17 results on '"Ramalho-Santos, J."'
Search Results
2. Geography of follicle formation in the embryonic mouse ovary impacts activation pattern during the first wave of folliculogenesis.
- Author
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Cordeiro MH, Kim SY, Ebbert K, Duncan FE, Ramalho-Santos J, and Woodruff TK
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- Animals, DEAD-box RNA Helicases genetics, Egg Proteins genetics, Female, Fluorescence, Genotype, Germ Cells physiology, Growth Differentiation Factor 9 genetics, Meiosis genetics, Membrane Glycoproteins genetics, Mice, Mice, Transgenic, Pregnancy, Receptors, Cell Surface genetics, Reproduction genetics, Reproduction physiology, Sexual Maturation genetics, Sexual Maturation physiology, Zona Pellucida Glycoproteins, Ovarian Follicle embryology, Ovary embryology
- Abstract
During embryonic development, mouse female germ cells enter meiosis in an anterior-to-posterior wave believed to be driven by retinoic acid. It has been proposed that ovarian follicle formation and activation follow the same general wave of meiotic progression; however, the precise anatomic specification of these processes has not been delineated. Here, we created a mouse line using Mvh, Gdf9, and Zp3 promoters to drive distinct temporal expression of three fluorescent proteins in the oocytes and to identify where the first follicle cohort develops. The fluorescent profile revealed that the first growing follicles consistently appeared in a specific region of the ovary, the anterior-dorsal region, which led us to analyze if meiotic onset occurred earlier in the dorsal ovarian region. Surprisingly, in addition to the anterior-to-posterior wave, we observed an early meiotic entry in the ventral region of the ovary. This additional anatomic stratification of meiosis contrasts with the localization of the initial follicle formation and activation in the dorsal region of the ovary. Therefore, our study suggests that the specification of cortical and medullar areas in the ventral and dorsal regions on the ovary, rather than the onset of meiosis, impacts where the first follicle activation event occurs., (© 2015 by the Society for the Study of Reproduction, Inc.)
- Published
- 2015
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3. Sins of the fathers: sperm DNA damage in the context of assisted reproduction.
- Author
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Ramalho-Santos J
- Subjects
- Female, Humans, Male, Pregnancy, DNA Damage, Embryo Implantation genetics, Embryonic Development genetics, Infertility genetics, Spermatozoa metabolism
- Published
- 2014
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4. p,p'-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations.
- Author
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Tavares RS, Mansell S, Barratt CL, Wilson SM, Publicover SJ, and Ramalho-Santos J
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- Benzimidazoles pharmacology, Calcium Channels metabolism, Calcium Signaling drug effects, Cell Survival drug effects, Cyclopropanes, Humans, In Vitro Techniques, Male, Mibefradil pharmacology, Naphthalenes, Spermatozoa physiology, Calcium metabolism, Calcium Channels drug effects, Dichlorodiphenyl Dichloroethylene toxicity, Endocrine Disruptors toxicity, Spermatozoa drug effects
- Abstract
Study Question: Is the environmental endocrine disruptor p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) able to induce non-genomic changes in human sperm and consequently affect functional sperm parameters?, Summary Answer: p,p'-DDE promoted Ca(2+) flux into human sperm by activating CatSper channels even at doses found in human reproductive fluids, ultimately compromising sperm parameters important for fertilization., What Is Known Already: p,p'-DDE may promote non-genomic actions and interact directly with pre-existing signaling pathways, as already observed in other cell types. However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known., Study Design, Size, Duration: Normozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several p,p'-DDE concentrations for 3 days at 37°C and 5% CO2 in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p'-DDE incubation periods were also performed in order to monitor sperm rapid Ca(2+) responses. All experiments were repeated on a minimum of five sperm samples from different individuals., Participants/materials, Setting, Methods: All healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively., Main Results and the Role of Chance: p,p'-DDE rapidly increased [Ca(2+)]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of response up to 200%, without affecting sperm viability, except after 3 days of continuous exposure to the highest concentration tested (P < 0.05). Furthermore, experiments performed in a low Ca(2+) medium demonstrated that extracellular Ca(2+) influx was responsible for this Ca(2+) increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p'-DDE-induced [Ca(2+)]i rise, suggesting the participation of CatSper in this process (P < 0.05). In fact, whole-cell patch-clamp recordings confirmed CatSper as a target