Cauda Epididymidis Research Articles

The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase Cζ, a Mammalian Egg-activating Factor

Sperm-specific phospholipase C-zeta (PLCzeta) causes intracellular Ca(2+) oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCzeta, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCzeta mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374-381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg(376), Lys(377), Arg(378), Lys(379), or Lys(381) with glutamate, whereas Ca(2+) oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys(299) and/or Lys(301) in the C terminus of X domain, or Trp(13), Phe(14), or Val(18) in the N terminus of EF1. Ca(2+) oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374-383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296-309 or 1-19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2-4 or C2 domain. Thus, PLCzeta is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCdelta1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.

Comparison Between Epididymosomes Collected in the Intraluminal Compartment of the Bovine Caput and Cauda Epididymidis1

During their transit along the epididymidis, mammalian spermatozoa acquire new proteins involved in the acquisition of male gamete fertilizing ability. We previously described membranous vesicles called epididymosomes, which are secreted in an apocrine manner by the epididymal epithelium. Some selected proteins associated with epididymosomes are transferred to spermatozoa during epididymal transit. The present study compared epididymosomes collected from caput epididymal fluid with vesicles from the cauda epididymidis in the bull. Two-dimensional gel electrophoresis revealed major differences in protein composition of epididymosomes isolated from the caput and cauda epididymidis. LC-QToF analysis of major protein spots as well as Western blot analysis confirmed the differences in proteins associated with these two populations of epididymosomes. Biotinylated proteins associated with caput and cauda epididymosomes also revealed differences. When incubated with caput epididymal spermatozoa, epididymosomes prepared from these two segments transferred different protein patterns. By contrast, cauda epididymosomes transferred the same pattern of proteins to spermatozoa from the caput and cauda epididymidis. Transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa decreased in a dose-dependent manner when biotinylated epididymosomes were diluted with unbiotinylated vesicles. Caput epididymosomes added in excess were unable to inhibit transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa. Following transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa, addition of unbiotinylated cauda epididymosomes was unable to displace already transferred biotinylated proteins. These results established that epididymosomes from caput and cauda epididymidis have different protein composition and interact differently with maturing spermatozoa.

The Role of Potassium Chloride Cotransporters in Murine and Human Sperm Volume Regulation1

Spermatozoa need to undergo regulatory volume decrease (RVD) upon ejaculation to counteract swelling due to the hypo-osmolality of female tract fluids. Defects in sperm RVD lead to failure in both cervical mucus penetration in humans and utero-tubal junction passage in mice. The role of K/Cl cotransporters (KCCs) in RVD was investigated by incubation of spermatozoa from the murine cauda epididymidis and from human ejaculates in media mimicking female tract fluid osmolalities in the presence of KCC inhibitors. Furosemide at 100 microM or more caused swelling of murine spermatozoa as detected with a flow cytometer by increased laser forward scatter over 30 to 75 min of incubation. Bumetanide, known to have low affinity for KCCs, was effective at 1 mM, whereas 10 microM and 20 microM of the specific inhibitor DIOA (dihydroindenyl-oxy alkanoic acid) increased cell volume. These drug doses were ineffective in human spermatozoa, which, however, responded to quinine, confirming the occurrence of RVD under control conditions. The molecular identity of the murine KCC isoform involved was determined at both mRNA and protein levels. Conventional RT-PCR indicated the presence of transcripts from Slc12a4 (KCC1), Slc12a6 (KCC3), and Slc12a7 (KCC4) in the testis, whereas RT-nested PCR revealed the latter two isoforms in sperm mRNA. Of these three isoforms, only SLC12A7 (KCC4) was detected in murine sperm protein by Western blotting. Therefore, besides organic osmolyte efflux and KCl release through separate K(+) and Cl(-) ion channels, SLC12A7 also is involved in murine but not human sperm RVD mechanisms.

Proteinaceous diet inhibits gossypol-induced spermatotoxicity.

