Kento Miura

Biological activities of non-protein chromophores of antitumor protein antibiotics: auromomycin and neocarzinostatin.

Summary A non-protein chromophore(s), λmax 350 nm, was extracted by methanol from auromomycin. The methanol extracts of auromomycin and neocarzinostatin blocked growth of lymphoblastoma L5178Y cells. The degree of inhibition was similar to that by the original drugs. PM2 DNA was cleaved by auromomycin chromophore in the absence of reducing agents, whereas neocarzinostatin chromophore required 10 mM 2-mercaptoethanol for DNA strand scission. The DNA-cutting activities of the chromophores were slightly weaker than those of the native antibiotics. The protein fractions, left after methanol extraction, showed little activity on cell growth and isolated DNA. The results suggest that the chromophore moiety plays a predominant role in the inhibition of cell growth and cleavage of DNA.

Production of Sry knockout mouse using TALEN via oocyte injection

Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.

Experimental study on microbubble ejection method for frictional drag reduction

The formation of air bubbles ejected through a single hole in a flat plate was observed in uniform flow of 2–10 m/s It was confirmed that the size of the air bubbles was governed by main flow velocity and air flow rate. According to previous experiments, the size of the bubbles is an important factor in frictional drag reduction by microbubble ejection. Usually bubbles larger than a certain diameter (for example 1 mm) have no effect on frictional drag reduction. Three different methods were proposed and tested to generate smaller bubbles. Among them, a 2D convex (half body of an NACA 64-021 section) with ejection holes at the top was the best and most promising. The diameter of the bubbles became about one-third the size of the reference ejection on a flat plate. Moreover, the bubble size did not increase with increasing flow rate. This is a favorable characteristic for practical purposes. The skin friction force was measured directly with a miniature floating element transducer, and decreased drastically by microbubble ejection from the top of the 2D convex shape.

Early gonadogenesis in mammals: significance of long and narrow gonadal structure.

In mammalian embryogenesis, the gonadal primordium arises from the thickening of the coelomic epithelium, which results in a pair of extremely long and narrow gonadal structures along the anteroposterior axis. These gonadal structures are conserved in various mammalian species, suggesting a great advantage in properly receiving migrating primordial germ cells (PGCs) that are widely scattered throughout the hindgut tube. Soon after the PGCs settle, the bipotential gonads undergo sex determination into testes or ovaries by the sex‐determining gene, Sry, which is expressed in supporting cell precursors in a center‐to‐pole manner. Such a long, narrow gonadal structure bestows a considerable time lag on Sry expression between the center and pole regions, but testiculogenesis with cord formation and Leydig cell differentiation occurs synchronously throughout the whole organ. This synchronous testiculogenesis could be explained by a positive‐feedback mechanism between SOX9 (another SRY‐related transcription factor) and FGF9 downstream of Sry. FGF signals are likely secreted from the center region, rapidly diffuse into the poles, and then induce the establishment of SOX9 expression in Sertoli cells in the pole domains. This work focuses on recent knowledge of the molecular and cellular events of PGC migration, gonadogenesis, and testiculogenesis, and their biological significance in mammalian embryogenesis. Developmental Dynamics 242:330–338, 2013.

Heterogeneity in sexual bipotentiality and plasticity of granulosa cells in developing mouse ovaries

Summary In mammalian sex determination, SRY directly upregulates the expression of SOX9, the master regulatory transcription factor in Sertoli cell differentiation, leading to testis formation. Without SRY action, the bipotential gonadal cells become pre-granulosa cells, which results in ovarian follicle development. When, where and how pre-granulosa cells are determined to differentiate into developing ovaries, however, remains unclear. By monitoring SRY-dependent SOX9 inducibility (SDSI) in an Sry-inducible mouse system, we were able to identify spatiotemporal changes in the sexual bipotentiality/plasticity of ovarian somatic cells throughout life. The early pre-granulosa cells maintain the SDSI until 11.5 d.p.c., after which most pre-granulosa cells rapidly lose this ability by 12.0 d.p.c. Unexpectedly, we found a subpopulation of the pre-granulosa cells near the mesonephric tissue that continuously retains SDSI throughout fetal and early postnatal stages. After birth, these SDSI-positive pre-granulosa cells contribute to the initial round of folliculogenesis by the secondary follicle stage. In experimental sex reversal of 13.5-d.p.c. ovaries grafted into adult male nude mice, the differentiated granulosa cells re-acquire the SDSI before other signs of masculinization. Our data provide direct evidence of an unexpectedly high sexual heterogeneity of granulosa cells in developing mouse ovaries in a stage- and region-specific manner. Discovery of such sexually bipotential granulosa cells provides a novel entry point to the understanding of masculinization in various cases of XX disorders of sexual development in mammalian ovaries.

