注射hCG后不同时间猪所排卵泡内COC的形态和卵母细胞减数分裂进程

研究了超排处理注射 h CG后不同时间猪卵丘卵母细胞复合体 (COC)的回收率和形态、卵母细胞减数分裂进程以及 COC形态与生发泡 (GV)染色质构型之间的关系。结果表明 :(1)注射 h CG后 4 h COC的回收率为 5 3.1% ,明显低于注射 h CG后 18、2 2、2 4 h(71.2 %、76 .5 %、70 .0 % ,P<0 .0 5 ) ;(2 )注射 h CG后 18、2 2、2 4 h收集的 COC分别有98.9%、98.0 %、91.4 %的卵丘已经发生扩展 ,而注射 h CG后 4 h收集的 COC则无一发生卵丘扩展 ;(3)注射 h CG后 4h,有少量卵母细胞开始恢复减数分裂 ,至 18h左右开始发生生发泡破裂 (GVBD) ,到 2 2～ 2 4 h有 5 8.4 %～ 6 0 .0 %的卵母细胞发生 GVBD;(4)外层卵丘扩展、放射冠轻微扩展的卵母细胞处于 GV- 1期的比例 (6 9.6 % )明显高于卵丘完全扩展的卵母细胞 (47.8% )和放射冠部分扩展、卵丘已经脱落的卵母细胞 (2 9.3% ) ,而后 2者发生 GVBD的比例(40 %、4 4 % )则略高于前者 (2 7% )。     

Collection Rates and Morphology of Equine Oocytes Obtained from Immature Follicles after Treatment with Equine Pituitary Extract (EPE) and Human Chorionic Gonadotropin

This study examined the effect of treating mares with equine pituitary extract (EPE) in combination with human chorionic gonadotropin (hCG; EPE/hCG) on the recovery rate of immature oocytes by ovum pick-up (OPU) and on oocyte morphology. Ten mares were subjected to each of two treatments in a random sequence: superstimulated with EPE (25 mg, twice daily) and treated with hCG (2,500 IU) or control (no exogenous treatment). The cytoplasmic morphology of oocytes recovered was evaluated through transmission electron microscopy. Follicular fluid was collected at aspiration for progesterone analysis, which was performed by radioimmunoassay. The EPE/hCG did not increase the oocyte recovery rate from immature follicles when compared with the controls (15.5% and 16.7%, respectively). A significantly higher oocyte recovery rate per mare was observed (70% versus 50%). However, precocious granulosa cell expansion was observed with EPE/hCG treatment in contrast to the control (64.4% and 33% of follicles with expanded cumulus, respectively), and increased intrafollicular progesterone concentration was also seen (158.80 ng/mL versus 82.05 ng/mL). The ultrastructural analysis of oocytes from both groups showed morphologic features related to immaturity. Numerous vesicles containing cortical granules were found, distributed in clusters into the cytoplasm, and junctional complexes were still seen between oocyte and granulosa cells. In conclusion, EPE/hCG treatment induced some follicular modifications, but the recovery rate was not increased. All oocytes examined presented signs of immaturity.     

