows the characteristics of secon- dary spermatocytes, a round or oval nucleus with high electron-dense chromatin and several small mitochondria were de- tected in the cytoplasm. (c) Histological image shows several secondary spermatocytes distributed in the spermatogenic cyst. (D) Ultrastructural image shows telophase II secondary spermatocytes, which were dividing into two early sperma- tids. (d) Histological image shows the presence of several secondary spermatocytes during meiosis II, in which some sper- matocytes have developed into the early spermatid. N, nucleus; Nu, nucleolus; M, mitochondria; L, Leptotene spermato- cyte; Z, Zygotene spermatocyte; P, Pachytene spermatocyte; D, Diplotene spermatocyte; PS, primary spermatocyte; SS, secondary spermatocyte; M1, metaphase I spermatocyte; M2, metaphase II spermatocyte; T2, telophase II spermatocyte; ST1, early spermatid.

3.3.2.2 Secondary spermatocytes

The secondary spermatocytes were rarely encountered due to their short lifespan, because they rapidly enter the secondary meiotic division. Nonetheless, there were still a few cysts that contained the secondary spermatocytes that showed the metaphasic or telophasic figures of the meio- tic division (Figs.6C, D, c, d). The secondary spermatocytes, which had just occurred from the meiosis I, were clearly smaller than the primary spermatocytes, measuring 2.61 μm ± 0.12 μm in nuclear diameter (= 20), with a round or oval nucleus, containing chromatin with higher electron density. Meanwhile several round but small mitochondria were ob- served in the cytoplasm (Figs.6C, c). In the metaphase or telophase of meiosis II, the secondary spermatocytes exhi- bited a smaller cell and nucleus size and more condensed chromatin compared to those observed in meiosis I, as they quickly developed into the more compacted spermatids (Figs.6D, d). Notably, the development of spermatocytes was not fully synchronous in the same cyst, as some of them were in the metaphase or telophase, while few of them had finished dividing (Figs.6B, D, b, d).

3.3.3 Spermatid stage

The spermatids were undergoing a final differentiation period, known as spermiogenesis. The spermatids ofcan be divided into three stages, including early, mid- dle, and late spermatids, based on the changes in the nu- cleus, chromatin, mitochondria, and cytoplasm.

3.3.3.1 Early spermatid stage

The early spermatids showed a slightly smaller cell size compared with that of the secondary spermatocytes, with a 2.03 μm ± 0.03 μm nuclear diameter (= 20). They had a regular round nucleus with relatively lower density and evenly distributed chromatin. In the cytoplasm, there were several round mitochondria randomly distributed around the nucleus. In addition, the flagellum formation had begun in this stage (Figs.7A, a).

3.3.3.2 Middle spermatid stage

Middle spermatids were smaller than the early sperma- tids, measuring 1.79 μm ± 0.11 μm in nuclear diameter (Figs. 7B1, B2, b) (= 20). The nuclei of the middle spermatids were slightly deformed and reshaped into oval or nearly oval, one side of the nucleus was slightly depressed to form a shallow nuclear fossa, and the chromatin in the nucleus was more condensed (Figs.7B1, B2). Meanwhile, the mi- tochondria were fused into a larger size and had migrated to one side of the nucleus, near the nuclear fossa. The mid- piece would form later on this side. Furthermore, the ini- tial part of flagellum was elongated from the centriolar com- plex which was located outside the nuclear fossa (Figs. 7B1, B2).

3.3.3.3 Late spermatid stage

Late spermatids were more compacted, their nuclei were further deformed and reshaped into a kidney-like shape, measuring 1.87 μm ± 0.10 μm in length and 1.08 μm ± 0.07 μm in width (Figs.7C1, C2, c) (= 20). The chromatin in the nucleus gathered into numerous small patches and were attached to each other closely. The volume of cytoplasm was decreased by the discharge of residual cytoplasm, and the mitochondria were further fused and showed larger size (Figs.7C1, C2). The remaining cytoplasm and fused mito- chondria were concentrated in the back of nucleus to form the midpiece, which showed a sleeve-like structure, and the flagellum was located in the central space of the sleeve (Fig.8C1). In addition, many vesicles were present in the sleeve, which may be related to the discharge of residual cytoplasm (Fig.7C1).

3.3.4 Mature spermatozoa stage

The mature spermatozoa were the most compacted germ cells in the testes, consisting of an ellipsoidal head, a short midpiece, and a long flagellum (Figs.8a – e).

