RESUMEN
OBJECTIVE: To investigate whether expression of regulatory components of the cell division cycle can be used independently to predict survival and response to adjuvant therapy in glioblastomas. METHOD: A tissue micro-array, constructed using glioblastomas (n = 66), was stained using antibodies against minichromosome maintenance protein-2 (Mcm-2), expressed throughout the cell-division cycle; geminin, a protein that prevents re-initiation of DNA replication; and cyclin A, an S-phase cyclin. A semi-quantitative labelling index (LI) was calculated using an average of 18 high-power fields (hpf) in three replicate cores. The patients were divided into two groups: Group 1 (n = 50) underwent surgery and radiotherapy with 24 patients receiving temozolomide, and Group 2 (n = 16) received surgical treatment only. RESULTS: The LIs (median +/- IQR) for Group 1 were as follows: Mcm-2, 36.7% (22.9%-51.8%); geminin, 7.8% (5.8%-10.5%); and cyclin A, 4.2% (2.4%-6.9%). Elevated LIs, higher than the median, for geminin and cyclin A correlated with prolonged survival when the tumours received adjuvant therapy (Kaplan-Meier curves, p = 0.0046 and p = 0.0063 for geminin and cyclin A, respectively). Linear regression analysis revealed positive correlations with survival for Mcm-2 (p = 0.0376), geminin (p = 0.0006) and cyclin A (p = 0.004). In Group 2, there was no relationship between the patient survival and the LI for any marker. CONCLUSIONS: Geminin and cyclin A, each show potential as independent prognostic markers in glioblastomas receiving adjuvant therapy. This may reflect the fact that both geminin and cyclin A estimate proliferating tumour cell subpopulations sensitive to radio/chemotherapy. These markers could provide valuable prognostic information, even in small biopsies, especially if combined with O(6)MGMT expression and 1p;19q deletion status.
Asunto(s)
Neoplasias Encefálicas/patología , Ciclo Celular/fisiología , División Celular/fisiología , Glioblastoma/patología , Anciano , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor , Biopsia , Neoplasias Encefálicas/cirugía , Proteínas de Ciclo Celular/análisis , Quimioradioterapia , Terapia Combinada , Ciclina A/análisis , Ciclina A/metabolismo , Femenino , Geminina/análisis , Geminina/metabolismo , Glioblastoma/cirugía , Humanos , Inmunohistoquímica , Estado de Ejecución de Karnofsky , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Componente 2 del Complejo de Mantenimiento de Minicromosoma/análisis , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Recurrencia Local de Neoplasia/epidemiología , Recurrencia Local de Neoplasia/patología , Pronóstico , Fase S/efectos de los fármacos , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
We previously developed an immunohistochemical method for estimating cell cycle state and phase in tissue samples, including biopsies that are too small for flow cytometry. We have used our technique to examine whether primary abnormalities of the cell cycle exist in laryngeal neoplasia. Antibodies against the markers of cell cycle entry, minichromosome maintenance protein-2 (Mcm-2) and Ki67, and putative markers of cell cycle phase, cyclin D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis) were applied to paraffin-embedded sections of normal larynx (n = 8), laryngeal dysplasia (n = 10) and laryngeal squamous cell carcinoma (n = 10). Cells expressing each marker were determined as a percentage of total cells, termed the labelling index (LI), and as a percentage of Mcm-2-positive cells, termed the labelling fraction (LF). The frequency of coexpression of each putative phase marker was investigated by confocal microscopy. There was a correlation between Mcm-2 and Ki67 LIs (rho = 0.93) but Mcm-2 LIs were consistently higher. All cells expressing a phase marker coexpressed Mcm-2, whereas Ki67 was not expressed in a proportion of these cells. The putative phase markers showed little coexpression. Labelling index values increased on progression from normal larynx through laryngeal dysplasia to squamous cell carcinoma for Mcm-2 (P = 0.001), Ki67 (P = 0.0002), cyclin D1 (P = 0.015), cyclin A (P = 0.0001) and cyclin B1 (P = 0.0004). There was no evidence of an increase in the LF for any phase marker. Immunohistochemistry can be used to estimate cell cycle state and phase in laryngeal biopsies. Our data argues against primary cell cycle phase abnormalities in laryngeal neoplasia.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias Laríngeas/patología , Biomarcadores de Tumor/biosíntesis , Ciclo Celular , Proteínas de Ciclo Celular/biosíntesis , Fase G1 , Fase G2 , Humanos , Inmunohistoquímica , Antígeno Ki-67/biosíntesis , Microscopía Confocal/métodos , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Mitosis , Proteínas Nucleares/biosíntesis , Adhesión en Parafina , Fase S , Sensibilidad y EspecificidadRESUMEN
