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In vivo studies on folliculogenesis have documented a relation among intrafollicular steroid content, follicle growth, and oocyte development. This study examined how profound changes in androgen/estrogen ratio would affect mouse in vitro follicular development. Arimidex, a potent follicular aromatase inhibitor was used for this purpose. Early preantral follicles were cultured for 12 days up to the preovulatory stage. Oocyte's meiotic maturation, spindle and chromosome configurations, in vitro fertilization and preimplantation embryo development were evaluated. Compared to controls, Arimidex reduced E2 concentration in follicle culture medium by a factor 1000, and an expected simultaneous accumulation of testosterone was measured in the conditioned medium. Arimidex treatment provoked a dose-dependent earlier differentiation of the granulosa cells as judged by an earlier antrallike cavity formation and slightly elevated basal progesterone secretion. Follicle survival exceeded 98% in all groups and all follicles responded normally to HCG/EGF addition on day 12 by cumulus mucification. By the HCG ovulatory challenge, progesterone output was reduced in Arimidex supplemented groups suggesting preovulatory luteinization. These results indicate that in vitro mouse follicles can develop normally under very low levels of estrogens and that a local androgen increase by a factor 3 is not atretogenic. Oocyte growth did not differ among culture conditions. Arimidex treatment induced a dose dependent enhancement of GVBD and polar body formation rate in response to HCG at the end of culture. Spindle and chromosome analyses demonstrated that in all groups, 90% of the oocytes which extruded a polar body had also reached the MII stage. While most of the cultured MII oocytes had a normal spindle and well aligned chromosomes, significantly less oocytes were fertilized in the groups cultured in the presence of Arimidex. Once fertilized, however, there was found to be no difference for preimplantation embryo development between controls and Arimidex treatment. These data suggest that in mice a pronounced estrogenic environment is not essential for in vitro folliculogenesis. Drastic changes in the intrafollicular steroid concentrations do not disrupt meiotic maturation nor compromise early preimplantation development, but adversely affect fertilization of in vitro grown oocytes.
Mol Reprod Dev 2002 Apr
PMID:Effects of aromatase inhibition on in vitro follicle and oocyte development analyzed by early preantral mouse follicle culture. 1189 27

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of treatment with fertility drugs. Using human lung microvascular endothelial cells (HUMEC-L) as an in-vitro model of OHSS, we have tested the hypothesis that the endothelium is a target of HCG in the pathogenesis of OHSS. Since OHSS is characterized by increased capillary permeability, we have investigated the production and action of vasoactive agents. When HUMEC-L were cultured with high doses of estradiol (E(2)), no significant changes were observed in the secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-6 or IL-1 beta. However, the addition of HCG resulted in a significant increase in the secretion of VEGF and IL-6. Time-course experiments showed that VEGF was secreted within minutes of HCG addition, whereas IL-6 was significantly increased only after 48 h in culture. The secretion of IL-1 beta was unchanged by these hormonal conditions. The presence of HCG receptors was demonstrated in HUMEC-L in basal conditions as well as after the addition of E(2). The expression of VEGF receptors was also investigated. High doses of E(2) were unable to increase the expression of KDR, flt-1 and sfl-t, but the addition of HCG significantly upregulated the KDR concentration in endothelial cells, while no change was observed for flt. Permeability assays demonstrated that while E(2) alone did not change the arrangement of HUMEC-L in vitro, the presence of HCG caused changes in the actin fibres corresponding to increased capillary permeability. Anti-human VEGF antibodies were able to overcome these changes. In conclusion, these experiments show that the endothelium may be a primary target of HCG, causing an acute release of VEGF and a significant increase in IL-6 and resulting in an autocrine-paracrine action that may increase vascular permeability.
Mol Hum Reprod 2002 May
PMID:The role of endothelial cells in the pathogenesis of ovarian hyperstimulation syndrome. 1199 37

This chapter presents the summary of two molecular cytogenetic techniques--FISH and CGH--with their applications and limitations in the studies of pregnancy loss. These molecular techniques clearly represent a significant advantage over the traditional cytogenetic technique and likely will become the predominant cytogenetic techniques in reproductive cytogenetics of the future.
Methods Mol Biol 2002
PMID:Molecular cytogenetics in reproductive pathology. 1239 6

