The rodent carcinogens 1,4-dioxane and thiourea induce meiotic non-disjunction in Drosophila melanogaster females

The ability of the rodent carcinogens 1,4-dioxane (DX) and thiourea (TU) to induce meiotic non-disjunction (ND) was assessed in 3- and 6-day-old Drosophila melanogaster females. The chemicals were administered orally and three 24 h and one 48 h broods were obtained after mating, to sample oocytes treated in increasingly earlier stages of development. The broods represent mainly mature oocytes (brood I), nearly mature oocytes (brood II), early oocytes (brood III) and very early oocytes (brood IV). The toxicity of DX increased with dose (1% (not toxic), 1.5, 2, 3, 3.5%) as well as a reduction in fecundity which was moderate. Induction of ND in mature oocytes was positive with 2, 3 and 3.5% concentrations and was not related to dose. In immature oocytes it was also positive though already at the lowest concentration tested (1%), suggesting a sensitivity higher than that of mature oocytes. TU at 0.10-10%, did not affect viability, but since fecundity was seriously impaired at high doses, ND was not assessed beyond the 1.5% concentration. TU also induced ND in mature and in immature oocytes; neither a threshold nor a dose effect was detected. The response of mature oocytes was lower than that of immature oocytes. TU induced increases of ND in the earliest cells tested in a more consistent fashion than DX. The data clearly show that both chemicals induced ND in mature oocytes and in the three subsets in which immature oocytes were fractionated. Though toxicity may play a significant unspecific role in the induction of chromosome malsegregation by DX and TU, the induction of ND at low doses, moderately toxic to the oocytes, suggests that the interaction with specific targets contributed to the results obtained.     

Chromosome malsegregation induced by the rodent carcinogens acetamide,pyridine and diethanolamine in Drosophila melanogaster females

The effect of the rodent carcinogens acetamide (AC), pyridine (PY) and diethanolamine (DEA) on meiotic chromosome segregation was assessed in 4-day-old Drosophila melanogaster females. After oral treatment with 0.05, 0.1, 0.2 and 0.3% PY; 0.5, 1, 1.5, 2 and 4% AC; or 5, 10, 20, 40 and 80% DEA, the females were mated to 7-day-old males and three 24h broods were obtained to sample cells exposed mainly as mature oocytes (brood I), and nearly mature oocytes (brood II) with an increasing proportion of early oocytes (brood III). Viability was not affected at the two (PY) or three (AC, DEA) lowest concentrations, decreasing thereafter. PY increased the frequency of nondisjunction exclusively in brood II suggesting its interaction with specific targets. AC and DEA (the most active of the three) induced similar frequencies of nondisjunction in all broods suggesting unspecific cell division perturbations probably due to toxicity. No clear dose effect relationships were observed.     

The Effect of a Wasp's Age on Its Cuticular Hydrocarbon Profile and Its Tolerance by Nestmate and Non-Nestmate Conspecifics (Polistes fuscatus, Hymenoptera: Vespidae)

The effect of a wasp's age on its cuticular hydrocarbon profile and its tolerance by nestmate and non-nestmate conspecifics was investigated in a laboratory study of newly eclosed wasps ( Polistes fuscatus ) that were isolated from their comb at eclosion. In blind observations, mature females did not discriminate between young nestmates and non-nestmates that were ≤ 48 h-old. However, mature females did discriminate between young nestmates and non-nestmates that were 72 h old. Specifically, mature females were significantly more tolerant of (and significantly more likely to accept) 72 h-old nestmates than 72 h old non-nestmates. The abundance, relative abundance, and colony specificity of cuticular hydrocarbons changed significantly between 24 h-old and 72 h-old wasps. Our behavioral and chemical evidence indicates that wasps begin to develop a colony signature between 2 and 3 d of age.     

