RESUMO
BACKGROUND: Children with autism spectrum disorder are at risk of a compromised dietary intake and nutritional status that could impact growth over both the short and long term. The limited body of published research addressing this concern has been contradictory and inconclusive to date. METHODS: This case-control study investigated the height, weight, body mass index (BMI) and other anthropometric measurements of children diagnosed with autism spectrum disorder (ASD). Eighty-six children with ASD and 57 healthy controls participated in the study. Caregivers of participants who met the inclusion criteria completed a health history questionnaire, provided information on dietary intake and feeding behaviour, and completed a nutrition physical with a healthcare professional, which provided all of the anthropometric measurements required for the study. RESULTS: Body mass index and BMI Z-scores for females with ASD and corresponding healthy controls were significantly different. Female participants with ASD had significantly lower BMI and BMI Z-scores than control participants. The prevalence of risk for failure-to-thrive status was consistent across ASD subjects and controls. The prevalence of overweight and obesity was consistent across ASD subjects and controls. Children with ASD comprised 60% of the total number of children across BMI categories and mid-arm muscle circumference percentile ranges, which is consistent with the proportion of children in the overall sample. CONCLUSIONS: More research is needed to fully assess physical status and potential growth concerns of children with ASD. A full physical assessment should be a component of primary care for all children with ASD.
Assuntos
Transtorno do Espectro Autista/epidemiologia , Desenvolvimento Infantil , Sobrepeso/epidemiologia , Obesidade Infantil/epidemiologia , Transtorno do Espectro Autista/complicações , Estatura , Índice de Massa Corporal , Peso Corporal , Estudos de Casos e Controles , Criança , Pré-Escolar , Dieta , Feminino , Seguimentos , Humanos , Masculino , Estado Nutricional , Sobrepeso/complicações , Obesidade Infantil/complicações , Prevalência , Inquéritos e QuestionáriosRESUMO
Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.
Assuntos
Fertilização in vitro/efeitos adversos , Fertilização/fisiologia , Zigoto/citologia , Animais , Ciclo Celular , Núcleo Celular , Feminino , Infertilidade Masculina/terapia , Macaca mulatta , Masculino , Microinjeções , Oócitos/fisiologia , Interações Espermatozoide-Óvulo , Espermatozoides/patologiaRESUMO
Primates that are identical in both nuclear and cytoplasmic components have not been produced by current cloning strategies, yet such identicals represent the ideal model for investigations of human diseases. Here, genetically identical nonhuman embryos were produced as twin and larger sets by separation and reaggregation of blastomeres of cleavage-stage embryos. A total of 368 multiples were created by the splitting of 107 rhesus embryos with four pregnancies established after 13 embryo transfers (31% versus 53% in vitro fertilization controls). The birth of Tetra, a healthy female cloned from a quarter of an embryo, proves that this approach can result in live offspring.
Assuntos
Blastômeros/fisiologia , Fase de Clivagem do Zigoto/fisiologia , Clonagem de Organismos/métodos , Desenvolvimento Embrionário e Fetal , Macaca mulatta/embriologia , Animais , Apoptose , Blastocisto/fisiologia , Transferência Embrionária , Feminino , Gravidez , Gêmeos Monozigóticos , Zona Pelúcida/fisiologiaRESUMO
During mammalian fertilization, the zygotic centrosome organizes a large sperm aster, critical for uniting the male and female pronuclei prior to first mitosis. Fluorescent imaging of inseminated human oocytes has shown that centrosomal defects may result in abnormal microtubule nucleation preventing genomic union, suggesting a novel cause of fertilization failure. Working with rhesus monkey gametes, we have developed a preclinical model for understanding the cell biological basis of intracytoplasmic sperm injection (ICSI). Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear theca, structures normally removed at the oolemma during IVF; this is turn causes a delay in the onset of DNA synthesis. These unusual modifications raise concerns that the ICSI procedure itself may result in chromatin damage during DNA decondensation and further highlight the need for a more rigorous assessment of methods of assisted reproduction prior to their global application.
