RESUMO
Reduced serum testosterone (T), or hypogonadism, affects millions of men and is associated with many pathologies, including infertility, cardiovascular diseases, metabolic syndrome, and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However, TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus, there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs), proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs, the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively, human induced pluripotent stem cells (hiPSCs), which are expandable in culture and have the potential to differentiate into all somatic cell types, have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis, synthesized T rather than cortisol, secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol, and displayed ultrastructural features resembling LCs. By contrast, hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Células Intersticiais do Testículo/enzimologia , Testosterona/metabolismo , Expressão Gênica , Humanos , Células Intersticiais do Testículo/ultraestrutura , Masculino , TranscriptomaRESUMO
This review article provides a critical analysis of the structure and molecular mechanisms of the microtubule axoneme of cilia and sperm flagella and their associated elements required for male fertility.A broad range of genetic and molecular defects (ciliopathies) are considered in the context of human diseases involving impaired motility in cilia and sperm flagella, providing provocative thought for future research in the area of male infertility.
Assuntos
Infertilidade Masculina/patologia , Técnicas de Reprodução Assistida , Espermatozoides/ultraestrutura , Axonema/patologia , Axonema/ultraestrutura , Cílios , Flagelos/patologia , Flagelos/ultraestrutura , Humanos , Infertilidade Masculina/genética , Masculino , Espermatozoides/patologiaRESUMO
CONTEXT: Pheochromocytomas and paragangliomas (pheo/pgl) are neuroendocrine tumours derived from chromaffin cells. Although mostly benign, up to 26% of pheo/pgl will undergo malignant transformation. Reliable histological signs to differentiate benign pheo/pgl from malignant tumours are currently lacking. Increased IGF-1R expression has been shown during progression to metastatic phenotypes of several types of cancer. OBJECTIVE: To analyse the distribution and expression of the IGF-1R in pheo/pgl of different genetic origin and degree of malignancy. MEASUREMENTS: We studied the expression of the IGF-1R protein by immunohistochemistry, in 40 primary tumours from patients with pheo/pgl from different genetic aetiology (11 of 29 metastatic/nonmetastatic diseases). RESULTS: We found a strong association between increased expression of IGF-1R and malignant behaviour regardless of the age at diagnosis and the genetic aetiology. IGF-1R labelling was mostly weak in primary tumours from patients with nonmetastatic pheo/pgl. Conversely, intense IGF-1R labelling was predominant in cases of pheo/pgl with confirmed metastatic disease. The risk of metastases was 11·7 times higher if tumour IGF-1R labelling was intense independently of age at diagnosis. The probability of remaining free of metastases was higher in patients with pheo/pgl scored weak for IGF-1R at 60 months and more than twofold higher at 120 months of follow-up than in patients with intense IGF-1R labelling in their primary tumours. CONCLUSIONS: Our results strongly suggest that IGF-1R is associated with malignancy in familial pheo/pgl and that IGF-1R expression in the primary tumour might be a useful tool to detect those patients harbouring pheo/pgl who have an increased risk of metastasis.
Assuntos
Paraganglioma/metabolismo , Paraganglioma/patologia , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Receptor IGF Tipo 1/metabolismo , Adolescente , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Acrosome biogenesis is a key event in sperm differentiation that depends on the proper interaction between the Golgi complex and the nuclear envelope of early spermatids. We studied the development, structure and biochemical characteristics of human acrosomes in germ cells and spermatozoa from testicular biopsies and semen samples of fertile men and patients with acrosomeless spermatozoa (globozoospermia). A set of proteins collectively known as the perinuclear theca (PT), which has been related to acrosomal development in many mammalian species, were also investigated. METHODS: We evaluated spermatozoa from five males with globozoospermia and six fertile men, and immature germ cells from testicular biopsies of one globozoospermic patient and three men with obstructive azoospermia. Samples were assessed by transmission electron microscopy, immunofluorescence microscopy, ultrastructural immunocytochemistry and proteomic analysis by western blot. RESULTS: In normal spermiogenesis, the development of the acrosome depends on the correct formation of Golgi-derived proacrosomal vesicles and simultaneous modifications in the nuclear envelope. PT proteins are consistently found in proacrosomic vesicles, localize underneath the acrosome and expand over the nuclear surface along acrosome biogenesis. In fertile men, the PT is composed of six proteins, similar to those previously described for other mammals (16, 22, 29, 34, 50 and 68 kDa). In globozoospermia, abnormal proacrosomal vesicles and paranuclear multivesicular and multilamellar structures were observed that resulted in acrosomes insufficiently developed or detached from the nuclear envelope. PT proteins, dissociated from the acrosomes, were ectopically localized in the cytoplasm. Proteomic analysis showed a significant decrease in all six PT proteins. CONCLUSIONS: The alterations observed during early acrosome biogenesis in globozoospermia are due to anomalous development of Golgi-derived proacrosomic vesicles, failure of PT proteins to properly associate with the nuclear surface and significant deficiencies in specific PT components that are necessary for proper acrosome formation, implantation and expansion over the spermatid nucleus.
