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Biomedical subjects

G D Berkovitz

Publications and source records attributed to G D Berkovitz.

At least 37 records · Page 2Linked to original sources
PubMed1995

Assay of plasma testosterone during the first six months of life: importance of chromatographic purification of steroids.

Determination of the plasma concentration of testosterone (T) is important in evaluating infants born with ambiguous genitalia and micropenis, and several commercially available kits provide a direct assay of T in unextracted plasma. Using plasma samples obtained from 36 subjects < 6 months old, we compared the concentration of plasma T measured by RIA after extraction and purification by column chromatography with the T concentration measured in a direct assay. When aliquots of samples were purified before RIA, the concentration of T was markedly lower than in the direct assay. In the first 3 weeks postpartum, results of the direct assay were 3.8-fold greater than those obtained after purification. This difference decreased over time, and by age 2 months there was fairly good agreement between the two methods. These data indicate that some direct assays of plasma T are inappropriate during the first 2 months postpartum.

Carbon Tetrachloride↗
PubMed1994

Embryonic testicular regression sequence: a part of the clinical spectrum of 46,XY gonadal dysgenesis.

We report on a group of 9 subjects who had a 46,XY karyotype, ambiguous genitalia, abnormalities of sexual duct formation, and lack of gonadal tissue on one or both sides. This is sometimes referred to as "embryonic testicular regression." Previous investigators have suggested that this condition results from loss of testes at a critical stage in development. We examined the possibility that the "embryonic testicular regression" is part of the clinical spectrum of 46,XY gonadal dysgenesis. Four subjects totally lacked gonadal tissue, three of them having ambiguous genitalia, and one a micropenis. The development of incongruous sexual ducts (presence of Müllerian ducts in the subject with micropenis, and absence of Müllerian and Wolffian ducts in two subjects with ambiguous genitalia) suggests that the embryonic gonads were intrinsically functionally abnormal before their disappearance. Five subjects had unilateral gonadal tissue, ambiguous genitalia, and a mix of Wolffian and Müllerian structures. The development of incongruous sexual ducts in 3 of them, the presence of ambiguous external genitalia in 5, and the presence of abnormal gonadal histology in 2 patients all indicate an underlying abnormality of gonadal differentiation in these subjects. The occurrence of testicular regression in several subjects in the family of one patient suggests a genetic basis for the condition. The presence of multiple congenital anomalies in other subjects in our study suggests either a mutation in a single gene that functions in several developmental pathways, or a defect of multiple genes that might be the result of a chromosome deletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Multiple↗
PubMed1994

Nonrandom inactivation of the Y-bearing X chromosome in a 46,XX individual: evidence for the etiology of 46,XX true hermaphroditism.

We previously reported a subject with 46,XX true hermaphroditism who had a 46,X,del(X) karyotype and Y-chromosomal sequences in genomic DNA. We hypothesized that the Y-chromosomal sequences were translocated to the deleted X chromosome and that the incomplete testis determination of this individual was the result of inactivation of the translocated X chromosome. In situ hybridization studies demonstrated that the Y-chromosomal sequences were located on the distal portion of the short arm of the deleted X chromosome. Investigation of the replication of the X chromosome, using a modified R-banding technique and localization of Y-chromosomal sequences by in situ hybridization, showed that the translocated X chromosome was late replicating in all 100 EBV-transformed lymphoblasts that were examined. By contrast, when cells from a subject with 46,XX maleness were studied, the translocated X chromosome was late replicating in only 21 of 47 cells. As the late-replicating X chromosome is presumed to be the inactive X chromosome, selection of cells in which the Y-bearing X chromosome has been inactivated may play a role in the incomplete testis determination in subjects with "Y-positive" 46,XX true hermaphroditism.

Chromosome Mapping↗
PubMed1993

The effect of epidermal growth factor in human granulosa cells varies with follicle size.

