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.

Hitting the wall: Human sperm velocity recovery under ultra-confined conditions.

Infertility is a common medical condition encountered by health systems throughout the world. Despite the development of complex in vitro fertilization techniques, only one-third of these procedures are successful. New lab-on-a-chip systems that focus on spermatozoa selection require a better understanding of sperm behavior under ultra-confined conditions in order to improve outcomes. Experimental studies combined with models and simulations allow the evaluation of the efficiency of different lab-on-a-chip devices during the design process. In this work, we provide experimental evidence of the dynamics of sperm interacting with a lateral wall in a shallow chamber. We observe a decrease in average sperm velocity during initial wall interaction and partial recovery after the alignment of the trajectory of the cell. To describe this phenomenon, we propose a simple model for the sperm alignment process with a single free parameter. By incorporating experimental motility characterization into the model, we achieve an accurate description of the average velocity behavior of the sperm population close to walls. These results will contribute to the design of more efficient lab-on-a-chip devices for the treatment of human infertility.

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).

Continuous behavioural 'switching' in human spermatozoa and its regulation by Ca2+-mobilising stimuli.

Human sperm show a variety of different behaviours (types of motility) that have different functional roles. Previous reports suggest that sperm may reversibly switch between these behaviours. We have recorded and analysed the behaviour of individual human sperm (180 cells in total), each cell monitored continuously for 3-3.5 min either under control conditions or in the presence of Ca2+-mobilising stimuli. Switching between different behaviours was assessed visually (1 s bins using four behaviour categories), and was verified by fractal dimension analysis of sperm head tracks. In the absence of stimuli, ~90% of cells showed at least one behavioural transition (mean rate under control conditions = 6.4 ± 0.8 transitions.min-1). Type 1 behaviour (progressive, activated-like motility) was most common, but the majority of cells (>70%) displayed at least three behaviour types. Treatment of sperm with Ca2+-mobilising agonists had negligible effects on the rate of switching but increased the time spent in type 2 and type 3 (hyperactivation-like) behaviours (P < 2*10-8; chi-square). Treatment with 4-aminopyridine under alkaline conditions (pHo = 8.5), a highly-potent Ca2+-mobilising stimulus, was the most effective in increasing the proportion of type 3 behaviour, biasing switching away from type 1 (P < 0.005) and dramatically extending the duration of type 3 events (P < 10-16). Other stimuli, including 300 nM progesterone and 1% human follicular fluid, had qualitatively similar effects but were less potent. We conclude that human sperm observed in vitro constitutively display a range of behaviours and regulation of motility by [Ca2+]i, at the level of the single cell, is achieved not by causing cells to adopt a 'new' behaviour but by changing the relative contributions of those behaviours.

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.

Involvement of fibroblast growth factor 2 (FGF2) and its receptors in the regulation of mouse sperm physiology.

Fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) have been described in several tissues, where they regulate cellular proliferation, differentiation, motility and apoptosis. Although FGF2/FGFRs expression in the male reproductive tract has been reported, there is scarce evidence on their presence in the female reproductive tract and their involvement in the modulation of sperm function. Therefore, the objective of this study was to determine the expression of FGF2 in the female reproductive tract and to assess the role of the FGF2/FGFRs system in the regulation of sperm physiology using the murine model. FGF2 was detected in uterus and oviduct protein extracts, and it was immunolocalized in epithelial cells of the uterus, isthmus and ampulla, as well as in the cumulus oophorus-oocyte complex. The receptors FGFR1, FGFR2, FGFR3 and FGFR4 were immunodetected in the flagellum and acrosomal region of sperm recovered from the cauda epididymis. Analysis of testis sections showed the expression of FGFRs in germ cells at different stages of the spermatogenesis, suggesting the testicular origin of the sperm FGFRs. Sperm incubation with recombinant FGF2 (rFGF2) led to increased sperm motility and velocity and to enhanced intracellular Ca2+ levels and acrosomal loss compared to the control. In conclusion, this study shows that FGF2 is expressed in tissues of the female reproductive tract. Also, the fact that functional FGFRs are present in mouse sperm and that rFGF2 affects sperm motility and acrosomal exocytosis, suggests the involvement of this system in the in vivo regulation of sperm function.

