HISTOLOGICAL STUDIES ON GONADAL DEVELOPMENT IN THE ENDEMIC TIBETAN FISH PTYCHOBARBUS DIPOGON

ZENG Ben-He， RAO Chang-Wei and LIU Hai-Ping

(1. Institute of Fisheries Science， Tibet Academy of Agricultural and Animal Husbandry Sciences， Lhasa 850002， China; 2. Pangduo Township， Linzhou County， Lhasa 850000， China)

Abstract: The histology of gonadal development in Ptychobarbus dipogon was studied by using conventional paraffin sections and HE staining， in order to obtain information on its reproductive systems useful for its artificial breeding. The results showed that the development of oocytes can be divided into five phases， and ovarian development can be divided into six stages. In stage V ovaries， ovum worship was found to occur， indicated by the 1.38∶1 ratio of small ova to large ova. Phase 3 oocytes were characterized by appearance of yolk granules and follicles; phase 4 oocytes by rapid increase of yolk granules， and the movement of the nucleus toward the animal poles; phase 5 by the fusion of yolk granules， and the separation of oocytes from follicular membranes to float in the ovarian cavity. Male P. dipogen had lobular testes， and their germ cells can be categorized into spermatogonia， spermatocytes; spermatids; and spermatozoa. Their testes also developed in six stages. The species followed a batch-synchronous spawning pattern.

Key words: Ptychobarbus dipogon; Gonadal development; Histological structure; Spawning pattern

Ptychobarbus dipogonis a species of the genusPtychobarbus， subfamily Schizothoracinae， family Cyprinidae， order Cypriniformes. It is a benthic cold water fish of the Tibetan Plateau， distribute only in the middle trunk and branches of the Yarlung Zangbo River， where it is usually found in clear waters with gravel bottoms and slow water flows[1]. Muscles of the species were rich in polyunsaturated fatty acids[2]，giving it some potential as a food source. Following recent economic growth in Tibet Autonomous Region， factors like overfishing and invasive species have sharply reduced theP. dipogonpopulation in the Yarlung Zangbo River， particularly the number of large individuals. In 2016， the China Species Red List includedP. dipogonas a vertebrate under threat[3].Preservation and sustainable exploitation of resources were an important part of China’s national strategy to development its western regions， and the need to studyP. dipogonis urgent. Extant studies on the species have been surveys of natural resources[4]， and research on its growth and feeding habits[5]， muscle quality[3]， and phylogeny[6]. No report has been made on histology of its gonadal development. Tang，et al.[7]analyzed tissues ofGymmocypris przewalskiito find its gonadal development can be categorized into six periods， with five developmental phases for germ cells in both testes and ovaries. In this study， we investigated the gonadal development ofP. dipogonvia tissue microanalysis， in hope to obtain information useful for further biological research， preservation，and proliferation of the species， and contribute to histological knowledge about gonadal development in highland fishes.

1 Materials and Methods

1.1 Experimental material

From February to March in 2013， at the Xaitongmoin segment (area A1 in Fig. 1) of the Yarlung Zangbo trunk in Xigaze region (about 4000 m in elevation)， we collected 50 females (body length 36.8—55.6 cm， weight 148.4—1704.6 g)， and 65 males(body length 18.6 —39.4 cm， weight 52.4—1208.3 g)using fishing nets (5 m-long， 1 m-tall trammels with 3 cm meshes) and electrofishing (direct current). Specimens were delivered by live fish transport vehicles to the Tibetan Fish Nurturing Station at School of Agriculture and Animal Husbandry， Tibet University for analysis.

1.2 Histological study

Each specimen was dissected to extract its gonads. The gonads were weighed， cut into pieces about 0.5 cm3in size， fixed in Bouin’s solution， and transferred to 70% ethanol solution after 24h. Later， the pieces were dehydrated， decolored， and waxed in a dehydrator， and wrapped using a paraffin embedding machine. An Elex slicer was used to make tissue sections at 5 μm thickness. Slices were HE stained and packed in Canadian gum. Lastly， they were observed and photographed using an Olympus microscope.

1.3 Statistical analysis

The reported staging methods were used as references for identifying the gonadal stages and germ cell phases ofP. dipogon[7—9]. Diameters of germ cells and their nuclei were measured by Motic 2.0 software. Test data were first tested by one-way ANOVA in SPSS 19.0， and analyzed by Duncan’s multiple range test if differences were significant. Diameters of male germ cells were processed in SPSS 19.0 using two-way ANOVA， General Linear Model-Univariate，with significance level set to 0.05. The test results were expressed as “mean ± standard deviation”.

