RESUMO
The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whether in vitro maturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI, in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, although in vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation by in vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu of in vitro-matured oocytes and to replicate the molecular changes associated with in vivo capacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.
Assuntos
Doenças dos Bovinos/terapia , Núcleo Celular/patologia , Montagem e Desmontagem da Cromatina , Infertilidade Masculina/veterinária , Cabeça do Espermatozoide/patologia , Injeções de Esperma Intracitoplásmicas/veterinária , Interações Espermatozoide-Óvulo , Animais , Sinalização do Cálcio , Bovinos , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/patologia , Núcleo Celular/metabolismo , Células Cultivadas , Técnicas de Cultura Embrionária/veterinária , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Infertilidade Masculina/terapia , Masculino , Camundongos , Partenogênese , Especificidade da Espécie , Capacitação Espermática , Cabeça do Espermatozoide/metabolismoRESUMO
This work evaluates sperm head morphometric characteristics in adolescents from 12 to 18 years of age, and the effect of varicocele. Volunteers between 150 and 224 months of age (mean 191, n = 87), who had reached oigarche by 12 years old, were recruited in the area of Barranquilla, Colombia. Morphometric analysis of sperm heads was performed with principal component (PC) and discriminant analysis. Combining seminal fluid and sperm parameters provided five PCs: two related to sperm morphometry, one to sperm motility, and two to seminal fluid components. Discriminant analysis on the morphometric results of varicocele and nonvaricocele groups did not provide a useful classification matrix. Of the semen-related PCs, the most explanatory (40%) was related to sperm motility. Two PCs, including sperm head elongation and size, were sufficient to evaluate sperm morphometric characteristics. Most of the morphometric variables were correlated with age, with an increase in size and decrease in the elongation of the sperm head. For head size, the entire sperm population could be divided into two morphometric subpopulations, SP1 and SP2, which did not change during adolescence. In general, for varicocele individuals, SP1 had larger and more elongated sperm heads than SP2, which had smaller and more elongated heads than in nonvaricocele men. In summary, sperm head morphometry assessed by CASA-Morph and multivariate cluster analysis provides a better comprehension of the ejaculate structure and possibly sperm function. Morphometric analysis provides much more information than data obtained from conventional semen analysis.
Assuntos
Cabeça do Espermatozoide/patologia , Espermatozoides/citologia , Varicocele/patologia , Adolescente , Forma Celular/fisiologia , Criança , Análise por Conglomerados , Humanos , Masculino , Análise Multivariada , Análise do Sêmen/métodos , Motilidade dos EspermatozoidesRESUMO
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
A specific cause of infertility cannot be identified in at least 25% of men referred to a specialized clinic. Diagnosis of infertile men is based mainly on standard semen analysis and the observation of sperm under light microscope. The aim of our study was to find the subcellular sperm characteristics that could explain infertility in a group of teratozoospermic infertile men. Morphological characteristics of sperm from non-teratozoospermic (control donors) and teratozoospermic infertile men were analyzed by transmission electron microscopy (TEM) and quantified. Our analysis showed that sperm cells from control donors presented a higher number of normal heads and tails than infertile men. Regarding subcellular characteristics of nucleus and tails, only the percentage of vacuolated nucleus, the absence of at least one pair of microtubules of the axoneme and the total distortion of the tail were statistically higher in infertile men than in control donors. There were no differences in the number of normal acrosomes between the groups. Although the ultrastructural sperm defects overlapped between control donors and infertile men, TEM permits the identification and differentiation of a larger amount of defects than light microscopy. Vacuolated nucleus and gross alterations of the tail are the major sperm defects that seem to have prognostic value in teratozoospermic men.
Assuntos
Infertilidade Masculina/fisiopatologia , Espermatozoides/anormalidades , Espermatozoides/ultraestrutura , Adulto , Feminino , Humanos , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/etiologia , Infertilidade Masculina/patologia , Masculino , Gravidez , Sêmen/citologia , Cabeça do Espermatozoide/patologia , Cabeça do Espermatozoide/ultraestrutura , Injeções de Esperma Intracitoplásmicas , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Adulto JovemRESUMO
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
We analyzed the appearance and localization of the sub-acrosomal perinuclear theca (PT) during human spermiogenesis. The PT is tightly associated with acrosomal biogenesis.
Assuntos
Cabeça do Espermatozoide/fisiologia , Cabeça do Espermatozoide/ultraestrutura , Espermatogênese/fisiologia , Acrossomo/fisiologia , Acrossomo/ultraestrutura , Azoospermia/patologia , Núcleo Celular/fisiologia , Humanos , Masculino , Microscopia de Fluorescência , Morfogênese/fisiologia , Cabeça do Espermatozoide/patologiaRESUMO
Sperm pathology is presented as the discipline of characterizing structural and functional deficiencies in abnormal spermatozoa. This concept complements that of sperm morphology mainly concerned with the appearance of spermatozoa. These two notions collaborate in providing correlations of prognostic value with sperm fertilizing capacity, explaining the mechanisms of sperm inefficiency, suggesting strategies to improve fertilization and opening a door to molecular genetic studies. Phenotypes of genetic origin involving sperm heads, flagella and the neck region are presented describing their clinical manifestations, sperm structure, cytochemistry and genetic background. When available, animal models are used to highlight possible genetic mechanisms. Sperm pathologies secondary to andrological conditions or environmental factors are described, stressing the non-specific nature of the sperm response to noxious agents. The available literature on the prognostic value of sperm pathologies in ICSI is also reviewed. Flagellar anomalies bear a good prognosis, but those affecting the acrosome, sperm chromatin and the neck region entail an increasing chance of failure, which highlights the differential roles played by specific sperm components in fertilization, implantation and early embryonic development. A final discussion is devoted to genetic counselling and the risks involved in using immotile or abnormal spermatozoa in assisted reproduction.
Assuntos
Infertilidade Masculina/patologia , Espermatozoides/patologia , Espermatozoides/fisiologia , Reação Acrossômica , Cromatina/genética , Cromatina/patologia , Humanos , Infertilidade Masculina/terapia , Masculino , Prognóstico , Cabeça do Espermatozoide/patologia , Injeções de Esperma Intracitoplásmicas/métodos , Motilidade dos Espermatozoides/genéticaAssuntos
Infertilidade Masculina/genética , Oligospermia/genética , Espermatozoides/patologia , Humanos , Infertilidade Masculina/patologia , Masculino , Oligospermia/patologia , Cabeça do Espermatozoide/patologia , Cabeça do Espermatozoide/ultraestrutura , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Espermatozoides/ultraestruturaRESUMO
Protamine crosslinking by disulphide (-SS-) bonds in the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine-rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatic. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smear from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet-blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro- and macro-spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho-/metachromatic ration seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in -SS- crosslinking.