The inhibitory effect of progesterone on lactogenesis during pregnancy is already evident by mid- to late gestation in rodents

Lactogenesis is a very complex process highly dependent on hormonal regulation. In the present study the time-course of the inhibitory actions of progesterone on prolactin secretion, mammary gland morphology and lactogenesis from mid- to late gestation in rodents was investigated. Groups of pregnant rats were luteectomised or administered with mifepristone on Day 10, 13, 15 or 17 of gestation and decapitated 28 or 48h later. Whole-blood samples and the inguinal mammary glands were taken for determinations of hormone levels and for measurement of mammary content of casein and lactose and for tissue morphology analyses, respectively. Luteectomy or mifepristone evoked prolactin increases only after Day 17 of gestation. Mammary content of casein was increased by both treatments regardless of timing or duration. Mifepristone was less effective than luteectomy in inducing lactose production and the effect was only observed after Day 15 of gestation. Analysis of mammary gland morphology confirmed the observed effect of progesterone on lactogenesis. Both treatments triggered remarkable secretory activity in the mammary gland, even without a parallel epithelial proliferation, demonstrating that the mammary epithelium is able to synthesise milk compounds long before its full lobulo-alveolar development is achieved, provided that progesterone action is abolished. Thus, the present study demonstrates that progesterone is a potent hormonal switch for the prolactin and prolactin-like effects on mammary gland development and its milk-synthesising capacity during pregnancy, and that its inhibitory action is already evident by mid-pregnancy in rodents.Fil: López Fontana, Constanza Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Maselli, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Salicioni, Ana M.. University Of Massachusetts Amherst; Estados UnidosFil: Caron, Ruben Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

Functional human sperm capacitation requires both bicarbonate dependent-PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/PKA pathway. Recent studies in mice revealed however that a Src Family Kinase (SFK) induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (p<0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6 h-incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster eggs. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analogue and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (p<0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species-specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.Fil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Da Ros, Vanina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Salicioni, A.. University Of Massachussets; Estados UnidosFil: Navarrete, F.. University Of Massachussets; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Visconti, P. E.. University Of Massachussets;Fil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas -conicet. Instituto de Biología y Medicina Experimental (i); Argentin

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

Testis-specific serine kinase protein family in male fertility and as targets for non-hormonal male contraception

Male contraception is a very active area of research. Several hormonal agents have entered clinical trials, while potential non-hormonal targets have been brought to light more recently and are at earlier stages of development. The general strategy is to target genes along the molecular pathways of sperm production, maturation, or function, and it is predicted that these novel approaches will hopefully lead to more selective male contraceptive compounds with a decreased side effect burden. Protein kinases are known to play a major role in signaling events associated with sperm differentiation and function. In this review, we focus our analysis on the testis-specific serine kinase (TSSK) protein family. We have previously shown that members of the family of TSSKs are postmeiotically expressed in male germ cells and in mature mammalian sperm. The restricted postmeiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggests 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 knockout (KO) mice and of the double Tssk1 and Tssk2 KO mice and by the male subfertile phenotype observed in a Tssk4 KO mouse model

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

Biphasic Role of Calcium in Mouse Sperm Capacitation Signaling Pathways

Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up-regulation of a cAMP-dependent pathway, changes in intracellular pH, intracellular Ca2+, and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca2+ ions have a biphasic role in the regulation of cAMP-dependent signaling. Media without added Ca2+ salts (nominal zero Ca2+) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca2+. However, chelation of the extracellular Ca2+ traces by EGTA induced both cAMP-dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca2+ media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca2+ ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild-type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ media. Therefore, sperm lacking Catsper Ca2+ channels behave as wild-type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca2+ involved in the regulation of cAMP-dependent and tyrosine phosphorylation pathways required for sperm capacitation.Fil: Navarrete, Felipe A.. University of Massachusetts; Estados UnidosFil: García Vázquez, Francisco A.. University of Massachusetts; Estados Unidos. Universidad de Murcia; EspañaFil: Alvau, Antonio. University of Massachusetts; Estados UnidosFil: Escoffier, Jessica. University of Massachusetts; Estados UnidosFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México; MéxicoFil: Salicioni, Ana M.. University of Massachusetts; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Visconti, Pablo E.. University of Massachusetts; Estados Unido

