Trisomic pregnancy and earlier age at menopause

We tested the hypothesis that the connection between advanced maternal age and autosomal trisomy reflects the diminution of the oocyte pool with age. Because menopause occurs when the number of oocytes falls below some threshold, our hypothesis is that menopause occurs at an earlier age among women with trisomic pregnancies than it does among women with chromosomally normal pregnancies. To determine their menstrual status, we interviewed women from our previous study of karyotyped spontaneous abortions who, in 1993, were age >/=44 years. Premenopausal women completed interviews every 4-5 mo, until menopause or until the study ended in 1997. The primary analyses compare 111 women whose index pregnancy was a trisomic spontaneous abortion with two groups: women whose index pregnancy was a chromosomally normal loss (n=157) and women whose index pregnancy was a chromosomally normal birth (n=226). We used a parametric logistic survival analysis to compare median ages at menopause. The estimated median age at menopause was 0.96 years earlier (95% confidence interval -0.18 to 2.10) among women with trisomic losses than it was among women with chromosomally normal losses and chromosomally normal births combined. Results were unaltered by adjustment for education, ethnicity, and cigarette smoking. Our results support the hypothesis that trisomy risk is increased with decreased numbers of oocytes. Decreased numbers may indicate accelerated oocyte atresia or fewer oocytes formed during fetal development.     

Synaptonemal complex analysis in spermatocytes and oocytes of tertiary trisomic Ts(512)31H mice with male sterility

Whole-mount preparations of silver-stained spermatocytes and oocytes from Ts(512)31H mice were examined in the electron microscope. The 5(12) chromosome was associated with the XY bivalent in the large majority of spermatocytes, whereas in about one-half of the oocytes, the 5(12) was associated with either unpaired chromosomes or heterochromatic parts of chromosomes or showed self-synapsis. There was a tendency for 5(12) chromosomes to be more fully heterochromatic in oocytes than in spermatocytes. A large proportion of oocytes (50%) and a much smaller proportion of spermatocytes exhibited various errors of chromosome pairing, but these proportions were only marginally greater than in control gametocytes from mice with normal karyotypes. It is concluded that the observed errors of pairing bear no simple relation to the almost complete breakdown of spermatogenesis and the marked impairment of oogenesis that occur in tertiary trisomic Ts(5(12))31H mice.     

Meiosis in trisomic female mice with Robertsonian translocations. II. Chromosome behaviour at first and second meiotic metaphases

First and second meiotic metaphases (MI and MII, respectively) from female mice of Robertsonian translocation (Rb) stock, trisomic for chromosome 16 (Ts16) or 19 (Ts19), were studied. The mature trisomic oocytes were derived from explanted fetal ovaries that had been cultured and then transplanted so as to mature heterotopically. Multivalent configurations involving the Rb chromosomes and the additional trisomic acrocentric were analysed. Pentavalent configurations occurred in 74.5% of 98 Ts16 MI and 44.2% of 249 Ts19 MI oocytes; quadrivalents (with a univalent acrocentric) were found in 9.2% of Ts16 MI and 10.8% of Ts19 MI oocytes. In 1% of Ts16 MI and 4% of Ts19 MI oocytes, there were two Rb bivalents and a univalent trisomic acrocentric. Rb trivalents and Rb bivalents occurred together in 14.3% of Ts16 MI and 39.4% of Ts19 MI oocytes. Chiasma frequencies were similar in trisomic and chromosomally balanced MI. Chiasma position, distribution, and localization were nearly identical, whether they were found in Rb multivalents or acrocentric bivalents, but one control group (from chromosomally balanced Ts19 littermates) had significantly more terminal chiasmata. Within the triple homologous region of 8% of Rb pentavalents, two chiasmata were observed in the same relative position in the two sister chromatids of one of the three homologs, suggesting a lapse in chiasma position interference. Assortment at MI anaphase was influenced by secondary nondisjunction of the Rb. The ratio of balanced to unbalanced MII oocytes was 1:4 in both trisomies.     

