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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes a
b
a
Ai Tsuji , Toshinobu Nakamura & Katsumi Shibata a
Department of Nutrition, School of Human Cultures, The University of Shiga Prefecture, Hikone, Japan b
Department of Animal Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan Published online: 10 Oct 2014.
Click for updates To cite this article: Ai Tsuji, Toshinobu Nakamura & Katsumi Shibata (2015) Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes, Bioscience, Biotechnology, and Biochemistry, 79:2, 292-299, DOI: 10.1080/09168451.2014.968090 To link to this article: http://dx.doi.org/10.1080/09168451.2014.968090
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 2, 292–299
Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes Ai Tsuji1, Toshinobu Nakamura2 and Katsumi Shibata1,* 1 2
Department of Nutrition, School of Human Cultures, The University of Shiga Prefecture, Hikone, Japan; Department of Animal Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan
Received July 25, 2014; accepted September 12, 2014
Downloaded by [University of Birmingham] at 10:29 20 March 2015
http://dx.doi.org/10.1080/09168451.2014.968090
Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice. Key words:
biotin; deficiency; mouse; oocyte quality
No good-quality oocytes (chromosomal and spindle abnormalities oocytes) are the major factors in infertility, fetal loss (miscarriage), and conceptions, resulting in birth defects such as trisomy 21 (Down syndrome).1) Recent studies have shown that macronutrients affect the oocyte maturation2,3) and that high-fat diets in mice induce the formation of abnormal oocytes that has chromosome misalignment and spindle defects.2) Caloric restriction without malnutrition, depressed the aging-related increase in the oocyte aneuploidy and chromosomal misalignment.4) Biotin deficiency affects fetal development and growth.5) Biotin is a water-soluble vitamin that acts as a prosthetic group of carboxylases,6) regulates gene expression,7,8) and has a wide range of effects on the systemic processes such as development of embryo,9,10)immunity,11) growth,12) and metabolism.13) Biotin deficiency in experimental animals causes
growth retardation, alopecia, dermatitis, and neurological impairment.14) Several studies in rodents have reported that biotin deficiency also affects fetal development,5) with biotin deficiency during pregnancy being found to increase the rates of abnormal fetal development and growth, as well as affect the rates of absorption and embryonic death in rodents.5,15,16) Báez-Saldaña et al.17) also reported that biotin deficiency in young female mice induced ovary atrophy, estrus arrest, reduction in the oocyte of primordial and graafian follicles, and increase in serum estradiol, a hormone that was involved in oocyte growth. This study suggests that biotin deficiency decreased oocyte growth in the ovaries, but the effect of biotin deficiency on oocyte quality remained unclear. To our knowledge, however, no study has directly examinedthe effects of biotin deficiency on oocyte quality. Here, we investigated the direct effect of biotin deficiency and its subsequent restoration on oocyte quality.
Materials and methods Mice. Animals were allowed free access to food and water, and body weight was measured for every two days. Food intake of mice in metabolic cages was measured daily. Temperature was maintained at approximately 20 °C with 60% humidity and a 12-h light/dark cycle (lights on at 6:00 and off at 18:00). The care and treatment of the experimental animals conformed to the guidelines of the ethical treatment of laboratory animals set by the University of Shiga Prefecture (Shiga, Japan). Deficiency experiment. Female ICR mice (3 weeks old) were obtained from Charles River Laboratories (Tokyo, Japan) and immediately divided into two groups (control group, n = 21; biotin-deficient group, n = 30). For each group, approximately half (control group, n = 11; biotin-deficient group, n = 15) were individually housed in metabolic cages (LC-0335; CLEA Japan, Tokyo, Japan) to collect 24 h urine samples and
*Corresponding author. Email: [email protected] Abbreviations: 3-HIA, 3-hydoroxyisovaleric acid; DAPI, 4′6-diamidino-2-phenylindole dihydrochloride. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Downloaded by [University of Birmingham] at 10:29 20 March 2015
Biotin deficiency induces abnormal oocytes
293
half (control group, n = 10; biotin-deficient group, n = 15, five mice/plastic cage) in plastic cages. On day 22, mice in metabolic cages (n = 5 per group) were sacrificed to analyze the oocyte quality, while those in plastic cages (n = 5 per group) were sacrificed to analyze the biotin concentrations in the liver, ovaries, and uterus.
of cumulus cells by 5% hyaluronidase, (Sigma-Aldrich Inc., Tokyo, Japan) for 5 min at room temperature. Oocytes were counted and classified using a Hoffman light microscope as mature metaphase II (MII) or dead (condensed and fragmented cytoplasm). Oocytes from the experimental groups were analyzed in the blind study.