猪卵丘卵母细胞复合体和壁层颗粒细胞上LHR/hCGR的检测及性腺激素的分泌

用取自 ３～５ ｍ ｍ 卵泡的处于减数分裂阻抑状态的猪卵丘卵母细胞复合体（ｐ Ｃ Ｅ Ｏ），培养 ４８ ｈ，用 Ｆ Ｓ Ｈ（１００ Ｉ Ｕ／ Ｌ）或 ｈ Ｃ Ｇ（０、５０、１００、２００、５００ Ｉ Ｕ／ Ｌ）刺激 ｐ Ｃ Ｅ Ｏ 分泌甾体激素；用放射受体测定法（ Ｒ Ｒ Ａ）比较 ｐ Ｃ Ｅ Ｏ 的卵丘细胞及壁层颗粒细胞（ Ｍ Ｇ Ｃ）上促黄体激素受体／人绒毛膜促性腺激素受体（ Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ）的数量； Ａ Ｍ ｅ Ｘ 石蜡切片、免疫组织化学染色，检测 Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ 在 ｐ Ｃ Ｅ Ｏ 以及 Ｍ Ｇ Ｃ 的分布情况。在 Ｆ Ｓ Ｈ 作用下，ｐ Ｃ Ｅ Ｏ 分泌的孕酮明显高于 ｈ Ｃ Ｇ 作用组和对照组 （ Ｐ ＜ ００５）； 平均每个壁层颗粒细胞上的 Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ是每个卵丘细胞的 １０５ 倍； Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ 在基膜两侧分布较多，且卵母细胞上没有 Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ，临近卵母细胞的卵丘细胞膜上较少被染色。以上结果表明，在体外试验中，可能由于 Ｃ Ｅ Ｏ 上 Ｌ Ｈ Ｒ／ｈ Ｃ Ｇ Ｒ 的数量不足或生理活性降低， Ｌ Ｈ／ｈ Ｃ Ｇ 不能促进卵母细胞恢复减数分裂。     

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis)

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.     

Exposure to L-Ascorbic Acid or α-Tocopherol Facilitates the Development of Porcine Denuded Oocytes from Metaphase I to Metaphase II and Prevents Cumulus Cells from Fragmentation

It is known that alpha-tocopherol (vitamin E) and L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidants. The objective of the present study was to investigate the effects of alpha-tocopherol and L-ascorbic acid on porcine oocyte meiotic maturation, viability and the functions of cumulus cells. In two independent experiments, porcine oocytes with or free from cumulus cells were exposed to different levels of alpha-tocopherol (0, 10, 100 and 200 microM) or L-ascorbic acid (0, 50, 250 and 750 microM). Cumulus expansion, cumulus cell DNA fragmentation, meiotic maturation and degeneration of oocytes were assessed 48 h after in vitro culture. The results showed that: (1) neither alpha-tocopherol nor L-ascorbic acid influenced cumulus expansion but both prevented cumulus cell DNA fragmentation. (2) Alpha-tocopherol lowered the percentage of denuded oocytes (DOs) arrested at germinal vesicle stage (GV). Among the oocytes undergoing germinal vesicle breakdown (GVBD) proportion, fewer DOs treated by alpha-tocopherol were at metaphase I (MI) and more at metaphase II (MII). L-ascorbic acid caused lower percentage of DOs arrested at GV stage and higher percentage of DOs undergoing GVBD, especially at MII. The influences of alpha-tocopherol and L-ascorbic acid were not obvious in cumulus-enclosed oocytes (CEOs). (3) Both vitamins compromised the viability of CEOs and DOs. These results indicate that exposure to alpha-tocopherol or L-ascorbic acid promotes the development of porcine DOs from MI to MII and prevents cumulus cell DNA fragmentation at certain levels, especially 10 microM alpha-tocopherol or 250 microM L-ascorbic acid.     

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.     

Timing of sequential changes in chromosome configurations during the 1st meiotic division of pig oocytes cultured in vitro

This study examines the timing of changes in chromosome configurations of pig oocytes derived from small antral follicles of follicular and/or inactive stage donors using modified 199 medium supplemented with gonadotropins (Follicle Stimulating Hormone (FSH), 10 IU/ml; Human Chorionic Gonadotropin (hCG), 10 IU/ml) and Glucosamine (0.539 mg/ml). Oocytes (n = 1,215) were fixed at the end of 3 hourly intervals from 0-48 hr of culture. Results were expressed as the percentage of oocytes at each stage of maturation for each time point. The germinal vesicle (GV) stage was observed for the first 17.6 hr; germinal vesicle breakdown (GVBD) stage between 17.6-26.4 hr; metaphase I (M-I) from 26.4-30.9 hr; anaphase I (A-I) ranged from 30.9-33.4 hr; telophase I (T-I) at 33.4-34.4 hr; and metaphase II (M-II) at 34.4-48 hr.     

