Incidence of four major osteoporotic fragility fractures among older individuals in Sado, Japan, in 2020.

INTRODUCTION: This study compared the 2020 incidence of fragility fractures in Sado City with those from 2004 to 2015. MATERIALS AND METHODS: Data from patients aged ≥ 60 years living in Sado City with fragility fractures in the hip, vertebral, distal radius, and proximal humerus between January 1 and December 31, 2020, were collected. We examined the number and incidence of four types of osteoporotic fractures in the older population aged ≥ 60 years living in Sado City in 2020. We compared the results with those of the 2004, 2010, and 2015 surveys, examining the temporal change and trend in the incidence of the four fracture types in this population. We investigated the use rate of anti-osteoporotic medications and the relationship between their administration and the occurrence of fragility fractures. RESULTS: The age-specific incidence of hip fractures slightly decreased from 2015. However, the incidence of the other three fractures slightly increased, although the difference was not statistically significant. The incidence of hip fractures markedly increased in the 80 s. In 2020, the percentage of patients taking anti-osteoporotic agents before the occurrence of fractures decreased to 12.4% from 14.5% in 2015; it increased from 4% in 2004 to 7.6% in 2010. CONCLUSION: The 2020 incidence of the four fractures did not decrease, and the percentage of patients receiving anti-osteoporotic agents did not increase. A higher frequency of osteoporosis treatment is necessary to reduce the incidence of fragility fractures. We recommend using anti-osteoporotic agents to prevent hip fractures among individuals in their mid-70 s and above.

Transcriptome characterization of gonadal sex differentiation in Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel).

Tunas (genus Thunnus) are one of the most ecologically and commercially important fish worldwide. To establish a biological basis for reproduction in this globally essential species, we have recently studied crucial reproductive aspects of the Pacific bluefin tuna (T. orientalis; PBT), as a model of tuna species, based on our closed-cycle aquaculture technology. In this study, we clarified the global expression profile of the genes regulating gonadal sex differentiation in PBT, as this developmental process is vital to sexual reproduction. Based on the results of our comparative (RNA-sequencing) and temporal (qRT-PCR) transcriptome analyses using the updated genome dataset, we propose the molecular mechanisms of gonadal sex differentiation in PBT. In female gonads, foxl2 and cyp19a1a (coding aromatase) are expressed at the onset of sex differentiation. Active aromatase-mediated estrogen biosynthesis, which includes positive regulation of cyp19a1a expression by Foxl2, induces ovarian differentiation. By contrast, dmrt1 and gsdf are upregulated in differentiating male gonads lacking active estrogen synthesis. Dmrt1 and Gsdf would mainly promote testicular differentiation. Furthermore, androgen biosynthesis is upregulated in differentiating male gonad. Endogenous androgens may also be vital to testicular differentiation. This study provides the first comprehensive data clarifying the molecular basis for gonadal sex differentiation in tunas.

Prediction of the Sex-Associated Genomic Region in Tunas (Thunnus Fishes).

Fish species have a variety of sex determination systems. Tunas (genus Thunnus) have an XY genetic sex determination system. However, the Y chromosome or responsible locus has not yet been identified in males. In a previous study, a female genome of Pacific bluefin tuna (T. orientalis) was sequenced, and candidates for sex-associated DNA polymorphisms were identified by a genome-wide association study using resequencing data. In the present study, we sequenced a male genome of Pacific bluefin tuna by long-read and linked-read sequencing technologies and explored male-specific loci through a comparison with the female genome. As a result, we found a unique region carrying the male-specific haplotype, where a homolog of estrogen sulfotransferase gene was predicted to be encoded. The genome-wide mapping of previously resequenced data indicated that, among the functionally annotated genes, only this gene, named sult1st6y, was paternally inherited in the males of Pacific bluefin tuna. We reviewed the RNA-seq data of southern bluefin tuna (T. maccoyii) in the public database and found that sult1st6y of southern bluefin tuna was expressed in all male testes, but absent or suppressed in the female ovary. Since estrogen sulfotransferase is responsible for the inactivation of estrogens, it is reasonable to assume that the expression of sult1st6y in gonad cells may inhibit female development, thereby inducing the individuals to become males. Thus, our results raise a promising hypothesis that sult1st6y is the sex determination gene in Thunnus fishes or at least functions at a crucial point in the sex-differentiation cascade.

