Normal Haploid Research Articles

Chromosomes of three gall wasps of the tribe Aylacini (Hymenoptera, Cynipidae).

Chromosomes of two species of the tribe Aylacini (Cynipidae), Isocolus jaceae (Schenck, 1863) and I. scabiosae (Giraud, 1859) (both have 2n = 18) were studied for the first time. In addition, 2n = 20 is confirmed in a member of the same tribe, Aulacidea hieracii (Bouché, 1834). All chromosomes of these gall wasps are biarmed; however, they gradually decrease in size in the case of A. hieracii, whereas a pair of large metacentrics is characteristic of karyotypes of both Isocolus Förster, 1869 species. Chromosomes of the two latter gall wasps are either metacentric or submetacentric, but elements with lower centromeric indices prevail in the karyotype of A. hieracii. Chromomycin A3 (CMA3)/DAPI staining revealed single CMA3-positive bands on a particular pair of chromosomes of all species, and these bands apparently refer to the nucleolus organizing regions (NORs). However, localization of CMA3-positive bands differs substantially between the studied members of Isocolus and Aulacidea Ashmead, 1897. Together with normal haploid and diploid mitotic divisions, several metaphase plates with 2n = 17 containing a peculiar dicentric chromosome were found in a single male specimen of I. scabiosae; this appears to be the first report of an obvious dicentric in the order Hymenoptera in general. Certain aspects of the chromosome diversity and karyotype evolution within the family Cynipidae and the tribe Aylacini in particular are briefly discussed.

Corrigendum to: 131 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED 4-DAY-OLD CATTLE EMBRYOS: INFLUENCE OF THE OOCYTE MATURATION MEDIA

Chromosomal abnormalities were related to embryo developmental failures in cattle, being highly increased in IVF-produced compared with multiple-ovulation embryo transfer-produced embryos. The origin of this difference remains unclear, but they were related to the suboptimal environmental culture laboratory conditions. We studied the influence of 3 different maturation media in the appearance of chromosomal abnormalities in early in vitro-produced embryos. A total of 562 oocytes classified as Class A were collected by follicular aspiration in slaughterhouse ovaries and matured by 20 h in 3 different culture media supplemented with 10% FCS and antibiotics as follows: TCM-199 (T, 193 oocytes); DME (D, 178 oocytes); and RPMI-1640 (R, 191 oocytes). Matured oocytes were fertilized by 16 h with 1 × 106 spz mL–1 in IVF-TALP media and cultured by 72 h in SOF media. Later, 48 h after fertilization, 562 presumptive embryos were evaluated showing a normal cleavage rate of 64.7, 55.2, and 54.9% in T, D, and R groups, respectively. After culture, only 181 early embryos (90 hpi) showing normal development were correctly karyotyped following our standard laboratory techniques. Individual blastomeres were stained with Giemsa and assessed by direct observation at ×1250 magnification in a brightfield microscope. The percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Only embryos showing almost 2 different blastomeres correctly karyotyped were included in this study. Cleavage rates were statistically higher (P < 0.05) in the oocytes matured in T media in comparison with the oocytes matured in D or R media. The percentage of diploid embryos were almost equals in the 3 groups evaluated (Table 1). There was some variation when each kind of chromosomal abnormality was assessed individually, but no statistical differences were observed. These results are in consonance with our previous studies in which we demonstrated the maturation time and morphological quality are the 2 main oocyte-derived factors influencing the ploidy of early embryos. It was also demonstrated that chromosomal complements were affected, in a much lesser extent, by the maturation media supplementation. However, in the present study, the maturation media did not statistically affected embryo ploidy. However, the higher percentage of cleaved embryos using TCM-199 observed agree with fact that this maturation media is one of the most widely used in IVF procedures in cattle. Based on our data, we suggest that oocyte maturation, a well established technique in cattle, could be performed using different maturation media without expecting major differences in the embryo ploidy, and therefore, the differences observed in cleavage rate must be originated in other physiological factors. Table 1.Results

Corrigendum to: 131 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED 4-DAY-OLD CATTLE EMBRYOS: INFLUENCE OF THE OOCYTE MATURATION MEDIA

