Growth hormone and gene expression of in vitro‐matured rhesus macaque oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010. © 2009 Wiley‐Liss, Inc.     

Functions of Fyn kinase in the completion of meiosis in mouse oocytes

Oocyte maturation invokes complex signaling pathways to achieve cytoplasmic and nuclear competencies for fertilization and development. The Src-family kinases FYN, YES and SRC are expressed in mammalian oocytes but their function during oocyte maturation remains an open question. Using chemical inhibitor, siRNA knockdown, and gene deletion strategies the function of Src-family kinases was evaluated in mouse oocytes during maturation under in vivo and in vitro conditions. Suppression of Src-family as a group with SKI606 greatly reduced meiotic cell cycle progression to metaphase-II. Knockdown of FYN kinase expression after injection of FYN siRNA resulted in an approximately 50% reduction in progression to metaphase-II similar to what was observed in oocytes isolated from FYN (−/−) mice matured in vitro. Meiotic cell cycle impairment due to a Fyn kinase deficiency was also evident during oocyte maturation in vivo since ovulated cumulus oocyte complexes collected from FYN (−/−) mice included immature metaphase-I oocytes (18%). Commonalities in meiotic spindle and chromosome alignment defects under these experimental conditions demonstrate a significant role for Fyn kinase activity in meiotic maturation.     

体外培养小鼠卵母细胞及其生长分化因子-9基因表达

体外培养小鼠的窦前卵泡以得到第二次减数分裂中期(MⅡ)卵母细胞,比较体外发育卵母细胞与体内生长的卵母细胞生长分化因子-9(GDF-9)的基因表达量,探讨GDF-9的表达对卵母细胞体外发育成熟的影响。选择体外培养第2天(D2)、D4、D6、D8、D10、D12卵母细胞作为体外发育组;同窝雌性小鼠出生后D12、D14、D16、D18、D20、D22卵母细胞作为体内发育组;半定量逆转录多聚酶链反应技术分别检测两组MⅠ卵母细胞GDF-9基因表达量。结果体外培养小鼠窦前卵泡可以得到MⅡ期卵母细胞,卵泡成活率、窦腔形成率、卵母细胞成熟率分别达到89·5%、51·8%和56·6%。小鼠卵母细胞GDF-9基因表达量随发育时间的改变而发生变化,而体外发育D8—12卵母细胞GDF-9表达量显著低于同期体内发育卵母细胞(P<0·05)。体外发育D8—12卵母细胞GDF-9基因表达量低于同期体内发育的卵母细胞的原因之一可能是其发育潜能较低。     

Growth of mouse oocytes to maturity from premeiotic germ cells in vitro

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.     

Unravelling the mysteries of virulence gene regulation in Salmonella typhimurium

Salmonella typhimurium, which causes gastroenteritis in calves and humans as well as a typhoid-like disease in mice, uses numerous virulence factors to infect its hosts. Genes encoding these factors are regulated by many environmental conditions and regulatory pathways in vitro. Many virulence genes are specifically induced at particular sites during infection or in cultured host cells. The complex regulation of virulence genes observed in vitro may be necessary to restrict their expression to specific locations within the host. In vitro and in vivo studies provide clues about how virulence genes might be regulated in vivo. Future studies must assess the actual environmental signals and regulators that modulate each virulence gene in vivo and determine how multiple regulatory pathways are integrated to co-ordinate the appropriate expression of virulence factors at specific sites in vivo.     

Close packing of Listeria monocytogenes ActA, a natively unfolded protein, enhances F-actin assembly without dimerization

Studies of the biochemistry of Listeria monocytogenes virulence protein ActA have typically focused on the behavior of bacteria in complex systems or on the characterization of the protein after expression and purification. Although prior in vivo work has proposed that ActA forms dimers on the surface of L. monocytogenes, dimerization has not been demonstrated in vitro, and little consideration has been given to the surface environment where ActA performs its pivotal role in bacterial actin-based motility. We have synthesized and characterized an ActA dimer and provide evidence that the two ActA molecules do not interact with each other even when tethered together. However, we also demonstrate that artificial dimers provide superior activation of actin nucleation by the Arp2/3 complex compared with monomers and that increased activation of the Arp2/3 complex by dimers may be a general property of Arp2/3 activators. It appears that the close packing ( approximately 19 nm) of ActA molecules on the surface of L. monocytogenes is so dense that the kinetics of actin nucleation mimic that of synthetic ActA dimers. We also present observations indicating that ActA is a natively unfolded protein, largely random coil that is responsible for many of the unique physical properties of ActA including its extended structure, aberrant mobility during SDS-PAGE, and ability to resist irreversible denaturation upon heating.     