Squamous dysplasia of the oral cavity indicates increased risk of progression to squamous cell carcinoma (SCC). An important advance would be the development of a minimally invasive assay for identification of oral SCC and dysplasia. We have investigated the suitability in this context of immunostaining oral smears for minichromosome maintainance proteins (MCMs), sensitive and specific biomarkers of cell cycle entry. Immunohistochemical examination of 66 oral tissue samples showed a greater frequency of Mcm-2 expression in surface layers of moderate/severe dysplasia and SCC compared to benign keratosis/mild dysplasia. Immunocytochemistry for Mcm-2/Mcm-5 was performed on 101 oral smears. Conventional smears included 23 from normal mucosa, benign proliferative disease and mild dysplasia, all of which were MCM negative. Of 52 conventional smears of SCC tissue samples, 18 were inadequate. However, MCM-positive cells were present in 33/34 adequate samples. Of 26 liquid-based cytology smears, 19 out of 20 smears from SCC were adequate and all were MCM positive. Six smears from benign lesions were adequate and MCM negative. We conclude that MCMs are promising markers for early detection of oral SCC and dysplasia, particularly in a liquid-based cytology platform. Detection of MCMs would be amenable to automation and potentially applicable in the developing world. Further studies are now warranted.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/diagnóstico , Proteínas de Ciclo Celular/análisis , Neoplasias de la Boca/diagnóstico , Proteínas Nucleares/análisis , Lesiones Precancerosas/diagnóstico , Biomarcadores de Tumor/biosíntesis , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/biosíntesis , Humanos , Inmunohistoquímica , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Mucosa Bucal/química , Mucosa Bucal/patología , Neoplasias de la Boca/patología , Proteínas Nucleares/biosíntesis , Adhesión en Parafina , Lesiones Precancerosas/patología , Sensibilidad y Especificidad , Coloración y Etiquetado , Recolección de Tejidos y Órganos/métodosRESUMEN
The complete androgen insensitivity syndrome (CAIS), caused by mutations in the androgen receptor (AR) gene, is associated with abnormal testicular development and an increased risk of germ cell malignancy. Previous histological studies in CAIS have selected patients purely on the basis of clinical diagnosis and were mostly based on small numbers, many of whom were post-pubertal. Here, we present 44 cases of CAIS, each with molecular pathological confirmation of an AR mutation. The median age at gonadectomy was 5.5 years (5.5; IQR 1-13). We have been able, therefore, to investigate testicular development in infancy, childhood and puberty, and estimate the incidence of premalignant change in this series. In addition, we have investigated whether the presence of epididymides and/or vasa deferentia in CAIS, previously shown to be associated with residual activity of mutant ARs, is related to a particular testicular phenotype. Epididymides/vasa deferentia were present in 36% of cases and these patients showed varying degrees of seminiferous tubule maturation at puberty above those without epididymides/vasa deferentia (p = 0.003). There were no other histological differences between these patient groups. In both groups, features of testicular degeneration and dysgenesis were present and germ cell development was delayed, with prolonged expression of the gonocyte markers, placental-like alkaline phosphatase and activator protein-2gamma. Germ cell numbers rapidly declined after the first year of life (R(2) = 0.42). Only two cases of carcinoma in situ were identified in our study and both patients were postpubertal (17 and 53 years). From these results and the literature, we conclude that the risk of premalignant change in germ cells is low before and during puberty. Patients can be advised, therefore, that gonadectomy can be delayed to allow for a natural puberty, with low risk of malignant transformation. Our study only included one patient over 18 years, so we cannot comment on the risk of malignant transformation in later life.
