RESUMEN
The chromatoid body is a cytoplasmic male germ cell structure that plays a role in the regulation of mRNA transcription during spermatogenesis. A proteomic analysis of this structure has identified the presence of its classic molecular markers (MVH and MIWI), as well as a significant number of transient proteins. Circadian locomotor output cycles protein kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), which are molecular components of the circadian clock, are likely located in the chromatoid body in a transient fashion. This study sought to determine whether aging produces morphological changes in the chromatoid bodies of round spermatids similar to those previously observed in BMAL1 knockout mice. A sample of 30 male mice was divided into three groups: juvenile mice (45 days old), adult mice (120 days old), and old mice (+180 days old). Aging was confirmed by viability and sperm count analyses and testosterone dosage. Squash slides prepared with fragments of seminiferous tubules were immunostained for MVH, MIWI, BMAL1, and CLOCK detection. In juvenile and adult specimens, single round chromatoid bodies were observed using MVH/BMAL1 and MIWI/CLOCK immunostaining. In old specimens, many chromatoid bodies displayed changes in number and morphology, as well as an increase in the interactions between MVH and BMAL1; MIWI and CLOCK. Changes in chromatoid body morphology increased interactions between the proteins analyzed herein, and decreased amounts of these proteins in seminiferous tubules of older mice may indicate that aging influences the assembly and physiology of chromatoid bodies, which may, in turn, affect fertility. Impact statement The results discussed in this paper indicate that aging compromises the structure and physiology of chromatoid bodies (CBs) in post-meiotic male cells. Since CB is a fundamental structure for the differentiation of the mature male germ cell it is possible that this imbalance in CB physiology may play a role in the reduction of fertility in older men. It is important to note that not only the classic CB markers (such as the MIWI and MVH proteins) were used to showcase the structural changes in the CBs but also the main components of circadian cycle control (the CLOCK and BMAL1 proteins), indicating that the reduction of circadian control in aged males may contribute to these changes in CBs as well. Therefore, it is intriguing to evaluate the hypothesis that controlling these physiological/structural changes in CBs may be a way of delaying the effects of aging in males.
Asunto(s)
Envejecimiento/patología , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Espermátides/patología , Factores de Transcripción ARNTL/análisis , Factores de Edad , Animales , Proteínas Argonautas/análisis , Proteínas CLOCK/análisis , ARN Helicasas DEAD-box/análisis , Masculino , Ratones , Microscopía Fluorescente , Nucleoproteínas/metabolismoRESUMEN
Rb translocations are chromosomal rearrangements frequently found in natural populations of the house mouse Mus musculus domesticus. The standard diploid karyotype of the house mouse consisting of 40 telocentric chromosomes may be reduced by the emergence of metacentric Rb chromosomes. Multiple simple Rb heterozygotes form trivalents exhibiting higher anaphase nondisjunction frequency and consequently higher number of unbalanced gametes than in normal males. This work will attempt to establish whether frequencies of aneuploidy observed in heterozygote spermatids of the house mouse M. musculus domesticus show differences in chromosomes derived from different trivalents. Towards this goal, the number and distribution frequency of aneuploidy was assessed via FISH staining of specific chromosomes of spermatids derived from 2n = 32 individuals. Our results showed that for a given set of target chromosomes, 90% of the gametes were balanced, resulting from alternate segregation, and that there were no differences (approx. 10%) in aneuploidy frequencies in chromosomes derived from different trivalents. These observations suggest that segregation effectiveness does not depend on the type of chromosomes involved in trivalents. As a consequence of the trivalent's configuration, joint segregation of the telocentric chromosomes occurs thus favoring their appearance together in early spermatids. Our data suggest that Rb chromosomes and their telocentric homologs are subject to architectural constraints placing them close to each other. This proximity may ultimately facilitate fusion between them, hence contributing to a prevalence of Rb metacentric chromosomes.
Asunto(s)
Aneuploidia , Cromosomas/genética , Meiosis/genética , Translocación Genética/genética , Animales , Heterocigoto , Cariotipificación , Masculino , Ratones , No Disyunción Genética , Espermátides/patologíaRESUMEN
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.
