Caput Sperm Research Articles

Sperm Heterogeneity Accounts for Sperm DNA Methylation Variations Observed in the Caput Epididymis, Independently From DNMT/TET Activities

Following their production in the testis, spermatozoa enter the epididymis where they gain their motility and fertilizing abilities. This post-testicular maturation coincides with sperm epigenetic profile changes that influence progeny outcome. While recent studies highlighted the dynamics of small non-coding RNAs in maturing spermatozoa, little is known regarding sperm methylation changes and their impact at the post-fertilization level. Fluorescence-activated cell sorting (FACS) was used to purify spermatozoa from the testis and different epididymal segments (i.e., caput, corpus and cauda) of CAG/su9-DsRed2; Acr3-EGFP transgenic mice in order to map out sperm methylome dynamics. Reduced representation bisulfite sequencing (RRBS-Seq) performed on DNA from these respective sperm populations indicated that high methylation changes were observed between spermatozoa from the caput vs. testis with 5,546 entries meeting our threshold values (q value <0.01, methylation difference above 25%). Most of these changes were transitory during epididymal sperm maturation according to the low number of entries identified between spermatozoa from cauda vs. testis. According to enzymatic and sperm/epididymal fluid co-incubation assays, (de)methylases were not found responsible for these sperm methylation changes. Instead, we identified that a subpopulation of caput spermatozoa displayed distinct methylation marks that were susceptible to sperm DNAse treatment and accounted for the DNA methylation profile changes observed in the proximal epididymis. Our results support the paradigm that a fraction of caput spermatozoa has a higher propensity to bind extracellular DNA, a phenomenon responsible for the sperm methylome variations observed at the post-testicular level. Further investigating the degree of conservation of this sperm heterogeneity in human will eventually provide new considerations regarding sperm selection procedures used in fertility clinics.

Chromatin alterations during the epididymal maturation of mouse sperm refine the paternally inherited epigenome

BackgroundPaternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit.ResultsUsing proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation.ConclusionsThese data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Low Sperm Motility Is Determined by Abnormal Protein Modification during Epididymal Maturation.

PurposeDuring epididymal sperm maturation, spermatozoa acquire progressive motility through dynamic protein modifications. However, the relationship between sequential protein modifications during epididymal sperm maturation and sperm motility and fertility has not yet been investigated. This study investigated whether sequential changes in fertility-related protein expression including that of enolase 1 (ENO1), ubiquinol-cytochrome c reductase core protein 1 and 2 (UQCRC1 and UQCRC2), and voltage-dependent anion channel 2 (VDAC2) in spermatozoa during epididymal maturation are related to bovine sperm motility. Moreover, we found that mitochondrial metabolism is closely related to fertility-related proteins. Therefore, we investigated how the sequential modification of mitochondrial proteins during epididymal maturation regulates sperm motility.Materials and MethodsTo determine the differential protein expression in caput and cauda epididymal spermatozoa from low and high motility bulls, western blot analysis was performed. Moreover, signaling pathways were identified to understand the mechanisms of regulation of sperm motility through the differential protein expression associated with fertility-related proteins.ResultsWe found that ENO1 was substantially higher in the caput spermatozoa from low motility bulls than the caput and cauda spermatozoa from high motility bulls. However, ENO1 expression in low motility bull spermatozoa was downregulated to a level comparable to that in the high motility bull spermatozoa during epididymal maturation. Moreover, there was a lack of modification of mitochondrial proteins, including glutathione peroxidase 4 and NADH:Ubiquinone Oxidoreductase Core Subunit S8, in low motility bull spermatozoa during epididymal maturation, whereas active changes were detected in high motility bull spermatozoa.ConclusionsIrregular modifications of mitochondrial proteins during epididymal sperm maturation may increase excessive ROS production and premature activation of spermatozoa during epididymal maturation. Consequently, spermatozoa may lose their motility by the earlier consumption of their energy source and may be damaged by ROS during epididymal maturation, resulting in a decline in sperm motility and bull fertility.

Polarized epithelium-sperm co-culture system reveals stimulatory factors for the secretion of mouse epididymal quiescin sulfhydryl oxidase 1.

