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

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

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

Human fertilization: epididymal hCRISP1 mediates sperm zona pellucida binding through its interaction with ZP3

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm?egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein?protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved notonly in sperm?egg fusion but also in the previous stage of sperm?ZP binding through its specific interaction with human ZP3.Fil: Maldera, Julieta Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Heidelberg University. Center for Molecular Biology; AlemaniaFil: Weigel Muñoz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Chirinos, M.. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubiran; MéxicoFil: Busso, Dolores. Pontificia Universidad Católica de Chile; Chile. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Raffo, F. G. E.. Centro Médico Fertilab; ArgentinaFil: Battistone, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Blaquier, J. A.. Centro Médico Fertilab; ArgentinaFil: Larrea, F.. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubiran; MéxicoFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

A Phenomenological Study on The Lived Experience of First and Second Year Teachers in Standards-Based Grading Districts

228 leavesABSTRACT Problem: There is an existing cycle of questionable grading practices at the K-12 level. As a result, districts continue to search for innovative methods of evaluating and reporting student progress. One result of this effort has been the adoption of a standards-based grading approach. Research concerning standards-based grading implementation has resulted in an abundance of strategies to change existing practices used by educators. Very little attention has been given to the experience of new educators, who are still developing their educational pedagogy and practice for the first time. Procedures: This hermeneutic phenomenology (Heidegger, 2008; Van der Zalm & Bergum, 2000) explored the lived experience of 11 first or second year teachers in districts currently utilizing a standards-based grading approach. The research question for this study was: What is the lived experience of first or second year K-12 educators in districts using standards-based grading policies? This overarching research question was supported by five supporting subquestions, which utilized Senge’s (2006) Five Disciplines of the Learning Organization as a conceptual framework. Purposeful, criterion, and stratified purposive sampling were all used for recruiting participants for this study (Creswell, 2014; Salmons, 2010; Van der Zalm & Bergum, 2000). Data was collected using a three-interview approach recommended by Seidman (2013), including life history, details of experience, and reflection on the meaning. Additional data gathered included supplemental materials and researcher reflections. The data analysis process included coding, triangulation, member-checking, and inter-coder agreement. Findings: Findings were communicated in a descriptive and interpretive method, in order to gain understanding of a specific phenomenon (Heidegger, 2008; Van der Zalm & Bergum, 2000). Data analysis resulted in the following findings: (a) inconsistencies with grading and assessment preservice training, (b) student teaching experiences, (c) professional development practice concerning grading and assessment, (d) focusing on connections with students, (e) navigating the traditional K-12 assessment model, (f) experiences of new teachers as K-12 students, (g) grading based on standards, (h) inconsistencies in SBG implementation, (i) balancing theory and practice, (j) the role of mentors, (k) the function of PLCs, (l) the role of new educators on teacher teams, (m) the responsibility of teachers to grade student work, (n) holding students accountable for their work, and (o) translating a number to a letter grade. Conclusions: Varying experiences in preservice programs, the presence of a mentor, a lack of consistent SBG implementation, and fundamental misunderstandings of the standards-based grading system create a unique lived experience for the new educators. Recommendations: Preservice programs should be providing more specific training for new educators in the areas of assessment and grading. Preservice educators would benefit from more explicit instruction concerning assessment and grading; specifically, preservice programs should explore implementing a specific assessment and grading course. Districts that are currently utilizing a standards-based model should ensure consistent implementation of SBG building and district-wide, as well as adopt a competency-based professional development model for their teachers concerning grading and assessment practice. It is essential for districts to consistently and accurately communicate the rationale for SBG to their respective faculties

Environmental surveillance of poliovirus and non-polio Enterovirus in Milan (Northern Italy) in 2011 and 2012

Introduction. The WHO Strategic Plan of the Global Polio Eradication Initiative indicates the environmental surveillance of poliovirus (PV) as a key activity to supplement the standard acute flaccid paralysis (AFP) surveillance. In this framework, other human Enteroviruses (EVs) are monitored due to their high circulation in human population and their involvement in outbreaks of meningitis, encephalitis, hand-foot-mouth disease and other acute and chronic manifestations. This study aimed at analyzing the circulation of PVs (wild, Sabin-like, and vaccine-derived) and non-polio Enteroviruses (NPEVs) in wastewater samples collected in the Milan area (Northern Italy) in 2011 and 2012. Methods. This study was carried out according to WHO guidelines for environmental surveillance of PV (WHO/V&B/03.03). Wastewater samples were collected regularly twice a month at the inlet of 3 different wastewater treatment plants in the Milan area in 2011 and 2012. EV identification was carried out by viral isolation in cell cultures (RD and L20b), followed by PCR specific for the 5\u2032 noncoding region (5\u2032NCR) (nt. 179-575) common to all EVs. EV characterization was performed by sequence analysis of the VP1 region (nt. 2628-2976). Result. Overall, 128 sewage samples were collected during the study period. No PVs were isolated. NPEVs were isolated in 70% and 76.5% of wastewater specimens analyzed in 2011 and 2012, respectively. All isolates belonged to the EV-B group and those circulating more intensely were Echovirus type 11 (48.8% in 2011; 65.4% in 2012) and Echovirus type 6 (34.8% in 2011; 13.5% in 2012), representing 81% of all EVs identified. The circulation of Echovirus type 11 significantly (p<0.05) increased during the study period, while Echovirus type 6 presented a decreasing trend. Conclusion. During environmental surveillance in Milan area no PV were isolated, supporting the epidemiological data deriving from AFP surveillance. NPEVs had shown an high circulations rate and Echovirus type 11 and 6 were the predominant strains, similarly to other studies. In countries where no surveillance system is implemented for the diagnosis of NPEV infections and hospitalizations, environmental surveillance are a useful tool to monitor the circulation of these viruses and their possible emergency in the population

