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NR5A1 or steroidogenic factor 1 (SF1) is an autosomal gene, which encodes a protein that is a member of nuclear receptor family. NR5A1 regulates the transcription of numerous genes that are expressed in hypothalamic-pituitary-gonadal axis and adrenal cortex which in turn, coordinate the gonadal development, steroidogenesis and sex differentiation. Several mutations in NR5A1 have been reported to cause gonadal dysgenesis with adrenal insufficiency in individuals with 46,XY karyotype. However, studies in the past few years have shown that NR5A1 mutations can also contribute to primary ovarian insufficiency and impaired spermatogenesis. As there is no genetic study on NR5A1 in Indian infertile men, we have sequenced the entire coding region (exons 2-7) of NR5A1 in 502 infertile men of which, 414 were non-obstructive azoospermic and 88 severe oligozoospermic, along with 427 ethnically matched fertile controls. Interestingly, none of the mutations reported to be associated with male infertility were found in our study, except one polymorphism, rs1110061. However, it was not significantly different between infertile and fertile groups (p = .76). In addition, we have identified six intronic variants; but none of them was significantly associated with male infertility.

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Calcium/calmodulin-dependent protein kinase IV (CAMK4) is a multifunctional serine/threonine protein kinase, which plays an important role in the spermatogenesis by phosphorylating protamines. It has been shown to be involved in the regulation of human sperm motility. Moreover, the Camk4 knockout mice were infertile because of severely reduced sperm count and morphological abnormalities. As no study is available on the association of this gene with male infertility, we analysed all the exons of CAMK4 gene in ethnically matched 283 infertile and 268 fertile Indian men. We identified twenty nucleotide substitutions, of which twelve were novel. Of these novel variants, eight were exclusively detected in infertile men. Moreover, two infertile men-specific mutations were non-synonymous replacing amino acids at the highly conserved region. In silico analysis predicted both of these mutations as 'deleterious'. In addition to nucleotide substitutions, we identified five novel insertion-deletion mutations; of these, g.150264_66delGCG was exclusively found in two oligoasthenoteratozoospermic men. In silico analysis of infertile men exclusive mutations predicted that they can alter/diminish the potential binding sites of splicing factors, which may affect the mRNA splicing and protein translation. Our study suggests that the mutations in CAMK4 may lead to abnormal semen parameters.

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Transition nuclear proteins (TNP1 and TNP2) are the major nuclear proteins that replace somatic histones during spermatogenesis. TNPs are required for the maintenance of normal spermatogenesis. Moreover, spermatogenesis was found to be compromised in both Tnp1 and Tnp2 null mice. As no study is available on the role of these genes in Indian infertile men, we have sequenced the entire TNP1 and TNP2 genes in 320 infertile men and 280 control fertile men drawn from two states in India. We identified 18 variants, including 8 previously known and 10 novel. Of the 10 novel variants, 3 were found only in azoospermic men, of which 2 (g.-688A>T in TNP1 and g.1030G>A in TNP2) were predicted to affect the transcription factor binding sites and therefore can cause deregulation of gene expression. Haplotype association analysis showed a significant omnibus association (omnibus χ(2) = 7.87, p = 0.0195) for the single nucleotide polymorphisms (SNPs) in the TNP1 gene with azoospermia. The frequency of the haplotype GCG (H3) was increased in azoospermic men (53.1%) compared with fertile men (43%; χ(2) = 7.964, p = 0.005). However, similar analysis of the TNP2 gene did not show any association with infertility. Furthermore, expression analysis of the TNP1 gene in obstructive azoospermic men showed that haplotypes of the TNP1 gene do not affect its expression level. Our results suggest that the individual SNPs of the TNP1 and TNP2 genes are not associated with infertility; however, the haplotype GCG of the TNP1 gene is a risk factor for azoospermia.

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Recent studies suggest that estrogens play an important role in male fertility. Estrogen signaling is mediated by Estrogen Receptors (ERalpha and ERbeta). Association of ERbeta with male infertility has not been analyzed to date except for genotyping of known polymorphisms in two different studies, which yielded controversial interpretation. Hence, we performed sequencing of all the exons and untranslated regions of ERbeta gene in 300 infertile and 255 fertile control Indian men. We identified eight novel mutations and four known single nucleotide polymorphisms (SNPs). Of the eight novel mutations, four were non-synonymous, of which one was detected only in infertile men, whereas the other three mutations were detected only in fertile men. Using different bioinformatics tools, we predicted that non-synonymous mutations were benign and they neither altered the structure nor the function of the protein. Among synonymous novel mutations, one was detected in both fertile and infertile men, two were exclusive to infertile men and one was exclusive to fertile men. None of the known SNPs or novel mutations showed statistically significant difference between infertile and fertile men. Moreover, infertile men having ERbeta mutations had normal reproductive tract and serum hormone levels. Our results suggest that the SNPs and mutations in ERbeta gene are not a common cause of spermatogenesis failure in Indian men, although mutations specifically found in infertile men can affect transcription, translation or have synergic effect with other variants in causing infertility.

