RNA SYNTHESIS IN THE MOUSE OOCYTE

RNA synthesis in the oocyte and granulosa cell nuclei of growing follicles has been studied in the mouse ovary. The RNA precursor [³H]uridine was administered intraperitoneally to adult mice and the amount of label incorporated into ovarian RNA was quantitated autoradiographically using grain-counting procedures. Uridine incorporation into the nucleus is low in oocytes of small, resting follicles but increases during follicle growth and reaches a peak prior to the beginning of antrum formation. Thereafter uptake rapidly declines and is very low in the oocytes of maturing follicles. Uridine incorporation into granulosa cell nuclei, in contrast to that found in the oocyte, increases gradually during most of the period of follicle growth. Qualitative studies of the activity of endogenous, DNA-dependent RNA polymerases have also been made in fixed oocytes isolated from follicles at different stages of growth. Polymerase activity is demonstrable in the nucleolus and nucleoplasm of oocytes from growing follicles, but is absent from maturing oocytes of large follicles.     

草鱼产卵类型的研究

本文是对已达性成熟年龄的池养和江河雌性草鱼产卵前后卵巢组织学结构的研究。实验结果证明草鱼是一次产卵类型。经人工催产后的雌性草鱼,由于亲鱼的成熟程度存在个体间的差异,有的全产,有的部分产。在湘江天然产卵场捕得的雌性草鱼,也有全产和部分产的。人工催产全产后的卵巢组织学结构是Ⅰ、Ⅱ时相,部分产后的卵巢组织学结构是Ⅰ、Ⅱ和Ⅳ(Ⅳ+、Ⅳ++)时相,已达满熟阶段但未经人工催产的卵巢组织学结构是Ⅰ、Ⅱ和Ⅳ(Ⅳ+++)时相,以上都未发现有处于Ⅲ时相的卵母细胞。从江河天然产卵场捕得的全产、部分产和尚未产卵的雌性草鱼的卵巢组织学结构,与上述结果一致。证实由Ⅲ时相到Ⅳ时相是同步性的。5月全产后的雌性草鱼,其卵巢组织学结构在6-9月内处于第Ⅱ期,没有新的Ⅳ时相卵母细胞。因此,夏季全产后的雌性草鱼,不可能在当年夏季或秋季完成由Ⅱ-Ⅲ-Ⅳ时相的发育程序。草鱼的卵巢成熟系数在繁殖季节只出现一次高峰。       

Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes

In order to determine whether differentiated somatic cells maintain genetic totipotency, nuclear transplantations from several differentiated somatic cell types into eggs and oocytes were performed previously in Rana pipiens and Xenopus laevis. The formation of postneurula embryos and tadpoles under the direction of the test nuclei demonstrated their genetic multipotency. In addition, Rana erythrocyte nuclei transplanted to oocytes directed more extensive tadpole development than those injected into eggs. We have extended our studies of the genomic potential of differentiated somatic nuclei from the peripheral blood of Rana pipiens. First, we show that the developmental potential of erythrocyte nuclei injected into oocytes at first meiotic metaphase was greater than those injected into diplotene oocytes. Second, we demonstrate that erythroblast and leukocyte nuclei transplanted to oocytes at first meiotic metaphase promoted more advanced tadpole development than those previously injected into Xenopus eggs. Third, erythrocyte nuclei were more successful in promoting advanced tadpole development compared with erythroblast and leukocyte nuclei. The results show that differentiated somatic nuclei transferred to the cytoplasm of oocytes at first meiotic metaphase display enhanced genomic and developmental potential over those transplanted to diplotene oocytes and eggs, at least for the three nuclear cell types tested from the peripheral blood.     