An intact acrosome is required for the chemotactic response to progesterone in mouse spermatozoa.

Mammalian sperm become fertilization-competent in the oviduct, during a process known as capacitation that involves the acquisition of the ability to exocytose the acrosome but also the chemotactic responses-both of which contribute to successful fertilization. Chemotaxis is used by spermatozoa to orient and to locate the egg; the acrosome reaction facilitates sperm binding to and fusing with the egg membrane. Mammalian spermatozoa are able to sense picomolar concentrations of progesterone, which drives chemotactic behavior. The state of the acrosome during the chemotactic response, however, is unknown. Genetically modified mouse spermatozoa were employed in a chemotaxis assay under fluorescence microscopy to evaluate their acrosome status while swimming, allowing us to elucidate the acrosome integrity of sperm responding to progesterone-induced chemotaxis. We first showed that wild-type mouse spermatozoa chemotactically respond to a gradient of progesterone, and that the genetic modifications employed do not affect the chemotactic behavior of sperm to progesterone. Next, we found that acrosome-intact, but not acrosome-reacted, spermatozoa orient and respond to picomolar concentrations of progesterone and that chemotaxis normally occurs prior to the acrosome reaction. Our results suggest that premature commitment to acrosome exocytosis leads to navigation failure, so proper control and timing of the acrosome reaction is required for fertilization success and male fertility.

Variability in sperm form and function in the context of sperm competition risk in two Tupinambis lizards.

In polyandrous species, sperm morphometry and sperm velocity are under strong sexual selection. Although several hypotheses have been proposed to explain the role of sperm competition in sperm trait variation, this aspect is still poorly understood. It has been suggested that an increase in sperm competition pressure could reduce sperm size variation or produce a diversity of sperm to maximize male fertilization success. We aim at elucidating the variability of sperm morphometric traits and velocity in two Tupinambis lizards in the context of sperm competition risk. Sperm traits showed substantial variation at all levels examined: between species, among males within species, and within the ejaculate of individual males. Sperm velocity was found to be positively correlated with flagellum: midpiece ratio, with relatively longer flagella associated with faster sperm. Our results document high variability in sperm form and function in lizards.

Spermatozoa characterization in the one-sided livebearing Jenynsia multidentata (Cyprinodontiformes: Anablepidae).

Several sperm parameters have been employed as useful tools to evaluate fish fertility. Within teleosts, approximately 3% of fish species are known to be viviparous. The Order Cyprinodontiformes includes several species with internal fertilization, and within this group most of the studies about sperm quality have been mainly focused on the Poeciliidae family. The livebearing fish Jenynsia multidentata (Anablepidae) inhabits an extensive area of the Neotropical region and it has been used as a useful fish laboratory model to evaluate the effects of xenobiotics through different biomarkers. The present work characterized the sperm of this species through a simple protocol of semen collection. Sperm population showed linearity greater than 89% and 70% of fish have a straight line and curvilinear velocity valued between 50 and 100 µm/s. Although 85% of individuals showed a proportion of live sperm higher than 60%, the male population had a high degree of heterogeneity in its sperm count. Morphometry analyses showed a total sperm and head lengths of 46.66 ± 2.06 µm and 3.46 ± 0.41 mm, respectively. A rather long midpiece region (9.12 ± 0.65 µm) was registered, which may indicate high energy-producing capabilities of the spermatozoa. This study established basic parameter values which could be useful for evaluating reproductive potential of J. multidentata populations.

