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The idea of using a sperm cell to introduce exogenous DNA into an oocyte at the time of fertilization is of interest for simple production of transgenic mice. Since 1989, contradictory reports have appeared in the literature, but now this technology, so-called sperm-mediated gene transfer (SMGT), is considered reproducible. Concomitantly, in vivo transfection of sperm cells has also proceeded, including direct gene delivery into a testis, so-called testis-mediated gene transfer (TMGT), and gene transfer into seminiferous tubules. This review summarizes what has been achieved in the field of gene transfer via sperm cells.
Cross-talk between the embryo and mother is necessary for the establishment of successful pregnancy. In the mouse, cervical stimuli during mating induce prolactin secretion from the pituitary and formation of the corpus luteum (CL) of pregnancy. Placental lactogen supports the maintenance of pregnancy after implantation. In the human, the embryo secretes chorionic gonadotropin (hCG), which has lenbinizing hormone (LH) activity and maintains pregnancy before the luteal-placental shift occurs. In cattle and sheep, the mechanism underlying the establishment of pregnancy is unique. Their embryos secrete interferon (IFN)-τ, which is related to type I IFNs. IFN-τ prevents the secretion of prostaglandin F2α(PGF2α) from the uterine endometrium by suppressing the expression of estrogen and oxytocin receptor. In the pig, estrogen secreted from the conceptus is a factor for maternal recognition of pregnancy. Estrogen leads to a shift in the direction of PGF2α secretion from endocrine to exocrine and inhibits the regression of CL. Thus, each species has specific mechanisms for the establishment of pregnancy.
Sperm and oocytes must undergo several steps for successful fertilization, including sperm capacitation, the acrosome reaction and completion of oocyte maturation. During these steps, it is believed that specific molecules interact with precise timing. Although an increasing body of information on the fertilization-related molecules of sperm or oocytes has been accumulated through biological and gene targeting analyses, the information on these molecular interactions remains limited. Nonetheless, the current molecular information is the basis for future advances in the understanding of the mechanisms underlying fertilization. In this review, we introduce molecules that are involved in sperm-oocyte interactions at the site of fertilization, and address the molecular events during the sperm-cumulus, sperm-zona, and sperm-oolemmal interactions. Although the information introduced in this review has been obtained primarily from mice, similar molecules are likely engaged in analogous processes in other speicies.
In the mouse, maturing oocytes and zygotes until the late 1-cell stage are transcriptionally inert. The development of early preimplantation embryos, including reprogramming of differentiated germ cells into totipotent embryos, is regulated by the translation of mRNAs in oocytes preliminarily stored during oocytogenesis (maternal mRNAs). In the period of oocyte to zygote transition in mammals, the translational promotion or repression of maternal mRNAs does not start in unison. For example, a set of maternal mRNAs that are translationally quiescent in the growing stage become translated after the start of maturation and/or fertilization, but another group of maternal mRNAs that are actively translated in the growing stage become inactivated during maturation. This selective and temporal translational profile of maternal mRNAs seems to be regulated by RNA-binding proteins that bind to maternal mRNAs and decide the timing of their entry into the ribosome and by special short sequences in maternal mRNAs that recognize specific RNA-binding proteins. In this review, we focus on the mechanisms that modulate the profile of post-transcriptional regulation in the period of oocyte to zygote transition in mammals.
Analysis at the individual level using genetically-engineered mice has allowed conclusions to be reached regarding the actual function of a target gene. In recent years, the “knockdown” method using RNA interference (RNAi) has been established as a powerful tool for analyzing gene function. In this review, we focus on RNAi knockdown technology for producing genetically-engineered mice and describe the value of this approach from the perspective of both basic research and therapeutic potential. First, we introduce the basic mechanism of RNAi and development of knockdown animals from worms to mice. Next, we describe strategies to produce knockdown mice using DNA-based expression vectors introduced into zygotes or embryonic stem cells. Finally, we refer to the trends of research for clinical application. By way of illustration, we show the production of knockdown mice for treatment of neurodegenerative disease and mention the prospect of therapeutic potential of RNAi technology.
The progress of nuclear transfer technology, we can create cloned animals. And recently from this same technology we can possible to establish nuclear transfer embryonic stem cells (ntES cell). From our experiments we can establish mouse ntES cells, which any kind of cell type and strain and sex. And ntES cell has very similar ability like ES cells that has capacities for in vitro differentiation and in vivo germline transmission. The ntES cell made from donor somatic cells, which are very useful for therapeutic cloning Because of no immune rejection. Especially for human, it have high expectation from this new opportunity for rejuvenation of the ageing or diseased body. However, even for cloned animals the efficiency is so low and that have many problems. So, it necessary to do more analysis of ntES cell can work normal and safe before clinic.
The present study investigated the effect of separation of spermatozoa by sonication or Piezo-pulse on in vitro development of oocytes injected with sperm heads in the rat. We also examined development to term of rat oocytes injected with sperm heads. Rat frozen-thawed spermatozoa were separated into heads and tails by sonication for 10 sec or Piezo-pulse in KRB medium, and each treated sperm head was injected into an ooplasm. The oocytes were observed for formation of two pronuclei and development to 2-cell embryos. The percentages of formation of two pronuclei and development to the 2-cell stage did not significantly (P>0.05) differ between the two groups. Oocytes injected with sonicated sperm heads that reached the pronuclear stage at 10 h after injection of sperm heads were transferred into 7 recipients. Five recipients became pregnant, and 8 living pups were obtained. The results indicate that rat oocytes injected with sonicated sperm heads can develop to term in vivo. Furthermore, no difference was observed in the development in vitro between rat oocytes injected with sperm heads separated by sonication or by Piezo-pulse.
