The aim of this study was to develop a serum-free culture system that could support high levels of cleavage and blastocyst formation from sheep zygotes developed in vitro. To this end, we investigated the effects on sheep zygote development of amino acids, ammonium, vitamins, and culture of embryos in groups in Synthetic Oviduct Fluid (SOF) medium supplemented with BSA (32 mg/ml). The inclusion of amino acids in the culture medium had no effect on the percentage of embryos arrested at the 8-16-cell stage when embryos were cultured singly in the same drop of medium for 6 days (43% in SOF; 41% in SOF+amino acids). However, in medium containing all Eagle's amino acids, replacing the culture medium every 48 h to alleviate ammonium toxicity significantly decreased the number of arrested embryos (6%; p < 0.05) and significantly increased blastocyst cell number (52 cells in SOF; 105 cells in SOF+amino acids; p < 0.01) and the number of embryos developing to the blastocyst stage (29% in SOF; 67% in SOF+amino acids; p < 0.05). When the medium was renewed every 48 h, nonessential amino acids and glutamine also significantly decreased the number of arrested embryos (p < 0.05). Culturing embryos singly or in groups in SOF medium with all Eagle's amino acids that was renewed every 48 h resulted in significant increases in blastocyst hatching and mean cell number (47%, 31%, and 79%; 105, 136, and 173 cells for embryos cultured singly, in groups of 2, and in groups of 4, respectively). After culture in groups of 4, blastocyst cell numbers were equivalent to in vivo-developed controls (160 cells) and significantly greater than those developed in serum (103 cells; p < 0.01). Analysis of blastocyst metabolism, expressed on a per-cell basis, revealed that amino acids did not affect either glucose uptake or lactate production, whereas the addition of amino acids and vitamins resulted in a significant increase in both parameters (p < 0.01). A similar response was observed in serum-derived blastocysts. Ammonium production by sheep blastocysts after culture in the presence of amino acids was significantly greater than that produced by mouse blastocysts, indirect evidence that ruminant embryos utilize amino acids to a greater extent than do rodent embryos.
This presentation reviews current information on the events that lead to rupture of an ovarian follicle. It contains a summary of the morphological changes that occur at the apex of a follicle wall during ovulation. Existing information shows that the tenacious connective tissue layers of the tunica albuginea and theca externa must be weakened before the follicle wall can dissociate and break open under the force of a modest intrafollicular pressure. These changes are probably dependent on transformation of quiescent thecal fibroblasts into proliferating cells in a manner that is characteristic of tissue responses to inflammatory reactions. The metabolic factors that initiate transformation of the fibroblasts are uncertain, but they are probably generated by gonadotropin-induced changes in the theca interna and granulosa of a follicle as these layers begin to luteinize during the ovulatory process.
Noninvasive fecal assays were used to study steroid metabolism and ovarian activity in several felid species. Using the domestic cat (Felis catus) as model, the excretory products of injected [14C]estradiol (E2) and [14C]progesterone (P4) were determined. Within 2 days, 97.0 +/- 0.6% and 96.7 +/- 0.5% of recovered E2 and P4 radioactivity, respectively, was found in feces. E2 was excreted as unconjugated estradiol and estrone (40%) and as a non-enzyme-hydrolyzable conjugate (60%). P4 was excreted primarily as non-enzyme-hydrolyzable, conjugated metabolites (78%) and as unconjugated pregnenolone epimers. A simple method for extracting fecal steroid metabolites optimized extraction efficiencies of the E2 and P4 excretion products (90.1 +/- 0.8% and 87.2 +/- 1.4%, respectively). Analysis of HPLC fractions of extracted fecal samples from the radiolabel-injected domestic cats revealed that E2 immunoreactivity coincided primarily with the unconjugated metabolized [14C]E2 peak, whereas progestogen immunoreactivity coincided with a single conjugated epimer and multiple unconjugated pregnenolone epimers. After HPLC separation, similar immunoreactive E2 and P4 metabolite profiles were observed in the leopard cat (F. bengalensis), cheetah (Acinonyx jubatus), clouded leopard (Neofelis nebulosa), and snow leopard (Panthera uncia). Longitudinal analyses demonstrated that changes in fecal E2 and P4 metabolite concentrations reflected natural or artificially induced ovarian activity. For example, severalfold increases in E2 excretion were associated with overt estrus or exogenous gonadotropin treatment, and elevated fecal P4 metabolite concentrations occurred during pregnant and nonpregnant (pseudopregnant) luteal phases. Although overall concentrations were similar, the duration of elevated fecal P4 metabolites during pseudopregnancy was approximately half that observed during pregnancy. In summary, steroid metabolism mechanisms appear to be conserved among these physically diverse, taxonomically related species. Results indicate that this hormone-monitoring approach will be extremely useful for elucidating the hormonal regulatory mechanism associated with the reproductive cycle, pregnancy, and parturition of intractable and endangered felid species.
