In vivo dynamic volumetric imaging of mouse testis and epididymis with optical coherence tomography†.

The implementation of live imaging in reproductive research is crucial for studying the physiological dynamics. Sperm transport is a highly dynamic process regulated by tubular contractions and luminal flows within the male reproductive tract. However, due to the lack of imaging techniques to capture these dynamics in vivo, there is little information on the physiological and biomechanical regulation of sperm transport through the male reproductive tract. Here, we present a functional in vivo imaging approach using optical coherence tomography, enabling live, label-free, depth-resolved, three-dimensional, high-resolution visualization of the mouse testis and epididymis. With this approach, we spatiotemporally captured tubular contractility in mouse testis and epididymis, as well as microstructures of these reproductive organs. Our findings demonstrated that the contraction frequency varies significantly depending on the epididymal regions, suggesting the spatial regulation of epididymal contractility. Furthermore, we implemented quantitative measurements of the contraction wave and luminal transport through the epididymal duct, revealing the physiological dynamics within the male reproductive tract. The results show that the contraction wave propagates along the epididymal duct and the wave propagation velocity was estimated in vivo. In conclusion, this is the first study to develop in vivo dynamic volumetric imaging of the male reproductive tract, which allows for quantitative analysis of the dynamics associated with sperm transport. This study sets a platform for various studies investigating normal and abnormal male reproductive physiology as well as the pharmacological and environmental effects on reproductive functions in mouse models, ultimately contributing to a comprehensive understanding of male reproductive disorders.

Effects of a high-fat diet on rat epididymis.

The epididymis plays an essential role in reproduction, promoting sperm cell maturation. In this study, we investigated the effects of a high-fat diet (HFD) in the three regions of the epididymis of rats, including caput, corpus, and cauda. Our results showed an increase in malondialdehyde and a decrease in superoxide dismutase, which indicated an increase in oxidative stress in all segments of the epididymis. The cellular response mechanisms were mostly detected in the corpus/cauda regions, which showed an increase in apoptosis, probably for eliminating dysfunctional cells arising from HFD-induced oxidative stress, and a decrease in mitophagy. Additionally, an increase in lipophagy to prevent lipid accumulation and a decrease in cell proliferation were recorded in the corpus.

Host defense functions of the epididymal amyloid matrix.

The epididymal lumen is an immunologically distinct environment. It maintains tolerance for the naturally antigenic spermatozoa to allow their maturation into functional cells while simultaneously defending against pathogens that can ascend the male tract and cause infertility. We previously demonstrated that a nonpathological amyloid matrix that includes several cystatin-related epididymal spermatogenic (CRES) subgroup family members is distributed throughout the mouse epididymal lumen but its function was unknown. Here, we reveal a role for the epididymal amyloid matrix in host defense and demonstrate that the CRES amyloids and CD-1 mouse epididymal amyloid matrix exhibit potent antimicrobial activity against bacterial strains that commonly cause epididymal infections in men. We show the CRES and epididymal amyloids use several defense mechanisms including bacterial trapping, disruption of bacterial membranes and promotion of unique bacterial ghost-like structures. Remarkably, these antimicrobial actions varied depending on the bacterial strain indicating CRES amyloids and the epididymal amyloids elicit strain-specific host defense responses. We also demonstrate that the CRES monomer and immature assemblies of the epididymal amyloid transitioned into advanced structures in the presence of bacteria, suggesting their amyloid-forming/shape-shifting properties allows for a rapid reaction to a pathogen and provides an inherent plasticity in their host defense response. Together, our studies reveal new mechanistic insight into how the male reproductive tract defends against pathogens. Future studies using a mouse model for human epididymitis are needed to establish the epididymal amyloid responses to pathogens in vivo. Broadly, our studies provide an example of why nature has maintained the amyloid fold throughout evolution.

Exogenous glucocorticoid treatment affects Sertoli cell load and epididymal sperm quality in domestic cats (Felis catus).