Recovery experiment. On day 22, the remaining mice of the biotin-deficient group in metabolic cages (n = 10) and plastic cages (n = 10) were randomly divided into two groups. Group 1 consisted of mice in individual metabolic cages (n = 5) and one plastic cage (n = 5) that maintained a biotin-deficient diet. Group 2 (the recovery group) consisted of mice in individual metabolic cages (n = 5) and one plastic cage (n = 5) that were fed a control diet for 20 days. The remaining mice in individual metabolic cages (n = 6) and one plastic cage (n = 5) were continuously fed the control diet. On Day 42, the mice housed in metabolic cages were sacrificed to analyze the oocyte quality, and five mice housed in plastic cages were sacrificed to analyze the biotin concentrations in the liver, uterus, and ovaries.
Immunofluorescence. Oocytes were collected from the control group (oocyte number; n = 101) and the biotin-deficient group (oocyte number; n = 123) on day 22, and from the control group (oocyte number; n = 240), the recovery group (oocyte number; n = 134), and the biotin-deficient group (oocyte number; n = 158) on day 42. Oocytes were collected by inducing superovulation and then puncturing the oviducts with forceps at which point they were washed in PBS and then moved in 5% protease (Sigma–Aldrich Inc., Tokyo, Japan) to soften and remove the zona pellucida. Oocytes were then extensively washed with PBS and fixed in PBS containing 4% paraformaldehyde for 15 min at room temperature. Oocytes were permeabilized with 0.2% Triton X-100 in PBS for 20 min at room temperature and blocked for 1 h in 5% goat serum (Sigma-Aldrich Inc., Tokyo, Japan) in PBST (0.05% Tween20, PBS) at room temperature. Oocytes were then washed with PBST and incubated for 1 h in a 1:4000 dilution of mouse anti-α-tubulin antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) in PBST containing 5% goat serum at room temperature, after which they were washed again and incubated for 1 h in a 1:500 dilution of goat anti-mouse IgG with Alexa Flour-488 and 4′ 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Dojindo Laboratories, Kumamoto, Japan). After washing, oocytes were mounted using PermaFluor aqueous mounting medium (Thermo Fisher Scientific Inc. Kanagawa, Japan) and analyzed by confocal fluorescence microscopy (FV10i; Olympus, Inc., Tokyo, Japan). Oocytes were classified as abnormal if: (1) the chromosomes failed to align on an otherwise normal meiotic spindle; (2) the spindle exhibited serious malformations; or (3) both spindle and chromosome alignment were normal, but one more pair of chromosomes was far removed from the spindle equator, most commonly behind one spindle pole. Oocytes with barrel-shaped bipolar spindles and distinct and wellorganized microtubule fibers, along with tightly aligned chromosomes on the metaphase plate, were classified as normal.
Diet. The control group was fed 30% egg white solids diet supplemented with 0.004 g biotin/kg diet, while the biotin-deficient group was fed 30% egg white solids diet (Table 1), as previously described.17) Egg white solids were obtained from CLEA Japan, Inc. (Tokyo, Japan), which contains avidin that forms a non-absorbable complex with biotin in the alimentary tract and results in biotin deficiency. So a sufficient quantity of biotin was therefore added to the control diet. Oocyte retrieval and classification. To get oocytes, mice which were randomly selected on day 20 or day 40 were superovulated with an intraperitoneal injection of 5 IU pregnant mare serum gonadotrophin (PMSG; product No. E164A; ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) followed by 5 IU human chorionic gonadotrophin (hCG; product No. E801A; ASKA Pharmaceutical) after 46 to 48 h. Oocytes were collected 18 h after hCG injection in EmbryoMax® FHM HEPES buffered medium (FHM; Millipore Corp., Billerica, MA, USA). Retrieved oocytes were denuded Table 1.
