Effect of retinoids on rat spleen

It has been shown in experiments on Wistar male rats with the use of light and electron microscopy and morphometry that a single intraperitoneal injection of high doses of all-trans-methylretinoate and methyl-7,8- dihydroretinoate leads to an increase in the spleen weight, the appearance in the spleen of red cells changed in the shape, activation of phagocytic intensity of macrophages, intercellular interaction, lymphocyte proliferation and formation of new lymph follicles.     

Effect of cysteamine during in vitro maturation on buffalo embryo development

The purpose of this study was to evaluate whether the addition of cysteamine during in vitro maturation (IVM) of buffalo oocytes enhances embryo development. Cumulus-oocyte complexes (COC) from slaughterhouse ovaries were matured in vitro in TCM 199 supplemented with 10% fetal calf serum (FCS), 0.5 microg mL(-1) FSH, 5 microg mL(-1) LH, 1 microg mL(-1) 17 beta estradiol and 0 (control), 50, 100 or 200 micromol L(-1) of cysteamine for 24 hours. The matured oocytes then were fertilized and cultured for 7 days. No beneficial effect on maturation and cleavage rate was related to the addition of cysteamine. However, the percentage of embryos that developed to compact morula and blastocyst stage was significantly higher (P < or = 0.01) for oocytes matured in medium containing 50 micromol L(-1) of cysteamine than it was for oocytes matured with 0, 100 and 200 micromol L(-1) cysteamine (22.6% vs 14.9%, 15.7% and 13% respectively); moreover, the addition of 50 micromol L(-1) of cysteamine during IVM significantly (P < or = 0.01) increased the proportion of transferable quality (Grades 1 and 2) embryos (19.3% vs 11.3%, 11.6% and 11.2% respectively). The present study showed that adding a thiol compound (such as cysteamine) to the IVM medium improves buffalo in vitro embryo production (IVEP) efficiency, which so far has been unsatisfactory.     

Effect of various growth-promoting factors on preimplantation bovine embryo development in vitro

One-cell bovine embryos produced by in vitro oocyte maturation (IVM) and in vitro fertilization (IVF) were cultured in chemically defined medium (CDM) to which the following growth-promoting factors were added separately: transferrin (Tf, 10 ug/ml), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), transforming growth factor-betal (TGF-beta1), insulin-like growth factor-one (IGF-I), insulin-like growth factor-two (IGF-II), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), all at 10 ng/ml. The embryos were cultured for a total of 10 days and were assessed. The culture medium was changed every 2 days. There were no beneficial effects of growth factors on embryo development to morula or hatched blastocyst stages of development. However, supplementation with EGF approached significance (P<0.08) when compared with that of CDM alone at the blastocyst stage of development. The results suggest that supplementation with growth factors does not improve bovine IVM-IVF embryo development when cultured under chemically-defined conditions.     

Effect of individual heifer oocyte donors on cloned embryo development in vitro

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.     

Effects of lysosomal proteinase inhibition on the development of the rat embryo in vitro

Altered lysosomal function in the visceral yolk sac can result in abnormal development. As proteolysis is an important function of the rodent visceral yolk sac during early and mid-gestation, we characterized the lysosomal proteolytic enzyme activity of this extraembryonic membrane and determined the effects of inhibitors of protein degradation on embryonic development. Constituent activities of cysteine and aspartic acid proteinases were measured in rat visceral yolk sac on gestation day 12, and the effects of the cysteine proteinase inhibitors leupeptin, E-64 [trans-epoxysuccinyl-l-leucylamido(4-guanido)butane] and N-ethylmaleimide and the aspartic acid proteinase inhibitor pepstatin were determined in Sprague-Dawley rat embryos cultured in vitro from gestation days 10-12. It was determined that only cysteine proteinases, primarily cathepsins B and L, are active in the mid-gestation visceral yolk sac. The cysteine proteinase inhibitors leupeptin and E-64 both produced a concentration-related decrease in embryonic growth, as measured by crown-rump length, somite number, and embryonic protein content, and a concentration-related increase in incidence of abnormalities. A characteristic pattern of abnormalities was produced which involved a decrease in neural tube volume and the formation of a subectodermal blister opposite the point of attachment of the vitelline vessels. At high concentrations, anophthalmia was also observed. The decreased neural tube volume was associated with increased osmolality of the exocoelomic fluid, the major extraembryonic fluid compartment. It is possible that the osmotic change decreased neural tube volume by causing water to move to the compartment with a higher solute concentration, out of the embryo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of osteopontin (OPN) on in vitro embryo development in cattle