Genotoxic damage induced by isopropanol in germinal and somatic cells of Drosophila melanogaster

Isopropanol (isopropyl alcohol, 2-propanol, IPA) is a volatile solvent widely used in domestic or industrial environments and reported as innocuous in various test systems. The aim of this work was to search for in vivo genotoxic effects of IPA in Drosophila melanogaster, studying its ability to induce nondisjunction (ND) in females, sex linked recessive lethals (SLRL) in males, and somatic mutation and/or recombination (SMART) in larvae. Treatments were acute (60min) and were administered via inhalation. IPA had low toxicity in adult flies (75% IPA mortality index, MI=12.7% (females) and 2.6% (males)) and larvae (MI=14.3%, 75% IPA). Female fertility was severely affected during the first 24h (brood I, BI) after treatment, but, afterwards, control values were recovered. IPA induced a 50-fold increase of ND (%) in 24h old females, and a six-fold rise in 4-5 d old BI offspring. Nondisjunction frequencies (%) in the offspring of broods II to V (24h in each case) were similar to control values. IPA doses of 25% and 50% (v/v), tested in 24h old females, showed a significant dose-dependent increase of ND(%)in BI only, with control values in subsequent broods. Flies gave normal offspring when kept in regular media for 24h before mating. The eye spot test (SMART) showed a significant increase at 50% IPA (p<0.05, m=2), but the response was not dose-dependent. IPA failed to induce SLRL in any of the spermatogenesis stages tested. These findings suggest that the main effect of IPA is to induce chromosomal malsegregation; IPA must be present at the resumption of M-phase I after fertilization, to exert these effects. The alcohol does not affect DNA directly, but perturbations of the nuclear membrane may be responsible for induction of ND.     

In vitro fertilization and embryo development in vitro and in vivo in the tiger (Panthera tigris)

A study was conducted to evaluate the adaptability to the tiger of an in vitro fertilization/embryo culture system previously developed in the domestic cat. In Trial I (July 1989), 10 female tigers were treated with either 2,500 (n = 5) or 5,000 (n = 5) IU eCG i.m. and with 2,000 IU hCG i.m. 84 h later. In Trial II (January 1990), 6 females (5 of which were treated in Trial I) were given 2,500 IU eCG i.m. and 2,000 IU hCG i.m. 84 h later. Twenty-four to twenty-six hours after hCG treatment, all tigers were subjected to laparoscopy, and oocytes were aspirated transabdominally. On the basis of follicular development (follicles greater than or equal to 2 mm in diameter), all females responded to exogenous gonadotropins (range, 6-52 follicles/female). Follicle number and oocyte recovery rate were unaffected (p greater than 0.05) by eCG dose or time of year. A total of 456 oocytes were collected from 468 follicles (97.4% recovery; mean, 28.5 +/- 3.4 oocytes/female). Of these, 378 (82.9%) qualified as mature, 48 (10.5%) as immature, and 30 (6.6%) as degenerate. During Trial I, 8 electroejaculates were collected from 7 male tigers, and in Trial II, 3 semen samples were collected from 3 males. Motile sperm were recovered on each occasion; the overall mean (+/- SEM) ejaculate volume was 7.5 +/- 0.7 ml, the number of motile sperm/ejaculate was 105.9 +/- 20.6 x 10(6), and the percentage of structurally normal sperm/ejaculate was 81.4 +/- 2.0%. After swim-up processing, 0.05 x 10(6) motile sperm were co-cultured with 10 or fewer tiger oocytes in a humidified atmosphere (38 degrees C) of 5% CO2 in air. Of the 358 mature oocytes inseminated, 227 (63.4%) were fertilized. Oocytes from 2 females became contaminated in culture and, therefore, were excluded from embryo cleavage calculations. Of the remaining 195 fertilized oocytes, 187 (95.9%) cleaved to the two-cell stage. No parthenogenetic cleavage was observed in noninseminated control oocytes (n = 20). Eighty-six good-to-excellent-quality two- to four-cell embryos were transferred surgically into the oviducts of 4 of the original oocyte donors in Trial I and 2 females in Trial II. A pregnancy occurred in 1 female in Trial II, and 3 live-born cubs were delivered by Caesarean section 107 days after embryo transfer. Of the 56 cleaved embryos cultured in vitro in Ham's F10 for 72 h, 14 (25.0%) were at the sixteen-cell stage, and 15 (26.8%) were morulae.(ABSTRACT TRUNCATED AT 400 WORDS)     

