The effect of cryopreservation media on the quality of β-thalassemia mouse spermatozoa

Background: The mouse model of human diseases is commonly used for biomedical study, including β-thalassemia (β-thal), an inherited hemoglobin disorder. Maintaining the mice strain by natural mating systems is costly and seems impractical, especially during the COVID-19 pandemic. Sperm-freezing is a cost-effective solution for β-thal mouse colony management. Aim: To determine appropriate cryopreservation media for β-thal mouse spermatozoa to establish a β-thal mouse sperm bank. Methods: The epididymal spermatozoa of C57BL/6 wild-type (WT) and β-globin gene knockout thalassemia (BKO) mice were frozen in four freezing media: I) raffinose–skim milk–monothioglycerol (MTG), II) raffinose–skim milk– glutamine, III) raffinose–egg yolk–glycerol, and IV) egg yolk–TES–Tris. The sperm quality was assessed prior to and following freeze-thawing. Results: Compared with WT counterparts, the viable spermatozoa before freezing exhibiting elevated levels of oxidative stress were significantly greater in BKO (p = 0.01). After thawing, the membrane integrity of BKO spermatozoa preserved in I was significantly lower (p = 0.001). The sperm viability and membrane integrity of BKO males were also inferior when media III and IV were used (p = 0.008–0.027). The amount of oxidative stress in the spermatozoon of BKO mice was significantly greater when preserved in I, III, and IV (p = 0.002–0.044). Comparing freezing media, the motility and acrosome integrity of WT and BKO spermatozoa preserved in IV were significantly higher than those in other media (p < 0.001 to p = 0.01). Spermatozoa with the highest mitochondrial membrane potential were observed in I in both genotypes (p = 0.012 to p > 0.05). The viability, membrane integrity, and oxidative stress of post-thaw BKO spermatozoa did not significantly differ among freezing solutions. Conclusion: Irrespective of freezing media, spermatozoa of BKO males are rather more sensitive to cryopreservation than those of WT. Raffinose–skim milk–MTG/glutamine, raffinose–egg yolk–glycerol, and egg yolk–TES–Tris can all be used to preserve BKO mouse spermatozoa. However, with slightly better sperm characteristics, egg yolk–TES– Tris may be a diluent of choice for BKO mouse sperm cryopreservation. The addition of a reducing agent to thawing media is also strongly recommended to efficiently prevent oxidative stress and therefore improve frozen-thawed sperm survival

The Roles of Mitophagy and Autophagy in Ineffective Erythropoiesis in β-Thalassemia

β-Thalassemia is one of the most common genetically inherited disorders worldwide, and it is characterized by defective β-globin chain synthesis leading to reduced or absent β-globin chains. The excess α-globin chains are the key factor leading to the death of differentiating erythroblasts in a process termed ineffective erythropoiesis, leading to anemia and associated complications in patients. The mechanism of ineffective erythropoiesis in β-thalassemia is complex and not fully understood. Autophagy is primarily known as a cell recycling mechanism in which old or dysfunctional proteins and organelles are digested to allow recycling of constituent elements. In late stage, erythropoiesis autophagy is involved in the removal of mitochondria as part of terminal differentiation. Several studies have shown that autophagy is increased in earlier erythropoiesis in β-thalassemia erythroblasts, as compared to normal erythroblasts. This review summarizes what is known about the role of autophagy in β-thalassemia erythropoiesis and shows that modulation of autophagy and its interplay with apoptosis may provide a new therapeutic route in the treatment of β-thalassemia. Literature was searched and relevant articles were collected from databases, including PubMed, Scopus, Prospero, Clinicaltrials.gov, Google Scholar, and the Google search engine. Search terms included: β-thalassemia, ineffective erythropoiesis, autophagy, novel treatment, and drugs during the initial search. Relevant titles and abstracts were screened to choose relevant articles. Further, selected full-text articles were retrieved, and then, relevant cross-references were scanned to collect further information for the present review

Dysregulation of microRNA in Thalassemia

生体の働きは，遺伝子の発現によりタンパク質が合成されて支えられている．その異常を捉えることは，遺伝性疾患や後天的な遺伝子異常で引き起こされる腫瘍のみならず様々な病態を的確に診断し最適の治療法を選択できることを可能にした．さらに，多くの遺伝子の発現が細胞内や様々な体液中に存在するmicroRNA（miRNA）により調節されていることが明らかになってきた．miRNA は個体発生，細胞増殖・分化，アポトーシス，腫瘍化に関与していること，腫瘍や心血管疾患，代謝疾患などで病態特異的に変化していることが報告され，新世代のバイオマーカーや治療法として注目されている．サラセミア（地中海貧血）は全世界で罹患者が最も多い遺伝性疾患の一つであり，主に熱帯・亜熱帯地域に居住している人々が高率に罹患している先天性貧血である．毎年6 万人の重症型サラセミアの新生児が出生している．死産，流産が多い上に，出生しても重度の鉄過剰症，輸血依存性貧血となり熱帯・亜熱帯地域の国民の健康を著しく損ねている．サラセミアを代表とするヘモグロビンの合成や構造異常は，1970 年代に分子診断が導入されて臨床応用された初めての疾患であるが，治療は生涯にわたる赤血球輸血と鉄過剰症の予防のための徐鉄療法に依存しており，分子レベルの決定的な根治療法は未だ開発されるに至っていない．我々は過去10 年間，タイ国マヒドン大学などASEAN 諸国の大学との共同研究として，いわばアジア保健学の課題の一つであるサラセミアのmiRNA を解析し，新たなバイオマーカー・治療法の開発を試みてきた．本稿では，サラセミアを概説しmiRNA解析の有効性について紹介する

Microparticles from β-thalassaemia/HbE patients induce endothelial cell dysfunction

Abstract Thromboembolic complication occurs frequently in β-thalassaemia/HbE patients, particularly in splenectomised patients. Endothelial cells play an important role in thrombosis. There is strong evidence of endothelial cell activation and dysfunction in β-thalassaemia. Microparticles (MPs) are associated with thrombosis and endothelial cell dysfunction in many diseases including β-thalassaemia. However, the effect of thalassaemic-MPs on endothelial cells mediating thrombus formation has not been elucidated. In this study, the effects of circulating MPs from β-thalassaemia/HbE patients on endothelial cell functions were investigated. The results showed that MPs directly induce tissue factor, interleukin (IL)-6, IL-8, intracellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin expression in human umbilical vein endothelial cells (HUVECs). Notably, the levels of these endothelial cell activation markers were significantly increased in HUVECs treated with MPs obtained from splenectomised β-thalassaemia/HbE patients when compared to MPs from non-splenectomised patients or normal subjects. The increased endothelial cell activation ultimately lead to increased monocyte-endothelial cell adhesion. THP-1 and HUVECs adhesion induced by MPs from normal subjects, non-splenectomised and splenectomised patients increased to 2.0 ± 0.4, 2.3 ± 0.4 and 3.8 ± 0.4 fold, respectively when compared to untreated cells. This finding suggests that MPs play an important role on thrombosis and vascular dysfunction in β-thalassaemia/HbE disease, especially in splenectomised cases