Chromosomal abnormalities were related to embryo developmental failures in cattle, being highly increased in IVF-produced compared with multiple-ovulation embryo transfer-produced embryos. The origin of this difference remains unclear, but they were related to the suboptimal environmental culture laboratory conditions. We studied the influence of 3 different maturation media in the appearance of chromosomal abnormalities in early in vitro-produced embryos. A total of 562 oocytes classified as Class A were collected by follicular aspiration in slaughterhouse ovaries and matured by 20 h in 3 different culture media supplemented with 10% FCS and antibiotics as follows: TCM-199 (T, 193 oocytes); DME (D, 178 oocytes); and RPMI-1640 (R, 191 oocytes). Matured oocytes were fertilized by 16 h with 1 × 106 spz mL–1 in IVF-TALP media and cultured by 72 h in SOF media. Later, 48 h after fertilization, 562 presumptive embryos were evaluated showing a normal cleavage rate of 64.7, 55.2, and 54.9% in T, D, and R groups, respectively. After culture, only 181 early embryos (90 hpi) showing normal development were correctly karyotyped following our standard laboratory techniques. Individual blastomeres were stained with Giemsa and assessed by direct observation at ×1250 magnification in a brightfield microscope. The percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Only embryos showing almost 2 different blastomeres correctly karyotyped were included in this study. Cleavage rates were statistically higher (P < 0.05) in the oocytes matured in T media in comparison with the oocytes matured in D or R media. The percentage of diploid embryos were almost equals in the 3 groups evaluated (Table 1). There was some variation when each kind of chromosomal abnormality was assessed individually, but no statistical differences were observed. These results are in consonance with our previous studies in which we demonstrated the maturation time and morphological quality are the 2 main oocyte-derived factors influencing the ploidy of early embryos. It was also demonstrated that chromosomal complements were affected, in a much lesser extent, by the maturation media supplementation. However, in the present study, the maturation media did not statistically affected embryo ploidy. However, the higher percentage of cleaved embryos using TCM-199 observed agree with fact that this maturation media is one of the most widely used in IVF procedures in cattle. Based on our data, we suggest that oocyte maturation, a well established technique in cattle, could be performed using different maturation media without expecting major differences in the embryo ploidy, and therefore, the differences observed in cleavage rate must be originated in other physiological factors. Table 1.Results

87 EVALUATION OF THE NUMERICAL CHROMOSOMAL ABNORMALITIES ON IN VITRO EARLY BOVINE EMBRYOS: EFFECT OF THE CELL CO-CULTURE WITH GRANULOSA CELLS

Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in in vitro-produced (IVP) embryos. It has been described that CNA are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affects the CNA rates. The addition of granulosa cells during early embryo development is a well-known procedure to simplify the culture of bovine IVP and cloned embryos. This technique avoids the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of IVP cattle embryos. Cumulus–oocyte complexes (COC) were matured in TCM-199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin during 20 h at 38.5°C in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1 × 106 spermatozoa mL–1, selected through a Percoll gradient centrifugation. After fertilization, zygotes were divided in 2 groups and cultured in TCM-199 medium for 48 h, with (TCM-GC) or without (TCM) the addition of 1 × 106 granulosa cells. These cells were obtained by centrifuging and washing the follicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a brightfield microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were: D = 81.4%, H = 7.2%, P = 7.2%. and A = 3.6% in TCM and D = 84.3%, H = 3.9%, P = 9.8%, and A = 1.9% in TCM-GC. No significant differences (P > 0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in co-culture during embryo development did not influence the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques used in the culture of bovine embryos until Day 3.

Zmynd15 Encodes a Histone Deacetylase-dependent Transcriptional Repressor Essential for Spermiogenesis and Male Fertility

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

Meiosis in Solanum phureja

Meiosis and pollen mitosis were studied in a clone of Solanum phureja in order to elucidate the generally high frequency (about 90 %) of tetraploid progeny in tetraploid X diploid crosses involving S. phureja. The first meiotic division was normal with 12 bivalents in metaphase and apparently normal chromosome disjunction. The second meiotic division showed normal metaphases with 12 chromosomes in each of the two spindles, along with cells showing a bi- or tripolar spindle (cell wall formation was simultaneous). At the end of the second division those disturbances resulted in unreduced cells either with two diploid nuclei or with one diploid and two haploid nuclei along with the normal tetrads with four haploid nuclei. The frequency of unreduced gametes was about 50 %. First and second pollen mitoses were also represented by normal haploid as well as unreduced diploid cells. Hence, the high frequency of tetraploid offspring in Solanum tuberosum X S. phureja crosses could be caused by selective fertilization with unreduced diploid pollen grains.

Mitotic and meiotic chromosomes of the variable hare (Lepus timidus L.), the common hare (Lepus europaeus Pall.) and their hybrids.