The acrosomeless head can be differentiated into an an- terior and posterior region. It mainly consisted of a kidney- shaped nucleus and the centriolar complex. The nucleus was enclosed by double nuclear membranes, and contain- ed a highly condensed filamentous cluster of chromatin in- terspersed by electron lucent areas, measuring 1.63 μm ± 0.04 μm in length and 1.01 μm ± 0.06 μm in width (= 10; Figs.8a, a1, A, B). In the posterior region of the nucleus, it was slightly depressed and shaped into a shallow nuclear fossa. The centriolar complex lay outside the nuclear fossa and ran parallel to the nucleus. The centriolar complex con- sisted of the proximal and distal centriole. The centriole complexes were oriented perpendicularly to each other (Figs. 8C, D, d; Figs.9a – c).

The midpiece was short and measured 0.37 μm ± 0.03 μm in length, and was located in the posterior region of the head (Figs.8a – c). It mainly contained the initiating part of the flagellar axis and the sleeve (Figs.8e, E – G; Figs.9a, f). The flagellar axis extended from the distal centriole and began with a transitional structure termed the base body. The base body lacked the central microtubules and showed a ‘9 + 0’ axoneme pattern (Figs.8D, E; Figs.9a, c, d). The sleeve was located in the posterior region of the head, with 4 – 6 sphe- rical mitochondria. It surrounded the initial portion of fla- gellar axis and was separated from it by the cytoplasmic channel (Figs.8e, E – G; Figs.9a, d – f).

The flagellum was a long and fine structure, measuring 0.17 μm ± 0.02 μm in diameter (= 20). It presented a ty- pical ‘9 + 2’ axoneme pattern and had two irregular short fins shaped by an extension of the flagellum membrane (Figs.8a, H, I; Fig.9g).

4 Discussion

4.1 Testicular Type and Morphological Features

The classification of male Osteichthyes gonads is ambi- guous, and two types of testes are commonly distinguish-ed in the literature. Billard. (1982) divided the testi- cular structure of Osteichthyes into tubular and lobular ones. The tubular testes consist of several tubules, which are re- gularly oriented between the external tunica propria (blind end), and the central cavity in which the spermatozoa are released. The spermatogonia are only distributed in the blind end of tubules, and they are wrapped in the spermatogenic cysts. As the spermatogonia develop into mature sperm, the spermatogenic cysts gradually migrate to the central cavity and release the mature sperms into it. In contrast, the lo- bular testes consist of many lobules, with a lobular lumen in every lobule. The spermatogonia and other germ cells are scattered along the lobules, the spermatogenic cysts remain roughly at the same place, and the mature sperm are released into the lobular lumen. To use gonad morphology routine- ly in phylogenetic systematic analyses, Parenti and Grier (2004) reviewed previous researches on the structure and morphology of Osteichthyes male gonads and re-classified them into two main types: anastomosing tubular and lobu- lar testes. Basal taxa of Osteichthyes, such as Cypriniformes and Salmoniformes, have anastomosing tubular testes, whe- reas the derived taxa of Osteichthyes (Neoteleostei) have lobular testes. Lobular testes have been subdivided into re- stricted lobular testes and unrestricted lobular testes accord- ing to the distribution of spermatogonia. In restricted lobu- lar testes, the spermatogonia are ‘restricted’ to the terminal end of the lobule, while the spermatogonia in the unrestrict- ed lobular testes are distributed along the lobule. In our study, we observed that L. polyactis testes consist of many lobules, and the spermatogonia are distributed along the lo- bule. Consequently, the testes of L. polyactis can be classi- fied as unrestricted lobular type, similar to those of most of the perciform fishes, such as L. crocea (Lin et al., 1992), R. canadum (Brown-Peterson et al., 2002), K. pelamis (Ashi- da et al., 2010), T. obesus (Guan et al., 2011), and N. albi- flora (Ma et al., 2014). In addition, some scholars divided the testes into ampullar- or radial-type according to the ar- rangement of seminiferous tubules or lobules. Ampullar type testes are identified frequently in Cypriniformes, while the radial-type are identified more frequently in Perciformes (Meng et al., 1987; Lin et al., 1992; He et al., 2001; Guan et al., 2011; Chen et al., 2013). The seminiferous lobules in L. polyactis testis are arranged radially from the central sperm duct to the peripheral connective tissue of the testes. Therefore, we can also classify the L. polyactis testes as an unrestricted lobular type with a radial arrangement of se- miniferous lobules. Furthermore, in this study, we selected different areas (anterior, middle, and posterior) of the tes- tes for paraffin sectioning and to observe the histological structure. The results showed that the central sperm duct was detected in all regions of the testes, indicating that the length of the sperm duct is similar to that of the testis and the sperm duct is longitudinally distributed.