Diet compositions.
Egg white solids Gelatinized cornstarch Sucrose Corn oil Dextrin Cellulose Choline bitartrate Mineral mixture (AIN-93-G-MX)* Vitamin mixture (biotin free)* Biotin
Control diet (g/kg diet)
Biotin-deficient diet (g/kg diet)
300 309.3 154.2 80 50 50 2.5 42 12 0.004
300 309.3 145.2 80 50 50 2.5 42 12 –
*Composition of mineral and vitamin mixtures formulated to meet AIN-76A39–46).
294
A. Tsuji et al.
Downloaded by [University of Birmingham] at 10:29 20 March 2015
Biotin assay in urine, liver, plasma, uterus, and ovaries. Twenty-four hour urine samples on day 19 and day 39 were collected from mice in metabolic cages with amber bottles containing 1 mL of 1 mol/L HCl and were stored at −30 °C until use. To avoid the effect of superovulation on metabolism in mice, we collected a 24 h urine sample prior to PMSG injection. Biotin concentrations in urine samples on day 19 and day 39, and in the liver, uterus, and ovaries at day 22 and day 42 were assayed via a microbiological method with Lactobacillus plantarum ACTT 8014.18) Measurement of 3-hydoroxyisovaleric acid in urine. In rodents and humans, urinary excretion of 3-hydroxyisovaleric acid (3-HIA) is an early and sensitive indicator of biotin deficiency.19–23) Concentrations of 3-HIA, a metabolite of L-leucine in 24 h urine samples on day 19 and day 39 were measured via high-performance liquid chromatography (HPLC) in accordance with the method described by Watanabe et al.19) The 3-HIA (Tokyo chemical industry Co., Ltd,. Tokyo, Japan) derivatized with 2-nitiophenylhydrazine hydrochloride (Tokyo chemical industry Co., Ltd. Tokyo, Japan) was detected using the HITACHI HPLC system (Hitachi, Ltd. Tokyo, Japan) and the HPLC column was a YMC-Pack FA (particle size 5 μm, 250 × 6.0 mm) (YMC Co., Ltd., Kyoto, Japan). Serum estradiol analyses. On day 22 and day 42, blood samples were collected into EDTA-2Na tubes (Terumo Co., Ltd., Tokyo, Japan) from the carotid artery. The collected samples were centrifuged at 1700 × g for 30 min at 4 °C to obtain plasma samples which were stored at −80 °C until use. Estradiol concentration was determined in mice plasma on day 22 and day 42 using an enzyme-linked immunosorbent assay (ELISA) kit (catalog No. 582251; Cayman Chemical Company, Ann Arbor, MI, USA) in accordance with the manufacturer’s instructions. Estrus cycle. The estrus cycle of mice in the metabolic cages was evaluated by daily vaginal smears at 9:00 from Day 22 to Day 40. The bulb was gently depressed to expel a ~25–50 μL water at the opening of vaginal canal. This step was repeated for four to five times. Placed the fluid on glass slide, and allowed the smear to completely dry at room temperature. The dry glass slide was stained with 1.6% Giemsa stain solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 10 min, and dried at room temperature. The vaginal smear was detected at the estrus stage with a microscope (Carl Zeiss Japan). Estrus cycle is composed of “proestrus → estrus → metestrus → diestrus.” Estrus stages were defined as proestrus (60–100% nucleated epithelial cells), estrus (70–100% cornified squamous epithelial cells), metestrus (~50% cornified epithelial cells and 50% leukocytes), and diestrus (60–100% leukocytes).24) Mice that exhibited at least three consecutive four- or five-day cycles were considered to have a regular cycle, while those that displayed a prolonged cycle (≥6 days) had an irregular cycle.