Mitotic and meiotic chromosome studies in the variable hare (Lepus timidus L.), the common hare (Lepus europaeus Pall.) and their hybrids have shown a diploid chromosome number of 48. No differences in chromosome structure could be established between the two species. There was a normal meiotic behaviour of the chromosomes in the male hybrids with the formation of secondary spermatocytes with a normal haploid chromosome complement. The hybrids appeared to have a normal fertility and the reproductive barrier existing between the two species was apparently not built up by sterility factors.

Meiotic segregation patterns and ICSI pregnancy outcome of a rare (13;21) Robertsonian translocation carrier: a case report

t(13;21) is an uncommon Robertsonian translocation (RT) with limited information in the literature. Hereby, we assessed the meiotic segregation and interchromosomal effect (ICE) in sperm nuclei from a t(13;21) carrier. The pregnancy outcome following ICSI was also included as reference for physicians and patients. Dual-colour fluorescent in situ hybridization (FISH) was carried out to analyse the segregation pattern of chromosomes 13 and 21, while triple-colour FISH was used to investigate the possible concurrence of ICE. With respect to chromosomal constitutions of 13 and 21, 88.39% of the spermatozoa were normal or balanced due to alternative segregations, and 11.08% showed nullisomy or disomy as a result of adjacent segregations. However, for chromosome 18 and sex chromosomes, the proportion of normal haploids was 98.79%. The rate of disomy was not significantly higher than the controls for either chromosome 18 or X/Y. The rare t(13;21) case exhibited a similar pattern of meiotic segregation as in the common RTs. ICEs were not observed in the current case.

Meiotic abnormalities in in vitro-matured marmoset monkey (Callithrix jacchus) oocytes: development of a non-human primate model to investigate causal factors

Meiotic abnormalities are thought to be a major causal factor of low embryo development rates, for embryos developed from in vitro-matured oocytes. A new non-human primate model, in the common marmoset, is being developed to facilitate investigation of the mechanisms involved. Oocytes were dissected from antral follicles from three size classes. They were allowed to mature in vitro for only 24 h, in order to focus the investigation on the rapidly maturing oocytes. Chromosome spreads were visualized with Giemsa staining, and spindles /chromosomes with fluorescently labelled anti-alpha-tubulin antibody combined with a DNA fluorochrome. 40% of the oocytes had reached metaphase II (MII) after 24 h. Of the MII oocytes selected for karyotyping, readable chromosomal spreads were obtained from 64%. Overall, 63% of these presented a normal haploid chromosome number of 23,X, with all abnormal karyotypes occurring in the oocytes from small follicles. For another group of MII oocytes, where meiotic spindles were visualized, only half of the MII oocytes displayed well-formed spindles and apparently correct chromosomal alignment. This work provides the first information on the normal and aneuploid MII meiotic chromosome sets for the marmoset oocyte, and demonstrates a high rate of chromosomal and spindle abnormality among rapidly maturing oocytes from small antral follicles.

Do morphological anomalies reflect chromosomal aneuploidies?*: Case Report

In cases of severe teratozoospermia, the current morphological criteria used to assess chromosomal status is insufficient for the selection of spermatozoa for intracytoplasmic sperm injection (ICSI). Case histories are reported of four patients presenting 100% teratozoospermia, and the integrity of their individual chromosomal statuses is determined using a three-colour fluorescence in-situ hybridization (FISH) technique. Patient 1 presented shortened flagella syndrome, patient 2 globozoospermia, patient 3 spermatozoa with irregular acrosomes, and patient 4 macrocephalic spermatozoa with associated multiple flagella. Three-colour FISH analysis using chromosome X, Y and 1-specific probes showed that approximately 95% of the spermatozoa analysed from patients 1, 2 and 3 presented X,1 and Y,1 signals, X,Y ratios and aneuploidy/diploidy rates comparable with those observed in normal controls. In contrast, patient 4 showed a highly elevated Y to X sex ratio and a highly elevated aneuploidy/diploidy rate. Three-colour FISH analysis thus demonstrates an increased incidence of chromosomal abnormalities in association with macrocephalic spermatozoa. Moreover, the analysis shows that in patients affected with either globozoospermia, shortened flagella syndrome or a condition of abnormal acrosomal spermatozoa, no association exists between chromosomal status and phenotype. Since these patients display normal haploid, sex chromosome and aneuploidy status, ICSI can be conceivably offered as a treatment for their infertility.