Fig.7 Histological (a, b, c) and ultrastructural (A, B1, B2, C1, C2) features of spermatids in Larimichthys polyactis. (a) Histological image shows a large number of early spermatids with round nucleus distributed in the same cysts. (A) Ultra- structure of early spermatid shows round nucleus, perinuclear mitochondria, and flagellum. (b) Histological image shows the slight deformation of the middle spermatids. (B1, B2) Ultrastructure of middle spermatid, the nucleus was depressed and formed the initial structure of nuclear fossa, and the enlarged mitochondria were located near to the nuclear fossa. (c) Histological image shows the further deformation of late spermatids, their nuclei in a kidney-like shape. (C1, C2) Ultra- structure of late spermatid, the nuclei were highly condensed, and the chromatin concentrated into numerous small pat- ches, the remaining cytoplasm and fused mitochondria were concentrated in the midpiece, the flagellum was elongated from the nuclear fossa. N, nucleus; F, flagellum; M, mitochondria; NF, nuclear fossa; V, vesicle; S, sleeve; ST1, early spermatid; ST2, middle spermatid; ST3, late spermatid.

Fig.8 Histological (a1) and ultrastructural (SEM, a – e; TEM, A – I) features of spermatozoa in Larimichthys polyactis. (a) The fine structure of spermatozoa, showing the head, midpiece, and flagellum. (b, c, d, e) Different perspectives show the morphology and structure of spermatozoa, including the head, midpiece, flagellum, nuclear fossa, and mitochondrion. (a1) Histological image shows the kidney-shaped nucleus of spermatozoa. (A, B) Longitudinal sections show the kidney-shaped nucleus, spherical mitochondria, and flagellum. (C, D, E, F) A series of cross sections show the structure of centriolar complex and the initiating portion of flagellar axis, along with the morphology of proximal centriole, distal centriole, base body, and flagellar axoneme. (G) The cross section of midpiece, showing four spherical mitochondria surrounding the flagellar axoneme separated from them by the cytoplasmic channel. (H, I) Longitudinal and cross sections of flagellum, presented the lateral fins and ‘9 + 2’ pattern of axoneme. H, head; F, flagellum; M, mitochondria; MP, midpiece; NF, nu- clear fossa; SP, spermatozoa; N, nucleus; NM, nuclear membrane; PC, proximal centriole; DC, distal centriole; BB, base body; CC, cytoplasmic channel; S, sleeve; AX, axoneme; CDM, central doublets of microtubules; LF, lateral fin; PDM, peri- pheral doublets of microtubules.

Fig.9 Diagram of mature spermatozoa in Larimichthys polyactis. (a) Longitudinal section shows the features of mature sper- matozoa. (b) – (g) A series of cross sections show the features of mature spermatozoa. PM, plasma membrane; PC, proximal centriole; N, nucleus; NF, nuclear fossa; DC, distal centriole; BB, base body; M, mitochondria; CC, cytoplasmic channel; F, flagellum; S, sleeve; PDM, peripheral doublets of microtubules; LF, lateral fin; CDM, central doublets of microtubules.

4.2 Features of Testis Development

In this study, we observed the developmental process oftestes from the age of 4 to 16 months. In Feb- ruary of the following year, a small section of the testes in the 10-month-old males had developed to stage V, indicat- ing the beginning of sexual maturity. In April, all testes of the 12-month-old males were in stage V, and the mature sperms in the testes were the most abundant, because the lo- bular lumen and sperm duct were filled with mature sperms. In May, part of the testes in the 13-month-old males had re- gressed and were in stage VI, suggesting that the cultured malecan reach maturity within 1 year and March – May can be confirmed as the breeding season for male. Furthermore, in our study, the females were ma- ture at April and gradually degraded at May (unpublish data), indicating that April – May is the breeding season for, and April is the best time for artificial breeding. In the wild, the testis development ofis noticea- bly different. Wu (1982) reported on the testis development ofin the Bohai Sea, the spermiogenesis reach- ed a peak in May, and there were abundant mature sperms distributed throughout the sperm duct and lobular lumen, while most of testes regressed in June, revealing that May is the peak reproductive period for male. Han. (2010) reported on the reproductive cycle ofin waters of the Korean south coast. The results show- ed that the testes were mature from April to June, and that May was the best reproductive period for male. In our study, we found that the maturation time of culturedwas approximately one month earlier thanin the Bohai Sea or the waters off the Korean south coast. this difference may be attributable to different envi- ronmental water temperatures, as we increase the water tem- perature of the tanks during November – April in this study.

