Developmental potential of aged oocyte rescued by nuclear transfer following parthenogenetic activation and in vitro fertilization

Mouse oocyte aged in vitro cannot develop normally following activation. To investigate the roles of nucleus or cytoplasm elements in oocyte aged in vitro process and their subsequent development capability following activation, we reconstructed oocytes with MII chromosome spindle and cytoplasm from aged and fresh oocytes by nuclear transfer. The subsequent developmental potential after parthenogenetic activation (PA) or in vitro fertilization (IVF) was evaluated. After nuclear transfer, more than 75.6% of karyoplast and cytoplast pairs can be fused and reconstructed oocytes have a normal haploid karyotype. Following PA, aged oocytes cannot develop beyond four-cell stage, reconstructed oocytes from fresh nucleus and aged cytoplasm developed to blastocyst with a low percentage (9.1%). Instead, blastocyst formation rate of reconstructed oocyte from aged nucleus and fresh cytoplasm was higher (60.0%). Following IVF, zygote with diploid karyotype can be formed from zona pellucida (ZP)-free oocyte. After cultured in vitro, aged oocytes cannot develop beyond two-cell; reconstructed oocytes from fresh nucleus and aged cytoplasm developed to blastocyst with low percentage (15.0%). However, high blastocyst formation rate (86.2%) can be obtained from reconstructed oocytes from aged nucleus and fresh cytoplasm. Furthermore, after embryo transfer, three viable pups have been obtained, although the efficiency is very low. These observation demonstrated that cytoplasm is more crucial than nucleus to aging process. Fresh cytoplasm could partly rescue nucleus susceptibility to apoptosis from aging in vitro.     

Ultrastructural localisation of calcium deposits in pig oocytes maturing in vitro: effects of verapamil

The culture of pig oocytes in the presence of the calcium channel blocker verapamil (0.02 mM) resulted in the blocking of meiosis at the metaphase I stage, and only a small fraction (about 28%) of the oocytes were able to continue their maturation to the stage of metaphase II. Hence, meiotic maturation in pig oocytes is a calcium-dependent process. After isolation of the pig oocytes from their follicles, the intracellular calcium deposits in the oocyte and granulosa cells, detectable using the combined oxalate-pyroantimonate method, are depleted. The amount of calcium deposits in the oocyte and granulosa cells increased during oocyte meiotic maturation in vitro, especially in the nucleus, mitochondria, vacuoles and cytoplasm. The replenishment of calcium deposits is significantly changed under the effect of verapamil. The increase in calcium deposits in the oocyte nucleus was delayed, a much larger amount of deposits was formed in the mitochondria, and the amount of deposits in the vacuoles was demonstrably smaller. A significant peak in the accumulation of calcium deposits was observed in the cytoplasm of verapamil-treated oocytes after 16 h of in vitro culture. We propose that an altered pattern in the replenishment of calcium deposits can disturb intracellular signalling and prevent the exit of oocytes from the metaphase I stage.     

SPINDLES, SPINDLE PLAQUES, AND MEIOSIS IN THE YEAST SACCHAROMYCES CEREVISIAE (HANSEN)

The intranuclear spindle of yeast has an electron-opaque body at each pole. These spindle plaques lie on the nuclear envelope. During mitosis the spindle elongates while the nuclear membranes remain intact. After equatorial constriction there are two daughted nuclei, each with one spindle plaque. The spindle plaque then duplicates so that two side-by-side plaques are produced. These move rapidly apart and rotate so that they bracket a stable 0.8 µm spindle. Later, during mitosis, this spindle elongates, etc. Yeast cells placed on sporulation medium soon enter meiosis. After 4 hr the spindle plaques of the more mature cells duplicate, producing a stable side-by-side arrangement. Subsequently the plaques move apart to bracket a 0.8 µm spindle which immediately starts to elongate. When this meiosis I spindle reaches its maximum length of 3–5 µm, each of the plaques at the poles of the spindle duplicates and the resulting side-by-side plaques increase in size. The nucleus does not divide. The large side-by-side plaques separate and bracket a short spindle of about 1 µm which elongates gradually to 2 or 3 µm. Thus there are two spindles within one nucleus at meiosis II. To the side of each of the four plaques a bulge forms on the nucleus. The four bulges enlarge while the original nucleus shrinks. These four developing ascospore nuclei are partially surrounded by cytoplasm and by a prospore wall which originates from the cytoplasmic side of the spindle plaque. Eventually the spore nuclei pinch off and the spore wall closes. In some of the larger yeast cells this development is completed after 8 hr on sporulation medium.     