2 Results and analysis

2.1 Morphology, staging and characteristics of ovaries and female germ cells

Through observation， 50 female specimens were determined to be 3 in stage Ⅰ (body length 36.8—42.6 cm)， 8 in Ⅱ (37.2—45.3 cm)， 14 in Ⅲ (41.6—54.3 cm)， 20 in Ⅳ (46.2—55.1 cm)， 3 in Ⅴ (45.6—55.6 cm)， and 2 in Ⅵ (45.8—49.2 cm).

Morphology of female germ cell phasesPhase 1 denotes the earliest phase of oocyte development， during which the cell was still an oogonium， or was in transition from oogonium into primary oocyte.In this phase， the protoplasm in the cell was starting to increase， and the nucleus was highly visible. 5 to 19 nucleoli can be seen in the same section of the nucleus. A phase 1 cell had a diameter from 85 to 148 μm， which was roughly half of normal cells， and a nucleus diameter from 35 to 110 μm (see Plate Ⅰ-1 and 2).

Phase 2 denotes the growth and meiosis of the primary oocyte. The cell appears round with an irregular angular surface early in phase 2， and became round or oval-shaped later. In the early period， Increasing of protoplasm caused the cell to grow in size significantly， and its nucleus also expands. Lamellar structures， or ovarian boards where ova were produced， begin to form in ovaries (Plate Ⅰ-4). In the late period， the oocyte continued to expand， while 3 to 16 nucleoli can be seen in the same nucleus section. A phase 2 oocyte has a diameter from 101 to 142 μm，and a nucleus diameter from 39 to 79 μm (PlateⅠ-3 and 4).

Fig. 1 Sampling area of P. dipogon

Phase 3 denotes the growth and meiosis of the secondary oocyte. Phase 3 oocytes were larger than phase 2 oocytes. In such a cell， nucleoli were seen scattered in the nucleus， with a tendency to be closer to the center than before， and their number increased dramatically from 12 to 26 (Plate Ⅰ-5 and 7). Yolk granules begin to appear in cytoplasm around the nucleus， and spread out from there (Plate Ⅰ-6). Follicles begin to appear and increase in the cortical layer(Plate Ⅰ-5). A phase 3 oocyte had grown significantly，and can be as large as 148—341 μm， with a nucleus diameter of 55—173 μm.

Phase 4 denotes the growth of the ovum. The cell continues to expand in volume， and sees rapid accumulation of yolk， as vacuoles were pushed to peripheral cytoplasm of the cell (Plate Ⅰ-7). The zona radiata outside its cellular membrane had thickened， becoming highly visible. Late in phase 4， yolk granules had filled up the space outside the nucleus， and cytoplasm becomes only noticeable around the ootid membrane and the nucleus. Both cytoplasm and nucleus were moving toward the animal pole， until they arrived in the protoplasm plate of the animal pole， directly below the micropyle (Plate Ⅰ-8). A phase 4 cell had a diameter from 202 to 351 μm， with a nucleus diameter from 61 to 210 μm.

Phase 5 denotes the maturation of the ovum. Its cytoplasm filled with large yolk granules， the ovum had separated from the follicular membrane and floats freely in the ovarian cavity (Plate Ⅰ-9). A phase 5 ovum had a diameter from 251 to 418 μm， with a nucleus diameter from 63 to 217 μm.

Staging of ovarian developmentStage Ⅰovaries appeared as translucent wires on both sides of the abdomen close to the swim bladder， indistinguishable from testes to naked eyes. They contained predominantly oogonia and a minority of phase 1 oocytes.This was the stage in which oogonia transform into oocytes (Plate Ⅰ-1 and 2).

Stage Ⅱ ovaries appeared as flattened， semitranslucent ribbons， with a slight yellow hue， distinct from testes in naked eyes. In individuals undergoing a second sex maturation， ovaries appeared spongier and larger， with more and thicker-appearing blood vessels.They contain predominantly phase 1 and 2 oocytes，and fewer oogonia than stage Ⅰ. Cytoplasmic vacuolization begins to occur throughout the ovary (PlateⅠ-3 and 4).