Chromosomal analysis of multipronuclear zygotes obtained after partial zona dissection of the oocytes.

We have attempted to analyse the chromosome constitution of multipronuclear 1-cell zygotes obtained after partial zona dissection (PZD) of the oocytes. Six cells with three pronuclei could not be evaluated whereas another one was characterized by the presence of a normal haploid and two uninterpretable metaphases. Complete karyotypes were established for 21 tripronuclear cells, taking the varying arrangement of the chromosome sets into consideration. Of the zygotes, 10 showed three separated haploid metaphases (distribution pattern n/n/n), eight zygotes had one haploid and one diploid chromosome set (n/2n) and in three cells the individual sets were not distinguishable (3n). The sex chromosome ratio XXX:XXY:XYY was 7:9:5. Chromosome abnormalities were found in eight of the completely or partially analysable tripronuclear zygotes (36.4%) and included numerical (4 cells), structural (2 cells) as well as combinations of numerical and structural alterations (2 cells). Three out of 11 zygotes with four pronuclei could not be evaluated at all. In three cases, only two chromosome sets were analysable and another cell displayed one uninterpretable set. Three out of eight completely or partially analysable zygotes with four pronuclei (37.5%) had chromosomal abnormalities. Excluding the four cells with one or two uninterpretable metaphases, the sex chromosome distribution XXXX:XXXY:XXYY:XYYY in the zygotes with four pronuclei was 0:1:1:2. Compared with previously analysed multipronuclear zygotes obtained after conventional in-vitro fertilization (IVF), the rate of aberrant zygotes as well as the incidence of aberrant (male + female) chromosome sets were not significantly changed after PZD.

Postmeiotic restitution in 2n-egg formation of diploid potato

Four diploid (2n=2x=24) interspecific F1 hybrids of tuberous Solanum species were tested for the modes of origin of 2n-eggs. The four hybrids were heterozygous for the genetic marker amylose-free starch (Amf/amf) on chromosome 8. By crossing these hybrids with tetraploid S. tuberosum parents (2n=4x=48) that were nulliplex for this marker, i.e. Amf/amf×amf/amf/amf/amf crosses, tetraploid progenies were generated and classified for starch phenotypes of microspores. Based on the segregation of the amf marker gene, the tetraploid progenies were classified into nulliplex, simplex and duplex genotypes. In the progenies of three F1 hybrids, the simplex genotypes predominated and the origin of 2n-eggs could be explained as the result of second division restitution (SDR). But in the progeny of one F1 hybrid (a S. microdontum×S. tuberosum hybrid), there were only nulliplex and duplex genotypes, indicating complete homozygosity of the 2n-eggs (Amf/Amf or amf/amf) for this locus. In order to genotype the 2n-eggs also for other loci on the same chromosome, a tetraploid progeny was generated from a cross between this hybrid and a tetraploid S. tuberosum parent (Tetra 4) and analysed for four RFLP loci on chromosome 8. This analysis showed that all 2n-eggs of the S. microdontum×S. tuberosum hybrid were homozygous for all four loci, as was observed for the amf locus. From the same analysis it was evident that crossovers had occurred between the two genomes of this F1 hybrid. These homozygous and recombinant genotypes indicated that the 2n-eggs had originated from the doubling of the chromosome number in the normal haploid products of meiosis. Following the terminology of first and second division restitution (FDR and SDR), this new mode of origin of 2n-eggs in diploid potato is called post-meiotic restitution (PMR).

Zytogenetische Analyse von Zygoten mit drei Vorkernen nach partieller Zona dissection (PZD) der Eizellen

Objective : Tripronuclear zygotes obtained after partial zona dissection (PZD) of the oocytes were analysed cytogenetically to determine the rate of chromosomal abnormalities and to allow a comparison with previously published data after conventional IVF. Material and Methods: ollowing incubation in hypotonic solution preparations were made from a total of 28 cells. Chromosomes were stained homogeneously with iemsa. Results: Six cells could not be evaluated whereas another one was characterised by the presence of a normal haploid and two non-interpretable metaphases. Complete karyotypes were established for 21 cells, taking the varying arrangement of the chromosome sets into consideration. Ten zygotes showed three separated haploid metaphases (distribution pattern n/n/n), eight zygotes had one haploid and one diploid chromosome set (n/ 2n) and in three cells the individual sets were not distinguishable (3n). The sex chromosome ratio XXX:XXY:XYY was 7:9:5. Cytogenetic abnormalities were found in eight zygotes including four with numerical and two with structural chromosome alterations. In two cells, aneuploidy and structural aberrations appeared simultaneously. Compared with previously analysed tripronuclear zygotes obtained after conventional IVF, the rate of aberrant zygotes (38.1% vs. 20%) as well as the incidence of aberrant (male + female) chromosome sets (14.1 % vs. 6.7%) were increased after PZD. However, these differences were not significant (probability of error=5%). Conclusion: The transmission of the abnormality could be ascribed to spermatozoa in two cases. The results indicate the need for further investigation of a possibly increased development of chromosomally aberrant zygotes after microinsemination. In addition, more information on the parental origin of the observed aberrations will be necessary.

Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes.

Chromosomal aberrations are the major cause of pre- and post-implantation embryo wastage and some studies suggest that half of all human conception have a chromosomal abnormality. Analysis of gametes provides information on the origin of these chromosomal aberrations. The purpose of this study was to develop a reliable multi-probe fluorescence in-situ hybridization (FISH) procedure that would enable us to investigate aneuploidy in unfertilized oocytes subjected to intracytoplasmic sperm injection (ICSI). Oocytes were spread with HCl and Tween 20 solution, and then two rounds of triple-probe FISH were performed on each oocyte using directly-labelled centromeric probes: chromosomes 1, 7, 15 (overnight hybridization); chromosomes 1, X, Y (2 h hybridization). After the first round, the slides were counterstained and evaluated, and the positions of FISH signals were recorded. For the second round, the counterstain was removed and the second probe cocktail was applied. The chromosome 1 probe was an internal control for the two hybridization procedures, while the Y chromosome probe was used to detect sperm DNA. To evaluate the method, a total of 79 oocytes from 27 patients were studied. Of these, 67 (84.8%) were successfully spread and 97% of these oocytes exhibited discernible FISH signals. Upon lysis, oocytes exhibited one or more DNA fragments (mean 1.9, range 1-3). Of the 65 analysable oocytes, 17 (26.2%) displayed a normal haploid chromosome constitution with paired spots for the two chromatids. A further 23 oocytes (35.4%) showed an ambiguous chromosome complement due to an abnormal number of DNA fragments which may have resulted from loss of DNA during spreading or to an abnormal oocyte, while 25 oocytes (38.4%) displayed aneuploidy for one or more of the chromosomes studied. In conclusion, this new approach is a quick and efficient method with which numerical chromosomal abnormalities in human oocytes can be studied; interpretation of the patterns of DNA fragments and FISH signals requires further clarification.

Cytology of 2N pollen formation and pollen morphology in diploid lotus tenuis (fabaceae)

Two genotypes of Lotus tenuis Waldst & Kit. ex Willd. PI 204882, a diploid (2n = 2x = 12), were identified as producing 2n pollen (maximum = 6%). The objectives of this research were: (1) to determine the mechanism(s) of 2n pollen formation in the L. tenuis genotypes and (2) to morphologically describe n and 2n pollen using light and scanning electron microscopy. Meiotic studies revealed that 2n pollen resulted from tripolar spindles during anaphase II of microsporogenesis. The 2n pollen germinated well, although abnormal pollen tubes were observed. The genetic constitution of 2n pollen resulting from tripolar spindles is equivalent to first division restitution (FDR) of meiosis. Fresh and air‐dried pollen samples exhibited differences in size and shape under light and scanning electron microscopy. The size of diploid (2n) pollen was larger than that of haploid (1n) pollen. Normal haploid (1n) pollen was globose‐prolate in shape, while diploid (2n) pollen was tetrahedral in shape.

Analysis of the chromosome complement of frozen‐thawed mouse oocytes after parthenogenetic activation

Frozen-thawed mouse oocytes were artificially activated with Sr2+ and analyzed cytogenetically at the first cleavage division to examine the behavior of the maternal chromosomes independently of the paternal complement. There was no significant difference in the rate of activation between frozen-thawed and freshly collected oocytes and the majority of oocytes (> 90%) had a normal haploid chromosome constitution. The incidence of second polar body retention in frozen-thawed oocytes was low and did not differ significantly from that observed in fresh oocytes and oocytes exposed to dimethylsulfoxide (DMSO) at 0 degree C or 37 degrees C for extended periods beyond those required for protection. The frequency of aneuploidy was similar for frozen-thawed and fresh oocytes but oocytes held at 0 degree C without DMSO or held at 37 degrees C with DMSO for 1 hr showed a 2.5 and 12-fold increase in the frequency of aneuploidy compared with oocytes subjected to a conventional oocyte/embryo freezing regime. It is concluded that the procedures used in successful oocyte cryopreservation do not increase the incidence of chromosomal abnormalities of maternal origin in the resulting embryos.