The present study was designed to investigate the effect of a proteinaceous dietary supplement, fishmeal, on gossypol-induced spermatotoxicity. Twenty-five adult male Wistar rats, averaging 205 g b.w., were randomly sorted into four experimental groups (I-IV) of 5 animals each, and a control group. Crude cottonseed oil was administered orally to each animal in groups I-IV at a rate that provided 14 mg/kg/d free gossypol; in addition, 3 g/d, 7 g/d, and 10 g/d of fishmeal was provided as meal supplement to each animal in groups I, II and III respectively. The control group received rat pellets and water freely. At the end of the 53-day treatment period, all animals were placed under chloroform anaesthesia; the caudal epididymides were removed, minced and placed in Ham's F10 solution for the evaluation of sperm count and motility. The testes were also processed for histological studies using the eosin and haematoxylin (H & E) method. Our findings revealed a dose-dependent inhibition of gossypol-induced spermatotoxicity by the supplemented fishmeal; this suggests that proteinaceous diets are protective against gossypol-induced male infertility.

Impairment of adult male reproductive function in rats exposed to ethanol since puberty

The objective of this work was to evaluate reproductive function in adult male rats exposed to ethanol since puberty. Male Wistar rats, 50 days old, received a liquid diet with 36% of the daily calories derived from ethanol or an isocaloric control diet for 55 days. The ethanol treatment impaired sexual behavior and only 22% of these rats reached ejaculation. The fertility of ethanol-treated animals was significantly reduced, mainly after natural mating. Serum testosterone levels, daily sperm production and sperm count in the epididymis were also significantly diminished after ethanol treatment, associated with an acceleration of the sperm transit time in the cauda epididymidis, decrease in sperm motility and increased percentage of abnormal shaped sperm cells. The results showed that chronic consumption of ethanol beginning at puberty impairs the reproductive function of adult male rats.

Different expression of lipocalin-type prostaglandin D synthase in rat epididymidis

This study was designed to explore the different expression of L-PGDS (lipocalin-type prostaglandin D synthase) in rat epididymidis and to gain further insight into the potential function of L-PGDS in male reproduction. The expression of L-PGDS in rat epididymidis was assessed using real-time quantitative PCR and immunoblotting. The distribution of L-PGDS in rat epididymidis was explored by immunohistochemical methods. The result of immunohistochemistry displayed that L-PGDS was mainly distributed in epididymidis and localized within the cytoplasm and the cilia of the epithelial cells. Real-time quantitative PCR and immunoblotting showed that L-PGDS was strikingly expressed in the caput epididymidis, while a moderate to weak expression was observed in the corpus and cauda epididymidis, the level of mRNA was 0.52 ± 0.02 in the caput, 0.48 ± 0.03 in the corpus and 0.32 ± 0.01 in the cauda epididymidis, the level of protein expression in caput, corpus and the cauda groups was 1, 0.89 ± 0.03 and 0.62 ± 0.01, which suggested that L-PGDS may play certain kind of role during the process of the spermatozoa maturation.

Pathogenetic transition in the morphology of abnormal sperm in the testes and the caput, corpus, and cauda epididymides of male rats after treatment with 4,6-dinitro- o-cresol

In order to elucidate the pathogenesis of tailless sperm, 4,6-dinitro- o-cresol (DNOC) was administered to Jcl:SD male rats at daily oral doses of 0, 10 or 15 mg/kg for 5 days. Sperm were collected from the caput, corpus, and cauda epididymides on days 1, 7 and 14 after the last dosing (D1, D7 and D14, respectively), counted and examined morphologically by phase-contrast and scanning electron microscopy. The incidence of abnormal sperm was significantly increased in the DNOC 15 mg/kg group. On D1, peeled sperm (loss of mitochondrial sheath at the proximal end of the middle piece) was frequently observed in the caput epididymides, whereas sperm in the corpus and cauda epididymides had normal morphology. Distribution of the peeled sperm changed as time passed and the corpus epididymides showed a peak incidence on D7. On D14, the highest incidence of abnormal sperm was observed in the cauda epididymides, where the major abnormality was tailless. Similar effects were also found in the 10 mg/kg group but were less potent. Transmission electron microscopy of testicular sperm on D1 revealed the presence of elongated spermatids that lacked the mitochondrial sheath at the proximal end of the middle piece, although the round and elongating spermatids looked normal. These results suggest that DNOC exposure of male rats primarily causes partial loss of the mitochondrial sheath in the testicular elongated spermatids, and that the affected sperm become tailless by D14 after reaching the cauda epididymides.

