The maintenance of transgenes in rats is reviewed with special references to embryo cryopreservation (transgene banking) and intracytoplasmic injection of spermatozoa or round spermatids (transgene passage). Production of transgenic rats by pronuclear DNA microinjection is easily performed, particularly using the micro-vibration method. Two-cell stage embryos derived from the mating of transgenic rats with wild-type rats can be successfully cryopreserved by conventional two-step freezing in the presence of 10% dimethylsulfoxide. However, the transmission rates of transgenes into newborn offspring are lower than the 50% which is expected in simple Mendelian inheritance. Furthermore, some transgenic rats are unable to transmit their transgenes to subsequent generations. When the inability of male transgenic rats to transmit the transgene is not associated with mosaicism, microinsemination techniques such as intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) can be used for passage of the transgenes. Our ICSI procedure includes the use of a piezo-driven small-sized injection pipette (2-4 μm in diameter), isolated sperm heads, and freshly ovulated oocytes. In the case of ROSI, round spermatids are injected into Sr2+-activated oocytes using a piezo-driven middle-sized pipette (5-6 μm in diameter). These microinsemination techniques can be combined with cryopreservation of sperm and spermatid cells, leading to successful transmission of transgenes carried in male gametes.
Integrins are heterodimeric transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix adhesion. They also participate in cytoskeletal rearrangement, co-regulation of growth factor activities and activation of signal transduction. This review describes the available information regarding the role of integrins in human reproductive physiology and discusses their clinical implications. Integrins play important roles in several reproductive processes including fertilization, embryogenesis, and implantation. Disturbance of integrin expression in reproductive organs can be the cause, or the result of such reproductive disorders as endometriosis, unexplained infertility, hydrosalpinx, ectopic pregnancy, and preeclampsia. Further knowledge of the integrin-mediated regulation of cell growth and cell survival may facilitate our understanding of many key aspects of development and physiology in reproduction.
Angiogenesis, the development of the new capillaries by endothelial cell proliferation and outgrowth from pre-existing vessels, is one of the prominent features of early corpus luteum (CL). The process of angiogenesis is an important component of normal development and function of CL. Of the numerous promoters of angiogenesis and maintenance of new established capillaries that have been identified, the most important factors appear to be vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF). The biological effects of VEGF and FGF factor families are mediated by signaling through specific tyrosine kinase receptors. The VEGF, FGF and IGF family members in bovine CL are discussed with the literature available for large domestic animals. The highest expression for VEGF, VEGFR-2, FGF-2, FGFR, IGF-1, IGF-2 and IGFR1 were found during the early luteal phase (Ld 1-4) followed by a significant decrease afterwards. The VEGF and IGF-1 protein was localized predominantly in luteal cells. FGF-1 mRNA expression peaked (P<0.05) during mid-luteal stage and FGF-1 protein was localized in cytoplasm of luteal cells, but endothelial cells are always negative. FGF-2 protein during angiogenesis (d 1-5) was found in cytoplasm of endothelial cells and changed thereafter exclusively to the cytoplasm of luteal cells. In contrast, the IGF-2 protein is exclusively localized in pericytes and few endothelial cells. The results obtained (mainly in ruminant) suggest an important role of these growth factors for angiogenesis and furthermore for maintenance and function of the bovine corpus luteum.