Extensión 'network-aware' del GUI de Windows

El objetivo es proveer un mecanismo que permita ejecutar cualquier aplicación Windows 3.1 existente en una computadora remota y que su Interfase Gráfica de Usuario (GUI) esté en la workstation local. En otras palabras, un usuario debería poder interactuar con un programa Windows en una máquina (a la que nos referiremos indistintamente como máquina, computadora o nodo local), pero ese programa no necesariamente tendría que estar ejecutándose en la computadora en la que el usuario se encuentra, sino que podría estar ejecutándose en un nodo remoto conectado con el primero mediante alguna tecnología de red. Para lograr esto, en el nodo local debería existir minimamente un UIMS (User Interface Management System), que en principio no necesariamente debería ser el de Windows. Actualmente la mayoría de los sistemas operativos disponen de un entorno gráfico; por ejemplo el entorno gráfico de cualquier plataforma UNIX está basado en el sistema de ventanas X Window System, las computadoras Apple Macintosh tienen su propio sistema de ventanas, el UIMS de OS/2 es Presentation Manager, etc. Ademas, debido a que Windows no implementa un UIMS basado en red, es necesario definir un protocolo de comunicación a nivel de aplicación para trasladar todos los elementos de la GUI desde el nodo remoto (donde se ejecuta la aplicación Windows) hacia el nodo local. Por último, en el nodo local deberían resolverse los requerimientos gráficos que transporta el protocolo antes mencionado y generar los requerimientos correspondientes sobre el UIMS local. Por otro lado, como consecuencia de la separación de la GUI de las aplicaciones, hay que disponer de un mecanismo para que el usuario en el nodo local pueda iniciar la ejecución de aplicaciones en el nodo remoto, que presenten su interfase en el nodo local.Tesis digitalizada en SEDICI gracias a la colaboración de la Biblioteca de la Facultad de Informática.Facultad de Ciencias Exacta

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

Human Sperm Remain Motile After a Temporary Energy Restriction but do Not Undergo Capacitation-Related Events

To acquire fertilization competence, mammalian sperm must undergo several biochemical and physiological modifications known as capacitation. Despite its relevance, the metabolic pathways that regulate the capacitation-related events, including the development of hyperactivated motility, are still poorly described. Previous studies from our group have shown that temporary energy restriction in mouse sperm enhanced hyperactivation, in vitro fertilization, early embryo development and pregnancy rates after embryo transfer, and it improved intracytoplasmic sperm injection results in the bovine model. However, the effects of starvation and energy recovery protocols on human sperm function have not yet been established. In the present work, human sperm were incubated for different periods of time in medium containing glucose, pyruvate and lactate (NUTR) or devoid of nutrients for the starving condition (STRV). Sperm maintained in STRV displayed reduced percentages of motility and kinematic parameters compared to cells incubated in NUTR medium. Moreover, they did not undergo hyperactivation and showed reduced levels of ATP, cAMP and protein tyrosine phosphorylation. Similar to our results with mouse sperm, starvation induced increased intracellular Ca2+ concentrations. Starved human sperm were capable to continue moving for more than 27 h, but the incubation with a mitochondrial uncoupler or inhibitors of oxidative phosphorylation led to a complete motility loss. When exogenous nutrients were added back (sperm energy recovery (SER) treatment), hyperactivated motility was rescued and there was a rise in sperm ATP and cAMP levels in 1 min, with a decrease in intracellular Ca2+ concentration and no changes in sperm protein tyrosine phosphorylation. The finding that human sperm can remain motile for several hours under starvation due to mitochondrial use of endogenous metabolites implies that other metabolic pathways may play a role in sperm energy production. In addition, full recovery of motility and other capacitation parameters of human sperm after SER suggests that this treatment might be used to modulate human sperm fertilizing ability in vitro

Compartmentalization of distinct cAMP signaling pathways in mammalian sperm.

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. While the HCO3--dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this manuscript, we used Western blotting and cholera toxin-dependent ADP ribosylation to show Gs presence in the sperm head. Also, we showed that forskolin, a tmAC specific activator, induces cAMP accumulation in sperm from both WT and Adcy10 null mice. This increase is blocked by the tmAC inhibitor SQ-22536 but not by the Adcy10 inhibitor KH7. While Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome reacted sperm, and forskolin is able to increase intracellular Ca2+ and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.Fil: Wertheimer Hermitte, Eva Victoria. University Of Massachussets; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Krapf, Dario. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: de la Vega Beltran, José L.. Universidad Nacional Autónoma de México; MéxicoFil: Sánchez Cárdenas, Claudia. Universidad Nacional Autónoma de México; MéxicoFil: Navarrete, Felipe. University Of Massachussets; Estados UnidosFil: Haddad, Douglas. University Of Massachussets; Estados UnidosFil: Escoffier, Jessica. University Of Massachussets; Estados UnidosFil: Salicioni, Ana M.. University Of Massachussets; Estados UnidosFil: Levin, Lonny R.. Cornell University; Estados UnidosFil: Buck, Jochen. Cornell University; Estados UnidosFil: Mager, Jesse. University Of Massachussets; Estados UnidosFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Visconti, Pablo E.. University Of Massachussets; Estados Unido