Fertility-related phosphoprotein osteopontin (OPN) is present in the bovine oviduct epithelium and fluid. The objectives were to determine the effects of OPN on percentages of cleavage and embryo development in vitro in cattle, and to assess the ability of OPN to induce in vitro capacitation of bovine sperm. In vitro-matured bovine oocytes were fertilized in the presence of 0, 10, 20, or 40 microg/mL OPN. There were greater percentages (P<0.01) of cleavage and compact morulae-blastocysts (79.7 and 43.3%, respectively) with 10 microg/mL OPN than in the control group (without OPN; 71.2 and 32.1%, respectively). Furthermore, percentages of advanced blastocysts were greater in the group receiving 40 microg/mL OPN versus control (56.4% vs. 42.0%, P<0.05). Capacitation was assessed by the ability of sperm to undergo the acrosome reaction after incubation with lysophosphatidylcholine. Semen from three bulls was incubated for 2h in either TALP medium alone (control) or with TALP medium containing 0.01 mM heparin, or with TALP medium containing 10 or 20 microg/mL OPN. Incubation with 10 and 20 microg/mL OPN produced more (P<0.01) capacitated sperm (14.4 and 13.6%, respectively) than the untreated control group (8.3%), but both untreated sperm and those treated with OPN had significantly fewer capacitated sperm than those treated with 0.01 mM of heparin (30.5%). In conclusion, OPN improved the efficiency of bovine in vitro embryo production and influenced sperm capacitation.     

Cadherin mRNAs during rat embryo development in vivo and in vitro.

Whole rat embryo cultures are being used in increasing numbers of laboratories to study the mechanisms by which teratogens disturb development. The development of early somite stage embryos in vitro is very similar morphologically to that in vivo, yet few biochemical comparisons have been made. The purpose of this study was to determine the steady-state mRNA concentrations of a family of Ca(2+)-dependent cell adhesion molecules, the cadherins, during rat embryonic development in vivo and in vitro. Embryos and yolk sacs were collected on days 10, 11, and 12 of gestation (in vivo); they were also obtained from day 10 embryos after growth in culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). Total RNAs isolated from embryos and yolk sacs were studied by Northern blot analysis using specific cDNA probes for three cadherins, E-cadherin, N-cadherin, and P-cadherin. Although E-cadherin mRNA was detected in embryos, it was present at much higher concentrations in yolk sacs. In addition, multiple species of E-cadherin mRNA ranging from 3.0 to 13 kb were detected. Interestingly, the concentration of the major 4.5-kb E-cadherin mRNA species in yolk sac after 45 hr in culture was increased 2.8-fold over that on day 12 of gestation in vivo. Second, two species (4.3 and 3.5 kb) of N-cadherin mRNA were detected, almost exclusively in embryos. In yolk sac, N-cadherin mRNA was detected only after 45 hr in culture. Third, P-cadherin mRNA was detected as a single 3.5-kb species, mainly in embryos. P-cadherin mRNA concentrations in yolk sac after 45 hr in culture were 5.6-fold higher than in vivo. Thus, these results demonstrate that there is a differential distribution of cadherin mRNAs in rat embryos and yolk sacs. Further, there appear to be multiple species of mRNAs for E-cadherin and N-cadherin. Finally, while whole embryo culture in vitro did not significantly alter the steady-state concentrations of cadherin mRNAs in the embryo, these concentrations were dramatically increased in the yolk sac.     

Effects of acute exposures to elevated temperatures on rat embryo growth and development in vitro

Day 10 rat embryos (8-12 somites) cultured in vitro were exposed to elevated temperatures (41-43 degrees C) for varying lengths of time. A 15-minute exposure to a temperature of 43 degrees C (109.4 degrees F) was sufficient to produce malformed embryos when observed on day 11. Longer exposures at this temperature produce higher incidences of malformed embryos and also more severely affected embryos. Temperatures of 42 degrees C (107.6 degrees F) or 41 degrees C (105.8 degrees F) also produced malformed embryos, but the required length of exposure was increased compared to 43 degrees C. The minimal length of exposure at 42 degrees C was 60 minutes, while at 41 degrees C it was increased to 4 hours. The central nervous system was particularly sensitive to increased temperatures, and embryos exposed to "teratogenic doses" of hyperthermia exhibited primarily microcephaly and microphthalmia. In addition, histological analyses revealed that at 4.5 hours after a 30-minute exposure to 43 degrees C, necrotic debris was prevalent in the neuroepithelium, less prevalent in the surrounding mesenchyme and surface ectoderm, and absent in the tissues of the heart.     