In the human, mosaicism may occur before implantation; but, to determine when it first occurs, it is necessary to study the chromosomal complement of all blastomeres. Full karyotypes of blastomeres from 2- to 8-cell human embryos by conventional karyotyping of metaphase spreads are difficult to obtain. The aim of this study was to assess the stage at which mosaicism occurred in preimplantation human embryos through use of fluorescence in situ hybridization (FISH) with multiple probes. All or most blastomeres from 2- to 12-cell human embryos were analyzed by FISH using probes for gonosomes and chromosome 18. FISH was performed on blastomeres from 117 morphologically normal monospermic embryos that were not transferred after preimplantation diagnosis because of their risk of carrying X-linked disease; 20 (17.1%) of these embryos were mosaic. Another group of 163 arrested or morphologically abnormal monospermic embryos were also analyzed by FISH; 47 (28.8%) of these embryos were mosaic. In addition, 37 dispermic embryos were analyzed, and 28 (75.7%) of these were found to be mosaic. Mosaicism first occurred at the second cleavage division when the monospermic embryo was mostly diploid and at the first cleavage division when the embryo was mostly haploid, polyploid, or dispermic.
Vascular endothelial growth factor (VEGF; also known as vascular permeability factor) is a secreted angiogenic growth factor. It is highly specific for endothelial cells, and its receptor, the fms-like tyrosine kinase (flt), has been localized only to endothelial cells in vivo. Here we describe the expression of mRNA encoding flt in human trophoblast as revealed by in situ hybridization. This mRNA is highly expressed in the cytotrophoblast shell and columns and also highly expressed by the extravillous trophoblast (EVT) in the maternal decidua both in the first trimester and at term. The trophoblast-like choriocarcinoma cell line BeWo also expresses this receptor and the related receptor, kinase domain-containing receptor (KDR), which is also a receptor for VEGF. Treatment of the cell line BeWo with VEGF165 stimulated 3H-thymidine incorporation and tyrosine phosphorylation of MAP (mitogen-activated protein) kinase in a time- and dose-dependent fashion. This study is the first demonstration of the presence of flt on non-endothelial cells in vivo and suggests a role for VEGF in the growth and differentiation of cytotrophoblast at implantation.
Fertilization of the immature, prophase I-arrested mouse oocyte produces multiple Ca2+ transients similar to those of the mature, metaphase II egg; however, the first Ca2+ transient is much lower in amplitude and shorter in duration. In contrast to prophase I-arrested oocytes, maturing oocytes fertilized after germinal vesicle breakdown have first Ca2+ transients similar to those of mature fertilized eggs. Immature, prophase-arrested oocytes release less Ca2+ in response to injection of inositol 1,4,5-trisphosphate (IP3) than eggs. At high concentrations, the sulfhydryl reagent, thimerosal (200 microM), causes Ca2+ oscillations in eggs and produces similar oscillations in oocytes. A lower concentration of thimerosal (25 microM) does not cause Ca2+ oscillations, but does sensitize IP3-induced Ca2+ release in both eggs and oocytes, since IP3-induced Ca2+ release is enhanced in the presence of 25 microM thimerosal. Incubation of oocytes in 25 microM thimerosal before injection of 2.2 microM IP3 causes oocytes to release as much Ca2+ as is released in eggs injected with 2.2 microM IP3. These results indicate that immature mouse oocytes possess intracellular stores of releasable Ca2+ similar in size to Ca2+ stores in eggs; however, these stores are less sensitive to IP3. Development of the IP3-induced Ca2+ release mechanism may be an important component of maturation; at fertilization of the egg, Ca2+ must be elevated to levels sufficient to activate further development and establish a block to polyspermy. Mouse oocytes appear to develop an increased sensitivity to IP3 during the course of oocyte maturation.