Spermatozoa characterization in the one-sided livebearing Jenynsia multidentata (Cyprinodontiformes: Anablepidae) / Caracterización de parámetros espermáticos del pez vivíparo Jenynsia multidentata (Cyprinodontiformes: Anablepidae)

Several sperm parameters have been employed as useful tools to evaluate fish fertility. Within teleosts, approximately 3% of fish species are known to be viviparous. The Order Cyprinodontiformes includes several species with internal fertilization, and within this group most of the studies about sperm quality have been mainly focused on the Poeciliidae family. The livebearing fish Jenynsia multidentata (Anablepidae) inhabits an extensive area of the Neotropical region and it has been used as a useful fish laboratory model to evaluate the effects of xenobiotics through different biomarkers. The present work characterized the sperm of this species through a simple protocol of semen collection. Sperm population showed linearity greater than 89% and 70% of fish have a straight line and curvilinear velocity valued between 50 and 100µm/s. Although 85% of individuals showed a proportion of live sperm higher than 60%, the male population had a high degree of heterogeneity in its sperm count. Morphometry analyses showed a total sperm and head lengths of 46.66±2.06µm and 3.46±0.41mm, respectively. A rather long midpiece region (9.12±0.65µm) was registered, which may indicate high energy-producing capabilities of the spermatozoa. This study established basic parameter values which could be useful for evaluating reproductive potential of J. multidentata populations.

Diversos parámetros espermáticos han sido utilizados para evaluar la fertilidad de peces. Dentro de los peces teleósteos, aproximadamente el 3% de las especies son vivíparas. El orden Cyprinodontiformes incluye varias especies con fecundación interna. Dentro de este orden la mayor parte de los estudios sobre la calidad del esperma se han centrado principalmente en la familia Poeciliidae. El pez vivíparo Jenynsia multidentata (Anablepidae) habita una extensa área de la región Neotropical y ha sido utilizado como un exitoso modelo de laboratorio. El objetivo del presente trabajo fue caracterizar los espermatozoides de esta especie a través de un simple protocolo de recolección de esperma. La población de espermatozoides mostró una linealidad superior al 89% y el 70% de los peces tienen una velocidad lineal y curvilineal entre 50 y 100µm/s. Aunque el 85% de los individuos mostró una proporción de espermatozoides vivos de más del 60%, se observó una alta heterogeneidad en el recuento espermático. Los análisis morfométricos mostraron una longitud total de espermatozoides de 46.66±2.06µm y una longitud de la cabeza de 3.46±0.41µm. Los espermatozoides presentan una pieza media larga (9.12±0.65µm) lo que puede indicar una alta capacidad de producción de energía. El presente estudio establece valores básicos de parámetros que pueden ser útiles para evaluar el potencial reproductivo de las poblaciones de J. multidentata.

Infertility treatment, a matter of a lovely sperm?

Nowadays, the efficiency of the infertility treatment is relatively low. One of the cues to counteract this problem relies on the optimum selection of spermatozoa. We developed a new method (sperm selection assay (SSA)) based on the chemical attraction of spermatozoa that are at the best functional state. Additionally, the SSA leads spermatozoa to complete and/or acquire the competence to fertilize the egg. These effects are equally observed either in normal or subfertile semen samples. Those capabilities of SSA may improve the success of current infertility treatment.

Impairments in aromatase expression, reproductive behavior, and sperm quality of male fish exposed to 17ß-estradiol.