Statistical analyses. Data on day 22 were analyzed by student t-test, and those on day 42 by one-way ANOVA, followed by Tukey’s multiple comparison tests. p
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes a
b
a
Ai Tsuji , Toshinobu Nakamura & Katsumi Shibata a
Department of Nutrition, School of Human Cultures, The University of Shiga Prefecture, Hikone, Japan b
Department of Animal Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan Published online: 10 Oct 2014.
Click for updates To cite this article: Ai Tsuji, Toshinobu Nakamura & Katsumi Shibata (2015) Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes, Bioscience, Biotechnology, and Biochemistry, 79:2, 292-299, DOI: 10.1080/09168451.2014.968090 To link to this article: http://dx.doi.org/10.1080/09168451.2014.968090
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 2, 292–299
Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes Ai Tsuji1, Toshinobu Nakamura2 and Katsumi Shibata1,* 1 2
Department of Nutrition, School of Human Cultures, The University of Shiga Prefecture, Hikone, Japan; Department of Animal Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan
Received July 25, 2014; accepted September 12, 2014
Downloaded by [University of Birmingham] at 10:29 20 March 2015
http://dx.doi.org/10.1080/09168451.2014.968090
Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice. Key words:
biotin; deficiency; mouse; oocyte quality
No good-quality oocytes (chromosomal and spindle abnormalities oocytes) are the major factors in infertility, fetal loss (miscarriage), and conceptions, resulting in birth defects such as trisomy 21 (Down syndrome).1) Recent studies have shown that macronutrients affect the oocyte maturation2,3) and that high-fat diets in mice induce the formation of abnormal oocytes that has chromosome misalignment and spindle defects.2) Caloric restriction without malnutrition, depressed the aging-related increase in the oocyte aneuploidy and chromosomal misalignment.4) Biotin deficiency affects fetal development and growth.5) Biotin is a water-soluble vitamin that acts as a prosthetic group of carboxylases,6) regulates gene expression,7,8) and has a wide range of effects on the systemic processes such as development of embryo,9,10)immunity,11) growth,12) and metabolism.13) Biotin deficiency in experimental animals causes
growth retardation, alopecia, dermatitis, and neurological impairment.14) Several studies in rodents have reported that biotin deficiency also affects fetal development,5) with biotin deficiency during pregnancy being found to increase the rates of abnormal fetal development and growth, as well as affect the rates of absorption and embryonic death in rodents.5,15,16) Báez-Saldaña et al.17) also reported that biotin deficiency in young female mice induced ovary atrophy, estrus arrest, reduction in the oocyte of primordial and graafian follicles, and increase in serum estradiol, a hormone that was involved in oocyte growth. This study suggests that biotin deficiency decreased oocyte growth in the ovaries, but the effect of biotin deficiency on oocyte quality remained unclear. To our knowledge, however, no study has directly examinedthe effects of biotin deficiency on oocyte quality. Here, we investigated the direct effect of biotin deficiency and its subsequent restoration on oocyte quality.
Materials and methods Mice. Animals were allowed free access to food and water, and body weight was measured for every two days. Food intake of mice in metabolic cages was measured daily. Temperature was maintained at approximately 20 °C with 60% humidity and a 12-h light/dark cycle (lights on at 6:00 and off at 18:00). The care and treatment of the experimental animals conformed to the guidelines of the ethical treatment of laboratory animals set by the University of Shiga Prefecture (Shiga, Japan). Deficiency experiment. Female ICR mice (3 weeks old) were obtained from Charles River Laboratories (Tokyo, Japan) and immediately divided into two groups (control group, n = 21; biotin-deficient group, n = 30). For each group, approximately half (control group, n = 11; biotin-deficient group, n = 15) were individually housed in metabolic cages (LC-0335; CLEA Japan, Tokyo, Japan) to collect 24 h urine samples and
*Corresponding author. Email: [email protected] Abbreviations: 3-HIA, 3-hydoroxyisovaleric acid; DAPI, 4′6-diamidino-2-phenylindole dihydrochloride. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Downloaded by [University of Birmingham] at 10:29 20 March 2015
Biotin deficiency induces abnormal oocytes
293
half (control group, n = 10; biotin-deficient group, n = 15, five mice/plastic cage) in plastic cages. On day 22, mice in metabolic cages (n = 5 per group) were sacrificed to analyze the oocyte quality, while those in plastic cages (n = 5 per group) were sacrificed to analyze the biotin concentrations in the liver, ovaries, and uterus.
of cumulus cells by 5% hyaluronidase, (Sigma-Aldrich Inc., Tokyo, Japan) for 5 min at room temperature. Oocytes were counted and classified using a Hoffman light microscope as mature metaphase II (MII) or dead (condensed and fragmented cytoplasm). Oocytes from the experimental groups were analyzed in the blind study.
