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Fertilization mode may affect sperm characteristics, such as morphology, velocity, and motility. However, there is little information on how fertilization mode affects sperm evolution because several factors (e.g., sperm competition) are intricately intertwined when phylogenetically distant species are compared. Here, we investigated sperm characteristics by comparing seven externally and four internally fertilizing marine fishes from three different groups containing close relatives, considering sperm competition levels. The sperm head was significantly slenderer in internal fertilizers than in external fertilizers, suggesting that a slender head is advantageous for swimming in viscous ovarian fluid or in narrow spaces of the ovary. In addition, sperm motility differed between external and internal fertilizers; sperm of external fertilizers were only motile in seawater, whereas sperm of internal fertilizers were only motile in an isotonic solution. These results suggest that sperm motility was adapted according to fertilization mode. By contrast, total sperm length and sperm velocity were not associated with fertilization mode, perhaps because of the different levels of sperm competition. Relative testis mass (an index of sperm competition level) was positively correlated with sperm velocity and negatively correlated with the ratio of sperm head length to total sperm length. These findings suggest that species with higher levels of sperm competition have faster sperm with longer flagella relative to the head length. These results contradict the previous assumption that the evolution of internal fertilization increases the total sperm length. In addition, copulatory behavior with internal insemination may involve a large genital morphology, but this is not essential in fish, suggesting the existence of various sperm transfer methods. Although the power of our analyses is not strong because of the limited number of species, we propose a new scenario of sperm evolution in which internal fertilization would increase sperm head length, but not total sperm length, and change sperm motility.

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In this study, the authors report the first record of egg masses deposited in solitary tunicates by the snubnose sculpin, Orthonopias triacis, from the Northeastern Pacific. Four egg masses were discovered in the tunicate Ascidia ceratodes that were genetically determined to be O. triacis. Female O. triacis had long ovipositors that allow deposition of their eggs inside the atrium of the tunicates. A comparison of host-tunicate size with ovipositor length of sculpins from the Northwestern Pacific, including the genera Furcina and Pseudoblennius, revealed that O. triacis had shorter ovipositors and spawned in the atrium of smaller species of tunicates. Ancestral state reconstruction of egg deposition in solitary tunicates using 1.86Mbp RNAseq data of 20 sculpin species from Northeastern and Northwestern Pacific revealed that this unusual spawning behaviour may have evolved convergently in different species occurring in the Northeastern vs. the Northwestern Pacific.

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Sperm production is costly, and males are expected to strategically allocate this potentially limiting resource to maximize their fitness. Sperm allocation theory predicts that males should adjust their sperm expenditure in relation to female quality. However, the available empirical evidence is limited. In this study, we assessed whether wild male medaka (Oryzias latipes) would allocate their sperm depending on female quality under controlled conditions. Behavioral observations revealed that spawning behavior could be classified into four stages: male quivering, female quivering, sperm release, and stay. Of these behaviors, only the duration of sperm release was positively correlated with the number of sperm ejaculated in the aquarium, suggesting that males may adjust the sperm number expended for each mating by controlling the duration of sperm release. The estimated amount of sperm released per spawning was positively and significantly correlated with the body depth and weight of paired females. This result is consistent with the prediction arising from the sperm allocation hypothesis, and suggests that male medaka may allocate their sperm depending on paired female body depth and weight. However, the number of released sperm did not correlate with the number of spawned eggs, which was positively correlated with body depth and weight of females. These results imply that medaka females may have counter-strategies against sperm allocation by males, and consequently, males might not be able to adjust the number of sperm efficiently. We disscuss our results from the perspective of sexual conflict over sperm as a limited resource.

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To investigate the effects of preventing temperature decrease on the reproductive activity of the male cold-water teleost, Cottus pollux SE, testicular development, serum 11-ketotestosterone (11-KT) levels, and physiological responses associated with nesting behavior (i.e., elevation of serum 11-KT levels and accumulation of urine in the urinary bladder) were observed from November to January. Specifically, males were exposed to three different cooling regimes (control, 16 to 6 °C; H1, 16 to 11 °C; H2, 16 to 14 °C), and the results were compared. In addition, the effects of temperature on male reproductive behavior were also clarified. At higher water temperature regimes, the rate of testicular development and serum 11-KT levels were both higher from November to mid-December than from mid-December to January. However, the results showed that high water temperature regimes in the coldest period of winter did not suppress spermatogenesis completely. Conversely, the physiological responses to nesting were affected by high water temperatures, with serum 11-KT levels increasing and urine accumulation in the urinary bladder being suppressed. Furthermore, frequencies of two behaviors associated with nesting, i.e., body undulation and face displays, were also suppressed under high water temperatures (~ 14 °C) compared with normal temperatures (~ 7 °C) during the breeding season. Based on the physiological and behavioral responses to nesting, findings showed that preventing a water temperature decrease during winter suppresses reproductive activity in Cottus pollux SE.