Elevated glucocorticoid (GC) concentrations associated with captivity-related stress have been linked to impaired testicular function and low sperm quality in felids, but direct physiological evidence is lacking. This study assessed the effects of exogenous GC treatment on felid testicular function using the domestic cat (Felis catus) as a model species. Sixteen intact male cats aged 2.4 ± 0.8 years (mean ± SEM) were divided randomly into treatment (n = 8) and control (n = 8) groups. Treatment cats were given 1 mg kg-1 oral prednisolone daily for 50 days. Blood samples were taken on Days 0 (first prednisolone treatment), 2, 4, 7, 10, 20, 30, 40, 50 (prior to neutering) and 60 of the trial. All cats were orchiectomised on day 50, epididymal sperm assessed, and the testes fixed for histological assessment. Testosterone concentrations did not differ between the two groups. While sperm motility was similar between the treatment and control groups, cats given prednisolone had a higher proportion of morphologically abnormal sperm in both the caput (72.5% vs. 59.6%, P < 0.001) and cauda (56.7% vs. 35.8%, P < 0.001) epididymis. Testicular histomorphometric data and total number of germ cells per seminiferous tubule cross section did not differ between groups, nor did the relative abundance of spermatogonia, spermatocytes, and spermatids. Cats given prednisolone had fewer Sertoli cells per tubule cross-section than those in the control group (17.1 ± 0.9 vs. 19.7 ± 0.8, P = 0.04), which was likely related to higher rates of Sertoli cell apoptosis in treatment versus control cats (0.25 ± 0.02 vs. 0.10 ± 0.02 apoptotic Sertoli cells per tubule, respectively; P < 0.001). Sertoli cell load (number of germ cells per Sertoli cell) was also higher in the treatment group than in the control group (11.5 ± 0.8 vs. 9.4 ± 1.2 germ cells per Sertoli cell, respectively; P < 0.001), and was positively correlated with the percentage of morphologically abnormal sperm in the epididymis (r2 = 0.78, P < 0.001). Prednisolone treatment resulted in an increase in the proportion of abnormal sperm in the epididymis, which may be explained by an increased nurturing demand on a reduced Sertoli cell population. These findings provide novel evidence to support the hypothesis that elevated GC concentrations, such as those resulting from captivity-related stress, have the potential to impair testicular function and sperm quality in felids.

Evaluation of sperm membrane functionality during epididymal transit in red-rumped agouti (Dasyprocta leporina).

We studied the sperm membrane functionality through the epididymal transit by comparing different hypoosmotic solutions and verifying possible associations among osmotic response and functional parameters of sperm in red-rumped agouti (Dasyprocta leporina). For this purpose, epididymal sperm from six sexually mature male agoutis were collected via flotation. Then, analyses of sperm parameters and hypoosmotic swelling test using different hypoosmotic solutions (0, 50 and 200 mOsm/L) in different regions of the epididymis (caput, corpus and cauda) were performed. There was an increase (p < .05) in the values for sperm concentration, the total number of sperm recovered, total and progressive motility, average path velocity, straight-line velocity, curvilinear velocity, and rapid and medium subpopulations following the caput-corpus-cauda direction. Regardless of the hypoosmotic solution, the agouti sperm membrane presented similar functional integrity in all the epididymal regions. Moreover, the highest (p < .05) osmotic responses were reached with the use of 50 mOsm/L solution in comparison to 0 and 200 mOsm/L for all the regions. Significant correlations among osmotic response and some sperm kinetic parameters were observed, especially in epididymal caput, while no correlations were found in the region of the cauda. In summary, red-rumped agouti sperm present similar membrane functionality during epididymal transit, but there are evident correlations among such functionality and sperm kinetic parameters, especially in the caput region. Moreover, we indicate the use of a 50 mOsm/L hypoosmotic solution for the analysis of this parameter through the hypoosmotic swelling test.

Aldose Reductase B1 in Pig Sperm Is Related to Their Function and Fertilizing Ability.

Aldose reductase B1 (AKR1B1) has been reported to participate in the modulation of male and female reproductive physiology in several mammalian species. In spite of this, whether or not AKR1B1 could be related to sperm quality, functionality and fertilizing ability is yet to be elucidated. The present study, therefore, aimed to investigate: i) the presence of AKR1B1 in epididymal and ejaculated sperm; ii) the relationship between the AKR1B1 present in sperm and the physiology of the male gamete; iii) the liaison between the relative content of AKR1B1 in sperm and their ability to withstand preservation for 72 h; and iv) the potential link between sperm AKR1B1 and in vitro fertility outcomes. Immunoblotting revealed that AKR1B1 is present in both epididymal and ejaculated sperm with a similar relative content. Moreover, the relative levels of AKR1B1 in sperm (36 kDa band) were found to be negatively related to several kinematic parameters and intracellular calcium levels, and positively to the percentage of sperm with distal cytoplasmic droplets after storage. Finally, AKR1B1 amounts in sperm (36 kDa band) were negatively associated to fertilization rate at two days post-fertilization and embryo development at six days post-fertilization. The results of the present work suggest that AKR1B1 in sperm is probably acquired during maturation rather than at ejaculation and could play a role in that process. Moreover, AKR1B1 seems to be related to the sperm resilience to preservation and to their fertilizing capacity, as lower levels of the 36 kDa band (putative inactive form of this protein) result in better reproductive outcomes.