Assuntos
Citoesqueleto/fisiologia , Técnicas Reprodutivas , Acrossomo/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Centrossomo/ultraestrutura , Dano ao DNA , Feminino , Humanos , Macaca mulatta , Masculino , Modelos Animais , Injeções de Esperma Intracitoplásmicas/efeitos adversosRESUMO
Intracytoplasmic sperm injection (ICSI) has heralded an era of tremendous improvements in treating male infertility leading to the births of thousands of babies. However, recent concerns over possible long-term effects of ICSI on offspring has prompted the development of a preclinical, nonhuman primate model to assess the safety of ICSI. Fluorescent imaging of rhesus macaque IVF zygotes revealed that this species shares many similarities with humans in terms of cytoskeletal and chromatin dynamics during fertilization. However, rhesus monkey zygotes fertilized by ICSI resulted in abnormal nuclear remodeling leading to asynchronous chromatin decondensation in the apical region of the sperm head, delaying the onset of DNA synthesis. The persistence of the acrosome and perinuclear theca on the apex of sperm introduced into the oocyte by ICSI may constrict the DNA in this region. Despite these differences, normal rhesus monkey ICSI embryos have been produced and have lead to several births after transfer. The irregularities described in this paper raise concerns that the ICSI procedure may result in chromatin damage during DNA decondensation and further highlight the need for devising improved pre-clinical assessment prior to global acceptance of this, and other, novel methods of assisted reproduction.
Assuntos
Fertilização in vitro/efeitos adversos , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Animais , Cromatina/ultraestrutura , Transferência Embrionária , Feminino , Humanos , Macaca mulatta , Masculino , Gravidez , Zigoto/ultraestruturaRESUMO
For medical students and residency programs alike, the residency application process is time-consuming. This paper examines one program's experience with a computerized system designed by the Association of American Medical Colleges (AAMC) to simplify and standardize the filing and receipt of applications over the Internet. A large-scale pilot test of the Electronic Residency Application Service (ERAS) was implemented in 1995-1996 for applicants to first-year residency positions in OB-GYN. Each student completed the computerized application, which was transmitted, along with other documents, to student-specified programs by the dean's office via the AAMC "electronic post office." ERAS will be extended to family practice residencies in 1997-1998. A major advantage of ERAS to residency programs is that materials were received in a well-organized, complete, and consistent format. Built-in filters allow grouping of applicants according to various criteria. Opening envelopes and filing documents is no longer necessary. Each student completes one application, and faculty write one letter of recommendation per student. Disadvantages of the 1995-1996 system related to the software, which had an inflexible interface and did not allow a spreadsheet view of the database. Personal statements and recommendation letters were often sent as unformatted ASCII text and were difficult to read. Dean's offices reported problems scanning documents such as transcripts and photographs. These problems led to resubmission of materials and receipt of duplicate copies. With the standard application format, ASCII-style personal statements and "generic" recommendation letters caused applicants to lose individuality. Specific recommendations to the AAMC for improving ERAS include providing a spreadsheet view, allowing students and faculty to write personal statements and letters in standard word processing formats, and allowing faculty to send unique letters to specific residency programs.
Assuntos
Redes de Comunicação de Computadores , Internato e Residência/organização & administração , Seleção de Pessoal/métodos , Controle de Formulários e Registros , Humanos , Estados UnidosRESUMO
Authors discuss the possible genetic and cell biological risks to offspring conceived by ICSI in relation to the lack of fundamental research using relevant animal models.
Assuntos
Fertilização/fisiologia , Injeções de Esperma Intracitoplásmicas , Experimentação Animal , Animais , Embrião de Mamíferos/fisiologia , Feminino , Doenças Genéticas Inatas , Humanos , Infertilidade Masculina , Masculino , Gravidez , Medição de Risco , Segurança , Responsabilidade SocialAssuntos
Reação Acrossômica/fisiologia , Proteínas de Membrana/metabolismo , Injeções de Esperma Intracitoplásmicas , Acrossomo/fisiologia , Acrossomo/ultraestrutura , Animais , Feminino , Macaca mulatta , Masculino , Microscopia Confocal , Proteínas R-SNARE , Cabeça do Espermatozoide/fisiologia , Cabeça do Espermatozoide/ultraestruturaRESUMO
The two cell populations of the day 5 mouse blastocyst--the inner cell mass (ICM) and trophectoderm (TE)--were isolated by immunosurgery and TE biopsy. The uptake of glucose and pyruvate and the production of lactate were measured in the isolated cell populations and in single day 5 blastocysts, using a non-invasive ultramicrofluorescence technique. The number of cells in isolated ICMs, TE vesicles, and intact blastocysts were counted after differential labeling with fluorochromes. The uptake of glucose and the formation of lactate were approximately three and five times greater, respectively, in the ICM cells compared with the TE cells. All the glucose consumed by the ICM could be accounted for by lactate formation, whereas the value for TE was 55%. The uptake of pyruvate was low in both cell types. The results are consistent with the proposition that the TE acts as a transporting epithelium, sparing nutrients for metabolism by the ICM.
