Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary

When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.YTIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.YTIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.     

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.     

Nonrandom segregation of the mouse univalent X chromosome: evidence of spindle-mediated meiotic drive

A fundamental principle of Mendelian inheritance is random segregation of alleles to progeny; however, examples of distorted transmission either of specific alleles or of whole chromosomes have been described in a variety of species. In humans and mice, a distortion in chromosome transmission is often associated with a chromosome abnormality. One such example is the fertile XO female mouse. A transmission distortion effect that results in an excess of XX over XO daughters among the progeny of XO females has been recognized for nearly four decades. Utilizing contemporary methodology that combines immunofluorescence, FISH, and three-dimensional confocal microscopy, we have readdressed the meiotic segregation behavior of the single X chromosome in oocytes from XO females produced on two different inbred backgrounds. Our studies demonstrate that segregation of the univalent X chromosome at the first meiotic division is nonrandom, with preferential retention of the X chromosome in the oocyte in approximately 60% of cells. We propose that this deviation from Mendelian expectations is facilitated by a spindle-mediated mechanism. This mechanism, which appears to be a general feature of the female meiotic process, has implications for the frequency of nondisjunction in our species.     

Sex, not genotype, determines recombination levels in mice

Recombination, the precise physical breakage and rejoining of DNA between homologous chromosomes, plays a central role in mediating the orderly segregation of meiotic chromosomes in most eukaryotes. Despite its importance, the factors that control the number and placement of recombination events within a cell remain poorly defined. The rate of recombination exhibits remarkable species specificity, and, within a species, recombination is affected by the physical size of the chromosome, chromosomal location, proximity to other recombination events (i.e., chiasma interference), and, intriguingly, the sex of the transmitting parent. To distinguish between simple genetic and nongenetic explanations of sex-specific recombination differences in mammals, we compared recombination in meiocytes from XY sex-reversed and XO females with that in meiocytes from XX female and XY male mice. The rate and pattern of recombination in XY and XO oocytes were virtually identical to those in normal XX females, indicating that sex, not genotype, is the primary determinant of meiotic recombination patterns in mammals.     

Progesterone-stimulated meiotic cell division in Xenopus oocytes. Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase.

Ripe Xenopus oocytes in first meiotic prophase when incubated with progesterone in vitro progress synchronously in 3 to 5 h without interphase to second meiotic metaphase where they remain until fertilization or activation. Using highly purified preparations of regulatory and catalytic subunits of adenosine 3':5'-monophosphate-dependent protein kinase from muscle, this progesterone-stimulated cell division sequence was found to be inhibited by microinjection of the catalytic subunit and induced directly in the absence of progesterone after microinjection of regulatory subunit. Dose-response curves revealed that half-maximal effects of regulatory and catalytic subunits occurred at an internal concentration of approximately 0.1 muM. These results indicate that the catalytic subunit is necessary and sufficient to block progesterone-stimulated meiotic cell division. Other experiments revealed that the catalytic subunit was inhibitory only during the first hour after progesterone exposure, suggesting that initial steps in meiotic cell division are affected. Control experiments demonstrate that the muscle cAMP-dependent protein kinase subunits may interact with the endogenous oocyte protein kinase. The results support a model in which meiotic cell division is regulated by a phosphoprotein subject to control by cAMP-dependent protein kinase.     

Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging

Two forms of oocytes termed SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus) differing for the spatial distribution of nuclear and nucleolar-associated chromatin have been described within the antral compartment of the ovary of a number of mammals. The biological significance of these two kind of oocytes is as yet not completely clear. In previous studies we have shown that prior to ovulation, mouse SN oocytes isolated from the antral compartment, matured and fertilized in vitro have a far better meiotic and developmental competence than NSN oocytes. Immediately after ovulation SN and NSN oocytes remaining in the antral compartment do not develop beyond the 2-cell stage. To further examine the correlation between chromatin distribution and meiotic competence of mouse antral oocytes, in the present study we have analyzed chromosome segregation at the first meiotic division in antral (SN and NSN) and in ovulated oocytes. SN and NSN oocytes were isolated before (48 h post PMSG injection) or after (15 h post–hCG injection) ovulation from ovaries of females of increasing age, they were cultured in vitro to metaphase II, and their aneuploidy rate was examined. Comparison of data obtained before and after ovulation highlights two main points: 1. Following ovulation a statistically significant increase of aneuploidy is observed in antral oocytes in most age groups and it is attributable to SN oocytes. 2. The aneuploidy rate of ovulated oocytes does not increase during female aging. We have found a correlation between chromatin distribution, hormonal status, and the incidence of aneuploidy during the oocyte first meiotic division. Mol. Reprod. Dev. 50 :305–312, 1998. © 1998 Wiley-Liss, Inc.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.     

Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol-induced parthenogenones

A high frequency of parthenogenetic activation occurs when ovulated mouse oocytes are briefly exposed to a dilute solution of ethanol in vitro. Cytogenetic analyses of parthenogenones at metaphase of the first cleavage division have confirmed that parthenogenetic activation, per se, does not increase the incidence of chromosome segregation errors during the completion of the second meiotic division. Ethanol-induced activation, however, significantly increases the incidence of aneuploidy. The ultrastructural changes that occur in the morphology and organization of the second meiotic spindle apparatus in ethanol- and hyaluronidase-activated oocytes is reported here. Abnormalities in the arrangement of microtubule arrays and chromosome position were principally observed in ethanol-activated oocytes at anaphase and telophase of the second meiotic division, but were only rarely observed in hyaluronidase-activated oocytes. It is proposed that the abnormalities in spindle morphology and chromosome displacement observed in ethanol-activated oocytes represent the initial events that lead to chromosome segregation errors following exposure to this agent.     

