Sexual discrimination and attraction through scents in the water vole, Arvicola terrestris.

In mammals, especially rodents, social behaviours, such as parenting, territoriality or mate attraction, are largely based on olfactory communication through chemosignals. These behaviours are mediated by species-specific chemosignals, including small organic molecules and proteins that are secreted in the urine or in various fluids from exocrine glands. Chemosignal detection is mainly ensured by olfactory neurons in two specific sensory organs, the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). This study aimed to characterise the olfactory communication in the fossorial ecotype of the water voles, Arvicola terrestris. We first measured the olfactory investigation of urine and lateral scent gland secretions from conspecifics. Our results showed that water voles can discriminate the sex of conspecifics based on the smell of urine, and that urinary male odour is attractive for female voles. Then, we demonstrated the ability of the VNO and MOE to detect volatile organic compounds (VOCs) found in water vole secretions using live-cell calcium imaging in dissociated cells. Finally, we evaluated the attractiveness of two mixtures of VOCs from urine or lateral scent glands in the field during a cyclical outbreak of vole populations.

Three-dimensional imaging of vascular development in the mouse epididymis.

Long considered an accessory tubule of the male reproductive system, the epididymis is proving to be a key determinant of male fertility. In addition to its secretory role in ensuring functional maturation and survival of spermatozoa, the epididymis has a complex immune function. Indeed, it must manage both peripheral tolerance to sperm antigens foreign to the immune system and the protection of spermatozoa as well as the organ itself against pathogens ascending the epididymal tubule. Although our knowledge of the immunobiology of this organ is beginning to accumulate at the molecular and cellular levels, the organization of blood and lymphatic networks of this tissue, important players in the immune response, remains largely unknown. In the present report, we have taken advantage of a VEGFR3:YFP transgenic mouse model. Using high-resolution three-dimensional (3D) imaging and organ clearing coupled with multiplex immunodetections of lymphatic (LYVE1, PDPN, PROX1) and/or blood (PLVAP/Meca32) markers, we provide a simultaneous deep 3D view of the lymphatic and blood epididymal vasculature in the mature adult mouse as well as during postnatal development.

Loss of Nuclear/DNA Integrity in Mouse Epididymal Spermatozoa after Short-Term Exposure to Low Doses of Dibutyl Phthalate or Bisphenol AF and Its Mitigation by Oral Antioxidant Supplementation.

Routine exposure to chemicals omnipresent in the environment, particularly the so-called endocrine-disrupting chemicals (EDCs), has been associated with decreased sperm quality and increased anomalies in testis. The decline in semen quality and testicular abnormalities have been attributed to the disruption of endocrine signaling as well as oxidative stress. The present study set out to examine the effect of short-term exposure of two common EDCs widely used in the plastic industry: Dibutyl Phthalate (DBP) and Bisphenol AF (BPAF). Our research objective was to focus on the post-testicular compartment of the epididymis, where spermatozoa acquire their functional capacity and are stored. The data obtained indicated no significant effect for either chemicals on sperm viability, motility or acrosome integrity. Neither of the EDCs had a noticeable effect on the structures of the testis and epididymis. However, substantial impact on the integrity of the sperm nucleus and DNA structure was evidenced by a significant increase in nuclear decondensation and DNA base oxidation. The damage observed was postulated to arise from the pro-oxidant properties of the EDCs generating excess of reactive oxygen species (ROS) and triggering a state of oxidative stress. This hypothesis was confirmed when the observed damage was largely blocked by co-administering EDCs with an evidenced-based antioxidant formulation.

Spermatozoa isolation with Felix™ outperforms conventional density gradient centrifugation preparation in selecting cells with low DNA damage.

BACKGROUND: The optimization of spermatozoa preparation techniques in order to obtain cell fractions enriched with structurally and functionally "superior" spermatozoa is a key objective of the assisted reproduction industry. OBJECTIVES: The purpose of this study was to evaluate a recent development of an electrophoretic spermatozoa separation device (Felix™, Memphasys Ltd, Sydney, Australia) and to compare its performance with conventional spermatozoa preparation by density gradient centrifugation (DGC). Particular attention was paid to the evaluation of sperm DNA/nuclear integrity. MATERIALS & METHODS: A cohort of 29 human semen samples was studied. Semen samples were analyzed fresh and after DGC or Felix™ preparation. Semen parameters monitored included sample volume, sperm count, total motility, progressive motility, sperm DNA fragmentation using the Sperm Chromatin Structure Assay and sperm DNA oxidation. RESULTS: Spermatozoa preparation with Felix™ resulted in significantly improved spermatozoa fractions with higher progressive motility, lower sperm DNA fragmentation, and lower sperm DNA oxidation compared with raw semen and DGC-prepared spermatozoa. DISCUSSION & CONCLUSION: The data collected in this study support the preparation of spermatozoa by the Felix™ system as it allows selection of spermatozoa with the highest progressive motility as well as the lowest nuclear/DNA damage. These improved sperm parameters, along with the fact that the Felix™ separation process is very fast and highly standardized, should be of great interest to the assisted reproduction technologies industry.

