Serafín Pérez-Cerezales

Involvement of opsins in mammalian sperm thermotaxis

A unique characteristic of mammalian sperm thermotaxis is extreme temperature sensitivity, manifested by the capacity of spermatozoa to respond to temperature changes of <0.0006 °C as they swim their body-length distance. The identity of the sensing system that confers this exceptional sensitivity on spermatozoa is not known. Here we show that the temperature-sensing system of mammalian spermatozoa involves opsins, known to be G-protein-coupled receptors that act as photosensors in vision. We demonstrate by molecular, immunological, and functional approaches that opsins are present in human and mouse spermatozoa at specific sites, which depend on the species and the opsin type, and that they are involved in sperm thermotaxis via two signalling pathways—the phospholipase C and the cyclic-nucleotide pathways. Our results suggest that, depending on the context and the tissue, mammalian opsins act not only as photosensors but also as thermosensors.

Behavioral mechanism of human sperm in thermotaxis: a role for hyperactivation

STUDY QUESTION What is the behavioral mechanism underlying the response of human spermatozoa to a temperature gradient in thermotaxis? SUMMARY ANSWER Human spermatozoa swim up a temperature gradient by modulating their speed and frequencies of hyperactivation events and turns. WHAT IS KNOWN ALREADY Capacitated human spermatozoa are capable of thermotactically responding to a temperature gradient with an outcome of swimming up the gradient. This response occurs even when the gradient is very shallow. STUDY DESIGN, SIZE, DURATION Human sperm samples were exposed to a fast temperature change. A quantitative analysis of sperm motility parameters, flagellar wave propagation, and directional changes before, during, and after the temperature change was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS The swimming behavior of 44 human sperm samples from nine healthy donors was recorded under a phase-contrast microscope at 75 and 2000 frames/s. A temperature shift was achieved by using a thermoregulated microscope stage. The tracks made by the cells were analyzed by a homemade computerized motion analysis system and ImageJ software. MAIN RESULTS AND THE ROLE OF CHANCE A temperature shift from 31 to 37°C resulted in enhanced speed and a lower frequency of turning events. These were reflected in a 35 ± 1% (mean ± SEM) increase of the straight-line velocity, 33 ± 1% increase of the average path velocity, 11 ± 1% increase of the curvilinear velocity, 20 ± 1% increase of the wobble, and 4 ± 1% increase of the linearity. Qualitatively, the inverse trend was observed in response to a 37-to-31°C shift. In addition, the amplitude of flagellar waves increased close to the sperm head, resulting in higher side-to-side motion of the head and, often, hyperactivation. This increase in the extent of sperm hyperactivation was reflected in an increase in the average (mean ± SEM) fractal dimension from 1.15 ± 0.01 to 1.29 ± 0.01 and in the percentage of hyperactivated spermatozoa from 3 ± 1% to 19 ± 2%. These changes in hyperactivation were observed less often in sperm populations that had not been incubated for capacitation. All these changes partially adapted within 3-10 min, meaning that following the initial change and while being kept at the new temperature, the values of the measured motility parameters slowly and partially returned toward the original values. These results led us to conclude that spermatozoa direct their swimming in a temperature gradient by modulating the frequency of turns (both abrupt turns as in hyperactivation events and subtle turns) and speed in a way that favors swimming in the direction of the gradient. LIMITATIONS, REASONS FOR CAUTION The conclusions were made on the basis of results obtained in temporal and steep temperature gradients. The conclusions for spatial, shallow gradients were made by extrapolation. WIDER IMPLICATIONS OF THE FINDINGS This is the first study that reveals the behavior of human spermatozoa in thermotaxis. This behavior is very similar to that observed during human sperm chemotaxis, suggesting commonality of guidance mechanisms in mammalian spermatozoa. This study further substantiates the function of hyperactivation as a means to direct spermatozoa in guidance mechanisms. STUDY FUNDING/COMPETING INTERESTS The authors have no conflict of interest and no funding to declare.

Effect of liver growth factor on both testicular regeneration and recovery of spermatogenesis in busulfan-treated mice

BackgroundSome adult stem cells persist in adult tissue; however, we do not know how to stimulate stem cells in adults to heal injuries. Liver growth factor (LGF) is a biliprotein with hepatic mitogen activity. Its concentration increases markedly in the presence of any type of liver injury, and it shows in vivo therapeutic biological activity at extrahepatic sites.MethodsWe have analyzed the effect of LGF on the replenishment of germinal cells in the testes of mice injected with busulfan, a common cancer drug that also specifically affects germ line stem cells and spermatogonia. We determined the testicular and epididymal weight, spermatozoal concentration in the epididymis and sperm motility, and performed a histological analysis.ResultsIntraperitoneal administration of LGF was able to partially restore spermatogenesis, as well as sperm production and motility, in mice sterilized with busulfan. LGF treatment in busulfan-treated animals that have suffered a disruption of spermatogenesis can accelerate the reactivation of this process in most of the tubules, as shown in the histological analysis.ConclusionsOur results suggest a potential use of LGF in the mobilization of testicular stem cells and in the restoration of spermatogenesis after busulfan-induced damage to the testicular germinal epithelium.