of p,p'-DDE action by monitoring an increase in CatSper currents of >100% (P < 0.01). Finally, acrosomal integrity was adversely affected after 2 days of exposure to p,p'-DDE concentrations, suggesting that [Ca(2+)]i rise may cause premature acrosome reaction (P < 0.05)., Limitations, Reasons for Caution: This is an in vitro study, and caution must be taken when extrapolating the results., Wider Implications of the Findings: A novel non-genomic p,p'-DDE mechanism specific to sperm is shown in this study. p,p'-DDE was able to induce [Ca(2+)]i rise in human sperm through the opening of CatSper consequently compromising male fertility. The promiscuous nature of CatSper activation may predispose human sperm to the action of some persistent endocrine disruptors., Study Funding/competing Interest(s): The study was supported by both the Portuguese National Science Foundation (FCT; PEst-C/SAU/LA0001/2011) and the UK Wellcome Trust (Grant #86470). SM was supported by the Infertility Research Trust. RST is a recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). None of the authors has any conflict of interest to declare.
- Published
- 2013
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5. A sperm's tail: the importance of getting it right.
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Ramalho-Santos J
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- Animals, Female, Humans, Male, Oocytes physiology, Sperm Tail physiology, Sperm-Ovum Interactions
- Published
- 2011
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6. Human procreation in unchartered territory: new twists in ethical discussions.
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Ramalho-Santos J
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- Cloning, Organism ethics, Embryonic Stem Cells cytology, Humans, Induced Pluripotent Stem Cells cytology, Pluripotent Stem Cells cytology, Reproductive Techniques, Assisted ethics, Nuclear Transfer Techniques ethics, Reproductive Medicine ethics
- Abstract
Since their validation in mammals, there have been profound ethical discussions on the possible applications of somatic cell nuclear transfer, human embryonic stem cells and induced pluripotent stem cells to reproductive medicine. This has been the case whether these technologies were considered as direct (i.e. when procreation is the ultimate goal) or indirect applications. In most countries, the majority of these approaches have been either stringently regulated, or regulation has been strongly and consensually suggested. However, this is not necessarily the case for possibilities such as same-sex chimaeras or the direct differentiation of gametes from somatic cells, skipping a pluripotent cell intermediate. The author suggests that the field of reproductive medicine should be more proactive in discussing both current and emerging developments with possible implications for human reproduction, even those reaching beyond current paradigms.
- Published
- 2011
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7. Dual use of Diff-Quik-like stains for the simultaneous evaluation of human sperm morphology and chromatin status.
- Author
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Sousa AP, Tavares RS, Velez de la Calle JF, Figueiredo H, Almeida V, Almeida-Santos T, and Ramalho-Santos J
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- Cell Nucleus ultrastructure, DNA Damage, Embryonic Development, Humans, In Situ Nick-End Labeling, Male, Spermatozoa abnormalities, Spermatozoa ultrastructure, Azure Stains, Chromatin ultrastructure, Coloring Agents, Methylene Blue, Spermatozoa cytology, Xanthenes
- Abstract
Background: Sperm chromatin status and nuclear DNA damage can be detected using well-established assays. However, most techniques are time-consuming and/or involve elaborate protocols and equipment. We have recently developed a simple and fast method to monitor sperm chromatin status in field conditions using the Diff-Quik assay which is employed in fertility clinics to assess sperm morphology with standard bright field microscopy. In the present study, we demonstrate that any Diff-Quik-like stain can easily, reproducibly and routinely monitor human sperm chromatin status as well., Methods: Different Diff-Quik-like stains were used to assess sperm morphology and the presence of abnormal dark nuclear staining in human sperm from four ART centres. The TUNEL assay was performed in the same samples, and fertility outcomes were assessed., Results: A significant correlation was found between TUNEL-positive sperm and dark sperm nuclei. Moreover, associations were also found between the percentage of dark sperm nuclei and seminal parameters, embryo development rate, embryo quality and clinical pregnancy, as well as with cryptorchidism, and there was a tendency towards an association with age. A value of 32% abnormal staining is suggested as a predictive threshold for embryo development and pregnancy., Conclusions: Our results show that any Diff-Quik-like stain, already implemented in most laboratories to assess sperm morphology, can be adapted as an indicator for chromatin status in human sperm.
