Intracytoplasmic sperm injection and potential transmission of genetic disease.
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Chromosome Aberrations↗
Biomedical subjects
D J Lamb
Publications and source records attributed to D J Lamb.
The disappearing Y chromosome--"I told you so!".
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Gene Deletion↗
Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice.
Prostate cancer mortality results from metastasis to bone and hormone-independent tumor growth. Models to study these progressive changes are lacking. Here we describe the propagation of advanced human prostate cancer by direct transfer of surgical samples from patients into immune-deficient male SCID mice. Explants from six of eight patients formed prostate tumors and two showed unique cytogenetic, biologic and molecular features that were retained through six or more passages. One grew in an androgen-independent fashion, whereas the second formed tumors that regressed following castration then regrew. Micrometastatic disease was detected in the hematopoietic tissues of half of the recipient mice. Thus selected specimens of advanced human prostate cancer can be propagated in SCID mice in a manner that recapitulates the clinical transition from androgen-sensitive to androgen-independent growth, accompanied by micrometastasis.
Androgens↗
Molecular biology and function of the androgen receptor in genital development.
PURPOSE: The rapidly growing field of molecular biology has caused exponential growth in our knowledge of the processes of embryogenesis. Since the cloning of the androgen receptor gene in 1988, investigators have been able to clarify many of the molecular events of male sexual differentiation that are mediated through the androgen receptor. We reviewed the current state of knowledge of the androgen receptor and its role in male genital development. MATERIALS AND METHODS: An intensive literature search was conducted to review reports on the androgen receptor and sexual differentiation since 1988. This review also includes ongoing research from our laboratory on the role of the androgen receptor in human genital development, as well as collaboration with other investigators. RESULTS: We reviewed the basic molecular biology of androgenic action mediated through the androgen receptor. This information has been integrated into the current understanding of human male sexual differentiation to clarify how androgens virilize the undifferentiated embryo. Defects in function of the androgen receptor may be manifested as a spectrum of phenotypes of the androgen insensitivity syndrome, and these phenotypes of male pseudohermaphroditism have been reviewed on a clinical and molecular basis. New molecular techniques have augmented the evaluation and diagnosis of the androgen insensitivity syndrome, and some groups have successfully diagnosed the condition prenatally. CONCLUSIONS: Basic scientific research of androgen receptor function and its role in male sexual development has provided a clearer understanding of the mechanisms responsible for the spectrum of defects secondary to the androgen insensitivity syndrome. This knowledge will enable clinicians to offer more accurate diagnosis and insightful counseling to affected patients and their families.
Disorders of Sex Development↗
Molecular determinants of sexual differentiation.
The processes of sexual differentiation have been greatly clarified by molecular biologic discoveries over the past five years. Gonadal differentiation into a testis or ovaries is controlled by a multitude of genes beginning with SRY which is believed to represent the testis determining factor. Other genes involved include SF-1, WT-1, DAX-1, and SOX9. The fully developed testis produces Mullerian inhibiting substance and testosterone to create the male phenotype; the female phenotype develops in their absence. This hormonally-driven process also requires additional factors and appropriate receptors. Errors in this pathway may be manifested clinically as intersex disorders, and the study of these disorders has helped to further elucidate the molecular mechanisms of sexual differentiation.
Animals↗
Androgen receptor gene mutations are rarely associated with isolated penile hypospadias.
PURPOSE: Hypospadias has no known single etiology but it has been linked to androgen insensitivity caused by mutations of the androgen receptor gene. The purpose of this study was to search for such mutations in cases of various degrees of isolated hypospadias to determine whether such an association exists and, if so, with any particular anatomical subgroup. MATERIALS AND METHODS: Isolated deoxyribonucleic acid from the penile tissue of 40 patients undergoing reconstructive surgery was screened for mutations of the coding regions of the androgen receptor gene using single strand conformational polymorphism analysis. In cases with abnormal single strand conformational polymorphism findings sequence analysis of the deoxyribonucleic acid was performed to define the mutation. RESULTS: A missense mutation of exon 2 of the androgen receptor gene was noted in 1 patient with isolated distal penile shaft hypospadias. Sequence analysis revealed that the mutation changed amino acid residue 546 from proline to serine. No abnormalities were detected in the other 39 patients. CONCLUSIONS: Isolated distal shaft hypospadias is associated with mutations of the androgen receptor gene but these mutations appear to be a rare cause of hypospadias.
Child↗
Transition metal ions within human atherosclerotic lesions can catalyse the oxidation of low density lipoprotein by macrophages.