Assuntos
Acrossomo/fisiologia , Imuno-Histoquímica/métodos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Proteômica/métodos , Espermatozoides/anormalidades , Animais , Biópsia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Feminino , Células Germinativas/citologia , Complexo de Golgi/metabolismo , Humanos , Infertilidade Masculina/diagnóstico por imagem , Masculino , Microscopia Eletrônica de Transmissão/métodos , Espermátides/patologia , Espermatozoides/patologia , Testículo/patologia , UltrassonografiaRESUMO
Transgenic mice bearing a construct in which the expression of the SV40 oncogene is directed by the AMH promoter (AT mice) develop testicular tumours in adult life. We aimed to study early steps of tumour development and characterize tumours at different ages by histological, morphometric, and immunohistochemical techniques. One- to 3-month-old AT mice depicted multifocal Leydig cell hyperplasia. The testicular volume occupied by interstitial tissue was significantly higher in 3-month-old AT mice in comparison with littermate controls. Between 5 1/2 and 7 months, microscopic interstitial tumours developed that progressively evolved to form large confluent areas of high mitotic index in 7- to 14-month-old AT mice. Tumour cells had the characteristics and histoarchitecture of Leydig cells, or formed solid cord-like structures reminiscent of those seen in Sertoli cell tumours. Hyperplastic areas and tumours diffusely expressed 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in Leydig cell areas. AMH expression was negative in Leydig cell conglomerates and tumours and variable in cord-like tumours. The SV40 T antigen and markers of cell proliferation (PCNA) were intensely positive in hyperplastic cells and tumours. Control mice of similar ages showed neither hyperplasia nor tumours, and SV40 T expression was always negative. In conclusion, transgenic mice develop large testicular tumours that are preceded by interstitial hyperplasia and microtumours. The histological and immunohistochemical phenotype of tumours (Leydig and Sertoli cell differentiation, positive 3beta-HSD, and variable AMH) suggests a mixed differentiation of somatic cells of the specialized gonadal stroma. The finding that an oncogene directed by a promoter specifically active in fetal Sertoli cells has given rise to testicular tumours of mixed differentiation is compatible with a common origin of Leydig and Sertoli cells from the specific stroma of the gonadal ridge, as supported by double labelling experiments in fetal mice showing co-localization of the transgene with Sertoli and Leydig cell markers.
Assuntos
Tumor de Células de Leydig/patologia , Neoplasias Testiculares/patologia , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Diferenciação Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Hiperplasia/patologia , Tumor de Células de Leydig/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Camundongos , Camundongos Transgênicos , Células de Sertoli/patologia , Neoplasias Testiculares/metabolismoRESUMO
Fertilization in mammals occurs via a series of well-defined events in the secluded environment of the female reproductive tract. The mode of selection of the fertilizing spermatozoon nevertheless remains unknown. As has become evident during in vitro fertilization by sperm microinjection into the oocyte, abnormal spermatozoa can successfully fertilize oocytes. Under these extreme conditions, post-fertilization events, early embryonic development and implantation are significantly compromised indicating that the contribution of spermatozoa extends beyond sperm penetration. Microscopic identification of normal spermatozoa is a well-standardized procedure but insights into the mechanisms that lead to aberrant sperm differentiation and into the subcellular nature of sperm abnormalities have only recently begun to be obtained. The spermatozoon is the result of a complex development in which spermatid organelles give rise to various structural components with characteristic functions. Similar to other differentiated cells, the spermatozoon has a specific pathology that is most clearly identified by ultrastructural evaluation coupled with immunocytochemistry and molecular techniques. This multidisciplinary approach allows the precise characterization of sperm abnormalities, including structural, molecular and functional aspects. We summarize here studies of the physiopathology of spermiogenesis in two abnormal sperm phenotypes of infertile men: dysplasia of the fibrous sheath and acephalic spermatozoa/abnormal head-tail attachment. The characterization of the abnormalities of the tail cytoskeleton and centrioles has uncovered aspects of the subcellular basis of pathological spermiogenesis, has suggested experimental approaches to explore the nature of these anomalies and has opened the way for genetic studies that will ultimately lead to the design of the therapeutic tools of the future.