中华大蟾蜍卵母细胞质膜存在钙依赖性的氯离子通道

本文采用双微电极电压钳方法研究了中华大蟾蜍卵母细胞内源性电压门控型离子通道的成分及其生理特性。卵母细胞去极化至 -30 mV 及更正电压时,有一持续的电压依赖性外向电流出现。钾离子通道拮抗剂四乙基氯化氨(tetraethy-lammonium chloride, TEA, 10 mmol/L)和 4- 氨基吡啶(4-aminopyridine, 4-AP, 10 mmol/L)协同作用时,该电流只能被抑制到最大电流幅度的(23.4±0.72)%。但是,上述浓度的TEA和4-AP 与氯离子通道拮抗剂5- 硝基-2, 3- 苯酚丙胺苯甲酸盐 (5-nitro-2,3-phenypropylamino benzoate, NPPB, 30 μmol/L)、无钙 Ringer 氏液或钙离子通道拮抗剂维拉帕米(40 μmol/L)协同作用时,可分别将此外向电流抑制到最大电流幅度的(2.1±0.08)%、(2.2±0.04)% 和(3.1±0.15)%。结果表明,中华大蟾蜍卵母细胞质膜上除有钾离子电流之外,还存在钙依赖性的氯离子电流。     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

The time interval between FSH-P administration and slaughter can influence the developmental competence of beef heifer oocytes

Superovulation alone may not be enough to result in developmentally competent oocytes. The objective of this study was to determine if a time interval between FSH administration and slaughter and between slaughter and oocyte recovery could increase the percentage of embryos. Beef heifers (n = 20) were superovulated with 1 bolus injection of 25 mg, im FSH-P diluted in saline and then slaughtered at 24, 48 or 72 h after FSH injection and the ovaries transported to the laboratory at 30 degrees C. For 6 of the heifers that received FSH-P and were then culled at 48 h post treatment, oocytes were recovered 1 to 2 h post slaughter from the first ovary and 4 to 5 h from the second ovary. Ovaries from untreated cows were collected and served as controls. The results indicated that FSH-P and culling at 48 h produced 35% >/= 32-cell embryos, significantly more than FSH-P and culling at 24 and 72 h (19 and 14%, respectively; P < 0.05). Furthermore, FSH-P and culling at 48 h produced 25% >/= 64-cell embryos, significantly more than FSH-P and culling at 24 and 72 h and the nontreatment control group (5, 7 and 15%, respectively; P < 0.05). The FSH-P group culled at 48 h produced more >/= 32-cell embryos, with an average of 84 +/- 5 cells/embryo, than the treated groups culled at 24 and 72 h and the untreated group (52 +/- 6, 60 +/- 5 and 63 +/- 3, respectively; P < 0.01). Finally, oocytes left in the postmortem ovaries for 4 to 5 h resulted in higher rates (51% and 41%) of >/= 32- and >/= 64-cell embryos, respectively, compared with that of the untreated control animals (29 and 18%; P < 0.05), but these rates were not different from oocytes left in ovaries for 1 to 2 h (33 and 24%). It is concluded that culling at 48 h after FSH treatment, as well as the conditioning effect on oocytes in warm postmortem ovaries for 4 to 5 h, increases the number of competent oocytes.     