Asunto(s)
Síndrome de Resistencia Androgénica/patología , Testículo/patología , Adolescente , Adulto , Envejecimiento , Síndrome de Resistencia Androgénica/metabolismo , Síndrome de Resistencia Androgénica/cirugía , Niño , Preescolar , Epidídimo/patología , Células Germinativas/patología , Humanos , Inmunohistoquímica/métodos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Orquiectomía , Receptores Androgénicos/análisis , Riesgo , Túbulos Seminíferos/patología , Neoplasias Testiculares/patología , Conducto Deferente/patologíaRESUMEN
We have investigated whether immunohistochemical markers can identify differences in cell cycle phase distribution in ovarian serous neoplasms, including borderline tumours of different grades. Sections of normal ovary (n=18), serous cystadenoma (n=21), borderline serous tumours (n=21) and serous cystadenocarcinoma (n=15) were analysed by immunohistochemistry using markers of cell cycle entry (Mcm-2) and cell cycle phase, including cyclin D1 (mid-to-late G1), cyclin A (S phase), cyclin B1 (G2 phase) and phosphohistone H3 (mitosis). Double-labelling confocal microscopy confirmed marker phase specificity and phase estimations were corroborated by flow cytometry. On progression from normal ovary through serous cystadenoma and borderline tumours to cystadenocarcinomas, expression of Mcm-2 (P<0.0001), cyclin D1 (P=0.002), cyclin A (P<0.0001), cyclin B1 (P<0.0001) and phosphohistone H3 (P<0.0001) increased, paralleled by an increase in the S-phase fraction (cyclin A : Mcm-2 ratio; P=0.002). Borderline tumours of increasing grade also showed increased Mcm-2 and cyclin A expression, together with an increase in the S-phase fraction. Immunohistochemistry can be used to estimate cell cycle phase distribution in ovarian serous neoplasms, giving results similar to flow cytometric analysis and enabling direct assessment of tumour heterogeneity. Immunohistochemical estimates of the S-phase fraction may identify serous borderline tumours likely to exhibit malignant progression and/or select serous cystadenocarcinomas likely to respond to adjuvant therapy.
Asunto(s)
Biomarcadores de Tumor/análisis , Proteínas de Ciclo Celular/análisis , Ciclo Celular , Cistadenocarcinoma Seroso/patología , Neoplasias Ováricas/patología , Proteínas de Ciclo Celular/biosíntesis , Transformación Celular Neoplásica , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica/métodos , Microscopía Confocal , Lesiones Precancerosas , Manejo de EspecímenesRESUMEN
OBJECTIVE: Minichromosome maintenance protein 2 (Mcm2) is an accurate indicator of cell cycle entry in tissue samples, but its expression in inflammatory bowel disease (IBD) has not previously been investigated. We have used immunohistochemistry to assess the expression of Mcm2, in comparison to the existing proliferation marker Ki-67, in active IBD and IBD without inflammatory activity. MATERIALS AND METHODS: For this experimental study, sections from colonic biopsy and resection specimens of 48 patients with IBD (5 inactive/quiescent Crohn's disease (CD), 13 active CD, 19 inactive/quiescent ulcerative colitis (UC) and 11 active UC) and 15 normal controls were immunostained with antibodies to Mcm2 and Ki-67. The percentage of immunopositive epithelial nuclei was determined by calculating a labelling index (LI) for entire glands and for gland thirds (superficial, middle and basal). RESULTS: The Mcm2 LI was significantly increased in the superficial third of glands in active vs. inactive/quiescent UC (P < 0.0001) and active vs. inactive/quiescent CD (P = 0.001). The Mcm2 LI was significantly greater than the Ki-67 LI in active IBD, both in entire glands (P < 0.0001) and in the superficial third of glands (UC, P = 0.001; CD, P = 0.0002). Mcm2 LIs for entire glands were significantly higher in UC (all cases) compared to CD (all cases) (P = 0.032). CONCLUSIONS: There is increased cell cycle entry, as indicated by expression of Mcm2 and to a lesser extent Ki-67, in the superficial third of colonic glands in active IBD compared to inactive/quiescent IBD. Detection of Mcm2 may contribute to improved histological assessment of small tissue biopsies and may enable the development of a direct stool-based test for detection of active IBD and potentially for assessment of disease activity.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Colon/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Humanos , Antígeno Ki-67/biosíntesisRESUMEN