Assuntos
Espermatogênese/fisiologia , Espermatozoides/anormalidades , Espermatozoides/patologia , Animais , Feminino , Humanos , Infertilidade Masculina/etiologia , Infertilidade Masculina/patologia , Masculino , Cabeça do Espermatozoide/patologia , Cabeça do Espermatozoide/ultraestrutura , Espermatozoides/citologia , Espermatozoides/ultraestruturaRESUMO
Understanding the cellular events during fertilization in mammals is a major challenge that can contribute to the improvement of future infertility treatments in humans and reproductive performance in farm animals. Of special interest is the role of the oocyte and sperm cytoskeleton during the initial interaction between gametes. The aim of this chapter is to describe methods for studying cytoskeletal features during in vitro fertilization after intracytoplasmic sperm injection (ICSI) in humans. The following protocols will provide a detailed description of how to perform immunodetection and imaging of human eggs, zygotes, and sperm by fluorescence (confocal and epifluorescence) and electron microscopy.
Assuntos
Citoesqueleto/metabolismo , Injeções de Esperma Intracitoplásmicas/métodos , Citoesqueleto/ultraestrutura , DNA/metabolismo , Fertilização , Humanos , Imuno-Histoquímica , Masculino , Microscopia de Fluorescência , Oócitos/citologia , Oócitos/metabolismo , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Fixação de Tecidos , Zigoto/citologia , Zigoto/metabolismoRESUMO
CONTEXT: Although gonadotropins and testosterone are high in the fetal/early postnatal periods, Sertoli cells remain immature and spermatogenesis does not progress. We hypothesized that Sertoli cells do not respond to testosterone because they do not express the androgen receptor. OBJECTIVE: The objective of the study was to describe the precise ontogeny of androgen receptor expression in the human testis from fetal life through adulthood. DESIGN: This was an immunohistochemical study on testicular biopsies from fetal, neonatal, prepubertal, pubertal, and adult human testes. MAIN OUTCOME MEASURES: Quantification of androgen receptor expression in Sertoli cells was measured. Evaluation of androgen receptor expression in peritubular and interstitial cells as well as anti-Müllerian hormone and inhibin-alpha was also performed. RESULTS: Androgen receptor expression was first observed in the nuclei of few Sertoli cells at the age of 5 months. Labeling was weak in 2-15% of Sertoli cells until 4 yr of age and progressively increased thereafter. High levels of androgen receptor expression were observed in more than 90% from the age of 8 yr through adulthood. Androgen receptor was positive in peritubular cells and variable in interstitial cells. Anti-Müllerian hormone immunolabeling was strong in all Sertoli cells from fetal life throughout prepuberty and weakened progressively as spermatogenesis developed. Inhibin-alpha expression was detected in all Sertoli cells from fetal life through adulthood. CONCLUSIONS: A lack of androgen receptor expression could explain a physiological Sertoli cell androgen insensitivity during fetal and early postnatal life, which may serve to protect the testis from precocious Sertoli cell maturation, resulting in proliferation arrest and spermatogenic development.