Expression and distribution of OCTN2 in mouse epididymis and its association with obstructive azoospermia in juvenile visceral steatosis mice

L-carnitine, an essential cofactor for mitochondrial, beta-oxidation of long-chain fatty acids, is known to play important roles in sperm maturation and metabolism when spermatozoa pass and acquire motility in the epididymis. We reported that obstructive azoospermia occurred in the epididymis in the juvenile visceral steatosis (JVS) mice, which are OCTN2 dysfunction mice caused by mutations in the gene encoding OCTN2, have been used for animal models of primary systemic carnitine deficiency. The aim of present study is to investigate the expression of OCTN2 protein in the mouse epididymis and its relation between the localization of OCTN2 and obstructive azoospermia in JVS mice as animal models for human male infertility. Animals used in this study were wild-type (C57BL/6 J) mice (n = 4) and JVS mice (n = 4). We made a specific polyclonal antibody against OCTN2 and examined immunohistochemically the localization of OCTN2 in the mouse epididymis. OCTN2 was localized on the apical membrane of the principal cells of distal corpus and cauda epididymides. Immunocytochemistry demonstrated that OCTN2 was localized on the surface of microvillus upon the principal cells. In JVS mice, immunoreactivity started in a region immediately distal to where the sperm obstruction occurred. Our results suggest that OCTN2 functions as a carnitine transporter between the epithelium and the lumen in distal corpus and cauda epididymides and provides a clue as to why obstructive azoospermia is induced in distal parts of epididymis.

Cells positive for microtubule-associated protein 1B (MAP 1B) are present along rat and human efferent ductules and epididymis

Microtubule-associated protein 1B (MAP 1B) is a neuronal cytoskeleton marker with predominant expression in the developing nervous system. The present study provides evidence for the expression of this cytoskeleton protein in non-neuronal and neuronal cells along rat and human efferent ductules and epididymis (initial segment, caput, and cauda). Reverse transcription/polymerase chain reaction and Western blot analysis were used to confirm the presence of MAP 1B (mRNA and protein) in rat tissues. Immunohistochemical studies revealed MAP-1B-positive staining in columnar ciliated cells present in efferent ductules and in narrow cells located in the initial segment, in both rat and human. MAP-1B-positive basal cells, located underneath the columnar cells, were only identified in the initial segment and caput epididymidis of the rat. Qualitative analysis of tissues from 40-day-old and 120-day-old rats indicated that the number of MAP-1B-positive ciliated, narrow, and basal cells per tubule increased with sexual maturation. These immunoreactive cells did not stain for dopamine beta-hydroxylase or acetylcholinesterase, indicating that they were not adrenergic or cholinergic in nature. Immunohistochemical studies also revealed the presence of MAP-1B-positive staining in interstitial nerve fibers in caput and cauda epididymidis from both rat and human. Thus, the expression of MAP 1B is not confined to a specific cell type in rat and human efferent ductules and epididymis. The functional significance of this cytoskeleton protein in tissues from the male reproductive tract requires further investigation.

Lipid Remodeling of Murine Epididymosomes and Spermatozoa During Epididymal Maturation1