Assuntos
Blastocisto/metabolismo , Metabolismo Energético , Trofoblastos/metabolismo , Animais , Blastocisto/citologia , Técnicas de Cultura , Ectoderma/metabolismo , Feminino , Glucose/metabolismo , Lactatos/metabolismo , Ácido Láctico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Microscopia de Fluorescência , Piruvatos/metabolismo , Ácido Pirúvico , Trofoblastos/citologiaRESUMO
During fertilization in humans, the sperm introduces the centrosome, the oocyte's microtubule organizing centre (MTOC), restoring centrosome function. The newly activated oocyte initiates extrusion of the second polar body and begins pronuclear formation. Apposition of the male and female pronuclei requires microtubule-mediated motility in the form of an aster of microtubules emanating from the sperm centrosome. The centrosome then duplicates and separates, forming the two poles of the mitotic spindle, upon which the parental genomes intermix, completing fertilization. The restoration and function of the centrosome is critical for successful fertilization suggesting that a defective sperm centrosome will lead to fertilization arrest and may be a new cause of male infertility. Several types of fertilization failure, associated with the sperm centrosome, are documented. These include: i) failure of the sperm to nucleate microtubules after sperm incorporation; ii) detachment of the sperm centrosome from the sperm head; and iii) failure in microtubule elongation after successful sperm aster formation. Although some types of severe male infertility can be overcome with the use of intracytoplasmic sperm injection (ICSI), men with defective sperm centrosomes are unlikely to benefit. The rate of sperm aster formation, size and organization during bovine fertilization has been used as a measurement of bovine sperm quality. Sperm from bulls which developed large highly organized sperm asters resulted in a higher rate of fertilization in vitro. The development of an assay using Xenopus laevis oocyte extracts has also been used to test centrosome function and sperm aster formation using sperm from men with questionable fertility. In general, these sperm were incapable of forming sperm asters and when used for in vitro fertilization, resulted typically in fertilization failure. These discoveries on the inheritance and function of the sperm centrosome have revealed a new cause of fertilization failure linked to male infertility which may not be circumvented using ICSI.
Assuntos
Centrossomo/fisiologia , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Animais , Centrossomo/ultraestrutura , Humanos , Masculino , Mutação/genética , Mutação/fisiologiaRESUMO
The concentrations of glucose, pyruvate, and lactate have been measured in the blastocoel fluid of single rat and mouse blastocysts, using the technique of micropuncture combined with an ultramicrofluorescence assay. When cultured in the presence of 5.55 mM glucose, 11.5-12.5 mM L-lactate and 0.25-0.33 mM pyruvate, concentrations in the blastocoel fluid of mouse and rat were 2.30 and 2.75 mM glucose, 14.6 and 19.6 mM L-lactate, and 0.13 and 0.50 mM pyruvate, respectively. When cultured in the presence of 1.0 mM glucose and 1.0 mM L-lactate, concentrations in the blastocoel fluid were 0.50 and 0.59 mM glucose and 2.22 and 3.70 mM L-lactate, respectively. These results suggest that (1) the blastocyst is capable of maintaining considerable concentration gradients of substrates across the trophectoderm, (2) the microenvironment of the blastocoel is adequately supplied with energy substrates for the development of the inner cell mass, and (3) the inner cell mass is capable of developing in both high and low glucose and lactate concentrations.
