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Cadmium (Cd), an environmental heavy metal pollutant, is one of the high risk factors for human diseases due to the exponential increase of its use in industrial processes and products during the last 50 years. It deposits mainly in the liver and leads to various diseases including cancer. Currently, there is a growing attention to explore natural compounds for the prevention of unfavorable effects of Cd in humans.

Cadmium (Cd), an economically valuable metal, is widely used in various industrial processes. Although it is of economic value, it is hazardous to human health. Cd accumulates in vital organs where it causes various diseases. Natural compounds with chelating or antioxidant properties have been tested to reduce the toxic effect of Cd. The anti-oxidant, anti-diabetic and hypocholesterolemic properties of fenugreek (Trigonella foenum-graecum) leaves make it a candidate for investigation as protective agent against Cd-induced toxicity. In the present study, the protective effects of fenugreek leaf extract (FLE) on cell viability, morphology, and whole genomic transcription in cadmium chloride (CdCl2)-treated rat liver cells were analyzed. The cells were treated with 25 µM CdCl2 alone, or co-treated with 5 µg/ml FLE for 48 h. The co-treated cells were pretreated with FLE for 2 or 4 h, followed by CdCl2 treatment. Genomic transcription analysis was performed in the CdCl2-treated cells following treatment for 6 h. The CdCl2 caused a significant decrease in viability (35.8±4.1%) and morphological distortion of the cells, compared with the untreated control cells; whereas 4 h pretreatment with FLE (5 µg/ml) reversed the Cd-induced morphology alteration and increased the cell viability to 102±3.8%. Genomic transcription analysis of the CdCl2 only-treated cells showed 61 upregulated and 124 downregulated genes, compared with 180 upregulated and 162 downregulated genes in the FLE pretreated cells. Furthermore, 37 and 26% of the affected total genomic genes in the CdCl2 only-treated cells were involved in binding and catalytic activities, respectively, whereas 50 and 20% of the genes in the FLE pretreated cells were involved in binding and catalytic activities, respectively. In conclusion, these results suggested that genome transcriptome modulation may be important in the protective effect of FLE against Cd-induced toxicity in normal rat liver cells.

Variation in genome structure is an important source of human genetic polymorphism: It affects a large proportion of the genome and has a variety of phenotypic consequences relevant to health and disease. In spite of this, human genome structure variation is incompletely characterized due to a lack of approaches for discovering a broad range of structural variants in a global, comprehensive fashion. We addressed this gap with Optical Mapping, a high-throughput, high-resolution single-molecule system for studying genome structure. We used Optical Mapping to create genome-wide restriction maps of a complete hydatidiform mole and three lymphoblast-derived cell lines, and we validated the approach by demonstrating a strong concordance with existing methods. We also describe thousands of new variants with sizes ranging from kb to Mb.

Androgens play key roles in spermatogenesis, and they exert their effect via the androgen receptor (AR). The AR gene has a repetitive DNA sequence in exon 1 that encodes a polyglutamine tract. Instability in the glutamine (CAG) repeat unit length is polymorphic across ethnic groups. Previous studies of the relationship between the repeat unit length and male infertility have been contradictory. To establish the range of wild-type alleles in Egyptian men, we determined the range of repeat lengths in a population of normally fertile, ethnically selected Egyptian men. We also investigated the association between trinucleotide repeat length within the AR gene and male factor infertility in a population of ethnically selected Egyptian infertile men, who were compared with fertile, ethnic group-matched and age-matched controls. The study included 129 clinically selected infertile Egyptian men who were scheduled for intracytoplasmic sperm injection and 52 ethnically matched fertile controls. The experimental population was grouped according to sperm counts ranging from nonobstructive azoospermia to normozoospermia. The CAG repeat N-terminal domain region of the AR gene was amplified in peripheral blood DNA, and allele size was determined by fragment analysis. Allele size and single-nucleotide polymorphism and mutation rates were determined by sequencing individual amplified alleles. The mean CAG repeat length in the azoospermia group was 18.55 +/- 2.0; in the severe oligozoospermia group it was 18.21 +/- 3.42; in the oligozoospermia group it was 18.27 +/- 2.93; and in the infertile with normal sperm count group it was 17.72 +/- 2.0. In the control group, the mean CAG repeat length was 18.18 +/- 3.63. No significant correlation was found between CAG repeat length and the risk of male factor infertility in an ethnically defined population of Egyptian men. However, a significant and positive correlation between CAG repeat length and serum testosterone concentration was demonstrated. This suggests the involvement of epigenetic regulation linked to this region.