Epididymal embryonic development harbors TLR4/NFKB signaling pathway as a morphogenetic player.

The Wolffian duct (WD) is an embryonic tissue that undergoes androgen-induced morphological changes to become the epididymis. Toll-like receptor 4 (TLR4)- and nuclear factor kB (NFKB)-induced effectors are expressed in the adult epididymis and represent important players in epididymal innate immune responses. TLR4/NFKB signaling pathway is evolutionarily conserved and plays a critical morphogenetic role in several species; however, its function during WD morphogenesis is unknown. We hypothesized that TLR4/NFKB pathway plays a role during WD development. Here we examined TLR4 expression and regulation of TLR4-target genes during rat WD morphogenesis between embryonic days (e) 17.5-20.5. The functionality of TLR4/NFKB signaling was examined using WD organotypic cultures treated with lipopolysaccharide (LPS) from E. coli (TLR4 agonist) and PDTC (NFKB inhibitor). TLR4 was detected at mRNA level in e17.5 (uncoiled duct) and e20.5 (coiled duct) WDs, and spatio-temporal changes in TLR4 immunoreactivity were observed between these two time points. Expression level analysis of a subset of TLR4-regulated genes showed that TLR4/NFKB pathway was activated after exposure of cultured WD to LPS (4 h), an event that was abrogated by PDTC. Long-term exposure of cultured WDs to LPS (96 h) resulted in dysregulations of morphogenetic events and LAMA1 immunodistribution changes, suggesting the extracellular matrix at the intersection between WD morphogenesis and balance of innate immune components. Our results unveil the epididymal morphogenesis as an event equipped with TLR4/NFKB signaling components that may serve developmental functions, and eventually transition to host defense function when the fetus is exposed to an infectious or noninfectious threat.

Caput Ligation Renders Immature Mouse Sperm Motile and Capable to Undergo cAMP-Dependent Phosphorylation.

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.

Mechanisms underlying spontaneous phasic contractions and sympathetic control of smooth muscle in the rat caudal epididymis.

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5 mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300 µm dia.) exhibited SPCs, while distal segments (350-500 µm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1 µM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1 µM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10 µM). SPCs were also inhibited by caffeine (1 µM), 2-APB (100 µM), niflumic acid (100 µM), or by lowering extracellular [Cl-] from 134.4 to 12.4 mM but not by ryanodine (25 µM) or tetracaine (100 µM). Electrical field stimulation (EFS) at 2 Hz for 60 s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.

Contribution of epididymal epithelial cell functions to sperm epigenetic changes and the health of progeny.

BACKGROUND: Spermatozoa acquire their motility and fertilizing abilities during their maturation through the epididymis. This process is controlled by epididymal epithelial cells that possess features adapted to sense and respond to their surrounding environment and to communicate with spermatozoa. During the past decade, new intercellular communication processes have been discovered, including the secretion and transport of molecules from the epithelium to spermatozoa via extracellular vesicles (EVs), as well as sensing of the intraluminal milieu by cellular extensions. OBJECTIVE AND RATIONALE: This review addresses recent findings regarding epididymal epithelial cell features and interactions between spermatozoa and the epididymal epithelium as well as epigenetic modifications undergone by spermatozoa during transit through the epididymal microenvironment. SEARCH METHODS: A systematic search was conducted in Pubmed with the keyword 'epididymis'. Results were filtered on original research articles published from 2009 to 2021 and written in the English language. One hundred fifteen original articles presenting recent advancements on the epididymis contribution to sperm maturation were selected. Some additional papers cited in the primary reference were also included. A special focus was given to higher mammalian species, particularly rodents, bovines and humans, that are the most studied in this field. OUTCOMES: This review provides novel insights into the contribution of epididymal epithelium and EVs to post-testicular sperm maturation. First, new immune cell populations have been described in the epididymis, where they are proposed to play a role in protecting the environment surrounding sperm against infections or autoimmune responses. Second, novel epididymal cell extensions, including dendrites, axopodia and primary cilia, have been identified as sensors of the environment surrounding sperm. Third, new functions have been outlined for epididymal EVs, which modify the sperm epigenetic profile and participate in transgenerational epigenetic inheritance of paternal traits. WIDER IMPLICATIONS: Although the majority of these findings result from studies in rodents, this fundamental research will ultimately improve our knowledge of human reproductive physiopathologies. Recent discoveries linking sperm epigenetic modifications with paternal environmental exposure and progeny outcome further stress the importance of advancing fundamental research on the epididymis. From this, new therapeutic options for infertile couples and better counseling strategies may arise to increase positive health outcomes in children conceived either naturally or with ART.