We have isolated vesicular structures from mouse epididymal fluid, referred to as epididymosomes. Epididymosomes have a roughly spherical aspect and a bilayer membrane, and they are heterogeneous in size and content. They originate from the epididymal epithelium, notably from the caput region, and are emitted in the epididymal lumen by way of apocrine secretion. We characterized their membranous lipid profiles in caput and cauda epididymidal fluid samples and found that epididymosomes were particularly rich in sphingomyelin (SM) and arachidonic acid. The proportion of SM increased markedly during epididymal transit and represented half the total phospholipids in cauda epididymidal epididymosomes. The cholesterol:phospholipid ratio increased from 0.26 in the caput to 0.48 in the cauda epididymidis. Measures of epididymosomal membrane anisotropy revealed that epididymosomes became more rigid during epididymal transit, in agreement with their lipid composition. In addition, we have characterized the membrane lipid pattern of murine epididymal spermatozoa during their maturation. Here, we have shown that mouse epididymal spermatozoa were distinguished by high percentages of SM and polyunsaturated membranous fatty acids (PUFAs), principally represented by arachidonic, docosapentanoic, and docosahexanoic acids. Both SM and PUFA increased throughout the epididymal tract. In particular, we observed a threefold rise in the ratio of docosapentanoic acid. Epididymal spermatozoa had a constant cholesterol:phospholipid ratio (average, 0.30) during epididymal transit. These data suggest that in contrast with epididymosomes, spermatozoal membranes seem to become more fluid during epididymal maturation.

Actions of 5α-reductase inhibitors on the epididymis

Testosterone is converted to the more biologically active androgen, dihydrotestosterone (DHT), by steroid 5α-reductase. Two isozymes of 5α-reductase, types 1 and 2, are abundantly expressed in the epididymis. DHT is the androgen found in the nuclei of epididymal cells and is essential for the maturation of spermatozoa. Thus, one approach to block androgen action in the epididymis is to inhibit DHT formation. Several compounds have been reported to inhibit either one or both forms of 5α-reductase in many tissues. The first commercially available inhibitor of 5α-reductase, finasteride, has a predominant effect on the type 2 isozyme, while more recently developed agents, such as dutasteride, PNU157706 and FK143, act as dual inhibitors. We found that the treatment of adult rats with such agents results in pronounced effects on the expression of genes essential to the formation of the optimal luminal microenvironment that is required for proper sperm maturation. Furthermore, drug treatment caused a significant decrease in the percentage of progressively motile and morphologically normal spermatozoa in the cauda epididymides. Mating females to treated males resulted in fewer successful pregnancies and a higher rate of pre-implantation loss. Thus, there may be a role for dual 5α-reductase inhibitors as potential components of a male contraceptive.

Effect of storage media and storage time on survival of spermatozoa recovered from canine and feline epididymides

The aim of this study was evaluate the survival ability of canine and feline spermatozoa maintained within epididymides stored at 4 °C for 24, 48 or 72 h in sterile isotonic saline solution (SAL) or a Tris–egg yolk (TEY) storage medium. Fifteen domestic dogs and 15 cats were neutered and their testes were placed in TEY or SAL and stored at 4 °C for either 24, 48 or 72 h. Sperm samples were obtained by cutting the cauda epididymides into a Tris extender and were evaluated for motility, velocity, viability, plasma membrane integrity, and acrosome morphology. In dogs, there were no significant differences between storage media for motility, plasma membrane integrity, viability and velocity. However, dog sperm stored in TEY had better acrosome morphology compared to sperm stored in SAL ( P < 0.05). Dog sperm recovered at 72 h had a reduction in all parameters studied compared to those recovered at 24 h ( P < 0.05). In cats, sperm recovered from epididymides stored in TEY had higher motility, plasma membrane integrity and velocity at all times compared to those stored in SAL ( P < 0.05). Cat sperm recovered at 72 h had reduced motility, acrosome morphology, viability and velocity compared to those recovered at 24 h ( P < 0.05). The addition of TEY to canine epididymal sperm, thus, had a better protective effect than SAL only on acrosome morphology. In cats, in contrast, TEY had a better protective effect than SAL on all epididymal sperm parameters studied. In both species, sperm recovered at 72 h had a significant reduction in all parameters studied compared to those recovered at 24 h.