The localization and activity of mitochondria in hamster oocytes during maturation and fertilization were monitored by using three different fluorescent probes: rhodamine 123 (Rh123), MitoTracker Red (MT-Red) and 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)). Oocytes aged in vivo were also examined. The germinal vesicle (GV) oocytes exhibited a range of mitochondrial organizations, from a uniform distribution to a more cortical restriction. Metaphase I (MI) and MII oocytes included numerous mitochondria, among which the fluorescence intensity increased two-fold over that of GV oocytes. About half (54.5%) of the MII oocytes exhibited a mitochondria free zone at the cortex. An approximately 10 % decline in the density of mitochondria was observed in the oocytes aged 10 h post-ovulation, despite no significant changes. After fertilization, mitochondria became progressively aggregated and localized in the perinuclear region of all eggs examined. These comparative studies show that the signals for Rh123 and MT-Red were virtually coincident, but that for DiOC6(3) displayed wider areas of bright organizations regardless of the very low concentration used in this study (5 ng/ml). In addition, transition from a relatively homogeneous distribution of active mitochondria to perinuclear clustering may reflect energy production and utilization during oocyte maturation and fertilization in hamsters, as reported in other species.
We evaluated the clinical efficacy of the transport fresh embryo frozen-thawed embryo transfer method, whereby fresh embryos are transported from the satellite center for cryopreservation at the main ART center. In the Transport group (T group), surplus embryos from the satellite center were transported to the main ART center for frozen-thawed embryo transfer in 28 cycles in 15 patients. In the Center group (C group), oocytes were collected for frozen-thawed embryo transfer at the main ART center in 256 cycles in 165 patients. The slow freezing method was used. No significant differences were seen between groups in rates of embryo viability, embryo transfer, pregnancy, IVF embryo viability, ICSI embryo viability, pronuclear phase embryo viability, and cleavage phase embryo viability, or the numbers of transferred embryos. The transport fresh embryo frozen-thawed embryo transfer method is suitable for clinical application because there were no adverse effects from either transport or freeze/thawing.
The purpose of this study was to cryopreserve bovine oocytes for subsequent blastocyst production by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). A vitrification procedure using gel-loading tips as containers was applied to cryopreserve in vitro-matured and denuded oocytes. In Experiment 1, oocytes were vitrified-warmed in vitrification solution (VS) containing 25, 28, 31, or 40% ethylene glycol (EG) and 1.0 M sucrose. The proportions of survived oocytes that appeared to be morphologically normal after warming, and cleaved oocytes after IVF were lower with 25% EG-based VS when compared with 28–40% EG-based VS. Blastocyst yields 8 days after IVF of oocytes vitrified-warmed in 28 and 31% EG-based VS (12 and 17%, respectively) were not significantly different from those of the fresh control group (32%). Day-7 blastocysts derived from vitrified oocytes were composed of a smaller number of inner cell mass (ICM) and trophectoderm cells than the fresh Day-7 blastocysts. In Experiment 2, oocytes vitrified-warmed in 31% EG-based VS were subjected to enucleation and SCNT. The proportions of oocytes fused and cleaved in the vitrified group were comparable to those in the fresh control group. Blastocyst yields 6 and 7 days after SCNT of vitrified oocytes were lower than those of control oocytes, but 8 days after the SCNT, the difference became statistically comparable (45 versus 58% in control group). ICM and trophectoderm cell numbers in Day-7 blastocysts derived from vitrified oocytes were smaller than those of control blastocysts due to a slower developmental rate. In conclusion, bovine oocytes cryopreserved by vitrification in gel-loading tip were capable of developing into blastocysts after conventional IVF and SCNT, with slightly smaller cell numbers and a slower developmental rate.
An experiment was conducted to investigate pH, osmolality, and the concentration of ammonia, total protein, glutamine and glutamic acid in follicular fluid at different developmental stages (<2, 3–4 and 5–6 mm in diameter) and serum of porcine. The concentrations of ammonia and total protein content were determined with the catalyzed indophenols reaction and the Bradford assay method and read on a spectrophotometer set at 625 and 595 nm, respectively. Glutamine and glutamic acid concentrations were determined by HPLC. The pH value was lower (P<0.05) but osmolality was higher (P<0.05) in follicular fluid than in serum. The concentration of ammonia was lower (P<0.05) in follicular fluid than in serum. On the other hand, glutamine and glutamic acid concentrations were higher (P<0.05) in the follicular fluid than in serum. The pH increased and osmolality decreased with increasing follicle size, and protein content was almost similar in small, medium and large follicular fluid. Ammonia, glutamine and glutamic acid concentrations decreased (P<0.001) as follicular size increased. During early follicular development ammonia and amino acids were synthesized for high metabolic breakdown of protein and gradually decreased due to metabolism of ammonia and glutamic acid to glutamate.