Effect of sperm exposure time on in vitro fertilization and embryo development of bovine oocytes matured in vitro

This study was designed to investigate the effect of sperm exposure time on the fertilization rate and subsequent developmental capacity of bovine oocytes matured in vitro. Cumulus oocyte complexes (COCs) obtained from 2 to 6 mm follicles were matured for 24 h in TCM-199 supplemented with fetal bovine serum (FBS) and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed by incubating 15 to 20 matured oocytes with 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium for either 4, 8, 12, 16, 20, 24 or 28 h. Following sperm exposure for different periods of times, the presumptive zygotes were co-cultured with Buffalo Rat Liver cells (BRLC) monolayers in CZB medium without glucose, a simple semi-defined medium developed for mouse embryo culture, for 3 d post-insemination and then in M199/FBS (TCM-199-HEPES supplemented with 20% heat-treated FBS and 1 mM sodium pyruvate) for 5 d. The fertilization rates differed significantly among the 7 treatment groups, with higher frequencies obtained by co-incubation of gametes for 20, 24 or 28 h (67 to 76%) than for 4, 8 and 12 h (26 to 54.5%), with 16 h (57%) being intermediate. However, the length of sperm exposure time did not significantly affect subsequent embryo development, although an increasing trend was noted from 4 h to 20 h. The number of fertilized oocytes at 3 d post-insemination cleaving to 2- to 4-cell vs 8-cell stage was not different among treatment groups. Development of 8-cell embryos to morulae and blastocysts did not differ among the treatment groups. These data suggest that the optimum duration of sperm-oocyte incubation is 24 h, and periods shorter than 16 h may result in a reduced fertilization rate.     

Biphasic action of retinoids on gonadotropin receptor induction in rat granulosa cells in vitro

Vitamin A (retinol) has been held to be uniquely essential for normal vision and reproduction, all other functions being served by its metabolite retinoic acid. The inability of retinoic acid to maintain adequate serum progesterone is implicated as the cause of fetal resorption. The availability of lipoproteins is a major limiting factor in progesterone production and the ovarian expression of lipoprotein receptors is dependent on the action of luteinizing hormone (LH). Therefore, we investigated the effects of retinol and retinoic acid on LH receptor induction by ovarian cells in an attempt to determine the basis for the reported differences in the gonadal action of these two retinoids. Our results indicate that retinoic acid (10(-10) M) and retinol (10(-8) M) each synergistically enhance the ability of follicle stimulating hormone (FSH) to induce LH-receptors and to stimulate the formation of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone. However, at higher concentrations, both retinoids inhibited these effects of FSH. For every measured effect, retinoic acid was more potent than retinol. Since retinol is metabolized to retinoic acid in other tissues, these results suggest that retinoic acid may be the mediator of the action of retinol on the ovary and that retinol's unique effect on reproduction needs to be investigated further.     

Effect of concentration of spermatozoids on fertilization of oocytes and human embryo development in vitro

The correlation between sperm insemination concentrations, rates of normal and abnormal fertilization and embryo development was investigated. For male factor patients fertilization rates are significantly lower than for female factor. We have found the increased fertilization rate for male factor, if insemination concentration increased from 10 x 10(4) to 15 x 10(4) per 1 ml. In cases of severe male factor infertility the concentration of sperm of 30 x 10(4) per 1 ml had no effect. We have found no difference in abnormal rates of fertilization, when the number of sperm increased in male factor. The correlation between the frequency of polysperm zygote and slightly increased insemination concentration was observed in patients with normal sperm.     

Oxidative stress on mouse embryo development in vitro.