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- 2009
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8. The expression of polymerase gamma and mitochondrial transcription factor A and the regulation of mitochondrial DNA content in mature human sperm.
- Author
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Amaral A, Ramalho-Santos J, and St John JC
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- Cyclooxygenase 1 analysis, Cyclooxygenase 1 genetics, Cyclooxygenase 1 metabolism, DNA Polymerase gamma, DNA, Mitochondrial analysis, DNA-Binding Proteins analysis, DNA-Directed DNA Polymerase analysis, Electron Transport Complex IV analysis, Electron Transport Complex IV genetics, Electron Transport Complex IV metabolism, Gene Dosage, Humans, Immunohistochemistry, Male, Mitochondrial Proteins analysis, Oligospermia genetics, Oligospermia metabolism, Polymerase Chain Reaction, Spermatozoa chemistry, Transcription Factors analysis, Trinucleotide Repeats, DNA, Mitochondrial metabolism, DNA-Binding Proteins metabolism, DNA-Directed DNA Polymerase metabolism, Mitochondrial Proteins metabolism, Spermatogenesis, Spermatozoa metabolism, Transcription Factors metabolism
- Abstract
Background: Human mitochondrial DNA (mtDNA) encodes 13 polypeptides of the electron transfer chain. Its replication is dependent on the nuclear-encoded polymerase gamma (POLG) and mitochondrial transcription factor A (TFAM). For POLG, only the polyglutamine tract, characterized by a series of CAG repeats, has been investigated in human sperm. However, TFAM is associated with the reduction in mtDNA content of testicular sperm. We have determined whether POLG and TFAM have functional roles in post-ejaculatory sperm mtDNA., Methods: Sperm samples were categorized as: normals, samples with one or two abnormal sperm parameters and oligoasthenoteratozoospermics (OATs). These were analysed by fluorescent PCR to determine the number of CAG repeats, real-time PCR for mtDNA copy number and immunocytochemistry and western blotting for patterns of expression for POLG, TFAM and the mtDNA-encoded COXI., Results: Only the OAT group presented with a significantly higher incidence of heterozygosity for CAG repeats, higher mtDNA content and a lower percentage of sperm expressing POLG and TFAM. Paradoxically, good-quality sperm had fewer mtDNA copies but significantly more sperm expressed POLG, TFAM and COXI., Conclusions: Our data support the original findings that an association between sperm quality and POLG CAG repeats does exist. However, the biological significance of these variants in male infertility remains unclear, as these do not seem to affect mtDNA maintenance. The reduction in mtDNA content in normal samples likely reflects normal spermiogenesis, whereas increases in POLG and TFAM expression possibly compensate for the low mtDNA content, maintaining mitochondrial homeostasis.
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- 2007
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9. Aberrant nucleo-cytoplasmic cross-talk results in donor cell mtDNA persistence in cloned embryos.