Growing evidence shows that environmental estrogen can reach levels that are high enough to exert adverse reproductive effects on wild fish populations. The authors report different parameters of male reproductive behavior, brain, and gonadal aromatase expression, as well as sperm quality in an internally fertilizing fish species (Jenynsia multidentata, Jenyns) exposed to environmentally relevant concentrations of 17ß-estradiol (E(2) ). Adult males were exposed to 0, 50, 100, and 250 ng/L E(2) over 28 d. The authors' findings demonstrate that E(2) exposure resulted in a very clear increase in brain aromatase transcript abundance at all assayed concentrations compared with control; however, no effects on gonadal aromatase expression were observed. Behavioral measures revealed increased sexual activity at 50 ng/L but not 100 or 250 ng/L E(2) . In contrast to the molecular and behavioral responses, the condition factor, gonadosomatic index, and sperm quality were unaltered by E(2) exposure. The results from the present work suggest that E(2) affects some aspects of the reproductive biology of J. multidentata. These modifications in the reproductive biology caused by exposure to E(2) could potentially lead to long-term effects at population levels that may not always be immediately evident. To the best of the authors' knowledge, this is the first report on the combined effect of E(2) on aromatase expression, sexual behavior, and sperm parameters in fish.

Diagnóstico y optimización de la calidad de muestras de semen humano sobre la base de la respuesta quimiotáctica de los espermatozoides / DIagnosis and optimization of human semen sample quality based on the sperm chemotactic response

INTRODUCCIÓN La infertilidad es un problema de salud humana. Es indispensable realizar un diagnóstico correcto del hombre, pero no se dispone de un test que permita evaluar las características fisiológicas del espermatozoide. OBJETIVOS Determinar si el Ensayo de Selección Espermática (ESE) permite evaluar el estado fisiológico de una muestra de semen, definir valores para diagnosticar muestras normales y patológicas, y correlacionar los resultados con los valores de la Organización Mundial de la Salud (OMS). MÉTODOS Se utilizó el ESE como método de diagnóstico para valorar la calidad seminal funcional de un amplio rango de muestras. Luego se evaluó la posible correlación entre los valores hallados y los parámetros espermáticos, a fin de establecer valores umbrales obtenidos con el ESE que sean indicadores de la fisiología de la muestra. RESULTADOS El índice neto de acumulación de espermatozoides después del ESE en presencia de progesterona fue de alrededor del 9%. El nivel de espermatozoides capacitados aumentó después del ESE en presencia de progesterona, mientras que el nivel de espermatozoides fragmentados y con descondensación de la cromatina se redujo. No se observó correlación para cada uno de los parámetros espermáticos con respecto al ESE. DISCUSIÓN Los resultados obtenidos corroboran que el ESE permite seleccionar espermatozoides capacitados, con el ADN intacto y bajo nivel de condensación de la cromatina. No se observó correlación entre los valores de acumulación espermática obtenidos con el ESE y los parámetros clásicos de la OMS.

Human sperm chemotaxis depends on critical levels of reactive oxygen species.

OBJECTIVE: To verify whether chemotaxis is in part an oxidative process mediated by reactive oxygen species (ROS). DESIGN: In this prospective study, after removal of seminal plasma, the sperm suspension received no treatment (control), ROS formation by stimulation with phorbol 12-myristate 13-acetate (PMA), antioxidant treatment (with catalase), or PMA stimulus in the presence of catalase. At time zero and after 3 hours of incubation, the percentage of capacitated and oriented spermatozoa and the ROS levels were determined. SETTING: Andrology laboratory in a medical research institution. PATIENT(S): Normal semen was obtained from eight men. INTERVENTION(S): The semen samples were evaluated to determine the effect of ROS production by stimulation with PMA and/or antioxidant treatment (with catalase) on the percentage of capacitated and oriented spermatozoa. MAIN OUTCOME MEASURE(S): The sperm capacitation, chemotaxis and reactive oxygen species were assessed before and after PMA and/or antioxidant treatment. RESULT(S): Prolonged exposure to high quantities of ROS decrease the sperm chemotactic response, probably because of oxidative damage of the cell. However, this effect may be reduced by the addition of antioxidants like catalase. CONCLUSION(S): Similar to capacitation, chemotaxis seems to depend on the production of ROS, but in the latter process there may be a critical level of ROS necessary for chemotaxis to occur.

Molecular mechanism for human sperm chemotaxis mediated by progesterone.