Quantitative and spatial differences in the expression of tryptophan-metabolizing enzymes in mouse epididymis

Previous reports have suggested that indoleamine 2,3-dioxygenase (IDO) activity is particularly important in mouse epididymis tissue. We show here, using reverse transcription/polymerase chain reaction assays, Northern assays, Western blotting experiments, and immunohistochemistry that IDO is indeed highly expressed in mouse epididymis, and that IDO mRNA distribution and protein location are precisely regionalized within the organ and within sub-territories of the proximal part of the epididymal duct, the so-called caput epididymidis. Within the caput epididymidis, both the principal and the apical cells have been shown to express IDO. On the contrary, tryptophan dioxygenase (TDO), a sister enzyme of IDO, is weakly and uniformly expressed in mouse epididymis and, in contrast to IDO, is also expressed in testis. In the epididymis, TDO protein expression has been found in a totally different cell type in the smooth muscle layer surrounding the epididymal tubules. Finally, IDO is not secreted into the epididymal lumen, whereas the testis-expressed TDO is present on the head of spermatozoa retrieved from the cauda epididymidis. On the basis of the various functions that have been associated with IDO/TDO, we discuss the putative impacts of IDO/TDO expression on the physiology of mammalian epididymis and spermatozoa.

Localização histoquímica de algumas hidrolases no epidídimo do gerbilo

Neste estudo verificou-se, principalmente, que a expressão tissular da FoAl esteve presente nas regiões proximais do epidídimo de gerbilo. As reatividades da FoAc e da ATPase foram intensas (fortes), na maioria das regiões do epidídimo caracterizadas, marcadamente ao nível do citoplasma apical das células do epitélio tubular, exceto no corpo epididimal um delgado ístmo de conexão da cabeça à cauda epididimárias nesta espécie, com notória hiporeatividade enzimática a praticamente todas as enzimas aqui estudadas. A SDH mostrou também fraca atividade em todas as regiões, e estruturas do ducto epididimário do gerbilo, exceto no compartimento intraluminal onde se verificou atividade SDH-intensa nos espermatozóides estocados no lúmen. As reações enzimáticas caracterizadas foram correlacionadas com alguns papéis histofisiológicos tais como a mediação enzimática das fosfatases e ATPase em processos como endocitose, secreção, absorção e processo de transporte ativo das células do epitélio tubular. Uma possível marcação específica de mitocôndrias da peça média dos espermatozóides intraluminais pela SDH foi sugerida, com embasamento em processos metabólicos das mitocôndrias.

Proteomic Profiling of Accessory Structures from the Mouse Sperm Flagellum

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie Blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin, and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, validating this proteomic approach; 2) proteins that had not been shown to localize to any accessory structure but would be predicted to be present, e.g. glycolytic enzymes; 3) proteins known to be part of the flagellum but not localized to a specific site, e.g. adenylate kinase; 4) proteins not expected to be part of the accessory structures based on their previously reported locations, e.g. tektins; and 5) unknown proteins for which no information is available to make a determination as to location. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum.

Measuring mouse sperm parameters using a particle counter and sperm quality analyzer: A simple and inexpensive method

This study examined a method for analyzing the count, motility, and morphology of mouse epididymal sperm, optimizing the diluent, incubation time, sample concentration, and temperature, using a particle counter (CDA-500) to count and size sperm and a sperm quality analyzer (SQA-IIC) to measure sperm motility, quantified as the sperm motility index (SMI). The optimal conditions consisted of a 30-min incubation in D-MEM (Dulbecco's modified Eagle's medium; considering cost and availability) at 37 °C, with 5 × 10 6 cells mL −1 in the original solution. Furthermore, the influence of formalin fixation, and the correlation between the automated counter and a manual method were investigated. The sample fixation had no marked effect on the sperm count or morphology assessment. A linear correlation was observed between the manual and automated methods ( y = 0.920 x + 0.276; r 2 = 0.571; p < 0.001; range: (3–6) × 10 6). The suitability of the proposed method was confirmed using spermatozoa prepared from mice treated with the reproductive toxin diethylstilbestrol (DES). Using sperm from the cauda epididymidis on one side per mouse, we confirmed that measurement of these sperm parameters using the two devices was simple, rapid, inexpensive, and reproducible.

