Embryo production by intracytoplasmic injection of sperm retrieved from Meishan neonatal testicular tissue cryopreserved and grafted into nude mice

Testicular xenografting, combined with cryopreservation can assist conservation of the genetic diversity of indigenous pigs by salvaging germ cells from their neonatal testes. Using Meishan male piglets as an example, we examined whether testicular tissue would acquire the ability to produce sperm after cryopreservation and grafting into nude mice (MS group). For comparison, testicular tissue from neonatal Western crossbreed male piglets was used (WC group). Sixty days after xenografting (day 0 = grafting), MS grafts had already developed seminiferous tubules containing sperm, whereas in the WC grafts, sperm first appeared on day 120. The proportion of tubules containing spermatids and sperm was higher in the MS group than in the WC group between days 90 and 120. Moreover, in vitro‐matured porcine oocytes injected with a single sperm obtained from the MS group on day 180 developed to the blastocyst stage. The blastocyst formation rate after injection of the xenogeneic sperm was 14.6%, whereas the ratio in the absence of such injection (attributable to parthenogenesis) was 6.7%. Thus, cryopreserved Meishan testicular tissue acquired spermatogenic activity in host mice 60 days earlier than Western crossbreed tissue. Such xenogeneic sperm are likely capable of generating blastocysts in vitro.     

Synthetic microRNA‐205 exhibited tumour suppression in spontaneous canine malignant melanoma by intratumoral injection

MicroRNAs (miRNA) are small, noncoding RNA molecules consisting of 18 to 25 nucleotides. Malignant melanomas (MMs) are one of the most common malignancies in both dogs and humans. We previously reported that chemically modified synthetic miRNA‐205 (miR‐205BP/S3) inhibits melanoma growth in vitro and in vivo. The present study aimed to evaluate the efficacy of intratumoral administration of synthetic miR‐205 for spontaneous CMMs and to evaluate its potential as systemic therapy. Ten dogs with various stages of MM were treated with miR‐205BP/S3 injected into tumours. Adverse effects (AEs) were assessed in accordance with the Veterinary Cooperative Oncology Group‐Common Terminology Criteria for Adverse Events (VCOG‐CTCAE) v1.1 guidelines. Five cases attained complete remission (CR), three attained stable disease (SD), and two cases displayed characteristics of progressive disease (PD). In all cases, no changes were observed in the blood parameters upon miRNA administration, and miR‐205BP/S3 administration did not yield any side effects. The present results suggest that intratumoral administration of miR‐205BP/S3 is a potentially applicable treatment for canine melanoma.     

Changes in circulating and testicular levels of inhibin A and B during postnatal development in bulls

We investigated testicular and circulating levels of dimeric inhibins in Holstein bulls from the infantile to postpubertal periods (5 to 50 weeks of age) and examined the relationship between the profiles of circulating dimeric inhibins and FSH. Concentrations of total inhibin and inhibin B in the testis were highest at 4 to 5 weeks of age but decreased gradually as the bulls aged. Testicular inhibin A levels showed a gradual decline to a nadir at 15 to 26 weeks of age, but by 39 weeks, they were high again. The contents of total inhibin, inhibin A, and inhibin B per testis generally increased with age. Fractionation of testicular homogenates obtained from 15-week-old bulls by a combination of immunoaffinity chromatography and SDS-PAGE confirmed the presence of two major molecular weight forms (32 and 45 kDa) of dimeric inhibins in the testes. Circulating levels of total inhibin and inhibin A showed a significant increase in bulls at around 10 to 14 weeks of age compared to the levels between 5 and 7 weeks of age but decreased thereafter. However, immunoreactivity for inhibin B was not detected in the peripheral circulation, probably because of low sensitivity of the inhibin B assays. The concentrations of plasma FSH were high at 5 weeks of age but declined to lower levels between 11 and 40 weeks, and then increased from 41 weeks onward. There was no significant correlation between the plasma levels of FSH and inhibin A or total inhibin. The results clearly indicate that the bull testis produces inhibin A and B and secretes at least inhibin A into the circulation during postnatal development. However, the profile of circulating FSH in bulls shows no reciprocal relationship with the inhibin A or total inhibin profile during the postnatal period.     

