Youichi Hayakawa

Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae.

Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.

Parasperm: morphological and functional studies on nonfertile sperm

Sperm polymorphism, a phenomenon in which more than one type of sperm is produced within a species, occurs widely in animals from invertebrates to vertebrates. Sperm in this phenomenon can be categorized into fertile sperm and nonfertile sperm on the basis of fertilization ability. Nonfertile sperm can be further classified into parasperm and aberrant deformed sperm. Parasperm are sperm produced through a constant developmental process along with normal fertile eusperm, and they are readily distinguished from deformed sperm, which are irregularly crippled by unpredictable errors at certain stages during spermatogenesis. Sperm identified as parasperm occur widely in invertebrates but are presently quite limited in vertebrates. This may be the result of the deficiency both of studies on parasperm and of clear criteria to identify parasperm in vertebrates. Some vertebrates show unique spermatogenesis, such as symplastic spermatid and semicystic spermatogenesis. Thus, parasperm must be identified by comparing cells in each cyst and in semen, because irregularly shaped cells in the seminal duct could be either parasperm or normal spermatids. Although parasperm are identified by clear criteria in vertebrates, only in cottoid fishes to date, it is possible for parasperm to be discovered in other vertebrates. Recently, roles related to sperm competition have been reported in several species (e.g., the marine cottoid fish Hemilepidotus gilberti), and, with some of them, parasperm production is influenced by an intraspecific factor such as a sex ratio or the density of a population. Sperm competition is one of the important candidates for influencing evolution of parasperm functions, but not all parasperm seem to have a relation to sperm competition. Parasperm function may relate to the ecological conditions of each species that produces parasperm. Studies on parasperm function will be advanced by an ecological approach concerning male fertilization success as well as cytological investigation for parasperm.

Sexual bipotentiality of behavior in male and female goldfish

It is known that in goldfish Carassius auratus, a non-sex changing fish, prostaglandin (PG) treatment can induce female-typical sex behavior in males, and androgen treatment can induce male-typical sex behavior in females. These facts suggest that goldfish have a sexually bipotential brain even after attaining sexual maturity unlike mammals which have sexually differentiated brain. In the present study, in order to further characterize the brain function of goldfish, whether hormonal treatments which induce heterotypical sexual behavior suppress the occurrence of sex-typical behavior and whether sex-typical and heterotypical behavior can be induced in a relatively short time were examined. In the first series of experiments, male goldfish were shown to retain their ability to perform male-typical sex behavior within a week after being induced to perform female-typical behavior. Likewise, female goldfish were also shown to retain their female-typical sex behavior a week after being induced to perform male-typical behavior. In the second series of experiments, when PG-injected experimental males were placed with both PG-injected females and sexually mature males, the experimental males performed male- and female-typical behavior alternately with the females and the males, respectively during 90 min test period. When methyltestosterone-treated experimental females were injected with PG and placed with both PG-injected females and mature males, the experimental females performed male- and female-typical behavior alternately during 90 min test period. The results of the present study are consistent with the current knowledge that goldfish possess a sexually bipotential brain that can regulate both male and female-typical sex behaviors.

Annual changes in testicular development and occurrence of parasperm in the male reproductive organs of fourspine sculpin, Cottus kazika

Annual changes in testicular development and occurrence of parasperm were investigated using 2-year-old male fourspine sculpins Cottus kazika, based on the histological observation of testes. The male reproductive organ of fourspine sculpins comprised a pair of testes and a sperm duct that functioned as a sperm-storage organ. Male maturity was divided into the following periods: spermatogonial proliferation period (September), early spermatogenic period (October), mid-spermatogenic period (November), late spermatogenic period (December and January), functional maturation period (February and March), and recovery period (April to August). Spermatogenesis rapidly progressed from October to January and continued until the functional maturation period. Parasperm formation, which is known in some cottidae species, was observed in fourspine sculpins. Testicular regression of cultured fourspine sculpins progressed slowly during the recovery period when residual parasperm and empty spaces occupied the testis. The parasperm were immotile and oval and slightly concave on one side; additionally, they stained strongly with hematoxylin and PAS. Seminal lobules of the testis were filled with parasperm during the spawning period; in contrast, the sperm duct was filled with eusperm. These findings were observed in both cultured and wild fish. In this study, the functions of parasperm with regard to reproduction in fourspine sculpins are discussed.