Stage Ⅲ ovaries had expanded. Filled with thick vessels， each ovary was cylindrical in the middle， and appearing as thick， flattened stripes in both ends， light yellow in color， visually distinct from testes. Germ cells begin to show accumulation of yolk. Ovaries contain predominantly phase 3 oocytes， with some phase 1 and 2 oocytes. Phase 3 oocytes accounted for 34.13%—58.42% in number of cells in a section， and 61.22%—85.63% in total area of a section (Plate Ⅰ-5 and 6).

Stage Ⅳ ovaries continued to swell， taking up about a third of the body cavity. Eggs displayed significant yolk deposition， and appeared round and orange-colored. Ovaries contain predominantly phase 4 ova and small numbers of phase 2 and 3 oocytes.Phase 4 cells accounted for 51.33%—68.44% in cell number， 76.85%—91.66% in total area (Plate Ⅰ-7 and 8).

Stage Ⅴ ovaries continued to swell， taking up a half to a third of the body cavity. They appeared spongy; eggs will leak out from the cloaca if the fish was lifted by its head， or the abdomen was lightly suppressed. Eggs were round and translucent， floating freely in the ovarian cavity. Ovaries contain predominantly phase 5 ova and small numbers of phase 2， 3 and 4 cells. Phase 5 cells accounted for 62.48%—76.25% in cell number， 89.29%—94.37% in total area of the section (Plate Ⅰ-9).

Stage Ⅵ ovaries had shrunk and loosened after spawning mature eggs. Suffused by blood， they were deep red in color， and contain mostly small white eggs， with only small amounts of mature eggs. Cellular sections showed that in addition to the small eggs，and a few unspawned mature eggs， they also contained empty follicles， large amounts of phase 2 and 3 oocytes， and small amounts of phase 1 cells. These cells were irregular in shape and size， generally round or spindle-shaped (Plate Ⅰ-10 and 11).

Changes in diameter, nucleus diameter, and nucleoli of germ cells in ovarian development of P.dipogonAs seen in Tab. 1， the diameter， nucleus diame ter， and number of nucleoli of germ cells display a trend of increase from stage Ⅰ to Ⅴ (P＜0.05)， and a trend of decrease from stage Ⅴ to Ⅵ (P＜0.05).

Tab. 1 Diameter and number of nucleoli of germ cells in ovarian developmental stages of P. dipogon ovaries (N=30)

Number of germ cells and comparison of small and large eggs in ovarian development of P. dipogonAs seen in Tab. 2 and 3， the number of germ cells show significant increase before stage III(P＜0.05)， and significant decrease after stage III(P＜0.05). In sexually mature specimens， the number ratio of small to large eggs was 1.38∶1， and the weight ratio was 0.21∶1 (PlateⅠ-18).

2.2 Morphology, staging and characteristics of testes and male germ cells

Through observation， 65 male specimens were determined to be 2 in stage Ⅰ (body length 18.6—19.8 cm)， 10 in Ⅱ (22.4—31.7 cm)， 18 in Ⅲ (28.6—36.5 cm)， 14 in Ⅳ (29.7—39.2 cm)， 10 in Ⅴ (31.2—39.4 cm)， and 11 in Ⅵ (30.6—38.5 cm).

Morphology of male germ cell phasesA spermatogonium was produced from a primary germ cell. It was the larger of the phases， with a diameter of 13—23 μm， and a 9—16 μm nucleus located in the middle， round or fusiform in shape. Weakly alkaline，spermatogonia and their nuclei were both stained purple by H&E， with the nuclei having a deeper hue(Plate Ⅰ-12).

Primary spermatocytes were developed from spermatogonia. They were smaller than spermatogonia at 11—15 μm， with deeply stained nuclei at 7—10 μm， round or oval-shaped without obviously visible nucleolus (Plate Ⅰ-13).

Secondary spermatocytes were produced by the meiosis of primary spermatocytes. They were 7—11 μm in diameter， with 3—6 μm nuclei， round in shape(Plate Ⅰ-14).

Spermatids were produced by the meiosis of secondary spermatocytes. They were even smaller at 4—5 μm， round or square in shape， with a deeper stained color. Cytoplasm was not significantly visible in them (Plate Ⅰ-15).

Spermatozoa were morphed from spermatids，and the smallest cells in testes， with a head 2 to 3 μm in diameter， and had very clear boundaries (Plate Ⅰ-16).