Meiotic segregation analysis in cows carrying the t(1;29) Robertsonian translocation

Heterozygous carriers of Robertsonian translocations generally have a normal phenotype but present reproductive failure. In cattle, the t(1;29) Robertsonian translocation is very common and carriers show a 3-5% decrease in fertility. Some data suggest that female carriers have a higher decrease than male carriers but no direct studies of the chromosome content of oocytes from a t(1;29) carrier cow have been performed so far. Four heterozygous carrier cows underwent hormonal stimulations and follicles punctions and about 800 oocytes were matured in vitro. Six hundred metaphase II preparations were obtained and analysed by fluorescent in situ hybridization with bovine chromosome 1 and 29 painting probes. Proportions of different kinds of oocytes were assessed: 74.11% (292/394) were normal and balanced, 4.06% (16/394) unbalanced and 21.83% (86/394) diploid. For all cows, the number of normal oocytes was not significantly different from the number of translocated oocytes but the diploidy and unbalanced rate were significantly different between them. As found in bulls, the meiotic segregation pattern in cows has shown a preponderance of alternate products. However, the frequency of unbalanced gametes determined in females (4.06%) was significantly higher than the frequency observed in males (2.76%). The divergence in the rate of diploid gametes (0.04% vs. 21.83%) is mainly explained by the difference between males and females.     

Meiosis in trisomic female mice with Robertsonian translocations. I. Prophase pairing

The prophase oocytes of two murine Robertsonian translocation (Rb) trisomies of chromosomes 16 and 19 were investigated using electron microscopy and a whole-cell micro-spreading technique after silver staining. About 20% of fetuses of each type were trisomic. They were obtained by mating animals heterozygous for two Rb's, monobrachially homologous for either chromosome 16 or 19, to an entirely acrocentric stock. Because of the almost inevitable prenatal mortality of the trisomic embryos, their fetal ovaries were "rescued" by an in vitro method for prophase studies. Analysis of the recovered oocytes showed frequent, close pairing associations of the three trisomic axes and evidence suggesting that the closely apposed axes coincided with the side-by-side formation of parallel, complete, true synaptonemal complexes; hence, the cytogenetic dogma that pairing is always two-by-two was contradicted. The presence of two parallel complexes has implications for crossing-over recombination. Triple associations of axes were found in almost half the trisomy 19 (Ts19) and in about 70% of the trisomy 16 (Ts16) prophases. The extent of triple associations varied and was greater in Ts16 than in Ts19 oocytes. Other relevant observations concerned the proportions of univalents and of univalence of the trisomic axes (21% in Ts16 and 46% in Ts19) and the distinctive, thickened appearance of all univalent axes. The pairing behaviour observed in balanced heterozygotes confirms what appears to be nonhomologous pairing and synaptic adjustment within the short-arm axes of the Rb trivalents.     

Duplication/deficiency mapping of situs inversus viscerum (iv), a gene that determines left-right asymmetry in the mouse.

A recessive mutation in the mouse, situs inversus viscerum (iv), results in randomization of organ position along the left-right body axis: approximately 50% of the progeny of homozygous matings exhibit situs solitus and 50% exhibit situs inversus. Recent studies have established genetic linkage between iv and the immunoglobulin heavy chain gene complex (Igh-C), located on distal mouse chromosome 12. In the present study, we have refined the genetic map location of iv relative to the breakpoint of a reciprocal translocation, T(5;12)31H, involving the telomeric region of chromosome 12 distal to Igh-C and the proximal region of chromosome 5. The translocation results in a large 12(5) derivative chromosome and a small 5(12) derivative chromosome. Because mice with either monosomy or tertiary trisomy for the 5(12) chromosomal region are viable, duplication/deficiency mapping is possible. Deficiency mapping was performed by mating iv/iv homozygotes and T31H heterozygotes. Two animals monosomic for distal mouse chromosome 12 were produced. One of the animals with cytogenetically confirmed monosomy for distal chromosome 12 exhibited situs inversus, indicating that the iv mutation is located at or distal to the T31H breakpoint. For duplication analysis, matings were initially carried out between iv/iv homozygotes and unbalanced T31H animals trisomic for distal chromosome 12. Cytogenetically verified tertiary trisomic progeny were identified and backcrossed with iv/iv homozygotes. The resulting trisomic progeny, 50% of which are expected to carry the iv mutation on both cytogenetically normal copies of chromosome 12, were scored for phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytogenetic analysis of caprine 2- to 4-cell embryos produced in vitro