Mutagenicity of sodium azide and its metabolite azidoalanine in Drosophila melanogaster

The mutagenic and toxic activities of sodium azide (NaN(3) ) and its organic metabolite L-azidoalanine [N(3)-CH(2)-CH(NH)(2)-COOH] were examined in the different stages of spermatogenesis in Drosophila melanogaster. Both azide and azidoalanine were toxic to the injected males, but azidoalanine was significantly less toxic than sodium azide. Following the injection with 0.2 microl of these compounds in the hemocoel of young adult wild-type males, the minimum concentrations of these compounds with complete toxic effects (zero survival) were 40 mM sodium azide and 160 mM azidoalanine. Sex-linked recessive lethals were scored by the Muller-5 method in three successive broods, representing sperms (brood A), spermatids (brood B), and a compiled group of meiotic and premeiotic germ cell stages (brood C). The results provide strong experimental evidence that azidoalanine is significantly (p<0.01) mutagenic to all stages of spermatogenesis in Drosophila melanogaster. Sodium azide, however, was not significantly (p>0.05) mutagenic and did not increase the rate of sex-linked recessive lethals over those produced by the control group injected with 0.45% NaCl. These results indicate the requirement of metabolic activation of azide in Drosophila as a prerequisite for its mutagenic effects.     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Pattern of sea bass oocyte development after ovarian stimulation by LHRHa

The cyclic pattern of oocyte development in the sea bass, Dicentrarchus labrax L., was studied after induction of spawning by two injections, 24 h apart, of a luteinizing hormone releasing-hormone analog (LHRHa) administered at the end of vitellogenesis. The first difference in the developmental stage of the ovary and in the size-frequency distribution of oocytes between the LHRHa treated group and the control group, was detected 32 h after the first injection, the LHRHa group showing a higher proportion of the 900 μm diameter oocyte class (maturing oocytes) ( P <0.01). At 48 h LHRHa-treated females showed an increase in the 1000 and 1100 μm classes (maturing oocyte and ovulated eggs) ( P <0.01) and at 72 h these females exhibited a bimodal pattern, reaching the highest proportions in the 1100 (27.4%) and the 600 (14.7%) μm classes (ovulated eggs and advanced vitellogenic oocytes, respectively). Bimodal distributions were present in 80% of the LHRHa-treated females. Once oocyte final maturation was triggered by LHRHa the time needed for ovulation was about 48 h and the interval between consecutive ovulations and spawnings seemed to be 48–72 h.     

Reduced expression of MAD2, BCL2, and MAP kinase activity in pig oocytes after in vitro aging are associated with defects in sister chromatid segregation during meiosis II and embryo fragmentation after activation

This study was conducted to examine expression of centromere protein B (CENPB), spindle checkpoint protein MAD2 (mitotic arrest deficient protein), and antiapoptotic protein BCL2; activities of MAPK (mitogen-activated protein kinase) and mitochondria distribution in pig oocytes during aging, and their relationship with sister chromatid separation during meiosis II and embryo fragmentation and apoptosis after activation. After immature oocytes were cultured for 40-72 h, CENPB, MAD2, tubulin, BCL2, and MAPK in the oocytes were examined by immunoblotting. Spindles, chromosomes, kinetochores, and mitochondria were examined by immunofluorescence staining and apoptosis was examined by TUNEL assay. It was found that tubulin and CENPB was not changed during 40-72 h of culture. However, the expression of MAD2 and BCL2 and the activity of MAPK were gradually reduced during oocyte aging. The percentages of oocytes with normal spindle, chromosomes, and kinetochores were also reduced as oocyte aged from 9.5% at 40 h to 17.3%, 34.6%, and 42.9% at 48, 60, and 72 h, respectively. Aggregated mitochondria were found in the aged oocytes as compared with the uniform distribution in young oocytes. After activation, the proportions of oocytes with abnormal anaphase II were significantly increased in aged oocytes. More (P<0.001) oocytes cultured for 60-72 h fragmented and showed apoptosis after activation as compared with the oocytes cultured for 40-48 h. This study indicates that aging reduces expression in spindle checkpoint protein and antiapoptosis protein and MAPK activity in pig oocytes. These events in turn cause abnormal sister chromatid segregation during meiosis II, embryo fragmentation, and apoptosis.     