Timing of nuclear maturation and postovulatory aging in oocytes of in vitro-grown mouse follicles with or without oil overlay

Meiotic maturation of the oocyte is a timed sequence of events induced by the ovulatory LH surge. In vitro maturation of oocytes is known to alter the meiotic time course. This study documented the timing of meiosis in oocytes grown in vitro for 12 days, from the preantral follicle stage onward, and the influence of an oil overlay. In the oil-free culture, the stability of the metaphase II spindle was further explored to determine the postovulatory aging events. After the maturation stimulus, in vitro-grown oocytes were collected at 2-h intervals spanning the period of meiosis (0-18 h) and at 3-h intervals during early postovulatory aging (18-27 h). Stage of maturation was assessed both morphologically and by detailed spindle analysis and chromosome alignment. Results revealed that oil overlay did not impair the competence of cultured oocytes to proceed to meiosis II, but delayed meiosis I progression. Oil overlay during culture causes a different hormonal exposure of the follicles by a differential segregation into the oil overlay. The use of a progesterone receptor antagonist, however, did not induce a delay in meiotic progression. Aging effects in oil-free cultured follicles were detected 5 h after the establishment of the metaphase II spindle, comparable to their in vivo grown counterparts. The predominant effect of aging was an interphase-like appearance of the cytoskeleton. So an optimal time window for fertilization after in vitro follicular growth was determined to be 16-21 h after maturation induction.     

In vitro parthenogenetic development of mouse oocytes following reciprocal transfer of the chromosome spindle between in vivo-matured oocytes and in vitro-matured oocytes

Mouse follicles grown in vitro from preantral to mature stages yield oocytes that can be fertilized in vitro, but embryonic development is poor. To investigate whether this poor development is due to a nuclear or a cytoplasmatic factor, we designed an experiment in which the MII chromosome spindle was exchanged between in vitro-matured oocytes and in vivo-matured oocytes by electrofusion. Subsequent embryo development was evaluated by blastocyst formation rate and blastocyst cell number after parthenogenetic activation. Electrofusion was successful in 62-78% of the oocytes. Transfer of the spindle apparatus from in vitro-matured oocytes to the in vivo MII cytoplasmic environment resulted in a high rate of blastocyst development, whereas in the reverse situation (transfer of the nucleus from in vivo-matured oocytes into in vitro-matured MII cytoplasm) poor quality embryos and a low rate of blastocyst formation was observed. These results indicate that the low developmental competence of in vitro-matured oocytes from mouse preantral follicles after activation is caused by the cytoplasmic component rather than the nuclear component.     

Development of mouse embryos derived from oocytes reconstructed by metaphase I spindle transfer

Abnormal oocyte spindle is frequently associated with the infertility of aged women. Directly manipulating the metaphase I (MI) spindle may be a feasible method to overcome this kind of problem. Here, we report that the MI meiotic spindle can be removed from MI mouse oocytes and will autonomously divide into two daughter cells with the same size, morphology and an equal number of chromosomes after culture for 5 h in maturation medium. The division rate of the MI spindle reached 56% after 10-15 h of culture. After transferring the MI meiotic spindle into synchronous ooplasm by electrofusion, about 61% of the reconstructed oocytes continued to complete the first meiosis and extruded a normal first polar body. The matured reconstructed oocytes can also be fertilised. Approximately 50% of the 2-cell embryos developed to the morula stage after in vitro culture.     

Somatic nucleus remodelling in immature and mature Rassir oocyte cytoplasm

Successful production of cloned animals derived from somatic cells has been achieved in sheep, cattle, goats, mice, pigs, rabbits, etc. But the efficiency of nuclear transfer is very low in all species. The present study was conducted to examine somatic nucleus remodelling and developmental ability in vitro of rabbit embryos by transferring somatic cells into enucleated germinal vesicle (GV), metaphase I (MI) or metaphase II (MII) oocytes. Microtubules were organized around condensed chromosomes after the nucleus had been transferred into any of the three types of cytoplasm. A bipolar spindle was formed in enucleated MII cytoplasm. Most of the nuclei failed to form a normal spindle within GV and MI cytoplasm. Some chromosomes scattered throughout the cytoplasm and some formed a monopolar spindle. Pseudopronucleus formation was observed in all three types of cytoplasm. Reconstructed embryos with MI and MII cytoplasm could develop to blastcysts. Nuclei in GV cytoplasm could develop only to the 4-cell stage. These results suggest that (1) GV material is important for nucleus remodelling after nuclear transfer, and (2) oocyte cytoplasm has the capacity to dedifferentiate somatic cells during oocyte maturation.     