Progesterone effects on mouse sperm kinetics in conditions of viscosity

The spermatozoa delivered to the female genital tract need to swim towards the oocyte through viscous secretions. Once close to the oocyte, the spermatozoa are guided by a gradient of progesterone (P4) and other unknown chemoattractants via a process known as chemotaxis. Using polyvinylpyrrolidone to establish the conditions of viscosity, we examined the response of mouse spermatozoa to P4 Herein, we show that in low-viscous media, P4 induces hyperactive-like motility whereby sperm show erratic trajectories and non-progressive movement. However, an opposite response is produced in viscous medium in that trajectories are linear and motility is more progressive and less erratic. Our observations provide a behavioural explanation for the chemotaxis of spermatozoa swimming under viscous conditions in a spatial gradient of the chemoattractant P4 They also highlight the importance of using viscous solutions to mimic in vivo conditions when analysing sperm behaviour in response to any stimulus.

New challenges in the analysis of gene transcription in bovine blastocysts.

In the last years, enormous progress has been made in the analysis of gene transcription at the blastocyst stage. The study of gene expression at this early stage of development is challenging because of the very small amount of starting material, which limits the use of traditional mRNA analysis approaches such as Northern blot. Another problem is the difficulty for data normalization, particularly the identification of the best housekeeping gene with the lowest fluctuation under different developmental conditions. Moreover, the transcriptional analysis of embryo biopsies or individual embryos needs to take into consideration that the blastocyst is a transitional stage of development, which is composed of three different types of cells (trophoblast, epiblast and primitive ectoderm) with different patterns of gene expression, and that there are large differences between male and female blastocysts. In this review, we analyse the different specific and sensitive tools available to compare mRNA expression levels of specific genes at the blastocyst stage, and how the protocol and the analytical method used can influence the results dramatically. Finally, we describe future research challenges to identify candidate genes related to developmental competence of bovine blastocysts, not only in terms of pregnancy rates but also in relation to adverse long-term consequences in the adult animal.

Elimination of methylation marks at lysines 4 and 9 of histone 3 (H3K4 and H3K9) of spermatozoa alters offspring phenotype

The contribution of the contents of spermatozoa to the development of the embryo is currently being considered wider than was previously thought. Recent findings point to the participation of epigenetic marks present in the retained histones of mature spermatozoa on embryo and fetal development. Here we created a novel conditional transgenic mouse that expresses lysine (K) demethylase 1a (Kdm1a) during spermatogenesis when the testicles are subjected to heat stress. Using these animals under these conditions we were able to reduce the methylation level of histone 3 at lysines 4 and 9 (H3K4 and H3K9, respectively) in mature spermatozoa. The offspring of these transgenic mice were followed for correct development and growth after birth. We found that the offspring of males expressing Kdm1a suffered 20% of reabsorptions at Day 15 after implantation (vs 0.3% in the control). In addition, 35% of the offspring sired by these males showed some kind of abnormality (suckling defects, lack of movement coordination, dropping forelimbs, abnormal body curvature, absence of eyes, gigantisms and neuromuscular defects) and 25% died before postnatal Day 21. Some abnormalities were maintained to adulthood. These results show that alteration of epigenetic marks present in the retained histones of mature spermatozoa affect fetal development and have phenotypic consequences in the newborn.

The oviduct: from sperm selection to the epigenetic landscape of the embryo

Abstract The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of preimplantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed. Summary Sentence Herein we review the influence of the oviduct on spermatozoa and embryo with special emphasis on the composition of the oviductal flow, sperm selection and remodelling of embryo epigenetics.

Transgenic mouse offspring generated by ROSI.

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes.

Sperm selection by thermotaxis improves ICSI outcome in mice

The ejaculate is a heterogeneous pool of spermatozoa containing only a small physiologically adequate subpopulation for fertilization. As there is no method to isolate this subpopulation, its specific characteristics are unknown. This is one of the main reasons why we lack effective tools to identify male infertility and for the low efficiency of assisted reproductive technologies. The aim of this study was to improve ICSI outcome by sperm selection through thermotaxis. Here we show that a specific subpopulation of mouse and human spermatozoa can be selected in vitro by thermotaxis and that this subpopulation is the one that enters the fallopian tube in mice. Further, we confirm that these selected spermatozoa in mice and humans show a much higher DNA integrity and lower chromatin compaction than unselected sperm, and in mice, they give rise to more and better embryos through intracytoplasmic sperm injection, doubling the number of successful pregnancies. Collectively, our results indicate that a high quality sperm subpopulation is selected in vitro by thermotaxis and that this subpopulation is also selected in vivo within the fallopian tube possibly by thermotaxis.

Experimental Studies on Sperm DNA Fragmentation and Reproductive Outcomes

Most of the assisted reproductive technologies (ARTs) overcome the selection of spermatozoa occurring within the female genital tract in mammals. This selection ensures the fertilization with spermatozoa containing chromatin of high integrity for supporting the ulterior embryo development. Consequently the use of nonselected spermatozoa porting fragmented DNA has been related to developmental and postnatal effects in animal models. The present chapter provides an overview on the implications of using sperm porting fragmented DNA in the reproductive outcome focusing in the knowledge acquired through experimentation in animal models. This is highly relevant given that intracytoplasmic sperm injection (ICSI) has been widely used in fertility treatments in humans. However, experimentation in mouse has demonstrated the risk of using DNA-fragmented spermatozoa in ICSI reducing the embryo and fetal performance as well as provoking long-term deleterious effects in the offspring at adulthood. In addition, the use of storage techniques for spermatozoa such as freezing-thawing and vitrification has been shown to increase the risk of fertilizing with fragmented DNA. Together with mammalian studies, the use of other nonmammalian animal models like fish could help in understanding the consequences of these treatments and to develop new strategies for sperm evaluation and selection before applying ARTs.