Assuntos
Blastocisto/metabolismo , Glucose/metabolismo , Lactatos/metabolismo , Piruvatos/metabolismo , Animais , Líquidos Corporais/metabolismo , Feminino , Fluorometria , Ácido Láctico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Gravidez , Punções , Ácido Pirúvico , Ratos , Ratos WistarRESUMO
Fertilization in humans follows a complex series of events including binding of the sperm to the oocyte plasma membrane, oocyte activation, the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei and the restoration of the sperm centrosome. This duplicates and separates, forming two mitotic spindle poles upon which the parental genomes can intermix to complete fertilization. The use of intracytoplasmic sperm injection (ICSI) has been highly effective as a treatment for severe male infertility and thousands of ICSI babies have been born world-wide. Working with rhesus monkey gametes, we have developed a preclinical animal model for understanding the cell biological basis of ICSI. Typically, ICSI results in abnormal nuclear remodeling during sperm decondensation due to the presence of the sperm acrosome and perinuclear structures normally removed at the oolemma during in vitro fertilization. These unusual modifications raise concerns that the ICSI procedure itself might lead to the observed increase in chromosome anomalies reported for
Assuntos
Fertilização in vitro , Infertilidade/terapia , Oócitos/ultraestrutura , Espermatozoides/ultraestrutura , Animais , Aberrações Cromossômicas , Feminino , Macaca mulatta , Masculino , Microscopia Eletrônica , Modelos Animais , Injeções de Esperma Intracitoplásmicas/efeitos adversosRESUMO
The perinuclear theca (PT) is a unique cytoskeletal structure whose anterior part is intercalated between the inner acrosomal membrane and the nuclear envelope of the mammalian sperm head and is important for spermiogenesis and stabilization of sperm structures (Oko and Maravei, Biol. Reprod. 50, 1000-1014, 1994; Oko and Maravei, Microsc. Res. Tech. 32, 520-532, 1995). Using immunofluorescence labeling of inseminated bovine oocytes and serial sectioning-ultrastructural analysis, we demonstrate that the PT is removed from the sperm nucleus following the loss of the sperm plasma membrane and the interaction of oocyte cortex with the PT. These events precede the development of the male pronucleus. The removal of the PT involves the elongated oocyte microvilli, rich in actin microfilaments, since it can be blocked by the microfilament-disrupting drug cytochalasin B. Reduction of disulfide bonds, which is a major factor supporting the disassembly of the sperm nucleus and accessory structures during mammalian fertilization, seems to exert little effect on the PT in vitro, as evidenced by the treatment of isolated bull sperm with the disulfide bond-reducing agent dithiothreitol. In vivo, intact bull sperm microinjected into mature oocytes do not undergo disassembly of the PT. Consequently, the decondensation of the sperm nucleus does not occur. These data suggest that the binding of the PT to the oocyte microvillar region and its removal from the sperm nucleus constitute an early step in mammalian fertilization, which is required for the conversion of the sperm nucleus into a male pronucleus.
Assuntos
Citoesqueleto/fisiologia , Fertilização , Oócitos/fisiologia , Cabeça do Espermatozoide/fisiologia , Animais , Anticorpos/imunologia , Bovinos , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Corantes Fluorescentes , Masculino , Microscopia Eletrônica , Microscopia de Fluorescência , Microvilosidades/fisiologia , Microvilosidades/ultraestrutura , Oócitos/ultraestrutura , Cabeça do Espermatozoide/química , Cabeça do Espermatozoide/ultraestrutura , Espermatozoides/fisiologiaRESUMO
Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.
Assuntos
Núcleo Celular/ultraestrutura , Técnicas Reprodutivas , Espermatozoides/fisiologia , Animais , Núcleo Celular/genética , Centrossomo , Cromatina/ultraestrutura , Feminino , Fertilização , Humanos , Macaca mulatta , Masculino , Microtúbulos , Óvulo/ultraestrutura , Coelhos , Injeções de Esperma Intracitoplásmicas , Espermatozoides/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
The effects of two metabolic inhibitors, methyl palmoxirate (MP) and amino-oxyacetate (AOA), on mouse preimplantation embryo development and cell number, and inner cell mass. (ICM) cell metabolism have been examined. Two-cell embryos were cultured in media supplemented with either MP, which inhibits fatty acid oxidation, or AOA, which inhibits the transamination of glutamate into alpha-ketoglutarate. Embryos were scored for development daily. On day 5, expanded blastocysts were differentially labeled with fluorochromes to visualize TE and ICM cell nuclei, or the ICMs isolated by immunosurgery and their energy metabolism determined using microfluorometric methods. Embryos exposed to the two inhibitors developed into fully expanded blastocysts, although cell numbers of both the TE and ICM cells were significantly reduced compared to controls. The uptake of glucose in the presence of 1 mM MP or AOA did not differ from the controls, but less glucose was accountable for by lactate production. MP significantly reduced lactate production. In the presence of 4 mM AOA, the amount of glucose oxidized and the amount of lactate formed by ICMs were significantly reduced. The results indicate that the fuels used by isolated mouse ICMs vary in response to substrate availability and that fatty acids may be a potential energy source.