In vitro fertilisation of mouse oocytes reconstructed by transfer of metaphase II chromosomes results in live births

The interaction between nucleus and cytoplasm can be explored through nuclear transfer. We describe here another tool to investigate this interaction: MII meiotic apparatus transfer (MAT) between mouse oocytes. In this study, the MII oocyte meiotic apparatus or spindle from C57BL/6 mice, a black strain, was transferred into an enucleated metaphase oocyte from Kunming mouse, a white strain. The results showed that the enucleation rate by treating oocytes with 3% sucrose was 100%, but the electrofusion efficiency was very low, with only 17.6% of reconstructed karyoplast-recipient cytoplasm pairs fused. When the fused oocytes were exposed to spermatozoa from C57BL/6 mice, 9 of 11 (82%) were fertilised. Eight reconstructed embryos at 1- to 4-cell stages were transferred into the oviducts of two synchronously pregnant Kunming strain fosters and one delivered two normal C57BL/6 offspring. This study indicates that MII meiotic apparatus or spindle sustains normal structure and function after micromanipulation and electrofusion. MAT provides a model for further research on the application of this technique to assisted human reproduction.     

Nuclear origin of aging-associated meiotic defects in senescence-accelerated mice

Factors of both cytoplasmic and nuclear origin regulate metaphase chromosome alignment and spindle checkpoint during mitosis. Most aneuploidies associated with maternal aging are believed to derive from nondisjunction and meiotic errors, such as aberrations in spindle formation and chromosome alignment at meiosis I. Senescence-accelerated mice (SAM) exhibit aging-associated meiotic defects, specifically chromosome misalignments at meiosis I and II that resemble those found in human female aging. How maternal aging disrupts meiosis remains largely unexplained. Using germinal vesicle nuclear transfer, we found that aging-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model. Cytoplasm of young hybrid B6C3F1 mouse oocytes could partly rescue aging-associated meiotic chromosome misalignment, whereas cytoplasm of young SAM was ineffective in preventing the meiotic defects of old SAM oocytes, which is indicative of a deficiency of SAM oocyte cytoplasm. Our results demonstrate that both nuclear and cytoplasmic factors contribute to the meiotic defects of the old SAM oocytes and that the nuclear compartment plays the predominant role in the etiology of aging-related meiotic defects.     

Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes

The present study aimed to analyze detailed morphological and functional characteristics of mouse in vitro matured oocytes after a pre-maturation culture (PMC) by temporary nuclear arrest with the specific phosphodiesterase 3-inhibitor (PDE3-I) Cilostamide. In a first experiment the lowest effective dose of Cilostamide was determined. Cumulus-oocyte complexes (COCs), isolated from small antral follicles, were exposed to different concentrations of Cilostamide (ranging from 0 (control) to 10 microM) for 24 hr. Afterwards, oocytes were removed from PDE3-I-containing medium and underwent in vitro maturation (IVM) for 16-18 hr. A concentration of 1 microM Cilostamide was the lowest effective dose for maximum level of inhibition and reversibility of meiosis inhibition. This concentration was used in further experiments to evaluate oocyte quality following IVM in relation to different parameters: kinetics of meiotic progression, metaphase II (MII) spindle morphology, aneuploidy rate, fertilization, and embryonic developmental rates. The results were compared to nonarrested (in vitro control) and in vivo matured oocytes (in vivo control). Following withdrawal of the inhibitor, the progression of meiosis was more synchronous and accelerated in arrested when compared to nonarrested oocytes. A PMC resulted in a significant increase in the number of oocytes constituting a MII spindle of normal morphology. None of the oocytes exposed to PDE3-I showed numerical chromosome alterations. In addition, fertilization and embryonic developmental rates were higher in the PMC group compared to in vitro controls, but lower than in vivo controls. These results provide evidence that induced nuclear arrest by PDE3-I is a safe and reliable method to improve oocyte quality after IVM.     

