Identifying infertile homozygous Inverdale (FecXI) ewe lambs on the basis of genotype differences in reproductive hormone concentrations

Introduction of the Inverdale prolificacy gene (FecX^I) could markedly improve reproductive efficiency in commercial flocks, but as homozygous carrier Inverdale ewes are infertile, it is imperative that these animals are identified at an early age and excluded from breeding stock. As the ovaries of homozygous carrier ewes are nonfunctional, there are wide differences in reproductive hormone levels between these and other Inverdale genotypes. This study assesses the accuracy of using hormone concentrations alone, to identify infertile homozygous ewe lambs. Ewe lambs were blood sampled at 2, 5 and/or 8 months of age, and plasma analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH) and inhibin content. These animals were either the offspring of both known carrier rams and known carrier ewes, and therefore would be either homozygous (II) or heterozygous (I +) for the Inverdale gene (group 1, N = 122), or had one parent that was a carrier and therefore would be either heterozygous or noncarriers (+ +) of the gene (group 2, N = 32). Animals were designated as either II or I + / + + on the basis of their plasma hormone concentrations. Inverdale genotype was also assigned from laparoscopic observation of the ovaries at each of these occasions. Definitive assignment of genotype was made at laparoscopy as adults during the breeding season. On the basis of laparoscopy as adults, 62 (51%) lambs in group 1 were identified as homozygous and 60 (49%) as heterozygous. At all three ages, both mean FSH and mean LH concentrations were significantly higher in II than in I + lambs. Mean inhibin concentrations were significantly lower in II lambs at 8 months, but did not differ significantly between genotypes at 2 or 5 months of age. The use of discriminant analysis techniques to segregate individual animals in group 1 on the basis of their plasma FSH and LH concentrations, correctly identified Inverdale genotype in 50/52 (96%) lambs at 2 months, 75/79 (95%) at 5 months and 118/122 (97%) at 8 months of age. Discriminant analysis was equally effective for segregating II ewe lambs (group 1) from fertile ewe lambs of I + and + + genotype (group 2, 97% correct at 5 months and 98% at 8 months). At no stage did inclusion of inhibin concentrations into the discriminant function alter the number of homozygous ewes misclassified. This demonstrates that infertile homozygous ewe lambs can accurately be distinguished from their fertile flockmates by using plasma concentrations of gonadotrophins alone, and that this can be achieved from as early as 2 months of age.     

Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.     

Meiotic disjunction,sex-determination and embryonic lethality in an X-linked “Simple” translocation of the onion fly,Hylemya antiqua (Meigen)

It was shown that the translocation in study is X-linked. After testcrossing translocation heterozygous males they generally only produce translocation heterozygous daughters and normal sons. The small acrocentric chromosomes involved in the translocation appeared to be the sex-chromosomes. The X-chromosome has a secondary constriction which is missing in the (male determining) Y-chromosome. Meiotic orientation was studied in translocation heterozygous males and females. The alternate and adjacent I orientations were found in about equal frequencies. Further, numerical meiotic non-disjunction (two types) occurred in translocation heterozygous males (about 2%), but is much higher in females (18.7%). In (achiasmate) males the homologous centromeres predominantly regulate meiotic pairing, coorientation and disjunction, apparently independently of the chromosomal rearrangement. Disturbed telomere pairing in particular leading to reduced chiasma frequency most probably explains the high numerical non-disjunction in chiasmate females. A rather good relationship exists between the percentage “semi”-sterility (28%), scored as late embryonic lethals (eggs, 72 hrs.) and the percentage karyotypes (20%) in young eggs (8–16 hrs.) with a large chromosomal deficiency. The remaining sterility (8%) can be explained by the somewhat decreased viability of tertiary trisomics and duplication karyotypes at the end of the egg stage. This translocation behaves like a “simple” one.     

Kinetics of oogenesis in mice heterozygous for Robertsonian translocations

The total number of oocytes at different postmating time intervals (18-40 days) was determined in mice homozygous and heterozygous for different Robertsonian (Rb) translocations, of both laboratory and feral origin. The number of oocytes was lower in heterozygous than in homozygous mice throughout the period studied. Independently of the genetic background (i.e. laboratory or feral), structural heterozygosity had a progressive detrimental effect on oocyte numbers: open, or chain diakinetic configurations had a greater detrimental effect than close, or ring, configurations. The genetic background, however, affected the ovarian constitution in terms of the total number of germ cells, which are more numerous in laboratory than in feral mice. The kinetics of oogenesis seems to be faster in feral than in laboratory mice. At the light of the data here presented, and of those already available from the literature on male and female gametogenesis in conditions of structural heterozygosity, it appears that factors other than unsaturation of pairing sites or interference with pachytene X-chromosome inactivation have to be considered. In the wild, the reduced oocyte numbers in Rb heterozygous female can contribute to the retention of isolated populations in contact zones.     

