Modified guanidinium thiocyanate method for human sperm DNA isolation.

Mammalian sperm chromatin is highly condensed, so isolating DNA from such chromatin can be a formidable task. The procedures that produce high quality DNA from somatic cells fail to yield quality sperm DNA. In this study we have modified the previously used guanidinium method to make it simple and efficient in isolating human sperm DNA. In our method, the lysis buffer contained guanidinium, sodium citrate, sarkosyl, proteinase K and mercaptoethanol. Proteinase K was not used in the original guanidinium method but was included in our protocol. CsCl centrifugation of the lysate, as described in the original procedure, was omitted. Instead, isopropyl alcohol was added directly to the lysis buffer to harvest the DNA. This modified guanidinium method generated high molecular weight DNA while the other two methods resulted in considerable DNA degradation. There was no difficulty in restriction enzyme digestion of DNA prepared by the modified method as revealed by Southern blot analysis. Since the modified guanidinium method is a simple one-step procedure which avoids homogenization, organic solvents, centrifugation and, more importantly, produces degradation-free DNA, it could be the method of choice when DNA from mature germ cells is needed.

Evidence for a partial deletion in the androgen receptor gene in a phenotypic male with azoospermia.

Androgen resistance is thought to vary phenotypically from a normal female to an infertile male. Previous evaluation of infertile males has been limited to androgen receptor-binding affinity. The androgen receptor gene has been isolated, cloned, and studied extensively in patients with complete androgen insensitivity syndrome, but no comparative data are available on infertile males. To address this matter, the androgen receptor gene was studied in seven azoospermic males by use of the polymerase chain reaction and Southern blot hybridization. A partial gene deletion was found in one patient. This study provides the first molecular evidence of an abnormality in the androgen receptor gene in a phenotypic male with azoospermia.

Probing genomic deoxyribonucleic acid for gene rearrangement in 14 patients with androgen insensitivity syndrome.

Androgen insensitivity appears to involve mutations in the X-linked androgen receptor (AR) gene in genetic males. In this study; 14 patients with androgen insensitivity syndrome (unrelated patients [n = 6]; related patients [n = 8]) were studied. Ten patients had complete and 4 had partial insensitivity to androgens. Deoxyribonucleic acid samples from controls and study subjects were examined with probes specific for the AR gene domains (hAR1, hAR2, hAR3). In one subject with complete androgen insensitivity syndrome, a reduction in size of the 2.4 kilobase band hybridizing to hAR1 was noted. Southern blot analysis of these subjects, however, did not detect deletions or gene rearrangement. These results suggest that deletions detectable by Southern method are infrequent mutants of the AR gene in patients with androgen insensitivity syndrome.

Correlation of the testicular determinant factor sequence zinc finger Y with varying gonadal phenotypes in a series of 13 subjects with gonadal dysgenesis due to Y aneuploidy.

Deoxyribonucleic acid samples from a series of 13 subjects with 45,X/46,X,altered Y, and varying gonadal phenotypes (streak-streak, n = 9; streak-testis, n = 2; testis-testis, n = 2) were analyzed for the presence of the candidate testicular determinant factor sequence zinc finger Y. The Y-specific probes Y97 mapped to Y centromere, pDP105 A,B mapped to Yp and distal Yq11, respectively, hybridized with the deoxyribonucleic acid from all the 13 study subjects. The same deoxyribonucleic acid samples were analyzed for the presence of the zinc finger Y sequence. Eleven of the 13 subjects were positive for the zinc finger Y sequence. Four zinc finger Y-positive subjects had unilateral (n = 2) or bilateral (n = 2) testicular differentiation. Among the nine subjects with bilateral streak gonads, seven showed the presence of this sequence. The lack of testicular differentiation in the presence of quantitatively normal or almost normal zinc finger Y bands could not be explained by mosaicism alone. Mutations not detectable by analysis with the method of Southern with pDP1007, may occur in the testicular determinant factor gene vitiating testicular development.

Deoxyribonucleic acid analysis of the zinc finger Y gene in 45,X/46,XY subjects.