Oxygen radicals are involved in the in vitro block phenomenon of embryo development, because a low oxygen tension and superoxide dismutase (SOD) have been shown to promote the in vitro development of mouse embryos. One of the target molecules damaged by oxygen radicals may be the thiol (SH) group of proteins because it is readily oxidized. In this study, we evaluated the effects of thioredoxin, which is a powerful protein disulfide reductase, on mouse (Institute of Cancer Research, ICR) preimplantation embryo development. Culture of mouse pronuclear embryos recovered 17 h after human chorionic gonadotrophin (hCG) administration in the presence of thioredoxin (200 micrograms/mL) significantly increased the blastulation rate (75.3%) when compared to the control culture system (8.9%). The effects of thioredoxin were observed only from the pronuclear stage to the two-cell stage (17-48 h after hCG administration). An additive effect of thioredoxin and SOD, or thioredoxin and a low oxygen tension, was observed. These results suggest that the oxidation of the SH group of proteins is one of the causes of developmental blockage of embryos in vitro. The target protein for reduction by thioredoxin has not been identified yet, but thioredoxin will be a new clue for clarifying the mechanism of blocking development in vitro.     

The effects of plasminogen on in vitro ovine embryo development

Plasminogen activator production by ovine embryos and the effects of plasminogen on ovine embryo development and zona pellucida integrity were evaluated. Eight-cell to sixteen-cell embryos were cultured in Whitten's medium containing 0, 60, or 120 micrograms/ml plasminogen. Plasmin and plasminogen activator concentrations in the medium were determined by a caseinolytic assay. More blastocysts hatched in medium containing 60 and 120 micrograms/ml plasminogen (33 and 21%, respectively) than 0 microgram/ml plasminogen (0%; p less than 0.05). Zona pellucida dissolution time in acidified phosphate-buffered saline was less after incubation in medium with 60 and 120 micrograms/ml plasminogen (7.2 and 5.9 min, respectively) than 0 microgram/ml plasminogen (9.4 min; p less than 0.05). Plasminogen activator production was low until the morula stage, increased during morula-blastocyst transition, and remained elevated through blastocoelic expansion and hatching. Zona pellucida solubility, plasminogen activator production, and plasminogen conversion to plasmin increased as embryonic stage advanced; however, plasminogen activator production and plasmin conversion to plasmin were poorly correlated with zona pellucida solubility. The results indicate that ovine embryos produce plasminogen activator, and plasmin can increase zona pellucida solubility; however, other factors may also be involved in altering zona pellucida integrity prior to hatching.     

Effect of short periods of sperm-oocyte coincubation during in vitro fertilization on embryo development in pigs

The present study was conducted to determine if the efficiency of in vitro pig embryo production could be improved by a reduction in the period of time that oocytes are exposed to sperm during in vitro fertilization. A total of 1596 immature cumulus-oocyte complexes from five replicates were matured in vitro and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) for 10, 30, 60 min or 6h (control group). The oocytes from short coincubation times were washed three times in fertilization medium to remove spermatozoa not bound to the zona and transferred to another droplet of the same medium (containing no sperm) for 6h. After 6h, the oocytes from each group were cultured in embryo culture medium for another 6h to assess fertilization parameters and for 7 days to assess embryo development. After each period of coincubation, some oocytes were stained with Hoechst-33342 to count zona-bound sperm. Although the number of zona-bound sperm increased with the coincubation time (34.1 +/- 1.7, 46.8 +/- 2.8, 62.8 +/- 3.8 and 139.5 +/- 6.1 for 10, 30, 60 min and 6h, respectively, P < 0.02), the penetration rate was not significantly different among groups (61.3-68.2%). However, the efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) increased (P < 0.04) as the coincubation time was increased (26.6 +/- 2.9%, 29.0 +/- 4.4%, 39.5 +/- 6.2%, and 49.3 +/- 3.0% for 10, 30, 60 min and 6h, respectively). Nevertheless, there were no significant differences among groups in blastocyst formation rates (17.5-25.5%). These results demonstrate that although a sperm-oocyte coincubation time of as little as 10 min results in fertilization rates similar to a 6-h coincubation, the reduction in the period of time of sperm-oocyte coincubation does not improve the efficiency of in vitro pig embryo production.     

Effect of anti-sperm antibody on the in vitro development of rat embryos

Since we previously proved that the fertilized rat eggs in early developmental stage have antigen(s) cross-reacting to spermatozoa, the effect of antibody to spermatozoa on the cleavage of fertilized rat eggs was examined in vitro. Fertilized eggs from Fisher rats in the morula stage were cultured in vitro for 15 to 39 hr in the medium containing antibody to rat spermatozoa and rabit complement, and the developmental rates of morulae to blastocysts were compared with those cultured in the presence of either antibody or the complement alone. When rat morulae were cultured in the medium containing rabbit complement and IgG from rabbit antiserum to rat spermatozoa (heteroantibody) or from rat antiserum to rat spermatozoa (isoantibody), the development of moralae to blastocysts was markedly suppressed, whereas those cultured in the medum containing rabbit complement and IgG from the control rabbit serum or rabbit antibody IgG to rat spermatozoa alone without complement normally developed to the blastocysts. These results indicate that the antibody to spermatozoa in presence of complement can impair the in vitro development of fertilized rat eggs.     

Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes

The aim of this study was to analyze different culture systems on embryo development of prepubertal goat oocytes. We compare (i) the effect of the age of donor (goat) of oocytes on in vitro maturation, fertilization and subsequent embryo development, (ii) the effect of the origin of oviduct cells from coculture of prepubertal goat embryo development, and (iii) the effect of in vivo culture in rabbit oviducts for 1, 2 and 3 days on the development of prepubertal goat embryos produced in vitro. In Experiment 1, at 24 h post-insemination (hpi), oocytes from adult goats were allocated in TCM199 with oviduct cells from adult goats, and oocytes from prepubertal goats were randomly placed in drops with oviduct epithelial cells from adult (aOEC) or prepubertal (pOEC) goats. Cleavage rate and embryo development were evaluated at 48 hpi and after 7 days coculture, respectively. In Experiment 2, at 24 hpi, prepubertal oocytes were allocated in TCM 199 with pOEC. At 40-42 hpi, a group of embryos remained in the coculture (control group), and the rest were transferred to rabbit oviducts (three rabbits for replicate) for culturing in vivo for 24, 48 and 72 h. After these in vivo cultures, embryos were recovered, evaluated and placed in TCM199 with pOEC until Day 8 post-insemination. The maturation, fertilization and blastocyst rates did not differ significantly between oocytes obtained from adult and prepubertal goats. The percentage of blastocysts obtained from prepubertal goat embryos cocultured with aOEC or pOEC was also similar (12.1% versus 12.2%). The transfer of prepubertal goat embryos to rabbit oviducts for 1, 2 and 3 days did not improve the blastocyst rate compared to the control group (9.7, 10.9, 4.1 and 11.5%, respectively). In conclusion, in our conditions, there were no significant differences in embryo development between oocytes obtained from prepubertal and adult goats, and the embryo development from prepubertal goat oocytes were similar in the different culture systems compared.     

Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to 8-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to 8-cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<0.05) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14%). These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors.     

Effect of co-administration of varenicline and antidepressants on extracellular monoamine concentrations in rat prefrontal cortex

Since a substantial proportion of smokers have comorbid mood disorders, the smoking cessation aid varenicline might occasionally be prescribed to patients who are simultaneously treated with antidepressants. Given that varenicline is a selective nicotinic acetylcholine receptor partial agonist and not a substrate or inhibitor of drug metabolizing enzymes, pharmacokinetic interactions with various classes of antidepressants are highly unlikely. It is, however, conceivable that varenicline may have a pharmacodynamic effect on antidepressant-evoked increases in central monoamine release. Interactions resulting in excessive transmitter release could cause adverse events such as serotonin syndrome, while attenuation of monoamine release could impact the clinical efficacy of antidepressants. To investigate this we examined whether varenicline administration modulates the effects of the selective serotonin reuptake inhibitor sertraline and the monoamine oxidase inhibitor clorgyline, given alone and combined, on extracellular concentrations of the monoamines serotonin, dopamine, and norepinephrine in rat brain by microdialysis. Given the important role attributed to cortical monoamine release in serotonin syndrome as well as antidepressant activity, the effects on extracellular monoamine concentrations were measured in the medial prefrontal cortex. Responses to maximally effective doses of sertraline or clorgyline and of sertraline plus clorgyline were the same in the absence as in the presence of a relatively high dose of varenicline, which by itself had no significant effect on cortical monoamine release. This is consistent with the binding profile of varenicline that has insufficient affinity for receptors, enzymes, or transporters to inhibit or potentiate the pharmacologic effects of antidepressants. Since varenicline neither diminished nor potentiated sertraline- or clorgyline-induced increases in neurotransmitter levels, combining varenicline with serotonergic antidepressants is unlikely to cause excessive serotonin release or to attenuate antidepressant efficacy via effects on cortical serotonin, dopamine or norepinephrine release.