Assuntos
Ácido Amino-Oxiacético/farmacologia , Antimetabólitos/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Propionatos/farmacologia , Animais , Contagem de Células , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , GravidezRESUMO
Fertilization is a precisely orchestrated cascade of events that results in the union of paternal and maternal genomes and in the establishment of mitotic potential of the zygote. To initiate embryonic development, the structures of the fertilizing sperm have to be disassembled and transformed into zygotic components by interactions with the cytoplasm of the egg. These interactions include the decondensation of the sperm nucleus into male pronucleus, the assembly of the zygotic centrosome, and the gathering of centrosomal proteins and sperm aster microtubules around the sperm centriole. Both the formation of the male pronucleus and the assembly of the zygotic centrosome are crucial steps required for pronuclear apposition and genomic union. The discovery of previously undetected fertilization failures that are due to defects in the assembly of the zygotic centrosome, abnormal pronuclear development, and compromised cytoskeletal dynamics enforces the development of new diagnostic strategies. Moreover, the introduction of new methods of infertility treatments, such as intracytoplasmic sperm injection and round spermatid nucleus injection into assisted human reproductive technology programs, emphasizes our lack of understanding of the cellular and molecular basis of human fertilization and evokes the need for additional experimentation. These efforts, however, are compromised by the sensitive nature of human embryo research and thus are severely restricted. Animal models that are reliable and cost-effective and that feature the characteristics of human fertilization have therefore been sought. Rodents such as the rat, mouse, and hamster are poor models owing to their maternal inheritance of the zygotic centrosome that is in strong contrast with the biparentally contributed assembly of the human zygotic centrosome during fertilization. Although rabbits are similar to humans from the standpoint of mitotic potential inheritance, information on postfertilization events in rabbits are lacking. Nonhuman primates represented by the rhesus monkey proved to be a reliable model for human in vitro fertilization and intracytoplasmic sperm injection, an advantage that is further emphasized by phyllogenetic similarity. In situations in which the high cost of primate research does not allow for large-scale experimentation (i.e., when large numbers of oocytes and embryos are needed), ruminants would be an ideal solution. Represented by the cow and sheep, domestic ruminants feature a fertilization strategy similar to that of the human. In addition, large numbers of gametes can be obtained wherever farms and slaughterhouses are accessible. Moreover, the detailed information on ruminant fertilization is strengthened by years of research and well-defined reproductive technology aimed at increasing the productivity of farm animals. Ruminants and rhesus monkeys have been extensively studied, and the data from these studies have been extrapolated in order to propose new strategies for the diagnosis and treatment of human infertility.
Assuntos
Infertilidade , Animais , Cricetinae , Modelos Animais de Doenças , Feminino , Fertilização in vitro , Humanos , Infertilidade/genética , Infertilidade/patologia , Infertilidade/terapia , Masculino , Camundongos , Gravidez , Coelhos , Ratos , Interações Espermatozoide-ÓvuloRESUMO
In addition to functional nuclear pore complexes engaged in nucleo-cytoplasmic transport, the cytoplasmic stacks of pore complexes, called annulate lamellae, exist in numerous cell types. Although both annulate lamellae and nuclear pore complexes are present in fertilized mammalian oocytes, their relative roles in the process of fertilization and preimplantation development are not known. Using epifluorescence and electron microscopy, we explored their fate during bovine fertilization. The assembly of annulate lamellae in bovine oocytes was triggered by sperm-oocyte binding and continued concomitantly with the incorporation of the nuclear pores in the nuclear envelopes of the developing male and female pronuclei. This process was also induced by the parthenogenetic activation of metaphase-II-arrested oocytes. Depletion of Ca2+, previously implicated in oocyte activation and in the insertion of pore complexes into the nuclear envelope, prevented the formation of nuclear pore complexes, but not the assembly of annulate lamellae in oocyte cytoplasm. Injection of the nuclear pore antagonist, wheat germ agglutinin, into the cytoplasm of mature oocytes that were subsequently fertilized caused the arrest of pronuclear development, indicating the requirement of nuclear pore complexes for normal pronuclear development. Treatment of the fertilized oocytes with the microtubule inhibitor, nocodazole, prevented gathering of annulate lamellae around the developing pronuclei, insertion of nuclear pores into their nuclear envelopes, and further pronuclear development. The formation of the male pronuclei was reconstituted in Xenopus egg extracts and reflected the behavior of nuclear pores during natural fertilization. These data suggest that nuclear pore complexes are required for normal pronuclear development from its beginning up until pronuclear apposition. Annulate lamellae may be involved in the turnover of nuclear pore complexes during fertilization, which is in turn facilitated by the reorganization of oocyte microtubules and influx of Ca2+ into oocyte cytoplasm.