WHAMM is required for meiotic spindle migration and asymmetric cytokinesis in mouse oocytes

WASP homolog associated with actin, membranes and microtubules (WHAMM) is a newly discovered nucleation-promoting factor that links actin and microtubule cytoskeleton and regulates transport from the endoplasmic reticulum to the Golgi apparatus. However, knowledge of WHAMM is limited to interphase somatic cells. In this study, we examined its localization and function in mouse oocytes during meiosis. Immunostaining showed that in the germinal vesicle (GV) stage, there was no WHAMM signal; after meiosis resumption, WHAMM was associated with the spindle at prometaphase I (Pro MI), metaphase I (MI), telophase I (TI) and metaphase II (MII) stages. Nocodazole and taxol treatments showed that WHAMM was localized around the MI spindle. Depletion of WHAMM by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in failure of spindle migration, disruption of asymmetric cytokinesis and a decrease in the first polar body extrusion rate during meiotic maturation. Moreover, actin cap formation was also disrupted after WHAMM depletion, confirming the failure of spindle migration. Taken together, our data suggest that WHAMM is required for peripheral spindle migration and asymmetric cytokinesis during mouse oocyte maturation.     

Morphogenesis of Ascospores in Saccharomyces cerevisiae

Ultrastructural changes associated with ascospore formation in Saccharomyces cerevisiae were investigated by using freeze-etching and thin-sectioning techniques. The first nuclear division (meiosis I) is indicated by the appearance of spindle fibers within the nucleus. The nucleus subsequently elongates and eventually assumes a barbell shape; the second nuclear division (meiosis II) occurs before nuclear separation. The spindle fibers involved in meiosis II appear to be oriented perpendicular to those observed in meiosis I. A discrete bilaminar structure (forespore wall) progressively delineates each ascospore nucleus and encloses cytoplasmic material including mitochondria and endoplasmic reticulum. The forespores then elongate, close off, and become separated from the ascus cytoplasm by membranes. The ascospores assume a spherical shape as spore coat material is laid down; the latter stages of ascospore formation are characterized by thickening of the ascospore wall and disintegration of the ascus cytoplasm. No structures which could be identified as chromosomes were observed.     

Cdc25C expression in meiotically competent and incompetent goat oocytes

Change in Cdc25C expression and localization during maturation and meiotic competence acquisition was investigated in goat oocytes. Western blot analysis revealed that Cdc25C is constitutively expressed throughout meiosis in competent goat oocytes, with changes in its phosphorylation level. Cdc25C was detected at 55 and 70 kDa, representing the nonphosphorylated form and the hyperphosphorylated active form, respectively. During the G2-M transition at meiosis resumption, Cdc25C was hyperphosphorylated as evidenced by a clear shift from 55 to 70 kDa. Okadaic acid which induced premature meiosis resumption associated with MPF activation also involved a premature shift from 55 to 70 kDa in goat competent oocytes. After artificial activation of goat oocytes, Cdc25C returned to its 55 kDa form. By indirect immunofluorescence, Cdc25C was found essentially localized in the nucleus at the germinal vesicle stage, suggesting that Cdc25C functions within the nucleus to regulate MPF activation. Concomitantly with germinal vesicle breakdown, Cdc25C was redistributed throughout the cytoplasm. The amount of Cdc25C, very low in incompetent oocytes, increased with meiosis competence acquisition. On the other hand, during oocyte growth while the expression of Cdc25C increased, its phosphorylation level increased concomitantly as well as its nuclear translocation. These results suggest that meiosis resumption needs a sufficient amount of Cdc25C which must be completely phosphorylated and nuclear and that the amount of Cdc25C may be a limiting factor for meiotic competence acquisition. We could consider that Cdc25C nuclear translocation and phosphorylation, during oocyte growth, prepare the oocytes in advance for the G2-M phase transition occurring during meiosis resumption.     

Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture

Trichlorfon (TCF), an organophosphate insecticide and potent inhibitor of choline esterases, was previously shown to induce first meiotic nondisjunction and spindle aberrations in isolated, follicle cell-denuded mouse oocytes maturing in vitro. To explore dose-response and direct and indirect, potentially synergistic effects of TCF on the somatic cells and the oocyte within a follicle, we presently employed preantral follicle culture. 100 microg/ml TCF added at the time of hormonally stimulated resumption of meiosis of follicle cell-enclosed mouse oocytes, 16 h before in vitro ovulation, induced significant rises in first meiotic nondisjunction in oocytes from preantral follicle culture. Lower concentrations (6 microg/ml TCF) disturbed polar body formation. Nuclear maturation to meiosis II in absence of cytokinesis resulted in significant increases in polyploidy. Oocytes maturing in follicles in the presence of TCF had aberrant second meiotic spindles. Influences of TCF on somatic cell function were evident by reduced follicular mucification in vitro and deceased progesterone production. In contrast to TCF, acetylcholine (0.1-100 microM) increased progesterone production. The observations therefore suggest that TCF influences oocyte maturation and folliculogenesis directly and indirectly. High TCF is aneugenic at first meiotic division in oocytes, irrespective of the presence or absence of follicle cells. At lower concentrations TCF interferes with spindle formation, chromosome congression at meiosis II, and coordination of nuclear and cytoplasmic maturation, posing risks for second meiotic errors. The observations suggest that chronic TCF exposure during maturation in the follicle may predispose oocytes to the formation of chromosomally unbalanced preimplantation embryos after fertilization.     

绵羊卵泡液和次黄嘌呤对卵母细胞体外减数分裂的影响

目的　利用在培养液中添加绵羊卵泡液和次黄嘌呤 ,抑制卵母细胞GVBD发生 ,延长转录活性 ,从而使卵母细胞真正成熟 ,提高胚胎质量及生产效率。方法　利用体外成熟技术对有屠宰采集的绵羊卵母细胞进行培养 ,培养液中添加卵泡液及次黄嘌呤 ,检查成熟效果。结果　将卵母细胞培养在 5 0 %和 10 0 %的卵泡液中 ,2 4h后处于GV期的卵母细胞分别为 19% (8 4 2 )和 33 3% (13 39)。在含有 4mmol L次黄嘌呤的培养液中 ,2 4h后有2 1 6 % (16 74 )的卵母细胞处GV期 ,而对照组中只有 6 % (3 5 0 ) ,经过次黄嘌呤处理的卵母细胞多数都停滞于PⅠ期(44 6 % ,33 74 )。在 4mmol L次黄嘌呤培养液中添加FSH并未使受到抑制的卵母细胞诱导成熟。结论　卵泡液和次黄嘌呤只能在有限的程度上抑制减数分裂的重新启动 ,并对减数分裂的全过程都有影响 ,这种影响程度与抑制因子的浓度相关 ,存在明显的剂量效应。     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

MONOPLASTIDIC CELL DIVISION IN LOWER LAND PLANTS

In many bryophytes and vascular cryptogams mitosis and/or meiosis takes place in cells containing a single plastid. In monoplastidic cell division plastid polarity assures that nuclear and plastid division are infallibly coordinated. The two major components of plastid polarity are morphogenetic plastid migration and microtubule organization at the plastids. Before nuclear division the plastid migrates to a position intersecting the future division plane. This morphogenetic migration is a reliable marker of division polarity in cells with and without a preprophase band of microtubules (PPB). The PPB, which predicts the future division plane before mitosis, is a characteristic feature of land plants and its insertion into the cytokinetic apparatus marks the evolution of a cortical microtubule system and a commitment to meristematic growth. Microtubule systems associated with plastid division, the axial microtubule system (AMS) in mitosis and the quadripolar microtubule system (QMS) in meiosis, contribute to predictive positioning of plastids and participate directly in spindle ontogeny. Division polarity in monoplastidic sporocytes is remarkable in that division sites are selected prior to the two successive nuclear divisions of meiosis. Plastid arrangement prior to meiosis determines the future spore domains in monoplastidic sporocytes, whereas in polyplastidic sporocytes the spore nuclei play a major role in claiming cytoplasmic domains. It is hypothesized that predivision microtubule systems associated with monoplastidic cell division are early forming components of the mitotic apparatus that serve to orient the spindle and insure equal apportionment of nucleus and plastids. “Can it be supposed that cytoplasm would be intrusted with so important a task as the preparation of a chloroplast for each of the four nuclei that are later to preside over the spores before there is any indication that such nuclear division is to take place?” Bradley Moore Davis, 1899     

Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis

Background

During oocyte meiosis, the cytoskeleton dynamics, especially spindle organization, are critical for chromosome congression and segregation. However, the roles of the kinesin superfamily in this process are still largely unknown.