- Author
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Lloyd RE, Lee JH, Alberio R, Bowles EJ, Ramalho-Santos J, Campbell KH, and St John JC
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- Animals, Cloning, Molecular, DNA, Mitochondrial metabolism, Fertilization in Vitro, Fibroblasts metabolism, Goats, Membrane Potentials, Mitochondria metabolism, Oocytes metabolism, Organ Culture Techniques methods, Polymerase Chain Reaction, Sheep, Cell Nucleus metabolism, Cloning, Organism methods, Cytoplasm metabolism, DNA, Mitochondrial genetics
- Abstract
Mitochondrial DNA is an extranuclear genome normally maternally inherited through the oocyte. However, the use of nuclear transfer can result in both donor cell and recipient oocyte mitochondrial DNA persisting through to blastocyst and being transmitted to the offspring. The degree of donor mitochondrial DNA transmission appears to be random and currently no evidence exists to explain this phenomenon. To determine whether this is a dilution factor or directly related to the transcriptional status of the donor cell in respect of mitochondrial DNA transcription factors, we have generated sheep nuclear transfer embryos using donor cells: (1) possessing their full mitochondrial DNA complement, (2) those partially depleted, and (3) those depleted but containing residual levels. For each donor type, donor mitochondrial DNA persisted in some blastocysts. It is evident from the donor cells used that nuclear-encoded mitochondrial DNA transcription and replication factors persist even after mitochondrial DNA depletion, as do transcripts for some of the mitochondrial-encoded genes. These cells are therefore still programmed to drive mitochondrial DNA replication and transcription. In nuclear transfer-derived embryos, we have observed the persistence of these nuclear-encoded mitochondrial DNA transcription and replication factors but not in those embryos generated through in vitro fertilization. Consequently, nucleo-mitochondrial interaction following nuclear transfer is out of sequence as the onset of mitochondrial replication is a postimplantation event.
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- 2006
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10. WAVE1, an A-kinase anchoring protein, during mammalian spermatogenesis.
- Author
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Rawe VY, Ramalho-Santos J, Payne C, Chemes HE, and Schatten G
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- Animals, Cattle, Cell Compartmentation, Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit, Epididymis cytology, Epididymis physiology, Golgi Apparatus metabolism, Humans, Male, Mice, Papio, Proteins metabolism, Spermatozoa metabolism, Wiskott-Aldrich Syndrome Protein Family, rac1 GTP-Binding Protein metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Mammals physiology, Microfilament Proteins metabolism, Spermatogenesis physiology
- Abstract
Background: Proper compartmentalization of signalling cascades is paramount to many intracellular activities during spermatogenesis and sperm function. In the present study we focus on the A-kinase-anchoring protein (AKAP) WAVE1, a member of the Wiskott-Aldrich syndrome (WASP) family of adaptor proteins, to study its localization throughout mammalian spermatogenesis., Methods: Using transmission electron microscopy, immunocytochemistry and western blotting, we examined the distribution of WAVE1 and putative partners during mammalian spermatogenesis. The localization and association of PKA RII, the regulatory subunit II of protein kinase A, tyrosine kinase Abl, and small GTPase RAC1 were also explored., Results: WAVE1 localization in spermatocytes and round spermatids coincided with Golgi apparatus distribution, whereas in elongated spermatids and testicular sperm WAVE1 localized to the mitochondrial sheath. Following epididymal passage, WAVE1 was found exclusively on the mitochondrial sheath, suggesting that the protein may function in this region. WAVE1 and PKA RII co-localized along the mitochondrial sheath, PKA RII concentrates in the mid-piece, and RAC1 associated with the post-acrosomal region and the connecting piece. The distribution of WAVE1, PKA RII and RAC1 is conserved in mature mouse, bull, baboon and human sperm., Conclusions: The data support the possibility of a functional signalling unit established by WAVE1 and its associated proteins in the mid-piece of maturing sperm.
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- 2004
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11. Ubiquitination of prohibitin in mammalian sperm mitochondria: possible roles in the regulation of mitochondrial inheritance and sperm quality control.