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.
Mol Reprod Dev 2003 Feb
PMID:Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine. 1250 54

In making a selection of features of these technologies, it is inevitable that some will be omitted that other cytogeneticists feel should have been included. The author could probably justifiably be accused of bias. However, based on experience in a laboratory that has used almost every type of assay mentioned in this chapter, the following opinions are offered about their current value in providing a routine malignancy cytogenetics service: 1. The foundation is still a conventional cytogenetic study, preferably with the use of an automated karyotyping system. 2. Added to this, there should be the capability of performing FISH studies using chromosome paints and gene-specific probes. Cytogenetics and FISH form a powerful partnership when backed by experienced cytogeneticists. MFISH or SKY are also useful if the laboratory can afford the considerable extra expense. CGH and fibre FISH are generally better suited to research projects, and at present have few applications in a routine diagnostics service. 3. At present, molecular methods such as RT-PCR mostly tend to produce results that have a greater need of confirmation by other techniques before they can be used for clinical management.
Methods Mol Biol 2003
PMID:Some difficult choices in cytogenetics. 1274 18

Array CGH combined with mRNA microarray analyses was successfully applied for genome-wide screening of proto-oncogenes and tumor suppressor genes in 2002. The CCND1-ORAOV1-FGF19-FGF4-FGF3-FLJ10261-FADD-PPFIA1-EMS1 locus on human chromosome 11q13 is one of the most frequently amplified regions within the human genome. Here, we identified and characterized mouse Ppfia1 gene by using bioinformatics. Nucleotide sequence of mouse Ppfia1 cDNA was determined in silico by assembling nucleotide sequences of ESTs BY727670, CA327608, BU708520, BQ886535, and a 5'-truncated partial cDNA BC038349. Mouse Ppfia1 gene, consisting of 28 exons, was located between Fadd and Ems1 (also known as Cttn) genes on mouse chromosome 7. Mouse Ppfia1 (1201 aa) and human PPFIA1 (1202 aa), showing 95.8% total-amino-acid identity, were found to consist of MAH (myosin heavy chain tail and ATPase homologous) domain and three SAM (sterile alpha motif) domains. MAH domain is implicated in the homo- or hetero-oligomer formation through the coiled-coil interaction, while SAM domain is implicated in the interaction with other proteins. Mouse Ccnd1-Ems1 locus and human CCND1-EMS1 locus were evolutionarily conserved in the order and the orientation of genes therein. Nucleotide and amino-acid substitution rates of Ccnd1, Ppfia1 and Ems1 genes located near both ends of the Ccnd1-Ems1 locus were relatively lower than those in the middle part of the locus. This is the first report on mouse Ppfia1 gene as well as comprehensive comparison of CCND1-EMS1 locus within the human and mouse genomes.
Int J Mol Med 2003 Aug
PMID:Identification and characterization of mouse Ppfia1 gene in silico. 1285 29

Chromosomal abnormalities, such as deletions and duplications, are characterized by specific and often complex phenotypes resulting from an imbalance in normal gene dosage. However, routine chromosome banding is not sensitive enough to detect subtle chromosome aberrations (<5-10 Mb). Array-based comparative genomic hybridization (array CGH) is a powerful new technology capable of identifying chromosomal imbalance at a high resolution by co-hybridizing differentially labeled test and control DNAs to a microarray of genomic clones. We used a previously assembled contig of large-insert clones that span 10.5 Mb of the most distal region of 1p36 to design a microarray. The array includes 97 clones from 1p36, 41 clones from the subtelomeric regions of all human chromosomes, and three clones from each of the X and Y chromosomes. We used this microarray to study 25 subjects with well-characterized deletions of 1p36. All array CGH results agree with the deletion sizes and locations of the breakpoints in these subjects as determined previously by FISH and microsatellite analyses. Terminal deletions, interstitial deletions, derivative chromosomes and complex rearrangements were also identified. We anticipate that array CGH will change the diagnostic approach to many congenital and acquired genetic diseases such as mental retardation, birth defects and cancer.
Hum Mol Genet 2003 Sep 01
PMID:Development of a comparative genomic hybridization microarray and demonstration of its utility with 25 well-characterized 1p36 deletions. 1291 73