Sex Determination and Differentiation in Mammals

In most mammals, sex determination is initiated by transient expression of Sry, Sex-determining region Y gene, encoding an HMG-box transcription factor in bipotential gonadal supporting cells. In XY mouse gonads, SRY activates SOX9, another SRY-related HMG-box factor, during a critical time window (i.e. embryonic days 11.0–11.5) in male supporting cells. The maintenance of high-level SOX9 expression consequently induces Sertoli cell differentiation, leading to testis formation during the early organogenic stages. In XX gonads without Sry expression, bipotential supporting cells express FOXL2, a forkhead transcription factor, shortly after this time window, resulting in pre-granulosa cell differentiation and its subsequent contribution to ovarian folliculogenesis after birth. At later fetal stages, after cessation of SRY expression, the balance between masclinizing FGF9 and feminizing WNT4 signals affects the maintenance of high-level SOX9 expression in the supporting cells. During the perinatal and postnatal stages, each sex of the supporting cells is maintained by the balance between the masclinizing actions of SOX9 and DMRT1 and the feminizing actions of FOXL2, estrogen, and retinoic acid. In this chapter, we review recent knowledge regarding the SRY-dependent sex determination system during the critical time window and discuss the antagonistic interaction between testicular and ovarian factors during the late fetal, perinatal, and postnatal periods in mice.

CRISPR/Cas9-mediated knock-in of the murine Y chromosomal Sry gene

Mammalian zygote-mediated genome editing via the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is widely used to generate genome-modified animals. This system allows for the production of loss-of-function mutations in various Y chromosome genes, including Sry, in mice. Here, we report the establishment of a CRISPR-Cas9-mediated knock-in line of Flag-tag sequences into the Sry locus at the C-terminal coding end of the Y chromosome (YSry-flag). In the F1 and successive generations, all male pups carrying the YSry-flag chromosome had normal testis differentiation and proper spermatogenesis at maturity, enabling complete fertility and the production of viable offspring. To our knowledge, this study is the first to produce a stable Sry knock-in line at the C-terminal region, highlighting a novel approach for examining the significance of amino acid changes at the naive Sry locus in mammals.

Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ

In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

Defects in the first wave of folliculogenesis in mouse XO ovaries

In mouse ovaries, the first wave of folliculogenesis perinatally starts near the medullary region, which directs the initial round of follicular growth soon after birth. At the same time, cortical primordial follicles start forming in the ovarian surface region, and then some are cyclically recruited for the second and subsequent rounds of follicular growth. Recent studies suggest different dynamics between the first and subsequent waves of follicular growth in postnatal ovaries. However, the phenotypic differences between these phases remain unclear. Here, we show direct evidence that XO female mice, a murine model for Turner Syndrome, lack the first wave of folliculogenesis. Our histopathological analyses of XX and XO littermates revealed a lack of anti-Müllerian hormone (AMH)-positive primary follicles in the XO ovaries by 4 days post partum (dpp). This loss of first follicles was also confirmed by histological bioassay for SRY-dependent SOX9 inducibility, a specific marker for the first follicular granulosa cells. In contrast, cortical primordial follicles formed properly in XO ovaries, and some of them formed primary and secondary follicles in the subcortical region by 7 dpp. They rapidly developed into late antral follicles, showing similarities to XX littermate ovaries by 21 dpp. These results suggest distinct X-monosomy effects between the first and subsequent waves of follicular growth, highlighting the high susceptibility to elimination of XO oocytes in the first wave of mammalian folliculogenesis.