The aim of this study was to investigate the effect of Ca2+- and Mg2+-free culture conditions on induction of spontaneous first cleavage in rat oocytes. Oocytes recovered from superovulated Wistar females 17 h after hCG injection were exposed for 1.5 h to Ca2+/Mg2+-free, Ca2+-free, Mg2+-free, or control mKRB medium supplemented with 5 μg/ml cytochalasin-B (CB). The oocytes were then cultured for 4.5 h in control medium containing CB and for 28 h in CB-free control medium. The oocytes exposed to the Ca2+/Mg2+-free medium (23%) cleaved better than those exposed to Ca2+-free (8%), Mg2+-free (0%), or control (0%) media. The cleavage rates of oocytes recovered 12, 17 and 22 h after hCG injection and exposed to the Ca2+/Mg2+-free medium were 4, 17 and 22%, respectively. The efficiency of oocyte activation in 1.25 mM Sr2+ treatment for 6 h was not influenced by the culture background of bivalent cations (cleavage rates: both 49% in Ca2+/Mg2+-free and Ca2+-free medium). These results indicate that Ca2+/Mg2+-free culture condition induced the spontaneous first cleavage of rat oocytes in an age dependent manner, but had no additional effect on Sr2+-induced oocyte activation.
In Experiment 1, bovine cumulus oocyte complexes (COCs) were preincubated for 24 h in M199 supplemented with 25-100 μM BL-I, an inhibitor of cdc2 kinase activity. Half of the COCs were fixed to assess rates of germinal vesicle breakdown (GVBD), and the rest were cultured for a further 20 h without BL-I to investigate whether BL-1 affects their meiotic competence. In Experiment 2, COCs treated with BL-I for 24 h or 48 h were matured (IVM), fertilized (IVF) and then cultured in vitro (IVC) for 8 days. Oocytes exposed to 100 μM BL-I showed a significantly higher rate of germinal vesicle (GV) (86%) stage than the others (4-72%). Oocytes exposed to 100 μM BL-I for 48 h showed equally high GV (95%) stage and maturation (84%) rates compared to oocytes treated for 24 h and control oocytes with no treatment. However, they showed significantly lower normal fertilization (27%), cleavage (27%) and blastocyst formation (6%) rates than oocytes treated for 24 h (66, 69 and 42 %, respectively) and control oocytes (65, 75 and 42%, respectively). The results indicate that BL-I can reversibly inhibit GVBD of bovine oocytes for at least 24 h but not for 48 h without compromising subsequent developmental competence after IVM-IVF-IVC.
Zygotic gene activation (ZGA) is initiated at the late 1-cell stage in the mouse. At this time a number of nuclear proteins involved in transcription regulation, RNA synthesis as well as RNA processing are translocated from the cytoplasm to the nuclei of the zygotes. In the present series of experiments, we demonstrated the developmentally regulated subcellular localization of p100prp1/zer1/prp6 (also called U5-102 kDa protein), which was previously identified by us as a human homologue of Prp1p/Zer1p of Schizosaccharomyces pombe and Prp6p of Saccharomyces cerevisiae, and found to be tightly associated with the U5 small nuclear ribonucleoprotein particle. Both Prp1p/Zer1p and Prp6p are important regulators of pre-mRNA processing and cell cycle progression in yeast. Immunocytochemical analysis revealed that p100prp1/zer1/prp6 was gradually translocated into the pronuclei from the cytoplasm in mouse 1-cell embryos. The nuclear translocation of p100prp1/zer1/prp6 was not prevented by treatment of 1-cell embryos with aphidicolin, indicating that the translocation is independent of DNA synthesis. These findings suggest that p100prp1/zer1/prp6 is involved in pre-mRNA processing during preimplantation development, including the onset of ZGA in the mouse embryo.
Plasma concentrations of immunoreactive (ir-) inhibin, FSH, LH, estradiol and progesterone were measured by RIA between -6 and 12 days after parturition in 6 mares. Follicular development was also monitored in the same animals after parturition by ultrasound scanner. Plasma concentrations of ir-inhibin were relatively low before parturition, then rose significantly after parturition, in accordance with the development of 10-30 mm follicles in diameter during the early period of postpartum estrus. In contrast, concentrations of both estradiol and progesterone remained high before parturition, then dropped significantly 1 day after parturition. The present study demonstrated that inhibin is more likely to be secreted from maternal ovaries into maternal circulation than the feto-placental unit during the prenatal period. This suggests that inhibin could be used as a potential indicator of follicular development around parturition in mares.
Developmental competence of in vitro matured porcine oocytes obtained by TCM199- and NCSU 23-based IVM systems after electrical activation were compared. When the nuclear phase of granulosa-cumulus-oocyte complexes (GCOCs) used in the TCM199-based IVM system and cumulus-oocyte complexes (COCs)used in the NCSU 23-based IVM system were observed, most of GCOCs were fully grown dictyate arrest oocytes (GV I, 68.6%), on the other hand, only 33.8% of COCs were at GV I stage. There were no differences in the rates of maturation (93.2%, 82/88 vs 91.3%, 63/69), normal cleavage (70.7%, 58/82 vs 65.1%, 41/63) and development to blastocysts (37.8%, 31/82 vs 34.9%, 22/63) between the in vitro matured oocytes obtained by the two IVM systems. However, the parthenogenetic blastocysts derived from the NCSU23-based IVM system had higher cell numbers than the blastocysts derived from the TCM199-based IVM system (35.6 ± 4.5 vs 22.6 ± 2.0; P<0.01). Transfer of parthenogenetic oocytes derived from the NCSU23-based IVM system resulted in a high developmental rate to somite stage fetuses (26.9%, 45/167). These data demonstrate that the activated porcine oocytes derived from the two IVM systems had equal ability of in vitro development, though there was a difference in the cell number of the parthenogenetic blastocysts. It was also shown that in vitro matured oocytes obtained by the NCSU23-based IVM system had excellent ability of parthenogenetic development to fetuses, indicating that they are usable in porcine developmental engineering.
Sperm-mediated gene transfer (SMGT), which uses sperm cells as vectors for DNA delivery during fertilization, is one of the methods used to achieve transgenesis in animals as an alternative to pronuclear microinjection. Despite the convenience of SMGT, its application in producing transgenic animals, especially domestic animals, is still limited due to its low success rate. In this study, to increase the efficiency of SMGT, we tested liposome-peptide-DNA (LPD) complex, a new reagent known to stabilize transfection in cultured cells. Two peptides, one designed from the amino acid sequence of human histone H1 (Hs) and the other from human protamine (Pr), were used with a CMV/β-actin/EGFP fusion gene to form LPD complex. Spermatozoa obtained from rat epididymis were incubated with LPD complex and used for artificial insemination, and the expression of EGFP in the morula-stage embryos was observed. Pr caused significantly more embryos to express EGFP, while Hs had no effect on the expression rate. Moreover, only Pr resulted in the production of offspring carrying foreign DNA. These results suggest that LPD complex with the aid of Pr could be useful in the transfer of foreign DNA by SMGT, probably by stabilizing liposome-DNA complex during fertilization.
To develop an in vitro culture system for Mongolian gerbil embryos, we examined the effect of co-culture with oviductal epithelial cells on the development of 1- and 2-cell embryos collected from superovulated gerbils. In the first experiment, 2-cell embryos were cultured in modified TCM 199 supplemented with pyruvate, lactate and fetal calf serum (mTCM199) with or without gerbil or mouse oviductal cells. In the second experiment, in 1-cell embryos were cultured with or without gerbil oviductal cells in mTCM199. Although, 2-cell gerbil embryos did not develop beyond the 16-cell stage without oviductal cells in both TCM199 and mTCM199, co-culture with gerbil or mouse oviductal cells supported the development of 2-cell gerbil embryos to blastocysts. Co-culture with gerbil oviductal cells in mTCM199, but not in TCM199, also supported the development of 1-cell embryos to blastocysts. There were no differences between the in vivo and in vitro developed blastocysts in morphological appearance and cell numbers. This co-culture system can be used for in vitro culture of Mongolian gerbil embryos.