Sperm Ion Transporters and Channels in Human Asthenozoospermia: Genetic Etiology, Lessons from Animal Models, and Clinical Perspectives.

In mammals, sperm fertilization potential relies on efficient progression within the female genital tract to reach and fertilize the oocyte. This fundamental property is supported by the flagellum, an evolutionarily conserved organelle that provides the mechanical force for sperm propulsion and motility. Importantly several functional maturation events that occur during the journey of the sperm cells through the genital tracts are necessary for the activation of flagellar beating and the acquisition of fertilization potential. Ion transporters and channels located at the surface of the sperm cells have been demonstrated to be involved in these processes, in particular, through the activation of downstream signaling pathways and the promotion of novel biochemical and electrophysiological properties in the sperm cells. We performed a systematic literature review to describe the currently known genetic alterations in humans that affect sperm ion transporters and channels and result in asthenozoospermia, a pathophysiological condition defined by reduced or absent sperm motility and observed in nearly 80% of infertile men. We also present the physiological relevance and functional mechanisms of additional ion channels identified in the mouse. Finally, considering the state-of-the art, we discuss future perspectives in terms of therapeutics of asthenozoospermia and male contraception.

When idiopathic male infertility is rooted in maternal malnutrition during the perinatal period in mice†.

Infertility represents a growing burden worldwide, with one in seven couples presenting difficulties conceiving. Among these, 10-15% of the men have idiopathic infertility that does not correlate with any defect in the classical sperm parameters measured. In the present study, we used a mouse model to investigate the effects of maternal undernutrition on fertility in male progeny. Our results indicate that mothers fed on a low-protein diet during gestation and lactation produce male offspring with normal sperm morphology, concentration, and motility but exhibiting an overall decrease of fertility when they reach adulthood. Particularly, in contrast to control, sperm from these offspring show a remarkable lower capacity to fertilize oocytes when copulation occurs early in the estrus cycle relative to ovulation, due to an altered sperm capacitation. Our data demonstrate for the first time that maternal nutritional stress can have long-term consequences on the reproductive health of male progeny by affecting sperm physiology, especially capacitation, with no observable impact on spermatogenesis and classical quantitative and qualitative sperm parameters. Moreover, our experimental model could be of major interest to study, explain, and ultimately treat certain categories of infertilities.

Photoperiod is involved in the regulation of seasonal breeding in male water voles (Arvicola terrestris).

Mammals living at temperate latitudes typically display annual cyclicity in their reproductive activity: births are synchronized when environmental conditions are most favorable. In a majority of these species, day length is the main proximate factor used to anticipate seasonal changes and to adapt physiology. The brain integrates this photoperiodic signal through key hypothalamic structures, which regulate the reproductive axis. In this context, our study aimed to characterize regulations that occur along the hypothalamo-pituitary-gonadal (HPG) axis in male fossorial water voles (Arvicola terrestris, also known as Arvicola amphibius) throughout the year and to further probe the implication of photoperiod in these seasonal regulations. Our monthly field monitoring showed dramatic seasonal changes in the morphology and activity of reproductive organs, as well as in the androgen-dependent lateral scent glands. Moreover, our data uncovered seasonal variations at the hypothalamic level. During the breeding season, kisspeptin expression in the arcuate nucleus (ARC) decreases, while RFRP3 expression in the dorsomedial hypothalamic nucleus (DMH) increases. Our follow-up laboratory study revealed activation of the reproductive axis and confirmed a decrease in kisspeptin expression in males exposed to a long photoperiod (summer condition) compared with those maintained under a short photoperiod (winter condition) that retain all features reminiscent of sexual inhibition. Altogether, our study characterizes neuroendocrine and anatomical markers of seasonal reproductive rhythmicity in male water voles and further suggests that these seasonal changes are strongly impacted by photoperiod.