Chromosome condensation activity in Rana pipiens eggs matured in vivo and in blastomeres arrested by cytostatic factor (CSF)

The ability of brain nuclei to give rise to condensed chromosomes was studied inRana pipiens eggs which had undergone meiotic maturation in vivo, in blastomeres of two-cell embryos which had been arrested at metaphase by the injection of cytostatic factor (CSF) from mature eggs, and in immature fully grown ovarian oocytes with and without prior CSF injection. Chromosomes from brain nuclei were found to condense within 4 h in mature eggs and this chromosome condensation activity was enhanced by the chelation of free Ca²⁺ in the nuclear isolation medium. Chromosomes also condensed in CSF-arrested blastomeres whether they were placed in the blastomere 30 min before the CSF injection or as long as 22 h after the CSF. Both the Ca²⁺-sensitive CSF, 1CSF, and the Ca²⁺-insensitive CSF, 2CSF, resulted in chromosome condensation within arrested blastomeres. The condensation was accompanied by the formation of multipolar spindles and asters. However, it was found that cytoplasm in CSF-arrested blastomeres does not arrest mitosis at metaphase when transferred into a cleaving blastomere. Other experiments demonstrated that chromosome condensation does not occur in ovarian oocytes even when supplied with CSF. The results are interpreted as indicating that CSF does not directly bring about chromosome condensation, but arrests the cell cycle at metaphase and stabilizes the cytoplasmic conditions of metaphase which, in turn, induce chromosome condensation in foreign nuclei as well as spindle and aster formation.     

青鱼性腺发育的研究

湖南地区生长于池塘环境的青鱼,性成熟年龄是5—6年,雄性比雌性普遍地早熟一年。卵母细胞和滤泡细胞是同源的,都来自于卵原细胞。池养青鱼的卵母细胞只能发育到初级卵母细胞阶段(Ⅳ时相),必须通过人工催情,才能进行染色体的减数分裂,使卵母细胞由第Ⅳ时相发育到第Ⅴ时相。精细胞的发生,能够完成由精原细胞到精子的全部发育过程。青鱼在第一次性周期内,雄性精巢在第5个冬季进入第Ⅳ期,雌性卵巢在第6个冬季进入第Ⅲ期,从此以后,每年冬季,雄性精巢回复到第Ⅳ期,雌性卵巢回复到第Ⅲ期,这种性腺季节周期变化的规律,为生产上选留亲鱼提供了理论依据。青鱼雌性卵母细胞由第Ⅲ时相到第Ⅳ时相是同步性的;经人工催情产卵或自然退化后,卵巢的组织学结构又回复到第Ⅱ期,证明青鱼是一次产卵类型。已经达到性成熟年龄的雌性青鱼,卵母细胞的卵黄形成有两种不同的类型。第一种类型是泡内卵黄,第二种类型是泡外卵黄。如果饲养管理工作如投饵、水质调节不适宜,卵母细胞不能正常形成卵黄,就会出现卵子的败育现象,这是生产上一个重要问题,必须进一步研究。       

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.     

The potential significance of binovular follicles and binucleate giant oocytes for the development of genetic abnormalities

Normal development of a fertilizable female gamete emanates from a follicle containing only one oocyte that becomes haploid after first meiotic division. Binovular follicles including two oocytes and binucleate giant oocytes that are diploid after first meiosis constitute notable exceptions from this rule. Data provided by programmes of human-assisted reproduction on the occurrence of both phenomena have been reviewed to evaluate possible implications for the formation of genetic abnormalities. To exclude confusion with oocytes aspirated from two adjacent individual follicles, true binovularity has been defined as inclusion of two oocytes within a common zona pellucida or their fusion in the zonal region. A total of 18 conjoined oocytes have been reported and one of the oocyte was normally fertilized in seven cases. Simultaneous fertilization of both female gametes occurred only once. No pregnancy was achieved after transfer of an embryo from a binovular follicle. Binucleate giant oocytes have been observed sporadically but a few reports suggest an incidence of up to 0.3% of all gametes retrieved. Extensive studies performed by two independent centres demonstrated that giant oocytes are diploid at metaphase II, can undergo fertilization in vitro with formation of two or three pronuclei and develop into triploid zygotes and triploid or triploid/mosaic embryos. In summary, giant binucleate oocytes may be responsible for the development of digynic triploidy whereas the currently available data do not support a role of conjoined oocytes in producing dizygotic twins, mosaicism, chimaeras or tetraploidy. However, more information on the maturity and fertilizability of oocytes from binovular follicles is needed. Future studies should also evaluate a possible impact of pharmaceutical and environmental oestrogens on the formation of multiovular follicles.     