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.     

Pachytene pairing in relation to sperm and oocyte numbers in a male-fertile reciprocal translocation in the mouse

In adult males carrying the male-fertile reciprocal translocation T(2;4)13H, body weights, testis weights, and sperm counts were higher in heterozygotes than in homozygotes. Heterozygotes whose mothers were C3H/He exceeded their reciprocal counterparts in the same criteria. At 3-4 days of age, no significant differences between homozygous and heterozygous females were found in body weight, ovarian volume, or oocyte numbers, although mean oocyte volumes were somewhat larger in heterozygotes than in homozygotes. In homozygous males and females the synaptonemal complexes of rearranged chromosomes appeared as bivalents that were indistinguishable from normal bivalents. In most gametocytes of heterozygotes, the translocation was present in the form of a quadrivalent. The degree of pairing failure was greater in oocytes than in spermatocytes. Terminal asynapsis of quadrivalents was very rare in spermatocytes, but it affected one quarter of the oocytes. Only very few translocation configurations were associated with the XY bivalent. It is concluded that the number of sperm produced in male heterozygotes can match the general increase in vigor by the formation of a high level of fully paired quadrivalents, whereas a greater degree of terminal asynapsis in the quadrivalents of oocytes may indicate a slightly more deleterious effect of this translocation on oogenesis.     

Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.     

The frequency of aneuploidy in the secondary spermatocytes of normal and Robertsonian translocation-carrying rams.

A detailed analysis was made of the chromosomes in 1008 M II figures from three different types of heterozygous Robertsonian translocation-carrying rams (53,xy,t1; 53,xy,t3) and 225 M II figures from homozygous Robertsonian translocation-carrying rams (52,xy,t1t1; 52,xy,t3t3) and rams of normal karotype (54,xy). No hypermodal cells were recorded in either the normal or the homozygous rams, but from 4-5% to 9-2% of M II cells from the heterozygous rams were hypermodal. The heterozygous rams also produced a significantly higher level of hypomodal cells suggesting that, in addition to non-disjunction, lagging at anaphase I may have occurred. There were also distinct differences in M II aneuploid spermatocyte frequency between heterozygous versus normal and homozygous rams. Fewer balanced translocation X-carrying M II cells were recorded than expected in three of the four 53,xy,t2 rams. This coincides with mating data which suggest that 26,x,t2 gametes may occur less frequently than expected. Since ewes of normal karotype mated to 53,xy,t rams conceive to first service at a rate equal to or better than normal mating groups, and because no blastocysts with unbalanced karotypes associated with the t1 translocation have been recorded, it is suggested that only euploid spermatozoa are involved in fertilization. In the sheep, aneuploid spermatocytes probably degenerate before sperm maturation.     

Estimating meiotic disjunction frequencies in chicken translocation heterozygotes based on embryonic mortality

Chickens heterozygous for a chromosomal translocation [MN t(1;4)] were intercrossed and the progeny were analyzed for their chromosome complement. A ratio of 1 homozygous translocation carrier to 4 heterozygous translocation carriers to 1 homozygous standard chromosome carrier was noted (n = 520), rather than the 1:2:1 ratio expected from Mendelian segregation. The excess of heterozygous carriers was apparently caused by union of complementary duplication/deficient gametes. Embryonic death occurred in 68% of fertile zygotes. This finding fits very closely to expectations if alternate and adjacent meiotic disjunctions occurred at equal frequencies. As alternate disjunction frequencies increase from 0.5 to 1.0 in inter se matings, the proportion of inviable zygotes among fertilized ova will decrease from 0.625 to 0 and the proportion of translocation heterozygotes among viable progeny will decrease from 0.667 to 0.5. In instances where alternate and adjacent disjunction occur at equal frequency, preferential recovery of translocation carriers will occur. This may contribute to chromosomal diversity within a species, and possibly lead to speciation.     

