RESUMO
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
The oocyte's meiotic spindle is a dynamic structure that relies on microtubule organization and regulation by centrosomes. Disorganization of centrosomal proteins, including the nuclear mitotic apparatus (NuMA) protein and the molecular motor complex dynein/dynactin, can lead to chromosomal instability and developmental abnormalities. The present study reports the distribution and function of these proteins in human oocytes, zygotes and early embryos. A total of 239 oocytes, 90 zygotes and discarded embryos were fixed and analyzed with confocal microscopy for NuMA and dynactin distribution together with microtubules and chromatin. Microtubule-associated dynein-dependent transport functions were explored by inhibiting phosphatase and ATPase activity with sodium-orthovanadate (SOV). At germinal vesicle (GV) stages, NuMA was dispersed across the nucleoplasm. After GV breaks down, NuMA became cytoplasmic before localizing at the spindle poles in metaphase I and II oocytes. Aberrant NuMA localization patterns were found during oocyte in vitro maturation. After fertilization, normal and abnormal pronuclear stage zygotes and embryos displayed translocation of NuMA to interphase nuclei. SOV treatment for up to 2 h induced lower maturation rates with chromosomal scattering and ectopic localization of NuMA. Accurate distribution of NuMA is important for oocyte maturation, zygote and embryo development in humans. Proper assembly of NuMA is likely necessary for bipolar spindle organization and human oocyte developmental competence.
Assuntos
Antígenos Nucleares , Embrião de Mamíferos/metabolismo , Proteínas Associadas à Matriz Nuclear , Oócitos/metabolismo , Fuso Acromático/metabolismo , Feto Abortado , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Adulto , Antígenos Nucleares/fisiologia , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cromossomos , Complexo Dinactina , Dineínas/fisiologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/ultraestrutura , Desenvolvimento Embrionário , Feminino , Fertilização , Humanos , Metáfase , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Associadas à Matriz Nuclear/fisiologia , Oócitos/crescimento & desenvolvimento , Oócitos/ultraestrutura , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Gravidez , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/fisiologia , Vanadatos/farmacologia , ZigotoRESUMO
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
Magnetic activated cell sorting (MACS) with annexin V microbeads recognizes externalized phosphatidylserine (PS) residues on the surface of apoptotic spermatozoa. The successful use of this novel technique applied to a highly apoptotic semen sample before performing intracytoplasmic sperm injection (ICSI) is reported here. The use of annexin V microbeads for selecting non-apoptotic spermatozoa seems to reduce the percentage of altered cells, improving the chance of pregnancy after ICSI.
Assuntos
Separação Celular/métodos , Fragmentação do DNA , Infertilidade Masculina/terapia , Injeções de Esperma Intracitoplásmicas/métodos , Adulto , Anexina A5 , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Recém-Nascido , Magnetismo , Masculino , Microesferas , Gravidez , Resultado da GravidezRESUMO
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
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
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 authors analyzed the cytoskeletal dynamics during human and bovine fertilization. Microtubules, microfilaments, and actin-related proteins are differently required for pronuclear migration and apposition.
Assuntos
Citoesqueleto/ultraestrutura , Fertilização/fisiologia , Animais , Bovinos , Citoesqueleto/química , Feminino , Humanos , MasculinoRESUMO
OBJECTIVE: To report assisted reproduction technologies (ART) outcome and characterize severe mitochondrial sheath (MS) anomalies in two infertile asthenoteratozoospermic patients. DESIGN: Case reports. SETTING: Private IVF clinic and academic research institution. PATIENT(S): Two infertile men with asthenoteratozoospermia. INTERVENTION(S): Intracytoplasmic sperm injection (ICSI) was performed in both cases. MAIN OUTCOME MEASURE(S): Clinical and laboratory evaluation were performed and spermatozoa studied by epifluorescence microscopy and transmission electron microscopy (TEM). RESULT(S): Patient 1 had sperm with acute bendings at the level of the narrow midpieces. Mitochondria were either scarce or absent. Three ICSI embryos were transferred. A pregnancy was achieved followed by a miscarriage at the end of the first trimester. Patient 2 had sperm with very long MSs. The number of gyres was increased to more than 30. Two ICSI cycles were performed with good fertilization rates and embryo quality, but no pregnancy was achieved. CONCLUSION(S): MS defects were studied by phase-contrast, epifluorescence microscopy, and TEM that afforded a detailed view of the sperm midpiece and the topography of the whole flagellum. The results indicate that midpiece defects, while causing severe asthenozoospermia and lower fertilizing potential, may not necessarily represent negative prognostic factors in ART.
Assuntos
Astenozoospermia/diagnóstico , Infertilidade Masculina/etiologia , Mitocôndrias/patologia , Injeções de Esperma Intracitoplásmicas/estatística & dados numéricos , Espermatozoides/anormalidades , Adulto , Feminino , Humanos , Masculino , Gravidez , Resultado da Gravidez , Espermatozoides/ultraestruturaRESUMO
BACKGROUND: Profilins are ubiquitous proteins widely distributed in animals, including humans. They regulate actin polymerization by sequestering actin monomers in association with other actin-related proteins (Arps). Actin remodelling is essential for oocyte maturation, fertilization and embryo development; yet the role of profilins in these events is not well understood. Here we investigate profilin distribution and function during bovine fertilization and early embryogenesis, and we examine profilin localization with respect to the co-distribution of other Arps. METHODS AND RESULTS: Western blotting, confocal microscopy with immunofluorescence and protein inhibition studies with antibodies were implemented. Profilin distributes inside interphase nuclei, throughout the cytoplasm and near the cell cortex at different stages of bovine oocyte maturation, fertilization and embryo development. Expression is detected through the blastocyst stage, where profilin localizes to the inner cell mass as well as trophectoderm. Profilin co-distributes with actin monomers and Arps vasodilator-stimulated phospho protein, p140mDia, Arp 3 and p80 coilin in pronucleate-stage zygotes. Antiprofilin antibodies inhibit normal embryo development by disrupting microfilaments, but not microtubules, and result in a higher concentration of profilin and p140mDia mislocalized to the cortex. CONCLUSIONS: These findings demonstrate that profilin regulates actin dynamics both within the cytoplasm and inside the nuclei of developing mammalian embryos and that its function is essential during fertilization to ensure successful development.