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Peripheral blood karyotype was performed as the only genetic examin ation for sexual differentiation disorders for a long time, but it was not capable of illustrating all the clinical manifestations of gonadal dysgenesis even us ing a high-resolution technique. It is now obvious that the genesis of the tes tis is closely related to the SRY (sexual related Y) gene located in the 1A1A pa rt of the short arm of the Y chromosome.［1］The SRY gene has been detected in some cases of gonadal dysgenesis. Our aim was to reveal the molecular genetic b asis of gonadal dysgenesis to develop a clear clinical classification for patien ts with gonadal dysgenesis.
METHODS
Clinical materials
Sixteen cases of gonadal dysgenesis from the Clinic of Reproductive Endocrinolog y, Peking Union Medical College Hospital, or transferred from other departments were studied. Some cases of gonadal dysgenesis were due to reasons other than Y chromosome: androgen insensitivity syndrome (5 cases) and 17-α-hydroxylase deficiency (1 case). Each case had a peripheral blood karyotype of 46,XY. Gonadal dysgenesis may be due to abnormalities in the Y chromosome in 4 cases o f true hermaphrodite, 2 cases of 45,X/46,XY gonadal dysgenesis, 1 case each of 4 5,X gonadal dysgenesis, XY pure gonadal dysgenesis, testicular regression and a 46,XY female who gave birth to a normal baby. Besides the history, physical examination, serum sex hormone levels, peripheral blood karyotype and peripheral blood SRY detection, surgery was performed to obt ain gonad specimens for pathological analysis. SRY detection was performed in g onad samples in 4 cases.
Methods
Isolation of the genomic DNA
White blood cells were isolated from total blood and digested with proteinase K and genomic DNA was isolated.［2］ Normal male and female genomic DNA was isola ted from the peripheral blood of normal adult males and females using the same m ethod.
Polymerase chain reaction
Primers used for PCR were SRY1-SRY2 and A1-A2. SRY1-SRY2 were provided by Dr .D.C.Page (White Head Institute, USA); the SRY motif can be amplified with a product of 472 bp. The sequences of the primers were as follows: SRY1: 5' -AAGCTGGTGCTCCATTCTTGAG-3' (upstream) and SRY2: 5'-AATATTCCCGCTCTCCGGA-3' (downstream).［3］A1-A2 could amplify the human X chromosome specific sequ ence wit h a product of 331 bp and was used as a positive control. Taq DNA polymerase an d 10×PCR buffer were provided by the Genetic Institute of the Chinese Acade my of Sciences. Four deoxyribonucleotide triphosphates (dATP, dCTP, dTTP and dG TP) were purchased from the Boehringer Company.
The reaction mixture consisted of 2.5 μl of 10 PCR buffer, 18 μl of ddH［2］O , 2 μl of dNTP (contained 2.5 mmol/L each of four deoxyribonucleotide tripho sphates), 80 ng of each primer, 500 ng of template DNA, 1.5 units of Taq polym erase for a total volume of 25 μl. The amplification reaction was performed i n a programmable temperature control system (Ezcycler, Ericomp Inc., USA). The samples were subjected to 30 cycles of denaturation at 94℃ for 30 s, annealin g at 55℃ for 30 s and extension at 70℃ for 1.5 min. The amplified product w as electrophoresed on 6% polyacrylamide gel, and visualized with silver staining .［4］
Direct sequencing of PCR product
The PCR product was electrophoresed and isolated on 2% agarose gels and γ-P32 labeled dideoxidize sequencing was performed.［5］
RESULTS
