The objectives of the present study were to compare the in vitro maturation (IVM), fertilization and early embryonic development of bovine oocytes recovered from ovaries during the follicular, metestrus and diestrus stages of the estrous cycle and at anestrus and pregnancy after maturation in a serum free culture medium. Cumulus oocyte complexes (COCs) collected from ovaries at different reproductive statuses were matured in medium 199 supplemented with 10 g/ml FSH, 10 g/ml LH, 1.5 g/m estradiol, 75 g/ml streptomycin, 100 IU/ml penicillin and 10 mM HEPES. COCs were incubated in 200 microliter droplets of maturation medium 199 under oil for 24 h at 39degrees c and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim up, heparin capacitated sperm from two bulls separately in each replicate (20 h, 39C, 5% CO2). After fertilization, the presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin-G, 75 g/ml streptomycin and 10 mM HEPES for 144 h at 39C and 5% CO2 without medium freshening or change. Oocytes/embryos were fixed, stained with DAPI and evaluated under fluorescent microscope. The IVM rates were almost similar among oocytes from all reproductive statuses (range: 89.8 to 95.4%). However, IVM rates for oocytes from the metestrus (90.6%) and pregnant (89.9%) phases were lower than the other groups. The fertilization rates were lower (p<0.05) for oocytes from the diestrus phase (72.4%) than from the other phases (range: 81.1 to 86.6%). Oocytes, recovered during the metestrus phase of the estrous cycle, resulted in the highest cleavage rate (60.0%), while oocytes from the diestrus phase had the poorest embryonic development (39.8%: p<0.05). Majority of the embryos from all reproductive phases showed a developmental arrest around 8-cell stage. Although the developmental competence of oocytes from pregnant and anestrus animals was lower than that from the other reproductive stages, they could be potentially used as oocyte donors. Long term, in vitro embryo culture without medium freshening or change was hypothesized to have caused the failure to overcome the 8-cell block to development.
Animals ; Cattle/*embryology/*physiology ; Ectogenesis/physiology ; Embryonic and Fetal Development ; Estrous Cycle/*physiology ; Female ; Fertilization in Vitro ; Male ; Oocytes/*growth&development/*physiology ; Pregnancy

Angiopoietin-1 ; chemistry ; physiology ; Angiopoietin-2 ; chemistry ; physiology ; Angiopoietins ; physiology ; Animals ; Cardiovascular Diseases ; therapy ; Embryonic and Fetal Development ; Humans ; Neoplasms ; blood supply ; Receptor, TIE-2 ; chemistry ; physiology ; Signal Transduction

BACKGROUNDIt is believed that estrogen plays pivotal roles in the regulation of follicle/oocyte maturation and oocyte fertilizability. It is also involved in the functional preparation of the fallopian tubes for subsequent gamete interaction, in early embryonic development occurring in the tubal microenvironment, and in the preparation of the uterus for implantation. This study was designed to determine whether estrogen is required for follicular and embryonic development.

METHODSThe biosynthesis of estrogen was blocked by a daily injection of the aromatase inhibitor, Arimidex, at a dose of 100 micro g/d, using 3 - 4 week old C57B6 F1 female mice. Injections were continued for 3 days in experiment 1 (n = 10) and for 5 days in experiment 2 (n = 23). Mice in the control group (n = 27) were given the same amount of saline. Exogenous gonadotrophin [7.5 IU pregnant mare serum gonadotrophin (PMSG)] was administered to induce follicular growth and development on the second day. In experiment 1, we tested estrogen and progesterone levels and examined ovary morphology two days later. In experiment 2, 47 hours after PMSG injection, 5 IU human chorionic gonadotropin (hCG) was given and two female mice were then caged with a male mouse overnight. Two days later, we measured estrogen and progesterone levels. We then removed the embryos, cultured them, and examined embryonic development every 24 hours for 3 days.

RESULTSBefore hCG injection, estrogen levels in mice from the Arimidex group were suppressed by 94%, and progesterone levels were suppressed by 75%. There was no difference between the two groups in mean number of total follicles found per animal (30.4 follicles/animal in the control group and 27 follicles/animal in the Arimidex group). Two days after hCG injection, estrogen levels in the Arimidex group were significantly lower than that in the control group (P < 0.01), while progesterone levels were not significantly lower (P > 0.05). The rate of development of embryos, morulae, blastocysts, and hatching blastocysts was not significantly different between the two groups (P = 0.20, 0.10, 0.44, and 0.38, respectively).