Assuntos
Androgênios/fisiologia , Receptores Androgênicos/genética , Células de Sertoli/fisiologia , Testículo/embriologia , Testículo/fisiologia , Adolescente , Adulto , Hormônio Antimülleriano/fisiologia , Divisão Celular , Núcleo Celular/fisiologia , Criança , Pré-Escolar , Feto , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Células de Sertoli/citologia , Espermatogênese , Testículo/anatomia & histologia , Testículo/crescimento & desenvolvimento , Adulto JovemRESUMO
Sertoli cells are necessary to provide adequate levels of lactate for germ cell development. Lactate production is hormonally regulated by follicle-stimulating hormone (FSH) and by a large set of intratesticular regulators such as interleukin-1 beta (IL1 beta) and basic fibroblast growth factor (bFGF). Little is known regarding the critical step in the production of this metabolite, viz., the entrance of glucose into the cell as mediated by GLUTs. The aim of the present study was to investigate the expression of the glucose transporters GLUT1 and GLUT3 and its possible regulation by FSH, IL1 beta, and bFGF in Sertoli cells at two different time-points in sexual development. Sertoli cells retaining the ability to undergo mitosis (obtained from 8-day-old rats) and in the process of terminal differentiation (obtained from 20-day-old rats) were examined. Testicular tissue sections and Sertoli cell monolayers obtained from 8- and 20-day-old rats showed positive immunostaining for GLUT1 and GLUT3 proteins. GLUT1 and GLUT3 mRNA levels were detected at the two ages analyzed. Treatment of Sertoli cells obtained from 8- and 20-day-old rats with FSH, IL1 beta, and bFGF for various periods of time (12, 24, and 48 h) increased GLUT1 without changing GLUT3 mRNA levels. Our results thus show that Sertoli cells express GLUT1 and GLUT3 throughout pubertal development, and that, in Sertoli cells, only GLUT1 is regulated by hormones during pubertal development. Hormonal regulation of GLUT1 expression and consequently glucose uptake and lactate production may be a key molecular event in the regulation of spermatogenesis by hormones.
Assuntos
Transportador de Glucose Tipo 1/biossíntese , Transportador de Glucose Tipo 3/biossíntese , Células de Sertoli/metabolismo , Animais , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/fisiologia , Transportador de Glucose Tipo 1/efeitos dos fármacos , Transportador de Glucose Tipo 3/efeitos dos fármacos , Interleucina-1beta/farmacologia , Interleucina-1beta/fisiologia , Ácido Láctico/biossíntese , Masculino , Ratos , Ratos Sprague-Dawley , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacosRESUMO
BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.
Assuntos
Fertilização/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Espermatozoides/enzimologia , Zigoto/crescimento & desenvolvimento , Acrossomo/química , Animais , Bovinos , Feminino , Fertilização in vitro/veterinária , Humanos , Imuno-Histoquímica , Leupeptinas/farmacologia , Masculino , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/imunologia , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide/química , Cauda do Espermatozoide/ultraestrutura , Espermatozoides/imunologia , Zigoto/químicaRESUMO
The present paper reviews in detail ultrastructural and molecular studies addressed to characterize different phenotypes of sperm pathology in sterile men. In each case ultrastructural, immunocytochemical, molecular and genetic information is provided to differentiate two main kinds of sperm pathologies: systematic phenotypes with known or suspected genetic origin and non-systematic ones, usually secondary to different pathologies of the male reproductive system. Special attention is paid to detailed ultrastructural features profusely illustrated with electron micrographs. Diagnostic and fertility prognostic values of these phenotypes are also discussed and, when possible, comparison with similar pathologies in mammals and birds are discussed.
Assuntos
Interação Gene-Ambiente , Infertilidade Masculina/etiologia , Espermatozoides/patologia , Teratozoospermia/patologia , Animais , Humanos , Masculino , Fenótipo , Motilidade dos Espermatozoides , Teratozoospermia/classificação , Teratozoospermia/genéticaRESUMO
True hermaphroditism usually appears with ambiguous genitalia requiring extensive evaluation during the neonatal period. There have been occasional cases with better differentiation of external genitalia, leading to delays in diagnosis. We report the case of an adolescent boy with true hermaphroditism who presented with normal external genitalia and no sexual ambiguity. He was referred due to progressive gynecomastia and arrest of puberty. He presented at the age of 16 years for gynecomastia of rapid progression with normal penile development and both gonads in scrotum and normal testosterone and increased gonadotropin levels. Gonadal ultrasound scan was compatible with testicular and ovarian tissues in scrotum, and the karyotype showed two cellular lines (46,XX/46,XY). Gonadal histology revealed bilateral ovotestes. A genotype polymerase chain reaction mediated analysis using seven microsatellite markers did not confirm chimerism. Clinical findings and mechanism of generation are discussed.
