Antioxidant vitamins and lysophospholipids are critical for inducing mouse spermatogenesis under organ culture conditions.

In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.

Simultaneous Intake of Euglena gracilis and Vegetables Exerts Synergistic Anti-Obesity and Anti-Inflammatory Effects by Modulating the Gut Microbiota in Diet-Induced Obese Mice.

We determined whether the anti-obesity effect provided by the consumption of Euglena gracilis (Euglena), which is rich in insoluble dietary fiber, could be enhanced by the co-consumption of vegetables with an abundance of soluble dietary fiber. Nine-week-old male C57BL/6J mice were divided into five groups as follows: group 1 received a normal diet, group 2 received a high-fat diet, and groups 3, 4, and 5 received high fat diets containing 0.3% paramylon, 1.0% Euglena, or 1.0% Euglena plus 0.3% vegetables (barley leaf, kale, and ashitaba), respectively. Mice were fed ad libitum until 18 weeks of age. Euglena intake significantly reduced visceral fat accumulation in obese mice, and co-consumption of vegetables enhanced this effect. Consumption of Euglena with vegetables reduced adipocyte area, suppressed the expression of genes related to fatty acid synthesis, upregulated genes related to adipocyte lipolysis, and suppressed serum markers of inflammation. Notably, we also observed an increase in the fraction of short-chain fatty acid-producing beneficial bacteria, a reduction in harmful bacteria that cause inflammation, and an increase in short-chain fatty acid production. Therefore, the co-consumption of vegetables enhanced the anti-obesity and anti-inflammatory effects of Euglena, likely by modulating the gut microbiota composition.

Simultaneous Intake of Euglena Gracilis and Vegetables Synergistically Exerts an Anti-Inflammatory Effect and Attenuates Visceral Fat Accumulation by Affecting Gut Microbiota in Mice.

We determined whether the benefits provided by the consumption of Euglena gracilis (Euglena), which is a unicellular photosynthesizing green alga and rich in insoluble dietary fiber paramylon, can be enhanced by the co-consumption of vegetables that are rich in soluble dietary fiber. Nine-week-old male C57BL/6J mice were divided into four groups: group 1 received normal diet, whereas groups 2, 3 and 4 received normal diet containing 0.3% paramylon, 1.0% Euglena, or 1.0% Euglena plus 0.3% vegetables (barley leaf, kale and ashitaba), respectively. Mice were fed ad libitum until 18 weeks of age. Euglena intake significantly decreased serum markers of inflammation and co-consumption of vegetables enhanced this reduction. Notably, we observed an increase in the fraction of beneficial bacteria producing short-chain fatty acids, a reduction in harmful bacteria that cause inflammation and an increase in short-chain fatty acid production. Visceral fat accumulation was also reduced. Subsequent analyses showed that co-consumption of Euglena with vegetables reduced adipocyte area, suppressed the expression of genes related to fatty acid synthesis and increased the expression of genes related to adipocyte growth and lipolysis. Therefore, co-consumption of Euglena with vegetables enhanced its anti-inflammatory effect and the inhibitory effect on visceral fat accumulation likely by modulating the composition of gut microbiota.

In vitro mouse spermatogenesis with an organ culture method in chemically defined medium.

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

Animal-cell culture media: History, characteristics, and current issues.

Background: Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal-cell culture media. Methods: A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. Results: At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical-based synthetic media because naturally derived ingredients have their disadvantages such as large batch-to-batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum-containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Conclusions: Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.

Oral administration of Euglena gracilis Z and its carbohydrate storage substance provides survival protection against influenza virus infection in mice.

Euglena gracilis Z is a micro-algae that is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of Euglena gracilis Z has ß-1, 3-glucan structure. Euglena gracilis Z and paramylon are reported to affect the immune system. In this study, we investigated the protective effects of Euglena gracilis Z and paramylon against influenza virus infection in mice. Euglena gracilis Z and paramylon were administered to mice as a 2% dietary mixture ad libitum. At 2 weeks after initiation of dietary administration, mice were infected intranasally with influenza virus A/PR/8/34 (H1N1). Survival rate was monitored 10 days after infection. In addition, we performed virus titer and cytokine profiles in the lung. High survival rates were observed for Euglena gracilis Z and paramylon-treated groups compared to the control group. Significantly lower virus titer in the lung was observed in the Euglena gracilis Z and paramylon-treated groups compared to the control group from day 1 after infection. Higher amount of IL-1ß, IL-6, IL-12 (p70), IFN-γ, and IL-10 was observed in the paramylon groups compared to the control group. Our data therefore reveals a novel immunoregulatory role of the Euglena gracilis Z and paramylon which provides protection against influenza virus infection.