Effects of dipyrone and acetylsalicylic acid on contractions of distal cauda epididymis duct, serum testosterone and sperm count in rats.

The effects of dipyrone and acetylsalicylic acid (ASA) on male fertility are still not fully understood, mainly considering the epididymis as a putative target for their anti-fertility effects. Therefore, this study aimed to investigate the effects of dipyrone and ASA on the contractions of distal cauda epididymis duct, serum testosterone levels and sperm parameters in rats. Firstly, we checked the in vitro effects of dipyrone and ASA (10-1000 µM) on the contractions of distal cauda epididymis duct by pharmacological experiments. We also evaluated the effects of in vivo treatment with dipyrone and ASA 100 mg/kg (p.o.) for 15 days on epididymal duct contractions, serum testosterone levels and sperm parameters. In vitro dipyrone or ASA decreased the epididymal duct contractions induced by phenylephrine or carbachol. We observed that in vivo treatment with both drugs decreased the daily sperm production, serum testosterone levels and sperm count through epididymis without altering the epididymal duct contractions and sperm transit time through epididymis. In conclusion, in vitro dipyrone and ASA were able to diminish the contractions of epididymal duct, whilst in vivo administration decreased the sperm count throughout epididymis as a consequence of a low sperm production caused by reduced testosterone levels.

Comparative iTRAQ proteomics identified proteins associated with sperm maturation between yak and cattleyak epididymis.

BACKGROUND: During maturation, spermatozoa acquire motility and fertilizing capacity as they transit through the epididymis. In recent years, two-dimensional gel electrophoresis has been employed in proteomics studies conducted in rat, boar and human. However, there has not been a complete information regarding the proteins associated with sperm maturation in the epididymis. In this study, we employed iTRAQ proteomics to investigate proteins associated with sperm maturation between yak and cattleyak epididymis. RESULTS: After a successful sampling and protein extraction, the iTRAQ coupled with LC-MS/MS mass spectrometry and bioinformatics analysis were performed. We identified 288 differentially abundant proteins (DAPs) between yak and cattleyak epididymis; 151 were up-regulated while 137 were down-regulated in cattleyak relative to yak. Gene Ontology analysis identified that down-regulated DAPs in cattleyak were mostly enriched in the acetylation of protein component, along with negative and positive regulatory activities. iTRAQ proteomics data showed that the top up-regulated DAPs were mainly enriched in cell communication, cell adhesion, cytoskeleton organization, stress response, post-translational modifications and metabolic functions while the down-regulated DAPs were predominantly associated with sperm maturation, long-term sperm storage, sperm forward motility, sperm-oocyte fusion and regulatory functions. CONCLUSION: These results provide insight into the molecular mechanisms underlying male cattleyak sterility.

Self-renewal and differentiation of rat epididymal basal cells using a novel in vitro organoid model†.

The epididymis is composed of a pseudostratified epithelium that is comprised of various cell types. Studies have shown that rat basal cells share common properties with adult stem cells and begin to differentiate in vitro in response to fibroblast growth factor and 5α-dihydrotestosterone. The characterization of rat basal cells is therefore necessary to fully understand the role of these cells. The objectives of this study were to assess the ability of single basal cells to develop organoids and to assess their ability to self-renew and differentiate in vitro. We isolated basal cells from the rat epididymis and established three-dimensional cell cultures from the basal and nonbasal cell fractions. Organoids were formed by single adult epididymal basal cells. Organoids were dissociated into single basal cells, which were able to reform new organoids, and were maintained over 10 generations. Long-term culture of organoids revealed that these cells could be differentiated into cells expressing the principal cell markers aquaporin 9 and cystic fibrosis transmembrane conductance regulator. Electron microscopy demonstrated that organoids were composed of several polarized cell types displaying microvilli and the ability to form tight junctions. Additionally, organoids could be formed by basal cells from either the proximal or distal region of the epididymis and are able to secrete clusterin, a protein implicated in the maturation of spermatozoa. These data indicate that rat basal cells can be used to derive epididymal organoids and further support that notion that these may represent a stem cell population in the epididymis.