Assuntos
Membrana Nuclear/ultraestrutura , Zigoto/crescimento & desenvolvimento , Zigoto/ultraestrutura , Animais , Cálcio/metabolismo , Bovinos , Feminino , Fertilização/fisiologia , Técnicas In Vitro , Masculino , Microscopia Eletrônica , Microscopia de Fluorescência , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Membrana Nuclear/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oócitos/ultraestrutura , Partenogênese , Aglutininas do Germe de Trigo/farmacologia , Xenopus , Zigoto/metabolismoRESUMO
PROBLEM: Successful fertilization in humans follows a complex series of events, including the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei, the restoration of the sperm centrosome, and the nucleation of microtubule-mediated motility necessary to bring the male and female pronuclei into close apposition. These events occur after both fertilization in vitro and after intracytoplasmic sperm injection (ICSI), a new technique which is currently being applied in many clinics to overcome severe male infertility. Defects in any of the events leading to fertilization can be lethal to the zygote and may prove to be causes of infertility. METHODS: Imaging of inseminated human and rhesus oocytes using immunohistochemical techniques reveals several phases at which fertilization arrests. RESULTS: Oocytes from some infertile patients failed to complete fertilization due to failure of the sperm aster microtubules in uniting the sperm and egg nuclei. The rate of sperm aster formation, size, and organization during fertilization has been used as a measurement of bovine sperm quality. The development of an assay using Xenopus laevis oocyte extract can also be used to test sperm from various species for their ability to form esters and perform other centrosomal functions in vitro, as well as another indicator of sperm quality. Semen from men with questionable fertility was found to contain sperm which are generally incapable of producing sperm asters. In addition, the activity of centrosomal proteins such as gamma-tubulin and centrin have been detected in mammalian eggs and sperm. The levels of gamma-tubulin increase markedly after exposure to X. laevis egg extract. CONCLUSION: Defects in either male or female nucleus decondensation also resulted in the arrest of fertilization and was found to occur in both inseminated human oocytes and in rhesus oocytes fertilized by ICSI. These discoveries on the molecular basis of infertility in humans have important implications for infertility diagnosis and managing reproduction.
Assuntos
Centrossomo/fisiologia , Pai , Fertilização/fisiologia , Infertilidade Masculina/terapia , Animais , Humanos , MasculinoRESUMO
The cytoskeletal components of hamster oocytes, zygotes, and spontaneously activated parthogenotes were examined after immunocytochemical labeling. Microtubules were found only in the anastral, tangentially arranged second meiotic spindle of unfertilized oocytes. Taxol treatment of unfertilized oocytes greatly augmented astral microtubules in both the metaphase II spindle and the cortex. Disruption of the meiotic spindle microtubules with nocodazole resulted in cortical chromosomal scattering. During hamster sperm incorporation and pronuclear formation, no sperm aster was detected in association with the male DNA. Instead, a large overlapping array of microtubules assembled in the cortex. By mitosis, this interphase array disassembled and an anastral metaphase spindle formed. Microtubule and chromatin configurations were also imaged in hamster oocytes injected with human sperm. Astral microtubules were absent from the sperm centrosome. The implications of these results are discussed in relation to the hamster oocyte penetration assay, a test commonly used by in vitro fertilization clinics to demonstrate the fertilizing ability of human sperm. We conclude that since hamsters and humans follow different methods of centrosome inheritance, maternal and paternal, respectively, the hamster may be an inappropriate model for exploring microtubule and centrosomal defects in humans or for assaying postinsemination forms of human male fertility defects.