Chromosomal relationships among cultivars of Citrus reticulata Blanco, its hybrids and related species

Karyotypes of 93 individuals belonging to 18 accessions of mandarins, mandarin hybrids and two related species were analysed with the fluorochromes CMA and DAPI, to identify marker chromosomes. The karyotypes revealed highly differentiated banding patterns and could be classified in four groups (I–IV) according to the presence/absence of chromosomes with three bands (type A) or with two bands (one proximal and one terminal, type B, or both terminal, type C). The accessions of group I exhibited the simplest and homozygous karyotypes (lacking chromosome types A, B and C), represented by `Sunki' and `Cleopatra'. Group II (lacking chromosome types A and B) included three accessions of Mediterranean mandarins and `Cravo' mandarin, all of them with very similar and almost completely homozygous karyotypes. All other karyotypes of groups II and III (lacking chromosome type A) were heterozygous for one or more chromosome pairs and most of them seemed to be hybrid derivatives from non-mandarin accessions. Group IV (with chromosome types A and B) was represented only by two heterozygous hybrids (`Murcott' and `King'). The karyotype of most hybrids agrees with one of the possible combinations resulting from chromosome types segregation from their putative ancestor karyotypes, but at least `Orlando' seemed to be a more complex hybrid. Comparing with banding patterns of other Citrus species, those of group I and the Mediterranean mandarins (group II) are the best candidates to represent C. reticulata (sensu Swingle) as a true species.     

Transferrin types and reproduction in sheep

Transferrin types were determined for flocks of Finnish Landrace, Clun Forest, Soay and Merino sheep and gene frequencies were calculated. Analysis of ratios of transferrin types in segregating matings of Finnish Landrace and Clun Forest revealed a significant excess of heterozygotes in matings of heterozygous rams with heterozygous and with homozygous ewes. In Finnish Landrace, matings of sheep homozygous for Tf ^c to those heterozygous for Tf ^C gave a significant excess of homozygous male lambs and heterozygous female lambs. Finnish Landrace ewes of transferrin type BD had smaller litters than ewes of other types.     

Effects of the 7 21 Robertsonian translocation on fertilization rates and preimplantation development of bovine oocytes in vitro

A study was conducted to investigate the effect of the 7/21 Robertsonian translocation on fertilization and subsequent development of bovine oocytes matured in vitro. Semen from Japanese Black bulls, 2 with a normal karyotype (Bulls A and B) and 2 that were heterozygous for the 7/21 translocation (Bulls C and D), was used in this study. In vitro matured bovine oocytes were inseminated with frozen-thawed sperm capacitated with heparin. After insemination, oocytes were cultured at 38.5 degrees C on a monolayer of cumulus cells in TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate in an atmosphere of 2% CO2 in air. Cleavage rate was evaluated at 54 h after insemination, and development of embryos to the blastocyst stage was observed 7 to 10 d post insemination. There was no difference in the fertilization rate among the 4 bulls. Although the cleavage rate of oocytes inseminated with semen from Bull C (heterozygote) was lower (P < 0.05) than that obtained with semen from Bull B (normal), the blastocyst formation rate did not differ among the 4 bulls. These results indicate that the 7/21 Robertsonian translocation had no effect on the fertilization and blastocyst formation rates of bovine in vitro-matured oocytes.     

Expression of growth and differentiation factor-9 in the ovaries of fetal sheep homozygous or heterozygous for the inverdale prolificacy gene (FecX(I))

Abnormal follicular and oocyte growth in ovaries of sheep homozygous (II) for the Inverdale gene, FecX(I), suggest that this gene may influence a fundamental event in initiation of folliculogenesis, with two copies of the gene inhibiting growth at the primordial/primary stage. In addition, striking similarities in ovarian morphology between mice deficient in growth and differentiation factor-9 (GDF-9) and II sheep suggest a relationship between the FecX(I) gene and GDF-9 function in the ovary. Therefore, it was hypothesized that GDF-9 mRNA expression would be inhibited in ovaries of II fetal sheep. To test this hypothesis, in situ hybridization was used to characterize GDF-9 mRNA expression in ovaries of homozygous (II), heterozygous (I+), and control (++) fetal sheep at Day 135 of gestation. GDF-9 mRNA expression was localized exclusively to oocytes from the type 1 follicle stage onward in all genotypes and is the first demonstration of GDF-9 mRNA expression in ovaries of fetal sheep. In addition, GDF-9 mRNA expression was detected in oocytes of abnormal type 2 follicles in the ovaries of II sheep. Thus, it does not appear that inhibition of GDF-9 gene expression is the mechanism of action whereby the FecX(I) gene exerts its influence. However, the possibility of translation at specific stages of follicular development cannot presently be ruled out. In addition, the FecX(I) gene may be involved, either directly or indirectly, in regulating expression of receptors for GDF-9. At present, however, neither the FecX(I) gene product nor the GDF-9 receptor has been isolated or characterized.     