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down’s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.     

Effects of Protein Synthesis on the Maturation of Mouse Oocytes in vitro

卵母细胞成熟过程中伴随有多种蛋白质的合成与磷酸化,蛋白质的合成对卵细胞的成熟具有重要作用。本实验较系统地阐述小鼠卵母细胞体外成熟培养的不同阶段蛋白质合成对卵母细胞成熟的影响。放线菌酮是肽链延伸的抑制因子。将生发泡（ＧＶ）期的卵母细胞分别于Ｔ６成熟培养液中培养０、４、６、９小时后,转至含有１０ｍｇ／ｍｌ放线菌酮的Ｔ６成熟培养液继续培养１２１５小时。固定、染色、观察卵母细胞。结果如Ｔａｂｌｅ１。０小时实验组：抑制处理４小时,其生发泡破裂（ＧＶＢＤ）发生率与对照组无明显差异。表明：卵母细胞ＧＶＢＤ所需蛋白质（如：成熟促进因子ＭＰＦ等）是在卵巢的卵泡卵母细胞生长过程中完成的。４、６小时实验组：笫一极体的释放被完全抑制,卵母细胞不能达到ＭＩ期,染色质处于凝集状态（Ｆｉｇ．３＆４）。表明：ＧＶＢＤＭＩ期间所需蛋白质的合成对卵母细胞ＭＩ期中期纺缍体的形成与维持具有重要作用。９小时实验组：可能由于卵母细胞发育速度存在个体间的差异。没有进入ＭＩＩ期的便停滞于ＭＩ期以前。进入ＭＩ期的则能排出笫一极体。因此,笫一极体的释放总体上呈不完全抑制状态,其释放率低于对照组。但是,后者虽然弪过恢复培养至１５小时,可能由于微管蛋白的合成     

Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.     

The mechanism of secondary nondisjunction in Drosophila melanogaster females

Bridges (1916) observed that X chromosome nondisjunction was much more frequent in XXY females than it was in genetically normal XX females. In addition, virtually all cases of X nondisjunction in XXY females were due to XX <--> Y segregational events in oocytes in which the two X chromosomes had failed to undergo crossing over. He referred to these XX <--> Y segregation events as "secondary nondisjunction." Cooper (1948) proposed that secondary nondisjunction results from the formation of an X-Y-X trivalent, such that the Y chromosome directs the segregation of two achiasmate X chromosomes to opposite poles on the first meiotic spindle. Using in situ hybridization to X and YL chromosomal satellite sequences, we demonstrate that XX <--> Y segregations are indeed presaged by physical associations of the X and Y chromosomal heterochromatin. The physical colocalization of the three sex chromosomes is observed in virtually all oocytes in early prophase and maintained at high frequency until midprophase in all genotypes examined. Although these XXY associations are usually dissolved by late prophase in oocytes that undergo X chromosomal crossing over, they are maintained throughout prophase in oocytes with nonexchange X chromosomes. The persistence of such XXY associations in the absence of exchange presumably facilitates the segregation of the two X chromosomes and the Y chromosome to opposite poles on the developing meiotic spindle. Moreover, the observation that XXY pairings are dissolved at the end of pachytene in oocytes that do undergo X chromosomal crossing over demonstrates that exchanges can alter heterochromatic (and thus presumably centromeric) associations during meiotic prophase.     

Development and fertility of ovaries in the B6.YDOM sex-reversed female mouse

When the Y chromosome of Mus musculus domesticus (YDOM) was introduced onto the C57BL/6 (B6) mouse background, half of the XY progeny (B6.YDOM) developed bilateral ovaries and female internal and external genitalia. We examined the fertility of the B6.YDOM sex-reversed female mouse. The chromosomal sex of the individual mouse was identified by dot hybridization with mouse Y chromosome-specific DNA probes. The results indicated that all XY females lacked regular estrous cyclicity although most were able to mate and ovulate after treatment with gonadotropins. When they had been ovariectomized and grafted with ovaries from the XX female litter mate, they initiated estrous cyclicity. Reciprocally, the XX female that had received XY ovarian grafts did not resume estrous cyclicity. Development of the XY ovary was morphologically comparable to the XX ovary until 16 day of gestation (d.g.), when most germ cells had reached the zygotene or pachytene stage of meiotic prophase. However, by the day of delivery (19 or 20 d.g.), no oocyte remained in the medullary cords of the XY ovary. In the control XX ovary, the first generation of follicles developed in the medullary region, and 5 delta-3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) activity appeared first in the stromal cells around growing follicles by 10 days after birth. In contrast, in the XY ovary, follicles were not formed in the medullary region, and 3 beta-HSDH activity appeared in epithelial cells of the oocyte-free medullary cords. Primordial follicles in the cortex region continued development in both the XX and XY ovaries. These results suggest that the XY female is infertile due to a defect inside the XY ovary. The prenatal loss of oocytes in the medullary cords may be a key event leading to abnormal endocrine function, and thereby, the absence of estrous cyclicity.