Spermatozoa acquire fertilization ability through post-translational modifications. These membrane surface alterations occur in various segments of the epididymis. Quiescin sulfhydryl oxidases, which catalyze thiol-oxidation reactions, are involved in disulfide bond formation, which is essential for sperm maturation, upon transition and migration in the epididymis. Using castration and azoospermia transgenic mouse models, in the present study, we showed that quiescin sulfhydryl oxidase 1 (QSOX1) protein expression and secretion are positively correlated with the presence of testosterone and sperm cells. A two-dimensional in vitro epithelium-sperm co-culture system provided further evidence in support of the notion that both testosterone and its dominant metabolite, 5α-dihydrotestosterone, promote epididymal QSOX1 secretion. We also demonstrated that immature caput spermatozoa, but not mature cauda sperm cells, exhibited great potential to stimulate QSOX1 secretion in vitro, suggesting that sperm maturation is a key regulatory factor for mouse epididymal QSOX1 secretion. Proteomic analysis identified 582 secretory proteins from the co-culture supernatant, of which 258 were sperm-specific and 154 were of epididymal epithelium-origin. Gene Ontology analysis indicated that these secreted proteins exhibit functions known to facilitate sperm membrane organization, cellular activity, and sperm-egg recognition. Taken together, our data demonstrated that testosterone and sperm maturation status are key regulators of mouse epididymal QSOX1 protein expression and secretion.

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.

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

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. 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. 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. 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.

Stability of the cytosine methylome during post-testicular sperm maturation in mouse.

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA.

Vacuolar H(+)-ATPase is not restricted to clear cells of the epididymal epithelium in cattle.

Communication among epididymal epithelial cells creates the best luminal condition where spermatozoa mature, transport and are stored. Vacuolar ATPase (V-ATPase) and cytokeratin 5 (KRT5) have been used as signal indicators for clear and basal cells of the epididymal epithelium, respectively, in mice, rats, bats, and pigs; however, these two markers have not yet been described in the epididymis of bulls. Here, we examined the presence and distribution of the B1 subunit of V-ATPase (B1-VATPase) and KRT5 in the distinct regions of adult bovine epididymides, specifically, the caput, corpus, and cauda. Immunofluorescence staining and confocal microscopy showed that narrow shaped-clear cells were placed in the caput and corpus regions of the bovine epididymis; however, they were absent in the cauda epididymis. In addition, B1-VATPase was highly expressed in the cauda spermatozoa; however, it was rarely detected in the caput spermatozoa. On the other hand, KRT5-positive cells, basal cells, were maintained beneath the basal lamina and they had the traditional form with a dome-shaped morphology from the caput to cauda region of the bovine epididymis. The co-expression of B1-VATPase and KRT5 was confined to basal cells placed in the basal region of the epithelium. In summary, 1) clear cells were present with region-specific localization, 2) B1-VATPase was present in the corpus and cauda spermatozoa but absent in the caput, 3) co-expressed cells with B1-VATPase and KRT5 were present in the adult bovine epididymis, and 4) B1-VATPase was not a specific marker for clear cells in the bovine epididymis. Therefore, the perfect epididymal luminal condition created by the specific expression and localization patterns of B1-VATPase might be necessary to obtain fertilizing capacity of spermatozoa in the bovine epididymis.

Both Cauda and Caput Epididymal Sperm Are Capable of Supporting Full-Term Development in FVB and CD-1 Mice

Both Cauda and Caput Epididymal Sperm Are Capable of Supporting Full-Term Development in FVB and CD-1 Mice

Sperm Head Preparation and Genetic Background Affect Caput Sperm ICSI Embryo Viability: Cauda-Enriched miRNAs Only Essential in Specific Conditions

In 2018, we reported changes in preimplantation gene regulation, and defective implantation, in mouse embryos fertilized by intracytoplasmic sperm injection (ICSI) using sperm from the caput epididymis (Conine et al., 2018). We further showed that both the molecular defects and implantation were rescued by microinjection of purified epididymosomal microRNAs, revealing a functional role for sperm-delivered RNAs in the early embryo. At the time, we noted that one of our findings, the inability to recover viable offspring from caput sperm-fertilized embryos (Figure 3 in Conine et al., 2018), contradicted prior studies (Said et al., 2003; Suganuma et al., 2005) which documented successful production of such offspring.