Asunto(s)
Acrosoma/fisiología , Inmunohistoquímica/métodos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Proteómica/métodos , Espermatozoides/anomalías , Animales , Biopsia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Femenino , Células Germinativas/citología , Aparato de Golgi/metabolismo , Humanos , Infertilidad Masculina/diagnóstico por imagen , Masculino , Microscopía Electrónica de Transmisión/métodos , Espermátides/patología , Espermatozoides/patología , Testículo/patología , UltrasonografíaRESUMEN
This work characterizes the effects of ambient levels of urban particulate matter (PM(2.5)) from the city of Sao Paulo on spermatogenesis using mice exposed during the embryo-fetal and/or postnatal phases of development. Parental generations (BALB/c mice) were exposed to air pollution in chambers with or without filtering PM(2.5) for 4 months. Animals were mated, and half of the 1-day-old offspring were moved between chambers, which yielded prenatal and postnatal groups. Remaining offspring comprised the non-exposed and pre+postnatal exposed groups. After 90 days, the animals were sacrificed for testis collection and weighing. Optical microscopy was used for the morphometric analyses of the cell counts, spermatogenic cycle, proliferation, and apoptosis. Prenatally exposed animals presented reduced body and testicular weight with an increased gonadosomatic index (GSI). Testicular volume also decreased, as well as the tubular diameter in testes of the same animals. Proliferation, apoptosis, and spermatogenic cycle analyses showed no significant differences among groups. However, the tubules at stage VII of pre- and postnatal animals presented a reduced number of elongated spermatids. Pre+postnatal group presented higher spermatid head retention at stages VIII-XII. These results show that ambient levels of PM(2.5) from Sao Paulo city affect spermatogenesis by damaging sperm production.
Asunto(s)
Exposición por Inhalación , Material Particulado/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Espermátides/citología , Espermátides/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Contaminación del Aire/efectos adversos , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciudades , Desarrollo Embrionario/efectos de los fármacos , Femenino , Desarrollo Fetal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Reproducción/efectos de los fármacos , Espermátides/patología , Testículo/efectos de los fármacosRESUMEN
OBJECTIVE: To report the findings encountered in semen samples coming from two infertile men chronically exposed to carbofuran. METHODS: Semen samples were collected and analyzed as recommended by the World Health Organization. A morphological analysis was carried out by light microscopy. RESULTS: Seminal analysis revealed in the first case a total concentration of 42 million spermatozoa/mL with 17% motility and 20% normal shape. The second patient presented a total concentration of 5 million spermatozoa/mL with 6% motility and 2% normal shape. The patients presented a similar percentage of binucleated spermatozoa (28 and 26%) and of multinucleated spermatids (10 and 6%). CONCLUSION: To our knowledge, this is the first time that alterations in semen samples of men exposed to carbofuran are reported. More research in this area is needed to make conclusions on the subject.
Asunto(s)
Carbofurano/efectos adversos , Núcleo Celular/patología , Inhibidores de la Colinesterasa/efectos adversos , Exposición Profesional/efectos adversos , Oligospermia/patología , Espermátides/patología , Adulto , Agricultura , Humanos , Masculino , Oligospermia/etiología , Oligospermia/fisiopatología , Motilidad Espermática , Espermátides/fisiologíaRESUMEN
INTRODUCTION: The mechanism by which varicocele interferes in spermatogenesis has not been clearly defined. Germ cell apoptosis and oxidative stress appear to be involved in this process and the use of antioxidants has been proposed to counteract upon these effects. The present study evaluated the effects of N-acetylcysteine (NAC) on spermatogenesis and germ cell apoptosis in an experimental model of varicocele in rats. MATERIALS AND METHODS: Twenty 30-day-old animals were randomly divided into three groups: sham operation (Group 1), left experimental varicocele (Group 2) and left experimental varicocele group treated with NAC 50 mg/kg/day (Group 3). After 2 months, spermatogenesis was evaluated by absolute and true count of round spermatids, pachytenes, spermatocytes and Sertoli cells. The different cell relations were also analyzed. Germ cell apoptosis was quantified using the TUNEL method. The apoptotic index (AI) was calculated as the number of apoptotic cells per tubule. Statistical analysis was performed by analysis of variance considering P < 0.05. RESULTS: The absolute and true cell counts were similar among the groups (P > 0.05). The round spermatid/pachytene ratio was significantly smaller in Groups 2 and 3 compared to the Group 1 (P = 0.012). The AI values were 0.207 ± 0.09, 0.138 ± 0.11 and 0.298 ± 0.27, respectively (P = 0.256). CONCLUSION: Experimental varicocele in rats presented an association with the decreased round spermatid/pachytene ratio, suggesting the loss of germ cells during spermatogenesis. These effects were not influenced by the administration of NAC. Germ cell apoptosis was not influenced by experimental varicocele.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Células Germinativas/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Varicocele/fisiopatología , Animales , Recuento de Células , Células Germinativas/fisiología , Masculino , Ratas , Células de Sertoli/patología , Espermátides/patología , Testículo/patología , Varicocele/patologíaRESUMEN
The aim of this work was to assess the effect of metronidazole (MTZ) on the stages of the seminiferous epithelial cycle and spermatozoa morphology when the drug is administered in human therapeutic doses to 60-day-old CFW male mice. The frequency of the stages was established by counting spermatocytes in pachytene and spermatids. Abnormalities in the flagellum or the head, lack of maturity and multiple malformations, were considered in the morphological analysis. Murine control strain was compared with MTZ treated group (v.ip 130 mg/kg/bw) both kept in standard captivity conditions. Cellular composition or number of stages in the seminiferous tubules were not altered in MTZ exposed animals, though the number of cells in stages I, V and XII was increased. The sperm cell morphology was severely affected by the treatment with potentially serious consequences on the normal fertilization process. Thus, the MTZ has to be considered as a conceivable thread regarding male fertility.