Clasping behavior and the asymmetrically latitudinal structure of the testes in the male dwarf gourami Colisa lalia

During the course of spawning, the male dwarf gourami Colisa lalia exhibits a clasping behavior where the male bends his body around the female under the floating bubble nest. In this study, in order to investigate the role of this clasping behavior, we observed the mating behavior of C. lalia in aquaria and conducted examinations of the male’s reproductive system anatomically and histologically. Forcing two males to compete for spawning with a female resulted in one male becoming dominant over the other and ultimately clasping the female. The losing male did not attempt to “sneak” during spawning, and it was possible to confirm that the clasping behavior is indispensable for sperm emission and only performed by the dominant male. Although testes of teleosts generally longitudinally elongate (i.e., parallel to the body axis), the testes of C. lalia were latitudinally elongate (i.e., vertically with respect to the body axis) and located asymmetrically between the liver and gallbladder. The latitudinal structure of the testes was also confirmed histologically, as the main testicular duct develops vertically. The asymmetric testes did not appear to influence the way in which the body bent during the clasping maneuver because the male was observed to bend its body equally to the right and left during clasping. However, the testes were situated in the vicinity of the fulcrum formed when the male bends its body, suggesting that the latitudinal testes are structurally related to its clasping behavior (i.e., a mechanism analogous to that of a nutcracker). Following physical principles (i.e., levers and mechanical advantage), it can be proposed that male dwarf gourami squeeze semen out from their latitudinal testes by bending their bodies.

Comparison of ovarian functions for keeping embryos by measurement of dissolved oxygen concentrations in ovaries of copulatory and non-copulatory oviparous fishes and viviparous fishes

Abstract To investigate causes of anomalous development of embryos facultatively fertilized in the ovary of a non-copulatory oviparous fish Hemilepidotus gilberti (Scorpaeniformes: Cottdae), dissolved oxygen (DO) concentrations were measured in ovaries of copulatory oviparous ( Alcichthys alcicornis , Bero elegans ), non-copulatory oviparous ( H. gilberti , Hexagrammus otakii ), and viviparous ( Sebastes taczanowskii , Zoarces elongatus ) fishes. DO concentrations changed during vitellogenesis and ovulation cycles, and also before and after ovulation. DO concentrations in the ovary of H. gilberti and H. otakii at ovulation were 0.27±0.03 and 0.15±0.03 mg O 2 l −1 , respectively, whereas in A. alcicornis and B. elegans , the concentrations were 0.47±0.08 and 0.20±0.06 mg O 2 l −1 , respectively. In the ovaries of intralumenal gestation viviparous fishes, S. taczanowskii and Z. elongatus , DO concentration was from 0.01 to 0.11 mg O 2 l −1 . The average DO concentration during the artificial pregnancy of A. alcicornis was 0.97±0.19 mg O 2 l −1 , but all embryos showed deformity. DO concentrations recorded in oviparous fishes in this study were lower than the oxygen level at which most oviparous fish embryos exhibit retardation or death, and it probably caused the anomalous embryonic development. In contrast, the normal development of viviparous fish embryos at low oxygen level was attributed to the specialized structure of ovary, e.g. the dual arterial system to supply the developing embryos with the respiratory demands in Sebastes .

Comparative studies of testicular structure and sperm morphology among copulatory and non-copulatory sculpins (Cottidae: Scorpaeniformes: Teleostei)

Testicular structure of 9 species and sperm head morphology of 19 species of Cottidae were observed in order to clarify relationships between morphological characteristics of the male reproductive organ and reproductive mode (copulation or non-copulation). Morphological structure of the testis was divided into the following five types based on the sperm transfer and reservoir system: (1) a non-duct type in which the sperm duct is not a distinct exterior structure, but the tube for sperm transport traverses along the testis as an interior structure; (2) an anterior duct type with distinct anterior sperm ducts traversing along the testis; (3) a posterior duct type with distinct anterior sperm ducts traversing along the dorsal hilus of testis and posterior sperm ducts extending to the rear of the testis; (4) an anterior duct posterior vesicle type with distinct anterior sperm ducts traversing along the testis, and the right and left sperm ducts fusing in the rear of testis, forming the seminal vesicle; (5) a non-duct posterior vesicle type in which sperm ducts do not accompany the testis, and the testis and seminal vesicle are connected directly or through posterior sperm ducts. It is thought that in Cottidae the non-duct type of reproductive organ is primitive, and the anterior duct type is common to all non-copulating species. The testes and accompanying seminal vesicle were seen only in copulating species. Sperm head morphology was divided into three types according to the length/width ratio: oval type ≤2, intermediate type >2 and ≤3, and slender type >3. The type of sperm head corresponded closely to the reproductive mode; non-copulating species had oval sperm head, and copulating species had intermediate or slender ones. These results suggest that the structure of the testis and the morphology of the sperm head evolved from testes with anterior sperm ducts and oval sperm heads to testes with an associated seminal vesicle and slender sperm heads in association with the evolution from non-copulatory to copulatory reproduction in Cottidae.