Histological classification of laryngeal epithelial lesions is highly subjective, and methods of cytological detection are not well developed. Improved determination of aberrant cell cycle entry may allow increased objectivity in histological assessment and enable the development of less invasive diagnostic cytology tests. Sections of normal larynx (n=10), laryngeal dysplasia (n=20) and laryngeal squamous cell carcinoma (SCC) (n=10) were classified according to the Ljubljana classification and stained for markers of cell cycle entry, minichromosome maintenance protein-2 (Mcm-2) and Ki67. Expression patterns were compared using double labelling confocal microscopy. There was a correlation between Mcm-2 and Ki67 labelling indices (rho=0.93; 95% CI [0.84, 0.97]) and both markers showed increased expression from normal epithelium to SCC (Mcm-2, P=0.001; Ki67, P=0.0002). Importantly, there was minimal expression of Mcm-2 or Ki67 in the most superficial layers of normal larynx and abnormal or atypical hyperplasia, in contrast to carcinoma in situ and SCC. Clusters of Mcm-2/5-positive cells were present in cytological preparations from SCC, but not from those showing atypical hyperplasia or inflammation in non-neoplastic tissue. Minichromosome maintenance protein-2 staining may increase the objectivity and reliability of histological grading of laryngeal epithelial lesions. Laryngeal brushings, combined with immuno-enhanced liquid-based cytology, could be useful, as a less invasive approach, to the detection of laryngeal malignant and premalignant lesions.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Antígeno Ki-67/metabolismo , Neoplasias Laríngeas/metabolismo , Proteínas Nucleares/metabolismo , Antígenos de Neoplasias/metabolismo , Biopsia , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Carcinoma de Células Escamosas/patología , Ciclo Celular , Replicación del ADN , Humanos , Hiperplasia/metabolismo , Hiperplasia/patología , Técnicas para Inmunoenzimas , Neoplasias Laríngeas/patología , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patologíaRESUMEN
Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium (n=6), undifferentiated HPV positive VIN 1 (n=3), VIN 2 (n=8) and VIN 3 (n=20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of 'snapshots' of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Replicación del ADN , ADN de Neoplasias/metabolismo , Neoplasias de la Vulva/genética , Neoplasias de la Vulva/metabolismo , Biopsia , Carcinoma in Situ/patología , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas de Unión al ADN , Femenino , Histonas/genética , Humanos , Técnicas para Inmunoenzimas , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe , Neoplasias de la Vulva/patologíaRESUMEN
There are very few reports in the literature dealing with the neuropathology of infant head injury, and the question of whether diffuse traumatic brain damage [diffuse axonal injury (DAI)] occurs in such children has not yet been reliably established by detailed neuropathological studies. We report the findings in the brains of a series of 37 infants aged 9 months or less, all of whom died from inflicted head injuries, and 14 control infants who died of other causes. Axonal damage was identified using immunohistochemistry for beta-amyloid precursor protein. Full clinical details were available for each case, the most constant of which in the study cohort was an episode of significant apnoea at presentation, found to have been recorded in 75% of cases. Global hypoxic damage was the most common histological finding. Widespread axonal damage, interpreted as vascular, was present in 13 cases, but widespread traumatic axonal injury was found in only two children, both of whom had severe head injuries with multiple skull fractures. Epidural cervical haemorrhage and focal axonal damage to the brainstem and the spinal nerve roots, found in 11 cases but not in controls, indicate that the craniocervical junction is vulnerable in infant head injury, the neuropathology being that of stretch injury from cervical hyperextension/flexion. Damage to this region could account for the observed apnoea, which could in turn lead to hypoxic damage and brain swelling. The observation that the predominant histological abnormality in cases of inflicted head injury in the very young is diffuse hypoxic brain damage, not DAI, can be explained in one of two ways: either the unmyelinated axon of the immature cerebral hemispheres is relatively resistant to traumatic damage, or in shaking-type injuries the brain is not exposed to the forces necessary to produce DAI.