Gonadal sex differentiation and early ovarian/testicular development in cultured Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel).

Pacific bluefin tuna (PBT), Thunnus orientalis, is one of the most important species for aquaculture in Japan. Recently, the reduction in muscle fat content associated with sexual maturation in farmed PBT has become a serious problem. To develop technologies for inducing sterility, detailed and reliable data on gonadal development in PBT are needed. Here, we demonstrated the process of gonadal sex differentiation, and of early ovarian and testicular development during the immature stages in PBT. Gonadal sex differentiation was first characterized by the formation of the ovarian cavity in female and of the efferent ducts in male 57 days post hatching (dph). The gonads then differentiated into ovaries or testes according to the genotypic sex until 83 dph. During this period, primordial germ cells, oogonia, and type-A spermatogonia were solitarily distributed in the gonads, and the number of germ cells did not differ between sexes. After gonadal sex differentiation, gonads of PBTs developed in a sexually dimorphic manner: proliferation and differentiation of germ cells occurred earlier in the ovaries than in the testes. The oogonia in ovaries formed cysts at 185 dph, but the type-A spermatogonia were solitarily distributed in testes at this stage, and cysts of type-A spermatogonia were first observed at 247 dph. Moreover, the oogonia entered meiosis and differentiated into chromatin-nucleolus stage oocytes until 247 dph, and subsequently into peri-nucleolus stage oocytes until 285 dph, whereas the type-A spermatogonia differentiated into type-B spermatogonia, spermatocytes, spermatids, and spermatozoa from 446 dph onwards. We believe the results of this study provide the necessary basis for future studies on sterile PBT production.

Vision-based egg quality prediction in Pacific bluefin tuna (Thunnus orientalis) by deep neural network.

Closed-cycle aquaculture using hatchery produced seed stocks is vital to the sustainability of endangered species such as Pacific bluefin tuna (Thunnus orientalis) because this aquaculture system does not depend on aquaculture seeds collected from the wild. High egg quality promotes efficient aquaculture production by improving hatch rates and subsequent growth and survival of hatched larvae. In this study, we investigate the possibility of a simple, low-cost, and accurate egg quality prediction system based only on photographic images using deep neural networks. We photographed individual eggs immediately after spawning and assessed their qualities, i.e., whether they hatched normally and how many days larvae survived without feeding. The proposed system predicted normally hatching eggs with higher accuracy than human experts. It was also successful in predicting which eggs would produce longer-surviving larvae. We also analyzed the image aspects that contributed to the prediction to discover important egg features. Our results suggest the applicability of deep learning techniques to efficient egg quality prediction, and analysis of early developmental stages of development.

Insulin-like growth factors 1 and 2 regulate gene expression and enzymatic activity of cyp17a1 in ovarian follicles of the yellowtail, Seriola quinqueradiata.

There is accumulating evidence that insulin-like growth factors (IGFs), primary mediators of somatic growth, play an important role in fish reproduction. Previously, we showed that IGF-1 and IGF-2 are expressed in the ovarian follicle cells of the yellowtail (Seriola quinqueradiata) during the vitellogenic phase, suggesting that IGFs may be involved in ovarian steroidogenesis. In this study, we examined the effects of IGF-1 and IGF-2 on gene expression and activity of steroidogenic enzymes in yellowtail ovary in vitro. IGF-1 and IGF-2 had no effect on mRNA levels of several steroidogenesis-related genes (star, cyp11a1, hsd3b, cyp17a2, and cyp19a1). However, both IGFs enhanced the transcription of cyp17a1 in vitellogenic ovaries, although such up-regulation was not found in the ovary at the pre-vitellogenic stage. The stage-dependent effects of IGFs were correlated with changes in ovarian cyp17a1 mRNA levels during the reproductive cycle: transcript abundances increased in conjunction with ovarian development. In addition, IGF-induced cyp17a1 gene expression was significantly inhibited by wortmannin, suggesting that PI3 kinase plays an essential role in IGF-mediated ovarian steroidogenesis. Furthermore, IGF-1 and IGF-2 promoted the conversion of both progesterone and 17α-hydroxyprogesterone to androstenedione in vitellogenic ovaries, suggesting that both IGFs stimulated 17α-hydroxylase and C17-20 lyase activities. Taken together, these findings suggest that IGF-1 and IGF-2 act directly on follicle cells to stimulate steroid production through an increase in gene expression and enzymatic activity of cyp17a1 via induction of PI3 kinase.