Androgen insensitive syndrome (5 cases) and 17-α-hydroxylase deficiency (1 c ase) had a peripheral blood karyotype of 46,XY, and the SRY specific sequence wa s shown in all six cases to be SRY positive. Blood karyotype, SRY and pathology of the gonads were well correlated. The remaining 10 cases showed various discrepancies between blood karyotype, SRY and gonadal tissues. Clinical diagnoses were made from the peripheral blood ka ryotype. Four cases of true hermaphrodites were found in surgery to have ovotes tis in their gonads ( Table 1 ). Case 1 had a peripheral blood karyotype of 46,XX /47,XXY and blood SRY was positive. Cases 2 and 3 had a peripheral blood karyot ype of 46,XX with an SRY-specific sequence. The gonads of case 2 showed the ka ryotype 46,XY and were SRY positive. Case 4 had a peripheral blood karyotype of 46,XX and were SRY negative (Table 1, Fig. ). The presence of X chromosome se quences was shown in the PCR products of normal males and females. Male-specif ic SRY sequences were found in normal males only (Fig.). Case 5 had a periph eral blood karyotype of 45,X and was diagnosed as 45,X gonadal dysgenesis with p ositive peripheral blood SRY. Underdeveloped testes were found in both gonads ( Table 1). Both cases of XY pure gonadal dysgenesis and testicular regression (c ases 6 and 7) had the peripheral blood karyotype of 46,XY. Each showed underdev eloped testes in the gonads and positive SRY in the peripheral blood (Table 1) . The gonads of a 46,XY female who gave birth to a normal baby (case 8) were norm al ovaries; the peripheral blood had an SRY-specific sequence (Table 1) and sho wed no point mutation in the SRY motif using direct dideoxidize sequencing. Det ection of the SRY gene using PCR techniques was performed in peripheral blood an d gonads of two cases of 45,X/46,XY gonadal dysgenesis (cases 9 and 10). SRY wa s positive in peripheral blood in both cases. Case 9 had an ovarian stroma, 45, X karyotype and negative SRY in both gonads. Case 10, with the karyotype of 45, X in both gonads, showed that the left gonad was an underdeveloped testis on pat hological analysis and was SRY positive, the right gonad was fibrous tissue and SRY negative (Table 1).
Among the 16 cases of gonadal dysgenesis, 15 were blood SRY positive, among whic h 13 (87%) showed the presence of testicular tissue, and 2 showed ovaries withou t testicular tissue. One SRY negative case showed the presence of testicular ti ssue. The Y chromosome was present in the peripheral blood of 12/16 cases with positive SRY, among which 10 cases had testicular tissue in their gonads, but 2 cases had ovaries or ovarian stroma. The Y chromosome was absent in the periphe ral blood of the remaining 4 cases. They all had testicular tissue in their gon ads: 3 cases were SRY positive and 1 case SRY negative ( Table 2 ). The correlati on between peripheral blood SRY and gonad pathology was 81.25%, and the correla tion between peripheral blood Y chromosome and gonad pathology was 68.75%.
DISCUSSION
The causes of gonadal dysgenesis include: 1) abnormalities of the sex chromosome , 2) mutations of sex determining genes and 3) mutations of the genes controllin g the synthesis and metabolism of sex hormones and hormone receptors.［6］ The amazing progress in the field of molecular biology made it possible to analyze s exual development abnormalities on the molecular level. The SRY gene is located within the 1A1A part of the short arm of the Y chromosome. It has been cloned, sequenced and researched with deletion fragment since it was discovered in 1990 and is now considered the best candidate for testis determining factor (TDF) .［1,3,7,8］Testis will develop in female mice transgenic for SRY.［9］S RY w as detected in the 16 cases of gonadal dysgenesis in this study and its signific ance and problems were analyzed.
Clinical significance of SRY detection
The androgen insensitive syndrome and 17-α-hydroxylase deficiency are caused by abnormalities in the androgen receptor gene of the X chromosome and the 17- α-hydroxylase gene in autosomal DNA, respectively.［10］The SRY gene was detected in the blood and gonads of 6 cases of the above diagnoses and the resu lts were positive in each case.