三种绵羊BMP15基因第二外显子多态性及与产羔数的相关性分析

绵羊存在影响多胎性状的不同主效基因,选择影响Romney Hanna绵羊和Cambridge绵羊高繁殖力的骨形态发生蛋白15 (bone morphogenetic protein 15, BMP15)为候选基因,采用PCR-SSCP的方法检测BMP15基因外显子Ⅱ第747位点(T747→C)和755位点(T755→C)在蒙古羊、甘肃高山细毛羊、小尾寒羊三种绵羊母羊中的多态性,同时还研究了上述两处突变对三种绵羊产羔数的影响。表明:(1)一共检测到野生纯合型AA、突变杂合型AB (T747→C)、AC (T755→C)三种不同的基因型,AA为优势基因型,A为优势等位基因;(2)三种基因型在甘肃高山细毛羊中均被检测到,而蒙古羊和小尾寒羊中未检测出AB基因型;(3)突变杂合型蒙古羊(AC)比野生纯合型(AA)的平均产羔数多0.27只(p<0.05)。(4)AC的基因型频率,双羔母羊和多羔母羊均高于单羔母羊。根据以上实验推测,BMP15第755位点发生的T→C突变(AC型)对蒙古羊一胎产双羔影响十分显著,甘肃高山细毛羊中AC基因型的绵羊其产羔数有比AA基因型和AB基因型多的趋势,因此该位点可能是一个影响绵羊高繁殖力潜在的DNA标记。     

The generation of live offspring from vitrified oocytes

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P<0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P<0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits.     

Associations of PrP genotype with lamb production traits in three commercial breeds of British hill sheep

The National Scrapie Plan (NSP) was launched in Great Britain in 2001, with the aim of eventually eradicating scrapie, a small ruminant transmissible spongiform encephalopathy, from the national sheep flock. Specifically, a selective breeding programme, the Ram Genotyping Scheme, was devised enabling pedigree ram breeders to reduce the number of scrapie-susceptible genotypes from their flocks. The effect of large-scale manipulation of PrP genotypes on commercially important traits within the sheep industry is, however, unknown. We have therefore examined production traits in a total of 43 968 lambs from 32 pedigree breeders across three British hill breeds, comprising 8163 North Country Cheviot (Hill), 21 366 Scottish Blackface and 14 439 Welsh Mountain lambs. Traits examined included: weights at birth, 8 and 20 weeks; ultrasonic fat and muscle depth, and average daily weight gain from 8 to 20 weeks. Linear mixed models were fitted for each trait, including animal (direct) genetic effects and up to three maternal effects. Potential associations with the PrP gene were assessed by fitting either PrP genotype or number of copies of individual alleles as fixed effects. A number of breed-specific significant associations between production traits and the PrP gene were found, but no consistent significant effects were detected across the three breeds. Breed-specific effects were as follows: (i) 0.37 kg higher birth weights (BWTs) in AHQ homozygous North Country Cheviot (Hill) lambs (P < 0.01); (ii) 0.16 kg higher BWTs in ARR homozygous Scottish Blackface lambs (P < 0.05); (iii) 0.5 kg higher 8-week weights in VRQ heterozygous Scottish Blackface lambs (P < 0.01); (iv) a 0.72 kg decrease in scan weight associated with homozygous ARR Welsh Mountain lambs (P < 0.01); (v) 0.51 mm higher ultrasonic muscle depths in AHQ homozygous Welsh Mountain lambs (P < 0.01); (vi) 0.48 mm lower ultrasonic muscle depths in Welsh Mountain lambs carrying one or more copies of the ARR allele (P < 0.05) and (vii) 0.2 mm higher ultrasonic fat depths in heterozygous VRQ Welsh Mountain lambs (P < 0.05). The use of a Bonferroni correction to define appropriate significance thresholds across the three datasets, which account for the large number of independent comparisons made, resulted in breed-specific comparisons, with P < 0.01 becoming significant at P0.05, and the remaining breed-specific comparisons no longer being significant. The absence of a common effect across the three breeds suggests that any true association found may be due to breed-specific alleles of neighbouring genes in linkage disequilibrium with the PrP locus.     