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We found two patterns of parasitic mating behaviors by male in Japanese medaka, Oryzias latipes, in the spawning by one female and two males in the aquarium condition. In the first type of parasitic mating behavior, the unpaired male would rush toward the closely adhering female and paired male, and simultaneously perform emission behavior with close adhesion to the female (simultaneous emission). The second type of parasitic mating behavior was that the unpaired male would rush toward the female with spawned eggs hanging down from the genital pore several seconds after pair-spawning, perform emission behavior with close adhesion to the female without any prespawning behavior (post-spawn emission). The frequencies of "simultaneous emission" and "post-spawn emission" were 20 (19.6%) and 18 cases (17.7%), respectively, in 102 trials, and the average reproductive successes about 41% and 20%, respectively. The reproductive success of simultaneous emission was not correlated with the timing and duration of spawning behaviors of the paired/ unpaired male, while the reproductive success of post-spawn emission significantly decreased as the length of time during which the paired male adhered to female increased. Observations of two consecutive spawning behaviors using same combination of two males revealed that paired males always tend to become paired males. However, the role of both males was occasionally reversed, indicating flexibility in mating tactics.

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Males of the viviparous teleost fish Gambusia affinis copulate with females by using a specialized anal fin, or gonopodium. When female G. affinis were placed in a shallow transparent tank which was then floated on the surface of a larger aquarium housing male G. affinis , the males frequently attempted to copulate with females housed in the smaller quarantine tank. This copulatory behavior in male G. affinis was only observed to be elicited by visual stimuli; female G. affinis have a yellow spot and black anal spots around their urogenital opening. To investigate the function of the yellow spot of the female, we examined male copulatory behavior directed towards artificial female models having a yellow, black, gray, or white spot in the genital region. Of the differently colored females tested, males spent a significantly longer time in the vicinity of the artificial model with a yellow spot. In addition, males attempted to copulate with the yellow-spotted female model more frequently than with the models of different colors. These findings revealed that the yellow spot around the female urogenital opening of G. affinis attracts males and functions as a cue for copulation.

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We previously demonstrated the presence of three forms of vitellogenin (Vg), two 600 kDa Vgs (600Vg; VgA and VgB) and a 400 kDa Vg (400Vg; phosvitinless Vg) in plasma from maturing female viviparous mosquitofish, Gambusia affinis. For further quantitative elucidation of the accumulation and utilization of the multiple Vg-derived yolk proteins, two sandwich enzyme-linked immunosorbent assays (ELISA) were developed using antisera against 600Vgs and a 400 kDa yolk protein (400Yp; derived from 400Vg), respectively. Contents of 560 kDa yolk protein (560Yp; lipovitellins derived from 600Vg) and 400Yp measured by the ELISAs increased in accordance with the growth of vitellogenic oocytes, keeping their proportional ratio (mol/mol) at about 4:1. A similar ratio obtained for plasma Vgs suggests that the proportional accumulation of the multiple Vg-derived yolk proteins is regulated by the hepatic synthesis and secretion of their precursor Vgs. When egg homogenate was analyzed by gel chromatography, three peaks, consisting of 560Yp, 400Yp and 28 kDa native beta'-component, were observed. The elution profile showed no change until embryos reached the early neurula stage, however, the relative height of the 560Yp peak as compared to the 400Yp one decreased after retinal pigmentation. Results from measurements of 560Yp and 400Yp at each embryonic stage supported the occurrence of unequal utilization of the two yolk proteins. The proportional ratios (mol/mol) of 560Yp content versus 400Yp content gradually decreased from 4.1 fold in early neurula embryo to 1.4 fold in larva just before parturition. The present study thus demonstrated unequal utilization of the multiple Vg-derived yolk proteins in developing embryos of mosquitofish.