Recovery experiment. On day 22, the remaining mice of the biotin-deficient group in metabolic cages (n = 10) and plastic cages (n = 10) were randomly divided into two groups. Group 1 consisted of mice in individual metabolic cages (n = 5) and one plastic cage (n = 5) that maintained a biotin-deficient diet. Group 2 (the recovery group) consisted of mice in individual metabolic cages (n = 5) and one plastic cage (n = 5) that were fed a control diet for 20 days. The remaining mice in individual metabolic cages (n = 6) and one plastic cage (n = 5) were continuously fed the control diet. On Day 42, the mice housed in metabolic cages were sacrificed to analyze the oocyte quality, and five mice housed in plastic cages were sacrificed to analyze the biotin concentrations in the liver, uterus, and ovaries.
Immunofluorescence. Oocytes were collected from the control group (oocyte number; n = 101) and the biotin-deficient group (oocyte number; n = 123) on day 22, and from the control group (oocyte number; n = 240), the recovery group (oocyte number; n = 134), and the biotin-deficient group (oocyte number; n = 158) on day 42. Oocytes were collected by inducing superovulation and then puncturing the oviducts with forceps at which point they were washed in PBS and then moved in 5% protease (Sigma–Aldrich Inc., Tokyo, Japan) to soften and remove the zona pellucida. Oocytes were then extensively washed with PBS and fixed in PBS containing 4% paraformaldehyde for 15 min at room temperature. Oocytes were permeabilized with 0.2% Triton X-100 in PBS for 20 min at room temperature and blocked for 1 h in 5% goat serum (Sigma-Aldrich Inc., Tokyo, Japan) in PBST (0.05% Tween20, PBS) at room temperature. Oocytes were then washed with PBST and incubated for 1 h in a 1:4000 dilution of mouse anti-α-tubulin antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) in PBST containing 5% goat serum at room temperature, after which they were washed again and incubated for 1 h in a 1:500 dilution of goat anti-mouse IgG with Alexa Flour-488 and 4′ 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Dojindo Laboratories, Kumamoto, Japan). After washing, oocytes were mounted using PermaFluor aqueous mounting medium (Thermo Fisher Scientific Inc. Kanagawa, Japan) and analyzed by confocal fluorescence microscopy (FV10i; Olympus, Inc., Tokyo, Japan). Oocytes were classified as abnormal if: (1) the chromosomes failed to align on an otherwise normal meiotic spindle; (2) the spindle exhibited serious malformations; or (3) both spindle and chromosome alignment were normal, but one more pair of chromosomes was far removed from the spindle equator, most commonly behind one spindle pole. Oocytes with barrel-shaped bipolar spindles and distinct and wellorganized microtubule fibers, along with tightly aligned chromosomes on the metaphase plate, were classified as normal.
Diet. The control group was fed 30% egg white solids diet supplemented with 0.004 g biotin/kg diet, while the biotin-deficient group was fed 30% egg white solids diet (Table 1), as previously described.17) Egg white solids were obtained from CLEA Japan, Inc. (Tokyo, Japan), which contains avidin that forms a non-absorbable complex with biotin in the alimentary tract and results in biotin deficiency. So a sufficient quantity of biotin was therefore added to the control diet. Oocyte retrieval and classification. To get oocytes, mice which were randomly selected on day 20 or day 40 were superovulated with an intraperitoneal injection of 5 IU pregnant mare serum gonadotrophin (PMSG; product No. E164A; ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) followed by 5 IU human chorionic gonadotrophin (hCG; product No. E801A; ASKA Pharmaceutical) after 46 to 48 h. Oocytes were collected 18 h after hCG injection in EmbryoMax® FHM HEPES buffered medium (FHM; Millipore Corp., Billerica, MA, USA). Retrieved oocytes were denuded Table 1.