Developmental competence and apoptotic gene expression patterns of mature and immature human oocytes retrieved from controlled ovarian stimulation cycles

The purpose was to assess the developmental competence of the in vitro or in vivo matured human oocytes as well as the apoptotic genes expression of cumulus cells (CCs) regarding nuclear maturity status of associated oocytes retrieved from stimulated ICSI cycles. A total of 590 oocytes and the associated CCs were retrieved and divided into groups of test and control according to the nuclear maturity status in order to the developmental evaluation as well as expression patterns of apoptosis-related genes using real time PCR. The fertilization and embryo formation rates were 60.3% and 87.5% vs.69.1% and 92.8% in test and control groups, respectively. Good quality embryos on day 3 were 62.2% in test and 69.1% in control groups. There were significant differences in the rates of normal fertilized as well as unfertilized oocytes between the groups. Also, mRNA levels of some apoptotic genes were significantly higher in the CCs obtained from immature oocytes among patients with premature ovarian factors (POF) rather than other infertility etiologies (p?<?0.001). The data demonstrated the developmental competence of in vitro matured oocytes ?even to good quality cleavage embryos- is not completely consistent with molecular integrity and well-mannered gene expression patterns resulting to ICSI success. It seems that using immature oocytes could be helpful for patients at risk of ovarian hyperstimulation syndrome (OHSS) as the same as patients with diminished ovarian reserve.     

Abnormal platelet response to PAF and ADP in beta-thalassaemia

1. AGEPC (2 microM) caused a noticeable increment in platelet aggregation, in increasing order, in 9 heterozygous beta-thalassaemic subjects, 18 homozygous beta-thalassaemics and 12 splenectomized homozygous beta-thalassaemics. 2. Recombination experiments with "patient" platelets and "normal" plasma or the reverse, as well as hydrolysis of labelled AGEPC from "normal" and "patient" serum, suggested that the observed abnormalities were due to platelets rather than to the plasma PAF hydrolase. 3. A normal splenectomized subject showed also hyperaggregability and PAF serum levels in a splenectomized patient were found twice as high in a non-splenectomized patient. 4. ADP (5 microM) caused decreased or normal platelet aggregation in the homozygous patients, approximately normal in the heterozygous subjects and increased in the splenectomized patients.     

Offspring from one-month-old lambs: studies on the developmental capability of prepubertal oocytes

A wave of follicular growth in lamb ovaries occurs at about 4 weeks of age, generating a life-time peak in follicle numbers. In order to take advantage of the large number of oocytes available, and to substantially decrease the generation interval, embryos were derived from oocytes collected from 1-mo-old lambs. Animals were subjected to one of 3 regimes of hormonal stimulation: groups 1 and 2 were treated to obtain germinal vesicle-stage oocytes, and group 3 to produce mature metaphase II oocytes. Adult sheep stimulated by an appropriate dose of FSH served as control. The developmental ability of collected oocytes was evaluated by either in vivo or in vitro culture to the blastocyst stage after in vitro maturation and/or fertilization. Blastocysts were transferred immediately or after cryopreservation to suitable recipient sheep. In order to investigate the full developmental potential of these embryos, pregnancies were allowed to go to term. The results show significant differences (P < 0.001) between all experimental groups in blastocyst numbers produced. Embryos derived from group 1 animals produced the greatest number of blastocysts, under both in vivo (36. 7%), and in vitro (22.9%) culture systems. Group 2 gave lowest blastocyst production (5.0%), while group 3 yielded 13.2% blastocysts. The number of pregnant recipients carrying to term lamb-derived embryos was severely reduced for both in vivo- (2 of 9; 22.2%) and in vitro-cultured, fresh (3 of 10; 30.0%) and cryopreserved (1 of 6; 16.7%) lamb embryos. This study is the first report of the birth of live lambs derived from oocytes obtained from donors as young as 4 wk. Defects in the competence of lamb-derived embryos may account for the increased fetal loss during pregnancy and the occurrence of mummified fetuses delivered alongside normal healthy lambs.