The oxidation of low density lipoprotein (LDL) in the arterial wall may contribute to atherogenesis. The oxidation of LDL by cells usually requires catalytically active transition metal ions. We show here some that gruel samples from human advanced atherosclerotic lesions are capable of catalysing the oxidation of LDL by macrophages as measured by thiobarbituric acid-reactive substances, enhanced electrophoretic mobility and increased macrophage uptake. This catalysis could be inhibited by pretreatment of the gruel with Chelex-100, which binds transition metal ions. The presence of catalytically active transition metal ions in atherosclerotic lesions may help to explain why LDL oxidation occurs at these sites.
Animals↗
Genes involved in testicular development and function.
The development of the testis requires the highly regulated expression of a series of genes. Many of the genes involved are transcription factors, such as steroid hormone receptors and growth factors. Investigators have used gene cloning, mutation analysis, transgenic mice, and gene-deletion studies to define the role of specific genes in testicular development and function. In the past 5 years, investigators have defined a gene on the Y chromosome, SRY, thought to be required for testis determination. This protein is a member of a larger family of related transcription factors. Expression of this gene triggers a cascade of events that leads to the development of the Sertoli cell, Leydig cells, and the testis. The development of the male phenotype is dependent on the presence and action of androgens, which exert their effect after combining with a receptor in the nucleus of the target cell that stimulates gene transcription. Defects in the androgen receptor gene lead to a full spectrum of morphological defects in the male. Interestingly, mutations in other members of the steroid receptor superfamily, such as the estrogen receptor gene, also affect male fertility. A number of "orphan" receptors (i.e., receptors whose ligans have not been identified) are also required for normal testicular development and function, as are several genes normally thought to be tumor-suppressor genes (e.g., Wilms' tumor-suppressor gene). In contrast, alpha-inhibin has been thought to be an endocrine hormone, yet it functions as a tumor-suppressor gene in the testis. Testicular development and normal spermatogenesis require the proper function and coordination of a large number of transcription factors, steroid hormone and orphan receptors, and growth factors. There are likely to be a large number of other, as yet unidentified genes that are necessary for male gonadal development.
Animals↗
Steroid-regulated growth of DDT1MF-2 cells is profoundly influenced by culture conditions.
DDT1MF-2 cells provide an ideal model for studying tumor-growth-stimulation by steroids. These cells progress to a rapidly proliferating, androgen-independent state after prolonged culture without androgen. After brief culture in different lots of fetal bovine serum (FBS), some lots induced a permanent state of hormone-independence in cells that had been androgen-responsive. To test the hypothesis that factors influenced androgen-responsive growth even after removal of serum, hormone-responsive DDT1MF-2 cells (7000 cells/well) were plated in medium Dulbecco's Modified Eagle Medium/F-12 Nutrition Mixture (1:1)/1% ITS with (a) 0.1% FBS, (b) 0.1% NuSerum (c) 0.1% Hyclone, or (d) MCDB-110/0.1% ITS with 5 ng/ml bFGF. On Days 2-8, medium was replaced with D-MEM/F12/ITS with 10 nM testosterone (T), 10 nM triamcinolone acetonide (TA), or ethanol (control) and the cells counted. While testosterone induced a 1.4-fold increase in cell growth after exposure to FBS or NuSerum, maximal testosterone effect (3-6-fold increase) was observed after Hyclone. Hydroxyflutamide antagonized the fivefold increase in growth observed with testosterone, with a slight decrease of growth with cAMP for cells plated in Hyclone. Androgen-independent cells were unaffected by testosterone, hydroxyflutamide, or 8Br-cAMP [medium (a)]. Maximal inhibition by triamcinolone acetonide (0.25 of control) was observed with medium (d). The effect of testosterone and triamcinolone acetonide on secretion of mitogenic activity into conditioned medium was also evaluated. Although conditioned media from control and testosterone-treated cells were mitogenic in a dose-dependent manner, the media from cells treated with triamcinolone acetonide and testosterone+TA conditioned medium was not mitogenic--but, of note, it was not growth inhibitory.
Cell Division↗
A quality control system for the optimized sperm penetration assay.