Results

In the present study, Kif18a, a member of the kinesin-8 family, regulated spindle organization through its effects on tubulin acetylation in mouse oocyte meiosis. Our results showed that Kif18a is expressed and mainly localized in the spindle region. Knock down of Kif18a caused the failure of first polar body extrusion, dramatically affecting spindle organization and resulting in severe chromosome misalignment. Further analysis showed that the disruption of Kif18a caused an increase in acetylated tubulin level, which might be the reason for the spindle organization defects after Kif18a knock down in oocyte meiosis, and the decreased expression of deacetylase Sirt2 was found after Kif18a knock down. Moreover, microinjections of tubulin K40R mRNA, which could induce tubulin deacetylation, protected the oocytes from the effects of Kif18a downregulation, resulting in normal spindle morphology in Kif18a-knock down oocytes.

Conclusions

Taken together, our results showed that Kif18a affected Sirt2-mediated tubulin acetylation level for spindle organization during mouse oocyte meiosis. Our results not only revealed the critical effect of Kif18a on microtubule stability, but also extended our understanding of kinesin activity in meiosis.

     

Continuous exposure to bisphenol A during in vitro follicular development induces meiotic abnormalities

Bisphenol A (BPA), a widely used environmental contaminant, may exert weak estrogenic, anti-androgenic and anti-thyroidic activities. BPA is suspected to possess aneugenic properties that may affect somatic cells and mammalian oocytes. Oocyte growth and maturation depend upon a complex bi-directional signaling between the oocyte and its companion somatic cells. Consequently, disturbances in oocyte maturation may originate either from direct effects of BPA at the level of the oocyte or from indirect influences at the follicular level, such as alterations in hormonal homeostasis. This study aimed to analyze the effects of chronic BPA exposure (3 nM to 30 microM) on follicle-enclosed growth and maturation of mouse oocytes in vitro. Oocytes were cultured and their spindle and chromosomes were stained by alpha-tubulin immunofluorescence and ethidium homodimer-2, respectively. Confocal microscopy was utilized for subsequent analysis. Only follicles that were exposed to 30 microM BPA during follicular development showed a slightly reduced granulosa cell proliferation and a lower total estrogen production, but they still developed and formed antral-like cavities. However, 18% of oocytes were unable to resume meiosis after stimulation of oocyte maturation, and 37% arrested after germinal vesicle breakdown, significantly different from controls (p<0.05). Only 45% of the oocytes extruded a first polar body (p < 0.05). 30 microM BPA led also to a significant increase in meiosis I-arrested oocytes with unaligned chromosomes and spindle aberrations. Oocytes that were able to progress beyond meiosis I, frequently arrested at an abnormal telophase I. Additionally, in many oocytes exposed to low chronic BPA that matured to meiosis II chromosomes failed to congress at the spindle equator. In conclusion, mouse follicle culture reveals non-linear dose-dependent effects of BPA on the meiotic spindle in mouse oocytes when exposure was chronic throughout oocyte growth and maturation.     

Nuclear shuttling of the peptidase nardilysin

The metalloendopeptidase nardilysin contains a putative N-terminal nuclear localization signal. The functionality of this sequence was tested with nardilysin-GFP fusion constructs. Expression in NIH3T3 cells showed approximately 90-95% of nardilysin-GFP as cytoplasmic. However, 3-6% of transfected cells showed both cytosolic and nuclear staining, while 2-4% showed predominantly nuclear staining. A nuclear localization signal mutant and an N-terminally truncated nardilysin-GFP with the nuclear localization signal deleted were completely cytoplasmic. Although endogenous nardilysin was barely detectable in the nucleus, after treatment with leptomycin B, nuclear nardilysin rose to approximately 15% and to over 25% after addition of spermine. The ability of a methionine 49 to act as the sole initiator methionine, as previously proposed, was tested by inserting a c-myc epitope between leucine28 and glycine29. Expression in HEK293 cells showed the presence of the c-myc tag, demonstrating that the enzyme can be translated from the first methionine and contains the nuclear localization signal.