- Author
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Thompson WE, Ramalho-Santos J, and Sutovsky P
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- Animals, Cattle, Epididymis cytology, Epididymis metabolism, Female, Fertilization physiology, Humans, Male, Microscopy, Immunoelectron, Mitochondria metabolism, Prohibitins, Proteins chemistry, Spermatogenesis, Spermatozoa ultrastructure, Ubiquitin metabolism, Proteins metabolism, Repressor Proteins, Spermatozoa metabolism
- Abstract
Ubiquitination of the sperm mitochondria during spermatogenesis has been implicated in the targeted degradation of paternal mitochondria after fertilization, a mechanism proposed to promote the predominantly maternal inheritance of mitochondrial DNA in humans and animals. The identity of ubiquitinated substrates in the sperm mitochondria is not known. In the present study, we show that prohibitin, a highly conserved, 30- to 32-kDa mitochondrial membrane protein, occurs in a number of unexpected isoforms, ranging from 64 to greater than 185 kDa in the mammalian sperm mitochondria, which are the ubiquitinated substrates. These bands bind antiubiquitin antibodies, displaying a pattern consistent with polyubiquitinated "ladders." Immunoprecipitation of sperm extracts with antiprohibitin antibodies followed by probing of the resultant immunocomplexes with antiubiquitin yields a banding pattern identical to that observed by antiprohibitin Western blot analysis. In fact, the presumably nonubiquitinated 30-kDa prohibitin band shows no antiubiquitin immunoreactivity. We demonstrate that ubiquitination of prohibitin occurs in testicular spermatids and spermatozoa. Ubiquitinated prohibitin molecules also accumulate in the defective fractions of ejaculated spermatozoa, which are thought to undergo surface ubiquitination during epididymal passage. In such sperm fractions, ubiquitin also coprecipitates with tubulin and microtubule-associated proteins, presumably contributed by the axonemes of defective, ubiquitinated spermatozoa. The results of the present study suggest that prohibitin is one of the ubiquitinated substrates that makes the sperm mitochondria recognizable by the egg's ubiquitin-proteasome dependent proteolytic machinery after fertilization and most likely facilitates the marking of defective spermatozoa in the epididymis for degradation.
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- 2003
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12. Control of membrane fusion during spermiogenesis and the acrosome reaction.
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Ramalho-Santos J, Schatten G, and Moreno RD
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- Acrosome ultrastructure, Animals, Cytoplasmic Granules ultrastructure, Fertilization, Humans, Intracellular Membranes physiology, Male, Spermatozoa physiology, Spermatozoa ultrastructure, Acrosome Reaction, Membrane Fusion physiology, Spermatogenesis
- Abstract
Membrane fusion is important to reproduction because it occurs in several steps during the process of fertilization. Many events of intracellular trafficking occur during both spermiogenesis and oogenesis. The acrosome reaction, a key feature during mammalian fertilization, is a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome. Once the sperm has crossed the zona pellucida, the gametes fuse, but in the case of the sperm this process takes place through a specific membrane domain in the head, the equatorial segment. The cortical reaction, a process that prevents polyspermy, involves the exocytosis of the cortical granules to the extracellular milieu. In lower vertebrates, the formation of the zygotic nucleus involves the fusion (syngamia) of the male pronucleus with the female pronucleus. Other undiscovered membrane trafficking processes may also be relevant for the formation of the zygotic centrosome or other zygotic structures. In this review, we focus on the recent discovery of molecular machinery components involved in intracellular trafficking during mammalian spermiogenesis, notably related to acrosome biogenesis. We also extend our discussion to the molecular mechanism of membrane fusion during the acrosome reaction. The data available so far suggest that proteins participating in the intracellular trafficking events leading to the formation of the acrosome during mammalian spermiogenesis are also involved in controlling the acrosome reaction during fertilization.
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- 2002
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13. Golgi apparatus dynamics during mouse oocyte in vitro maturation: effect of the membrane trafficking inhibitor brefeldin A.
