Improvement in blastocyst quality by neurotensin signaling via its receptors in bovine spermatozoa during in vitro fertilization.

Previously, we reported that neurotensin (NT), which is expressed in the uterus and oviduct, enhanced bovine sperm capacitation and acrosome reactions. As NT mRNA expression in bovine oviducts increases dramatically in the follicular phase, we hypothesized that NT modulates fertilization and subsequent conception in cattle. The objective of this study was to evaluate the effect of NT on embryo development and blastocyst quality. The rate of embryo cleavage was significantly increased by the addition of NT to the fertilization medium. Furthermore, the total number of cells and numbers of cells in the inner cell mass of blastocysts were significantly increased by NT during in vitro fertilization (IVF). These results suggested that NT enhanced the efficiency of early bovine embryo development and blastocyst quality. The expression of NT receptors (NTRs) in sperm, testes, oocytes, and cumulus cells was evaluated to determine whether NT acted via NTRs in sperm alone or in both male and female reproductive cells during IVF. Immunocytochemistry and reverse transcription polymerase chain reaction revealed that NTR1 and NTR2 were expressed in sperm and testes, but not in oocytes and cumulus cells. We propose that NT selectively acts upon sperm via NTR1 and NTR2 during IVF to improve the cleavage rate and quality of blastocysts, which are important determinants of sperm quality for successful conception. This research supports our hypothesis that NT acts as a key modulator of fertilization and conception in cattle. Further studies are necessary to apply our findings to the industrial framework of bovine reproduction.

Glutathione treatment of Japanese Black bull sperm prior to intracytoplasmic sperm injection promotes embryo development.

Intracytoplasmic sperm injection (ICSI) was expected to enable more efficient use of sperm from sires with preferable genetic traits and result in a generation containing a larger number of offspring with superior genetic characteristics in livestock. However, the efficiency of the early development of embryos produced by ICSI is still far from satisfactory in cattle. The present study aimed to investigate the effects of the treatment of cryopreserved sperm with glutathione (GSH) on the early development of embryos produced by ICSI in Japanese Black cattle. Moreover, the disulfide bond state and mitochondrial function were investigated in the sperm treated with GSH to confirm the effectiveness of the abovementioned treatment. We also investigated the effect of 7% ethanol activation treatment on the developmental ability of ICSI embryos using GSH-treated sperm. There was no effect on the blastocyst rate from the activation treatment. When sperm-injected oocytes were cultured in vitro, the treatment with GSH significantly improved the early development of embryos. Specifically, the rates of embryos reaching the 4-8-cell stage and blastocyst stage were significantly higher in ICSI with GSH-treated sperm (71.4% and 31.0%, respectively) than that with the control sperm (36.6% and 7.0%, respectively). Moreover, the GSH-treated sperm treatment significantly decreased the number of disulfide bonds in the sperm head (as shown by monobromobimane staining) and enhanced the mitochondrial function in the sperm middle piece (as shown by Rhodamine 123 staining and the adenosine triphosphate-dependent bioluminescence assay). Based on these results, we suggest that the treatment of cryopreserved sperm with GSH might contribute to the improvement of ICSI techniques for the production of blastocysts in Japanese Black cattle.

[Usefulness of an anti-mouse cerebellar tissue-derived antigen antibody test in predicting immunotherapy efficacy in patients with idiopathic cerebellar ataxia].

BACKGROUND: Autoimmune cerebellar ataxia (AICA) is a general term for diseases in which the cerebellum is damaged by an autoimmune mechanism. For the diagnosis of the AICA, anti-thyroid antibodies (anti-thyroid peroxidase antibody and anti-thyroglobulin antibody), anti-glutamic acid decarboxylase (GAD) antibodies, and anti-gliadin antibodies are measured. Immunotherapy is known to be effective for AICA, but some patients with effective immunotherapy lack autoantibodies associated with cerebellar ataxia. The purpose of this study was to clarify whether the effectiveness of immunotherapy in patients with suspected AICA could be predicted by anti-mouse cerebellar tissue-derived antigen antibody tests. METHODS: This study was conducted on 25 patients with idiopathic cerebellar ataxia (excluding multiple system atrophy, hereditary spinocerebellar degeneration, cancer-bearing patients, and patients taking phenytoin) who received immunotherapy from 2005 to 2016 at Tokyo Medical University Hachioji Medical Center. The patients were suspected of having AICA because they were positive for cerebellar ataxia-related autoantibodies (anti-thyroid antibody, anti-GAD antibody, anti-gliadin antibody, or anti-transglutaminase 6 antibody) or other autoantibodies. Antibodies that bind to mouse cerebellar tissue-derived antigens were defined as "anti-mouse cerebellar tissue-derived antigen antibodies" in this study, and their IgG-class antibodies were comprehensively measured using a slot blot. RESULTS: Anti-mouse cerebellar tissue-derived antigen antibody test results were correlated with immunotherapy efficacy. Furthermore, the combination of anti-mouse cerebellar tissue-derived antigen and anti-GAD antibody tests could predict the effectiveness of immunotherapy with 83% sensitivity and 100% specificity, while the combination of the anti-mouse cerebellar tissue-derived antigen, anti-GAD, and anti-gliadin (IgA class) antibody tests could predict the effectiveness of immunotherapy with 94% sensitivity and 86% specificity. CONCLUSION: Anti-mouse cerebellar tissue-derived antigen antibody tests could help to provide useful information for immunotherapy administration to patients with idiopathic cerebellar ataxia suspected to be AICA.