Controlled spermatozoa-oocyte interaction improves embryo quality in sheep

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture

Graphene and Reproduction: A Love-Hate Relationship

Since its discovery, graphene and its multiple derivatives have been extensively used in many fields and with different applications, even in biomedicine. Numerous efforts have been made to elucidate the potential toxicity derived from their use, giving rise to an adequate number of publications with varied results. On this basis, the study of the reproductive function constitutes a good tool to evaluate not only the toxic effects derived from the use of these materials directly on the individual, but also the potential toxicity passed on to the offspring. By providing a detailed scientometric analysis, the present review provides an updated overview gathering all the research studies focused on the use of graphene and graphene-based materials in the reproductive field, highlighting the consequences and effects reported to date from experiments performed in vivo and in vitro and in different animal species (from Archea to mammals). Special attention is given to the oxidized form of graphene, graphene oxide, which has been recently investigated for its ability to increase the in vitro fertilization outcomes. Thus, the potential use of graphene oxide against infertility is hypothesized here, probably by engineering the spermatozoa and thus manipulating them in a safer and more efficient way

Chapter Membrane Dynamics of Spermatozoa during Capacitation: New Insight in Germ Cells Signalling

The study of germline stem cells and of germline cells has deep implications for the understanding of fertility, development and cancer. Nowadays, we are experiencing the very fascinating challenge of application of –OMICS technologies to this issue, which is opening new and unexpected horizons in virtually all the branches of biology. Here, we carried out a review of signalling systems involved in maturation of male germ cells and in the process that leads them to become fully fertile. In particular, we discuss the control mechanisms involved in capacitation and acrosome reaction that act at membrane level. Indeed, spermatozoa membranes play key roles in determining the achievement of fertility: they are the interface with the surrounding environment, they locate the signal transduction systems and they are active in recognizing and binding the oocyte. In addition, we discuss the effect of several compounds that could exert a negative effect on reproductive activity, by interfering with the endocrine axis, the so-called endocrine disruptors

Preliminary study on the effect of parenteral naloxone, alone and in association with calcium gluconate, on bone healing in an ovine "drill hole" model system

BACKGROUND: Several diseases affect bone healing and physiology. Many drugs that are commonly used in orthopaedics as "analgesics" or anti-inflammatory agents impair bone healing. Stressful conditions are associated with decreased serum osteocalcin concentration. High endorphin levels alter calcium metabolism, blocking the membrane channels by which calcium normally enters cells. The consequent decrease of intracellular calcium impairs the activities of calcium-related enzymes. Naloxone is a pure opioid antagonist. Morphine-induced osteocalcin inhibition was abolished when osteoblasts were incubated with naloxone. Naloxone restored the altered cellular and tissue physiology by removing beta-endorphins from specific receptors. However, this is only possible if the circulating Ca concentration is adequate. The aim of the present study was to evaluate the efficacy of parenteral naloxone administration in inducing fast mineralization and callus remodelling in a group of sheep with a standardised bone lesion. METHODS: Twenty ewes were randomly assigned to 4 treatment groups. Group A acted as control, group B received a solution of calcium gluconate, group C a solution of naloxone, and group D a solution of calcium gluconate and naloxone. A transverse hole was drilled in the left metacarpus, including both cortices, then parenteral treatment was administered intramuscularly, daily for four weeks. Healing was evaluated by weekly radiographic examination for eight weeks. For quantitative evaluation, the ratio of the radiographic bone density between the drill area and the adjacent cortical bone was calculated. After eight weeks the sheep were slaughtered and a sample of bone was collected for histopathology RESULTS: Group D showed a higher radiographic ratio than the other groups. Sheep not treated with naloxone showed a persistently lower ratio in the lateral than the medial cortex (P < 0.01). Histopathology of bone samples showed more caverns and fewer osteoblasts in group D than in the other groups (P </= 0.001). CONCLUSION: A low-dose parenteral regimen of naloxone enhances mineralization and remodelling of the callus in healing cortical defects in sheep, especially if associated with calcium gluconate

Membrane Dynamics of Spermatozoa during Capacitation: New Insight in Germ Cells Signalling

The study of germline stem cells and of germline cells has deep implications for the understanding of fertility, development and cancer. Nowadays, we are experiencing the very fascinating challenge of application of –OMICS technologies to this issue, which is opening new and unexpected horizons in virtually all the branches of biology. Here, we carried out a review of signalling systems involved in maturation of male germ cells and in the process that leads them to become fully fertile. In particular, we discuss the control mechanisms involved in capacitation and acrosome reaction that act at membrane level. Indeed, spermatozoa membranes play key roles in determining the achievement of fertility: they are the interface with the surrounding environment, they locate the signal transduction systems and they are active in recognizing and binding the oocyte. In addition, we discuss the effect of several compounds that could exert a negative effect on reproductive activity, by interfering with the endocrine axis, the so-called endocrine disruptors

Unraveling the Antimicrobial Effectiveness of Coridothymus capitatus Hydrolate against Listeria monocytogenes in Environmental Conditions Encountered in Foods: An In Vitro Study