Assuntos
Genitália Masculina , Transtornos Ovotesticulares do Desenvolvimento Sexual/diagnóstico , Puberdade , Adolescente , Quimera , Genitália Masculina/fisiologia , Ginecomastia/diagnóstico , Humanos , Masculino , Fenótipo , Puberdade/fisiologiaRESUMO
Testicular dysgenesis derives from abnormal gonadal development caused by chromosome aberrations/mosaicisms or mutations/deletions in SRY or other genes responsible for testicular differentiation. Dysgenetic male pseudohaermaphroditism has bilateral dysgenetic testes characterized by a cortical network of anastomosing seminiferous cords that penetrate a thin albuginea. In asymmetric gonadal differentiation (or Mixed Gonadal Dysgenesis) a dysgenetic testis associates with a streak gonad with primitive sex cords embedded in an ovarian-like stroma. Uni- or bilateral ovotestes identify true haermaphroditism. Fluorescent in situ hybridisation studies demonstrate that the sex chromosomes of mosaic patients do not distribute homogeneously in asymmetric gonads. 45,X lines predominate over 46,XY in streak gonads, while the relationship between these two is more equivalent in dysgenetic testes, suggesting that testicular or streak differentiation is related to the balance between X0 and XY lines. Testicular dys-genesis is more severe when there is a frank predominance of X0 or XX cells. Higher percentages of XY cells coincide with lesser degrees of dysgenesis. DNA densitometry indicate a higher incidence of neoplastic transformation than previously anticipated. Various specimens showed clear aneuploid histograms but no clear indication of a cytological CIS phenotype. There was a wide cytological variation in aneuploid germ cells, ranging from normally looking big infantile spermatogonia to gonocyte/CIS cells. Aneuploidy probably precedes the full expression of the CIS phenotype. In case of doubt we recommend DNA densitometry to either confirm or discard their neoplastic nature. The earliest recognizable change in germ cell tumorigenesis is probably the polyploidisation of fetal germ cells, followed by the expression of the CIS phenotype in isolated germ cells scattered along infantile seminiferous tubules that later proliferate to give an adult type CIS pattern.
Assuntos
Disgenesia Gonadal/patologia , Lesões Pré-Cancerosas/patologia , Neoplasias Testiculares/patologia , Testículo/anormalidades , Criança , Cromossomos Humanos X/genética , Cromossomos Humanos Y/genética , DNA/análise , Densitometria , Transtornos do Desenvolvimento Sexual/genética , Disgenesia Gonadal/genética , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Fenótipo , Lesões Pré-Cancerosas/genética , Neoplasias Testiculares/genética , Testículo/patologiaRESUMO
This work reports the effects of a previous injection of mitomycin-modified splenocytes from multiple-low dose streptozotocin-treated mice (mld-sz) on autoimmune diabetes produced by mld-sz. Our work shows that a previous inoculation of modified mononuclear splenocytes from mld-sz mice prevents alterations in glycemia, in insulin secretion (IS) pattern from isolated perifused islets, and in mass of pancreatic islets. Immunohistochemistry showed an alteration in the number of beta, but not of alpha or delta cells. While a mononuclear intra-islet infiltration was observed in mld-sz mice, a predominantly polar or peri-islet infiltration was seen in vaccinated mice. Islet-associated mononuclear cells from mld-sz mice produced diabetes and induced a diminished IS when transferred to normal receptors. Those cells from previously vaccinated mld-sz mice had no effect when injected into normal receptors. In addition, they also inhibited the damage induced in normal receptors by the islet-associated mononuclear cells from mld-sz animals. Cellular death was also prevented by previous vaccination. Our results suggest that vaccination with modified splenocytes from mld-sz mice is capable of shifting the islet cells infiltration pattern from an aggressive one toward a protective one and thus preventing the beta cell destruction observed in mld-sz mice.