Prepubertal goat in vitro matured/in vitro fertilised oocytes produce only a small percentage of blastocysts. The present study examines the incidence of chromosomal anomalies in 2- to 4-cell embryos in vitro produced (IVP) from prepubertal oocytes fertilised with the semen of two males. Cumulus-oocyte complexes were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% heat inactivated Donor Bovine Serum (DBS), 10 microg/ml FSH + 10 microg/ml LH + 1 microg/ml 17beta-oestradiol for 27 h at 38.5 degrees C in 5% CO2 in air. IVM oocytes were inseminated with the sperm from two males prepared using the swim-up and heparin-capacitation procedures. At 24 h postinsemination (hpi) the oocytes were transferred to 100 microl drops of SOF medium for a further 24 h. At 17 hpi a sample of oocytes was stained with lacmoid to evaluate the nuclear stage after fertilisation. The cleavage rate was determined at 24, 36 and 48 hpi and chromosome slides were prepared according to the gradual-fixation technique and stained with Leishman. A total of 1070 2- to 4-cell embryos from prepubertal goat oocytes were studied, but it was only possible to analyse 241 cytogenetically. Of these, 40% exhibited a normal diploid chromosome complement, 59% were haploid and 1% were triploid. There were significant differences between the two males in sperm oocyte penetration and oocyte cleavage but no differences were found in chromosomal anomalies. In conclusion, the low number of embryos karyotyped and the high number of haploid embryos found in this study suggested a high incidence of abnormal fertilised embryos and deficient cytoplasmic maturation of the oocyte which inhibits sperm head decondensation.     

Balanced structural changes involving the human X: Effect on sexual phenotype

Summary A normal cell line arising from a translocation, t(12;21), possibly by dissociation, was observed in two brothers in early life. Each was conceived as trisomic 21 by their 45,XY,-12,-21,+t(12;21) father, who was phenotypically normal. Each brother showed morphologic manifestations of trisomy 21 syndrome, and each was mildly mentally retarded. Dermatoglyphic indices were not diagnostic of trisomy 21 syndrome. At 4 months the younger brother had a 50:50 proportion of trisomic:normal blood cells which became 25:75 of trisomic 21:normal at 36 months. The older brother had a 25:75 proportion of trisomic 21:normal when first studied at 41/2 years. A similar t(12;21) has not previously been reported. The occurrence of an apparently normal cell line arising spontaneously is unique.     

Meiotic disjunction and embryonic lethality in trisomies derived from an X-linked translocation in the onion fly,Hylemya antiqua (Meigen)

Translocation- and tertiary trisomies (for the X-chromosomes) were obtained after testcrossing translocation heterozygous females of an X-linked “simple” translocation stock. Meiotic disjunction as judged from segregations at M II (males) and in young eggs of testcrosses (males and females) in translocation trisomics was studied. No progeny of tertiary trisomic males and females was found, but male M II could be studied. Six different orientation types appeared in translocation trisomie (2n + 1) males and these were present in equal frequencies. No adjacent II configurations were found. The small X- and Y-chromosomes and the large translocated X-chromosome of the translocation complex disjoin at random (n and n + 1 gametes) in both translocation- and tertiary trisomic males. In translocation trisomic females four different orientation types appeared. From the high frequency of two of these (together, 94.5%) it is concluded that the two normal X-chromosomes show preferential pairing and disjunction, while the translocated X-chromosome moves to either one of the two poles at random. Primary trisomic (for the X-chromosome) males (XXY) and females (XXX) were obtained from testerossed translocation trisomics. Cytological analysis of adult male progeny of testerossed XXY males showed that no random orientation for the X-, X- and Y-chromosomes occurred because half of the sons was disomic (XY) and half of them trisomic (XXY). A possible mechanism is discussed. Analysis of young eggs of testerossed XXX females indicated a segregation of 2X∶1X=1∶1. The level of “semi”-sterility as scored from testcrosses of translocation trisomies appeared to be as in translocation heterozygotes. Here again a close relation exists between “semi”-sterility and deficiencies in eggs for a large chromosomal segment. The possible use of this translocation for genetic control of insect pests is discussed.     

Correlation of sperm viability with gamete interaction and fertilization in vitro in the cheetah (Acinonyx jubatus).