Identification of Arvicola terrestris scherman Sperm Antigens for Immune Contraceptive Purposes.

The cyclical proliferation of the wild fossorial rodent Arvicola terrestris scherman (ATS) is critical in mid-mountain ecosystems of several European countries. Our goal is to develop an immunocontraceptive vaccine to control their fertility, as a sustainable alternative to chemical poisons currently used. Indeed, these chemicals cause the death of ATS predators and animals sharing their ecosystem, and current laws progressively limit their use, making the development of a targeted vaccination strategy an interesting and efficient alternative. In order to identify species-specific sperm antigens, male and female ATS received subcutaneous injections of whole ATS spermatozoa to elicit an immune response. The analysis of the immune sera led to the identification of 120 immunogenic proteins of sperm cells. Of these, 15 were strictly sperm-specific and located in different regions of the male gamete. Some of these antigens are proteins involved in molecular events essential to the reproductive process, such as sperm-egg interaction, acrosomal reaction, or sperm motility. This approach not only identified a panel of immunogenic proteins from ATS sperm cells, but also demonstrated that some of these proteins trigger an immune response in both male and female ATS. These spermatic antigens are good candidates for the development of a contraceptive vaccine.

Field study reveals morphological and neuroendocrine correlates of seasonal breeding in female water voles, Arvicola terrestris.

Seasonally breeding mammals display timely physiological switches between reproductive activity and sexual rest, which ensure synchronisation of births at the most favourable time of the year. These switches correlate with seasonal changes along the hypothalamo-pituitary-gonadal axis, but they are primarily orchestrated at the hypothalamic level through environmental control of KISS1-dependent GnRH release. Our field study shows that births of fossorial water voles, Arvicola terrestris, are concentrated between March and October, which indicates the existence of an annual reproductive cycle in this species. Monthly field monitoring for over a year further reveals dramatic seasonal changes in the morphology of the ovary, uterus and lateral scent glands, which correlate with the reproductive status. Finally, we demonstrate seasonal variation in kisspeptin expression within the hypothalamic arcuate nucleus. Altogether, this study demonstrates a marked rhythm of seasonal breeding in the water vole and we speculate that this is governed by seasonal changes in photoperiod.

Differential expression and localisation of TGF-ß isoforms and receptors in the murine epididymis.

Testes produce spermatozoa that transit through and are stored in the epididymis where they acquire their fertilising capacities. Spermatozoa appear in the genital tract at puberty, long after the immune system was trained to self-antigens. As a consequence, this organ has to set strategies to tolerate sperm antigens to avoid autoimmune responses that would specifically target and destroy them. A recent study pointed the Transforming Growth Factor-beta (TGF-ß) signalling in the dendritic cells as a crucial mechanism for epididymal tolerance to spermatozoa. In the mouse, TGF-ß exists under three isoforms, and three distinct receptors have been described. Using RT-qPCR, immunohistochemistry and ELISA techniques, we investigated the expression and spatial distribution of the epididymal TGF-ß isoforms and of their receptors in young and adult mice. We showed that both ligands and receptors were produced by immune and non-immune cells in the epididymis, whatever the age mice have. These data bring new clues as to the mechanisms of peripheral tolerance to sperm cells in the murine epididymis and raise potential other implications of the cytokine isoforms.

Dietary Cholesterol and Lipid Overload: Impact on Male Fertility.

Lipid metabolic disorders due to poor eating habits are on the rise in both developed and developing countries, with a negative impact of the "Western diet" on sperm count and quality. Dietary lipid imbalance can involve cholesterol, fatty acids, or both, under different pathophysiological conditions grouped under the term dyslipidemia. The general feature of dyslipidemia is the development of systemic oxidative stress, a well-known deleterious factor for the quality of male gametes and associated with infertility. Sperm are particularly rich in polyunsaturated fatty acids (PUFA), an important characteristic associated with normal sperm physiology and reproductive outcomes, but also targets of choice for oxidative thrust. This review focuses on the effects of dietary cholesterol or different fatty acid overload on sperm composition and function in both animals and humans. The links between oxidative stress induced by dyslipidemia and sperm dysfunction are then discussed, including possible preventive or therapeutic strategies to preserve gamete quality, longevity when stored in cryobanking, and male fertility.

The epididymal immune balance: a key to preserving male fertility.