Asunto(s)
Antiprotozoarios/toxicidad , Metronidazol/toxicidad , Espermatozoides/efectos de los fármacos , Animales , Recuento de Células , Fertilidad/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , Fase Paquiteno/efectos de los fármacos , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/patología , Espermátides/efectos de los fármacos , Espermátides/patología , Espermatozoides/patologíaRESUMEN
Diethylcarbamazine (DEC) had been proved to be highly effective against lymphatic filariasis, however its effect on vertebrate cells remains uncertain. After 12 days treatment with DEC, most of the Leydig cells were hypertrophied with several lipid droplets, and others had no nucleus and presented characteristic steatosis features. Vacuolization of Sertoli cells was also noted. Ultrastructural analyses of DEC-treated testes revealed spermatogonies with morphological characteristics of apoptosis, as shrinkage of cytoplasm and increased chromosomal density. In addition, Leydig cells showed numerous lipid droplets scattered throughout the cytoplasm, multivesicular bodies and giant whorl-like smooth endoplasmic reticulum. Several spermatids presented vacuolated mitochondriae, which were disorganized in relation to the microtubular axis of the flagellae. These results indicate that DEC probably affects the microtubular function, however the present data does not exclude the possibility that DEC also can act directly on enzymatic hormonal pathways.
Asunto(s)
Dietilcarbamazina/toxicidad , Células Intersticiales del Testículo/efectos de los fármacos , Células de Sertoli/efectos de los fármacos , Testículo/efectos de los fármacos , Administración Oral , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Dietilcarbamazina/administración & dosificación , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Filaricidas/administración & dosificación , Filaricidas/toxicidad , Líquido Intracelular/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/ultraestructura , Movilización Lipídica/efectos de los fármacos , Lípidos/química , Masculino , Ratones , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Orgánulos/efectos de los fármacos , Orgánulos/ultraestructura , Células de Sertoli/ultraestructura , Espermátides/efectos de los fármacos , Espermátides/patología , Espermátides/ultraestructura , Espermatogénesis/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Espermatogonias/ultraestructura , Testículo/citología , Testículo/ultraestructura , Factores de Tiempo , Vacuolas/efectos de los fármacos , Vacuolas/ultraestructuraRESUMEN
OBJECTIVE: To search and to identify spermatozoa and spermatids, present in the ejaculate of non-obstructive azoospermic patients. MATERIALS AND METHODS: 27 patients, aged between 18 and 48 years, with initial diagnosis compatible with non-obstructive azoospermia, underwent up to 3 seminal samples, with assessment of macroscopic and microscopic parameters differentiated for each sample. In the first sample, 5 microL of semen were analyzed in a Horwell chamber in order to assess the presence or absence of spermatozoa. The procedure was repeated with 2 other aliquots. In the absence of spermatozoa, the entire sample was transferred to a conic tube and following centrifugation the sediment was freshly analyzed. The second seminal sample was collected only when no spermatozoa were found in the first sample and the research was performed in the same way. In cases where spermatozoa were not seen, the sample was centrifuged and the obtained sediment was stained by the panoptic method and observed under common light microscopy (1250X). The third seminal sample was collected only in cases when patients had not shown spermatozoa in the first and second seminal samples. RESULTS: 4/27 (14.8%) patients presented spermatozoa in the first seminal sample and 6/23 (26.1%), in the second seminal sample. No spermatozoa were seen in the third sample, however, 11/17 (64.7%) presented spermatids. CONCLUSION: In clinical situations where the initial diagnosis is non-obstructive azoospermia, one single routine seminal analysis is not enough to confirm this diagnosis and the analysis of the centrifuged sediment can have relevant clinical consequences. Among patients considered non-obstructive azoospermic, when duly assessed, 37% presented spermatozoa and 64.7%, spermatids.