Because of the high incidence of tumor formation, underdeveloped testis must be removed. Among the sixteen cases showed in this series, SRY should be tested in cases of gonadal dysgenesis with a peripheral blood karyotype of 46,XX to asses s whether underdeveloped testes are present in the gonads. The existence of tes ticular tissue can not be ruled out in patients who are SRY negative, so cases o f clinical masculinization should also have exploratory surgery. If the Y chrom osome is present and SRY is positive, gonads might be ovaries. Case 8 (Table 1) was a XY female with SRY positive, but her gonads were normal ovaries and she g ave birth to a normal baby. Ye et al［11］reported a case of 45,X/46,XY go nadal dysgenesis with ovaries and primordial follicle on one side. This case in dicates that gonads can be ovaries and should be preserved in patients with an Y chromosome and a positive SRY.
Karyotype and SRY in gonadal tissue
A case of 45,X/46,XY gonadal dysgenesis with ovaries and primordial follicles in the gonads was reported by Ye et al in 1987.［11］A total of 9 cases of 4 5,X/46,XY gonadal dysgenesis were reported by Lou et al in 1993.［12］The pathological findings showed a variety of gonadal dysgenesis, ranging from ovari es with primordial follicles to testis with spermatogenesis. In this study, we showed that gonad karyotype might not be the same as peripheral blood.
Gonad specimens from 4 cases were karyotyped and an SRY determination was perfo rmed. Three of four gonads from 2 cases of 45,X/46,XY gonadal dysgenesis (cases 9 and 10) were pathologically ovarian stroma with a karyotype of 45,X and negat ive for SRY. Although the karyotype was also 45,X in the fourth gonad, SRY was positive and the gonad was an underdeveloped testis pathologically. SRY spe cific sequence was observed in 1 case of true hermaphrodite with peripheral bloo d karyotype of 46,XX, and further investigation of the gonad showed a karyot ype of 46,XY.
We conclude that the development of the gonad is controlled by the karyotype or genotype of the gonad rather than of peripheral blood, making it important to an alyze the karyotype of the gonad and assess the presence of the SRY gene in pati ents with gonadal dysgenesis.
Some rare cases
Mutations and deletions in other part (out of the motif) of the SRY gene
Jager et al［13］pointed out that mutation and deletion of the SRY gene may lead to testicular dysgenesis. Case 8 was a XY female who gave birth to a norma l baby. Peripheral blood DNA was assessed using PCR and direct dideoxidize sequ encing, and no deletion or mutation was revealed within the SRY motif. Detectio n of SRY using the PCR technique in cases of XY pure gonadal dysgenesis and test icular regression (cases 6 and 7) revealed no deletion. Further investigation should include sequencing and detection of more parts of the SRY gene.
Translocation of SRY gene
Chapelle［14］in 1987 proposed that one reason for testis development in pa tients with no Y chromosome was the translocation of the testis determining fact or in the Y chromosome to the X chromosome or other autosomes. This led to the discovery of the SRY gene.［1］The karyotype of cases 3 and 4 were 46,XX and 4 5,X, respectively, but testis were found and SRY was positive. Fluorescent in s itu hybridization should be done in these patients to search for the translocate d fragment after the mosaic could be ruled out.
Gonad determining gene other than SRY
A true hermaphrodite (case 4) had testicular tissue without the Y chromosome or the SRY gene. Chapelle［14］ proposed that two genes were involved in testi cular development: testis determining factor in the Y chromosome (SRY) and testi cular forming factor in one of the autosomes. The former acts as a promoter and regulator of the latter. Patients with variations in the autosomes forming an SRY-like gene or variations in testicular forming factor, may develop testis wi thout SRY. As to the XY female who gave birth to a baby, the situation may be m ore complicated. Some unknown factors may be involved. Further investigation m ust be done in such complicated rare case to verify the hypothesis proposed by Chapelle.
Conclusion
Detection of the SRY gene using polymerase chain reaction can determine the exis tence of testis determining factor quickly and specifically, and may be used as an indicator that underdeveloped testes should be resected. Detection of the ka ryotype and SRY gene of different tissue origin can clarify the clinical diagnos is and classification of this kind of patient with gonadal dysgenesis. The resu lts obtained from this series enlighten the route for further investigation: det ection of more parts of the SRY gene; fluorescent in situ hybridization (FISH) t o search for translocated fragments of the Y chromosome; and the searching for o ther testicular forming factors.
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