Asunto(s)
Encéfalo/patología , Maltrato a los Niños/diagnóstico , Traumatismos Craneocerebrales/patología , Hipoxia Encefálica/patología , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Edema Encefálico/epidemiología , Edema Encefálico/patología , Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Estudios de Cohortes , Comorbilidad , Traumatismos Craneocerebrales/epidemiología , Traumatismos Craneocerebrales/metabolismo , Lesión Axonal Difusa/epidemiología , Lesión Axonal Difusa/metabolismo , Lesión Axonal Difusa/patología , Femenino , Medicina Legal , Humanos , Hipoxia Encefálica/epidemiología , Inmunohistoquímica , Lactante , Recién Nacido , Masculino , Traumatismos del Cuello/epidemiología , Traumatismos del Cuello/patología , Fracturas Craneales/epidemiología , Fracturas Craneales/patología , Raíces Nerviosas Espinales/patología , Reino Unido/epidemiologíaRESUMEN
AIMS: Previous uncontrolled reports have suggested that appendiceal inflammation may occur as a discontinuous lesion in ulcerative colitis. This study aims semiquantitatively to compare the prevalence and histological features of appendiceal inflammation in patients with ulcerative colitis and Crohn's disease, using colonic carcinoma and acute appendicitis specimens as controls. METHODS AND RESULTS: Surgical pathology records and original histological slides for the period 1980-1994 were examined. The prevalence of appendiceal inflammation in ulcerative colitis (24/50, 48%), was higher than in colonic carcinoma (5/65, 8%, P < 0.001), but was similar to that in Crohn's disease (14/27, 52%). Appendiceal inflammation with caecal sparing was seen in nine out of 24 specimens with ulcerative colitis (37%), two out of nine (22%) with Crohn's disease and five out of 65 (8%) with colonic carcinoma. Inflamed appendixes from patients with inflammatory bowel disease showed histological features typical of ulcerative colitis and Crohn's disease rather than acute appendicitis and were significantly less likely to have transmural inflammation. There had been a previous appendicectomy in 3% of ulcerative colitis patients compared with 8% of colonic carcinoma specimens and 21% (P < 0.01) Crohn's disease controls. CONCLUSION: In ulcerative colitis, as in Crohn's disease, appendiceal inflammation commonly occurs as a skip lesion and histologically resembles the colonic disease rather than acute appendicitis. The low prevalence of appendicectomy supports the hypothesis that the appendix itself may have a central role in the pathogenesis of ulcerative colitis.
Asunto(s)
Apendicitis/epidemiología , Apendicitis/inmunología , Colitis Ulcerosa/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apéndice/inmunología , Carcinoma/epidemiología , Carcinoma/inmunología , Colitis Ulcerosa/inmunología , Neoplasias del Colon/epidemiología , Neoplasias del Colon/inmunología , Femenino , Humanos , Inflamación , Masculino , Persona de Mediana EdadRESUMEN
The hypogonadal (hpg) mouse has a congenital deficiency in gonadotrophin-releasing hormone and the gonads consequently lack exposure to endogenous gonadotrophins during development. To determine the effect of FSH on Leydig cell function in these animals adult hpg mice were injected twice daily with FSH (2 micrograms injections) or LH (40 ng injections, the presumed LH contamination of FSH used). Following FSH treatment there was a clear stimulation of the seminiferous epithelium and in animals injected with FSH plus [3H]thymidine, the incorporation of label was largely confined to the germ cells with no apparent uptake by the Sertoli cells. In FSH-treated testes the Leydig cells contained numerous large lipid droplets, similar to the unstimulated hpg testis. There was no evidence of the interstitial hyperplasia which is observed following injection of high doses of LH (2 micrograms twice daily). There was no change in basal androgen content of the testis in vivo following FSH treatment but injection of a maximal dose of human chorionic gonadotrophin (hCG), 1 h before death, markedly increased testicular androgen content only in the FSH-treated group. Testicular androgen production in vitro was significantly increased following FSH treatment both under basal conditions (FSH-treated, 17.4 pmol/testis; control, 1.46 pmol/testis) and during stimulation by hCG (FSH-treated, 940 pmol/testis; control, 81 pmol/testis). Associated with the increased androgen production following FSH treatment there were significant increases in the activities of three steroidogenic enzymes; cholesterol side-chain cleavage (186-fold increase over control), 17 alpha-hydroxylase (103-fold increase) and 17-ketosteroid reductase (177-fold increase).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hormona Folículo Estimulante/farmacología , Hipogonadismo/patología , Células Intersticiales del Testículo/efectos de los fármacos , Andrógenos/biosíntesis , Animales , Gonadotropina Coriónica/farmacología , Células Intersticiales del Testículo/patología , Hormona Luteinizante/farmacología , Masculino , Ratones , Ratones Mutantes , Estimulación Química , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