Diet compositions.
Egg white solids Gelatinized cornstarch Sucrose Corn oil Dextrin Cellulose Choline bitartrate Mineral mixture (AIN-93-G-MX)* Vitamin mixture (biotin free)* Biotin
Control diet (g/kg diet)
Biotin-deficient diet (g/kg diet)
300 309.3 154.2 80 50 50 2.5 42 12 0.004
300 309.3 145.2 80 50 50 2.5 42 12 –
*Composition of mineral and vitamin mixtures formulated to meet AIN-76A39–46).
294
A. Tsuji et al.
Downloaded by [University of Birmingham] at 10:29 20 March 2015
Biotin assay in urine, liver, plasma, uterus, and ovaries. Twenty-four hour urine samples on day 19 and day 39 were collected from mice in metabolic cages with amber bottles containing 1 mL of 1 mol/L HCl and were stored at −30 °C until use. To avoid the effect of superovulation on metabolism in mice, we collected a 24 h urine sample prior to PMSG injection. Biotin concentrations in urine samples on day 19 and day 39, and in the liver, uterus, and ovaries at day 22 and day 42 were assayed via a microbiological method with Lactobacillus plantarum ACTT 8014.18) Measurement of 3-hydoroxyisovaleric acid in urine. In rodents and humans, urinary excretion of 3-hydroxyisovaleric acid (3-HIA) is an early and sensitive indicator of biotin deficiency.19–23) Concentrations of 3-HIA, a metabolite of L-leucine in 24 h urine samples on day 19 and day 39 were measured via high-performance liquid chromatography (HPLC) in accordance with the method described by Watanabe et al.19) The 3-HIA (Tokyo chemical industry Co., Ltd,. Tokyo, Japan) derivatized with 2-nitiophenylhydrazine hydrochloride (Tokyo chemical industry Co., Ltd. Tokyo, Japan) was detected using the HITACHI HPLC system (Hitachi, Ltd. Tokyo, Japan) and the HPLC column was a YMC-Pack FA (particle size 5 μm, 250 × 6.0 mm) (YMC Co., Ltd., Kyoto, Japan). Serum estradiol analyses. On day 22 and day 42, blood samples were collected into EDTA-2Na tubes (Terumo Co., Ltd., Tokyo, Japan) from the carotid artery. The collected samples were centrifuged at 1700 × g for 30 min at 4 °C to obtain plasma samples which were stored at −80 °C until use. Estradiol concentration was determined in mice plasma on day 22 and day 42 using an enzyme-linked immunosorbent assay (ELISA) kit (catalog No. 582251; Cayman Chemical Company, Ann Arbor, MI, USA) in accordance with the manufacturer’s instructions. Estrus cycle. The estrus cycle of mice in the metabolic cages was evaluated by daily vaginal smears at 9:00 from Day 22 to Day 40. The bulb was gently depressed to expel a ~25–50 μL water at the opening of vaginal canal. This step was repeated for four to five times. Placed the fluid on glass slide, and allowed the smear to completely dry at room temperature. The dry glass slide was stained with 1.6% Giemsa stain solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 10 min, and dried at room temperature. The vaginal smear was detected at the estrus stage with a microscope (Carl Zeiss Japan). Estrus cycle is composed of “proestrus → estrus → metestrus → diestrus.” Estrus stages were defined as proestrus (60–100% nucleated epithelial cells), estrus (70–100% cornified squamous epithelial cells), metestrus (~50% cornified epithelial cells and 50% leukocytes), and diestrus (60–100% leukocytes).24) Mice that exhibited at least three consecutive four- or five-day cycles were considered to have a regular cycle, while those that displayed a prolonged cycle (≥6 days) had an irregular cycle.
Statistical analyses. Data on day 22 were analyzed by student t-test, and those on day 42 by one-way ANOVA, followed by Tukey’s multiple comparison tests. p