Sperm chemotaxis is a chemical guiding mechanism that may orient spermatozoa to the egg surface. A picomolar concentration gradient of Progesterone (P), the main steroidal component secreted by the cumulus cells that surround the egg, attracts human spermatozoa. In order to elucidate the molecular mechanism of sperm chemotaxis mediated by P, we combine the application of different strategies: pharmacological inhibition of signaling molecules, measurements of the concentrations of second messengers and activation of the chemotactic signaling. Our data implicate a number of classic signal transduction pathways in the response and provide a model for the sequence of events, where the tmAC-cAMP-PKA pathway is activated first, followed by protein tyrosine phosphorylation (equatorial band and flagellum) and calcium mobilization (through IP(3)R and SOC channels), whereas the sGC-cGMP-PKG cascade, is activated later. These events lead to sperm orientation towards the source of the chemoattractant. The finding proposes a molecular mechanism which contributes to the understanding of the signal transduction pathway that takes place in a physiological process as chemotaxis.

Sperm competition and reproductive mode influence sperm dimensions and structure among snakes.

The role of sperm competition in increasing sperm length is a controversial issue, because findings from different taxa seem contradictory. We present a comparative study of 25 species of snakes with different levels of sperm competition to test whether it influences the size and structure of different sperm components. We show that, as levels of sperm competition increase, so does sperm length, and that this elongation is largely explained by increases in midpiece length. In snakes, the midpiece is comparatively large and it contains structures, which in other taxa are present in the rest of the flagellum, suggesting that it may integrate some of its functions. Thus, increases in sperm midpiece size would result in more energy as well as greater propulsion force. Sperm competition also increases the area occupied by the fibrous sheath and outer dense fibers within the sperm midpiece, revealing for the first time an effect upon structural elements within the sperm. Finally, differences in male-male encounter rates between oviparous and viviparous species seem to lead to differences in levels of sperm competition. We conclude that the influence of sperm competition upon different sperm components varies between taxa, because their structure and function is different.

Sperm guidance in mammals - an unpaved road to the egg.

Contrary to the prevalent view, there seems to be no competition in the mammalian female genital tract among large numbers of sperm cells that are racing towards the egg. Instead, small numbers of the ejaculated sperm cells enter the Fallopian tube, and these few must be guided to make the remaining long, obstructed way to the egg. Here, we review the mechanisms by which mammalian sperm cells are guided to the egg.

Human sperm chemotaxis: both the oocyte and its surrounding cumulus cells secrete sperm chemoattractants.

BACKGROUND: Human sperm chemotaxis to pre-ovulatory follicular fluid is well established in vitro. However, it is not known whether the female's oocyte-cumulus complex secretes sperm chemoattractants subsequent to ovulation (for enabling sperm chemotaxis within the Fallopian tube) and, if so, which of these cell types--the oocyte or the cumulus oophorus--is the physiological origin of the secreted chemoattractant. METHODS: By employing a directionality-based chemotaxis assay, we examined whether media conditioned with either individual, mature (metaphase II) human oocytes or the surrounding cumulus cells attract human sperm by chemotaxis. RESULTS: We observed sperm chemotaxis to each of these media, suggesting that both the oocyte and the cumulus cells secrete sperm chemoattractants. CONCLUSIONS: These observations suggest that sperm chemoattractants are secreted not only prior to ovulation within the follicle, as earlier studies have demonstrated, but also after oocyte maturation outside the follicle, and that there are two chemoattractant origins: the mature oocyte and the surrounding cumulus cells.

Timing of sperm capacitation appears to be programmed according to egg availability in the female genital tract.

The time course of the level of A23187-induced acrosome reaction between human and rabbit spermatozoa was compared. It was extended in the former (a periodic ovulator) and short in the latter (an induced ovulator). This finding suggests that the capacitated state is programmed to maximize the prospects that an ovulated egg will meet spermatozoa in the best functional state.

Lack of species-specificity in mammalian sperm chemotaxis.

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.