Author Correction: Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis).

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.

Early development of primordial germ cells in Pacific bluefin tuna Thunnus orientalis.

Bluefin tuna is one of the most important aquaculture species in several countries; however, information regarding the primordial germ cell (PGC) development and migration in this species is scarce. This information is vital for application in reproductive biotechnology, for example, induced sterility through targeted cell ablation or PGC manipulation. Teleost PGC can be visualized by injecting an RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3) into eggs. In this study, we identified the PGC and its migratory pathway during early embryogenesis and larvae development by injecting the GFP-zf-nos3 3'UTR mRNA into the Pacific bluefin tuna (PBT, Thunnus orientalis). PBT PGCs were initially found around the marginal and dorsal regions of the blastodisc at 50%-epiboly stage. The PGCs were aligned as two elongated lines at the posterior part of the embryonic body during the early segmentation period, and eventually formed a single tight cluster underneath somites 10 to 15 of the embryonic body until the late segmentation period. Although the aggregated PGCs stayed at the same position during hatching, they started migrating anteriorly and were split into two populations at 3 days after hatching (DAH). Until 15 DAH, these PGCs settled in two bilateral lines at the apex of the peritoneal cavity. Histological analysis of PBT larvae revealed that at 3 and 5 DAH, the PGCs were not enclosed by the somatic cells, whereas at 15 DAH, they were entirely covered by the somatic cells, indicating the development of the primordial gonads. These results are essential for future experiments in germ line control technologies for bluefin tuna.

Changes in gene expression and cellular localization of insulin-like growth factors 1 and 2 in the ovaries during ovary development of the yellowtail, Seriola quinqueradiata.

A method of controlling the somatic growth and reproduction of yellowtail fish (Seriola quinqueradiata) is needed in order to establish methods for the efficient aquaculture production of the species. However, little information about the hormonal interactions between somatic growth and reproduction is available for marine teleosts. There is accumulating evidence that insulin-like growth factor (IGF), a major hormone related somatic growth, plays an important role in fish reproduction. As the first step toward understanding the physiological role of IGF in the development of yellowtail ovaries, we characterized the expression and cellular localization of IGF-1 and IGF-2 in the ovary during development. We histologically classified the maturity of two-year-old females with ovaries at various developmental stages into the perinucleolar (Pn), yolk vesicle (Yv), primary yolk (Py), secondary yolk and tertiary yolk (Ty) stages, according to the most advanced type of oocyte present. The IGF-1 gene expression showed constitutively high levels at the different developmental stages, although IGF-1 mRNA levels tended to increase from the Py to the Ty stage with vitellogenesis, reaching maximum levels during the Ty stage. The IGF-2 mRNA levels increased as ovarian development advanced. Using immunohistochemistry methods, immunoreactive IGF-1 was mainly detected in the theca cells of ovarian follicles during late secondary oocyte growth, and in part of the granulosa cells of Ty stage oocytes. IGF-2 immunoreactivity was observed in all granulosa cells in layer in Ty stage oocytes. These results indicate that follicular IGFs may be involved in yellowtail reproduction via autocrine/paracrine mechanisms.

Production of donor-derived offspring by allogeneic transplantation of spermatogonia in the yellowtail (Seriola quinqueradiata).