Cytomorphology of female and male gonads in Antarctic toothfish <Emphasis Type="Italic">Dissosctichus mawsoni</Emphasis> (Nototheniidae) from the Ross Sea in the summer period

Results of histological analysis of gonads of female and male Antarctic toothfish Dissostichus mawsoni caught in the Ross Sea of the Pacific sector in the period of Antarctic summer (December–February) 2004–2005 are presented. Morphological indices and index of gonad maturity are described and ecological criteria of assessment of maturity stages of ovaries, cytological indices of oocytes, and the type of toothfish oogenesis are determined. It was established that in the period of fishing Antarctic toothfish, females and males with gonads at maturity stage III dominate. Ovaries contain two groups of oocytes of the period of trophoplasmatic growth and large oocytes of the nearest spawning season that comprise the smallest proportion of total sex cells. In the testes of two male toothfish, the primary fusion of renal and generative tissues was revealed. It is suggested that the termination of gonad maturity of toothfish takes place from March to April, and spawning takes place from June to August.     

Studies of oogenesis in the rotifer,Asplanchna

Summary Oocyte development in Asplanchna brightwelli was studied by observation through the transparent body wall of living females and by electron microscopy. During oogenesis, which requires four to six hours, the oocyte increases in volume approximately 1000-fold. Most of its cytoplasm appears to be derived from the vitellarium by direct flow through the cytoplasmic bridge. This flow is rapid enough to be easily observed in the living female at low magnifications. Ribosomes, mitochondria, cortical granules, and lipid droplets were observed in the bridge area in electron micrographs.RNA synthesis during oogenesis was studied by means of autoradiography. Very little synthesis could be demonstrated in oocyte nuclei at any period of oogenesis, whereas the vitellarium nuclei show active incorporation of ³H-uridine throughout the reproductive life of the adult female. Most of this RNA is subsequently transferred to developing oocytes.This research was supported by USPHS Grant GM 121183 to Dr. C. W. Birky, Jr.     

Morphology,ultrastructure, and germ cell cluster formation in ovarioles of aphids

The structure of aphid ovaries, including ovipare and virginopare morphs of five species, was investigated by light and electron microscopy. Aphids contain telotrophic meroistic ovarioles. The amount and distribution of cytoplasmic components of nurse cells, nutritive cords, and young oocytes are nearly identical to those known from scale insects and heteropterans. Each ovariole has a constant number of nurse cells and oocytes. In ovaries of ovipare morphs, the nurse cell nuclei enlarge by endomitosis (n = 2⁸n?2¹⁰n), whereas in virginopare morphs the nurse cell nuclei remain small (n = 2²n?2⁴n). Furthermore, in virginoparae the previtellogenic growth of oocytes is highly reduced, and vitellogenesis and chorionogenesis are blocked totally. Embryogenesis starts immediately after the shortened previtellogenic growth. In each ovariole, all germ cell descendants belong to one germ cell cluster that follows the 2ⁿ rule. The cluster normally contains 2⁵ = (32) cells, but other mostly smaller numbers also occur. In contrast to polytrophic meroistic ovarioles, more than one cell of each cluster will develop into an oocyte. In Drepanosiphum platanoides, 16 (2^n?1) nurse cells and 16 (2^n?1) oocytes exist in each cluster, whereas, in Metopolophium dirhodum, 8 (2^n?2) oocytes and 24 (2^n?1 + 2^n?2) nurse cells are normally found. In many ovarioles of Macrosiphum rosae, 21 nurse cells nourish 11 oocytes. Models of germ cell cluster formation in aphid ovaries are discussed.     

Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells

Although it is generally accepted that relatively high efficiencies of somatic cell cloning in mammals can be achieved by using donor cells from the female reproductive system (e.g., cumulus/granulosa, oviduct, and mammary gland cells), there is little information on the possibility of using male-specific somatic cells as donor cells. In this study we injected the nucleus of immature mouse Sertoli cells isolated from the testes of newborn (Days 3-10) males into enucleated mature oocytes in order to examine the ability of their nuclei to support embryonic development. After activation of the oocytes that had received the freshly recovered immature Sertoli cells, some developed into the morula/blastocyst stage, depending on the age of the donor cells (22.0-37.4%). When transferred into pseudopregnant females, 7 (3.3%, 7 of 215) developed into normal pups at term. Nuclear transfer of immature Sertoli cells after 1 wk in culture also produced normal pups after embryo transfer (3.1%, 2 of 65). Even after cryopreservation in a conventional cryoprotectant solution, their ability as donor cells was maintained, as demonstrated by the birth of cloned young (6.7%, 7 of 105). Immature Sertoli cells transfected with green fluorescent protein gene also supported embryo development into morulae/blastocysts, which showed specific fluorescence. This study demonstrates that immature Sertoli cells, male-specific somatic cells, are potential donors for somatic cell cloning.     

An ultrastructural investigation of the early stages of oocyte differentiation in Actinia fragacea (Cnidaria; Anthozoa)

Individuals from a population of the intertidal sea anemone Actinia fragacea (Tugwell) were collected at approximately monthly intervals over an 18 month period. Samples of gonad were removed from each anemone and examined by light and electron microscopy. During late spring and early summer, large numbers of small cells were seen in the endoderm of the female gonads, lying close to the mesoglea. For convenience, these cells were classified into three types. Type I cells are 6–9 μm in diameter, with relatively very large nuclei, which may contain synaptinemal complexes, and scant cytoplasm containing few organelles. Type II cells are larger, reaching 15 μ m in diameter, with more abundant cytoplasm containing more organelles and inclusions. The nucleus is more dense, but may also contain synaptinemal complexes. Type III cells are less common. They are similar in size to Type II cells, but their nuclei contain irregular dense chromatin masses, and the nuclear envelope is incomplete or absent. The possible significance of the various cell types is discussed. It is suggested that Type I cells are oocytes at a very early stage of differentiation and that Type II cells are rather later oocytes. The status of the Type III cells is uncertain.     

高氏后异刺线虫肠道和生殖细胞超微结构观察

我们曾经发现雌性高氏后异刺线虫在生殖周期中,虫体的外表形态和器官结构以及细胞核内DNA含量均发生连续的变化。现在进一步对线虫细胞的超微结构进行分析,着重观察核内染色质结构的变化。前三期雌虫肠细胞核内有许多粗大的染色质团块和显著的核仁。Ⅳ期肠核内染色质团块融合成少数球体,而且不断减少,以致消失,单剩下染色质细粒(纤丝切面)。而核仁增大,结构更为复杂。卵巢细胞亦有类似改变,但末见染色质球体。雄虫并无生殖周期,也无上述变化。     

The ability of dehydrated hamster and human sperm nuclei to develop into pronuclei.

To determine whether the nuclei of mature mammalian spermatozoa are resistant to dehydrated conditions, nuclei of hamster and human spermatozoa were freeze-dried or treated with various dehydrating agents before injection into hamster oocytes. Freeze-dried nuclei remained capable of developing into pronuclei even after 12 mo of storage at 4 degrees C. The level of DNA synthetic activity in the sperm (male) pronucleus was comparable to that in the egg (female) pronucleus. Sperm nuclei that had been stored in 100% ethanol, 100% methanol, or chloroform-methanol (2:1) mixture for 20 days were also capable of developing into pronuclei. Even the nuclei that had been dehydrated ("fixed") with Carnoy's fluid could develop into morphologically normal pronuclei. However, the level of DNA synthesis in the pronuclei derived from these chemically dehydrated nuclei was generally lower than that in the female pronuclei. Although the genetic integrity of the dehydrated sperm nuclei is yet to be determined, nuclei of mature hamster and human spermatozoa appear to be fairly resistant to dehydrated conditions.     

Ability of in vitro maturing bovine oocytes to transform sperm nuclei to metaphase chromosomes.

