15 research outputs found
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Caput Ligation Renders Immature Mouse Sperm Motile and Capable to Undergo cAMP-Dependent Phosphorylation
Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation
The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm
Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro.Fil: Alvau, Antonio. University of Massachussets; Estados UnidosFil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gervasi, Maria Gracia. University of Massachussets; Estados UnidosFil: Navarrete, Felipe A.. University of Massachussets; Estados UnidosFil: Xu, Xinran. State University of Colorado - Fort Collins; Estados UnidosFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: De la Vega Beltran, José Luis. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Greer, Peter. Queens University; CanadáFil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; MéxicoFil: Krapf, Diego. State University of Colorado - Fort Collins; Estados UnidosFil: Salicioni, Ana María. University of Massachussets; Estados UnidosFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados Unido
TSSK3, a novel target for male contraception, is required for spermiogenesis
We have previously shown that members of the family of testis-specific serine/threonine kinases (TSSKs) are post-meiotically expressed in testicular germ cells and in mature sperm in mammals. The restricted post-meiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggest that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the TSSK6 knock-out (KO) mice and of the double TSSK1/TSSK2 KO. The aim of this study was to develop KO mouse models of TSSK3 and to validate this kinase as a target for the development of a male contraceptive. We used CRISPR/Cas9 technology to generate the TSSK3 KO allele on B6D2F1 background mice. Male heterozygous pups were used to establish three independent TSSK3 KO lines. After natural mating of TSSK3 KO males, females that presented a plug (indicative of mating) were monitored for the following 24 days and no pregnancies or pups were found. Sperm numbers were drastically reduced in all three KO lines and, remarkably, round spermatids were detected in the cauda epididymis of KO mice. From the small population of sperm recovered, severe morphology defects were detected. Our results indicate an essential role of TSSK3 in spermiogenesis and support this kinase as a suitable candidate for the development of novel nonhormonal male contraceptives.Fil: Nayyab, Saman. University of Massachussets; Estados UnidosFil: Gervasi, María G.. University of Massachussets; Estados UnidosFil: Tourzani, Darya A.. University of Massachussets; Estados UnidosFil: Caraballo, Diego Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Jha, Kula N.. No especifíca;Fil: Teves, Maria E.. University of Virginia; Estados UnidosFil: Cui, Wei. University of Massachussets; Estados UnidosFil: Georg, Gunda I.. University of Minnesota; Estados UnidosFil: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: Salicioni, Ana M.. University of Massachussets; Estados Unido
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Mouse sperm energy restriction and recovery (SER) revealed novel metabolic pathways
Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites
Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies
To become fertile, mammalian sperm must undergo a series of biochemical and physiological changes known as capacitation. These changes involve crosstalk between metabolic and signaling pathways and can be recapitulated in vitro. In this work, sperm were incubated in the absence of exogenous nutrients (starved) until they were no longer able to move. Once immotile, energy substrates were added back to the media and sperm motility was rescued. Following rescue, a significantly higher percentage of starved sperm attained hyperactivated motility and displayed increased ability to fertilize in vitro when compared with sperm persistently incubated in standard capacitation media. Remarkably, the effects of this treatment continue beyond fertilization as starved and rescued sperm promoted higher rates of embryo development, and once transferred to pseudo-pregnant females, blastocysts derived from treated sperm produced significantly more pups. In addition, the starvation and rescue protocol increased fertilization and embryo development rates in sperm from a severely subfertile mouse model, and when combined with temporal increase in Ca2+ ion levels, this methodology significantly improved fertilization and embryo development rates in sperm of sterile CatSper1 KO mice model. Intracytoplasmic sperm injection (ICSI) does not work in the agriculturally relevant bovine system. Here, we show that transient nutrient starvation of bovine sperm significantly enhanced ICSI success in this species. These data reveal that the conditions under which sperm are treated impact postfertilization development and suggest that this “starvation and rescue method” can be used to improve assisted reproductive technologies (ARTs) in other mammalian species, including humans.Fil: Navarrete, Felipe A.. University of Massachussets; Estados UnidosFil: Aguila, Luis. University of Massachussets; Estados UnidosFil: Martin Hidalgo, David. University of Massachussets; Estados Unidos. Universidad de Extremadura ; EspañaFil: Tourzani, Darya A.. University of Massachussets; Estados UnidosFil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ardestani, Goli. University of Massachussets; Estados UnidosFil: Garcia Vazquez, Francisco A.. Universidad de Murcia; EspañaFil: Levin, Lonny R.. Cornell University; Estados UnidosFil: Buck, Jochen. Cornell University; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biología; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Mager, Jesse. University of Massachussets; Estados UnidosFil: Fissore, Rafael A.. University of Massachussets; Estados UnidosFil: Salicioni, Ana M.. University of Massachussets; Estados UnidosFil: Gervasi, María G.. University of Massachussets; Estados UnidosFil: Visconti, Pablo E.. University of Massachussets; Estados Unido
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Regulation of Rac1 activation by the low density lipoprotein receptor–related protein
The low density lipoprotein receptor–related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1–deficient MEFs demonstrated increased Rac1 activation compared with LRP-1–expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK cells. Untreated uPAR MEFs demonstrated substantially increased Rac1 activation compared with uPAR MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal– regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked
Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.
The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI)-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI)-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform
Assay characteristics of the chromium release assay vs. BLI assay.
<p>The spontaneous death, maximal killing, range, and the signal to noise (max:min) ratios measured by <sup>51</sup>Cr release and BLI are shown. Results are presented as mean ± SD of three independent experiments.</p><p>* p<0.01 by Student t test.</p
Comparison of cytotoxicity obtained at 4 hours by the chromium release and BLI method at higher E:T ratios.
<p>Luciferase-transduced the YAC1 cell line was co-cultured with mouse effector cells for 4 hours at various E:T ratios ranging from 100∶1 to 20∶1. The % specific lysis obtained by the chromium release assay (closed circles) or the BLI assay (open circles) is plotted against multiple E:T ratios. Results are represented as mean ± SD of triplicate values. One representative of 2 independent experiments is shown. The p value obtained from the statistical analysis performed permutated two-way ANOVA is shown.</p