Ultrastructural Evidence for Dual Sperm Morphotypes in Hormone-Induced Japanese Eel (Anguilla japonica): Implications for Sperm Maturation

TL;DR

Japanese eel sperm can have two different shapes, which may help improve artificial breeding techniques.

Contribution

The discovery of two distinct sperm morphotypes in Japanese eel provides new insights into sperm maturation and reproduction.

Findings

Two sperm types were identified: round and eyebrow-shaped, with distinct nuclear and axonemal structures.

Round sperm had a '9 + 2' axoneme pattern, while eyebrow-shaped sperm had a '9 + 0' pattern.

The findings suggest morphological diversity in eel sperm, relevant to reproductive biology and artificial breeding.

Abstract

Two distinct morphological structures of Japanese eel sperm were observed by means of light microscopy and electron microscopes. The cell nucleus of one type of sperm was round, the type of sperm was smaller in size, and the axoneme displayed a typical “9 + 2” pattern. Another type was the eyebrow-shaped sperm, characterized by an elongated head and an axoneme with a “9 + 0” microtubule pattern. These findings demonstrated morphological diversity in Japanese eel sperm, providing fundamental data and scientific support for further research in sperm biology and artificial reproduction.

The microstructure and ultrastructure of the sperm of Japanese eel, Anguilla japonica, artificially induced with weekly injections with carp pituitary (CP) and human chorionic gonadotropin (HCG), was studied, and milt from 10 out of 20 mature fish was collected. Two distinct morphological structures of A. japonica sperm had been observed with optical microscopy. The cell nucleus of one type of sperm was round or nearly round, the sperm was smaller in size, with 2.57 ± 0.62 μm of the long diameter of the cell nucleus, 2.11 ± 0.59 μm of the short diameter, and 37.35 ± 7.71 μm of the flagellum length. Another type was the eyebrow-shaped sperm, the sperm was relatively larger in size, with 7.66 ± 1.09 μm of the long axis, 2.54 ± 0.46 μm of the short axis, and 38.26 ± 9.02 μm of the flagellum length. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the anterior end of the round sperm lacked an acrosomal structure. The implantation fossa was located in the center of the base of the sperm head, and it was in the shape of a channel along the long axis of sperm. The centriolar complex was situated within the implantation fossa. There were 2–3 mitochondria distributed at the basal end of the nucleus. Sperm flagellum prolongated from the sleeve cavity, and the initial part of axoneme connected to the distal end of basal body. The axoneme displayed a typical “9 + 2” pattern. There was a spherical structure in the curving area for eyebrow-shaped sperm, and the axoneme formed a “9 + 0” pattern. The discovery of two different types of sperm in the Japanese eel has provided new perspectives for research on its artificial reproduction.

Figures

Full text

1. Introduction

The study of the morphology and ultrastructure of fish sperm can provide important information, not only for the evaluation of sperm quality to optimize artificial reproduction, but to guide the understanding of taxonomic classifications, including relationships at the family, subfamily, and species levels, and to help establish phylogenetic relationships among fish species [1,2]. In the teleost species studied to date, diversity in sperm structure has been observed according to whether the species adopted internal or external fertilization. In particular, this diversity was reflected in the shape of the sperm head, the number, form, and location of mitochondria, and the length and structure of the flagella [3]. However, the “dual morphotypes” sperm structure has not been found in other fish species. Ultrastructural studies of sperm of the order Anguilliformes have been made on some species to date. These have clearly revealed the unique ultrastructures of these sperm [4,5,6], including their eyebrow-shaped nucleus [7,8], rootlet attached to the neck region [9,10], flagellum pattern [11,12], and pseudoflagellum extending from the proximal centriole [13,14]. However, some structural aspects of the flagellum and mitochondria, such as their location and number, have remained less clear. As a fish with a unique and mysterious life history, whether the Japanese eel exhibit diversity in sperm morphology, could such diversity consequently influence fertilization? This could represent a critical aspect worthy of investigation in the current research on the artificial propagation of eels.