Nuclear status of immature and mature stallion spermatozoa

’The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatozoa. Evaluation of the thiol-disulfide status (monobromobimane labeling) demonstrated that immature cells obtained from ductulli efferentes contained mostly thiol groups, whereas these groups were oxidized in mature cells collected from the cauda epididymidis. Based on atomic absorption spectrophotometry, maturation of stallion spermatozoa was accompanied by a 60% reduction in the Zn 2+ content of sperm cells, concomitant with increased concentrations of this ion in epididymal fluid. Furthermore, the degree of disulfide bonding was inversely correlated with susceptibility of chromatin to acid denaturation (SCSA). Collectively, these data were consistent with the hypothesis that maturation of stallion spermatozoa involves oxidation of sulphydryl groups to form intra- and intermolecular disulfide links between adjacent protamines, with loss of zinc as an integral feature. These changes endow mechanical and chemical resistance to the nucleus, ensuring efficient transmission of the paternal genome at fertilization.

Genome-wide profiling of segmental-regulated transcriptomes in human epididymis using oligo microarray

Sperm maturation during passage through the epididymis depends on regionalized gene expression which maintains the progressively changing environment within the epididymal tubule. Towards defining the genes that drive the sequential maturation of spermatozoa, we profiled regionally regulated gene expression pattern in the epididymis of a fertile young male donor using Affymetrix human genome U133 plus 2.0 microarray representing approximately the whole human genome. Over 15 000 transcripts, almost one-third of the total on the array were identified in whole epididymis. Among them, 65% were detected in all three regions of the epididymis, 410 or 2.6% were present only in one region and the remaining 32.4% were distributed in two regions. Region-specific transcripts observed in caput (264), corpus (61) and cauda (81) epididymides were further classified as empirically determined reported genes or ESTs. This study revealed for the first time, the expression in human epididymis of a number of region-specific genes. The original data will be made publicly available on the Shanghai Science and Technology Database ( http://www.scbit.org/human_epididymis_transcriptomes).

Evaluation of epididymal semen quality using the Hamilton–Thorne analyser indicates variation between the two caudae epididymides of the same bull

Epididymal semen is being more often considered as a potential source of valuable genes for genome resource banks. To utilize this resource as efficiently as possible, storage and freezing fertility and preservation characteristics of epididymal semen have to be examined. Because semen quality should be assessed as objectively as possible, we introduced computer assisted sperm analysis (CASA) of epididymal bull semen. The aims of this study were: to determine the quality of fresh cauda epididymal bull sperm, conventionally and by CASA (Hamilton–Thorne Ceros 12.1); to compare epididymal sperm movement with the motion characteristics of ejaculated semen; and to investigate whether equality of semen characteristics exists between both caudae epididymides of the same bull. In experiment 1, it is shown that epididymal sperm has a lower motility (total: 48.7% versus 79.9%, p < 0.0001 and progressive: 34.4% versus 58.4%, p < 0.0001) and moves less straight (80.5% versus 84.5%, p < 0.0009) with a higher amplitude (6.1 μm versus 5.0 μm, p < 0.0001) than ejaculated semen. The epididymal straight line velocity (85.2 μm/s versus 98.3 μm/s, p < 0.0001) is lower, but the curvilinear velocity (173.5 μm/s versus 156.4 μm/s, p < 0.0001) is higher than those of ejaculated semen. The data in experiment 2 are analysed to determine equality, rather than to find a difference. They illustrate that mean differences, for most semen parameters, between the semen from paired caudae epididymides, deviated more than 20% from the average values of these parameters from all bulls; the exceptions (those parameters within 20% of the average for all bulls) were the percentage of live spermatozoa, the linearity of sperm movement, the weights of testis and epididymis, the weights of the cauda epididymis alone, the volumes, and the amplitudes of movement of the semen ( p < 0.05). The mean differences between the percentage of live spermatozoa and the amplitude of movement of the epididymal semen of both epididymides of one bull, were the only values smaller than 10% of the average value of this parameter ( p < 0.05). This implies that sperm from one cauda epididymis should not be used as a control for the other because, for most of the semen parameters (concentration, morphology, motility, and beat cross frequency), equality between caudae epididymides of the same bull could not be established.

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.