Physiological and pharmacological impact of oxytocin on epididymal propulsion during the ejaculatory process in rodents and men.

During the emission phase of ejaculation, the sperm is driven from the cauda epididymidis, where it is stored, through the vas deferens by strong contractions. These contractions are thought of as being mainly induced by the sympathetic nervous system and the neurotransmitter noradrenaline. In the present study, we investigated the effect of oxytocin (suggested to exert effects during ejaculation as well) on defined segments of the rat and human epididymis using live imaging. Our results indicate that it is the very last part of the epididymis, segment 19 (S19) in rat and likewise segment 9 in human, which responds in a uniquely strong and rapid manner to oxytocin (similar to noradrenaline). Because of the complex nature of this contractile response, we developed an imaging analysis method, which allowed us to quantify multidirectional contractions and to display them using heat maps. The reaction of S19 to oxytocin was concentration-dependent and could be inhibited by pretreatment with oxytocin antagonists (atosiban and cligosiban), but not with an arginine vasopressin 1A antagonist (SR49059). In both rat and human tissue, pretreatment with the alpha-1 adrenoreceptor antagonist tamsulosin inhibited the response to noradrenaline, whereas the effect of oxytocin was unimpaired. Our data (from men and rodents) strongly suggest that the hormone oxytocin is involved in the ejaculatory process. Thus, oxytocin-based medications might be a promising non-adrenergic treatment option for ejaculatory disorders. Additionally, we propose that S19 could be an advantageous model (detecting very low concentrations of oxytocin) to test the bioactivity of new oxytocin agonists and oxytocin antagonists.

Exposure to epididymal extracellular vesicles enhances immature sperm function and sustains vitality of cryopreserved spermatozoa in the domestic cat model.

PURPOSE: Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer key factors to maturing spermatozoa. Using an in vitro system previously developed in our laboratory, the objective was to (1) characterize the impact of EV exposure on the fertilizing ability and developmental potential of immature sperm cells from the caput epididymidis and (2) examine the benefit of EV exposure to restore vitality of mature spermatozoa from the cauda epididymidis after freezing-thawing. METHODS: EVs were isolated from entire epididymides and collected into pellets via ultracentrifugation. Immature spermatozoa from adult cats were isolated from the caput epididymis and incubated with EVs prior to in vitro fertilization. Similarly, mature spermatozoa were isolated from the cauda segment and cryopreserved prior to EV exposure and subsequent analysis of motility and developmental potential after fertilization. RESULTS: EV exposure did not affect the percentage of caput sperm penetration; however, it improved the fertilizing ability (faster pronuclear apposition) and the developmental potential (higher proportions of morula-blastocysts) of those immature sperm cells. While EV exposure was beneficial to the frozen-thawed sperm motility, it did not significantly improve the fertilizing ability and the developmental potential. CONCLUSIONS: Epididymal EVs contain multiple factors contributing to immature sperm function, specifically enhancing the ability to complete a faster pronuclear apposition with subsequently improved early embryonic development. Supplementation was also beneficial to the motility of spermatozoa that had undergone cryopreservation. Those new findings could lead to new options for male fertility treatment in animal models and humans.

In Vitro Contraction of Isolated Cauda Epididymal Duct Smooth Muscle as a Complimentary Approach to Physiological, Pathological, Toxicological, and Pharmacological Studies on Epididymal Function.

Contraction of cauda epididymal duct (CE) smooth muscle is one of the very first events of the seminal emission phase of ejaculation. The contraction of CE smooth muscle is governed by a complex interaction of hormones, autacoids, and by the neurotransmitters released from the epididymal intramural nerve endings, and any impairment in the CE smooth muscle contraction has the potential to impair male fertility. Apart the obvious pathophysiological and toxicological importance of CE smooth muscle contraction, modulation of CE contraction has pharmaceutical interest offering a druggable target to development of drugs to improve/impair male fertility. The in vitro contraction experiments constitute a valuable approach to an in-depth evaluation of functional and molecular changes resulting from pathologies or drug exposure. Therefore, this chapter consists in a description of in vitro pharmacological reactivity contractility of the epididymal duct in a controlled medium, maintained at 30 °C of temperature and continuously bubbled with 95% O2 and 5% CO2 to obtain cumulative concentration-response curves that has been fundamental to some of our investigations on epididymal physiology, toxicology, and pharmacology.

Effects of photoperiod on morphology and function in testis and epididymis of Cricetulus barabensis.