EGF decreases aromatase activity and stimulates the proliferation of granulosa cells. To determine if EGF affects follicles of different maturity differently, human granulosa cells obtained from 6 patients at the time of IVF were pooled according to size and cultured in serum free medium with or without EGF. Estrogen production at 72 h was determined by measuring tritiated water formed in a 1-h culture with 1 beta [3H] androstenedione. The change in estrogen production with EGF was compared for cells from the smallest and largest follicles for each patient. Estrogen production in response to EGF decreased with increasing follicular size in all six observations. EGF may mediate luteinized granulosa cell aromatase activity in vivo, and may be a factor responsible for the relative decline in estrogen production in the preovulatory follicle.

Aromatase↗
PubMed1993

The role of the sex-determining region Y gene in the etiology of 46,XX maleness.

The condition of 46,XX maleness is characterized by testicular development in subjects who have two X chromosomes but who lack a normal Y chromosome. Several etiologies have been proposed to explain 46,XX maleness: 1) translocation of the testis-determining factor from the Y to the X chromosome, 2) mutation in an autosomal or X chromosome gene which permits testicular determination in the absence of TDF, and 3) undetected mosaicism with a Y-bearing cell line. We evaluated 10 affected subjects who were ascertained for different reasons and who had several distinct phenotypes. Six subjects had inherited sequences from the short arm of the Y chromosome including the sex-determining region Y gene (SRY). Five of the subjects were pubertal at the time of evaluation and had a phenotype similar to that of Klinefelter syndrome with evidence of Sertoli cell and Leydig cell dysfunction. One subject had evidence from Southern blot analysis and in situ hybridization for the presence of an intact Y chromosome in approximately 1% of cells. Three subjects lacked Y sequences by Southern blot analysis and by polymerase chain reaction amplification of SRY. These subjects were ascertained in the newborn period because of congenital anomalies. One had multiple anomalies including cardiac abnormalities; one had cardiac anomalies alone; and one had ambiguous genitalia. Our data confirm the genetic heterogeneity of 46,XX maleness, in which some subjects have SRY while other subjects lack it. In addition, there is phenotypic heterogeneity among subjects who lack SRY suggesting that there is also genetic heterogeneity within this subgroup.

DNA Restriction Enzymes↗
PubMed1993

Report of a kindred with X-linked (or autosomal dominant sex-limited) 46,XY partial gonadal dysgenesis.

The condition termed 46,XY complete gonadal dysgenesis is characterized by the lack of testicular determination with resulting streak gonads, normal Mullerian structures, and female external genitalia. In the partial form, there is incomplete testicular determination with a wide range in the degree of ambiguous genitalia and sexual duct development. We evaluated a kindred in which a partial form of 46,XY gonadal dysgenesis occurred in four subjects from two generations. Pedigree analysis indicated an X-linked or possibly an autosomal sex-limited mode of inheritance. All affected subjects were ascertained because of ambiguous genitalia with minimal virilization. At 10 days of age, the proband had a subnormal plasma level of testosterone, and at 4 months, there was no rise in plasma T after stimulation with hCG. At laparotomy, a dysgenetic gonad was found on the right side, but no gonad was found on the left side. A vas deferens was present on the right, indicating the presence of functional Leydig cells early in fetal life. In the other affected subjects, gonadal tissue was also limited to one side of the abdomen and showed poorly developed seminiferous tubules. The sex-determining region Y gene, which encodes the testis-determining factor, was present and unaltered in the genomic DNA of all affected subjects. Duplication of the distal short arm of the X-chromosome has been associated with 46,XY complete gonadal dysgenesis in some patients. In our studies, Southern blot analysis revealed that sequences of the distal short arm of the X-chromosome (DXS9 to DXS84) were present in single copy, excluding a large duplication in this area of the X. Several kindreds with familial 46,XY complete gonadal dysgenesis have been reported; five of them had evidence of an X-linked mode of inheritance. Our study of a kindred with 46,XY partial gonadal dysgenesis further supports the role of an X chromosome gene in testicular determination. Evidence of some fetal Leydig cell function in the affected subjects of our report suggests that mutations of the putative X-chromosome gene can result in a partial as well as complete defect in testicular determination.

