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PubMed1994

Isolation and characterization of two isoforms of a retinoic acid-binding protein from rabbit epididymal secretions.

Two isoforms of a retinoic acid-binding protein have been purified from rabbit epididymal secretions using a combination of gel filtration and ion-exchange chromatography. The two polypeptides (EP21a and b) present similar molecular mass (21 kDa), under native or denaturing conditions and have very similar amino-acid composition and tryptic peptide maps but differ in net charge. Both isoforms are glycosilated though to a different extent (9.2% and 6.3% of carbohydrate content) and are major components of the epididymal fluid. Binding of retinoic acid to EP21s appears to be specific, since they do not bind retinol, but is non-saturable. EP21s seem to present some similarity to two retinoic acid-binding proteins from rat epididymal secretions (site of biosynthesis, androgen dependence, ligand specificity and association to the spermatozoa) but differ from the rat proteins in amino-acid composition and glycosilation.

Amino Acids↗
PubMed1988

Effects of exogenous melatonin prior to the breeding season on testis weight and epididymal androgen receptors in rams.

Rams were randomly assigned to an experiment that evaluated effects of treatment (2.5 mg melatonin/d for 45 d vs control; MEL vs CON) in mid-May through June on testis weight and concentration of epididymal androgen receptor in July or September (Sept). Mean testis weight of MEL and CON rams was not different in July (332 vs 283 g), but in Sept was less (P less than .05) for MEL rams than CON rams (268 vs 382 g). Testis weight of MEL rams was less (P less than .05) in Sept than in July. Caput, corpus and cauda epididymal tissues were used to prepare extracts which were analyzed for concentration of dihydrotestosterone (DHT) receptor using a new standard curve method. A standard extract was characterized using four independent 8-point Scatchard analyses and found to contain 6.0 fmol DHT receptor/mg wet tissue (Ka = 3.5 X 10(8).M-1); this extract was used to establish standard curves for assays of unknown samples. Data on concentration of DHT receptors measured by Scatchard analysis and the standard curve method were highly correlated (r = 0.99; P less than .01; n = 8). Concentrations of DHT receptor were not affected by treatment, month of castration, or their interaction. However, for data pooled across treatment and month, concentration (fmol/mg protein) of DHT receptor was greater (P less than .05) in caput or corpus (125 or 122) than in cauda (92) epididymidis. The regional distribution of epididymal DHT receptors in this study confirmed our previous findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Membrane changes in Chinese hamster spermatozoa during epididymal maturation.

Chinese hamster spermatozoa during epididymal maturation were examined by thin sectioning, freeze-fracture, and surface replica. Membrane-limited vesicles and tubules (MVTs) attach to the plasma membrane over the acrosome of the spermatozoa in the distal caput through proximal cauda epididymidis. The origin of these MVTs is likely to be spermatozoa degenerating in the epididymal lumen. The attachment of MVTs to the plasma membrane seems to be mediated by a paste-like substance covering the plasma membrane. A parallel striation pattern of intramembranous particles (IMPs) is seen in the plasma membrane of almost the entire postacrosomal region of the epididymal spermatozoa. The patterned domain begins to appear in the proximal caput epididymidis. The number and density of IMPs in the plasma membrane of the postacrosomal region increases with the development of striated pattern of IMPs in this region. In the cauda epididymidis, the redundant nuclear envelope elongates to form a shirt-like membrane covering the mitochondrial sheath. The elongated portion of the nuclear envelope is devoid of nuclear pores and has few IMPs.

Acrosome↗
PubMedDate not supplied

Epididymal white adipose tissue after cold stress in rats. I. Nonmitochondrial changes.

Epididymal adipose tissue in the rat is generally considered to be "pure" white adipose tissue (WAT) with a characteristic structure and function. Previous studies in cats have, however, indicated that adipose tissue with the morphological appearance of WAT could be converted into a tissue with the morphological appearance of brown adipose tissue (BAT) by intermittent cold stress. The present electron microscopic and morphometric study describes the effect of intermittent cold stress on the epididymal WAT of young rats. The tissue volume decreased markedly as did the lipid content. The mitochondrial volume increased dramatically. The extracellular matrix was vastly reduced as was the thickness of the plasma membrane, and the number of gap junctions between adipocytes increased markedly. Indications of neoinnervation and neovascularization were observed. A great abundance of preadipocytes indicated proliferative activity of the endothelium. The low amount of lipid droplets and a relative abundance of smooth and rough endoplasmic reticulum. Golgi apparatus, and lysosomes in the epididymal WAT of cold-stressed rats gave the cells the morphological appearance of young adipocytes or preadipocytes whereas the hypertrophic and hyperplastic mitochondria, the relative paucity of ribosomes on lipid droplet membranes, and the increased innervation and vascularization gave the cells the morphological characteristics of brown adipose tissue.

Adipose Tissue↗
PubMed2005

Molecular, biochemical, and cellular characterization of epididymal ADAMs, ADAM7 and ADAM28.

The mammalian epididymis is critical for sperm to acquire motility and fertilizing capacity. This maturation process involves the interaction of epididymal secretory proteins with sperm. We analyzed mouse a disintegrin and metalloprotease (ADAMs) 7 and 28 expressed specifically or predominantly in the epididymis. We found that these ADAM genes are expressed in an epididymal region-specific manner and their gene expression is regulated by both androgen and testicular factors (ADAM7) or only testicular factors (ADAM28). We identified an ADAM28 transcript isoform that lacks the transmembrane domain. Protein analysis revealed that ADAM7, but not ADAM28, is transferred from the epididymis to the sperm surface and redistributed in the sperm head during acrosome reaction. These processes were shown to occur without processing of the protein. Taken together, our results indicate that the two epididymal ADAMs closely related in phylogeny are differential in various characteristics and ADAM7 has unique secretory feature and interactive relationship with sperm.

ADAM Proteins↗
PubMed2006

Novel epididymal protease inhibitors with Kazal or WAP family domain.

The epididymal maturation of spermatozoa is regulated by changes in the luminal ion concentration and the processing of the sperm surface membrane by several glycosidases and proteases. In the present study, we identified five novel protease inhibitors that are highly expressed in the mouse epididymis. Four of the proteins were found to belong to the Kazal protease inhibitor family and were named SPINK8, SPINK10, SPINK11, and SPINK12, whereas one of the proteins, WFDC10, contained the WAP four-disulfide core domain structure. The novel genes showed very specific segmental expression patterns. The expression of all the five genes was regulated by testis-derived factors and decreased after gonadectomy. With the exception of Spink11, mRNA levels could be restored by testosterone replacement. We hypothesize that the protease inhibitors discovered represent a group of epididymal genes that contribute to the regulation of sperm maturation by regulating the proteolytic processing of the sperm membrane during epididymal transit.

Animals↗
PubMed2004

Immortalization by large T-antigen of the adult epididymal duct epithelium.

The SV40 large T-antigen has been widely used to convert various cell types to a transformed phenotype, and also to induce progressive tumours in transgenic animals. The objectives of this review are to compare and discuss three different approaches to generate epididymal epithelial cell lines using the large T-antigen. In the first approach, retroviral transfection of primary cultures was used to immortalize canine epididymal cells in vitro; the other two approaches used transgenic mice expressing the large T-antigen. In one of these in vivo approaches, a construct consisting of the coding sequence of a temperature sensitive (ts) SV40 large T-antigen was inserted in a mouse genome. When the cells are exposed to the permissive temperature of 33 degrees C, functional expression of the large T-antigen occurs and cells start to proliferate. In the second in vivo approach a tissue-specific promoter, the 5kb GPX5 promoter, was used to direct expression of the large T-antigen to the epididymal duct epithelium.

Animals↗
PubMed2006

Identification of novel epididymal genes by expression profiling and in silico gene discovery.

We have used both the UniGene RIKEN epididymal EST library and the Affymetrix microarray profiling for identifying novel epididymal genes in mouse. The use of ESTs is a complementary approach to Affymetrix arrays for identifying novel epididymal genes, while only 32% and 28% of ESTs of unknown genes were present in the U74v.2Set and MG 430 2.0 version Affymetrix arrays, respectively. As expected, the probe set for a notably larger proportion of known genes was present in the Affymetrix arrays, and the coverage was greatly improved by the newer array version. Furthermore, many genes with more than five ESTs in the UniGene library showed variable levels of expression in both versions of the Affymetrix arrays. However, both the Affymetrix and EST data correlated well with that obtained by quantitative RT-PCR, and thus, we conclude that the findings of high EST number but only limited expression in the arrays could be considered as false negatives in the Affymetrix arrays.

Animals↗
PubMed2006

Association between exposure to persistent organohalogen pollutants and epididymal and accessory sex gland function: multicentre study in Inuit and European populations.

Exposure to persistent organochlorine pollutants (POPs) may have negative impact on male reproductive function. We, therefore, investigated the association between serum levels of POPs and epididymal and accessory sex gland function. Serum levels of CB-153, p,p'-DDE and seminal markers of epididymal [neutral-alpha glucosidase (NAG)], prostatic [prostate specific-antigen (PSA)] and zinc, and seminal vesicle function (fructose) were measured from 135 Swedish fishermen and fertile men from Greenland (n=163), Warsaw, Poland (n=167) and Kharkiv, Ukraine (n=158). Multiple linear regression analyses, adjusting for potential confounders, were employed using both continuous and categorized exposure variables. Both exposure and outcome variables were log transformed. Considering the consistency between models with either continuous or categorized CB-153 levels, negative associations with the activity of NAG were found among Greenlandic men (mean difference 7.0 mU/ejaculate, 95% CI 3.0, 34), and in the aggregated cohort (mean difference 4.0 mU/ejaculate, 95% CI -0.2, 8.0). A positive association was observed between CB-153 and PSA as well as zinc among Kharkiv men. In the Swedish cohort, a negative association was found between CB-153 and fructose. In conclusion, the negative effects of POP on sperm motility, observed in the same study population might partly be caused by post-testicular mechanisms, involving a decreased epididymal function.

Adult↗
PubMed2006

Effects of the herbicide Roundup on the epididymal region of drakes Anas platyrhynchos.

Exposure to the Roundup has been shown to affect StAR protein and aromatase expression and activity, pointing out that this herbicide may cause adverse effects in animal reproduction by affecting androgen and estrogen synthesis. We tested this hypothesis by investigating the in vivo effects of the Roundup on the testis and epididymal region of drake Anas platyrhynchos. The exposure to the herbicide resulted in alterations in the structure of the testis and epididymal region as well as in the serum levels of testosterone and estradiol, with changes in the expression of androgen receptors restricted to the testis. The harmful effects were more conspicuous in the proximal efferent ductules and epididymal ducts, suggesting higher sensitivity of these segments among the male genital organs. The effects were mostly dose dependent, indicating that this herbicide may cause disorder in the morphophysiology of the male genital system of animals.

Administration, Oral↗
PubMed2005

Decay of sperm obtained from epididymes of wild ruminants depending on postmortem time.

We have carried out a study on the effect of postmortem time (PT) in some characteristics of epididymal sperm salvaged from hunted Iberian red deer and roe deer. Testis were collected, identified, refrigerated down to 5 degrees C, and sent to our laboratory by the wardens of the hunting reserves. This way, samples were delivered at different times postmortem. Sperm were extracted from the cauda epididymis by means of cuts. Analyzed parameters were: osmolality, pH, motility-both subjectively and with CASA, HOS test reactivity, acrosomal status and viability (assessed with propidium iodide). Osmolality and pH rose with prolonged postmortem time, possibly due to tissue decomposition. Most sperm quality parameters negatively correlated with PT. Besides, when comparing PT classes (groups of 24 h for red deer and 30 h for roe deer), we could appreciate that motility was more affected by PT than other quality variables. Progressive motility was especially impaired. We also classified the samples in high, medium and low quality for each PT group (considering progressive motility, intact acrosomes and reactivity to the HOS test), and it was clear that after 2 days the number of high quality samples was testimonial, and after several days, we almost found only low quality samples. In conclusion, epididymal sperm from Iberian red deer and roe deer undergo a decrease of quality with PT, but it could stay acceptable within many hours postmortem. There are implications for wildlife conservation programs, as epididymal sperm is a good source of germplasm. If valuable animals die and it is not possible to process their sperm immediately, it may still be possible to obtain viable spermatozoa many hours later.

Acrosome↗
PubMed2005

Unilateral intrauterine insemination with prostatic fluid-sensitized frozen caudal epididymal sperm in beagle dogs.

The effects of the absence or presence of prostatic fluid (PF) during cauda epididymal sperm retrieval were assessed as regards semen quality after freezing semen in egg yolk Tris-fructose citrate solution (EYT-FC). Epididymal spermatozoa from the left testis of each of 10 dogs was retrieved into PF, whereas that from the right testis into EYT-FC only. At sperm recovery, the only difference between the two groups was that the incidence of spermatozoa with cytoplasmic droplets (immature sperm) was lower in the PF group (P < 0.01). In contrast, after freezing-thawing, (mean +/- S.E.) sperm motility (32.0 +/- 1.4 versus 12.5 +/- 2.0%, P < 0.01) and viability (58.2 +/- 3.5 versus 41.8 +/- 5.6%, P < 0.05) were higher in the PF group than in the EYT-FC group, respectively. Furthermore, 25.6 +/- 2.7% spermatozoa in the PF group were still motile after being maintained at 20 degrees C for 6 h. The incidence of immature spermatozoa post-thaw was lower compared to that after recovery in the EYT-FC group (P < 0.01), but was still higher than that in the PF group (P < 0.05). Frozen-thawed spermatozoa (2 x 10(8)) were used for unilateral intrauterine AI. The conception rate of PF-unsensitized sperm was 20% (2/10), but that of PF-sensitized sperm was 80% (8/10; P < 0.01). Therefore, sperm recovered in PF and frozen-thawed were of good quality. Sensitization of epididymal sperm with PF before freezing clearly improved the conception rate to AI of spermatozoa derived from the cauda epididymus.

Acrosome↗
PubMed2005

Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS.

When collection of ejaculated sperm samples is not possible, as is the case with wild species, the epididymides of sacrificed wild males become the only possible source of spermatozoa. Mature cauda epididymal spermatozoa display characteristics similar to those of ejaculated sperm cells. The present work proposes a sperm staining technique suitable for the morphometric evaluation of red deer epididymal sperm using a new computerized system. Epididymides from wild animals were extracted no later than 2h post mortem. After epididymal sectioning, sperm samples were collected, cooled to and equilibrated at 5 degrees C, and frozen in liquid nitrogen. Before staining, sperm samples were thawed for 20s at 37 degrees C, and used for the preparation of slides. Three different sperm stains were tested: Hemacolor, Diff-Quik, and Harris' Hematoxylin. Morphometric analyses of sperm samples were performed using the morphologic module of the ISAS. Two hundred spermatozoa per sample and stain were captured at random and analyzed. Sperm morphometric values were significantly affected by the staining technique used. Moreover, significant differences were observed between animals. In our study, Diff-Quik could be considered to be the best sperm staining method, as it provided the highest percentage of well automatically analyzed cells by the ISAS, and discriminates better between animals. This sperm staining technique also proved to be a useful method for characterizing and discriminating between sperm samples of different animals.

Animals↗
PubMed2006

A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality.

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.

Acrosome↗
PubMed2006

Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm.

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.

Acrosome Reaction↗
PubMed2006

Utilization of frozen-thawed epididymal ram semen to preserve genetic diversity in Scrapie susceptible sheep breeds.

The European Union has introduced transmissible spongiform encephalopathy (TSE) resistance breeding programmes for several sheep breeds to cope with the genetic susceptibility to Scrapie infections. Due to the different allele frequencies among breeds, strong selection for ARR alleles is associated with a loss of genetic diversity in small populations and in larger populations with unfavourable ARR allele frequencies. To ensure maintenance of genetic diversity, an adhoc cryopreservation programme was initiated employing epididymal sperm from 109 rams representing 16 different breeds within one breeding season. Epididymal semen was chosen for this adhoc programme because time consuming training of rams for ejaculated semen collection via an artificial vagina was not possible. Prior to freezing, average sperm motility was 79.7% and acrosome integrity was 93.7%. After freezing, these levels were decreased to 60.5 and 72.8%, respectively. An insemination trial using frozen-thawed epididymal semen resulted in a lambing rate of 87.5%. Results show that this semen preservation method is robust and efficient and associated with high fertility. It may also be useful for other animal species.

Acrosome↗
PubMed2004

The development of signal transduction pathways during epididymal maturation is calcium dependent.

Capacitation has been correlated with the activation of a cAMP-PKA-dependent signaling pathway leading to protein tyrosine phosphorylation. The ability to exhibit this response to cAMP matures during epididymal maturation in concert with the ability of the spermatozoa to capacitate. In this study, we have addressed the mechanisms by which spermatozoa gain the potential to activate this signaling pathway during epididymal maturation. In a modified Tyrode's medium containing 1.7 mM calcium, caput spermatozoa had significantly higher [Ca2+]i than caudal cells and could not tyrosine phosphorylate in response to cAMP. However, in calcium-depleted medium both caput and caudal cells could exhibit a cAMP-dependent phosphorylation response. The inhibitory effect of calcium on tyrosine phosphorylation was also observed in caudal spermatozoa using thapsigargin, a Ca(2+)-ATPase inhibitor that increased [Ca2+]i and precipitated a corresponding decrease in phosphotyrosine expression. We also demonstrate that despite the activation of tyrosine phosphorylation in caput spermatozoa, these cells remain nonfunctional in terms of motility, sperm-egg recognition and acrosomal exocytosis. These results demonstrate that the signaling pathway leading to tyrosine phosphorylation in mouse spermatozoa is negatively regulated by [Ca2+]i, and that maturation mechanisms that control [Ca2+]i within the spermatozoon are critically important during epididymal transit.

Animals↗
PubMed2004

Developmental expression and function of Bmp4 in spermatogenesis and in maintaining epididymal integrity.

Bone morphogenetic proteins (BMPs) play essential roles in many aspects of developmental biology. We have previously shown that Bmp7, Bmp8a, and Bmp8b of the 60A class of Bmp genes have additive effects in spermatogenesis and in maintaining the epididymal integrity of the caput and caudal regions. Here we report that Bmp4 of the Dpp class has a unique expression pattern in the developing testis and epididymis. Bmp4 heterozygous males on a largely C57BL/6 background show compromised fertility due to degeneration of germ cells, reduced sperm counts, and decreased sperm motility. More interestingly, some of these males show extensive degeneration of the epididymal epithelium in the corpus region, rather than in the caput and cauda regions as for Bmp7 and Bmp8 mutants. Thus, these genetic data reveal a region-specific requirement of different classes of BMPs for epididymal epithelium to survive and have significant implications on male reproductive health and perhaps birth control.

Animals↗