Understanding new molecular and cell biology findings based on progressive scientific practices and interconnected activities in undergraduate students.

Nowadays Molecular Cell Biology (MCB) must be taught as science is practiced. Even though there are several approaches based on scientific practices, a key aspect is to define the purpose of each of these teaching strategies and, most importantly, their implementation. Our goal was to train students to acquire, understand, and communicate new scientific knowledge in the field. The main feature of our new teaching methodology was progressive training in scientific practices associated with a back-and-forward interplay between activities and assessments. The methodology was implemented over 4 years, in students attending the MCB course of the undergraduate degree in Biological Sciences. In the first two modules, the students were prepared to comprehend MCB concepts and techniques and to experience activities based on scientific practices. In the third module, the students analyzed a primary paper in-depth. They were assessed by midterm exams based on a primary paper, written laboratory reports, and the oral presentation of a scientific paper. Our teaching proposal was evaluated through the students' academic performance and by their opinion on the teaching methodology. Most students were satisfied since they improved their acquisition of concepts, their interpretation and integration of scientific knowledge, and developed skills to communicate scientific knowledge in writing and orally. The novelty of transversal interconnections and progressive training in scientific practices provides students with skills in acquiring and understanding new scientific information, even beyond the MCB course.

Chemotactic selection of frozen-thawed stallion sperm improves sperm quality and heterologous binding to oocytes.

The successful use of assisted reproduction techniques (ART) depends in part on the sperm physiological status. Several sperm selection procedures have been applied to improve quality of sperm population when using the ART. There has previously been development of a Sperm Selection Assay (SSA) for humans which is based on the attraction of capacitated sperm by chemotaxis towards progesterone (P), resulting in an enriched sperm population with an optimal physiological status similar to capacitated spermatozoa, with these cells having very little DNA fragmentation and optimal concentrations of reactive oxygen species (ROS). In the present study, the aim was to adapt the SSA for frozen-thawed stallion semen samples and evaluate the functional status of those sperm selected using the SSA procedure, and to determine whether this enriched sperm population has a greater capacity to bind to the zona pellucida of cattle oocytes. There were experimental conditions developed to conduct the SSA with stallion sperm. Using these conditions, the indexes of induced acrosome reaction, protein tyrosine phosphorylation, mitochondrial membrane potential, mitochondrial and cytoplasmic reactive oxygen species, and number of sperm bound to the zona pellucida of cattle were greater when the sperm population was selected using the SSA. Consistently, the DNA fragmentation and phospholipase C zeta indexes were less for the selected sperm. In conclusion, stallion sperm selected using chemotaxis utilizing the SSA provides a sperm population of greater quality, which when used may improve the outcomes with use of the ART.

Reactive oxygen species are involved in the signaling of equine sperm chemotaxis.

Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was increased with 0.1 and 0.2 mM hydrogen peroxide (H2O2), which was involved in the chemotactic signaling. By combining a concentration gradient of H2O2 with inhibitors/chelators of some of the signaling pathway elements, we showed that the activation of NOX (membrane NADPH oxidase) increases the intracellular ROS which activate the chemotaxis AMPc-PKA pathway. Our results provide evidence about the participation of ROS in the chemotactic signaling mediated by progesterone (P).

Sperm physiology varies according to ultradian and infradian rhythms.

The spermatozoon must be physiologically prepared to fertilize the egg, process called capacitation. Human sperm samples are heterogeneous in their ability to capacitate themselves, which leads to variability between samples from the same or different donors, and even along the seasons. Here we studied sperm variation in the capacitation state according to the ability of capacitated spermatozoa to acrosome react upon stimulation (% ARi) and to be recruited by chemotaxis (% Chex). Both indirect indicators of sperm capacitation increased along the incubation time with fluctuations. Those capacitated sperm recruited by chemotaxis showed an ultradian rhythm with a cycle every 2 h, which might be influenced by unknown intrinsic sperm factors. Two infradian rhythms of 12 months for the % ARi and of 6 months for % Chex were observed, which are associated with the joint action of temperature and photoperiod. Thus, to avoid false negative results, human sperm samples are recommended to be incubated for a long period (e.g. 18 h) preferably in spring time. This innovative point of view would lead to better comprehend human reproductive biology and to think experimental designs in the light of sperm cyclicity or to improve sperm aptitude for clinical purposes.

Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation.

STUDY QUESTION: Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? SUMMARY ANSWER: Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. WHAT IS KNOWN ALREADY: Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. STUDY DESIGN SIZE, DURATION: Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. MAIN RESULTS AND THE ROLE OF CHANCE: Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported DGAPA (IN203116 to C. Treviño), Fronteras-CONACyT No. 71 and Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH (RO1 HD38082) to P.E. Visconti and by a Lalor Foundation fellowship to M.G. Gervasi. A. Matamoros is a student of the Maestría en Ciencias Bioquímicas-UNAM program supported by CONACyT (416400) and DGAPA-UNAM. A. Moreno obtained a scholarship from Red MacroUniversidades and L. Giojalas obtained a schloarhip from CONICET and Universidad Nacional de Cordoba. The authors declare there are not conflicts of interest.