The Japanese eel, A. japonica, is a catadromous fish species between rivers and the sea. Every autumn and winter, its breeding populations migrate from rivers to the sea for reproduction. Their gonads mature during the migration and reproduce once they reach the spawning grounds. However, the eels mysteriously disappear after entering the ocean [15,16]. Therefore, the natural maturation of A. japonica sperm in their natural spawning grounds has not yet been achieved. Furthermore, A. japonica is the only fish species that requires hormone induction for sexual maturation, so the artificial breeding of eels remains a worldwide challenge. The present paper systematically examined the microscopic and ultrastructural features of A. japonica sperm using optical microscopy, SEM, and TEM. For the first time, sperm with a typical “9 + 2” axoneme structure have been obtained from Japanese eel in this study. This study can enrich the fundamental knowledge of eel reproductive biology and could provide a reference for further research on eel sperm activity and artificial breeding.

2. Materials and Methods

2.1. Collection of Fish Milt

Twenty wild males of Anguilla japonica (388–505 g) were captured in the Pearl River Estuary, China. After acclimatization for 1 week at salinity 15–20, the eels were kept in a water tank with a salinity of 32 at 20 °C. During the induction of maturation by hormonal treatment, all 20 male eels released sperm after being given weekly injections with 0.5 mg carp pituitary (CP) and human chorionic gonadotropin (HCG) at a dose of 50 IU/eel/week according to Liu et al. [17], and milt was collected from 10 of these fish. The milt was obtained by applying pressure on the abdomen after the eels matured, and sperm with over 90% motility was used as experimental material; sperm motility was defined as the percentage of motile sperm among the total sperm count in the field of view.

2.2. Sample Preparation for Optical Microscope Observation

A small amount of milt was taken to prepare sperm smears, with five smears made for each fish. The smears were stained with a combined Giemsa and Wright stain, then observed and photographed under a NIKON TE2000 inverted microscope (Nikon Corporation, Tokyo Metropolis, Japan), with measurements taken of the sperm size. The experiment was repeated three times, with 100 sperm cells observed per individual.

2.3. Sample Preparation for Scanning Electron Microscopy (SEM)

Milt samples of 5 mL were fixed for 3 h in fresh 2.5% glutaraldehyde in 0.1 M phosphate buffer solution (pH 7.4) at 0~4 °C, rinsed in the same buffer, and then immersed for 2 h in 1.0% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) at 20 °C. After rinsing in the buffer solution, sperm were dehydrated in a graded ethanol series and critical point dried. Samples were examined and photographed using a JEOL-6380LV scanning electron microscope (SEM) (JEOL Ltd, Akishima, Japan).

2.4. Sample Preparation for Transmission Electron Microscopy (TEM)

A total of 5 mL milt was collected from each individual and fixed with an equal volume of 2.5% glutaraldehyde (GA). The fixed milt was centrifuged at 3000 r/min for 5 min. After discarding the supernatant, an equal volume of 2.5% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.4) was added again. After being rinsed three times with PBS, the samples were fixed with 2% osmium tetroxide in phosphate buffer (pH 7.4) for 1 h. These were then dehydrated through a graded ethanol series and embedded in Epon812 resin (Shell Chemical Company of the United States, New York, NY, USA). Ultrathin sections (70~80 nm) were cut using an ULTRACUT ultramicrotome (Leica Microsystems, Vienna, Austria). The specimens were stained with uranyl acetate and lead citrate, and observed with a TEM (HITACHI H-600, Hitachi Ltd., Tokyo, Japan).

2.5. Statistical Analyses

Experimental data were analyzed using Excel software for graphical representation and SPSS 22.0 for statistical analysis. All data were presented as mean ± standard deviation (SD). Prior to conducting one-way ANOVA, all data were subjected to a one sample t-test, as well as normality and homogeneity tests. A 95% confidence interval was adopted as the criterion for significant differences. Statistical significance was defined as p < 0.05 for the test.