Staging of testicular developmentStage Ⅰtestes were translucent and wire-like， indistinguishable from ovaries by naked eyes. Two thirds of testes were spermatogonia， accompanied by only small numbers of primary spermatocytes.

Stage Ⅱ testes were thin ribbons， pale and semitranslucent. Their spermatogonia had decreased， and primary spermatocytes had increased to over 70%(Plate Ⅰ-13).

Stage Ⅲ testes were pale gray ribbons. Their stained color had become deeper. Their content was predominantly primary and secondary spermatocytes，with small amounts of spermatogonia， and some spermatids (Plate Ⅰ-14).

Stage Ⅳ testes were marble-colored， with a round or oval-shaped section， and more vessels on the surface. Their content were predominantly spermatids and spermatozoa. At the end of stage Ⅳ， some seminal capsules begin to break， releasing spermatozoa to fill up the lobule cavity (Plate Ⅰ-15).

Stage Ⅴ testes were marble-colored， soft and inflated. Sperms will leak out from the cloaca if the fish was lifted by its head， or the abdomen was lightly suppressed. In sections， the tissues were stained a deep purple. Each lobule cavity had notably expanded， filled with spermatozoa (Plate Ⅰ-16).

Stage Ⅵ testes had greatly shrunk， appeared to be pale red， and were suffused by blood. When the abdomen was lightly suppressed， only translucent mucous will leak out， rather than white semen. Testes were stained light blue in sections. Walls of testes had thickened， while seminal lobules and capsules had decreased in size. The inside of testes appeared irregular and empty， with some remaining spermatozoa and proliferating spermatogonia (Plate Ⅰ-17).

Tab. 2 Number of germ cells in ovarian developmental stages of P. dipogon (100 ind.， N=30)

Tab. 3 Comparison of small and large eggs in sexually mature P. dipogon

Changes in diameterof germ cells in testicular development of P. dipogonAs shown in Tab. 4，from stage Ⅰ to Ⅵ， the diameter of germ cells decreased before stabilizing (P＜ 0.05); the nucleus diameter showed a trend of gradual decrease from primary spermatocytes to spermatozoa. There was no significant interaction between influences on germ cell diameters from developmental stages and cell types (P＞0.05).

3 Discussion

3.1 Characteristics of yolk substances and nucleoli in female germ cells of P. dipogon

Three types of yolk were found during the maturation of fish oocytes: cortical vacuoles (containing carbohydrate yolk)， protein yolk granules， and lipid yolk drops[8]. In this study， the yolk ofP. dipogenwas found to comprise the former two types， with no lipid yolk drop. Cortical vacuoles occur earlier than protein yolk granules， allowing us to conclude this was the order in which the species accumulate yolk substances. Said order was different from what Cui’s[9]found aboutScatophagus argus. Protein granules inPtychobarbus dipogonoocytes start appearing in phase 3， and increases in number in later phases，which was similar to species likeScatophagus argus[9]，Coilia nasus[10]，Sparus macrocephalus[11]， andIlisha elongata[12]. However， in Gong’s[13]study of the ovarian cycle ofStromateoides argenteus， the species’ yolk granules were reported to appear in middle to late phase 2.

Studies had been done on nuclei in the oocytes of osteichthyes[7，14]. ForP. dipogenoocytes， nucleoli substances were built up during phase 1， forming several to dozens of large， round nucleoli on the inner surface of the nuclear membrane. These nucleoli were stained a deep color by alkaline dye. In middle to late phase 2， said nucleoli substances start being moved into the cytoplasm surrounding the nucleus， at a point later than the appearance of yolk granules. Gong，et al.[15]believed that the nucleolus effluents entering the cytoplasm may be predecessors to those yolk substances around the nucleus， or related to the formation of yolk substances. Tang，et al.[7]also found the two to be related in their research onGymmocypris przewalskii. Through observing sections， we also conclude the nucleolus effluents were related to yolk substances， consistent with the above findings.