- Author
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Moreno RD, Schatten G, and Ramalho-Santos J
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- Animals, Cell Division drug effects, Cell Division physiology, Female, Golgi Apparatus ultrastructure, Guanosine 5'-O-(3-Thiotriphosphate) pharmacology, Immunohistochemistry, Macaca mulatta, Membranes drug effects, Membranes metabolism, Mice, Microinjections, Microscopy, Confocal, Oocytes growth & development, Pregnancy, Brefeldin A pharmacology, Golgi Apparatus physiology, Oocytes physiology, Oocytes ultrastructure, Protein Synthesis Inhibitors pharmacology
- Abstract
We have studied Golgi apparatus dynamics during mouse oocyte in vitro maturation, employing both live imaging with the fluorescent lipid BODIPY-ceramide and immunocytochemistry using several specific markers (beta-COP, giantin, and TGN38). In germinal vesicle oocytes the Golgi consisted of a series of structures, possibly cisternal stacks, dispersed in the ooplasm, but slightly more concentrated in the interior than at the cortex. A similar pattern was detected in rhesus monkey germinal vesicle oocytes. These "mini-Golgis" were functionally active because they were reversibly disrupted by the membrane trafficking inhibitor brefeldin A. However, the drug had no visible effect if the oocytes had been previously microinjected with GTP-gamma-S. During in vitro maturation the large Golgi apparatus structures fragmented at germinal vesicle breakdown, and dispersed homogenously throughout the ooplasm, remaining in a fragmented state in metaphase-II oocytes. Similarly to what has been reported using protein synthesis inhibitors, the presence of brefeldin A blocked maturation at the germinal vesicle breakdown stage before the assembly of the metaphase-I spindle. These results suggest that progression of murine oocyte maturation may require functional membrane trafficking.
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- 2002
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14. ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.
- Author
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Ramalho-Santos J, Sutovsky P, Simerly C, Oko R, Wessel GM, Hewitson L, and Schatten G
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- Acrosome physiology, Animals, Bromodeoxyuridine metabolism, Cell Nucleus metabolism, Cell Nucleus ultrastructure, Centrosome physiology, Centrosome ultrastructure, DNA biosynthesis, Female, Macaca mulatta, Male, Microscopy, Electron, Microtubules ultrastructure, Spermatozoa ultrastructure, Zygote ultrastructure, DNA ultrastructure, Sperm Head ultrastructure, Sperm Injections, Intracytoplasmic
- Abstract
We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.
- Published
- 2000
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15. Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos.
- Author
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Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, and Schatten G
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- Animals, Antibodies pharmacology, Cattle, Cytoplasm metabolism, Endopeptidases metabolism, Fertilization, Lysosomes enzymology, Male, Oocytes ultrastructure, Spermatogenesis, Spermatozoa ultrastructure, Ubiquitins immunology, DNA, Mitochondrial genetics, Embryo, Mammalian metabolism, Gene Expression Regulation, Mitochondria metabolism, Spermatozoa metabolism, Ubiquitins metabolism
- Abstract
The strictly maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) in mammals is a developmental paradox promoted by an unknown mechanism responsible for the destruction of the sperm mitochondria shortly after fertilization. We have recently reported that the sperm mitochondria are ubiquitinated inside the oocyte cytoplasm and later subjected to proteolysis during preimplantation development (P. Sutovsky et al., Nature 1999; 402:371-372). Here, we provide further evidence for this process by showing that the proteolytic destruction of bull sperm mitochondria inside cow egg cytoplasm depends upon the activity of the universal proteolytic marker, ubiquitin, and the lysosomal apparatus of the egg. Binding of ubiquitin to sperm mitochondria was visualized by monospecific antibodies throughout pronuclear development and during the first embryonic divisions. The recognition and disposal of the ubiquitinated sperm mitochondria was prevented by the microinjection of anti-ubiquitin antibodies and by the treatment of the fertilized zygotes with lysosomotropic agent ammonium chloride. The postfecundal ubiquitination of sperm mitochondria and their destruction was not seen in the hybrid embryos created using cow eggs and sperm of wild cattle, gaur, thus supporting the hypothesis that sperm mitochondrion destruction is species specific. The initial ligation of ubiquitin molecules to sperm mitochondrial membrane proteins, one of which could be prohibitin, occurs during spermatogenesis. Even though the ubiquitin cross-reactivity was transiently lost from the sperm mitochondria during epididymal passage, likely as a result of disulfide bond cross-linking, it was restored and amplified after fertilization. Ubiquitination therefore may represent a mechanism for the elimination of paternal mitochondria during fertilization. Our data have important implications for anthropology, treatment of mitochondrial disorders, and for the new methods of assisted procreation, such as cloning, oocyte cytoplasm donation, and intracytoplasmic sperm injection.