Assuntos
Diabetes Mellitus Experimental/patologia , Baço/efeitos dos fármacos , Estreptozocina/administração & dosagem , Animais , Diabetes Mellitus Experimental/metabolismo , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Técnicas In Vitro , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Radioimunoensaio , Baço/citologiaRESUMO
Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis and sperm function. The development of the scanning electron microscope (SEM) extended these boundaries to the recognition of cell and organ surface features and the architectural array of cells and tissues. The merging of immunocytochemical and histochemical approaches with electron microscopy has completed a series of technical improvements that integrate structural and functional features to provide a broad understanding of cell biology in health and disease. With these advances the detailed study of the intricate structural and molecular organization as well as the chemical composition of cellular organelles is now possible. Immunocytochemistry is used to identify proteins or other components and localize them in specific cells or organelles with high specificity and sensitivity, and histochemistry can be used to understand their function (i.e., enzyme activity). When these techniques are used in conjunction with electron microscopy their resolving power is further increased to subcellular levels. In the present chapter we will describe in detail various ultrastructural techniques that are now available for basic or translational research in reproductive biology and reproductive medicine. These include TEM, ultrastructural immunocytochemistry, ultrastructural histochemistry, and SEM.
Assuntos
Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Espermatozoides/ultraestrutura , Testículo/ultraestrutura , Humanos , Imuno-Histoquímica/métodos , MasculinoRESUMO
El Síndrome de Turner es un desorden cromosómico causado por haploinsuficiencia completa o parcial de uno de los cromosomas sexuales. Incidencia 1: 2500 recién nacidas vivas. Clínicamente las pacientes presentan talla baja, un espectro amplio de anomalías somáticas y disgenesia gonadal. Desde el año 1968 hasta el presente se estudiaron clínica y citogenéticamente 630 niñas con fenotipo de Turner, sin ambigüedad genital y con cariotipos anormales, quienes consultaron en la División de Endocrinología del Hospital de Niños "Ricardo Gutiérrez". Se realizó cariotipo en sangre, al inicio con metodología estándar, luego con diferentes bandeos convencionales y de alta resolución. En casos especiales se aplicó la técnica FISH y el análisis molecular de los cromosomas X e Y. El número de metafases analizadas también varió con el tiempo, permitiendo evidenciar más de una línea celular. En casos de alta sospecha clínica, la lectura de 100 metafases permitió poner en evidencia mosaicismos bajos conteniendo la línea 45,X. En nuestra serie la monosomía de cromosoma sexual 45, X fue la más frecuente siguiendo los mosaicos numéricos y estructurales de uno de los cromosomas sexuales. Los diferentes hallazgos cromosómicos nos han permitido establecer una correlación fenotipo-cariotipo en regiones específicas de los cromosomas sexuales
Turner Syndrome is a common chromosomal disorder caused by total or partial haploinsufficiency of one of the sex chromosomes. Incidence: 1: 2500. Clinically is characterized by short stature, several typical somatic features, and gonadal dysgenesis. This is a retrospective study involving 630 girls with Turner phenotype and abnormal karyotype, evaluated at the Endocrinology Division of Children´s Hospital "Ricardo Gutiérrez" between 1968 and 2018. The karyotype was done in leucocytes from peripheral blood and the metaphases were analyzed at the beginning with standard methodology and then with different banding techniques, standard and high resolution. In special cases, the FISH technique and the molecular analysis of the X and Y chromosomes were applied. The number of metaphases analyzed also changed with time, allowing the finding of more than one cellular line. In cases where the clinical suspicion was strong, the analysis of 100 metaphases allowed us to put in evidence low mosaicisms containing the 45,X line. In our study the monosomy of sexual chromosome 45, X was the most frequent following the numerical and structural mosaics of one of the sex chromosomes. The different chromosomal constitutions have contributed to establish in our patients phenotype-karyotype correlation with specific regions of the sex chromosomes
Assuntos
Humanos , Cromossomos Sexuais , Síndrome de Turner , Fenótipo , Argentina , GenótipoRESUMO
This article presents an update on the variable prognostic significance of different sperm pathologies in patients with severe male factor infertility due to morphology and motility disorders. Severe asthenozoospermia is one of the leading causes of male infertility as spermatozoa cannot reach the oocyte and/or penetrate normally. Identifying structural causes of sperm immotility was of great concern before the advent of intracytoplasmic sperm injection (ICSI), because immotility was the limiting factor in the treatment of these patients. In these cases, in vitro methods are used to identify live spermatozoa or stimulate sperm motility to avoid selection of non-viable cells. With these advances, fertilization and pregnancy results have improved dramatically. The identification of genetic phenotypes in asthenozoospermia is important to adequately inform patients of treatment outcomes and risks. The one sperm characteristic that seriously affects fertility prognosis is teratozoospermia, primarily sperm head and neck anomalies. Defects of chromatin condensation and acrosomal hypoplasia are the two most common abnormalities in severe teratozoospermia. The introduction of microscopic methods to select spermatozoa and the development of new ones to evaluate sperm quality before ICSI will assure that ultrastructural identification of sperm pathologies will not only be of academic interest, but will also be an essential tool to inform treatment choice. Herein, we review the differential roles played by sperm components in normal fertilization and early embryo development and explore how assisted reproductive technologies have modified our concepts on the prognostic significance of sperm pathologies affecting the head, neck, mid-piece and tail.
