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.     

Transforming growth factor-β1 in reproduction

Expression patterns of TGF-βs during embryogenesis and in adult reproductive organs, as well as the activities of these molecules in in vitro assays of biological processes relating to reproduction and development, have suggested that TGF-βs may play a role in both reproductive function and embryonic development. To investigate the function of TGF-β1 in vivo, the murine TGF-β1 gene was disrupted by gene targeting, and animals that lacked TGF-β1 activity were generated. Homozygous mutant animals were obtained which exhibited a multifocal inflammatory disease. However, the observed numbers of homozygous mutant offspring were less than expected, suggesting the occurrence of some type of prenatal lethality. This paper reviews the proposed role of the TGF-βs in reproductive and developmental processes and discusses observations obtained from the TGF-β1 gene-targeting experiments as they relate to these processes. © 1994 Wiley-Liss, Inc.     

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12–14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me₂SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.     

Molecular and functional characterization of Anopheles gambiae inward rectifier potassium (Kir1) channels: A novel role in egg production

Inward rectifier potassium (Kir) channels play essential roles in regulating diverse physiological processes. Although Kir channels are encoded in mosquito genomes, their functions remain largely unknown. In this study, we identified the members of the Anopheles gambiae Kir gene family and began to investigate their function. Notably, we sequenced the A. gambiae Kir1 (AgKir1) gene and showed that it encodes all the canonical features of a Kir channel: an ion pore that is composed of a pore helix and a selectivity filter, two transmembrane domains that flank the ion pore, and the so-called G-loop. Heterologous expression of AgKir1 in Xenopus oocytes revealed that this gene encodes a functional, barium-sensitive Kir channel. Quantitative RT-PCR experiments then showed that relative AgKir1 mRNA levels are highest in the pupal stage, and that AgKir1 mRNA is enriched in the adult ovaries. Gene silencing of AgKir1 by RNA interference did not affect the survival of female mosquitoes following a blood meal, but decreased their egg output. These data provide evidence for a new role of Kir channels in mosquito fecundity, and further validates them as promising molecular targets for the development of a new class of mosquitocides to be used in vector control.     

猪卵巢体外保存温度对卵母细胞染色质构型和成熟能力的影响

Most porcine oocytes used in studies on embryo biotechnology and the in vitro production of embryos are currently obtained from the ovaries of slaughtered gilts. The duration and temperature during ovary transportation and handling might, therefore, affect the recovery of culturable COCs, chromatin configuration and developmental competence of oocytes. The effects of ovary storage temperature on chromatin configuration and in vitro maturation of porcine oocytes were examined in this study. Ovaries collected from a slaughterhouse were stored in vitro for 8 h under different temperatures. The results showed that more culturable COCs were isolated from the ovares stored at 39℃ than that from ovaries stored at 31℃ or 20℃ and before storage. Thirty-one centidegree was the best storage temperature in terms of cumulus expansion, nuclear maturation and morphology of the first polar body after in vitro maturation culture. The ability of cumulus expansion was completely lost in COCs derived from ovaries stored at 39℃ for 8 hours. Ovary storage （at both 31℃ and at 20℃ ） increased the proportion of oocytes with the GVc configuration in which chromatin condensed into a single big clump at the nucleolus and the functional significance of this configuration needs further investigations [ Acta Zoologica Sinica 51 （5）： 919 923, 2005].     

银鲫种系细胞标记分子Vasa: cDNA克隆及其抗体制备

种系细胞始自胚胎发育早期,是动物生殖及生殖工程的基础。为研究鱼类的种系细胞提供标记分子,我们克隆并鉴定了银鲫的vasacDNA即Cagvasa。CagvasacDNA全长2771碱基(nt),编码的蛋白为银鲫Vasa即CagVasa,全长701个氨基酸(aa)。CagVasa蛋白与已知Vasa蛋白的结构特征一致:在N端有14个RGG重复序列,在C端Vasa所特有的8个功能域俱全。银鲫Vasa与鲤鱼、斑马鱼、陆生脊椎动物和果蝇的Vasa蛋白分别有95%,89%,61%-66%和50%的同源性。卵巢切片的RNA原位杂交揭示,Cagvasa限于种系细胞,且表达水平呈现出低-高-低的动态变化:即两头低(卵原细胞跟Ⅳ期成熟卵子),中间高(Ⅱ-Ⅲ期卵子)。为分析鱼类种系细胞提供手段,我们用310aa的N端序列产生细菌的重组蛋白来免疫大白兔,获得了抗Vasa的多克隆抗体αVasa。Western免疫印迹表明,αVasa特异性地识别一个鱼类性腺的蛋白,该蛋白的分子量为75kD,仅见于银鲫的性腺和卵子。卵巢切片的组织免疫荧光共聚焦显微分析表明,抗体αVasa只对种系细胞染色:卵原细胞着色最深,卵母细胞和早期的卵子都浓染,成熟卵则浅染。类似情况亦见之于精子发生早期阶段的雄性种系细胞。卵巢和精巢的体细胞则不着色。因此,Cagvasa编码的当是Vasa同源蛋白,为银鲫种系细胞的第一个标记分子。我们的研究表明,抗体αVasa染色灵敏度高,特异性好,当是鉴别银鲫及其它鲤科鱼类的种系细胞的有效手段     

Co-expression pattern of dopamine beta-hydroxylase (DβH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.)

Co-expression of dopamine β-hydroxylase (DβH) and neuropeptide Y (NPY) has never been examined in ovary (OV) and umbilical cord (UC) of the European bison (Eb), the endangered wild species. The OV and UC samples were harvested from seasonally eliminated Eb females (45–120 days post coitum). Frozen histological sections were examined by double fluorescent immunohistochemistry (dF-IHC), using the primary mouse anti-DβH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DβH immunoreactive nerve fibers (DβH-IRs) and a little less frequent NPY-IRs were found in the bundle-like structures, innervating mainly perivascular regions of the OV. The NPY-IRs constantly co-expressed DβH, while some DβH-IRs did not express NPY. This specific pattern of innervation was observed both in the stromal and cortical regions of the OV. The simultaneous co-expression of DβH and NPY were also detected in the UC, in which specific single or bundle-like structures ran along the smooth muscles of blood vessels. The spatial-specific co-expression of DβH and NPY in OV and UC, may suggest that these markers are involved in the control of vascularization that regulates nourishing blood circulation required for proper pregnancy maintenance and efficient embryo/fetus development in the Eb.     

The Luteinizing Hormone Surge Regulates Circadian Clock Gene Expression in the Chicken Ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors’ previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary. (Author correspondence: stischkau@siumed.edu)     

Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.     

Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor™. Part I. Effect of the cell density on the process

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF‐ and ATF‐based cultures was shown at 20–35 × 10⁶ cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by‐product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 10⁸ cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 10⁸ cells/mL, achieved for the first time in a wave‐induced bioreactor, and 1.32 × 10⁸ cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO₂ level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 10⁸ cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013