OBJECTIVE: To develop a quality control system for the optimized sperm penetration assay (SPA) and to use this system to monitor interassay variability and stability over time. DESIGN: Four semen donors were tested consecutively for a period of weeks (7 to 139 weeks) with the SPA. Their average semen analyses and SPA scores were evaluated to monitor natural biologic variation. Intra-assay variation was obtained by dividing 11 semen samples into three aliquots and testing each separately in the SPA. A single ejaculate from seven individuals was aliquoted and frozen to be used as a control. They were tested on different assay days in 1986 and subsequently in 1991 to evaluate the assay stability over time. MAIN OUTCOME MEASURES: Results were expressed as a sperm capacitation index (mean number of sperm penetrations per ovum). RESULTS: Consecutive weekly semen analyses and SPAs on donors exhibited coefficients of variation ranging from 20% to > 40%. In contrast, these variations were much greater than intra-assay variability. Analysis of frozen semen specimens tested in several SPAs also displayed a low coefficient of variation. When aliquots of these frozen samples were tested in the SPA 5 years later, there were no differences in the observed values, showing the remarkable stability of this assay over time. The lower limit of the normal fertile range did not change over a period of 2 years. CONCLUSIONS: Results show that using fresh semen samples as a positive control in the SPA is inadequate. This deficiency has been overcome with the use of frozen semen controls. With frozen semen for quality control, the optimized SPA developed in this laboratory is a highly reproducible assay that meets the strict criteria required for clinical laboratory certification.
Animals↗
Iron released from transferrin at acidic pH can catalyse the oxidation of low density lipoprotein.
Low density lipoprotein (LDL) oxidation within the arterial wall may contribute to the disease of atherosclerosis. We have investigated the conditions under which transferrin (the major iron-carrying protein in plasma) may release iron ions to catalyse the oxidation of LDL. Transferrin that had been incubated at pH 5.5 released approximately 10% of its bound iron in 24 h, as measured by ultrafiltration and atomic absorption spectroscopy. Furthermore, transferrin co-incubated with LDL and L-cysteine at pH 5.5 resulted in the oxidation of the LDL as measured by thiobarbituric acid-reactive substances and electrophoretic mobility. This effect was observed at transferrin concentrations as low as 40% of its average plasma concentration. The release of iron from transferrin in atherosclerotic lesions due to a localised acidic pH may help to explain why LDL oxidation occurs in these lesions.
Humans↗
Acidic pH enables caeruloplasmin to catalyse the modification of low-density lipoprotein.
LDL oxidation within the arterial wall may contribute to the disease of atherosclerosis. There is some evidence that elevated plasma levels of copper are associated with an increased risk of coronary artery disease. We have investigated the conditions under which caeruloplasmin (the plasma copper carrier protein) can catalyse the macrophage-mediated modification of LDL. Low concentrations of CuSO4 (< 1 microM) could catalyse the macrophage-mediated modification of LDL. Native caeruloplasmin was unable to catalyse the modification of LDL at pH 7.4, but could do so after preincubation at acidic pH. After preincubation at acidic pH, concentrations of caeruloplasmin as low as 30 micrograms/ml (about one-tenth of the human plasma level) could catalyse significant LDL oxidation when added to macrophages. The activation of copper in caeruloplasmin in atherosclerotic lesions due to a localised acidic pH may help to explain why LDL oxidation occurs in these areas of the body.
Animals↗
Animal models that mimic human male reproductive defects.
All stages of male reproductive development and function are highly regulated, not only by the gonadotropins FSH, LH, and testosterone, but also by local control signals regulating cell function, mitosis, meiosis, and differentiation. The present approach to the treatment of many types of male infertility focuses on the use of suboptimal or defective sperm with protocols designed to overcome specific abnormalities. Elucidation of the molecular events required for normal male reproduction using animal model systems will eventually permit the design of new diagnostic procedures and therapeutic modalities. These advances may lead to enhancement or restoration of fertility in previously sterile men.
Animals↗
Evaluation of sperm function. What is available in the modern andrology laboratory?
Tests of sperm function markedly improve the clinician's ability to diagnose male factor infertility accurately. An understanding of the patient's sperm defect may improve the clinical management of male infertility. A wide spectrum of therapeutic options is available to the urologist treating the infertile male. IVF is now routinely employed for many types of male factor infertility with promising results. Micromanipulation has permitted men who, in the not so distant past, were considered infertile to experience fatherhood. In light of these promising therapeutic advances, accurate assessment of sperm function becomes crucial. Treatment options may be selected on the basis of specific functional defects of sperm. Sperm with an inability to bypass cervical mucus may be adequately treated with IUI. Oligospermic men with sperm capable of ova penetration can be treated with IVF, whereas men whose sperm are incapable of zona binding/penetration may be treated with micromanipulation. Sperm function assays provide a rationale for the selection of specific therapies to treat or bypass identified defects, and by providing comprehensive and accurate diagnosis, they make possible individually tailored therapies. Such individualized treatment choices may improve success rates for many male factor patients.
