Infestation of the Parasitic Isopod Mothocya parvostis on Juveniles of the Black Sea Bream Acanthopagrus schlegelii as an Optional Intermediate Host in Hiroshima Bay.

In Hiroshima Bay, parasitic isopods of the genus Mothocya infest the black sea bream Acanthopagrus schlegelii (Bleeker, 1854) and the Japanese halfbeak Hyporhamphus sajori (Temminck and Schlegel, 1846), two fish species that are abundant and commercially important in the Seto Inland Sea of Japan. Immature and mature Mothocya individuals can infect both juveniles and adults of H. sajori, while immature Mothocya are known to parasitize juveniles of A. schlegelii; i.e., no Mothocya parasites are found in adult A. schlegelii. The identification of the immature Mothocya parasitizing juveniles of A. schlegelii remains uncertain, because Mothocya species are morphologically identifiable only based on adult females. Also, the biological/ecological relationship between the hosts and parasites has not been studied. Here, we identified the parasites on A. schlegelii as Mothocya parvostis Bruce, 1986 by molecular sequence analyses along with other parasites obtained from H. sajori, the latter being morphologically confirmed by comparison with paratype materials of M. parvostis as well as the similar congener Mothocya sajori Bruce, 1986. The growth rates of the infected A. schlegelii juveniles from June to September in the years 2013-2015 and 2018 were significantly lower than those of the uninfected ones, suggesting a negative effect of the infection on the hosts. Our data on the prevalence and duration of the infection, as well as the body size gain of the hosts and parasites, corroborate a hypothesis that M. parvostis would utilize A. schlegelii as an optional intermediate host before it reaches the final host, H. sajori.

Responsiveness of rabbits to superovulation treatment by a single injection of follicle-stimulating hormone with aluminum hydroxide gel.

Aluminum hydroxide gel (Al-gel), which is used as an adjuvant, can absorb macromolecules. We investigated the applicability of Al-gel to the sustained release of follicle-stimulating hormone (FSH) as a simplified method of superovulation (SOV) in rabbits. The responsiveness of rabbits to SOV by a single injection of FSH dissolved in Al-gel suspension (3.2 mg Al/ml) and in 10% (w/v) polyvinylpyrrolidone (PVP), and by multiple injections of FSH in saline was examined. The numbers of total and fertilized eggs recovered from rabbits treated with FSH in Al-gel (40.5 and 26.3, respectively) were similar to multiple injections (47.4 and 28.6, respectively) and were significantly greater (P < 0.05) than single injection of FSH with PVP (17.3 and 11.5, respectively). We also compared the plasma FSH levels of rabbits which were induced SOV by multiple or a single injection of Al-gel. Al-gel provided sustained release of FSH to the blood stream at a high enough dose for SOV. Moreover, the developmental competence of the pups of DNA-injected embryos from rabbits treated with a single injection of FSH mixed with Al-gel (18%) was similar to that of DNA-injected embryos, recovered from rabbits treated with FSH dissolved in saline (21%). Two transgenic pups were obtained from embryos recovered from rabbits by a single injection of FSH with Al-gel. These results indicate that a single injection of FSH with Al-gel is an effective method for SOV of rabbit and that this technique is applicable to research requiring large numbers of rabbit embryos such as the production of transgenic rabbits.

Establishment and characterization of CAG/EGFP transgenic rabbit line.

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.

Small eye phenotypes observed in a human tau gene transgenic rat.

We developed a rat line showing small eye from transgenic rats that were obtained by microinjection of a DNA segment containing the human (h)tau cDNA (GenBank: BC000558: 31-677,774-1180) expressed under control of CAG promoter, which is related to Alzheimer disease, into the pronuclei rat embryos. The rat line was established by selective brother-sister mating of rats showing small eyes. Of 11 offspring in the 11th generation, there were eight animals with microphthalmia and the transgene. The remaining three rats without transgene did not show the small eyes phenotype. The globes of affected rats were 1.2 mm in length compared with normal globes (3.5 mm), and all other ocular structures were normal. The expression of hTau protein was evident immunohistochemically in the ciliary body, extraocular muscle, lens epithelium, and pigment epithelium. Cytogenetic analysis suggested that the chromosome location of the transgene was chromosome 1 (1p12). This region may include genes related to lens development, such as Cat5.

Refined porcine follicle stimulating hormone promotes the responsiveness of rabbits to multiple-ovulation treatment.