3. Results

3.1. The Microstructure of Sperm

Two morphological types of sperm were observed in Japanese eel under optical microscopy (Figure 1). One type featured sperm with round or nearly round nuclear. These sperm were relatively smaller, with measurements from 100 round sperm showing nuclear with a long diameter of (2.57 ± 0.62 μm), short diameter of (2.11 ± 0.59 μm), and flagellum length of (37.35 ± 7.71 μm). The round spermatic nuclear exhibited lighter staining, with darker staining at the junction between the nucleus and the flagellum (Figure 1b). Another type of sperm featured an “eyebrow-shaped” nucleus. These sperm were relatively larger, with the long axis of 100 nuclear measuring (7.66 ± 1.09 μm), the short axis (2.54 ± 0.46 μm), and the flagellum length (38.26 ± 9.02 μm). The eyebrow-shaped sperm nuclear showed darker staining (Figure 1c).

A morphological comparison of the two types of sperm is shown in Table 1. The long axis of the nucleus in “eyebrow-shaped” sperm was significantly greater than that in round sperm (p < 0.05), while there were no significant differences in the short diameter of the nucleus or the flagellum length between the two types of sperm (p > 0.05).

3.2. Scanning Electron Microscopy Observations of Sperm

Under scanning electron microscopy, two types of sperm were observed: round sperm and eyebrow-shaped sperm (Figure 2). The round sperm consisted of a head and a flagellar tail. The head contained a round nucleus, the flagellum was slender without lateral fins, and some mitochondria were distributed at the junction of the head and tail. The “eyebrow-shaped” sperm consisted of a head and a flagellar, and the head contained some spherical mitochondria distributed at the anterior end of the nucleus. The flagellum was slender and lacked lateral fins.

3.3. Transmission Electron Microscope Observations of Sperm

3.3.1. Round Sperm

Sperm consisted of two parts: the head and the flagellum (Figure 3a and Figure 4a). Structures such as the nucleus, centriole complex, mitochondria, and the sleeve could be observed in the head. The tail was structurally simple and elongated, composed of an axoneme.

3.3.2. Head Structure

The apex of the sperm head lacked an acrosome. The centriolar complex was located in the concave implantation fossa at the base of the nucleus, while the mitochondria and sleeve were situated at the lower end of the nucleus (Figure 3c,d,g and Figure 4c,d,g).

3.3.3. Nucleus

From the sagittal section of the sperm head, the sperm nucleus appeared circular or nearly circular. The nucleus was large and occupied almost the entire space of the head. The posterior end of the nucleus exhibited a deep medial depression, forming a groove-like structure known as the implantation fossa, which housed the centriolar complex (Figure 3c,d and Figure 4c,d). The position of the implantation fossa, where the basal body and the initial segment of the axoneme were located, was essentially parallel to the posterior end of the nucleus (Figure 3e and Figure 4e). In the transverse section of the sperm head, the nucleus was approximately elliptical, with a central cavity corresponding to the implantation fossa. This cavity contained the centriolar complex (Figure 3b and Figure 4b).

3.3.4. Centriolar Complex

The centriolar complex of sperm was located in the nuclear implantation fossa, comprising the proximal centriole and the basal body. The proximal centriole resided in the upper segment of the grooved implantation fossa, appearing as a ring-like structure composed of nine electron-dense patches where triplet microtubules were not clearly visible (Figure 3c,d and Figure 4c,d). The basal body was situated in the lower segment of the implantation fossa, parallel to the base of the nucleus (Figure 3d,e and Figure 4d,e). The proximal end (i.e., the end near the centriole) of the basal body exhibited a radial structure, while the distal end connected to the axoneme (Figure 3g and Figure 4g). In cross-sections at different levels of the basal body, the proximal end displayed a cylindrical ring structure formed of electron-dense material, where triplet microtubules were indistinct, and only nine electron-dense masses were visible. In cross-sections of the distal end, the nine ring-like electron-dense masses appeared split, revealing the inner and outer ring structures formed at the proximal and distal ends of the basal body (Figure 3e,f and Figure 4e,f). The proximal centriole and the basal body corresponded to each other (Figure 3d and Figure 4d).