Rotavirus genotypes in sewage treatment plants and in children hospitalized with acute diarrhea in Italy 2010 - 2011

Although the molecular surveillance network RotaNet-Italy provides useful nation-wide data on rotaviruses causing severe acute gastroenteritis in children in Italy, scarce information is available on rotavirus circulation in the general Italian population, including adults with mild or asymptomatic infection. This study investigated the genotypes of rotavirus present in urban wastewaters, and compared them with viral strains from clinical pediatric cases. During 2010 and 2011, 285 sewage samples from 4 Italian cities were tested by RT-PCRs specific for rotavirus VP7 and VP4 genes. Rotavirus was detected in 172 (60.4%) samples, 26 of which contained multiple rotavirus G (VP7 gene) genotypes, for a total of 198 G types. Thirty-two samples also contained multiple P (VP4 gene) genotypes, yielding 204 P types in 172 samples. Genotype G1 accounted for 65.6% of rotaviruses typed, followed by G2 (20.2%), G9 (7.6%), G4 (4.6%), G6 (1.0%), G3 (0.5%), and G26 (0.5%). VP4 genotype P[8] accounted for 75.0% of strains, P[4] for 23.0%, and the uncommon P[6], P[9], P[14], and P[19] for 2.0% of strains altogether. These rotavirus genotypes were also found in pediatric patients hospitalized in the same areas and years but in different proportions. Specifically, genotypes G2, G9, and P[4] were more prevalent in sewage samples than among patients, which suggests either a larger circulation of these latter strains through the general population not requiring medical care or their higher survival in wastewaters. A high nucleotide identity in the G1, G2, and G6 VP7 sequences was observed between strains from the environment and patients

Evidence for the involvement of Proline-rich Tyrosine Kinase 2 (PYK2) in tyrosine phosphorylation downstream of PKA activation during human sperm capacitation

Sperm capacitation involves an increase in intracellular Ca(2+) concentration as well as in protein kinase A (PKA)-dependent protein tyrosine (Tyr) phosphorylation. Interestingly, in humans, a decrease in extracellular Ca(2+) concentration ([Ca(2+)]e) during capacitation induces an increase in Tyr phosphorylation indicating the complexity of Ca(2+) signaling during this process. In view of this, in the present study we further investigated the Ca(2+)-mediated signaling pathways implicated in Tyr phosphorylation during human sperm capacitation. Results revealed that sperm incubation in a medium without added Ca(2+) (e Ca(2+)) increased Tyr phosphorylation but did not modify PKA-mediated phosphorylation. Moreover, inhibition of either PKA or Src family kinase signaling cascades in e Ca(2+) down-regulated both PKA substrate and Tyr phosphorylations, indicating that the [Ca(2+)]e effects on Tyr phosphorylation depend on PKA targets. Inhibition of calmodulin or Ser/Thr protein phosphatase 2B also increased Tyr phosphorylation without affecting PKA-mediated phosphorylation, supporting the potential role of these Ca(2+) downstream effectors in the increase in Tyr phosphorylation observed in e Ca(2+). Experiments aimed to identify the kinase responsible for these observations revealed the presence of proline-rich tyrosine kinase 2 (PYK2), a focal adhesion kinase (FAK) family member, in human sperm, and the use of PF431396, an FAK inhibitor, supported the involvement of PYK2 in Tyr phosphorylation downstream of PKA activation. Results also showed that PYK2 was activated in e Ca(2+) as well as during capacitation and that PF431396 affected capacitated sperm motility, acrosome reaction and ability to penetrate both mouse cumulus matrix and zona-free hamster eggs. Together, our observations support PYK2 as an intermediary component of Ca(2+) signaling between PKA-mediated and Tyr phosphorylations that is required for achieving functional human sperm capacitation.Fil: 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: Alvau, A.. University of Massachussets; Estados UnidosFil: Salicioni, A. M.. University of Massachussets; Estados UnidosFil: Visconti, P. E.. University of Massachussets; Estados UnidosFil: 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: 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; Argentin