Assuntos
Infertilidade Masculina/diagnóstico , Infertilidade Masculina/patologia , Cabeça do Espermatozoide/patologia , Cauda do Espermatozoide/patologia , Humanos , Infertilidade Masculina/terapia , Masculino , Prognóstico , Técnicas de Reprodução Assistida , Cabeça do Espermatozoide/fisiologia , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/fisiologia , Interações Espermatozoide-Óvulo/fisiologiaRESUMO
IGFs are involved in malignant transformation and growth of several tissues, including the adrenal medulla. The present study was designed to evaluate the impact of IGF-I on pheochromocytoma development. We used a murine pheochromocytoma (MPC) cell line (MPC4/30) and an animal model with a reduction of 75% in circulating IGF-I levels [liver-IGF-I-deficient (LID) mice] to perform studies in vitro and in vivo. We found that, in culture, IGF-I stimulation increases proliferation, migration, and anchorage-independent growth, whereas it inhibits apoptosis of MPC cells. When injected to control and to LID mice, MPC cells grow and form tumors with features of pheochromocytoma. Six weeks after cell inoculation, all control mice developed sc tumors. In contrast, in 73% of LID mice, tumor development was delayed to 7-12 wk, and the remaining 27% did not develop tumors up to 12 wk after inoculation. LID mice harboring MPC cells and treated with recombinant human IGF-I (LID+) developed tumors as controls. Tumors developed in control, LID, and LID+ mice had similar histology and were similarly positive for IGF-I receptor expression. The apoptotic index was higher in tumors from LID mice compared with those from control mice, whereas vascular density was decreased. In summary, our work demonstrates that IGF-I has a critical role in maintaining tumor phenotype and survival of already transformed pheochromocytoma cells and is required for the initial establishment of these tumors, providing encouragement to carry on research studies to address the IGF-I/IGF-I receptor system as a target of therapeutic strategies for pheochromocytoma treatment in the future.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Feocromocitoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Camundongos , Células PC12 , Ratos , Receptor IGF Tipo 1/metabolismoRESUMO
OBJECTIVE: To study expression of dysadherin in human testis, epididymis, and spermatozoa. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Testis, epididymis, and testicular spermatozoa from patients under treatment and semen from volunteer donors. INTERVENTION(S): Reverse transcription-polymerase chain reaction, immunohistochemistry, immunocytochemistry, and Western immunoblotting. MAIN OUTCOME MEASURE(S): Dysadherin messenger RNA (mRNA) analysis in testis, epididymis, and ejaculated spermatozoa, immunohistochemistry of both tissues, Western immunoblotting of tissue/cell extracts, and immunocytochemistry of spermatozoa. RESULT(S): Dysadherin mRNA was found in testis, epididymis, and ejaculated spermatozoa. Whereas testis and spermatozoa exhibited a distinctive 91-kDa protein form, the epididymis showed a 50-kDa moiety, also found in MDA-MB-231 breast cancer cells. Nucleotide sequence analysis revealed >99% homology between testicular and somatic cell mRNA, suggesting differential protein glycosylation. Dysadherin was immunodetected in round spermatids and testicular/ejaculated spermatozoa. It localizes to the acrosomal region and flagellum and colocalized with E-cadherin in the head and with the Na(+),K(+)-ATPase α4 subunit in the flagellum. CONCLUSION(S): This is the first report on expression of dysadherin in the male gonad and in spermatozoa. Its colocalization with E-cadherin and Na(+),K(+)-ATPase leads us to postulate a role for dysadherin as a modulator of sperm function.