Dedifferentiated and differentiated liposarcoma are characterized by 12q15 chromosomal amplification. Comparative genomic hybridization is a powerful tool able to detect DNA copy number changes in the genome. This technique has been widely used in frozen tumors and in some studies in paraffin-embedded tumors fixed with formalin. The purpose of this study was to demonstrate the ability of CGH to detect DNA copy number changes in the genome when the DNA was extracted from tissues fixed with Holland Bouin's fluid. Sixteen liposarcoma tumors both frozen and fixed in Holland Bouin's fluid were characterized by CGH. Eighty-one percent of the main chromosomal alterations detected in the frozen liposarcomas (amp 12q15, amp 6q23, amp 1p32, amp 16q22, +7, +8) were detected in the corresponding fixed tumors. The limitation of this technique when using Holland Bouin's fluid extracted DNA compared with formalin-extracted DNA was the yield of analyzable samples. Eighty-one percent of tumors fixed with Holland Bouin's fluid (13/16) were analyzable compared with 100% of formalin-fixed tumors (4/4). This study demonstrates that comparative genomic hybridization is a useful tool even if only fixed tissues (formalin and Holland Bouin's fluid tissues) are available, and that it allows more tumors to be analyzed in retrospective studies.
Diagn Mol Pathol 2003 Sep
PMID:Comparative genomic hybridization study of paraffin-embedded dedifferentiated liposarcoma fixed with Holland Bouin's fluid. 1296 Jun 99

The nature of genetic alterations in bilateral breast cancer (BBC) associated with the distinctive development of a second primary tumor or a metastatic lesion is not clearly established. In this study, patterns of promoter methylation and gene copy number changes were assessed for their utility in the distinction of two types of BBC (synchronous and metachronous). Seven cases of synchronous and five cases of metachronous breast cancer tissues were used in X chromosome inactivation assay to assess the methylation pattern of human androgen receptor gene. X chromosome inactivation assay alone did not provide enough information to distinguish the genetic origins of synchronous and metachronous BBC. When four pairs of paraffin-embedded BBC tissues were used in cDNA array-based CGH with placenta DNA as a reference, higher DNA copy number changes were observed from metachronous pairs (9.0%) than from synchronous pairs (3.1%). From the two cases of metachronous pairs tested, 44 genes were found to be commonly modulated in gene copy numbers in a cancer detected later.
Int J Mol Med 2004 Jan
PMID:The pattern of gene copy number changes in bilateral breast cancer surveyed by cDNA microarray-based comparative genomic hybridization. 1465 65

Deletions and duplications of genomic segments commonly cause developmental disorders. The resolution and efficiency in diagnosing such gene-dosage alterations can be drastically increased using microarray-based comparative genomic hybridization (array-CGH). However, array-CGH currently relies on spotting genomic clones as targets, which confers severe limitations to the approach including resolution of analysis and reliable gene-dosage assessment of regions with high content of redundant sequences. To improve the methodology for analysis, we compared the use of genomic clones, repeat-free pools of amplified genomic DNA and cDNAs (single and pooled) as targets on the array. For this purpose, we chose q11.2 locus on chromosome 22 as a testing ground. Microdeletions at 22q11 cause birth defects collectively described as the DiGeorge/velocardiofacial syndrome. The majority of patients present 3 Mb typical deletions. Here, we report the construction of a gene-dosage array, covering 6 Mb of 22q11 and including the typically deleted region. We hybridized DNA from six DiGeorge syndrome patients to the array, and show that as little as 11.5 kb non-redundant, repeat-free PCR-generated sequence can be used for reliable detection of hemizygous deletions. By extrapolation, this would allow analysis of the genome with an average resolution of 25 kb. In the case of cDNAs our results indicate that 3.5 kb sequence is necessary for accurate identification of haploid/diploid dosage alterations. Thus, for regions rich in redundant sequences and repeats, such as 22q11, a specifically tailored array-CGH approach is good for gene copy number profiling.
Int J Mol Med 2004 Feb
PMID:DNA copy-number analysis of the 22q11 deletion-syndrome region using array-CGH with genomic and PCR-based targets. 1471 34


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