Radiation-induced and transposon-induced chromosome damage in Drosophila: translocations and transmission distortion

The possible interaction between X-ray- and transposon-induced chromosome damage was monitored in the P-M system of hybrid dysgenesis in Drosophila melanogaster. One- to two-day-old F1 dysgenic males originating from a cross between M strain females and P strain males were irradiated with 5.5 Gy (550 rad) or used as controls to monitor X-Y translocations and transmission ratio distortion. Two 3-day sperm broods were sampled for the former and two 4-day broods for the latter to detect damage induced in the most radiosensitive cells. F1 nondysgenic males derived from M female to M male crosses (controls) were treated identically. X-Y chromosome translocations induced by P element mobility alone declined sharply with a decrease in temperature (18 versus 21 degrees C) and they were significantly reduced with aging of hybrid males from brood 2, 4-8 days of age, to brood 3, 7-11 days of age. No significant increase in translocations was observed when X irradiation and P-M dysgenesis were combined, showing no interaction between damages induced by the two mutator systems. In contrast, interaction was observed in transmission ratio distortion which was significantly increased by X irradiation of hybrid males derived from both reciprocal M X P and P x M crosses. The preferential elimination of P element-bearing autosomes occurred when either spermatocytes or spermatids were irradiated. An aging effect was also observed, resulting in less distortion in 9- to 14-day-old dysgenic males compared to 5- to 10-day-old hybrids.     

Influence of reproductive status on in vitro oocyte maturation in dogs

In the bitch, oocytes need 48-72 h to complete post-ovulatory maturation to the metaphase II stage in the isthmus of the oviduct, an interval similar to that found in in vitro studies. The effect of estrous cycle stage on in vitro meiotic competence of dog oocytes has been described in several studies. However, there are no reports evaluating the possible effects of pyometra or pregnancy on subsequent potential of oocytes recovered from such females to undergo in vitro maturation.In this study, immature cumulus-oocyte complexes (COCs) were recovered from fresh excised domestic dog ovaries in various reproductive states. The donor females were classified into groups based on stage of the estrous cycle: follicular (proestrus or estrus), luteal (diestrus) or anestrus or at the clinical conditions of pregnancy and pyometra. Grades 1 and 2 oocytes were cultured in vitro at 37 degrees C in TCM-199, supplemented with 25 mM Hepes/l (v/v), and with 10% heat inactived estrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium carbonate, 22 microg/ml pyruvic acid, 1.0 microg/ml estradiol, 0.5 microg/ml FSH and 0.03 IU/ml hCG. The nuclear maturation rate was evaluated at 72 h of incubation under Hoechst 33342 (10 microg/ml) staining for fluorescence microscopy. There was no statistical difference in nuclear progression to the MII stage among the various reproductive states (follicular phase, 5.4%; diestrus, 4.2%; anestrus, 4.4%; pyometra, 8.1% and pregnancy, 4.7%). Resumption of meiosis was 24.6% at the follicular phase, 19.6% for diestrus, 16.4% for anestrus, 37.1% for pyometra and 29.2% for pregnancy. Positive and higher numbers of residue above the expected value were observed for the pyometra and pregnancy conditions at the metaphase/anaphase I (MI/AI) stages.Our results indicate that in vitro nuclear maturation of dogs oocytes is not influenced by the in vivo reproductive status of the female. The quality of the oocyte is a more reliable indicator of its potential for meiotic maturation in vitro than the hormonal environment of the donor female at the time of oocyte retrieval.     