Blotting, Southern↗
PubMed1992

Partial gonadal dysgenesis in a patient with a marker Y chromosome.

We evaluated a patient with partial gonadal dysgenesis including a right dysgenetic testis and a left streak gonad with rudimentary fallopian tube and uterus. She had ambiguous external genitalia and was raised female. Although her height is normal (25th centile at age 12 years), she has some findings of Ullrich-Turner syndrome. Her karyotype was reported to be 46,X,+marker; subsequent molecular investigations showed the marker to be the short arm of the Y chromosome. Genomic DNA, isolated from leukocytes of the patient and her father, was digested with a variety of restriction endonucleases and subjected to Southern blot analysis. A positive hybridization signal was obtained with probes for the short arm of the Y chromosome (pRsY0.55, SRY, ZFY, 47Z, pY-190, and YC-2) in DNA from the patient, indicating the presence of most if not all of the short arm, while long arm probes (HinfA and pY3.4) indicated that at least 75% of the long arm of the Y chromosome was missing. The gene responsible for testicular determination (TDF) is on the distal portion of the short arm of the Y chromosome; Yq has no known influence on sex determination. Hence, the deletion of the long arm of the Y chromosome cannot explain the gonadal dysgenesis in this patient. One explanation for the gonadal dysgenesis and Ullrich-Turner phenotype in the patient could be undetected 45,X/46,X,+marY mosaicism but no such mosaicism was observed in peripheral lymphocytes. Several investigators have suggested the presence of an "anti-Turner" gene near TDF. Hence it is possible that the clinical phenotype in our patient results from a Y chromosomal defect in sequences flanking TDF, which reduces the function of both TDF and the "anti-Turner" genes.

Adult↗
PubMed1992

DNA binding activity of recombinant SRY from normal males and XY females.

The protein encoded by the human testis determining gene, SRY, contains a high mobility group (HMG) box related to that present in the T cell-specific, DNA-binding protein TCF-1. Recombinant SRY protein was able to bind to the same core sequence AACAAAG recognized by TCF-1 in a sequence dependent manner. In five XY females point mutations were found in the region encoding the HMG box. In four cases DNA binding activity of mutant SRY protein was negligible; in the fifth case DNA binding was reduced. These results imply that the DNA binding activity of SRY is required for sex determination.

Amino Acid Sequence↗
PubMed1992

The role of the sex-determining region of the Y chromosome (SRY) in the etiology of 46,XX true hermaphroditism.

The syndrome of 46,XX true hermaphroditism is a clinical condition in which both ovarian and testicular tissue are found in one individual. Both Mullerian and Wolffian structures are usually present, and external genitalia are often ambiguous. Two alternative mechanisms have been proposed to explain the development of testicular tissue in these subjects: (1) translocation of chromosomal material encoding the testicular determination factor (TDF) from the Y to the X chromosome or to an autosome, or (2) an autosomal dominant mutation that permits testicular determination in the absence of TDF. We have investigated five subjects with 46,XX true hermaphroditism. Four individuals had a normal 46,XX karyotype; one subject (307) had an apparent terminal deletion of the short arm of one X chromosome. Genomic DNA was isolated from these individuals and subjected to Southern blot analysis. Only subject 307 had Y chromosomal sequences that included the pseudoautosomal boundary, SRY (sex-determining region of Y), ZFY (Y gene encoding a zinc finger protein), and DXYS5 (an anonymous locus on the distal short arm of Y) but lacked sequences for DYZ5 (proximal short arm of Y) and for the long arm probes DYZ1 and DYZ2. The genomic DNA of the other four subjects lacked detectable Y chromosomal sequences when assayed either by Southern blotting or after polymerase chain reaction amplification. Our data demonstrate that 46,XX true hermaphroditism is a genetically heterogeneous condition, some subjects having TDF sequences but most not. The 46,XX subjects without SRY may have a mutation of an autosomal gene that permits testicular determination in the absence of TDF.