3.3.5. Sleeve

The sleeve was connected to the posterior end of the nucleus, forming a cylindrical structure. The central cavity, known as the sleeve cavity, exhibited a relatively large annular space. The initial segment of the axoneme was laid within the sleeve cavity. The sleeve contained mitochondria, which were arranged in a ring-shaped pattern, numbering approximately two to three (Figure 3g,i and Figure 4g,j).

3.3.6. Sperm Tail Structure

The sperm tail featured an elongated flagellum. The proximal end of the tail was situated within the sleeve cavity, from which it extended. The basal end of the axoneme connected to the basal body, located in the lower segment of the implantation fossa. The cytoplasmic membrane of the flagellum was continuous with the sleeve membrane (Figure 3a,h and Figure 4a,i,k). The core structure of the flagellum was the axoneme. Between the cytoplasmic membrane and the axoneme, uniformly distributed small cytoplasmic granules were observed (Figure 3i and Figure 4k). Cross-sectional views of the axoneme revealed an asymmetry in the plasma membrane on either side—one side longer than the other. The axoneme exhibited the typical “9 + 2” microtubule arrangement (Figure 3j,k and Figure 4l).

3.3.7. Eyebrow-Shaped Sperm

Under electron microscopy, it could be observed that the apex of the sperm head lacked an acrosome. In longitudinal sections of the head, the nucleus was curved with a large, spherical globule at the curvature. Vesicles were present on the nucleus (Figure 3l and Figure 4m). Some globules contained the centriole complex and mitochondria, which had not yet separated from the globule (Figure 3m,n and Figure 4n,o). In transverse sections of the sperm head, the nucleus and the globule were enclosed by a cytoplasmic membrane, which integrated them into a single unit (Figure 3m and Figure 4n). The sperm lacked implantation fossa, as well as independent structures such as the centriole complex and mitochondria. The axoneme of the sperm flagellum exhibited a “9 + 0” structure (Figure 3o and Figure 4l).

4. Discussion

In this study, two morphological types of sperm were observed in the Japanese eel, one with a round or nearly round head, and the other with an eyebrow-shaped head. This “eyebrow-shaped” sperm differed significantly from round sperm in both microscopic and ultrastructural features. Specifically, it was characterized by an elongated head nucleus, the absence of implantation fossa, independent centriolar complexes, and mitochondrial structures, as well as the presence of a spherical body at the curved nuclear end of the sperm head. Okamura [18] reported that the sperm head of the Japanese eel exhibited an “eyebrow-shaped” structure, with a spherical body at the curved upper end of the sperm head identified as the mitochondrion. This finding differed from the present observations. Within the spherules we observed, there were not only mitochondria but also centriole complexes. Neither the mitochondria nor the centriole complexes formed independent structures. Many scholars had reported that the morphology of eel sperm had an “eyebrow-shaped” structure [19,20,21,22], and they characterized this form as the mature sperm of eels [23,24]. In this study, the round sperm of Japanese eel consisted of the nucleus, centriolar complex, mitochondria, sleeve cavity, and “9 + 2” pattern flagellum, which was consistent with the structural features of sperm in most teleost fish [25,26,27,28]. This form of eel sperm had not been reported before. In contrast, the flagellum of eyebrow-shaped sperm of the Japanese eel exhibited a “9 + 0” microtubule pattern, which had also been reported in other fish species [10,18,29]. The round-head-type sperm could be a morphoabnormality or an immature form whose presence in the milt could be the product of the hormonal stimulation. These findings further confirmed the uniqueness of the species for Japanese eel, though the relationship between sperm morphological variation and fertilization/hatching remains to be further investigated.