Relationship between the peripheral concentrations of estradiol-17β (E2) and preovulatory characteristics of cumulus-oocyte complexes (COCs) during superovulation treatment in Japanese Black cows

The relationship between the peripheral concentrations of estradiol-17β (E(2)) and the preovulatory characteristics of cumulus oocyte complexes (COCs) during superovulation treatment was investigated in Japanese Black cows. A superovulation regimen with FSH treatment in a descending manner was commenced on day 7 (n=3) or day 10 (n=2) of the estrous cycle (day 0=estrus). Peripheral blood was collected to measure E(2) concentrations twice a day throughout the treatment. Ovariectomies were performed at 100 h after the initial FSH treatment in five cows. Every follicle more than 8 mm in diameter was isolated from the ovaries, and cumulus-oocyte complexes (COCs) were gently aspirated. The COCs were then separated into three groups based on the characteristics of the cumulus (compact, expanded and denuded) and subgrouped based on the stage of the nucleus in the oocytes (GV, GVBD). Plasma E(2) concentrations tended to increase gradually and reached the peak level at around 84 h (E(2)-84: n=3) or 96 h (E(2)-96: n=2) after the initial FSH treatment. The ratio of COCs with expanded cumulus was significantly higher in E(2)-84 than in E(2)-96 (P<0.01). However, there was no difference in the ratio of oocytes showing GVBD between E(2)-84 and E(2)-96 (P=0.73), and the characteristics of the cumulus did not affect the stage of the nucleus in the oocytes in either groups (compact, expanded and nude; P=0.61, 0.81 and 1.00). It was possible that the time until the peak plasma E(2) concentrations after the FSH treatment could become an indicator for the maturation of follicles and oocytes in preovulatory follicles during superovulation treatment in Japanese Black cows.     

Expression of nitric oxide synthase-3 in porcine oocytes obtained at different follicular development

The present study was designed to determine the localization of nitric oxide synthase-3 (NOS-3) in porcine follicles during follicular development. A 130-kDa NOS-3 protein was found with greater frequency much in the oocytes than in the cumulus cells, as revealed by Western blotting analysis. The content of NOS-3 in the oocyte was higher in large follicles (> 7-mm diameter) than in small follicles (< 2-mm). The data by Western blotting showed the same pattern as the observations obtained from the immunohistochemical studies, in which the periphery of the oocyte stained strong positive. The inner surface cell layer of granulosa cells and cumulus cells were positive staining, especially in large antral follicles. In the primordial follicles, NOS-3 was restricted to the cytoplasm of oocytes, and no stained product was observed in the nucleus of oocytes or granulosa cells. A significant synthesis of NO by oocytes was observed in the presence of ionomycin, but not in the absence of ionomycin, indicating that oocyte NOS-3 functions in response to transient elevations in the intracellular calcium level. We concluded that NOS-3 is expressed in the oocyte from the primordial follicular stage to antral follicular stage, and that it is functional at least in the antral follicles.     

Growth and maturation of follicles and oocytes following xenotransplantation of porcine ovarian tissues and in vitro maturation

This is the first report to show morphological evidence of in vitro maturation of oocytes recovered from xenotransplanted antral follicles. To develop a suitable tool for studing the growth and maturation of follicles and oocytes, we xenotransplanted small pieces of ovarian cortical tissue from sows, which contained small preantral follicles (primordial, primary, and secondary follicles; less than 0.05, 0.1 and 0.3 mm in diameter, respectively), under the capsules of kidneys of adult female severe combined immunodeficient (SCID) mice for 2 and 8 weeks, and then recovered cumulus-oocyte complexes from the growing tertiary follicles in xenografted tissues. The distribution of processes from cumulus cells to oocytes and the follicular growth, development, and maturation during xenotransplantation were histochemically analyzed. Tertiary follicles, 0.5 to 3.0 mm in diameter, were obtained from grafted tissues 2 (85%: 52 follicles/61 grafted tissues) and 8 (50%: 15/30) weeks after xenotransplantation, and then oocytes, which were tightly attached to cumulus cells, were collected from each tertiary follicle and cultured to assess their quality. At 2 weeks after grafting, 17.6% of the oocytes had matured to the metaphase II stage, but no such maturation was observed 8 weeks after grafting. Thus, in the 2 weeks group, preantral follicles rapidly grew in xenotransplanted porcine ovarian tissues to the tertiary stage, and oocytes could be recovered and matured from them by in vitro culture.     