We investigated whether refined follicle stimulating hormone (FSH) with only a little contaminating LH can promote the responsiveness of rabbits to multiple-ovulation treatment. One group of female rabbits was stimulated with refined porcine FSH (pFSH), an FSH source with low LH activity, and another group was treated with pFSH. The mean number of eggs recovered from donors stimulated with refined pFSH (27 +/- 3) was significantly greater (P<0.05) than that with pFSH (20 +/- 2). Furthermore, the mean number of remaining follicles of donors stimulated with refined pFSH (19 +/- 4) was significantly greater (P<0.05) than that with pFSH (12 +/- 1). To decrease the number of remaining follicles in donors treated with refined pFSH, the dose of human chorionic gonadotropin (hCG) was increased from 75 to 150. However, there were no differences in the numbers of eggs and remaining follicles. The results of the present study suggest that refined pFSH with little contaminating LH promotes the responsiveness of rabbits to multiple-ovulation treatment compared with pFSH.

Catalase in manipulation buffer enhances the developmental competence of DNA-injected embryos.

To improve the efficiency of transgenesis, we investigated the effects of a radical scavenger during microinjection on the development to blastocysts or pups of mouse pronuclear embryos, microinjected with the enhanced green fluorescent protein (EGFP) transgene. When embryos were microinjected in medium containing 0-1,000 units/ml catalase, the developmental rate to blastocysts was significantly higher (P<0.01) in 100-units/ml catalase (81%) than those in 0 and 1,000 units/ml (56 and 65%). To investigate the ontogenetic ability of DNA-injected embryos, EGFP-injected embryos manipulated under 0 or 100 units/ml catalase were transferred separately to recipient mice. The proportion of fetuses derived from EGFP-injected embryos manipulated under 100 units/ml catalase (29%) was significantly higher (P<0.05) than that manipulated under 0 units/ml catalase (19%). Furthermore, the numbers of transgenic pups were 17 in 100 units/ml catalase and 14 in 0 units/ml catalase. The results of the present study indicate that scavenging reactive oxygen species during in vitro micromanipulation is beneficial for the development of DNA-injected embryos.

Effect of a null mutation of the oviduct-specific glycoprotein gene on mouse fertilization.

The mammalian fertilization process takes place in a complex microenvironment within the female genital tract. A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), has been identified in oviductal fluid from various mammalian species, including humans. Although OGP is widely believed to be involved in the process of mammalian fertilization, including spermatozoon function and gamete interactions, based on experimental results obtained in vitro, its physiological significance remains controversial. The present study established OGP gene-null ( ogp (-/-)) mice, and primarily characterized their reproductive properties to study the physiological function(s) of OGP. Results obtained from studies using an in vivo or in vitro system showed that the fertility of ogp (-/-) females was within normal limits. These results indicate that OGP is not essential for the process of in vivo fertilization, at least in mice.

Successful cryopreservation of mouse ovaries by vitrification.

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.

Effects of cryopreservation of mouse embryos and in vitro fertilization on genotypic frequencies in colonies.

To evaluate the effects of cryopreservation and in vitro fertilization (IVF) on genotypic frequencies in mouse colonies, genotypic frequencies at 15 biochemical, 4 immunological and 20 microsatellite loci were examined in three colonies of MCH (ICR) mice derived from noncryopreserved embryos obtained by natural mating without the induction of superovulation, cryopreserved embryos obtained by natural mating with the induction of superovulation, and cryopreserved embryos obtained by the induction of superovulation and IVF. Three (Pgm-1, Ldr-1 and Hbb) out of the 15 biochemical loci, two (Thy-1 and H2K) out of four immunological loci and five (D5Mit18, D6Mit15, D12Mit5, D13Mit26, and D14Mit7) out of 20 microsatellite loci that showed polymorphisms in every colony were used for detection of genotypic frequencies. The genotypic frequencies of the loci in the three colonies did not differ from the predicted genotypic frequencies (P > 0.05). The results suggested that genetic drift does not occur among colonies established from treated and untreated embryos, and it was clear that the embryo banking by cryopreservation is suitable for preservation of outbred stock without genetic drift.

A testicular germ cell-associated serine-threonine kinase, MAK, is dispensable for sperm formation.

A member of the mitogen-activated protein kinase superfamily, MAK, has been proposed to have an important role in spermatogenesis, since Mak gene expression is highly restricted to testicular germ cells. To assess the biological function of MAK, we have established MAK-deficient (Mak(-/-)) mice. Mak(-/-) mice developed normally, and no gross abnormalities were observed. Spermatogenesis of the Mak(-/-) mice was also intact, and most of the mice were fertile. However, Mak(-/-) male-derived litter sizes and their sperm motility in vitro were mildly reduced. These data show that function of MAK is not essential for spermatogenesis and male fertility.