Generally, the sperm of teleost fish was divided into three parts: the head, the midpiece, and the tail. The main structure of the head was the nucleus; the midpiece was closely attached to the head and was primarily composed of structures, such as the centriolar complex and mitochondria; and the main structure of the tail was the flagellum, which consisted of an axoneme. Unlike most teleost fish, the centriolar complex of sperm in the Japanese eel was located within the implantation fossa, which was a concave depression at the lower end of the nucleus. Based on the morphological and structural characteristics of the Japanese eel sperm, the author suggested that the nucleus, centriolar complex, mitochondria, and sleeve could be collectively referred to as the head of the sperm. This means that the sperm of the Japanese eel could be divided into just the head and tail. The structural characteristics of the sperm head of the Japanese eel closely resembled to those of Larimichthys crocea [25]. The head of the sperm with an eyebrow-shaped contained not only the nucleus but also a spherical structure, and both were surrounded by a cytoplasmic membrane forming a single entity. Therefore, the “eyebrow-shaped” sperm were also divided into two parts: the head and the tail.

The flagellum of round sperm in Japanese eel had a typical “9 + 2” microtubule structure, which was consistent with that observed in most teleost sperm [25,26,27,28,29]. The doublet microtubules were clearly visible at the proximal end of the sperm axoneme, with central microtubules present in the center. This structural characteristic differed from the sperm of L. crocea [25], reflecting the diversity in sperm morphology among teleost species. Wang et al. [30] studied the ultrastructure of low-motility sperm in males and found abnormalities in the number of sperm microtubules, as the axoneme of the sperm tail exhibited a “9 + 0” structure with missing central microtubules. They suggested that microtubule abnormalities might affect or reduce sperm motility, potentially leading to male infertility. Woolley et al. [31] found that the axoneme of eel sperm with a “9 + 0” pattern only possessed inner dynein arms and lacked outer dynein arms. The authors proposed that this axonemal structure may represent an alternative morphological form during sperm maturation, though the underlying reasons for this phenomenon required further investigation.

The implantation fossa of round sperm had a deep groove formed by the inward concavity of the lower end of the nucleus toward the center, with its long axis paralleled to the long axis of the nucleus. The proximal centriole and the basal body were correspondingly located in the implantation fossa; this differed from the implantation fossa in carp sperm, which was situated on one side of the nucleus [32]. It also differed from the implantation fossa of L. crocea sperm, which was positioned on the dorsal side of the nucleus [25], but it was very similar to that of Pseudobagrus fulvidraco sperm, whose implantation fossa invaginated into the nucleus from the posterior to the anterior end, forming a well-like structure [26].

In the curved region of the eel sperm head, there existed a large spherical granule, a specialized structure that had not been observed in the sperm of many teleost fish. Observations revealed that this spherical granule underwent morphological changes during sperm differentiation: initially, it contained only several curved filamentous structures, with organelles such as the centriolar complex and mitochondria indistinguishable; later, the centriolar complex and mitochondria became clearly discernible as they formed within the granule.

This study only observed and described the morphological structure of two types of sperm in Japanese eels without verifying their motility or subsequent fertilization and hatching capacity. This is a limitation of the present research and warrants further in-depth investigation during future studies.

5. Conclusions

In summary, the sperm of Japanese eel had two distinct morphological structures. One type of sperm featured a spherical nucleus with a smaller head diameter and an axoneme following the typical “9 + 2” microtubule pattern. Another type was the eyebrow-shaped sperm, characterized by an elongated head and an axoneme with a “9 + 0” microtubule pattern. The morphological diversity of Japanese eel sperm further highlighted the uniqueness of this species. These findings has provided foundational data for the study of eel sperm biology and could offer scientific support for further research on artificial reproduction in eels.