Bovine Follicular Fluid Contains Antagonist Activity for Cumulus Expansion Promoting Effect by Epidermal Growth Factor

The present study was conducted to investigate the effects of bovine follicular fluid (bFF) fractions or epidermal growth factor (EGF) on cumulus cell expansion in bovine cumulus-oocyte complexes (COCs) in vitro. bFF derived from large follicles (15-20 mm) and its fractions (>100, 10-100 and <100 kDa) were added to the medium. Cumulus cell expansion was stimulated when COCs were incubated with the 10-100 and <100 kDa fractions or bFF; in contrast, the culture of COCs with the >100 kDa fraction resulted in the suppression of cumulus cell expansion. Although the >100 kDa fraction prohibited the expansion of cumulus cells in the medium with or without low concentration EGF, cumulus expansion was promoted when COCs were cultured with the >100 kDa fraction and high-concentration EGF. In conclusion, the results suggest that bFF contains promoting and inhibiting effect for expansion of cumulus cells in COCs in vitro. The inhibiting effect of bFF may act antagonistically against the effect of EGF for the event of expanding cumulus cells.     

Cumulus and oocyte maturation and in vitro and in vivo fertilization of oocytes in relation to follicular steroids,prolactin, and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge,no detectable LH surge,and progestin inhibition of LH surge

Crossbred heifers (n = 103) were synchronized to estrus with prostaglandin (PGF_2α) and superovulated with follicle stimulating hormone (FSH-P). Animals were ovariectomized every 12 hr after the PGF_2α injection (n = 7 to 9/time) up to 108 hr to monitor the follicular, hormonal, and oocyte changes associated with follicular development and ovulation. Twenty-eight animals were implanted with Norgestomet implants 12 hr before PGF_2α and ovariectomized at 72, 84, 96, and 108 hr post PGF_2α injection to monitor effects of progesterone and suppression of the luteinizing hormone (LH) surge on oocyte maturation and quality. Follicular fluid was collected and analyzed for progesterone, estradiol, prolactin, and glycosaminoglycan content in conjunction with cumulus maturation and nuclear stage of oocyte maturation. Analysis of in vivo matured oocytes by in vitro fertilization was carried out at 60, 72, 84, and 96 hr post PGF_2α and in vitro matured oocytes at 12 to 108 hr post PGF_2α. No developmental changes in cumulus cells surrounding the oocyte of small follicles was noted (≤ 4 mm dia) indicating a static population. Medium (> 4 ≤ 8 mm) and large size (> 8 mm) follicles developed to the corona radiata and loose cumulus stages in animals in which an LH surge was detected but cumulus status remained primarily in the tight cumulus stage for animals without an LH surge. The estradiol-to-progesterone ratio for tight cumulus (TC), corona radiata (CR), and loose cumulus (LC) stages was 1.8 ± .1, 1.0 ± .1, and .4 ± .2, respectively (P < .01). Nuclear maturation of oocytes in small follicles from animals without a detectable LH surge seem to indicate early maturation (48 to 72 hr post PGF_2α) in conjunction with a high percent of degenerate oocytes not seen in animals exhibiting an LH surge. Oocytes from medium size follicles matured to germinal vesicle breakdown (GVBD) and early meiosis (metaphase I; MI) stages of development in all treatments. Most oocytes were degenerate in Norgestomet-implanted animals. Oocytes from large follicles (> 8 mm dia) from animals exhibiting an LH surge were in MI and metaphase II (MII) stages (48 to 84 hr post PGF_2α) in preparation of ovulation whereas oocytes from animals not exhibiting an LH surge had oocytes that early matured to MII (48 to 72 hr post PGF_2α), later regressing to degenerate oocytes (84 to 108 hr). Follicular progesterone, estradiol, and prolactin increased with oocyte maturation, particularly in medium and large follicles. In vivo matured oocytes for fertilization (60, 72, 84, and 96 hr post PGF_2α) were nude (from the oviduct) and primarily CR from follicles. Tubal oocytes (37%) were fertilized more frequently by a single sperm than follicular oocytes (14.3%; P < .01) and single sperm penetration peaked at 72 hr post PGF_2α. Follicular hormone concentrations were not related to sperm penetration. Oocytes (n = 101) matured in vivo had lower fertilization potential from ovaries producing < 14 or > 50 follicles (39.3%) as compared to 21 to 45 aspirated follicles (68.2%; P < .05), with a peak penetration at 32 follicles (86.7% penetration). No treatment differences (LH surge or no detectable LH surge) were noted in relation to in vivo matured oocytes. Oocytes with single sperm penetration had the lowest estradiol/progesterone ratio of 2.2 vs polyspermic penetration of 13.7.     