Hypogonadal (hpg) mutant mice, with a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH), and testicular feminized (tfm) mice, which lack a functional androgen receptor, were used to study the effects of the potent GnRH agonist 'Zoladex' (ICI 118630; D-Ser (Bu(t))6, Azgly10-GnRH) on pituitary and gonadal function. Zoladex (0.5 mg) in a sustained-release lactide-glycolide copolymer depot was administered subcutaneously under anaesthesia and was left in place for 7 days, after which time the effects of the drug upon pituitary and serum gonadotrophin concentrations, glycoprotein hormone subunit mRNAs and testicular morphology were investigated. At the pituitary level, Zoladex treatment resulted in a substantial reduction in LH content in normal males, and LH content was depressed in hpg mice even below the basal levels normally found in these mutants. Pituitary LH content in the Zoladex-treated animals was depressed in the tfm groups, but not to the same levels as those found in the normal and castrated normal mice. Zoladex treatment at the time of castration prevented the post-operative elevation in serum LH associated with castration alone. In the androgen-deficient tfm mouse, Zoladex did not depress the normally elevated serum LH levels. Serum LH in the hpg animals was, in all cases, below the limit of detection of the assay. Pituitary FSH content was depressed into the hpg range in both the normal and castrated animals, but there was no further depression in the hpg mice. The pituitary content was reduced in the tfm mice, again the effects not being as dramatic as in the normal and castrated animals. Serum FSH content, as measured by radioimmunoassay, was depressed by 50% in normal mice; there was no reduction in the hpg mice, however. With regard to pituitary gonadotrophic hormone gene expression, Zoladex administration to normal mice caused a dramatic reduction in LH beta mRNA content, to a level approximating that found in untreated hpg mice. The drug also depressed LH beta mRNA in the castrated group to the hpg range when given at the time of castration, whereas in untreated castrated mice there was a significant increase in LH beta mRNA. In the tfm mouse, which can be considered as a model for long-term failure of androgen feedback, Zoladex again induced a fall in LH beta mRNA, but not to the same extent as in the normal and normal castrated group. Zoladex had no effect on the already low levels of LH beta mRNA found in hpg mice.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Buserelina/análogos & derivados , Feminización/metabolismo , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Hipogonadismo/metabolismo , Hipófisis/efectos de los fármacos , Testículo/efectos de los fármacos , Andrógenos/metabolismo , Animales , Buserelina/farmacología , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/metabolismo , Gonadotropinas/genética , Gonadotropinas/metabolismo , Goserelina , Hormona Luteinizante/sangre , Hormona Luteinizante/metabolismo , Masculino , Ratones , Orquiectomía , Tamaño de los Órganos , Hipófisis/metabolismo , Valores de Referencia , Testículo/anatomía & histología , Testículo/metabolismoRESUMEN
Abstract The effects of ovariectomy and oestrogen feedback for 10 days upon pituitary and serum luteinizing hormone (LH) content, pituitary glycoprotein subunit messenger ribonucleic acid (mRNA) and prolactin mRNA content in normal females, female hypogonadal mice and hypothalamic grafted female hypogonadal mice, bearing a graft of normal mouse preoptic area tissue into the third ventricle, have been investigated. In normal females ovariectomy resulted in a rise in serum LH, LHbeta-subunit and common alpha-subunit mRNAs with no significant change in pituitary LH content or follicle-stimulating hormone (FSH) beta-subunit mRNA. In the hypogonadal females, preoptic area grafting resulted in an elevation in all of the above parameters into the normal range. Ovariectomy in this group resulted in a further elevation of serum LH, LHbeta-subunit and alpha-subunit mRNAs with no change in pituitary LH content or FSHbeta-subunit mRNA, which in all cases were comparable to ovariectomized normal animals. Oestrogen treatment caused a fall in pituitary LH content and the serum LH fell below the detection of the assay. LHbeta-subunit and a-subunit mRNA mirrored this fall but there was no change in FSHbeta-subunit hybridization. These experiments suggest that even though normal neuronal input to the gonadotrophin-releasing hormone neurons is disrupted, oestrogen-induced negative feedback can still occur in grafted female hypogonadal animals. Gonadotrophin-releasing hormone neurons are reported to lack oestrogen receptors but feedback within this graft by co-transplanting oestrogen-sensitive neurons remains a possibility, as does feedback at the level of the host median eminence where graft axons extend to the pituitary portal vessels. The similarity of the response in normal and grafted animals indicates that these actions of oestrogen may be effected predominantly at the pituitary level.