Factors affecting follicular population, oocyte yield and quality in camels (Camelus dromedarius) ovary with special reference to maturation time in vitro

Three experiments were conducted to study a series of factors affecting in vitro reproductive parameters in camels. In Experiment 1, the effect of season and presence of a corpus luteum (CL) on ovarian follicular populations, oocyte yield and quality was studied using a total of 252 and 208 ovaries collected during the breeding and non-breeding season, respectively. Small, medium, large and the total number of ovarian follicles, oocyte yield and quality were measured. In Experiment 2, the effect of methods of oocyte retrieval and needle gauge on oocyte yield and quality was evaluated with oocytes recovered using slicing and aspiration with 18-, 19- or 20-gauge needle. Oocytes were evaluated microscopically and classified into three categories. The objective of Experiment 3 was to identify the optimum time for oocyte maturation in the dromedary camel. Oocytes were cultured in CR1aa medium at 38.5 degrees C under 5% CO(2) for 24, 32, 36, 48 and 72h. Maturation was calculated as the percentage of cumulus expansion and oocytes reaching metaphase II (MII). The number of small, medium, large and the total number of ovarian follicles were higher (P<0.01) during the breeding than non-breeding season. The recovery of total number of oocytes and Category I oocytes were also greater (P<0.01) during the breeding season. Ovaries without a CL possessed significantly (P<0.01) more ovarian follicles and more (P<0.05) small and large follicles. The total number of oocytes and Category I oocytes were also greater (P<0.01) in ovaries without CL. Slicing of camel ovaries increased (P<0.01) the yield of oocytes as compared to aspiration. The aspiration of follicles using a 20-gauge needle had greater yields of the total number of oocytes and Category I oocytes than when using 19- (P<0.05) and 18-gauge needle (P<0.01). The culture of camel oocytes for 36h produced higher (P<0.01) percentages of cumulus expansion and oocytes at MII. Increasing culture times up to 48 or 72h increased (P<0.01) the percentage of degenerated oocytes.In conclusion, the growth and development of ovarian follicles in the camel as well as yields of Category I oocyte were greater during the breeding season. Slicing or aspirations using a 20-gauge needle yielded greater numbers of total and Category I oocytes. Finally, maturation of oocytes in CR1aa medium for 36h produced higher percentages of cumulus expansion and oocytes at MII stage.     

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.     

Response of porcine oocytes to electrical and chemical activation during maturation in vitro

These studies were conducted to examine activation of in vitro-matured porcine oocytes in response to an electrical stimulus or to an ionophore. Cumulus-enclosed porcine oocytes were incubated in maturation medium supplemented with either FSH and LH (MM:Exp.1) or pregnant mare serum gonadotropin (PMSG; MM-P: experiments 2-4) at 39 degrees C in 5% CO2:95% air with high humidity. In experiment 1, groups of oocytes were stripped of cumulus and then shampulsed (control) or electrically pulsed with a Zimmerman Cell Fusion unit at 24, 31, 41, 48, and 65 h of incubation. Control oocytes were exposed to the activation medium for 20 sec, whereas oocytes to be pulsed were subjected to a single activation pulse (120 V, 30 microseconds). Oocytes were cultured for an additional 24 h and then fixed and examined. For oocytes pulsed at 24, 31, 41, 48, and 65 h, the proportions which activated were 0, 0, 87, 88, and 83%, respectively. In experiment 2, oocytes were electrically or sham-pulsed with a BTX 200 Embryomanipulation System at 24, 30, and 40 h of incubation and respective proportions of oocytes activating were 27%, 39%, and 72%. In experiment 3, oocytes were subjected to 0, 1, or 2 activation pulses after 41 h of incubation in MM-P. Double-pulsing halved the proportion of activated oocytes (P less than .0001). In experiment 4, oocytes were subjected to 0, 25, 50, or 100 microM ionophore at 48 h of incubation. Proportions of oocytes activated by ionophore were greater than for control (P less than .05), but activation was not increased by increasing dose of ionophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic toxicity of six carcinogens and six non-carcinogens in the Drosophila wing spot test