Up to 15% of male infertility has an immunological origin, either due to repetitive infections or to autoimmune responses mainly affecting the epididymis, prostate, and testis. Clinical observations and epidemiological data clearly contradict the idea that the testis confers immune protection to the whole male genital tract. As a consequence, the epididymis, in which posttesticular spermatozoa mature and are stored, has raised some interest in recent years when it comes to its immune mechanisms. Indeed, sperm cells are produced at puberty, long after the establishment of self-tolerance, and they possess unique surface proteins that cannot be recognized as self. These are potential targets of the immune system, with the risk of inducing autoantibodies and consequently male infertility. Epididymal immunity is based on a finely tuned equilibrium between efficient immune responses to pathogens and strong tolerance to sperm cells. These processes rely on incompletely described molecules and cell types. This review compiles recent studies focusing on the immune cell types populating the epididymis, and proposes hypothetical models of the organization of epididymal immunity with a special emphasis on the immune response, while also discussing important aspects of the epididymal immune regulation such as tolerance and tumour control.

Nuclear Integrity but Not Topology of Mouse Sperm Chromosome is Affected by Oxidative DNA Damage.

Recent studies have revealed a well-defined higher order of chromosome architecture, named chromosome territories, in the human sperm nuclei. The purpose of this work was, first, to investigate the topology of a selected number of chromosomes in murine sperm; second, to evaluate whether sperm DNA damage has any consequence on chromosome architecture. Using fluorescence in situ hybridization, confocal microscopy, and 3D-reconstruction approaches we demonstrate that chromosome positioning in the mouse sperm nucleus is not random. Some chromosomes tend to occupy preferentially discrete positions, while others, such as chromosome 2 in the mouse sperm nucleus are less defined. Using a mouse transgenic model (Gpx5-/-) of sperm nuclear oxidation, we show that oxidative DNA damage does not disrupt chromosome organization. However, when looking at specific nuclear 3D-parameters, we observed that they were significantly affected in the transgenic sperm, compared to the wild-type. Mild reductive DNA challenge confirmed the fragility of the organization of the oxidized sperm nucleus, which may have unforeseen consequences during post-fertilization events. These data suggest that in addition to the sperm DNA fragmentation, which is already known to modify sperm nucleus organization, the more frequent and, to date, the less highly-regarded phenomenon of sperm DNA oxidation also affects sperm chromatin packaging.

Slc26a3 deficiency is associated with epididymis dysplasia and impaired sperm fertilization potential in the mouse.

Members of the solute carrier 26 (SLC26) family have emerged as important players in mediating anions fluxes across the plasma membrane of epithelial cells, in cooperation with the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Among them, SLC26A3 acts as a chloride/bicarbonate exchanger, highly expressed in the gastrointestinal, pancreatic and renal tissues. In humans, mutations in the SLC26A3 gene were shown to induce congenital chloride-losing diarrhea (CLD), a rare autosomal recessive disorder characterized by life-long secretory diarrhea. In view of some reports indicating subfertility in some male CLD patients together with SLC26-A3 and -A6 expression in the male genital tract and sperm cells, we analyzed the male reproductive parameters and functions of SLC26A3 deficient mice, which were previously reported to display CLD gastro-intestinal features. We show that in contrast to Slc26a6, deletion of Slc26a3 is associated with severe lesions and abnormal cytoarchitecture of the epididymis, together with sperm quantitative, morphological and functional defects, which altogether compromised male fertility. Overall, our work provides new insight into the pathophysiological mechanisms that may alter the reproductive functions and lead to male subfertility in CLD patients, with a phenotype reminiscent of that induced by CFTR deficiency in the male genital tract.

Dual extraction of mRNA and lipids from a single biological sample.

The extraction of RNA and lipids from a large number of biological samples is time-consuming and costly with steps required for both transcriptomic and lipidomic approaches. Most protocols rely on independent extraction of nucleic acids and lipids from a single sample, thereby increasing the need for biological material and inducing variability in data analysis. We investigated whether it is possible to use a standard RNA extraction procedure to analyze not only RNA levels, but also lipids in a single liver sample. We show that the organic phase obtained when using standard reagents for RNA extraction can be used to analyze lipids, including neutral lipids and fatty acids, by gas chromatography. We applied this technique to an analysis of lipids and the associated gene expression pattern in mice with hepatic steatosis induced by pharmacological activation of nuclear receptor LXR.

Comprehensive overview of murine epididymal mononuclear phagocytes and lymphocytes: Unexpected populations arise.