The role and interaction of microtubules and microfilaments, which are important for progressing the events during oocyte maturation and activation, are not well understood. This study was designed to examine the cytoskeletal changes of the porcine oocyte activated electrically or by sperm with relation to the effects of oocyte aging and the paternal and maternal contributions. During electric activation, fusion of the first polar body (PBI) into the oocyte was attempted to evaluate changes in the cytoskeleton induced by incorporation of maternal chromatin in comparison with penetrated sperm (paternal) chromatin. Aged oocytes matured for 50-60 h displayed an elongated spindle and a less dense distribution of microfilaments compared to young oocytes matured for 44 h. Oocytes were effectively activated with double electric pulses regardless of aging (93-100%). Fusion of PBI into the oocyte declined with oocyte aging (from 52% to 22%). When fusion occurred, PBI chromatin was incorporated into the microtubule networks of the ooplasm and was frequently transformed into one "extra" nuclear-like structure. Young parthenotes possessed one microtubule-rich domain including one or more pronuclei. In aged parthenotes, however, the cortical and cytoplasmic microfilaments decreased in density, resulting in frequent fragmentation of eggs. In zygotes, male and female pronuclei were included in separate domains of microtubules, respectively, anchored by microfilaments. The present results suggest that the instability of cytoskeleton of the oocyte induced by aging may increase egg fragmentation and that there may be a difference between the paternal and maternal contributions to the cytoskeletal reorganization during pronuclear formation and migration.
The activities of hydroxysteroid dehydrogenases (HSDs) were histochemically demonstrated in cultured porcine oocytes, and the changes in steroid metabolism during meiotic maturation and also the relationship between nuclear maturation and changes in steroid metabolism in the cytoplasm were examined. The activities of Δ5-3β-HSD, 17 β-HSD, 20α-HSD and 20β-HSD were observed in 91 to 97% of porcine oocytes soon after collection. The percentages of oocytes showing the activities of Δ5-3β-HSD (using DHA as the substrate), 17β-HSD (testosterone) and 20β-HSD (20β-hydroxyprogesterone) did not change during maturation culture, while those showing the activities of Δ5-3β-HSD (pregnenolone and 17α-hydroxypregnenolone), 17β-HSD (estradiol-17β), 20α-HSD (20α-hydroxyprogesterone) and 20β-HSD (17α-hydroxyprogesterone) decreased as the time of maturation culture was prolonged and reached 4, 0, 0, 2 and 0%, respectively, after 44 h culture. In the oocytes cultured for 22 h with olomoucine, nuclei were all in the germinal vesicle stage, and the activities of Δ5-3β-HSD (pregnenolone and 17α-hydroxypregnenolone), 17β-HSD (estradiol-17β), 20α-HSD (20α-hydroxyprogesterone) and 20β-HSD (17α-hydroxyprogesterone) were observed in 57, 64, 65, 61 and 66% of the treated oocytes, respectively. On the other hand, 10, 2, 10, 7 and 2% of control oocytes respectively showed such HSD activities, demonstrating a significantly lower percentage of oocytes showing the HSD activities, compared with the olomoucine-treated oocytes. From the present findings, it was suggested in porcine oocytes that the metabolic abilities of progesterone, 17α-hydroxyprogesterone, 20α-hydroxyprogesterone, 17α,20β-dihydroxyprogesterone and estradiol-17β in the cytoplasm are closely related to nuclear maturation, and the disappearance of their metabolic abilities could be used as a characteristic for the resumption of meiotic maturation.
The aim of this study was to investigate in vivo viability of vitrified ovine embryos after direct transfer using a laparoscope. In vivo-derived ovine morulae and blastocysts were equilibrated in 25% glycerol and 25% ethylene glycol solution, loaded into a heat-pulled straw and vitrified. Thirty-eight embryos were transferred into 19 recipients (2 embryos per recipient) and pregnancy was examined at 54-59 days after transfer. The pregnancy, implantation, lambing, and survival rates were 15.8% (3/19), 10.5% (4/38), 15.8% (3/19) and 7.9% (3/38), respectively. The result indicates that viable lambs can be produced by direct transfer of vitrified ovine embryos.