Tab. 4 Diameter of germ cells by type in testicular developmental stages of P. dipogon (μm)

3.2 Spawning pattern of P. dipogon

Based on how oocytes develop， the ovarian development of most osteichthyes can be categorized as synchronous， group-synchronous， or asynchronous[16，17].Their spawning patterns can also be classified into three types: complete synchronous， batch-synchronous， and batch-asynchronous. In this study， stage V ovarian sections were found to contain some germ cells in phases 2， 3 and 4 in addition to the predominant phase 5， respectively accounting for 2.94%，5.38%， 21.83% and 69.85% of germ cells. The postspawning sections contained predominantly phase 2 and 3 oocytes， with small numbers of phase 1 cells，but no phase 4 cells. It was impossible for the phase 2 and 3 cells to mature or be spawned in the same year.This indicatesP. dipogento mature once per year， but spawn its mature eggs intermittently over several spawnings， rather than all at once， making it a batchsynchronous spawning species， like most Cyprinids[18—25].This may also be attributed to asynchronous growth of some ova due to the insufficient nutrition supply in highland waters; some asynchronously growing phase 6 ova may also be absorbed by the body as it builds new phase 1 and 2 oocytes.

3.3 Histological structure of P. dipogon testis

Many reports had been made on testicular structure and spermatozoa of osteichthyes[9，26]， butP. dipogenhas yet to be covered. This study shows that during its spermatogenesis， the male germ cells ofP.dipogenhad undergone three transformations: spermatogonia to spermatocytes， to spermatids， to spermatozoa， similar to other osteichthyes[19，27]. Based on how spermatogenic cells were distributed within， the testes of osteichthyes were generally divided into two types， lobular and tubular[28，29]. In a lobular testis，spermatogonia can be present in all parts of a lobule，while in a tubular testis， spermatogonia were only found in the front end of a seminiferous tubule. Like most Cyprinids[16，27，30—32]，P. dipogenhas lobular testes.

3.4 Differentiation of germ cells and ovum worship in P. dipogon

Sperms in most fish species were developed through the spermatogonia， primary spermatocytes，secondary spermatocytes， spermatids， and spermatozoa phases[18，19]. Spermatogonia concur with the first period of gonadal development， which was a proliferation stage in which stem cells generate spermatogonia through mitosis[33]. Spermatogonia undergo synapsis and form primary spermatocytes， which split into secondary spermatocytes through meiosis Ⅰ. Secondary spermatocytes become spermatocytes via meiosis Ⅱ， which morph into spermatozoa[34]. In this study， over six stages of testicular sections， diameters of germ cells show a trend of decrease before stabilization， consistent with most fish species (Tab. 5). This should be due to the decrease of cell matter over a series of mitosis and meiosis processes.

Tab. 5 Germ cell diameters of four Cyprinids (μm)

Tab. 6 Variations of cell and nucleus diameter in ovarian germ cells of seven Cyprinids (μm)

Oogenesis in fish usually consists of the ootogonia， primary oocytes， secondary oocytes， and ova stages[7，12，14]. Ootogonia were proliferated by the mitosis of reproductive stem cells[33]， then undergo synapsis to form primary oocytes. A primary oocyte splits into a polar body and a secondary oocyte in meiosis Ⅰ， which split into three polar bodies and an ovum in meiosis Ⅱ[34]. In this study， the number of germ cells was found to be the highest in stage Ⅲovaries， at 2.8 times that of stage Ⅱ， which indicates both meiosis processes had been completed before stage Ⅲ. Throughout the ovarian development， some polar bodies were absorbed， while the unabsorbed polar bodies morph into small eggs that cannot be fertilized， which was called ovum worship. In this study，ovarian sections show the diameters of both germ cells and their nuclei to increase from phase 1 to 5，consistent with most fish species (see Tab. 6). The ovum has enough energy， organelles， enzymes， nucleic acids， and other instruments to prepare for synthesizing yolk proteins. The ovum then enters the period of yolk formation， during which it also accumulates RNAs， glycogen particles， ribosomes， lipids and mitochondria， laying the ground for development after fertillization[34]. The ovum will continue to expand during this process.

4 Summary

ForP. dipogon， development of male germ cells occurs through the spermatogonia， primary spermatocytes， secondary spermatocytes， spermatids， and spermatozoa phases， and development of female germ cells occurs through the ootogonia， primary oocytes，secondary oocytes， and ova phases. Its ovarian and testicular development can both be divided into six stages， and both organs mature in stage Ⅴ. Through the gonadal development， male germ cells gradually become smaller， and female germ cells become larger. Males of the species had lobular testes. Females exhibit ovum worship in ootogenesis， and follow a batch-synchronous spawning pattern. This study should contribute to understanding ofP. dipogenreproduction， and provide theoretical knowledge for the artificial breeding of the species.