Despite increasing evidence that epididymal immune disorders can lead to infertility, the cells and mechanisms underlying epididymal immunity remain poorly understood. In this study, we propose a rather exhaustive overview of innate and adaptive immune cells present in the murine caput and cauda epididymis. Using flow cytometry and a wide set of markers, we screened the broadest panel of immune cells ever, in this organ. For the first time, we unequivocally quantified the innate populations of monocytes, macrophages, and dendritic cells subtypes. We also revealed the presence of B cells, gamma delta T cells, and double negative T cells in the murine epididymis. They were localized by immunofluorescence stainings, and appeared to be all present in the interstitium and epithelium along the organ, but with respective preferential regional distribution. Altogether, these findings provide new insights on the actors and potential mechanisms involved in the immune responses against genital tract ascending pathogens and in the setting and maintenance of tolerance toward the sperm cells.

Prostasomes, post-testicular sperm maturation and fertility.

Prostasomes are known as extracellular organelles originating from the epithelial cells of the prostate and detected in its secretions where they are mixed with sperm cells after ejaculation. They were first described in men but are also present in the semen of all mammals studied. Since their characterization by Gunnar Ronquist in the late 1970's studies have focused on different aspects which primarily include their molecular composition and structure, and secondly their ability to interact or even fuse with cells, particularly spermatozoa. They have the ability to bring molecules such as lipids or proteins to spermatozoa during their journey to the oocyte, and their role thus seem to be a sort of a "reservoir" that sperm may use depending on the surrounding conditions. Other properties have been suggested but this review will focus on the properties, acquired by sperm cells via prostasomes, that may influence fertility outcome.

Liver X Receptors (LXRs) Alpha and Beta Play Distinct Roles in the Mouse Epididymis.

After its production in the testis, a spermatozoon has to undergo posttesticular maturation steps to become fully motile and fertile. The first step is epididymal maturation, during which immature spermatozoa are transformed into biochemically mature cells ready to proceed to the next step, capacitation, a physiological process occurring in the female genital tract. The biochemical transformations include modification of sperm lipid composition during epididymal transit, with significant changes in fatty acids, phospholipids, and sterols between the caput and the cauda epididymal spermatozoa. Although quantitative aspects of these changes are well documented for several mammalian species, molecular mechanisms governing these steps are poorly understood. Transgenic male mice invalidated for the two liver X receptors (LXRalpha and LXRbeta, nuclear oxysterol receptors regulating cholesterol and lipid metabolism) become sterile when aging, showing an epididymal phenotype. We used single-knockout-model mice to characterize the role of each LXR isoform during sperm maturation in the epididymis. We show here that although a certain redundancy exists in the functions of the two LXR isoforms, some physiological processes are more under the influence of only one of them. In both cases, aging males showed slight subfertility, associated with dyslipidemia, emphasizing the importance of lipid metabolism in relation with male fertility.

Oxidative DNA damage in mouse sperm chromosomes: Size matters.

Normal embryo and foetal development as well as the health of the progeny are mostly dependent on gamete nuclear integrity. In the present study, in order to characterize more precisely oxidative DNA damage in mouse sperm we used two mouse models that display high levels of sperm oxidative DNA damage, a common alteration encountered both in in vivo and in vitro reproduction. Immunoprecipitation of oxidized sperm DNA coupled to deep sequencing showed that mouse chromosomes may be largely affected by oxidative alterations. We show that the vulnerability of chromosomes to oxidative attack inversely correlated with their size and was not linked to their GC richness. It was neither correlated with the chromosome content in persisting nucleosomes nor associated with methylated sequences. A strong correlation was found between oxidized sequences and sequences rich in short interspersed repeat elements (SINEs). Chromosome position in the sperm nucleus as revealed by fluorescent in situ hybridization appears to be a confounder. These data map for the first time fragile mouse sperm chromosomal regions when facing oxidative damage that may challenge the repair mechanisms of the oocyte post-fertilization.

Posttesticular sperm maturation, infertility, and hypercholesterolemia.

Cholesterol is a key molecule in the mammalian physiology of especial particular importance for the reproductive system as it is the common precursor for steroid hormone synthesis. Cholesterol is also a recognized modulator of sperm functions, not only at the level of gametogenesis. Cholesterol homeostasis regulation is crucial for posttesticular sperm maturation, and imbalanced cholesterol levels may particularly affect these posttesticular events. Metabolic lipid disorders (dyslipidemia) affect male fertility but are most of the time studied from the angle of endocrine/testicular consequences. This review will focus on the deleterious effects of a particular dyslipidemia, i.e., hypercholesterolemia, on posttesticular maturation of mammalian spermatozoa.