Cervix Mucus↗
Paracrine factors and the regulation of spermatogenesis.
MAIN PROBLEM: Although the gonadotropins and testosterone are required for normal spermatogenesis, it is believed that local control factors regulate spermatogenesis. For many years these regulatory factors had not been identified. Over the past five years, a number of growth factors have been identified in testis or isolated testicular cell types or secretions. Growth factors are key regulatory molecules which affect cell proliferation, meiosis, and differentiated function. These factors usually act in an autocrine (acting upon the cell which secreted it) or paracine (affecting another cell) manner and thus are involved in intercellular communications. METHODS: Growth factor secretion by testicular cell types or testis tissue has been analyzed using a variety of assays measuring cell proliferation in vitro, as well as assays using immunocytochemicals. Growth factor gene expression in testis has been analyzed by Northern blot analysis and in situ hybridization, which gives information concerning the stage and cell specific expression of the gene. Inbred strains of mice with mutations of deletions in a growth factor gene has been used to suggest the function of two specific factors in testicular development and growth. RESULTS: Among the growth factors expressed or secreted by testicular cell types, most are common to some other cell types in the body, such as transforming growth factors alpha and beta, epidermal growth factor, fibroblast-like growth factors, insulin-like growth factors, interleukins, endorphins, inhibin and activin, while others may be more testis specific such as mullerian inhibiting substance (anti-mullerian hormone) and Sertoli cell secreted growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Artificial intelligence in medicine and male infertility.
MAIN PROBLEM: fertility data is inadequately assessed by traditional statistical methods for a variety of reasons. First, the principal test of male fertility potential, the Semen Analysis (SA) is a composite of several dissimilar parameters, and the SA and other laboratory tests of fertility potential reflect physiological mechanisms that interact in complex ways. Second, patient data is often fragmented, obtained from multiple sources. Importantly, 2 patients are required for the final result. METHODS: Novel and powerful computational method, the neural network, was explored to analyze fertility data. An integrated series of programs was written in the C computer language to implement a back propagation algorithm. A model data analysis system was chosen, predicting the penetration of zona-free hamster ova by sperm (Sperm Penetration Assay (SPA)) and the distance travelled by the farthest swimming sperm (Penetrak Assay) from the SA, for these 2 assays are generally believed by the reproductive medical community to be independent of the SA. The classification accuracy of the neural network was compared to 2 standard statistical methods, linear discriminant function analysis (LDFA) and quadratic discriminant function analysis (QDFA). RESULTS: A neural network could be trained to correctly predict the Penetrak result in over 80% of assays it had not previously encountered, and another network could predict the SPA outcome in nearly 70%. The neural network was superior to LDFA and QDFA in predicting both assay outcomes (for Penetrak: LDFA = 64%, QDFA = 69%; for SPA: LDFA = 65%, QDFA = 45%).(ABSTRACT TRUNCATED AT 250 WORDS)
Algorithms↗
Tyrphostins inhibit Sertoli cell-secreted growth factor stimulation of A431 cell growth.
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Animals↗
A neural network to analyze fertility data.
OBJECTIVE: To program an artificial intelligence system, a neural network, and use it to predict results of sperm penetration in bovine cervical mucus (Penetrak assay; Serono Laboratories, Norwell, MA) and zona-free hamster egg penetration from the semen analysis. DESIGN: Results of 139 Penetrak assays, 1,416 zona-free hamster egg penetration assays, and the corresponding semen analyses were retrospectively analyzed by an artificial neural network. MAIN OUTCOME MEASURES: Classification errors of the neural network were compared with those of linear and quadratic discriminant function analyses. RESULTS: Data were separated into training and test sets. For the Penetrak result, linear and quadratic discriminant function analysis correctly predicted 58% and 74% of the training set results and only 64.1% and 69.2% of the test data, respectively. The neural network correctly predicted 92% of training set results and 80% of test set results. For the zona-free hamster egg penetration assay outcome, linear and quadratic discriminant function analysis correctly classified 66.3% and 46.0% of the training set and 64.9% and 44.7% of the test set, respectively. The neural network correctly classified 75.7% of the training data and 67.8% of the test data. CONCLUSIONS: Using the semen analysis, the neural network correctly classified 67.8% of zona-free hamster egg penetration assay results and 80% of Penetrak results it had not encountered previously, suggesting that this method of data analysis may be successfully employed to predict fertility potential.
Animals↗