4.3 Spermatogenic Cell Ultrastructural Characteristics

Two types of spermatogenesis (cystic and semi-cystic type) have been identified in teleosts (Mattei., 1993; An- drade., 2001). The cystic type of spermatogenesis oc- curs in most teleosts. In this type of spermatogenesis, the spermatogenic cells develop in the cysts during the whole spermatogenesis stage and the cysts open to release the ma- ture sperms into the lobular/tubular lumen after the com- pletion of spermatogenesis. In each cyst, the germ cells syn- chronously develop and are connected by cytoplasmic bri- dges. However, the development is not necessarily synchro- nous in different cysts (Andrade., 2001). In the semi- cystic type of spermatogenesis, the cysts open to release the germ cells into the lobular/tubular lumen before the end of spermatogenesis. This causes advanced break down of cyto- plasmic bridges and the release of germ cells in different stages into the lobular/tubular lumen, resulting in asynchro- nous spermatogenesis (Mattei., 1993). In the sperma- togenesis of, the germ cells displayed synchro- nous development in the same cyst until the completion of spermatogenesis. Only mature sperms were detected in the lobular lumen. Therefore, we can classify the spermatoge- nesis ofas cystic. Furthermore, we observed that the development of germ cells in the same cyst of the spermatocyte were not fully synchronous; some were at me- taphase I/II, but others had completed the division (Fig.6). A similar phenomenon was reported in, where the prophase and metaphase primary sperma- tocytes were observed in the same cyst, indicating sperma- togenesis may not be a precisely synchronized process (Tang., 2020).

The spermatogenesis ofincludes the sperma- togonium, spermatocyte, spermatid, and mature sperm. Two types of spermatogonia (primary/secondary or A/B sperma- togonia) were classified in, similar to most te- leosts, such as(You., 2001), tilapia species (Schulz., 2010),and(Camargo., 2017). It has been reported that part of primary sper- matogonia may act as stem cells to maintain the amount of spermatogonia, and the secondary spermatogonia usually differentiate into the primary (preleptotene) spermatocytes and finally generate the mature sperms (Guan., 1990; You., 2001, Schulz., 2010). In the testes of tila- pia, the type A spermatogonia can be further divided into undifferentiated (Aund) and differentiated (Adiff) type A sper- matogonia. While Aundspermatogonia show larger nuclear and cellular size, and are more frequently found close to the tunica albuginea, which results in high stemness capa- bility (Schulz., 2010). Two types ofspermatogonia have been distinguished in. The research found that the Aundspermatogonia were able to retain 5’-bromo- 2’-deoxyuridine (BrdU) over a long chase period, suggest- ing that these cells have a long cell cycle and there are po- tential stem cell candidates among them. Recently, the re- search concluded that Aundspermatogonia may be charac- terized as putative stem cells intestes (Ca- margo., 2017). In our study, although we have not de- tailed the primary spermatogonia into Aundand Adiffsper- matogonia, we found only a small number of spermatogo- nia located in the tunica albuginea of the testes at the end of first reproductive cycle, while the spermatogonia signi- ficantly proliferated and filled the whole testis in the re-de- velopment stage II testes. This indicated the similar stem cell role of the remaining spermatogonia (or a part of them), but the specific cell type or structure of spermatogonia need to be further investigated.

In the primary and secondary spermatogonia and primary spermatocytes of, a large number of electron- dense nuages are present in the cytoplasm. They either are adjacent to the nuclear envelope or adhere to the mitochon- dria. Nuages, a universal substance present in the early stages of germ cells, can gradually reduce as spermatoge- nesis progresses (Fishelson., 2006; Schulz., 2010; Fu., 2016). Current research on the composition and functions of this substance is mainly concentrated among model animals (Amikura., 2001; Chuma., 2009; Reunov and Reunova, 2016), such as,, and mice. In mice, the nuages function primarily in the postnatal germ cell differentiation in males, but not in the early embryonic stages. They are amorphous aggregated ribonucleoprotein and have many functions in RNA meta- bolism, retrotransposon regulation, and interplay with mito- chondria (Chuma., 2009). The functions of nuages in fish spermatogenesis has rarely been reported and needs further investigation.