Although the yellowtail (Seriola quinqueradiata) is the fish most commonly farmed in Japan, breeding of this species has not yet started. This is primarily due to the lack of sufficiently sophisticated methods for manipulating gametogenesis, which makes it difficult to collect gametes from specific dams and sires. If it were possible to produce large numbers of surrogate fish by transplanting germ cells isolated from donor individuals harboring desirable genetic traits, then the probability of acquiring gametes carrying the donor-derived haplotype would increase, and breeding programs involving this species might increase as a result. As a first step, we established a method for the allogeneic transplantation of yellowtail spermatogonia and the production of donor-derived offspring. Donor cells were collected from immature (10-month-old) yellowtail males with testes containing abundant type A spermatogonia, labeled with PKH26 fluorescent dye, and transferred into the peritoneal cavities of 8-day-old larvae. Fluorescence observation at 28 days post-transplantation revealed that PKH26-labeled cells were incorporated into recipients' gonads. To assess whether donor-derived spermatogonia could differentiate into functional gametes in the allogeneic recipient gonads, gametes collected from nine male and four female adult recipients were fertilized with wild-type eggs and milt. Analysis of microsatellite DNA markers confirmed that some of the first filial (F(1)) offspring were derived from donor fish, with the average contribution of donor-derived F(1) offspring being 66% and the maximum reaching 99%. These findings confirmed that our method was effective for transplanting yellowtail spermatogonia into allogeneic larvae to produce donor-derived offspring.

Chub mackerel gonads support colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells.

The production of xenogenic gametes from large-bodied, commercially important marine fish species in closely related smaller host fish species with short generation times may enable rapid and simple seed production of the target species. As a first step toward this goal, we assessed the suitability of chub mackerel, Scomber japonicus, as a small-bodied recipient species for xenogenic spermatogonial transplantation. Histological observation of the early gonadal development of chub mackerel larvae and transplantation of fluorescent-labeled spermatogonia from Nibe croaker, Nibea mitsukurii, revealed that 5.3-mm chub mackerel larvae were suitable recipients for successful transplantation. Intraperitoneally transplanted xenogenic spermatogonia efficiently colonized the gonads of these recipient larvae, and donor-derived Nibe croaker germ cells proliferated rapidly soon after colonization. Moreover, gonadal soma-derived growth factor (gsdf) mRNA, a gonadal somatic cell marker, was expressed in recipient-derived cells surrounding the incorporated donor-derived germ cells, suggesting that donor-derived germ cells had settled at an appropriate location in the recipient gonad. Our data show that xenogenic spermatogonial transplantation was successful in chub mackerel and that the somatic microenvironment of the chub mackerel gonad can support the colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells derived from a donor species of a different taxonomic family.

Development of spermatogonial cell transplantation in Nibe croaker, Nibea mitsukurii (Perciformes, Sciaenidae).

In a recently established system for intraperitoneal spermatogonial cell transplantation in salmonids, donor type A spermatogonia (type A SG) were microinjected into the peritoneal cavity of newly hatched larvae. Compared with salmonids, the larvae of marine teleosts are small and vulnerable to physiological and physical stresses, making it difficult to use them for cell manipulation. Herein, we developed type A SG cell transplantation in Nibe croaker (Nibea mitsukurii) by optimizing 1) the developmental stage of the donor testes used to prepare type A SG-enriched cell suspensions and 2) the timing and location of intraperitoneal cell transplantations to recipient larvae. Donor cells labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of 3-, 4-, 5-, and 6-mm larvae using glass micropipettes. Consequently, 20.6% of the 4-mm larvae recipients survived for 3 wk, and 36.3% of the survivors had donor-derived cells in their gonads. The incorporated donor cells were identified as germ cells by germ cell-specific nuclear morphology and expression of a germ cell marker. In contrast, no donor type A SG were incorporated into the gonads of 6-mm recipient larvae. These data indicate that there is a distinct narrow window in the developmental stages of recipient larvae when exogenous type A SG can be incorporated into the gonads. The establishment of this system in pelagic egg-spawning marine teleosts would allow the creation of a new broodstock system in which a target species with a large body size and long generation time could be produced from related species with a small body size and short generation time.