Chromosome bisection in the yeast Saccharomyces cerevisiae facilitated by yeast artificial chromosomes bearing a site-specific recombination system.

A chromosome bisection method was constructed using yeast artificial chromosomes (YAC) and a GAL1-promoted site-specific recombination system. This method was applied to bisect chromosome IV into the left and right parts of the centromere region. The bisection occurred at frequencies of about 10% when the recombination site DNAs were integrated onto YAC and chromosome IV in the same direction, but were less than 10(-3) when they were in opposite directions. Reconstruction of the original chromosome IV from the bisected chromosomes was also induced by galactose at high frequencies. Loss of the left part chromosome was found at the frequencies of 0.9 x 10(-3) after hybrid cells between the chromosome-bisected strain and a normal haploid were subcultivated in a complete medium. The bisection and reconstruction of chromosome IV and deletion of the left part chromosome were demonstrated by electrophoretical karyotyping.

Backcross (BC2‐BC4)‐Derived Fertile Plants from Glycine max and G. tomentella Intersubgeneric Hybrids

Wild perennial relatives of the soybean [Glycine max (L.) Merr.] have not been utilized to broaden the genetic base of the crop. This study provides information on production, morphology, cytology, and breeding behavior of backcross (BC2 to BC4)‐derived fertile plants from soybean (2n = 40, genome GG) and G. tomentella Hayata (2n = 78, genome DDEE). The main hurdle was to obtain BC~ plants (G. max cv. Altona) × (G. tomentella, Pl 483218)] → F1, 2n = 59, genome GDE → colchicine treatment (CT) → 2n = 118, GGDDEE × soybean cv. Clark 63 → BC1, 2,n = 76 (expected 2n = 79). Three sterile BC2 plants (2n = 58, 2n = 56, 2n = 55) were produced. Procedures for immature seed‐rescue were utilized to obtain F, amphiploid, BC1 and BC2 plants. Soybean cv. Clark 63 was used as the recurrent parent in the backcrossing programs. The range of chromosome number among BC3 plants was 2n = 41 to 2n = 52, and among BC4 plants was 2n = 40 to 64. A hypertriploid (2n = 64) in BC4 plant originated after fertilization of an unreduced egg (n = 44) by a normal haploid (n = 20) male gamete. Phenotypes of BC3 and BC4 plants were dissimilar but generally resembled closely to the soybean cv. Clark 63. We expect to isolate a series of aneuploid lines from BC3 to BC6 generations that may open up the path to introgress desired traits from wild perennial Glycine species to the cultivated soybean.

Cytogenetics of unfertilized or uncleaved oocytes within the scope of in vitro fertilization. Relations to hormone content of follicular fluid

Constituents of the follicular fluid (FF) have been suspected to influence fertilizability and cytogenetic constitution of human oocytes. Therefore, we analysed the FF concentrations of 17 beta-estradiol (E2), progesterone (P), testosterone (T), and prolactin (PRL) of 114 oocytes recovered for in vitro fertilisation (IVF). 46 of these oocytes were fertilised and transferred to the maternal uterus. Among the unfertilized gametes, 27 were not analysable. 30 were normal haploid, and 11 were classified as abnormal. There was no significant difference between fertilised and unfertilized oocytes for FF concentrations of E2, P, T, and PRL and for the E2/P ratios. Similarly, we detected no significant difference between normal and abnormal oocytes for these parameters.

Is there any correlation between follicular fluid hormone concentrations, fertilizability, and cytogenetic analysis of human oocytes recovered for in vitro fertilization?

Constituents of FF have been suspected to influence fertilizability and cytogenetic constitution of human oocytes. We therefore analyzed the FF concentrations of E2, P, T, and PRL for 114 oocytes recovered for IVF. Forty-six of these oocytes were fertilized and transferred to the maternal uterus. Among the unfertilized gametes, 27 were not analyzable, 30 were normal haploid, and 11 were classified as abnormal. There was no significant difference between fertilized and unfertilized oocytes for FF concentrations of E2, P, T, and PRL and for the E2:P ratios. Similarly, we detected no significant difference between normal and abnormal oocytes for these parameters.