Microdeletions of the long arm of the Y chromosome involving the azoospermia factor (AZF) region are associated with severe oligo- or azoospermia. Abnormal androgen receptor (AR) structure or function has also been implicated in male infertility. To assess the contribution of these genetic defects to male infertility, 61 Israeli men with severe oligo- (n = 15) or azoospermia (n = 46), were screened for Y chromosome microdeletions, and the AR-(CAG)n repeat length. Fifty fertile Israeli men were similarly analyzed. PCR amplification of 20-54 simple tag sequences (STSs) located at Yq was used to determine the rate and extent of Y chromosome microdeletions. PCR with primers flanking the AR-(CAG)n region and subsequent size fractionation on gradient acrylamide gels were used to determine AR-(CAG)n length. Five azoospermic individuals (5/61-8.2% and 5/46-10.8% of azoospermic patients) displayed Y chromosome microdeletions. The mean CAG repeat number in infertile men was 18.6 +/- 3.0 compared with 16.6 + 2.7 in fertile men (n = 50), a statistically significant difference (p = 0.003). Y chromosome microdeletions contribute to male infertility in our azoospermic population, and the mean length of the AR-CAG is significantly longer in our infertile population than in fertile men.

This chapter focuses on the history of cloning of cattle, the comparative methodology, successes, and failures, possible causes of failures with a particular emphasis on imprint maintenance and epigenetics, and applications of cloning of cattle.

The Y chromosome contains genes and gene families that play critical roles in the processes of testis determination and testis differentiation. Great strides have been made toward defining the genetic pathways associated with the determination of gender. The data are summarized and discussed and clinical ramifications are considered.

Cytogenetic and molecular deletion analyses of azoospermic and oligozoospermic males have suggested the existence of AZoospermia Factor(s) (AZF) residing in deletion intervals 5 and 6 of the human Y-chromosome and coinciding with three functional regions associated with spermatogenic failure. Nonpolymorphic microdeletions in AZF are associated with a broad spectrum of testicular phenotypes. Unfortunately, Sequence Tagged Sites (STSs) employed in screening protocols range broadly in number and mapsite and may be polymorphic. To thoroughly analyze the AZF region(s) and any correlations that may be drawn between genotype and phenotype, we describe the design of nine multiplex PCR reactions derived from analysis of 136 loci. Each multiplex contains 4-8 STS primer pairs, amplifying a total of 48 Y-linked STSs. Each multiplex consists of one positive control: either SMCX or MIC2. We screened four populations of males with these STSs. Population I consisted of 278 patients diagnosed as having significant male factor infertility: either azoospermia, severe oligozoospermia associated with hypogonadism and spermatogenic arrest or normal sperm counts associated with abnormal sperm morphology. Population II consisted of 200 unselected infertile patients. Population III consisted of 36 patients who had previously been shown to have aneuploidy, cytological deletions or translocations involving the Y-chromosome or normal karyotypes associated with severe phenotype abnormalities. Population IV consisted of 920 fertile (control) males. The deletion rates in populations I, II and III were 20.5%, 7% and 58.3%, respectively. A total of 92 patients with deletions were detected. The deletion rate in population IV was 0.87% involving 8 fertile individuals and 4 STSs which were avoided in multiplex panel construction. The ability of the nine multiplexes to detect pathology associated microdeletions is equal to or greater than screening protocols used in other studies. Furthermore, the data suggest a fourth AZF region between AZFb and AZFc, which we have termed AZFd. Patients with microdeletions restricted to AZFd may present with mild oligozoospermia or even normal sperm counts associated with abnormal sperm morphology. Though a definitive genotype/phenotype correlation does not exist, large deletions spanning multiple AZF regions or microdeletions restricted to AZFa usually result in patients with Sertoli Cell Only (SCO) or severe oligozoospermia, whereas microdeletions restricted to AZFb or AZFc can result in patients with phenotypes which range from SCO to moderate oligozoospermia. The panel of nine multiplexed reactions, the Y-deletion Detection System (YDDS), provides a fast, efficient and accurate method of assessing the integrity of the Y-chromosome. To date, this study provides the most extensive screening of a proven fertile male population in tandem with 514 infertile males, derived from three different patient selection protocols.

Background Some infertile men with azoospermia or severe oligospermia have small deletions in regions of the Y chromosome. However, the frequency of such microdeletions among men with infertility in general is unknown. We sought to determine the prevalence of Y-chromosome microdeletions among infertile men and to correlate the clinical presentation of the men with specific deletions. Methods We studied 200 consecutive infertile men. Each man was evaluated comprehensively for known causes of infertility, and Y-chromosome microdeletions were studied with use of the polymerase chain reaction to amplify specific regions of the chromosome. The Y chromosomes of 200 normal men were also analyzed. Results Fourteen infertile men (7 percent) and four normal men (2 percent) had microdeletions of the Y chromosome. Nine of the infertile men had azoospermia or severe oligospermia (sperm concentration