Reestablishment of transzonal projections and growth of bovine oocytes in vitro

Transzonal projections (TZPs) that maintain bidirectional communication between oocytes and granulosa cells or cumulus cells are important structures for oocyte growth. However, whether TZPs develop between TZP-free oocytes and granulosa cells, and whether reestablished TZPs support oocyte growth, is unknown. We first examined changes in TZPs after denudation of bovine oocytes collected from early antral follicles (0.5–0.7 mm). Twenty-four hours after denudation, almost all the TZPs disappeared. We also examined the reestablishment of TZPs by coculturing TZP-free denuded oocytes (DOs) with mural granulosa cells (MGCs) collected from early antral follicles. In addition, to confirm if the reestablished TZPs were functional, the reconstructed complexes (DO+MGCs) were subjected to in vitro growth culture and found that the MGCs adhered to TZP-free DOs and TZPs were reestablished. During in vitro growth culture, DO+MGCs developed and formed antrum-like structures. After culture, the number of TZPs in DO+MGCs increased, and the oocytes grew fully and acquired meiotic competence. These results suggest that reestablished TZPs are able to support oocyte growth.     

The influence of follicular factors on the in vitro maturation of bovine oocytes]

Immature oocytes from antral follicles of cattle were tested for the effect of follicular factors on maturation. In vitro maturation was accomplished by use of follicular fluid from small (2--5 mm) and large (above 15 mm) follicles and by addition to the medium of a granulose factor (GF) which had been isolated from the surface of granulosa cells. The parent material, with 84% (72/86) of oocytes at the germinal vesicle stage (GV-S) at the beginning of culturing, could be rated immature. 46% of all oocytes (41/89) had reached telophase I or metaphase II (full maturation) after 24 hours of maturation in hormone-free control medium (TCM 199 + 10% of foetal calf serum). 36% of oocytes (53/84), on the other hand, stayed between GV breakdown (GVBD) and anaphase I (incipient maturation). Full maturation was reached by as little as 14%. GF and follicular fluid from small antral follicles were found to inhibit GVBD in the oocytes. 59% (36/61) or 48% (61/127) of oocytes were blocked at GV stage. Positive determination of maturation inhibiting action of the above follicular components may provide a chance for their target-oriented use in control of the maturation process. The pool of immature oocytes of the ovaries, under such circumstances, might be more systematically utilised for in vitro manipulations.     

PI3-kinase and mitogen-activated protein kinase in cumulus cells mediate EGF-induced meiotic resumption of porcine oocyte