- Published
- 2000
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16. Vesicular traffic and golgi apparatus dynamics during mammalian spermatogenesis: implications for acrosome architecture.
- Author
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Moreno RD, Ramalho-Santos J, Sutovsky P, Chan EK, and Schatten G
- Subjects
- Animals, Autoantigens metabolism, Biological Transport, Brefeldin A pharmacology, Cell Compartmentation, Cell Line, Cells, Cultured, Coatomer Protein metabolism, Endoplasmic Reticulum metabolism, Fluorescent Dyes, Golgi Apparatus ultrastructure, Golgi Matrix Proteins, Macaca mulatta, Male, Membrane Proteins metabolism, Microscopy, Electron methods, Spermatids metabolism, Spermatids ultrastructure, Acrosome Reaction physiology, Coated Vesicles metabolism, Golgi Apparatus metabolism, Proteins, Spermatogenesis physiology
- Abstract
Vesicular membrane trafficking during acrosome biogenesis in bull and rhesus monkey spermatogenesis differs from the somatic cell paradigm as imaged dynamically using the Golgi apparatus probes beta-COP, giantin, Golgin-97, and Golgin-95/GM130. In particular, sorting and delivery of proteins seemed less precise during spermatogenesis. In early stages of spermiogenesis, many Golgi resident proteins and specific acrosomal markers were present in the acrosome. Trafficking in both round and elongating spermatids was similar to what has been described for somatic cells, as judged by the kinetics of Golgi protein incorporation into endoplasmic reticulum-like structures after brefeldin A treatment. These Golgi components were retrieved from the acrosome at later stages of differentiation and were completely devoid of immature spermatozoa. Our data suggest that active anterograde and retrograde vesicular transport trafficking pathways, involving both beta-COP- and clathrin-coated vesicles, are involved in retrieving Golgi proteins missorted to the acrosome and in controlling the growth and shape of this organelle.
- Published
- 2000
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17. On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.
- Author
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Sutovsky P, Ramalho-Santos J, Moreno RD, Oko R, Hewitson L, and Schatten G
- Subjects
- Animals, Cattle, Female, Fluorescent Antibody Technique, Macaca mulatta, Male, Microscopy, Electron, Cell Separation methods, Fluorescent Dyes, Mitochondria ultrastructure, Spermatids ultrastructure
- Abstract
The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility treatment, has been hampered by the ambiguous definition of a round spermatid and the lack of specific vital and non-vital markers. Using cells from rhesus monkey and bull, we describe a non-invasive method for the on-stage selection of individual round spermatids for ROSI, based on the polarized patterns of mitochondria, visualized in live round spermatid cells by epifluorescence microscopy after incubation with MitoTracker(TM), a vital, mitochondrion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosomal cap in parallel observations by Nomarski differential interference contrast microscopy. The existence of mitochondrial polarization was first established by the labelling of MitoTracker-tagged round spermatids with spermatid-specific antibodies against proteins of nascent sperm accessory structures combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope equipped with epifluorescence, the round spermatids can be individually selected from a heterogeneous population of testicular cells labelled with MitoTracker dyes. A major advantage of this approach is that the dyes are incorporated into the paternal mitochondria, destined for rapid elimination after fertilization. In addition, the relatively high excitation and emission wavelengths of MitoTracker dyes are less harmful to DNA after their photon excitation. Before the appropriate clinical testing is conducted, the MitoTracker-based round spermatid selection may be instrumental in the training of clinical staff.
- Published
- 1999
- Full Text
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