THE IMPACT OF SPERMATOZOA RETRIEVED FROM DIFFERENT REGIONS OF THE EPIDIDYMIS ON REPRODUCTIVE OUTCOMES

To determinate the area of the epididymis that yields spermatozoa most capable of supporting embryonic development. During the last 9 years, 46 men affected by obstructive azoospermia (OA) consented to have spermatozoa surgically retrieved from three areas of the epididymis: caput, corpus, and cauda. Sperm characteristic were analyzed and compared among the retrieval sites that were used for ICSI with their female partners. Clinical outcomes were compared among the different sources of spermatozoa. Consenting men underwent epididymal sperm retrieval, with specimens evaluated according to WHO 2010 criteria. A total of 36 specimens retrieved from the caput, 7 from the corpus, and 3 from the cauda were used for ICSI, controlling for maternal age (≤37 years old). Fertilization, implantation, and delivery rates were assessed and compared for all three areas of the epididymis. Unpaired t and Fisher’s exact tests were used to compare outcomes. A P value of <0.05 was considered significant. A total of 46 men were divided into three groups according to the spermatozoa source used: caput, corpus, and cauda. Paternal age was comparable among the three groups (39.7± 12). Average and standard deviation of spermatozoa concentration was 35.1± 39 in the caput, 40±29 in the corpus, and 30.2± 59 x106/ml in the cauda. The mean motility was 15.8±15% in the caput, rising to 40.1±29% in the corpus (P<0.01), and decreasing to 7±12% in the cauda (P<0.05). The mean numbers of injected oocytes were comparable at 12.7, 11, and 12.5, for the caput, corpus, and cauda, respectively. The fertilization rate with specimens from the caput was 65.8% (303/460), it rose to 77.6% (59/76) with specimens from the corpus (P<0.05), and reached the highest level at 88% (44/50, P<0.05) with spermatozoa from the cauda. The proportion of high-quality embryos was 73.5% (25/34) from sperm retrieved from the caput, 75% (3/4) from the corpus, and reached 100% (3/3) from the cauda. There was a trend of increasing implantation rates through the regions of the epididymis. With specimens retrieved from the caput, the implantation rate was 34.8% (23/66), it rose to 44.4% (4/9) from the corpus, and increased to 57.1% (4/7) from the cauda. The same trend was observed for delivery rates. In cycles using caput spermatozoa, the delivery rate was 48.4% (16/33) with 5.8% (1/17) pregnancy loss. In cycles using spermatozoa from the corpus, the delivery rate was 75% (3/4) with no pregnancy loss. Ultimately, the cycles with cauda had the highest delivery rate, with 100% (3/3). This study supports the current knowledge that while progressing through the epididymal regions, testicular spermatozoa undergo structural and metabolic changes that translate in phenotypic and functional processes. During the epididymal journey, spermatozoa appeared to enhance their ability to support embryonic development and implantation. Although they have poorer kinetic characteristics, we have found that spermatozoa retrieved from the cauda epididymis yield higher fertilization and pregnancy rates. Larger studies are needed to confirm these findings.

Successful ICSI in Mice Using Caput Epididymal Spermatozoa.

Spermatozoa undergo their last phase of spermiogenesis, known as maturation, as they pass through the epididymis. A recent report indicates that mouse immature spermatozoa retrieved from the caput epididymis for intracytoplasmic sperm injection (ICSI) give rise to embryos with multiple developmental defects. Further, these embryos were unable to develop to term after their transfer to surrogate mothers. Herein, we examined the potential of mouse caput spermatozoa to produce normal embryos by comparing the use of caput vs. cauda epididymal spermatozoa for in vitro fertilization (IVF) or ICSI. Two methods for the separation of sperm heads prior to ICSI were also compared: freezing/thawing or drawing through a syringe. We found that in contrast to caudal spermatozoa, caput spermatozoa failed to produce embryos via IVF, confirming their immature state. However, regardless of the method employed for the separation of sperm heads, similar efficiencies of blastocyst production in vitro and development to term after transfer to surrogate mothers were observed following ICSI using both caput and cauda epididymal spermatozoa. It therefore seems that mice spermatozoa recovered from the caput epididymis are as valid as caudal spermatozoa for the production of embryos and offspring by ICSI.

Susceptibility of epididymal sperm against reactive oxygen species in dogs

In order to add information to physiology of sperm maturation and help to underline future antioxidant supplementation treatment to epididymal sperm, the aim of this study was to evaluate susceptibility of caput, corpus and cauda epididymal sperm to different reactive oxygen species (ROS) in dogs. Epididymal sperm was separately collected from each segment (caput, corpus and cauda) and subjected to 4 different ROS-challenges: superoxide anion, hydrogen peroxide, hydroxyl radical, malondialdehyde (MDA) or maintained as control. After 30 min of incubation in each ROS, sperm was evaluated for sperm kinetics, plasma and acrossomal membrane integrity, mitochondrial activity and resistance to oxidative stress. Decreased total and progressive sperm motility and rapid velocity at epididymal corpus occurred after exposure to hydrogen peroxide, hydroxyl radical and MDA. However, for cauda epididymis, hydrogen peroxide and malondialdehyde promoted higher deleterious effect regarding sperm motility and velocity. Only at cauda epididymis MDA decreased sperm mitochondrial activity index and no kinetics alterations (motility or velocity) occurred after exposure to superoxide anion in corpus and cauda epididymis. In conclusion, corpus and cauda epididymal sperm are highly susceptible to deleterious effect of hydrogen peroxide, malondialdehyde and hydroxyl radical. In addition, epididymal canine sperm is relatively resistant to superoxide anion damage.