Single human sperm cryopreservation method using hollow-core agarose capsules.

OBJECTIVE: To develop an efficient cryopreservation method using a single sperm. DESIGN: Experimental study. SETTING: Laboratory of a private institute. PATIENT(S): A fertile donor. INTERVENTION(S): We produced hollow-core capsules with agarose walls. A single human sperm was injected into each capsule as per the conventional intracytoplasmic sperm injection (ICSI) method. The capsules that contained the spermatozoa were cryopreserved on polycarbonate or nylon mesh sheets using nitrogen vapor. Before their use, the capsules were thawed and recovered. The motile spermatozoa in the capsules were counted. MAIN OUTCOME MEASURE(S): The recovery rates of the agarose capsules and the spermatozoa in these capsules after thawing and the mortality and survival rates of the spermatozoa. RESULT(S): The recovery rates of the capsules were 91.5% (75/82) using polycarbonate sheets (PS) and 98.3% (59/60) using mesh sheets (MS) after thawing. The recovered capsules were not at all damaged. The recovery rates of the spermatozoa were 91.5% (75/82) using PS and 96.7% (58/60) using MS. Sperm motility rates were 85.3% (64/75) and 82.8% (48/58), whereas the survival rates of the immotile spermatozoa by the hypoosmotic swelling test were 81.8% (9/11) and 50.0% (5/10); furthermore, the total survival rates of the spermatozoa were 97.3% (73/75) and 91.4% (53/58) using PS and MS, respectively. There was no significant difference between the results obtained using PS and MS. CONCLUSION(S): A cryopreservation method for a single sperm using an agarose capsule has been developed. The method is expected to be useful in ICSI treatment in patients with few spermatozoa.

Oral administration of paramylon, a beta-1,3-D-glucan isolated from Euglena gracilis Z inhibits development of atopic dermatitis-like skin lesions in NC/Nga mice.

Paramylon is a beta-1,3-D-glucan isolated from Euglena gracilis Z. This study was designed to evaluate the suppressive effects of the oral administration of paramylon on the development of atopic dermatitis (AD)-like skin lesions induced by repeated application of 2,4,6-trinitrochlorobenzene (TNCB) in sensitized NC/Nga mice. The effects of paramylon were assessed by measuring macroscopical and histopathological findings of skin, ear swelling, serum levels of total IgE, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) and IL-18 and IL-12 contents in the skin lesions. Oral administration of paramylon inhibited the development of AD-like skin lesions as exemplified by a significant decrease in dermatitis scores for the back, ear swelling and hypertrophy of the skin, infiltration of inflammatory cells in the skin, and serum IgE levels. Oral administration of paramylon reduced serum levels of both IL-4 and IFN-gamma and IL-18 and IL-12 contents in the skin lesions. Oral administration of paramylon did not cause weight loss, as was observed with prednisolone. These results suggest that paramylon inhibits the development of AD-like skin lesions in NC/Nga mice by suppressing both the T-helper (Th) 1 and Th 2 cell responses. Our results indicate that paramylon treatment could provide an effective alternative therapy for the management of AD.

Atf1 is a target of the mitogen-activated protein kinase Pmk1 and regulates cell integrity in fission yeast.

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.

Rho2 is a target of the farnesyltransferase Cpp1 and acts upstream of Pmk1 mitogen-activated protein kinase signaling in fission yeast.

We have previously demonstrated that knockout of the calcineurin gene or inhibition of calcineurin activity by immunosuppressants resulted in hypersensitivity to Cl- in fission yeast. We also demonstrated that knockout of the components of the Pmk1 mitogen-activated protein kinase (MAPK) pathway, such as Pmk1 or Pek1 complemented the hypersensitivity to Cl-. Using this interaction between calcineurin and Pmk1 MAPK, here we developed a genetic screen that aims to identify new regulators of the Pmk1 signaling and isolated vic (viable in the presence of immunosuppressant and chloride ion) mutants. One of the mutants, vic1-1, carried a missense mutation in the cpp1+ gene encoding a beta subunit of the protein farnesyltransferase, which caused an amino acid substitution of aspartate 155 of Cpp1 to asparagine (Cpp1(D155N)). Analysis of the mutant strain revealed that Rho2 is a novel target of Cpp1. Moreover, Cpp1 and Rho2 act upstream of Pck2-Pmk1 MAPK signaling pathway, thereby resulting in the vic phenotype upon their mutations. Interestingly, compared with other substrates of Cpp1, defects of Rho2 function were more phenotypically manifested by the Cpp1(D155N) mutation. Together, our results demonstrate that Cpp1 is a key component of the Pck2-Pmk1 signaling through the spatial control of the small GTPase Rho2.