Sperm-oocyte interaction in vitro was studied in the cheetah, a species known to produce poor quality ejaculates and to experience low rates of fertility. Twelve female cheetahs were injected (i.m.) with eCG followed by hCG 84 h later. Twenty-four to 26 h post hCG, each was subjected to laparoscopic oocyte aspiration. A sperm motility index (SMI) was calculated for each of 9 cheetah sperm donors that produced ejaculates averaging 41.3 +/- 22.9 x 10(6) motile sperm and 28.4 +/- 4.9% structurally normal sperm. Each ejaculate was used to inseminate cheetah oocytes from 1 or 2 females and salt-stored, domestic cat oocytes. The presence of ovarian follicles (greater than or equal to 1.5 mm in diameter) showed that all females responded to exogenous gonadotropins (range, 11-35 follicles/female). A total of 277 cheetah oocytes was collected from 292 follicles (94.9% recovery; 23.1 +/- 2.2 oocytes/female). Of these, 250 (90.3%) qualified as mature and 27 (9.7%) as degenerate. Of the 214 mature oocytes inseminated, 56 (26.2%) were fertilized, and 37 (17.3%) cleaved to the 2-cell stage in vitro; but the incidence of in vitro fertilization (IVF) varied from 0 to 73.3% (p less than 0.001) among individual males. When oocytes from individual cheetahs (n = 5) were separated into two groups and inseminated with sperm from a male with an SMI greater than 0 after 6 h coincubation versus an SMI = 0 at this time, the mean fertilization rates were 28/44 (63.6%) and 0/37 (0%), respectively (p less than 0.05). Of the 117 domestic cat oocytes coincubated with cheetah sperm, 96.6% contained 1 or more cheetah sperm in the outer half of the zona pellucida (ZP). Although the mean number of cheetah sperm penetrating the outer ZP of the cat oocyte was similar (p greater than 0.05) among all males, there was a positive correlation between the number of sperm reaching the inner half of the ZP and fertilization rate in vitro (r = 0.82; p less than 0.05). Compared to IVF efficiency in the domestic cat and tiger as reported in earlier studies, IVF efficiency in the cheetah is low. Because oocytes from 11 of 12 cheetahs were fertilized in vitro, there is no evidence that the female gamete is incompetent. Although sperm pleiomorphisms may contribute to poor reproductive performance, examination of the data on the basis of individual sperm donors reveals that effective gamete interaction in the cheetah is dictated, in part, by sperm motility.     

Predictive variables of in vitro fertilization and pre-implantation embryo development in the mouse

The present study aims to analyze the cause-effect relationships among several in-vitro fertilization and pre-implantation embryo development variables in the mouse. Superovulation of hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of pregnant mare's serum gonadotropin at the estrus stage of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Ovulated cumulus-enclosed oocytes were inseminated with sperm from hybrid males of 12-16 weeks of age. The multiple linear regression analyses performed indicated that (a) total number of ovulated oocytes is a good predictor of both fertilization frequency and total number of cells in day-5 blastocysts; (b) fertilization frequency predicts percentage of day-5 blastocysts; (c) total number of cells in day-5 blastocysts is predicted by percentage of day-5 blastocysts; and (d) total number of cells in day-5 blastocysts predicts percentage of apoptotic cells, number of inner cell mass (ICM) and trophectoderm (TE) cells, and ICM/TE ratio in day-5 blastocysts. Mitotic index in day-5 blastocysts was positively correlated with total number of ovulated oocytes, percentage of ovulated cumulus-enclosed oocytes, fertilization frequency, percentage of day-5 blastocysts and total number of cells in day-5 blastocysts. On the contrary, it was negatively correlated with percentage of apoptotic cells in day-5 blastocysts.     

Improvement of fertilizing performance by normal and abnormal mouse semen after zona opening of mature oocytes.

The usefulness of opening the zona pellucida by partial zona dissection (PZD) to enhance fertilization of mature mouse oocytes was studied after insemination with three types of semen: normal and diluted semen and semen from long-term-vasectomized males. Zona opening did not by itself induce parthenogenetic cleavage of mature oocytes and did not significantly increase mono- and polyspermic fertilization of oocytes inseminated with normal semen. While a fertilization rate of 62% was obtained among intact oocytes, of which 4.5% were polyspermic, a 66.8% fertilization rate was observed among PZD oocytes, 6.3% of which were polyspermic. However, after using diluted semen, only 54 of 193 intact oocytes were fertilized (28%), and PZD improved the fertilization rate to 65.4%. Cleavage rate of nonmanipulated oocytes inseminated with abnormal semen from vasectomized males was dramatically decreased in comparison with those inseminated with normal semen (7.6% vs. 65%). PZD induced a moderate but significant improvement of fertilization performance when using this abnormal semen (19.6%).     