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos. Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF). Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days. The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state. Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this regard, is the analysis of gene expression patterns in nuclear transfer (NT) embryos to dissect the processes that failed and develop means to overcome the limitations imposed by these factors. In this study, we investigated expression patterns of histone deacetylase-1, -2, -3 (HDAC-1, -2, -3), DNA methyltransferase-3a (DNMT3A), and octamer binding protein-4 gene (OCT4) in donor cells with different cloning efficiencies and NT embryos derived from these cells employing a real-time RT-PCR assay. All genes investigated followed altered expression patterns in NT embryos when compared to IVF-derived embryos. In general, expression of HDAC genes was elevated especially at the compact morula stage and comparable to in vitro fertilized (IVF) embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than IVF embryos at all stages. Oct-4 transcript levels were also reduced in cloned compared to IVF embryos at the compact morula and blastocyst stages. This difference disappeared at the hatched blastocyst stage. There was a donor cell effect on the expression patterns of all genes investigated. These results demonstrate altered gene expression patterns for certain genes, in cloned cattle embryos from our donor cells of different efficiency in producing live offspring. Therefore we suggest that differences in expression of developmentally important genes during early embryo development may characterize the efficiency of donor cells in producing live offspring.

Accurate methods for measuring the biological effects of radiation are critical for estimating an individual's health risk from radiation exposure. We investigated the feasibility of using radiation-induced mutations in repetitive DNA sequences to measure genetic damage caused by radiation exposure. Most repetitive sequences are in non-coding regions of the genome and alterations in these loci are usually not deleterious. Thus, mutations in non-coding repetitive sequences might accumulate, providing a stable molecular record of DNA damage caused by all past exposures. To test this hypothesis, we screened repetitive DNA sequences to identify the loci most sensitive to radiation-induced mutations and then investigated whether these mutations were stable in vivo over time and after multiple exposures. Microsatellite repeat markers were identified that exhibited a linear dose response up to 1 Gy of 1 GeV/nucleon 56Fe ions and 137Cs gamma rays in mouse and human cells. Short tandem repeats on the Y chromosome and mononucleotide repeats on autosomal chromosomes exhibited significant increases in mutations ator= 0.5 Gy of 56Fe ions with frequencies averaging 4.3-10.3 x 10(-3) mutations/locus/Gy/cell, high enough for direct detection of mutations in irradiated cells. A significant increase in radiation-induced mutations in extended mononucleotide repeats was detectible in vivo in mouse blood and cheek samples 10 and 26 weeks after radiation exposure and these mutations were additive over multiple exposures. This study demonstrates the feasibility of a novel method for biodosimetry that is applicable to humans and other species. This new approach should complement existing methods of biodosimetry and might be useful for measuring radiation exposure in circumstances that are not amenable to current methods.

Tumors lacking DNA mismatch repair activity (MMR) from patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or those with sporadic colorectal cancer can be identified by the presence of high levels of instability in repetitive sequences known as microsatellites (MSI). The assessment of MSI phenotype in human tumors helps to establish a clinical diagnosis and is accomplished with a reference panel of five mononucleotide repeats. By contrast, detection of MSI in mouse tumors has proven to be problematic and lack of a uniform set of markers for classification of MSI has impeded comparison of results between studies. We tested for MSI in intestinal tumors from MMR-deficient mice with four mononucleotide repeats with polyA(24-37) tracts and three new markers with extended polyA(59-67) tracts. All seven markers were sensitive to MSI in MMR-deficient tumors, but those with extended mononucleotide tracts displayed larger deletions, which were easily distinguishable from the germline alleles. With a panel of the five most sensitive and specific mononucleotide repeats, a high level of MSI was detected in 100% of MMR-deficient tumors, but not in tumors with MMR activity. This novel panel is an improvement over existing combinations of mono- and dinucleotide repeat markers and should facilitate MSI screening and standardize results from different studies.

The aetiology of compromised spermatogenesis is often genetic in nature. There are only a few reports of father/son cohorts that have been evaluated to assess heritability of mutations associated with male factor infertility and the psychological well-being of the children. In the present study, multiple tissues were sampled from consenting male patients and their sons derived from intracytoplasmic sperm injection (ICSI) and underwent chromosomal and genetic analyses. Paediatric and psychological examinations were also conducted. In 87 men and 47 boys, 22 sequence tagged sites (STS) were analysed by multiplex PCR and deletion breakpoints were defined with additional loci. In one patient, the breakpoints map to the highly unstable palindrome-rich region within AZFb and proximal AZFc was investigated. A total of 86 blood, 26 semen, and 73 cheek cells samples were collected from adults, and 36 blood samples and 44 cheek cell specimens were obtained from the boys. Though all of the fathers had normal karyotypes, the incidence of chromosomal abnormalities in the somatic cells of male progeny was 8.3% (3/36). The incidence of germ line aneuploidy in these men was 0.5-2.8%. A CF mutation (Delta508) was detected in one of 87 men (1.2%) and microdeletions in Yq AZF were detected in 3.4% of 87 adults and in 2.1% of their sons (n = 47). In conclusion, screening for Y chromosome microdeletions provides crucial information in the counselling of couples seeking infertility treatment. Moreover, DNA extraction and Y deletion assessments of cheek cells provide a non-invasive approach. Inheritance of Yq deletions appears not to affect the psychological and physical development of children derived from ICSI.