The increased resistance of bacteria to antimicrobials, as well as the growing interest in innovative and sustainable alternatives to traditional food additives, are driving research towards the use of natural food preservatives. Among these, hydrolates (HYs) have gained attention as "mild" alternatives to conventional antimicrobial compounds. In this study, the response of L. monocytogenes ATCC 7644 exposed to increasing concentrations of Coridothymus capitatus HY (CHY) for 1 h at 37 °C was evaluated by means of Phenotype Microarray, modelling the kinetic data obtained by inoculating control and treated cells into GEN III microplates, after CHY removal. The results revealed differences concerning the growth dynamics in environmental conditions commonly encountered in food processing environments (different carbon sources, pH 6.0, pH 5.0, 1-8% NaCl). More specifically, for treated cells, the lag phase was extended, the growth rate was slowed down and, in most cases, the maximum concentration was diminished, suggesting the persistence of stress even after CHY removal. Confocal Laser Scanner Microscopy evidenced a diffuse aggregation and suffering of the treated cells, as a response to the stress encountered. In conclusion, the treatment with HY caused a stressing effect that persisted after its removal. The results suggest the potential of CHY application to control L. monocytogenes in food environments

Role and Modulation of TRPV1 in Mammalian Spermatozoa: An Updated Review

Based on the abundance of scientific publications, the polymodal sensor TRPV1 is known as one of the most studied proteins within the TRP channel family. This receptor has been found in numerous cell types from different species as well as in spermatozoa. The present review is focused on analyzing the role played by this important channel in the post-ejaculatory life of spermatozoa, where it has been described to be involved in events such as capacitation, acrosome reaction, calcium trafficking, sperm migration, and fertilization. By performing an exhaustive bibliographic search, this review gathers, for the first time, all the modulators of the TRPV1 function that, to our knowledge, were described to date in different species and cell types. Moreover, all those modulators with a relationship with the reproductive process, either found in the female tract, seminal plasma, or spermatozoa, are presented here. Since the sperm migration through the female reproductive tract is one of the most intriguing and less understood events of the fertilization process, in the present work, chemotaxis, thermotaxis, and rheotaxis guiding mechanisms and their relationship with TRPV1 receptor are deeply analyzed, hypothesizing its (in)direct participation during the sperm migration. Last, TRPV1 is presented as a pharmacological target, with a special focus on humans and some pathologies in mammals strictly related to the male reproductive system

Effect of electric current stimulation in combination with external fixator on bone healing in a sheep fracture model

Biophysical stimulations with electric and electromagnetic fields have been demonstrated to accelerate the bone-healing rate. This study has been designed to investigate the effects of electricity directly connected with the central pins of an external fixator in an experimental osteotomy model in sheep. Thirty mg/kg of tetracycline chloride were administered on the 30th and on the 45th day after surgery for histomorphometric studies. Plain radiographs were obtained in standard projections every 15 days after surgery and were analyzed with a software program (Corel Photo-Paint Pro X2, Corel Corporation, Ottawa, Canada). The specimens obtained after 60 days were examined with histological analysis. The results show that biophysical treatment with alternating electricity in combination with external fixator enhances new-bone formation. The translational value of this study, due to the similarities between ovine and human species, suggests that this treatment could be useful in speeding the bone-healing rate both in animals and humans

Graphene oxide affects in vitro fertilization outcome by interacting with sperm membrane in an animal model

We realized the exposure of boar spermatozoa to graphene oxide (GO) at concentration of 0.5, 1, 5, 10 and 50 μg/mL in an in vitro system able to promote the capacitation, i.e. the process that allows sperm cells to became fertile. Interestingly, we found that the highest GO concentration (5, 10 and 50 μg/mL) are toxic for spermatozoa, while the lowest ones (0.5 and 1 μg/mL) seem to significantly increase the sperm cells fertilizing ability (p >.05) in an in vitro fertilization experiment. To explain this finding, we investigated the effect of GO on sperm membrane structure (atomic force microscopy) and function (confocal microscopy and flow cytometry, substrate adhesion). As a result, we found that GO is able to interact with spermatozoa membranes and, in particular, it seems to be able to extract the cholesterol, which is a key player in spermatozoa physiology, from plasma membrane of boar spermatozoa incubated under capacitation conditions. In our opinion, these results are very important because they allow identifying either a plausible mechanism of GO toxicity on spermatozoa and new strategies to manage sperm capacitation

Pre-Treatment of Swine Oviductal Epithelial Cells with Progesterone Increases the Sperm Fertilizing Ability in an IVF Model

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional modification, called capacitation, in order to acquire their fertilizing ability. Since oviductal epithelial cells (SOECs) and progesterone (P4) are two major modulators of capacitation, here we investigated their impact on sperm functionality by using an IVF swine model. To that, we treated SOECs with P4 at 10, 100, and 1000 ng/mL before the coincubation with spermatozoa, thus finding that P4 at 100 ng/mL does not interfere with the cytoskeleton dynamics nor the cells' doubling time, but it promotes the sperm capacitation by increasing the number of spermatozoa per polyspermic oocyte (p &lt; 0.05). Moreover, we found that SOECs pre-treatment with P4 100 ng/mL is able to promote an increase in the sperm fertilizing ability, without needing the hormone addition at the time of fertilization. Our results are probably due to the downregulation in the expression of OVGP1, SPP1 and DMBT1 genes, confirming an increase in the dynamism of our system compared to the classic IVF protocols. The results obtained are intended to contribute to the development of more physiological and efficient IVF systems