Photoperiod regulates the seasonal reproductive rhythms of mammals by influencing the development and function of sexual organs; however, the underlying mechanism remains unclear. We examined the morphology and functioning of the main sex organs of striped dwarf hamsters (Cricetulus barabensis) under different photoperiods (short daylight [SD], moderate daylight [MD], and long daylight [LD]) and further investigated the underlying molecular mechanisms. There was an inverse correlation between blood melatonin levels and photoperiod in the order SD > MD > LD. Decreases in body and tissue weights were observed under SD, whereas testis and epididymis weights between MD and LD were comparable. The diameters of the spermatogenic tubules, thickness of the spermatogenic epithelium, and the number of spermatogonia and Sertoli cells decreased under SD, whereas the serum-luteinizing hormone, follicle-stimulating hormone, and fecal testosterone concentrations decreased under LD. In SD, bax/bcl2 protein expression increased in the testes and decreased in the epididymides, whereas LC3II/LC3I remained unchanged in the testes and increased in the epididymides compared with the MD group. In LD, bax/bcl2 and LC3II/LC3I protein expression levels were unchanged in the testes but were decreased in the epididymides. In SD and LD, adenosine triphosphate synthase and citrate synthase protein expression levels were unchanged in the testes but were decreased in the epididymides. Drp1 and Mff protein expression increased in the testes and decreased in the epididymides. Overall, different regulatory mechanisms in the testis and epididymis led to degeneration under SD and maintenance under LD, preferentially protecting mitochondrial function in the testis by regulating mitochondrial fission.

Roles of male reproductive tract extracellular vesicles in reproduction.

Extracellular vesicles (EVs) are secreted cell-derived membrane structures present in all organisms across animal, bacterial, and plant phyla. These vesicles play important roles in cell-cell communication in many processes integral to health and disease. Recent studies demonstrate that EVs and their cargo have influential and conserved roles in male reproduction. While EVs have been isolated from virtually all specialized tissues comprising the male reproductive tract, they are best characterized in the epididymis (epididymosomes) and seminal fluid (seminal fluid extracellular vesicles or prostasomes). Broadly speaking, EVs promote reproductive success through supporting sperm development and function, as well as influencing the physiology of female reproductive tract cells after mating. In this review, we present current knowledge on the composition and function of male reproductive tract EV populations in both normal physiology and pathology, and argue that their functions identify them as critical regulators of fertility and fecundity.

Epididymosomes: the black box of Darwin's pangenesis?

Darwin, in the pangenesis theory, imagined particles, named as 'gemmules', which are released from all ('pan') cells of the body. By cell-cell communication and also circulation through the body, they finally reach the germ cells to participate in the generation ('genesis') of the new individual. It has been shown that circulatory exosomes are affected by environmental stressors and they can reach the parental germ cells. Therefore, in the mirror of his theory, circulatory exosomes could interact with epididymosomes: epididymis-derived exosomes which have a wide spectrum of variation in content and size, are very sensitive to environmental stressors, and may be involved in translating external information to the germ cells. The protein and RNA cargo would be transferred by epididymosomes to sperm during sperm maturation, which would be then delivered to the embryo at fertilization and inherited by offspring. Therefore, in this study, we will briefly discuss Darwin's pangenesis theory and its possible relation with epididymosomes. We believed that epididymosomes could be considered as an attractive candidate for the storage of RNA contents, changing the epigenome of the next generations, and allowing the reappearance acquired characteristics of ancestors. Therefore, epididymosomes, as a black box of Darwin's pangenesis, may unravel parental life history and also disclose the historical events that affect the life of offspring.

Identification of a New QTL Region on Mouse Chromosome 1 Responsible for Male Hypofertility: Phenotype Characterization and Candidate Genes.

Male fertility disorders often have their origin in disturbed spermatogenesis, which can be induced by genetic factors. In this study, we used interspecific recombinant congenic mouse strains (IRCS) to identify genes responsible for male infertility. Using ultrasonography, in vivo and in vitro fertilization (IVF) and electron microscopy, the phenotyping of several IRCS carrying mouse chromosome 1 segments of Mus spretus origin revealed a decrease in the ability of sperm to fertilize. This teratozoospermia included the abnormal anchoring of the acrosome to the nucleus and a persistence of residual bodies at the level of epididymal sperm midpiece. We identified a quantitative trait locus (QTL) responsible for these phenotypes and we have proposed a short list of candidate genes specifically expressed in spermatids. The future functional validation of candidate genes should allow the identification of new genes and mechanisms involved in male infertility.