Bovine oocytes at the germinal vesicle stage were inseminated in Brackett & Oliphant's medium with bovine serum albumin, caffeine and heparin. Eight hours after insemination, oocytes were transferred into tissue culture medium-199 containing 10% fetal calf serum and cultured for 5-40 h at 39 degrees C in 5% CO2 in air. The proportions of unpenetrated and penetrated oocytes reaching metaphase II increased as the time of examination increased, reaching 70 and 65% 40 h after transfer, respectively. When oocytes were penetrated by more than four spermatozoa, meiotic maturation was greatly retarded. Sperm nuclei were decondensed in most (81%) penetrated oocytes 5 h after transfer. The decondensed sperm nuclei were recondensed and then transformed to metaphase chromosomes which were morphologically compacted at first but became slightly dispersed later. The formation of the metaphase chromosomes was observed in 86% of penetrated oocytes examined 40 h after transfer, and occurred in all metaphase II oocytes at that time. In oocytes penetrated by more than nine spermatozoa, no such transformation of sperm nuclei was observed. Well-developed male and female pro-nuclei were observed in only three (6%) of 51 oocytes penetrated 40 h after transfer.     

Reproductive cycles in oregon populations of the echinoid, Strongylocentrotus purpvratus (Stimpson). II. Seasonal changes in oocyte growth and in abundance of gametogenic stages in the ovary

During the annual reproductive cycle in the echinoid, Strongylocentrotus purpuratus (Stimpson) two major seasonal events in oogenesis have been demonstrated by several different quantitative methods. Both seasonal changes in size and frequency distributions of stages of the oocytes indicate that in the intertidal populations studied, there is an abrupt increase in the growth rate of oocytes during October, accompanied by an increase in the rate at which oogonia become primary oocytes. Between March, just after spawning is completed, and October, oocytes increase in volume at a rate of ≈ 2 % per day. In October and November, the growth rate of oocytes approaches 6 % per day, and thereafter declines for oocytes reaching maturity in December.The absolute numbers of oocytes and oogonial clusters/mm² of ovary wall were estimated and adjusted for changes produced by volume changes in the gonad. The lowest numbers of oogonial clusters occur from December to March. Renewed proliferation begins soon after spawning. There is a significant increase in number of clusters in March, and the absolute cluster number increases to a maximum in June, reaching approximately four times the initial number. The proliferation rate during this period is ≈ 180 clusters/mm²/month; some proliferation continues into September. The number of small oocytes present remains the same from March to August, when it begins to increase. The total number of oocytes produced during the fall period is approximately three times the number initially present at the start of the annual cycle in March. Two populations studied differ in the absolute numbers of oogonial clusters and oocytes produced in the annual cycle; environmental factors must influence the processes involved. In S. purpuratus the large majority of oocytes differentiate from oogonia during the same reproductive cycle in which they reach maturity as ova.     

Effect of telophase enucleation on bovine somatic nuclear transfer

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.     

Production of cloned goats after nuclear transfer using adult somatic cells.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.     

Differences in reproductive strategies between obscure puffer Takifugu obscurus and ocellated puffer Takifugu ocellatus during their spawning migration

Reproductive strategies were compared between obscure puffer Takifugu obscurus and ocellated puffer Takifugu ocellatus captured in waters near Yangzhong Island in the lower reaches of the Yangtze River during the spawning migration season from February to June in two consecutive years (1995 and 1996). Results showed that obscure puffer and ocellated puffer have adopted different reproductive strategies, including different spawning times, different size at maturity, and different number and size of oocytes, resulting in two different larval sizes. In detail, the timing of the spawning migration and status of gonad development of obscure puffer was about 1 month earlier than that of ocellated puffer; the obscure puffer was obviously longer and heavier than ocellated puffer in both mature male and female fish; mean GSI of obscure puffer females (15.8%) was significantly higher than that of ocellated puffer females (14.6%); the average diameter of ocellated puffer eggs (1.49 ± 0.12 mm) was significantly larger than that of the obscure puffer (1.22 ± 0.08 mm); and obscure puffer females (320.8 oocytes mg^?1 somatic wet weight) had significantly higher relative fecundity than ocellated puffer females (125.2 oocytes mg^?1 somatic wet weight). These differences in reproductive strategies between two closely related species of the Takifugu genus indicate that both obscure puffer and ocellated puffer fit the r/K dichotomy. Obscure puffer shows K‐selected characters with maturity at relatively large size and r‐selected characters with relatively many and small offspring, whereas ocellated puffer shows r‐selected characters with maturity at a relatively small size and K‐selected characters with relatively few and large offspring.