With the progress of the human gene mapping initiative, it is expected that the entire genome will be mapped within two years. A significant use for these data will centre on testing for genetic disease. Professionals associated with assisted reproduction are presented with a very special subset of the population, namely, couples suffering from infertility. Infertility may occur in the male, the female or both partners and may be heritable. Infertility, subfertility or recurrent spontaneous miscarriage is associated with chromosomal or genetic anomalies, suggesting that basic developmental genetics should be a part of the education of the physician or clinical embryologist. A review of the most common infertility-associated chromosomal and genetic diseases for which genetic testing has become routine in infertile parents and in the products of assisted reproduction through preimplantation genetic diagnosis (PGD) and prenatal testing follows. Less common genetic diseases that have compromising effects on reproduction and which are likely to be encountered by providers of assisted reproduction are also considered.

Background Some infertile men with azoospermia or severe oligospermia have small deletions in regions of the Y chromosome. However, the frequency of such microdeletions among men with infertility in general is unknown. We sought to determine the prevalence of Y-chromosome microdeletions among infertile men and to correlate the clinical presentation of the men with specific deletions. Methods We studied 200 consecutive infertile men. Each man was evaluated comprehensively for known causes of infertility, and Y-chromosome microdeletions were studied with use of the polymerase chain reaction to amplify specific regions of the chromosome. The Y chromosomes of 200 normal men were also analyzed. Results Fourteen infertile men (7 percent) and four normal men (2 percent) had microdeletions of the Y chromosome. Nine of the infertile men had azoospermia or severe oligospermia (sperm concentration

El compromiso espermático severo en el hombre infértil, puede ser el resultado de la presencia de deleciones en el cromosoma Y en la región AZFc (Factor de azoospermia región c). En este estudio se caracterizaron 97 hombres con infertilidad con una frecuencia del 6% para la presencia de deleciones en la población estudiada. De estos hombres positivos para deleción en la región Yq (AZFc), nacieron tres niños mediante el uso de inyección intracitoplasmática (ICSI) que heredaron la mutación en el cromosoma Y, fundamentando la importancia de crear un programa de tamizaje para detectar la presencia de deleción en este cromosoma en la población de hombres infértiles candidatos para técnica de fertilización asistida (ART) mediante ICSI. Palabras claves: hombres infértiles, delección, Zona Yq, fertilización asistida, inyección intracitoplasmática de espermatozoides.

Microdeletions linked to deletion intervals 5 and 6 of the Y chromosome have been associated with male factor infertility. Members from at least two gene families lie in the region containing azoospermia factor (AZF), namely YRRM and DAZ. With the advent of intracytoplasmic sperm injection (ICSI), it is possible for men with severe male factor infertility to produce a child. The genetic consequences of such a procedure have been questioned. This report describes the first study of a population (32 couples) of infertile fathers and their sons born after ICSI. The objectives were firstly to determine the incidence and map location of Y chromosome microdeletions and to compare the frequencies with other population studies involving severe male factor infertility, and secondly to formulate a working hypothesis concerning developmental aetiology of Y chromosome microdeletions. The incidence of microdeletions in the ICSI population was shown to be 9.4% (within the range 9-18% reported for populations of severe male factor infertility patients). Microdeletions in two out of three affected father/son pairs mapped in the region between AZFb and AZFc and the third involved a large microdeletion in AZFb and AZFc. Of three affected father/son pairs, microdeletions were detected in the blood of one infertile propositus father and three babies. Assuming that the gonomes of the ICSI-derived babies are direct reflections of those of their fathers germ lines, it is possible that two of three infertile fathers were mosaic for intact Y and microdeleted Y chromosomes. In such cases, the developmental aetiology of the microdeletion may be due to a de-novo microdeletion arising as a post-zygotic mitotic error in the infertile propositus father, thus producing a mosaic individual who may or may not transmit the deletion to his ICSI-derived sons depending on the extent of primordial germ cell mosaicism. In one of three affected fathers, the microdeletion detected in his blood was also detected in his ICSI-derived son. In this case the de-novo event giving rise to the microdeletion may have occurred due to a post- (or pre-) meiotic error in the germ line of this father's normally fertile father (i.e. the ICSI-derived baby's grandfather).