Six rodent carcinogens, 5 of which are also human carcinogens, and 6 compounds recognized as non-carcinogens were tested for their genotoxic activity in the Drosophila melanogaster wing spot test. 72-h-old larvae trans-heterozygous for the recessive wing cell markers 'multiple wing hairs' (mwh) and 'flare' (flr3) were fed various concentrations of the test compounds for a period of 48 h. With amitrole and 4-aminobiphenyl, larvae of the same age were also given an acute treatment of 6 h with higher concentrations, and, in addition, 48-h-old larvae were fed for a longer period of 72 h. Repeats of all experiments document the good reproducibility of the results in the wing spot test. Amitrole and 4-aminobiphenyl were genotoxic after both 48-h and 72-h treatments, but their activity could not be detected following acute exposure of only 6 h. Chlorambucil and melphalan were clearly genotoxic. The carcinogens sodium arsenite and sodium arsenate, however, which are highly toxic to Drosophila, could only be tested at low exposure levels and were negative under these treatment conditions. The 6 non-carcinogens (ascorbic acid, 2-aminobiphenyl, mannitol, piperonyl butoxide, stannous chloride and titanium dioxide) were all definitely non-genotoxic in the Drosophila wing spot test. The data for the non-carcinogens demonstrate that non-genotoxic compounds can be identified in the wing spot test with a reasonable experimental effort.     

In vitro maturation of domestic dog oocytes cultured in advanced preantral and early antral follicles

Initial studies in our laboratory demonstrated that a large proportion of domestic dog advanced preantral (APAN) and early antral (EAN) follicles contained grown oocytes that had acquired the dense cytoplasmic lipid characteristic of preovulatory oocytes. The objective of this study was to assess nuclear maturation of those oocytes after in vitro culture. Both APAN and EAN follicles (152 to 886 microns in diameter) were isolated from ovaries by treatment with collagenase and DNase. The follicles were cultured in Dulbecco's Modified Eagle's medium/nutrient mixture F-12 Ham culture medium supplemented with 20% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic, 1 microgram FSH/ml, 10 IU hCG/ml and 1 microgram estradiol/ml. Within each group (APAN or EAN), control follicles were not cultured (0 h), and 2 to 12 follicles per well were incubated under a humidified atmosphere of 5% CO2 in air at 37 degrees C for 24, 48 or 72 h. After 24 h of culture, significantly more (5.3%, 20/374; P < 0.05) oocytes from APAN follicles reached the metaphase I to metaphase II stages (MI to MII) than the percentage of control follicles observed at 0 h (0.9%, 3/318). Continued culture resulted in a further increase (P < 0.05) in the percentage of oocytes reaching MI to MII by 48 h (11.5%, 47/407), which remained unchanged at 72 h (9.9%, 40/404). The percentage of oocytes from EAN follicles reaching MI to MII did not significantly increase after 24 h of culture. However, there was an increase (P < 0.05) by 48 h of culture (8.7%, 11/126), which remained unchanged at 72 h (7.5%, 8/106). These results show that dog oocytes cultured within advanced preantral and early antral follicles in vitro are competent to resume meiosis to the metaphase stage.     

The effect of caffeine on repair systems in oocytes of Drosophila melanogaster. I

Young Drosophila females were treated with caffeine, then mated for 24 h to males that had been irradiated with 2000 R X-irradiation, so that only mature spermatozoa were sampled. The radiation-induced frequency of dominant lethals and sex chromosome loss in the paternal genome was determined. The results show that treatment of females with caffeine leads to an increase in the frequencies of radiation-induced dominant lethals and to sex-chromosome loss.When young virgin females of the radioresistant stock RöI₂ were treated with caffeine and then irradiated with 3000 R X-irradiation, a striking increasein dominant lethals (in the maternal genome) was observed; caffeine treatment increased the X-ray response of the radioresistant stock to the level of the normal (+60) stock. It is suggested that caffeine reduces the efficiency of a system in Drosophila oocytes that repairs X-ray-produced chromosome breaks in both the paternal and maternal genomes.