Abstract
Purpose
Materials and Methods
Results
Figures and Tables
Fig. 1
Characteristics of animals used in this study and impairment of the hypothalamus-pituitary-ovary axis in streptozotocin-induced type I diabetes mellitus. (A) PCR and restriction enzyme digestion were carried out to confirm the presence of the ins2 gene mutation. Control and AKITA mice showed different PCR products after digestion. (B) The streptozotocin (STZ)-injected mice had a lower body weight and (C) higher blood glucose levels, compared to control mice (n=15). Different letters indicate a statistically significant difference (p<0.05). (D) Ovary size was dramatically reduced 3 weeks after STZ injection, while AKITA mice did not show any reduction in ovary size (n=6). (E) In STZ-injected mice, the expression of FSHR and luteinizing hormone/chorionic gonadotropin receptor (LHCGR) was substantially diminished compared with control and AKITA mice. (F) The expression of anti-Mullerian hormone (AMH), anti-estrogen receptor-beta (ER-beta), and Ki-67 in the ovaries from control, AKITA, and STZ-injected mice (50 µm, ×200). The expression levels of these proteins, which are correlated with folliculogenesis and proliferation, were reduced in STZ-injected mice. Different letters (a, b, c) indicate a statistical significant difference. H, heterozygous; W, wild type; CON, control; FSHR, follicle stimulating hormone receptor.
![ymj-60-667-g001](/upload/SynapseData/ArticleImage/0069ymj/ymj-60-667-g001.jpg)
Fig. 2
Histological observation of oocyte quantity and quality after superovulation. (A) All stages of ovarian follicles and the corpus luteum (CL) were seen in control, AKITA, and streptozotocin (STZ)-injected mice. Black arrows indicate antral follicles, yellow arrows show CL, and the red arrow indicates trapped oocytes in CLs. CLs and oocytes are marked (400 µm, ×200). (B) Both diabetic mice models showed pyknotic nuclei of granulosa cells (orange arrows) and impairment of oocyte-granulosa cell connection (green arrows), while the growing follicles in control mice showed normal morphology (50 µm, ×200). (C) Ovarian tissue stained with oocyte-specific marker (MSY2) to demonstrate trapped oocytes in CL (100 µm, ×200). An inset indicates trapped oocytes in CL. Orange arrows and green signals indicate the oocytes of different follicle stages (B, C). The white arrow and letter ‘Oo’ indicate trapped oocytes in CLs, which are also shown in (A). (D and E) The numbers of oocytes and CLs in the ovary after superovulation were significantly lower in STZ-injected mice. Different letters (a, b) indicate a statistically significant difference (p<0.05, n=6). (F) Oocyte maturation in diabetic mice was also impaired, compared with control. * and † respectively indicate p<0.05 and p<0.001 (n=80). CON, control; Oo, oocyte; Deg-, degenerated; GV, germinal vesicle; MI, germnial vesicle breakdown; MII, metaphase II.
![ymj-60-667-g002](/upload/SynapseData/ArticleImage/0069ymj/ymj-60-667-g002.jpg)
Fig. 3
(A–E) Hormone profiles after superovulation. (A–E) Insulin, anti-Mullerian hormone (AMH), 17-beta-estradiol, testosterone, and progesterone were measured. Diabetic mice had lower insulin and progesterone levels, whereas 17-beta-estradiol and testosterone levels were comparable between control and diabetic mice. Different letters (a, b) indicate a statistically significant difference (p<0.05). STZ, streptozotocin; CON, control.
![ymj-60-667-g003](/upload/SynapseData/ArticleImage/0069ymj/ymj-60-667-g003.jpg)
Fig. 4
Microorganelles in mature oocytes and meiotic spindle formation. (A) Cortical granule (red) distribution in mature oocytes derived from wild-type, AKITA, and streptozotocin (STZ)-injected mice (n=20) (10 µm). Blue indicates DNA of chromsome which was stained by 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI). (B) Meiotic spindle (green) and DNA (blue) localized in mature oocytes. (C) The proportion of normal meiotic spindles. (C) Different letters (a, b) indicate statistically significant differences (p<0.05). CON, control.
![ymj-60-667-g004](/upload/SynapseData/ArticleImage/0069ymj/ymj-60-667-g004.jpg)
Fig. 5
Mitochondrial characteristics and exocytosis of calcium from mature oocytes. (A) A representative image of JC-1 staining of mature oocytes. Red and green indicate high and low mitochondrial membrane potential, respectively. Magnification was ×200. (B) The ratios of red/green fluorescence intensity were measured, and both diabetic mouse models had significantly lower mitochondrial membrane potential compared with controls. Different letters (a, b, c) indicate a statistically significant difference (p<0.05, n=14). (C) The quantity of mtDNA was similar between control and diabetic mouse groups (n=18). The exocytosis of calcium (D and E) was also comparable in oocytes from control and diabetic mice (n=15). STZ, streptozotocin; CON, control.
![ymj-60-667-g005](/upload/SynapseData/ArticleImage/0069ymj/ymj-60-667-g005.jpg)
Fig. 6
Embryonic developmental competence in type I diabetes mellitus mouse models was significantly lower compared with normal control mice. (A and B) Percentages of cleavage and blastulation after in vitro fertilization (IVF). The numbers of two-cell embryos and blastocysts were counted to compare pre-implantation embryonic development (n=85). (C and D) Total and apoptotic blastomeres of individual blastocysts were also evaluated by confocal microscopy (n=21). (E) Representative image of a hatching blastocyst after staining with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL). Magnification was ×200 and the images were obtained by confocal microscopy. Blue and Green signal respectively indicate nucleus and apoptotic DNA of blastomeres. Different letters (a, b) indicate statistically significant differences (p<0.05). STZ, streptozotocin; CON, control; MII, metaphase II.
![ymj-60-667-g006](/upload/SynapseData/ArticleImage/0069ymj/ymj-60-667-g006.jpg)
ACKNOWLEDGEMENTS
Notes
AUTHOR CONTRIBUTIONS Jaewang Lee performed the research and wrote the manuscript. Hoi Chang Lee performed research and conducted data analyses. So-Youn Kim contributed to discussion and reviewed/edited manuscript. Geum Joon Cho contributed to discussion and reviewed/edited manuscript. Teresa K. Woodruff reviewed and edited the manuscript.
References
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