The deoxyribonucleic acid from nine subjects with a 45,X/46,XY karyotype with a cytogenetically intact Y chromosome and phenotypically presenting with bilateral streak gonads, streak and testis, or bilateral scrotal testes along with a control male and female were analyzed for the presence of the zinc finger Y sequence through the molecular probe pDP1007. This particular probe is thought to constitute part of the putative testicular-determining factor gene. All the study subjects demonstrated the presence of zinc finger Y. Laser densitometry studies confirmed a correlation between the intensity of the zinc finger Y band and the percentage of Y cell lines. This study supports the fact that individuals with mixed gonadal dysgenesis and cytogenetically intact Y chromosomes will tend to have intact zinc finger Y sequences.

Screening of seven putative 45,X subjects with deoxyribonucleic acid probes to detect low-level mosaicism for Y cell lines.

The frequency of monosomy X in cytogenetically abnormal abortion material (10% to 15%) suggests that viable 45,X subjects might have covert mosaicism for X or Y cell lines. The deoxyribonucleic acid samples from seven 45,X subjects with Turner syndrome were examined with three Y-specific deoxyribonucleic acid probes. Successive hybridizations with each of these three sensitive deoxyribonucleic acid probes did not reveal any Y-specific band.

Molecular scanning of Yq11 (interval 6) in men with Sertoli-cell-only syndrome.

Data suggesting that probes pDP105/B and 50f2/C,E may identify sequences on distal Yq11 (interval 6) that are critical for spermatogenesis stimulated a study of this region by means of these two probes in azoospermic 46,XY men with biopsy-proved Sertoli-cell-only syndrome. Deoxyribonucleic acid samples from controls and study subjects were digested with the restriction enzymes TaqI, EcoRI, and BamHI. These samples were blotted and hybridized with pDP105/B, 50f2/C,E, and two more proximal Yq11 probes 4B-2 and pAS1. The sequence hydridizing to 50f2/C was absent in one study subject. No deletions were detected with pDP105/B and the two more proximal probes.

Use of human alpha-satellite deoxyribonucleic acid to detect Y-specific centromeric sequences.

The Y alphoid deoxyribonucleic acid probe Y97 has proved to be specific for the human Y centromere and to define a Y-specific 5.5 kb Eco RI fragment. Three experiments were designed to evaluate the sensitivity and the specificity of this Y alphoid probe Y97. In the first experiment the centromeric Y-specific 5.5 kb Eco RI fragment was clearly seen in the mixture of 0.050 microgram of male DNA with 4.950 micrograms of female DNA (1%). In the second experiment the same dilutional study was applied to the Yq11-related probe 4B-2 for comparison purpose. In the third experiment, hybridization with the Y97 probe was performed on 20 subjects with mosaic cell lines containing a cytogenetically identifiable Y (n = 10) and a cytogenetically unidentifiable minute (n = 10) fragment. Nineteen of the 20 subjects demonstrated the Y-specific 5.5 kb Eco RI hybridization band with the centromeric Y97 probe. These experiments demonstrated the utility of the Y97 probe to consistently identify cytogenetically altered Y chromosome fragments and confirm the mapping of the alphoid repeat sequences to the centromeric region of the Y chromosome.

Use of deoxyribonucleic acid probes to test for Yq11 deletions in males with spermatogenic arrest.

A cytogenetically detectable deletion in the area of Yq11 has been demonstrated in some men with spermatogenic arrest, leading to the suggestion that a spermatogenic factor(s) lies within this region. The probe pAS1 detects an argininosuccinate synthetase pseudogene 6 (ASSP6), which has been mapped to Ycen-q11. The 4B-2 (DYS 15) probe detects a single-copy 3.3 kb EcoRI fragment that maps to the proximal portion of the Y long arm located distal to the sequence detected by the pAS1 probe. Deoxyribonucleic acid (DNA) samples from normal males and females and ten males with spermatogenic arrest were digested with the restriction endonuclease EcoRI, electrophoresed on agarose gels, Southern blotted, and hybridized with the pAS1 and 4B-2 probes. All males tested, including the ten azoospermic males with spermatogenic arrest, exhibited 4.3 kb and 3.3 kb male specific fragments with the pAS1 and 4B-2 probes, respectively. From preliminary analyses, the authors conclude that the regions detected by these two probes are not absent in these azoospermic males and that the cause of their spermatogenic arrest may not involve deletion within this region. Molecular defects affecting spermatogenesis may involve loss of sequences at Yq11, which were not tested in the study, or they may derive from heterogenous causes.