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The objectives of this study were to characterize multiple forms of vitellogenin (Vg) in mosquitofish (Gambusia affinis) and to discover the fate of each Vg during its processing into product yolk proteins. Two Vg preparations, with apparent masses of 600 kDa (600 Vg) and 400 kDa (400 Vg), were isolated from the plasma of fish treated with estradiol-17beta (E(2)) by various chromatographic procedures. Immunological analyses verified the presence of two different Vg proteins (600 VgA and 600 VgB) in the 600 Vg preparation and of a single protein in the 400 Vg preparation. Three major yolk proteins (Yps) with apparent masses of 560, 400, and 28 kDa were observed in extracts of ovarian follicles from vitellogenic females. Immunological analyses demonstrated that the 400 Vg underwent no change in native mass after being incorporated into oocytes. The 600 Vgs gave rise to a 28 kDa beta'-component and a native 560 kDa Yp, which was heterodimeric in structure, consisting of two types of complexes between phosvitin (Pv) and lipovitellin (Lv) heavy- and light-chains. Full-length cDNAs encoding the 600 VgA, 600 VgB, and 400 Vg were isolated from a liver cDNA library of E(2) treated fish. Similar to the zebrafish vg3 gene, the 400 Vg cDNA lacked a Pv domain and was classified as an incomplete or phosvitinless (C-type) Vg. The deduced primary structures of 600 VgA and 600 VgB were complete, and these were categorized as type A and type B Vgs, respectively, according to our recent classification scheme. This is the first report on the characterization of three functional Vg genes and their circulating and yolk protein products in any vertebrate species.

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Changes in serum 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-DP) levels around the gestation time of normal pregnant and experimentally non-pregnant females were investigated in the black rockfish, Sebastes schlegeli, a viviparous teleost. The serum 17,20 beta-DP in both females showed similar changes and levels, increasing from the early to late gestation periods and declining just before parturition in pregnant females and egg-release in non-pregnant females, respectively. These results suggest that the maintenance of high serum levels of 17,20 beta-DP after oocyte maturation is not correlated with gestation or parturition, but occurs spontaneously in this species. The decline of 17,20 beta-DP levels prior to egg-release in non-pregnant females tended to occur one week earlier than those prior to parturition in pregnant females, suggesting that both a decline in 17,20 beta-DP levels in mothers and some response from embryos are needed for a smooth parturition. The post-ovulatory follicles were maintained throughout the gestation period and produced a considerable amount of 17,20 beta-DP in vitro (3.44-6.96 pg/ml/mg tissue), but little estradiol-17beta (0.92-1.66 pg/ml/mg tissue). The production of 17,20 beta-DP tended to be enhanced by the addition of a precursor steroid, pregnenolone, in the pre-, early and mid-gestation periods. These results strongly suggest that the follicle cells in black rockfish have the ability to synthesize 17,20 beta-DP during the post-ovulatory period, and high serum 17,20 beta-DP during gestation is supplied by the post-ovulatory follicles, which in Sebastes are considered to be functionally homologous to the mammalian corpus luteum.

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The ontogenetic development of gonads from embryo to adult was observed histologically in the viviparous teleost, Gambusia affinis. Primordial germ cells (PGCs) appeared in the subendodermal space of the embryo 14 days before birth, and then transferred to the dorsal mesentery to form paired genital ridges 12 days before birth. The PGCs proliferated in the genital ridge, forming gonadal primordia 10 days before birth. All gonadal primordia differentiated to the ovary containing oocytes 2 days before birth, but then redifferentiated to the ovary and testis just after birth. This indicates that the mosquitofish is a juvenile hermaphroditic species. The characteristics of gonadal sex differentiation just after birth were enlargement of the oocytes in females, and invasion of somatic cells from the hilar region to an inner portion of the gonad in males. The paired ovary fused at the basal area 5 days after birth, then on the ventral and dorsal portions, developing into a single ovary 10 days after birth. During this time a single ovarian cavity was formed on the dorsal portion of the ovary. The paired testes fused only at the basal area and became a single testis having two main lobes 10 days after birth. The oocytes gradually developed and began vitellogenesis 100 days after birth, but did not reach maturation until 110 days after birth. Spermatogenic cells formed cysts at 20 days, began meiosis at 70 days, and matured to form sperm balls 90 days after birth. The male fish sexually matured earlier than the female.

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Elkhorn sculpin, Alcichthys alcicornis, is a marine teleost with a unique reproductive mode called "internal gametic association," in which sperm introduced into the ovary by copulation enter the micropylar canal of ovulated eggs in the ovarian cavity, but actual sperm-egg fusion does not occur until the eggs have been released into sea water. It is also known that this fish is a multiple spawner, which spawns at intervals of a few days for one month, and the sperm introduced into the ovary at the beginning of the spawning season retain their fertilizability for the entire period. To clarify how the fertilizability of sperm is maintained internally, the ultrastructure of sperm, the morphological characteristics related to sperm storage in the ovary, and the characteristics of sperm motility were investigated. Mature sperm generally have the normal form of teleost sperm, devoid of acrosomal structures. However, it was found that the midpiece is comparatively elongated and has a compact aggregation of many small-size mitochondria. The intraovarian sperm remained floating in the ovarian fluid throughout the spawning season. The sperm showed high motility in isotonic and weak alkaline solution, containing sodium ions, which was similar to the ovarian fluid of this fish. Sperm continued to move in artificial ovarian fluid for 7-14 days. Considering these results together, it is thought that the intraovarian sperm move throughout the spawning season due to the plentiful energy generated by the many mitochondria.