CONCLUSIONSIn the present study, by depriving mice of normal estrogen support, we have been able to rule out the absolute need for rising levels of estrogen for the completion of the follicular maturation process and the development of embryos in vitro.

Animals ; Chorionic Gonadotropin ; pharmacology ; Embryonic and Fetal Development ; Estrogens ; physiology ; Female ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Nitriles ; pharmacology ; Oocytes ; physiology ; Ovarian Follicle ; physiology ; Pregnancy ; Triazoles ; pharmacology

Recent studies have suggested that the hydrosalpinx has a negative effect on pregnancy outcome, with markedly diminished implantation and increased early pregnancy loss. Fluid from the hydrosalpinx may leak into and accumulate in the uterine cavity. It is not clear, however if this creates a hostile local environment in the uterus for embryo implantation or exerts a direct embryotoxic effect. This study was conducted to investigate the detrimental effects of hydrosalpinx fluid (HSF) on the development of mouse embryos in vitro and to demonstrate whether Vero cells overcome these adverse effects. HSF was collected from three women with bilateral hydrosalpinx at the time of laparoscopic surgery. Collected fluid was centrifuged and the supernatant was frozen at -20degrees C. For co-culture, Vero cells were commercially obtained in a frozen state and cultured using Ham's F10 medium. Single-cell mouse embryos (B6CBAF1) were cultured for 5 days in 0, 0.4, 0.8, and 1.2% of HSF in media with and without Vero cells and examined daily to record the number of embryos reaching expanded blastocyst and hatching stage. Co-culture of mouse embryos with Vero cells at 0.8% HSF concentration significantly enhanced embryo development, but not at 1.2% hydrosalpinx fluid concentration. These results suggest that HSF is highly embryotoxic and Vero cells are likely to overcome these detrimental effects to some degree.
Animals ; Blastocyst/*physiology ; Body Fluids/*metabolism ; Cercopithecus aethiops ; Coculture Techniques ; Embryonic and Fetal Development ; Fallopian Tube Diseases/*metabolism ; Female ; Humans ; Infertility, Female/*metabolism ; Mice ; Mice, Inbred C57BL ; Vero Cells

The distribution of the nerve growth factor (NGF), the glial fibrillary acidic protein (GFAP) and the ciliary neurotrohic factor (CNTF) was performed in coronal sections of the mesencephalon, rhombencephalon and spinal cord in the developing Mongolian gerbils. Generally, NGF specifically recognizes neurons with the NGF receptor, whereas GFAP does the glia, and CNTF does the motor neurons. The receptor expression was examined separately in gerbils between embryonic days 15 (E15) and postnatal weeks 3 (PNW 3). The NGF-IR was first observed in the spinal cord at E21, which might be related to the maturation. The GFAP reactivity was peaked at the postnatal days 2 (PND2), while the highest CNTF-reaction was expressed at PNW 2. The GFAP stains were observed in the aqueduct and the spinal cord, which appeared to project laterally at E19. The CNTF was observed only after the birth and found in both the neurons and neuroglia of the substantia nigra, mesencephalon, cerebellum and the spinal cord from PND1 to PNW3. These results suggest that NGF, GFAP and CNTF are important for the development of the neurons and the neuroglia in the central nervous system at the late prenatal and postnatal stages.
Animals ; Brain Stem/enzymology/*metabolism ; Ciliary Neurotrophic Factor/*metabolism ; Embryonic and Fetal Development/physiology ; Female ; Gerbillinae/*embryology ; Glial Fibrillary Acidic Protein/*metabolism ; Immunohistochemistry/veterinary ; Mesencephalon/embryology/metabolism ; Nerve Growth Factor/*metabolism ; Pregnancy ; Rhombencephalon/embryology/metabolism ; Spinal Cord/embryology/*metabolism

For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos.
Adenine/administration & dosage/*analogs & derivatives/pharmacology ; Animals ; Cattle/*embryology/physiology ; Cell Fusion ; Electroporation/veterinary ; Embryonic and Fetal Development/*drug effects ; Enzyme Inhibitors/administration & dosage/pharmacology ; Female ; Inositol 1,4,5-Trisphosphate/administration & dosage/*pharmacology ; Microinjections/veterinary ; Nuclear Transfer Techniques ; Oocytes/drug effects/growth & development ; Parthenogenesis/*drug effects ; Protein Kinase Inhibitors ; Ryanodine/administration & dosage/*pharmacology ; Skin/cytology