The purpose of this study was to evaluate effects of cooling and rewarming on the meiotic spindle apparatus of bovine oocytes. In experiment 1, in vitro-matured bovine oocytes were either maintained at 39 degrees C or cooled abruptly to 4 degrees C or approximately 25 degrees C. Immunohistochemical and DNA staining for visualization of microtubules and chromosomes, respectively, revealed an anastral, barrel-shaped spindle in bovine oocytes. Exposure to 4 degrees C for 10-20 min caused complete disappearance of the spindle. Some chromosome dispersion occurred after 60 min at 4 degrees C. After exposure to approximately 25 degrees C for 30 min, 90% of oocytes appeared abnormal, having either an abnormal spindle or no spindle. In experiment 2, oocytes cooled to either approximately 25 degrees C or 4 degrees C for 30 min were rewarmed directly or in steps for 15 or 60 min. Spindles did not return to normal in most oocytes regardless of cooling temperature or rewarming scheme. Step-wise rewarming was no more beneficial than direct rewarming. More of the oocytes rewarmed directly contained dispersed chromosomes as time at 39 degrees C increased.
The lipid content of porcine 1-cell stage embryos was reduced (delipated) through the use of micromanipulation to remove the lipid layer formed after centrifugation. Of 94 delipated embryos chilled to 4 degrees C for 1 h at the 1-cell or 2- to 4-cell stage, 60 (64%) cleaved in culture with development to the morula-blastocyst stage, whereas all of the control embryos lysed within 24 h. Significantly more embryos developed beyond the 8-cell stage when they were chilled at the 2- to 4-cell stage compared with chilling at the 1-cell stage (44%, 20 of 45 vs. 18%, 9 of 49). Fewer embryos developed after chilling if they were only partially rather than fully delipated. Developmental rates of partially delipated embryos to the 8-cell and blastocyst stages were 33% (13 of 40) and 8% (3 of 40), rates significantly (p or = blastocyst: 40%, 19 of 48 vs. 57%, 17 of 30). These data demonstrate that the sensitivity of porcine embryos to chilling is related to their high lipid content and that they can become tolerant to chilling if their lipid content is reduced.
Many factors are implicated in the development of prostatic growth: androgens, growth factors, and stromo-epithelial interaction. This study examines the role of the sympathetic and parasympathetic branches of the autonomic nervous system control of different aspects of rat prostate growth and atrophy. Unilateral sympathectomy leads to decreases in ventral prostate weight, DNA, and protein content in the lesioned side. Unilateral parasympathectomy leads to increases in ventral prostate weight, DNA, and protein content in the intact side. The separate effects of sympathectomy and parasympathectomy are maintained across a diverse combination of neural manipulations. Significant re-innervation does not occur by 60 days after manipulation as assessed by tissue norepinephrine levels. There appears to be a differential effect of the autonomic nervous system on growth and maintenance of the ventral prostate. The mechanism of contralateral hyperplasia and ipsilateral atrophy has potential significance in understanding human abnormal prostate growth.
Leukemia inhibitory factor (LIF), a cytokine that induces macrophage differentiation in the murine M1 myeloid leukemia cell line, is essential for blastocyst implantation in mice. However, its expression and the role it plays in the human uterus are unknown. To clarify these issues, we examined LIF gene expression in the human uterus by Northern blot hybridization and by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Analysis of LIF mRNA showed two hybridization bands, with estimated mRNA sizes of about 4.0-kb pairs and 1.8-kb pairs. LIF mRNA was detected at high levels in endometrial tissue and decidua, but at low levels in the chorionic villus in first trimester and term placenta. In the secretory phase, the endometrial tissue showed higher LIF expression than in the proliferative phase (9.5-fold; p < 0.01). The endometrial tissues were separated into a stroma-enriched fraction (SF) and an epithelium-enriched fraction (EF), and the LIF mRNA levels in each fraction were examined by quantitative RT-PCR. These levels were higher in the EF than in the SF (3.3-fold; p < 0.05). These findings suggest that, in humans, LIF plays a role in uterine function during the menstrual cycle, as well as during pregnancy.