Base Sequence↗
PubMed1992

Use of granulosa-luteal cell culture to evaluate low and high clinical responses to menotropin stimulation.

The cause of a poor response to human menopausal gonadotropin (hMG) remains unexplained. To determine whether aromatase activity of cultured granulosa cells obtained from relatively low estradiol (E2) responders (serum E2 < 1000 pg/ml) to hMG therapy differed from that of good responders (E2 > or = 1000 pg/ml), we prospectively compared serum E2 on the day of human chorionic gonadotropin administration to in vitro aromatase activity following a 72-h culture. Granulosa cells were obtained from seven women undergoing hMG therapy and oocyte aspiration. Follicle stimulating hormone (FSH) was added to one-half of the cultures. Serum E2 was determined by radioimmunoassay, and aromatase activity was determined indirectly by measuring tritiated water formed by aromatization of 1-beta [3H] androstenedione to estrogen in 1 h. In this study, luteinized granulosa cells from patients with a relatively low serum E2 produced less estrogen in cultures when compared to cells from higher responders (p < 0.01). Aromatase activity was not significantly increased by FSH in the relatively high responders, whereas FSH stimulated a significant increase in aromatase activity in cells from lower responders (p < 0.001). Our results indicate that the clinical response to hMG is at least partly due to the "quality" of granulosa cell aromatase activity. A clinically relevant "block" to FSH action may be present in vivo in low responders which can be reversed in culture by addition of FSH.

Aromatase↗
PubMed1992

Induction and superinduction of messenger ribonucleic acid specific for aromatase cytochrome P-450 in cultured human skin fibroblasts.

Aromatase cytochrome P-450 (cytochrome P-450AROM) catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. Several studies indicate that cytochrome P-450AROM activity is induced by glucocorticoids such as dexamethasone (DEX) and superinduced by DEX plus cycloheximide (CHX). We have used cultured human skin fibroblasts as a model system to investigate the regulation of aromatase gene expression. Whereas Northern blot analysis of total cellular RNA or poly (A)+ RNA from untreated strains of normal human skin fibroblasts failed to demonstrate any hybridization with a specific human placental cytochrome P-450AROM complementary DNA, analysis of RNA from cells treated with DEX demonstrated hybridization of the cytochrome P-450AROM complementary DNA to two transcripts of about 2.5 and 3.0 kilobases. Incubation of cells with DEX plus CHX resulted in a further increase in levels of cytochrome P-450AROM messenger RNA (mRNA) when compared to cells treated with DEX alone, suggesting that inhibition of protein synthesis superinduces transcription of the cytochrome P-450AROM gene. By contrast, levels of beta-actin mRNA were not affected by treatment with DEX and CHX. Treatment of cells with CHX alone did not produce a change in either aromatase activity or levels of cytochrome P-450AROM mRNA transcripts. These results indicate that aromatase activity is regulated by changes in the concentration of cytochrome P-450AROM mRNA, and imply that control of cytochrome P-450AROM gene expression is at the level of gene transcription. We conclude that the cytochrome P-450AROM gene is regulated by a complex mechanism that includes both positive and negative transcription factors.

Aromatase↗
PubMed1992

Evidence for increased prevalence of SRY mutations in XY females with complete rather than partial gonadal dysgenesis.