Epidermal growth factor and three‐dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte‐like cells

Three‐dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold‐based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte‐like cells using embryoid body protocol in the two‐dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte‐like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or ?EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate‐based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene‐expression patterns, we can conclude that alginate‐based 3D coculture system provided a highly efficient protocol for oocyte‐like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte‐like cell differentiation.     

Nucleic acid enzymes as a novel generation of anti-gene agents

Recent molecular and cellular studies have highlighted the important role of some gene products in the cause and/or perpetuation of human pathological conditions including cancer and autoimmune diseases. The identification of such gene products has led to the development of new candidate therapies. The discovery of catalytic nucleic acid enzymes has provided researchers with a potentially important tool to block the expression of abnormal genes, provided that their sequences are known. The cleavage specificity of these compounds is determined by their hybridizing antisense arms, which anneal with the target mRNA in a complementary fashion. Nucleic acid enzymes can be delivered to cells either endogenously as gene encoding RNA enzymes (ribozymes) or exogenously as in vitro made agents. Given the progress reported during the last years, a wide range of molecular designs and chemical modifications can be introduced into these compounds, in particular the hammerhead type ribozyme. Here, we review the design, stability and the therapeutic application of these agents with the goals of illustrating relevant gene targets and signal pathways for molecular medicine. Relevant in vivo problems of the technology, mRNA repair by group I intron ribozymes and gene regulation by endogenous RNA will also be discussed.     

Expression and downregulation of WNT signaling pathway genes in rhesus monkey oocytes and embryos

Mammalian WNT genes encode secreted glycoproteins that are conserved homologues of the Drosophila Wingless gene, which plays a crucial role in Drosophila development. Recently, WNT pathway signaling has been implicated in ovarian development, oogenesis, and early development. We sought to evaluate whether these genes may contribute to the formation of healthy human oocytes or embryos, and whether the expression of these genes could provide informative markers of human oocyte and embryo quality. To do this, we employed the primate embryo gene expression resource (PREGER; www.preger.org) to examine expression of mRNAs encoding 38 components of the WNT signaling pathway in rhesus monkey oocytes and embryos as a nonhuman primate model. We observed considerable conservation between rhesus monkey and mouse of expression of WNT, FZD, and effector gene mRNAs, and a generalized downregulation of genes encoding key components of the WNT signaling pathway during preimplantation development. Our results support a role for WNT signaling during oocyte growth or maturation, but not during preimplantation development. Additionally, we observed differences between in vitro cultured and in vivo developing blastocysts, indicating possible effects of culture on WNT signaling during the peri-implantation period.     

Oocyte maturation: emerging concepts and technologies to improve developmental potential in vitro

Oocyte in vitro maturation (IVM) is an important reproductive technology that generates mature oocytes that are capable of supporting preimplantation embryo development and full development to term. There is great clinical and commercial incentive to improve the efficiency of the technology, however, progress has been slow over the past decade. A critical challenge is to understand what constitutes oocyte developmental competence and the mechanisms governing it. We have taken the approach of studying in detail oocyte-somatic cell interactions; including, oocyte-cumulus cell (CC) gap-junctional communication, and bidirectional paracrine signalling between the two cell types. It is becoming clear that, compared to oocytes matured in vivo, IVM oocytes undergo maturation prematurely as they are still in the process of acquiring developmental competence in vivo, and the molecular cascade reinitiating meiosis differs entirely to that in vivo. Attempts to enhance oocyte developmental competence by attenuating the spontaneous meiotic resumption of oocytes in vitro have been met with mixed success. Kinase inhibitors that prevent maturation-promoting factor activity have, in general, been ineffectual on promoting oocyte developmental potential post-IVM. In contrast, agents that modulate oocyte cAMP during IVM show greater potential, possibly as these compounds extend oocyte-CC gap-junctional communication. An important concept that is now emerging is that the oocyte secretes potent growth factors that regulate fundamental aspects of CC function and thereby determine the distinctive phenotype of the cumulus-oocyte complex. The capacity of an oocyte to regulate its own microenvironment by oocyte-secreted factors (OSFs) may constitute an important component of oocyte developmental competence. In support of this notion, we have recently demonstrated that supplementing IVM media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development.     

LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes

The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogen es EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes .