Cauda Epididymidis Research Articles

Changing P2X receptor localization on maturing sperm in the epididymides of mice, hamsters, rats, and humans: a preliminary study

To study using immunohistochemistry the localization of P2X receptor subtypes on the head of immature sperm in the human, mouse, hamster, and rat caput epididymidis. Basic research. University-based hospital. Three human epididymides were obtained from patients undergoing orchidectomy for metastatic prostate cancer. P2X(1), P2X(2), P2X(3), and P2X(4) receptor immunolocalization on sperm. In the present study, P2X(1,2, and 3) receptor localization was immunohistochemically demonstrated on the head of immature sperm in the human, mouse, hamster, and rat caput epididymidis. P2X(4) receptor immunostaining was also observed on the head of sperm in the caput epididymidis of mice, hamsters, and humans, but not rats. There was a subsequent loss of receptor staining on sperm in the cauda epididymidis, except in humans where staining of P2X(4) receptors persisted. Comparision with peanut agglutinin (PNA) binding studies suggested the P2X receptors were located on the acrosome membrane. P2X(5-7) receptors were examined but found to be absent. The change in localization of receptor subtypes is coincidental with the functionally essential morphologic and maturational changes seen in sperm as they travel through the epididymis, and is suggestive of a role for purinergic signaling in sperm maturation and possibly fertility.

Reversible antifertility effect of aqueous rhizome extract of Curcuma longa L. in male laboratory mice

Background The purpose of the present study was to evaluate the antifertility potential of Curcuma longa L . in the male laboratory mouse. Study Design Mice of the Parkes (P) strain were orally administered aqueous rhizome extract of C. longa (600 mg/kg body weight per day for 56 and 84 days), and effect of the treatment on various male reproductive endpoints and fertility was evaluated. Recovery studies were also performed. Results Histologically, testes in mice treated with the plant extract showed nonuniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same section; the affected tubules showed loosening of germinal epithelium, intraepithelial vacuolation and mixing of spermatids of different stages of spermatogenesis. Marked reductions in diameter of seminiferous tubules, height of germinal epithelium and number of germ cells in Stage VII tubules were also noted in testes of extract-treated mice. Epididymis and seminal vesicle also showed histological alterations. Furthermore, the treatment had adverse effects on motility, viability, morphology and number of spermatozoa in the cauda epididymidis, levels of sialic acid in the epididymis and fructose in the seminal vesicle, serum level of testosterone and on fertility and litter size. By 56 days of treatment withdrawal, however, the above parameters recovered to control levels. Conclusions The results show that in P mice C. longa treatment causes reversible suppression of spermatogenesis and fertility, thereby suggesting the potential of this plant in the regulation of male fertility.

Influence of the Time between Removal and Cooling of the Canine Epididymis on Post-Thaw Caudal Epididymal Sperm Quality

The effects of the time period between canine epididymis removal and cooling on post-thaw caudal epididymal sperm quality were investigated. Sperm recovered from the epididymis stored for 6 hr at 4 or 20 degrees C exhibited similar motility. However, when the epididymis was stored for 12 hr or longer at 20 degrees C, sperm motility was significantly lower than that at 4 degrees C (p<0.01). The post-thawed qualities of sperm recovered from the caudal epididymides that had been stored at 20 degrees C for 0 or 6 hr and then at 4 degrees C for 24 hr after removal were not significantly different. Therefore, leaving the canine epididymis at 20 degrees C for up to 6 hr after its removal may have little effect on the post-thaw quality of recovered caudal epididymal sperm.

Viability of Bali bull epididymal spermatozoa preserved at 3–5oC in Tris extender with different lactose concentrations

Cauda epididymal spermatozoa could be used as an alternative source of gamete in the application of various reproductive technologies, because the spermatozoa is motile and has ability for fertilizing the oocyte. The objective of this research was to examine the effectivity of lactose in maintaining viability of Bali bull epididymal spermatozoa preserved at 3–5 o C. Five testis with epididymides of Bali bulls were obtained from slaughterhouse. Epididymal spermatozoa was collected by the combination of slicing, flushing and tissues pressure of cauda epididymides with physiological saline (0.9% NaCl). Collected-spermatozoa was divided in equal volume into three tubes and diluted with Tris extender containing 20% egg yolk (control), Tris extender + 0.3 g lactose/100 ml (L0.3), and Tris extender + 0.6 g lactose/100 ml (L0.6), respectively. Diluted-spermatozoa was stored in refrigerator at 3–5 o C. Quality of diluted-spermatozoa including percentages of motile spermatozoa (MS), live spermatozoa (LS), and intact plasma membrane (IPM) were evaluated every day during storage at 3–5 o C for six days. Data were analyzed using completely randomized design with three treatments and five replications. Results of this study showed that mean spermatozoa concentration, percentage of MS, percentage of LS, percentage of abnormal spermatozoa, percentage of cytoplasmic droplet, and percentage of IPM of Bali bull fresh epididymal spermatozoa were 11,222.5 million cell/ml, 75, 86.75, 10.5, 14, and 86.75%, respectively. At day-7 storage, percentages of MS, LS, and IPM for L0.3 (39, 50.6, and 51.6%) and L0.6 (39, 51.4, and 51.8%) were significantly (P<0.05) higher than control (29, 41.8, and 42.4%). In conclusion, addition of lactose in Tris extender extended viability of Bali bull epididymal spermatozoa preserved at 3–5 o C. Key Words : Lactose, Preservation, Epididymal Spermatozoa, Bali Bull

9 BIRTH OF CALVES AFTER ARTIFICIAL INSEMINATION WITH CRYOPRESERVED BOVINE EPIDIDYMAL SPERMATOZOA HARVESTED FROM POSTMORTEM BULLS

Recently, interest in the preservation of epididymal sperm as a potential source of valuable genes for genome resource banks has increased. The ability to recover and cryopreserve postmortem epididymal sperm will allow the use of valuable gametes from a breeding male that dies unexpectedly. The objective of this study was to produce pregnancies and live births from AI by using frozen–thawed bovine caudal epididymal sperm. Paired testes were obtained from mature, mixed-breed beef bulls (n = 3) from a local abattoir and transported to the laboratory (within 5 h postmortem) in a Styrofoam box at a temperature that ranged between 28 and 29°C. The mean ± SEM weights of the pair of testes and caudae epididymides were 345 ± 9g and 10 ± 2g, respectively. Epididymal sperm from each bull were harvested by multiple incisions from the caudae epididymides, pooled, and then rinsed with 4 mL of egg yolk Tris-glucose citric acid monohydrate extender (EYT-GC). Harvested sperm samples were placed in a refrigerator at 4°C for 4 h before the addition of cryoprotectant. Samples were diluted slowly over a period of 30 min 1:1 in EYT-GC extender containing 14% glycerol to obtain a final concentration of 7% glycerol. Sperm concentration was adjusted to 35 million/straw and loaded into previously cooled 0.5-mL plastic straws. Straws were placed 2 cm above liquid nitrogen (LN2) vapors for 10 min and then plunged into LN2. Sperm were stored for 1 year before thawing. Luteal-phase crossbred beef cows were synchronized with a single 25-mg injection (i.m.) of prostaglandin, and those displaying standing estrus (n = 6) were each inseminated with 1 straw by a single technician from 1 bull 12 to 18 h after the onset of standing estrus. Straws were thawed for AI in a 37°C water bath for 40 s. Three of the 6 cows inseminated (50%) were diagnosed as pregnant by transrectal ultrasonography at 45 days post-AI. All 3 pregnant cows (100%) delivered healthy singleton calves (2 males and 1 female, with an average birth weight of 37 ± 2.5 kg), resulting in a mean gestation length of 286 ± 1.9 days (range: 282–288 days). We can conclude that epididymal sperm can be extracted by 5 h postmortem from bull testes, frozen, and subsequently used for the production of live offspring from AI. Further research is needed to improve this technology to optimize the utilization of valuable bovine male gametes.

168 POST-THAW VIABILITY OF EPIDIDYMAL SPERM FROM WHITE-TAIL DEER THAT WAS FROZEN USING GLYCEROL OR DMSO AS THE CRYOPROTECTANT

Cryoprotectants are compounds that protect and maintain the viability of cells when being subjected to cold environments. In addition to glycerol, DMSO has been evaluated for post-thaw epididymal sperm viability in various endangered species, but has not yet been assessed in White-tail deer. Testicles (in the scrotum) were collected from hunter harvested sexually mature White-tail bucks (n = 7) during the peak rutting season. Testicles within the scrotum, once removed from the postmortem buck, were placed in a plastic Ziploc bag and then into a Styrofoam ice chest (at ambient temperature) and transported to the Embryo Biotechnology Laboratory (EBL). Upon arrival at the EBL, sperm were flushed in a retrograde flow out of a small incision at the medial section of the cauda epididymides. Epididymal sperm were flushed from the epididymis using a non glycerol extender. Sperm from both epididymides of each buck were pooled and allowed a slow cool to 4°C. Upon reaching 4°C, the pooled epididymal sperm were equally divided and either 6% glycerol (A) or 6% DMSO (B) was added to each sperm sample. After the addition of the cryoprotectant sperm were loaded into 0.5-mL plastic straws and cryopreserved using liquid nitrogen vapor. Total motility (TM) was determined subjectively using an inverted Nikon Diaphot microscope. Membrane integrity (MI) was determined using SYBR 14 and propidium iodide staining under a microscope equipped with epiflurescence. Morphology was determined using an eosin-nigrosin staining. A paired t-test was used for statistical analyses. There were no differences between the glycerol and DMSO treatments for any of the three parameters measured (Table 1). However, post-thaw total motility and membrane integrity were significantly lower for both treatment groups (A and B) when compared with pre-freeze values. Furthermore, the DMSO treatment group had significantly lower normal morphology when compared with pre-freeze values, but was not different than the glycerol treatment group. Our results indicate that cryopreservation of epididymal sperm from White-tail deer with either 6% glycerol or 6% DMSO yield similar results for total motility, membrane integrity, and normal sperm morphology. Table 1.Pre-freeze and post-thaw total sperm motility, membrane integrity and normal sperm morphology for White-tail deer epididymal sperm Tony Vidrine of the Louisiana Department of Wildlife and Fisheries.

Generation of Live Rats Produced by In Vitro Fertilization Using Cryopreserved Spermatozoa1

In rats, the success of in vitro fertilization (IVF) was reported 40 years ago. Although it has been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported to date. Here, we report establishment of a successful IVF system using frozen/thawed rat spermatozoa. Our data showed that intracellular cAMP and free cholesterol levels in frozen/thawed rat sperm were maintained low, suppressing capacitation-associated tyrosine phosphorylation. The treatment of methyl-beta-cyclodextrin improved removal of free cholesterol from the membrane in frozen/thawed sperm but not induction of capacitation-associated tyrosine phosphorylation in the sperm. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), dramatically increased cAMP and tyrosine phosphorylation levels in frozen/thawed rat sperm. When the IBMX-treated frozen/thawed sperm were used for IVF, the proportions of pronuclear formation and blastocyst formation were significantly higher than those of frozen/thawed sperm treated without IBMX (P < 0.05). The embryos were developed to term at a high success rate equivalent to the rate obtained with IVF using fresh sperm. Thus, we established for the first time a successful IVF system in rats using cryopreserved spermatozoa.

Reversible antifertility effect of aqueous leaf extract ofAllamanda catharticaL. in male laboratory mice

The efficacy of oral administration of aqueous leaf extract of Allamanda cathartica (150 mg kg(-1) body weight day(-1) for 14, 28 and 42 days) in inducing infertility and changes in various male reproductive endpoints was evaluated in Parkes strain mice. The effect of the treatment on organ weight, histopathology, sperm parameters, testosterone level, haematology, serum biochemistry and on fertility indices was assessed. Histologically, testes in extract-treated mice showed nonuniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same sections. The treatment also had adverse effects on motility, viability, morphology and on number of spermatozoa in the cauda epididymidis. Serum levels of testosterone, alanine aminotransferase, aspartate aminotransferase and creatinine, haematological parameters and liver and kidney histoarchitecture were, however, not affected by the treatment. Fertility of the extract-treated males was also suppressed, although the libido remained unaffected. By 56 days of treatment withdrawal, however, the above parameters recovered to control levels. Our results thus suggest that A. cathartica treatment causes reversible suppression of fertility in male mice, without causing detectable toxic effects.

Seminal vesicle fluid reduces binding of porcine epididymal spermatozoa to oviduct explants

The purpose was to determine if exposure of porcine epididymal sperm to seminal vesicle fluid (SVF) would influence the number of sperm bound to oviduct explants. Study design and methods: Testes and seminal vesicles were obtained from five 12 month-old Large White boars at slaughter. Sperm were collected from the rete testis, caput, corpus, and cauda epididymidis; an aliquot (50 μL) was suspended in 1 ml of SVF and incubated at 39 °C for either 1 or 30 min. For controls, sperm samples were incubated in Androhep medium (Petrunkina AM, et al. Reproduction 2001;121:889–96). After incubation, the sample was centrifuged at 600 × g for 10 min and sperm suspended in modified Androhep medium at 5 × 106 mL−1. Before and after incubation in SVF, the capacitation status was determined by the chlortetracycline assay and viability by eosin–nigrosin staining. Oviduct explants were prepared from the isthmus and ampulla from non-cycling 20-week-old gilts using procedures modified from previous reports (Petrunkina AM, et al. Reproduction 2001;121:889–96; Suarez SS, et al. Biol Reprod 1991;56:447–53). Oviduct explants were set up as six replicates for each sperm sample and after incubation with sperm for 15 min at 39 °C in 5% CO2 in air, the explants were fixed, stained and number of sperm bound in area of 1.25 mm2 counted. Data were analysed with ANOVA. Results: There was no significant difference in the number of sperm that bound to explants after incubation for 1 min in either Androhep medium or SVF, nor was there any difference in sperm viability (average 90% viability), but SVF significantly reduced the percentage of uncapacitated sperm (70% vs. 80%). After incubation for 30 min in SVF, fewer sperm (P ≤ 0.05) from the cauda and corpus, but not the caput or rete testis, bound to both isthmus and ampulla explants. The number that bound to the isthmus and ampulla explants was 6.23 ± 0.87 and 4.0 ± 0.47, respectively, whereas the controls were 13.13 ± 1.74 and 6.65 ± 0.68. There was no significant difference between the treatments for sperm viability, but the percentage of uncapacitated sperm declined significantly after incubation in SVF compared to the control (35% vs. 60% for caudal sperm). Conclusion: The induction of capacitation by exposure to SVF was responsible for the reduced binding of sperm from the corpus and cauda to oviduct explants. This effect was not evident in sperm from the caput or rete testis. It was suggested that seminal plasma increases the binding of caudal sperm to the isthmus (Petrunkina AM, et al. Reproduction 2001;121:889–96); perhaps in vivo, components of seminal plasma other than SVF counter the capacitating effect, and add molecules to sperm to facilitate binding to the oviductal epithelium.

Segmental Expression of the Bradykinin Type 2 Receptor in Rat Efferent Ducts and Epididymis and Its Role in the Regulation of Aquaporin 91

Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.

Evaluation of antifertility potential of Brahmi in male mouse

Background The purpose of the present study was to evaluate the effect of Bacopa monnieri (Brahmi) on fertility of male laboratory mouse. Study Design Mice of the Parkes (P) strain were orally administered Brahmi (250 mg/kg body weight/day, for 28 and 56 days), and effect of the treatment on reproductive organs and fertility was investigated. Recovery and toxicological studies were also carried out. Results The treatment caused reduction in motility, viability, morphology, and number of spermatozoa in cauda epididymidis. Histologically, testes in mice treated with the plant extract showed alterations in the seminiferous tubules, and the alterations included intraepithelial vacuolation, loosening of germinal epithelium, exfoliation of germ cells and occurrence of giant cells. In severe cases, the tubules were lined by only Sertoli cells or Sertoli cells, spermatogonia and spermatocytes. Significant reductions were also noted in height of the germinal epithelium and diameter of the seminiferous tubules in Brahmi-treated mice compared to controls. Epididymis in treated males showed slight alterations in histological appearance. The treatment had no effect on levels of testosterone, alanine aminotransferase, aspartate aminotransferase and creatinine in blood serum, hematological parameters and on liver and kidney histoarchitecture. In Brahmi-treated males, libido remained unaffected, but fertility was notably suppressed. The alterations caused in the above reproductive endpoints by the plant extract were reversible, and by 56 days of treatment withdrawal, the parameters recovered to control levels. Conclusions The results in P mice thus suggest that Brahmi treatment causes reversible suppression of spermatogenesis and fertility, without producing apparent toxic effects.

β-Defensin 22 is a major component of the mouse sperm glycocalyx

Surface components of sperm isolated from the cauda epididymides were stabilized by whole sperm fixation for immunization of rabbits. The resulting immunoglobulins (Igs) recognized a single protein of 130 kDa (non-reduced) or 54-57 kDa (reduced) on western blots of cauda sperm. Igs recognized the same 54-57 kDa protein band on whole tissue blots of the corpus and cauda epididymidis and vas deferens. No immunoreactive bands were detected on blots of the prostate, seminal vesicles, testes, caput epididymis, or any of various non-reproductive tissues. Removal of sperm from the vas deferens prior to blotting eliminated the detection of the sperm antigen. Antibodies raised to synthetic peptides, identical in amino acid sequence to two unique spans of DEFB22, recognized the same 130/54-57 kDa antigen on western blots of both caudal sperm and the purified antigen isolated with the anti-sperm Ig. From indirect immunofluorescence, both the anti-sperm and anti-peptide Igs appeared to localize to the entire sperm surface, a pattern confirmed at the ultrastructural level. Real-time PCR identified the corpus epididymides as the major site of expression of DEFB22, with negligible expression in the testes, caput epididymides, and vas deferens. Immunostaining of epididymal sections showed DEFB22 being released into the lumen at the distal caput/proximal corpus, with sperm becoming intensely coated with DEFB22 as they reached the distal corpus. Most uterine sperm recovered from mice 4 h following copulation exhibited DEFB22 coating the entire sperm surface. By contrast, some sperm recovered from the oviduct and cumulus extracellular matrix showed loss of DEFB22 from the sperm head.

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, Bandicota indica

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.

Association of the Protein D and Protein E Forms of Rat CRISP1 with Epididymal Sperm1

Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.

Activation of Toll-Like Receptor 4 (TLR4) by In Vivo and In Vitro Exposure of Rat Epididymis to Lipopolysaccharide from Escherichia Coli1

This study provides the first evidence that rat epididymis is fully capable of initiating an inflammatory response to lipopolysaccharide (LPS) from Escherichia coli through activation of Toll-like receptor 4 (TLR4). TLR4 functionality was demonstrated by in vivo LPS challenge, which induced a time- and dose-dependent activation of the transcription factor nuclear factor kappa B (NFKB) in caput and cauda epididymides. NFKB activation by LPS in caput epididymidis was abrogated when rats were pretreated with the NFKB inhibitor PDTC, confirming the specificity of this response. Within 2 h of LPS treatment (0.01 and 1 mg/kg, i.v.), NFKB activation in caput and cauda was accompanied by upregulation of Il1b, Nfkbia, and Cd14, but not Tlr4, mRNA. These effects, however, were not sustained after 24 h of LPS treatment. Lipopolysaccharide systemic effects were not restricted to epididymides, since Il1b, Nfkbia, and Cd14 mRNAs were also upregulated in other male reproductive tissues from LPS-treated rats (1 mg/kg, i.v., 2 h). Constitutive TLR4 was immunolocalized in some, but not all, epididymal epithelial cells and in interstitial cells, some of them identified as resident ED2-positive macrophages. No change in TLR4 immunostaining pattern was observed when epididymides from control and LPS-treated rats were compared (1 mg/kg, i.v., 2 h and 24 h). Significant NFKB activation was also achieved within 1 min of in vitro incubation of caput epididymidis with LPS (0.01-5 mug/ml), confirming that components for TLR4 signaling cascade activation are fully active in this tissue. This study contributes to a better understanding of the innate immune response in the epididymis and other tissues from the male reproductive tract.

Fertilization in vitro with spermatozoa from different mice increased variation in the developmental potential of embryos compared to artificial parthenogenetic activation

Although successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr(2+) parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr(2+) parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that (1) epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; (2) like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; (3) parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male.

Apoptosis of sperm induced by murine cytomegalovirus infection and its mechanism regulated by sperm mitochondria: experiment with mice

To study the effects of murine cytomegalovirus (MCMV) infection on mature sperm apoptosis in mice, and to explore the mechanism of MCMV-induced apoptosis regulated by mitochondria. MCMV was inoculated into the testes of 15 BALB/c mice to establish acute MCMV infection models and 15 mice were used as controls. 1, 2, 4, 6, and 9 days after the infection 3 mice from each group were killed. Flow cytometry was used to observe the apoptosis of sperms. Laser scanning confocal microscopy was performed to examine the mitochondrial membrane potential (delta psi psi m) of sperm. The mitochondria ultrastructure of sperm was observed under electron microscope. The sperm apoptotic ratebeganto increase from day 2 after inoculation (D2 PI), peaked to the level of (39.3 +/- 1.0)% compared with that of the control group on D4 PI, and then fell-off (F = 362.822, P < 0.05). The delta psi m of sperm began to increase on D1 PI at the level of (74.0 +/- 1.4), began to decrease on D2 PI [( 63.0 +/- 2.2)], dropped to the minimum on D4 PI [(40.2 +/- 2.3)], then ascended slowly again (F = 32.257, P < 0.05). The mitochondria ultrastructure of sperm showed damage after MCMV infection that was especially severe from D2 PI to D6 PI. MCMV acute infection in reproductive organ induces apoptosis of mature sperm in the cauda epididymidis. Sperm mitochondria participate in and regulate initiatively the apoptosis of sperm.

Epididymal sperm storage in Rickett's big-footed bat (Myotis ricketti)

ABSTRACT Spermatozoa were present in the cauda epididymidis of male Rickett's big-footed bat, Myotis ricketti, from late September to early April but absent from the testes from mid November until late August. Males thus store spermatozoa in the cauda epididymidis for 4.5–6.5 months of the year. Assessments of sperm motility, movement pattern and computer-assisted sperm analysis (CASA) were carried out on spermatozoa which had been stored for over 3.5 months in the cauda epididymidis. The motility of sperm stored for the same period of time and taken from a male which died over 9 h before assessment was 86%. Four kinds of culture media were screened for their suitability for future studies of sperm motility, and a low calcium minimal capacitation medium and a hamster fertilization medium were selected. Serum testosterone (T) concentration increased dramatically in September and began to fall in October, before returning to baseline for the remainder of winter. These results indicate that high levels of T ...

Alteration of fertility endpoints in adult male F-344 rats by subchronic exposure to inhaled benzo(a)pyrene

The objective of this study was to evaluate the reproductive risk associated with exposure of adult male Fisher-344 rats to inhaled benzo(a)pyrene (BaP). Rats were assigned randomly to a treatment or control group. Treatment consisted of sub-chronic exposure of rats via inhalation to 75 μg BaP/m 3, 4 h daily for 60 days, while control animals were unexposed (UNC). Blood samples were collected immediately after the cessation of exposures (time 0) and subsequently at 24, 48, and 72 h, to assess the effect of bioavailable BaP on plasma testosterone and luteinizing hormone (LH) concentrations. Rats were sacrificed after the last blood collection. Testes were harvested, weighed and prepared for histology and morphometric analysis, and cauda epididymides were isolated for the determination of progressive motility and density of stored spermatozoa. BaP exposure reduced testis weight compared with UNC (mean±SE; 2.01±0.11 versus 3.04±0.16 g; P<0.025), and caused significant reductions in the components of the steroidogenic and spermatogenic compartments of the testis. Progressive motility and mean density of stored spermatozoa were reduced ( P<0.05). Plasma testosterone concentrations were decreased by two-thirds in BaP-exposed rats throughout the time periods studied compared with those of their UNC counterparts ( P<0.05), concomitant with increased concentrations of LH in BaP-exposed rats ( P<0.05). These data suggest that sub-chronic exposure to inhaled BaP contribute to reduced testicular and epididymal function in exposed rats.

Protein composition of human epididymosomes collected during surgical vasectomy reversal: a proteomic and genomic approach

The epididymal epithelium secretes membranous vesicles, called epididymosomes, with which a complex mixture of proteins is associated. These vesicles transfer to spermatozoa selected proteins involved in sperm maturation. Epididymosomes in the human excurrent duct have been described, but their protein composition and possible functions are unknown. Epididymosomes were collected during vasovasostomy procedures, purified and submitted to liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry. From all the mass spectra generated, 1022 peptides allowed the identification of 146 different proteins. Identification of some of these proteins was confirmed by western blots. Furthermore, western blot showed that the protein composition of epididymosomes differed from that characterizing prostasomes; membranous vesicles secreted by the prostate. Organization of the epididymosomes proteome according to common functional features suggests that epididymosomes have multiple functions. In order to understand the origin of epididymosomes collected distally, microarray databases of caput, corpus and cauda epididymidis were analysed to determine where along the excurrent duct the encoded proteins associated to epididymosomes are synthesised. Results suggest that some proteins synthesized in the caput and corpus epididymidis are associated with epididymosomes collected distally. Epididymosomes thus transit along the excurrent duct, and vesicles collected distally represent a mixed population.