Telomere Dynamics Throughout Spermatogenesis.

Telomeres are repeat regions of DNA that cap either end of each chromosome, thereby providing stability and protection from the degradation of gene-rich regions. Each cell replication causes the loss of telomeric repeats due to incomplete DNA replication, though it is well-established that progressive telomere shortening is evaded in male germ cells by the maintenance of active telomerase. However, germ cell telomeres are still susceptible to disruption or insult by oxidative stress, toxicant exposure, and aging. Our aim was to examine the relative telomere length (rTL) in an outbred Sprague Dawley (SD) and an inbred Brown Norway (BN) rat model for paternal aging. No significant differences were found when comparing pachytene spermatocytes (PS), round spermatids (RS), and sperm obtained from the caput and cauda of the epididymis of young and aged SD rats; this is likely due to the high variance observed among individuals. A significant age-dependent decrease in rTL was observed from 115.6 (±6.5) to 93.3 (±6.3) in caput sperm and from 142.4 (±14.6) to 105.3 (±2.5) in cauda sperm from BN rats. Additionally, an increase in rTL during epididymal maturation was observed in both strains, most strikingly from 115.6 (±6.5) to 142 (±14.6) in young BN rats. These results confirm the decrease in rTL in rodents, but only when an inbred strain is used, and represent the first demonstration that rTL changes as sperm transit through the epididymis.

Regulators of the protein phosphatase PP1γ2, PPP1R2, PPP1R7, and PPP1R11 are involved in epididymal sperm maturation.

The serine/threonine protein phosphatase 1 (PP1) inhibitors PPP1R2, PPP1R7, and PPP1R11 are evolutionarily ancient and highly conserved proteins. Four PP1 isoforms, PP1α, PP1β, PP1γ1, and PP1γ2, exist; three of them except PP1γ2 are ubiquitous. The fact that PP1γ2 isoform is present only in mammalian testis and sperm led to the notion that isoform-specific regulators for PP1γ2 in sperm may be responsible for its function. In this report, we studied these inhibitors, PPP1R2, R7, and R11, to determine their spatial and temporal expression in testis and their regulatory functions in sperm. We show that, similar to PP1γ2, the three inhibitors are expressed at high levels in developing spermatogenic cells. However, the transcripts for the regulators are expressed as unique sizes in testis compared with somatic tissues. The three regulators share localization with PP1γ2 in the head and the principal piece of sperm. We show that the association of inhibitors to PP1γ2 changes during epididymal sperm maturation. In immotile caput epididymal sperm, PPP1R2 and PPP1R7 are not bound to PP1γ2, whereas in motile caudal sperm, all three inhibitors are bound as heterodimers or heterotrimers. In caudal sperm from male mice lacking sAC and glycogen synthase kinase 3, where motility and fertility are impaired, the association of PP1γ2 to the inhibitors resembles immature caput sperm. Changes in the association of the regulators with PP1γ2, due to their phosphorylation, are part of biochemical mechanisms responsible for the development of motility and fertilizing ability of sperm during their passage through the epididymis.

Small RNAs Gained during Epididymal Transit of Sperm Are Essential for Embryonic Development in Mice

The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) versus distal (cauda) epididymis and then characterized the development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, subsequently implant inefficiently, and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects but also suppressed the post-implantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs.

Changes in Protein O-GlcNAcylation During Mouse Epididymal Sperm Maturation.

After leaving the testis, sperm undergo two sequential maturational processes before acquiring fertilizing capacity: sperm maturation in the male epididymis, and sperm capacitation in the female reproductive tract. During their transit through the epididymis, sperm experience several maturational changes; the acquisition of motility is one of them. The molecular basis of the regulation of this process is still not fully understood. Sperm are both transcriptionally and translationally silent, therefore post-translational modifications are essential to regulate their function. The post-translational modification by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) can act as a counterpart of phosphorylation in different cellular processes. Therefore, our work was aimed to characterize the O-GlcNAcylation system in the male reproductive tract and the occurrence of this phenomenon during sperm maturation. Our results indicate that O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation, is present in the testis, epididymis and immature caput sperm. Its presence is significantly reduced in mature cauda sperm. Consistently, caput sperm display high levels of O-GlcNAcylation when compared to mature cauda sperm, where it is mostly absent. Our results indicate that the modulation of O-GlcNAcylation takes place during sperm maturation and suggest a role for this post-translational modification in this process.