Previous studies have shown that epidermal growth factor (EGF) has the ability to promote in vitro cultured porcine oocyte maturation. However, little is known about the detailed downstream events in EGF-induced meiotic resumption. We designed this study to determine the relationship of EGF, EGFR, phosphatidylinositol 3-kinase (PI3-kinase), MAPK, and germinal vesicle breakdown (GVBD) during oocyte maturation. Our results showed that GVBD in cumulus-enclosed oocytes (CEOs) but not in denuded oocytes (DOs) was induced by EGF in a dose-dependent manner, which indicated that cumulus cells but not oocyte itself were the main target for EGF-induced meiotic resumption. Furthermore, we found that MAPK in cumulus cells rather than in oocyte was activated immediately after EGF administration. To explore whether EGF exerts its functions through MAPK pathway, the activities of EGF receptor (EGFR) and MAPK were inhibited by employing AG1478 and U0126, respectively. Inhibition of MAPK blocked EGF-induced GVBD, whereas inhibition of EGFR prevented MAPK activation. Both AG1478 and U0126 could lead to the failure of EGF-induced GVBD singly. Notably, we found that LY294002, a specific inhibitor of PI3-kinase, effectively inhibited EGF-induced MAPK activation as well as subsequent oocyte meiotic resumption and this inhibition could not be reversed by adding additional EGF. Thus, PI3-kinase-induced MAPK activation in cumulus cells mediated EGF-induced meiotic resumption in porcine CEOs. Together, this study provides evidences demonstrating a linear relationship of EGF/EGFR, PI3-kinase, MAPK and GVBD and presents a relatively definitive mechanism of EGF-induced meiotic resumption of porcine oocyte.     

Induction of superovulation using inhibin antiserum and competence of embryo development in wild large Japanese field mice (Apodemus speciosus)

Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild‐derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non‐reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non‐reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2‐ and/or 4‐cell stage using both fresh and frozen‐thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non‐reproductive seasons. This study is the first to develop IVF–IVC wild large Japanese field mice beyond the 2‐ and/or 4‐cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos.     

High developmental rates of mouse oocytes cryopreserved by an optimized vitrification protocol: the effects of cryoprotectants, calcium and cumulus cells

Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility.     

Serum progesterone and estradiol-17beta concentrations, and lapaloscopic observations of the ovary in the cheetah (Acinonyxjubatus) with pregnant mare serum gonadotropin and human chorionic gonadotropin treatments.

In 3 adult female cheetahs, induced-superovulation treatment was conducted, by means of 200 IU of pregnant mare serum gonadotropin (PMSG) and 100 IU of human chorionic gonadotropin (hCG) 80 hr after PMSG. The administration of PMSG created a sharp increase in the estradiol-17beta concentration, resulting in 232 pg/ml 8 hr later in one specimen out of three. The hCG administration showed an increase in the progesterone concentration of 2.29 ng/ml 46 hr later. In addition, after direct observation of the ovary surface by laparoscopy, 5 follicles in the right ovary over 2 mm in diameter, and 7 corpora lutea (5 in the right ovary and 2 in the left) were found. It is assumed that ovulation can be induced with hCG after 80 hr on PMSG during a cheetah's diestrus or proestrus.     

猪不同直径卵泡内卵母细胞胞质成熟及体外发育潜力差异原因的初探

试验根据直径将猪卵泡分为2组:G1组(4~7 mm)和G2组(2~4 mm),对2组卵泡内获取的卵母细胞体外成熟率和发育潜能进行了比较,利用相对定量PCR检测了卵丘细胞中卵丘扩展相关基因Has2、Ptgs2、Ptx31及Pgr的表达水平,应用绝对定量PCR检测了成熟培养前后卵母细胞线粒体拷贝数,并利用5,5'-二巯基-2-硝基苯酸(DTNB)酶循环法检测了体外成熟培养过程中卵母细胞谷胱甘肽(GSH)的含量。结果显示,G1组和G2组卵母细胞体外成熟率分别为95.06%和68.19%,G1和G2组排出第一极体的成熟卵母细胞孤雌激活后的囊胚率分别为51.47%和29.44%,2组卵母细胞在体外成熟率和孤雌发育率上均差异显著(P<0.05)。G1组卵丘细胞在体外成熟培养过程中的扩展程度明显高于G2组,G1组卵母细胞对应的卵丘细胞扩展相关基因Has2、Ptgs2、Ptx31、Pgr的表达水平高于G2组(Has2基因在卵母细胞成熟培养0、24 h除外);G1组卵母细胞线粒体数、谷胱甘肽含量均高于G2组。以上结果表明,大卵泡来源的卵母细胞体外成熟能力和发育潜力优于小卵泡来源的卵母细胞,这可能与卵母细胞成熟过程中卵丘扩展程度、卵丘扩展相关基因表达激活情况、卵胞质内谷胱甘肽含量和线粒体拷贝数有关。