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Oestrogen Receptor beta (ERbeta) gene plays an important role in the regulation of fertility in both males and females. Polymorphism in CA repeat located in the flanking region of ERbeta has been shown to be associated with several diseases, but its association with male infertility has not been analysed so far. However, RsaI polymorphism (rs1256049) in exon 5 of ERbeta has been shown to be associated with male infertility in Caucasian patients. Hence, we have analysed 695 Indian men, including 443 infertile and 252 fertile men to evaluate the association of CA repeat length and RsaI polymorphisms in male infertility. Our results revealed no significant difference in the distribution of CA repeat length between infertile (mean +/- SD 23.24 +/- 2.06, median 24) and fertile men (mean +/- SD 23.16 +/- 2.27, median 24). The analysis of dosage effect by classifying samples into SS (short/short), SL (short/long) and LL (long/long) groups also did not show any significant difference between infertile and fertile men. Similarly, RsaI polymorphism also did not show any significant difference between infertile and fertile men. Furthermore, the combined analysis of CA repeat and RsaI polymorphisms by haplotyping showed that the distribution of haplotypes was not significantly different between fertile and infertile men. Our results suggest that CA repeat length and RsaI polymorphisms in ERbeta are not associated with infertility in Indian men.

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The autosomal DAZL (Deleted-in-Azoospermic-Like) gene, mapped to the short arm of the human chromosome 3, is the precursor for the Y-chromosomal DAZ cluster, which encodes for putative RNA-binding proteins. Mutations in the DAZL have been reported to be associated with spermatogenic failure in Taiwanese population but not in Caucasians. As there was no study on Indian populations, we have analysed the entire coding sequences of exons 2 and 3 of DAZL in a total of 1010 men from Indian subcontinent, including 660 infertile men with 598 non-obstructive azoospermia, 62 severe oligozoospermia and 350 normozoospermic fertile control men, to investigate whether mutation(s) in the DAZL is associated with male infertility. Interestingly, none of our samples (1010) showed A386G (T54A) mutation, which was found to be associated with spermatogenic failure in Taiwanese population. In contrast, A260G (T12A) mutation was observed in both infertile and normozoospermic fertile control men, without any significant association with infertile groups (chi2= 0.342; p = 0.556). Similarly, we have found a novel A437G (I71V) mutation, which is also present in both infertile and normozoospermic fertile control men without any significant difference (chi2 = 0.476; p = 0.490). Our study clearly demonstrates the complete absence of the A386G (T54A) mutation in Indian subcontinent and the other two mutations --A260G (T12A) and A437G (I71V)--observed are polymorpic. Therefore, we conclude that these mutations in the DAZL gene are not associated with male infertility in Indian subcontinent.

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The genomic activity of nucleolar organizer regions (NORs) in eight human B-cell lymphoblastoid cell lines was studied following the routinely used Ag-NOR technique. The results demonstrate that (a) Ag-NORs are located in the short arms of D- and G-group chromosomes, (b) two out of eight cell lines have 66.7% and 49.0% of metaphases, respectively, with 9 to 10 active Ag-NORs, and (c) as a whole, Ag-NOR activity is much higher in B-cell lines as compared with conventional 72-hr peripheral blood lymphocyte (T-cell) harvests.

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Cytogenetic analyses of eight human lymphoblastoid cell lines that were established by a new procedure using B-cell growth factor (BCGF) and interleukin-4 (IL-4) revealed that three cell lines (37.5%) showed structural anomalies in more than 97% of their metaphases, whereas others were predominantly normal diploid. Our results indicate that a) the structural anomalies seen in three cell lines were not induced by culture technique but were constitutional defects present in donors' peripheral blood samples; b) one donor, whose blood gave rise to cell line BCDI, is a constitutional mosaic because both normal diploid and altered metaphases were present; and c) genetic instability should be monitored in regular blood donors because some of them may harbor blood-mediated clastogen or chromosome breakage factor(s) that could induce higher rates of recombinations in somatic cells of some recipients and thus may potentiate such individuals to develop certain types of malignancies.

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HindIII digested DNA from various mutant strains of Saccharomyces cerevisiae probed with a 340 bp nucleotide sequence in M13mp8 derived from a mouse liver cDNA clone p1581 showed strong hybridization to a 4.1 kb DNA fragment class. This was limited to the DNA of cells of alpha mating type but the fragments concerned apparently do not originate from chromosome III. The pattern of hybridization was modified in strains carrying the HO gene consistent with there being extra copies of the Bkm-homologous sequence in these cells. Northern analysis of RNA from cells synchronised in various stages of the mitotic and meiotic cell cycle probed with M13mp8/p1581 indicated related transcripts in meiotic cells.

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