Radiation-induced nondisjunction and Robertsonian translocation in Drosophila

In Drosophila melanogaster, gametes formed by oocytes in which Robertsonian translocations were induced in an immature stage usually show chromosomal imbalance. It is estimated that fewer than 20% of the gametes bearing newly induced Robertsonian translocations “fusing” X and fourth chromosomes are of balanced constitution. In contrast, when the two acrocentric pairs, X and fourth chromosomes, are replaced by an X-4 Robertsonian translocation, treatment of immature oocytes of homozygotes produces some 5–6-fold fewer sex-chromosome trisomics than do females of normal karyotype. In the place of such trisomics (having separate sex chromosomes), there is a much smaller number of compound-X chromosomes formed and a number of compound-fourth chromosomes as well. However, the production of “XO” males is not appreciably smaller in the translocation homozygotes. A number of possible mechanisms to account for this are suggested. The findings are consistent with the expectations of the hypothesis that radiation-induced nondisjunction results from improper conjunctions of heterologues, brought about by chromatid interchange^{7–12, 16}.     

Gamete segregation in female carriers of Robertsonian translocations

Eleven female carriers of either 45,XX,der(13;14) (q10;q10) or 45,XX, der(14;21)(q10;q10) underwent hormonal stimulation with the purpose of producing enough oocytes for in-vitro fertilization and preimplantation genetic diagnosis. Polar body biopsy was performed in those oocytes and FISH with painting probes was applied in their metaphase-like first polar body chromosomes. In this way, unbalanced, normal and balanced oocytes could be distinguished and segregation modes ascertained. der(14;21)(q10;q10) produced 42% unbalanced, 37% normal and 21% balanced oocytes (n = 86) while der(13;14)(q10;q10) generated 33% unbalanced, 51% normal and 16% balanced oocytes (n = 69). In both translocations the number of normal oocytes was significantly higher than the number of balanced oocytes. However, while the frequency of unbalanced events involving chromosome 13 and 14 was similar in der(13;14)(q10;q10), there were significantly more abnormalities involving chromosome 21 than 14 in the der(14;21) (q10;q10) cases. When comparing survival rates to term, trisomies from Robertsonian origin seem to survive more often than those originated by non-disjunction in non-translocation carriers. The meiotic segregation patterns found in female Robertsonian translocations are different from those described in male carriers, with higher rates of unbalanced gametes in females than in males.     

Nucleosome unit repeat size in aneuploid mice

Male 8-day-old mice that have part of chromosome 7 translocated to an X chromosome [T(X;7)1Ct] and that are chromosomally unbalanced for chromosome 7, and consequently trisomic for that part of chromosome 7, were found to have a smaller nucleosome repeat unit size than normal littermate males (Rake, A. V., and Edwards, R. H.,Biochem. Genet. 25:671, 1987). This smaller nucleosome size is maintained in adult trisomic males. Males with a balanced chromosomal translocation [T(X;7)1Ct] had a normal nucleosome size compared to their littermates. The nucleosome unit size is not altered in two other types of aneuploid mice studied (XO vs XX, 2n=39 and 40, respectively; and Ts12¹⁷ vs normal, 2n=40 and 41, respectively).     

Increased male reproductive success in Ts65Dn “Down syndrome” mice

The Ts65Dn mouse is trisomic for orthologs of about half the genes on Hsa21. A number of phenotypes in these trisomic mice parallel those in humans with trisomy 21 (Down syndrome), including cognitive deficits due to hippocampal malfunction that are sufficiently similar to human that “therapies” developed in Ts65Dn mice are making their way to human clinical trials. However, the impact of the model is limited by availability. Ts65Dn cannot be completely inbred and males are generally considered to be sterile. Females have few, small litters and they exhibit poor care of offspring, frequently abandoning entire litters. Here we report identification and selective breeding of rare fertile males from two working colonies of Ts65Dn mice. Trisomic offspring can be propagated by natural matings or by in vitro fertilization (IVF) to produce large cohorts of closely related siblings. The use of a robust euploid strain as recipients of fertilized embryos in IVF or as the female in natural matings greatly improves husbandry. Extra zygotes cultured to the blastocyst stage were used to create trisomic and euploid embryonic stem (ES) cells from littermates. We developed parameters for cryopreserving sperm from Ts65Dn males and used it to produce trisomic offspring by IVF. Use of cryopreserved sperm provides additional flexibility in the choice of oocyte donors from different genetic backgrounds, facilitating rapid production of complex crosses. This approach greatly increases the power of this important trisomic model to interrogate modifying effects of trisomic or disomic genes that contribute to trisomic phenotypes.     

Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.     

Fertilizing ability of spermatozoa from aged C57BL/6NNia mice

Spermatozoa from C57BL/6NNia mice (7- and 25-month-old males that produced offspring and 25-month-old males incapable of producing offspring which either mated or did not mate after being paired for 1 month with proven-fertile females) were tested in in-vitro fertilization studies. The 7-month-old males fertilized the largest number of oocytes (80-86%) in vitro and 79% of them subsequently developed into blastocysts in culture. Aged males which failed to mate fertilized the lowest number of oocytes (11-19%) with 48% developing to blastocysts. This group of mice had the lowest number of spermatozoa in the cauda epididymidis (3.2 +/- 0.4 x 10(5)/mg tissue) with fewer motile spermatozoa (22.3 +/- 5.1%) than younger males. The percentage of spermatozoa retaining their acrosome after 3 h in culture was higher in aged males which had not mated when compared to younger males that had mated. After 4 h in culture, however, the number of spermatozoa that had lost their acrosome was almost identical in the two groups. Superovulated mice which were artificially inseminated with spermatozoa from 25-month-old mice that had not mated did not become pregnant. Testosterone concentrations were lowest in aged mice not mating. These concentrations may explain the poor behavioural response of these males, but whether they account for the inability of spermatozoa to fertilize ova in vitro or in vivo after artificial insemination is not known.     

用FISH技术研究人类体外未受精卵的21号染色体非整倍体

采用荧光原位杂交技术,选用人类21号染色体端粒探针（21qter）,检测人类体外未受精卵的21号染色体非整倍体发生率,并比较非整倍体率与25－30岁和31－35岁这两个女性年龄组、IVF指征、超排方案之间的关系,在54个未受精卵中,正常21号单体30枚,二体16枚,三体4枚,缺体4枚,非整倍体率为44.4％（24／54）;25－30岁和31－35岁这两个年龄组、IVF指征、超排方案的患者的21号染色体非整倍体率之间的差异无显著性,卵母细胞21号染色体的非整倍性是造成体外受精失败的重要原因之一。     

Cadaveric sperm induces intergeneric androgenesis in the fish, Hemigrammus caudovittatus

Intergeneric androgenetic golden Buenos Aires tetra (BT), Hemigrammus caudovittatus was generated using sperm drawn from post-mortem males preserved at -20 degrees C for 10, 20, 30 and 40 days or fresh sperm to activate the UV-irradiated oocytes of black widow tetra (WT), Gymnocorymbus ternetzi. UV-irradiation (4.2 W/m(2)) of the oocytes for 3 min inactivated their nuclear genome. Fry hatched out from these activated oocytes were haploids; suffering haploid syndrome, they died before or within 48 h after hatching. Fresh BT sperm activated 95% oocytes; however, the sperm drawn from post-mortem males preserved at -20 degrees C for 60 (within glycerol packing) and 30 days (without glycerol packing) activated only 24 and 19% oocytes, respectively. Following activation, diploidy was restored by shocking the 25-min-old embryos at 41 degrees C for 2 min. Nuclear genomic inactivation of the oocytes was confirmed by (i) production of 100% haploids, (ii) karyotype and erythrocyte measurements, (iii) phenotypic markers, (iv) progeny testing and (v) species-specific marker. At hatching, survival of androgenotes decreased from 11% for those induced with fresh sperm to 4% for those generated using sperm from 30-day-old post-mortem males. Reproductive performance of the 'fresh' and 'cadaveric' F(0) and F(1) androgenetic males (Y(2)Y(2)) was superior to the control (X(1)Y(2)). Crosses involving homozygous (Y(2)Y(2)) 'fresh' F(0) androgenetic males with heterozygous females (X(1)X(2)) and F(0) homozygous males (Y(2)Y(2)) with females (X(2)X(2)) produced 2-4% unexpected female progenies. Paternal autosomes, inherited by the homozygous androgenetic female (X(2)X(2)), induced the production of female progenies in significantly less number of crosses than the crosses with heterozygous females (X(1)X(2)), which carried equal number of paternal and maternal autosomes. PCR analyses of the genomic DNA of normal male and unexpected F(1) and F(2) female progenies amplified by DMRT 1 specific primer produced bands of 237 and 300 bp length, and thereby confirmed that these unexpected females were genetic males. RAPD analyses of the androgenetic progenies showed that their genome was not contaminated with maternal genome.