4.4 Spermiogenesis and Mature Spermatozoa Features

Three types of spermiogenesis have been reported accord- ing to the presence/absence of nuclear rotation and the po- sition of the nucleus in relation to the flagellar axis (Qua- gio-Grassiotto., 2020). In a summary, in type I and type II spermiogenesis, the flagellar axis is parallel to the antero-posterior axis of the nucleus in the early stage, and the nucleus then rotates 90?, taking up an anterior medial position relative to the flagellar axis in type I spermioge- nesis, while the nuclear rotation does not occur in type II spermiogenesis. Thus, the flagellum is parallel to the antero- posterior axis of the nucleus in the mature sperm. In type III spermiogenesis, the flagellum axis is medial in relation to the nucleus and no nuclear rotation occurs during the whole spermiogenesis process (Quagio-Grassiotto., 2020).

Furthermore, spermatozoa can be classified into two types according to the differences of spermiogenesis. Type I and type III spermiogenesis result in formation of type I sperma- tozoa, and type II spermiogenesis results in formation of type II spermatozoa (Magalhaes., 2011). In our study, we found that the flagellum axis inwas paral-lel to the antero-posterior axis of the nucleus from the mid- dle spermatid to the mature sperm, and no nuclear rotation occurred during spermiogenesis. Consequently, the spermio- genesis ofcan be classified as type II, which re- sults in formations of type II spermatozoa. It is similar to that in most of the other Sciaenidae, such as(You., 2001),(Gusmao-Pompiani., 2005),(Gusmao-Pompiani., 2005), and(Miao., 2013). In Perciformes, type II spermio- genesis also seems to be dominant, as type II spermatozoa were found in 29 of the 41 families of Perciformes (Mat- tei, 1991). Hence, the spermiogenesis ofwas typical to Perciforms, with high similarity to Sciaenidae.

The ultrastructural characteristics of fish spermatozoon are useful data for fish phylogeny, as the morphology of fish spermatozoon is diverse but tends to be quite conserved within a given family or subfamily (Mattei, 1991; Quagio- Grassiotto., 2020). In order to learn the similarities be- tween the mature sperm ofand the sperm of related species, we compared the main ultrastructural cha- racteristics of mature sperm betweenand cer- tain other Perciformes (Table 2). The sperm ofexhibits several differences compared to the Sparidaes and Serranidaes, including the shape of the nucleus and head, direction of the flagellum, and the presence/absence of the lateral fin. Only a minor difference was observed between the sperm ofand the sperm of Thunnidae, which include the shape of the mitochondria (only in) and the presence or absence of a lateral fin (only in; Table 2). Surprisingly, almost no difference was observed betweenand other Sci- aenidae, barring the shape of the nucleus, such as theand(Table 2), indicating the high similarity of fish sperm morphology between closely relat- ed species of Sciaenidae. Many studies have reported that the morphology of fish sperm can provide the data for fish phylogeny (Mattei, 1991; Lahnsteiner and Patzner, 2008; Quagio-Grassiotto., 2020). It was identified in our study.

Table 2 Ultrastructural characteristics of L. polyactis and some other Perciforme species

Note: ‘–’ indicating absence of this data.

5 Conclusions

The male reproductive cycle and spermatogenesis ofwere studied with respect to anatomy, histology, and ultrastructure. A pair of unrestricted lobular testes were observed in, they were observed to attain ma- turity within 1 year. March to May was confirmed as the breeding season, with April as the ideal month for artificial breeding. The spermatogenesis ofdisplayed the cystic type of spermatogenesis. During spermiogenesis, no nuclear rotation occurred in the spermatid of, indicating the occurrence of type II spermiogenesis and the formation of type II spermatozoa. The spermiogenesis and sperm ultrastructure ofexhibit a high simila- rity to other members of the Sciaenidae, and also show certain similarity to other Perciformes. The results from the present study provide useful data for improving artificial breeding of the species.

Acknowledgements

We are thankful to all the members of the Fish Histology Laboratory at Ningbo University for valuable discussions. This study was supported by the NSFC-Zhejiang Joint Fund for the Integration of Industrialization and Informa- tization (No. U1809212), the Scientific and Technical Pro- ject of Zhejiang Province (Nos. 2021C02055, 2017C02013), the National Natural Science Foundation of China (No. 31272642), and Healthy Aquaculture, the K. C. Wong Mag- na Fund in Ningbo University and the Collaborative Inno- vation Center for Zhejiang Marine High-Efficiency.

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