The Y chromosome gene SRY (sex-determining region, Y gene) has been equated with the mammalian testis-determining factor. The SRY gene of five subjects with 46,XY complete gonadal dysgenesis (46,XY karyotype, completely female external genitalia, normal Müllerian ducts, and streak gonads) was evaluated for possible mutations in the coding region by using both single-strand conformation polymorphism (SSCP) assay and DNA sequencing. Mutations were identified in three subjects, of which two gave altered SSCP patterns. Two of them were point mutations causing amino acid substitutions, and the third was a single-base deletion causing a frameshift. All three mutations caused alterations in the putative DNA-binding region of the SRY protein. Genomic DNA was obtained from the fathers of two of the three mutant patients: one mutation was demonstrated to be de novo, and the other was inherited. The presence of SRY mutations in three of five patients suggests that the frequency of SRY mutations in XY females is higher than current estimates.

Base Sequence↗
PubMed1991

Clinical and pathologic spectrum of 46,XY gonadal dysgenesis: its relevance to the understanding of sex differentiation.

The condition termed "46,XY gonadal dysgenesis" is characterized by a 46,XY karyotype and incomplete testicular determination. It is likely the result of a mutation in the gene for the testicular determination factor or in another gene involved in the early stages of testicular differentiation. In view of the present interest in the identification of gene(s) initiating the differentiation of the embryonic gonads into testes, we have reviewed the phenotype of 15 patients with 46,XY gonadal dysgenesis to use this information for future molecular studies. Seven patients presented a complete form, 46,XY pure gonadal dysgenesis, including streak gonads, normal Müllerian structures, and normal female external genitalia. The structure of the streak gonads in these patients presented some variation. Eight patients presented an incomplete form, 46,XY partial gonadal dysgenesis, with ambiguous external genitalia and partial development of Müllerian and Wolffian structures. Among them, 3 had bilateral dysgenetic testes, and 4 had a streak gonad on one side with a contralateral dysgenetic testis. The streak gonads showed ovarian stroma with occasional primitive sex cords devoid of germ cells. However, a primordial follicle was observed in 1 streak gonad. The dysgenetic testes showed disorganized seminiferous tubules and ovarian stroma. In some patients, the ovarian stroma was intermixed with testicular tissue, while in others, distinct ovarian and testicular portions were present. In 1 patient, the dysgenetic testis contained a focus of well-differentiated ovarian tissue with primordial follicles. Our observations support the hypothesis that streak gonads in 46,XY pure gonadal dysgenesis arise from fetal ovaries and that dysgenetic testes in the partial form in 46,XY partial gonadal dysgenesis develop from ovotestis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1990

Testosterone lowers aromatase activity in cultured human genital skin fibroblasts.

The cytochrome P-450-dependent aromatase pathway utilizes the androgens testosterone (T) and androstenedione, as substrates for estrogen formation. In addition, androgens have been shown to influence the level of aromatase activity in various tissues. In cultured human skin fibroblasts, incubation with T for 14 h resulted in a dose-dependent decline in aromatase activity, the concentration of T producing a half-maximal decline being 6 nM. In the presence of T (50 nM), aromatase activity declined in a time-dependent fashion with maximal reduction occurring by 9 h. When aromatase kinetics were determined after preincubation of cells with T, there was a significant decline in the calculated Vmax with no significant change in the apparent Km, suggesting that incubation of cells with T reduced the number of active enzyme sites. Aromatase activity was unaffected by preincubation of cells with the synthetic androgen methyltrienolone. In addition, the decline in aromatase activity following preincubation with T was observed in cells derived from patients with complete androgen insensitivity demonstrating that the effect of T was not mediated by androgen receptors. Furthermore, new protein synthesis was not necessary for the T-mediated effect as the presence of cycloheximide (50 micrograms/ml) did not prevent it. When cells were incubated at low oxygen tension, the inhibition of aromatase activity by T was diminished. Testosterone is rapidly metabolized in genital skin fibroblasts to dihydrotestosterone, androstanedione, androsterone, 3 alpha-androstanediol, 3 beta-androstanediol and estradiol. To determine if a metabolite of T might be responsible for the repression of aromatase activity, aromatase activity was determined in cells following preincubation with various metabolites of T. Preincubation of cells with androstenedione, androstanedione or 3 alpha-androstanediol produced a small but significant decline in aromatase activity, whereas preincubation of cells with dihydrotestosterone, androsterone, or 3 beta-androstanediol did not have a significant effect. Aromatase activity was also unaffected by preincubation of cells with estradiol or diethylstilbestrol. When aromatase activity was assayed in microsomal preparations from cells preincubated with T, activity was reduced. Although cells preincubated with 50 nM [3H]T contained between 0.25 and 0.51 pmol of residual steroid/mg microsomal protein, the amount of [1-3H]androstenedione and T was insufficient to account for the observed decline in aromatase activity on the basis of competitive inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)