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The bluemouth rockfish, Helicolenus dactylopterus dactylopterus (De la Roche, 1809), is a zygoparous species with internal fertilization. The male urogenital papilla acts as the copulating organ, and the females retain the spermatozoa in their ovaries for up to 10 months. The objective of this study is to extend our knowledge of the mechanisms that allow the sperm to be retained in the ovaries for prolonged periods. To this end, we analyze the histochemical properties of: 1) the epithelium of the testicular sperm duct, 2) the sperm of the males, 3) the internal epithelium of the ovary wall, 4) the ovarian fluid, and 5) the spermatozoa storage crypts of females. The PAS (Periodic acid-Schiff) and bright Coomassie blue positive reactions of the epithelium of the spermatic duct point to the secretion of polysaccharides and proteins that could promote the bundling of the spermatozoa. The internal epithelium of the ovarian wall secretes polysaccharides, protein, and lipid compounds throughout the storage and spawning period. The acid nature of the ovarian fluid during the storage period may maintain the bundling of spermatozoa when they enter the ovary and may also inhibit sperm motility until the moment of fertilization. The polysaccharide granules that come from the cryptal epithelium into the cavity where spermatozoa are maintained may supply them with nutrients for the storage period. The presence of glucosaminoglycans on the surface of the sperm is probably related to the inhibition of spermatic motility produced by the acidic environment. They are absent in the spermatozoa located in the testicular ducts, relatively scarce in those of the duct of the copulating organ, and abundant in those within the intraovarian cryptal structures.

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The morphology of female sperm storage during the spawning period and the morphology of sperm degradation after the spawning period were investigated by electron microscopy in a copulating teleost, Alcichthys alcicornis. The spermatozoa were maintained in the ovarian cavity, floating in the ovarian fluid during the spawning period. The spermatozoa then degenerated and were phagocytized by macrophages invading the ovarian cavity after the spawning period. In the ovary during the spawning period, horseradish peroxidase used as a tracer revealed tight junctional complexes connecting adjacent cells of the inner ovarian epithelia (ovarian wall epithelium and ovigerous lamella epithelium). This indicates that a compartmentalization of the ovarian cavity occurs during the spawning period. The junctional complexes were breached after the spawning period, as shown by the fact that horseradish peroxidase penetrated the ovarian cavity via the intercellular space between the adjoining ovigerous lamella epithelia. These results suggest that the spermatozoa in the ovarian cavity are isolated from the maternal immune system by the tight junctional complexes between the adjoining inner ovarian epithelia during the spawning period, and then are eliminated by immune cells following the breakdown of the junctional complexes after the spawning period. J. Morphol. 233:153-163, 1997. © 1997 Wiley-Liss, Inc.

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Alcichthys alciocornis has a viscous ovarian fluid in the ovarian cavity, which plays an important role in its unique mode of reproduction called internal gametic association (i.e., internal insemination and sperm-egg association but a delay in the physiological fertilization until spawning). Seasonal changes in fine structure of the inner epithelial lining and capillary endothelium of the ovary revealed that ovarian fluid originated as a result of the secretory activity of the tissues. The ovarian cavity of A. alcicornis is lined with an ovigerous lamella epithelium and an ovarian wall epithelium. During the spawning period, both epithelia actively secreted proteinaceous substances which seemed to constitute the ovarian fluid. The substances appear to be synthesized in the rough-surfaced endoplasmic reticulum from the material which was transported from the blood capillary, taken into the epithelial cells by endocytosis, accumulated in secretory vesicles via Golgi apparatus in the cells, and finally released into the ovarian cavity by exocytosis. Microapocrine secretion was also observed to occur in both epithelia. Secretory activity of both epithelia by exocytosis and microapocrine secretion showed distinct seasonal changes. Active exocytosis and microapocrine secretion were observed during the spawning period (April-May). These activities slightly declined during the degeneration period (May-June) and were lost during the early recovery period (July). During the mid to late recovery period (October-March), there was some exocytosis but no microapocrine secretion. © 1995 Wiley-Liss, Inc.