Localization of MIF-II on mammalian spermatozoa: A study revealing its structure, function and motility inhibitory pathway

Motility of spermatozoa is a crucial factor for determining semen quality. Here we report motility inhibitory factor (MIF-II) from goat epididymal plasma, revealing its structure, function, localization and motility inhibitory pathway. Structural characterization with MALDI revealed novelty of this protein while circular dichroism data confirmed its alpha helical nature. Higher dilutions of MIF-II antibody increased cauda sperm motility and induced immature/immotile caput sperm motility as tested microscopically. Higher number of sperm cells and lower dilutions of antibody induced agglutination in cauda sperm showing surface localization. Indirect immuno-fluorescence showed MIF-II localization throughout the caput sperm surface which relocated more towards acrosomal region with maturation. ELISA assay revealed gradual increase and decrease in concentration of MIF-II in epididymal plasma and plasma membrane respectively from caput to cauda. Signaling cascade that leads to sperm motility inhibition elevates nitric oxide levels through cAMP dependent pathway. MIF-II treatment doesn't alter sperm surface morphology. Expression pattern of MIF-II during epididymal maturation goes hand-in-hand with gaining motility potential as well as dormancy of spermatozoa before ejaculation. Both MIF-II and its antibody inhibit fertilization in-vitro thus expected to open new gateway for future male infertility and contraceptive development research.

Epididymal α-l-fucosidase and its possible role in remodelling the surface of bull spermatozoa

The mammalian epididymis provides an appropriate environment for sperm maturation. During the epididymal transit, spermatozoa undergo biochemical and morphological changes that lead to the acquisition of the fertilizing capacity. In this study we analysed the fucosylation status of membrane glycoproteins in the spermatozoa obtained from different regions of the bull epididymis. High amounts of fucose were detected on caput spermatozoa (R.F.I. = 1010 ± 20.35), mostly located in the post-acrosome zone. A significant decrease in the fucose levels was detected toward the cauda (R.F.I. = 540.5 ± 49.93) (P < 0.05). This decrease was in line with the increased activity of α-l-fucosidase in the cauda fluid. In sperm from the cauda, the defucosylation occurred in some proteins, whereas others showed higher fucosylation rates. A significant decrease of fucose in the gametes was observed upon incubation of crude cauda fluid with caput spermatozoa (from R.F.I. = 1.45 ± 0.08 to 1.06 ± 0.03) (P < 0.05) indicating that the α-l-fucosidase present in the epididymal fluid is active on spermatozoa. Moreover, this effect was blocked with specific enzyme inhibitors. These results provide direct evidence that the α-l-fucosidase from epididymal fluid participates in the fucose removal from spermatozoa, as a step of sperm maturation in the bull epididymis.

Cytoplasmic droplet acting as a mitochondrial modulator during sperm maturation in dogs

Motility acquisition during sperm maturation and passage through the epididymis is closely related to mitochondrial function and appears to occur in parallel with cytoplasmic droplet (CD) migration. However, such mechanism remains unclear in dogs. Thus, the aim of this study was to characterize the influence of sperm CD in the mitochondrial functionality during epididymal sperm maturation in dogs. Twenty-one adult dogs were submitted to elective bilateral orchiectomy. Testicles were stored for 18–24h at 5°C and epididymal sperm samples were then collected from different segments of the epididymis (caput, corpus and cauda). Samples were evaluated for computer-assisted motility analysis (CASA), presence of CD (eosin/nigrosin stain), ultrastructural CD analysis and sperm mitochondrial activity (3,3' diaminobenzidine technique) and membrane potential (JC-1 probe). Samples collected from the corpus epididymis showed higher motility and mitochondrial activity in comparison to the caput sperm. Moreover, corpus sperm had lower percentage of proximal droplets compared to caput samples, while mitochondrial membrane potential remained unchanged. Cauda samples showed higher motility, mitochondrial activity and potential, however, lower presence of sperm droplets (proximal and distal). In conclusion, the CD is essential for epididymal sperm maturation in dogs, showing important functions along the transit in the epididymis. In the corpus segment, the migration of the CD along the sperm midpiece provides a high mitochondrial activity and the onset of sperm motility. On the other hand, sperm from cauda epididymis lack CD but suffered lipid membrane changes which allow a maximum mitochondrial membrane potential and motility.