RESUMEN
Hypogonadal (hpg) mice have a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH) and the gonads consequently lack exposure to gonadotrophins during development. We injected male hpg mice with LH for 10 days to investigate whether LH alone can stimulate normal steroidogenesis in these animals. Control animals had an inactive interstitium and very few germ cells. Testicular content of androgens was undetectable by radioimmunoassay in control animals unless a single injection of LH was given 1 h before death, when androgens were just detectable. Control testes incubated in vitro with [3H]pregnenolone demonstrated that without gonadotrophin stimulation pregnenolone was metabolized only to progesterone in significant amounts. Assay for cholesterol side-chain cleavage cytochrome P450 (P450scc) mRNA showed basal expression in saline-treated hpg mouse testis. LH treatment induced hypertrophy and hyperplasia of Leydig cells and division of germ cells. Testicular androgen content increased significantly, with testosterone and androstenedione as the major androgens. LH-treated testes incubated with [3H]pregnenolone in vitro had a greater synthetic capacity for testosterone, suggesting an increase in 17 alpha-hydroxylase/C17-20-lyase activity. Basal and human chorionic gonadotrophin-stimulated androgen production in vitro increased markedly following LH treatment to levels previously described in the normal adult animal. LH treatment caused a rapid and transient increase in the hybridization of P450scc mRNA which was sevenfold greater than that of saline-treated controls when the animals were killed 1 h after the last injection but fell to control levels within 24 h of cessation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Andrógenos/biosíntesis , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Hipogonadismo/metabolismo , Hormona Luteinizante/farmacología , ARN Mensajero/metabolismo , Testículo/metabolismo , Androstenodiona/biosíntesis , Animales , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Ratones , Ratones Mutantes , Testosterona/biosíntesisRESUMEN
In an analysis of the chromosomes in 332 metaphase II figures from 3 triple heterozygous rams (51,xy,t1t2t3) and a further 84 figures from 3 normal rams (54,xy), there were no hypermodal cells in the normal rams but 9 such cells were found in the triple heterozygotes, giving a mean aneuploid frequency of 5.42% which was similar to the levels previously found in the single heterozygotes. Forty hypomodal cells were found in the 6 rams of which a number would have lost chromosomes due to lagging at anaphase I. Individual variation in the aneuploid metaphase II frequency was observed in the triple heterozygotes. A significant surplus of secondary spermatocytes of normal karyotype and a deficit of 25,t1t3 were found in the metaphase II figures from the triple heterozygotes. There was also a significant increase of normal progeny and a deficit of 52,t1t3 and 53,t2 progeny from the matings of triple heterozygous rams and normal ewes. It is possible that the significantly uneven distribution of segregation products in the triple heterozygotes might have been the result of either cell selection or degeneration during spermatogenesis.
Asunto(s)
Aneuploidia , Ovinos/genética , Espermatozoides/ultraestructura , Translocación Genética , Animales , Heterocigoto , MasculinoRESUMEN
Meiotic preparations were made from testicular material obtained after surgical castration of eight stallions (Equus caballus) with normal spermatogenesis. The material was examined after conventional Giemsa staining and C-banding. C-banding demonstrated that the Y chromosome at diakinesis associated with the short arm of the X chromosome. In 315 cells at diplotene or diakinesis, 56 (17.7%) had univalents and 51 (16.1%) of these involved the sex chromosomes. The overall mean chiasma number was 54.4 +/- 1.8 SD, and the mean calculated nondisjunction (ND) frequency was 3.4%. These results are discussed in relation to other domestic species.