Asunto(s)
Eyaculación , Oligospermia/diagnóstico , Espermátides/patología , Espermatozoides/patología , Adolescente , Adulto , Centrifugación , Humanos , Masculino , Persona de Mediana Edad , Patología Clínica/métodos , Patología Clínica/normas , Reproducibilidad de los Resultados , Recuento de EspermatozoidesRESUMEN
OBJECTIVE: To search and to identify spermatozoa and spermatids, present in the ejaculate of non-obstructive azoospermic patients. MATERIALS AND METHODS: 27 patients, aged between 18 and 48 years, with initial diagnosis compatible with non-obstructive azoospermia, underwent up to 3 seminal samples, with assessment of macroscopic and microscopic parameters differentiated for each sample. In the first sample, 5 æL of semen were analyzed in a Horwell chamber in order to assess the presence or absence of spermatozoa. The procedure was repeated with 2 other aliquots. In the absence of spermatozoa, the entire sample was transferred to a conic tube and following centrifugation the sediment was freshly analyzed. The second seminal sample was collected only when no spermatozoa were found in the first sample and the research was performed in the same way. In cases where spermatozoa were not seen, the sample was centrifuged and the obtained sediment was stained by the panoptic method and observed under common light microscopy (1250X). The third seminal sample was collected only in cases when patients had not shown spermatozoa in the first and second seminal samples. RESULTS: 4/27 (14.8 percent) patients presented spermatozoa in the first seminal sample and 6/23 (26.1 percent), in the second seminal sample. No spermatozoa were seen in the third sample, however, 11/17 (64.7 percent) presented spermatids. CONCLUSION: In clinical situations where the initial diagnosis is non-obstructive azoospermia, one single routine seminal analysis is not enough to confirm this diagnosis and the analysis of the centrifuged sediment can have relevant clinical consequences. Among patients considered non-obstructive azoospermic, when duly assessed, 37 percent presented spermatozoa and 64.7 percent, spermatids.
Asunto(s)
Adolescente , Adulto , Humanos , Masculino , Persona de Mediana Edad , Eyaculación , Oligospermia/diagnóstico , Espermátides/patología , Espermatozoides/patología , Centrifugación , Patología Clínica/métodos , Patología Clínica/normas , Reproducibilidad de los Resultados , Recuento de EspermatozoidesRESUMEN
OBJECTIVE: To study hormonal and histological parameters of paediatric-adolescent varicocele in order to know certain aspects of its natural history, in an attempt to find prognostic markers of testicular damage. DESIGN AND METHODS: In a prospective cross-sectional study, we evaluated 93 children and adolescents with left unilateral varicocele and 29 healthy males as control group. All of them were classified according to Tanner stage. Scrotal Doppler in both testes and GnRH and human chorionic gonadotrophin (hCG) tests were performed in all subjects. Surgery was performed in 28 patients and homolateral testicular biopsy in 18. RESULTS: Hormonal measurements of patients with varicocele were compared with a control group for each Tanner stage. Testicular biopsy specimens were analysed by light and electron microscopy. We only observed statistical differences in Tanner III patients in basal FSH (median and range) controls=1.70 (1.10-3.70) IU/l vs varicocele=4.20 (1.00-7.50) IU/l, P<0.05 and in Tanner IV patients in LH post-GnRH: controls=11.0 (7.50-15.0) IU/l vs varicocele=18.0 (5.10-29.0) IU/l, P<0.05 and in testosterone post-hCG: controls=9.50 (7.7-10.0) ng/ml vs varicocele=12.0 (6.2-23.0) ng/ml, P<0.01. No correlation was found between the various clinical grades of varicocele and hormonal measurements for each Tanner stage. No statistically significant differences were found between pre- and post-operative hormonal findings, either in basal levels or in maximal responses. On the other hand, no morphological abnormalities were observed by electron microscopy in germ cells, tubular wall and interstice. CONCLUSIONS: There appears to be no reliable biochemical marker in children and adolescents that may predict impaired testicular function. A significant size discrepancy between both testes, testicular pain and a hyperresponse to GnRH stimulation should continue to be, for the time being, the indications for surgery.