Successful long term culture of immature porcine sertoli cells in the reconstructed testicular cell cord

The ultimate goal of this study was to establish an in vitro system to produce sperms. To pursue this goal, immature porcine testicular cells were cultured in stereostructural form and cultured testicular cord was investigated morphologically. At 4 weeks of age, the seminiferous tubules of the porcine testes consisted of undifferentiated germ cells (gonocytes and undifferentiated spermatogonia) and immature Sertoli cells. The interstitial tissue was largely occupied by Leydig cells. The testes were enzymatically digested, and the dispersed cells were encapsulated with alginate either immediately or after freeze-thawing. The resulting testicular cell cords were cultured for up to 10 weeks. After 2 weeks of culture, Sertoli cells, which were identified by their inhibin-positive reaction in immunohistochemistry, and Leydig cells, which were identified by their morphological characteristics, were observed in the cords. Neither undifferentiated nor differentiated types of germ cells were detected. The number of cells in the cords progressively decreased during the culture period. In order to discover the fate of the Sertoli cells, the level of inhibin in the spent media was determined. Inhibin in the media was at a detectable level after 2 days of culture. The levels increased and peaked at 2 weeks. When frozen-thawed testicular cells were applied to the culture, the peak level was maintained for over 8 weeks, in contrast to the gradual decrease of inhibin level when fresh cells were cultured. These results indicate that the culture conditions can sustain the survival of Sertoli cells. Further improvement is required for proliferation and differentiation of germ cells.     

Biochemical properties of the cell wall in the Arabidopsis mutant bor1‐1 in relation to boron nutrition

We examined concentrations of boron (B) and dimerization of rhamnogalacturonan II (RG‐II), a B‐binding polysaccharide, in the cell wall of a low‐B sensitive mutant of Arabidopsis thaliana, bor1‐1, to investigate possible effects of the bor1‐1 mutation on the biochemical form of pectins in the cell wall. In the bor1‐1 mutant, B concentrations in the cell wall from shoots were lower than those in the wild type at low B supply, whereas they were similar at sufficient B supply. The amount of B present as borate ester of the RG‐II dimer (dRG‐II‐B) in the bor1‐1 mutant was lower than that in the wild type at low B supply. In the wild type, about 90 % of RG‐II was present as dRG‐II‐B, both, at low and sufficient B supply. In the bor1‐1 mutant, about 60 % of RG‐II was in its monomeric form (mRG‐II) at low B supply, whereas more than 85 % of it was present as dRG‐II‐B at sufficient B supply. However, similar as the wild type, mRG‐II derived from the bor1‐1 mutant was able to form dRG‐II‐B in vitro in the presence of borate and lead. Sugar composition of cell wall fractions was similar in both genotypes. These results suggest that the polysaccharide composition in the cell wall was not strongly affected by the bor1‐1 mutation. The observed difference in dimerization of RG‐II at low B supply is most likely due to a reduced B concentration in the shoots of the bor1‐1 mutant.     

Introduction of Yeast Metallothionein Gene (CUP1) into Plant and Evaluation of Heavy Metal Tolerance of Transgenic Plant at the Callus Stage

For phytoremediation, it is necessary to use plants with a large biomass and ability to absorb and accumulate heavy metals. Attempts were made to produce heavy metal-tolerant plants by introducing a metallothionein gene, CUP 1, into plants with a large biomass. In the present study, we evaluated the heavy metal tolerance of sunflower plants at the callus stage in terms of 2,3,5-triphenyl-tetrazolium chloride (TTC)-reducing activity, measured the mRNA expression level of the introduced gene, and selected tolerant clones. The transgenic calli of several strains showed a high TTC-reducing activity even after treatment with Cd 200 μM for 10 d. In the transgenic calli of a few strains, which showed a low TTC-reducing activity, the amount of metallothionein was lower than that in the other transgenic strains. Conversely, some transgenic calli with a high TTC-reducing activity expressed a much higher amount of mRNA of the introduced gene.     

Intrinsic Biodegradation of Heavy Oil from Nakhodka and the Effect of Exogenous Fertilization at a Coastal Area of the Sea of Japan

We performed a field experiment in thebiodegradation of heavy oil spilled from the Russian tankerNakhodka on a beach in the Sea of Japan. We collectedoil-contaminated cobbles and treated half with nitrogen andphosphorus slow-release fertilizers to stimulate microbialdegradation of the oil; the other half acted as unfertilizedcontrols. The cobbles were placed in porous acrylic vessels andsubmerged. We monitored changes in the oils, macronutrients,microbial community structure and amount of chlorophyll a. There were no significant differences in these criteriabetween the fertilized and unfertilized vessels, apart from anincrease in chlorophyll a in the fertilized vessels.However, there was a major intrinsic degradation of semi-volatile oil compounds in the unfertilized vessels; this occurred at a rate similar to that in the fertilized vessels, despite the low concentration of macronutrients in the seawater at the site.