Aromatase↗
PubMed1990

Endocrine evaluation of adults with mild hypospadias.

The goal of our study was to determine the frequency of endocrine abnormalities in sexually mature men with mild hypospadias. For this purpose we evaluated 16 men 22 to 52 years old with mild hypospadias. All patients had a urethral meatus distal to the penoscrotal junction. Serum concentrations of luteinizing hormone, follicle-stimulating hormone and testosterone were measured in all subjects. Dihydrotestosterone and testosterone precursors also were determined in men who had abnormally elevated levels of luteinizing hormone. The level of serum luteinizing hormone in patients was significantly higher than that of normal men, whereas the levels of serum follicle-stimulating hormone and testosterone were similar to those of normal subjects. Of the 16 men with mild hypospadias 3 had abnormally elevated levels of gonadotropins: 2 had primary gonadal dysfunction (elevated luteinizing hormone and follicle-stimulating hormone with low or normal testosterone) and 1 had hormonal evidence of partial androgen insensitivity (elevated luteinizing hormone, normal follicle-stimulating hormone with high normal testosterone and dihydrotestosterone). These patients are rather unique, since they did not have other compromising factors to account for the gonadal failure. In summary, our data demonstrated the presence of endocrine dysfunction in men with mild hypospadias.

Adult↗
PubMed1990

Abnormal induction of aromatase activity by dexamethasone in fibroblasts from a patient with cortisol resistance.

Cortisol resistance is a rare condition due to abnormal glucocorticoid receptor function. Stimulation of aromatase activity by dexamethasone (DEX) in cultured human skin fibroblasts provides a model for studying the biological activity of glucocorticoid receptors in cells. Skin fibroblasts derived from an affected father and his less severely affected son with cortisol resistance were used for this study. Saturation analysis of DEX receptor binding was performed after incubation of cells with various DEX concentrations (1-50 nmol/L). In normal cells, the mean maximal binding capacity (Bmax) and dissociation constant (Kd) were 24 +/- 3 pmol/mg DNA and 14 +/- 3 nmol/L, respectively. Although the Bmax in cells of the father (33 +/- 2 pmol/mg DNA) was normal, the Kd (31 +/- 7 nmol/L) was abnormally elevated. By contrast, both the Bmax and Kd in cells of the son were normal. The dose response of aromatase activity to DEX stimulation was determined by assay of aromatase activity after incubation of cells in the absence or presence of DEX (0.25-500 nmol/L) for 14 h. In three strains of normal fibroblasts, the mean concentration of DEX that produced a half-maximal response was 6 +/- 1 nmol/L). In cells from the father, the mean concentration of DEX that produced half-maximal stimulation of aromatase (27 +/- 4 nmol/L) was abnormally elevated. By contrast, the concentration of DEX that half-maximally stimulated aromatase activity (6.0 and 5.9 nmol/L) was normal in cells from the son. These data provide additional evidence of abnormal glucocorticoid action in the father, but not in his son, and demonstrate the potential usefulness of determining aromatase induction by DEX as a means of assessing the biological activity of the glucocorticoid receptor.

Aromatase↗