Sperm malformations throughout the boar epididymal duct

Taking into account the importance of the sperm epididymal maturation process, and the consequential changes in the spermatozoa, we studied eight different sperm malformations in the caput, corpus, and cauda regions of the epididymis of healthy and sexually mature Landrace boars in order to determine the origin of these sperm abnormalities. Epididymal sperm characteristics were examined using light microscopy, scanning and transmission electron microscopy. The incidence of each type of malformation investigated was established after counts of 10 000 spermatozoa in each of the three epididymal regions. The different sperm malformations studied were: (1) spermatozoa with tail folded at the connecting piece; (2) spermatozoa with tail folded at the midpiece; (3) spermatozoa with tail folded at the Jensen's ring; (4) spermatozoa with tail folded at the principal piece; (5) coiled tail spermatozoa; (6) spermatozoa with two fused tails; (7) macrocephaly; and (8) microcephaly. The count performed in each epididymal region indicated that, whereas significant differences (P ≤ 0.01) existed between the frequencies of some types of sperm malformations and the epididymal region from where the sperm originate, other sperm malformations were more uniformly distributed along the epididymal duct. Among the eight different sperm malformations studied, three were found to be of secondary origin: spermatozoa with tail folded at the Jensen's ring (originated in the epididymal cauda); spermatozoa with coiled tail; and spermatozoa with two fused tails (originated in the epididymal corpus). Knowing the origin of spermatozoa abnormalities will assist research into the study of infertility and reproductive pathology.     

Effect of culture conditions on the obtention of boar epididymal epithelial cell monolayers

The goal of this study was to investigate the effect of the collagenase digestion time, the initial density of fragments and the culture temperature on the obtention of a boar epididymal epithelial cell culture, which is a useful methodology for the study of epididymal functions. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was only obtained when an adequate enzymatic digestion of the connective tissue surrounding the epididymal tubule was performed. For the correct digestion of caput and corpus fragments two collagenase digestions of 2 and 1h, respectively, were enough. Cauda fragments, however, needed two collagenase digestions of 3h each. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained regardless of the initial density tested (15, 30, 60 and 90fragments/well). However, cultures originated from 15 and 30fragments/well showed higher cell concentration during the first 2 weeks of culture than cultures originated from 60 and 90fragments/well. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained at both 32 and 37 degrees Celsius, but at 32 degrees Celsius cells grew very slowly and confluence was not reached until a week later than it was with cells growing at 37 degrees Celsius. In conclusion, we have observed that the time of digestion with collagenase is an important factor for the successful establishment of boar epididymal cell monolayers, and that the initial density of fragments and the culture temperature should be taken into account.     

Evaluation of boar sperm maturation after co-incubation with caput, corpus and cauda epididymal cultures [corrected

Boar sperm from the proximal caput epididymis were co-incubated with 1, 4, 7, 10 and 14-day old caput, corpus and cauda epididymal cultures for 24, 48 and 72 h. Boar kidney epithelial cells (LLC-PK1) and ECM alone were used as negative controls. Sperm motility, morphology and membrane integrity were studied to evaluate boar sperm maturation in vitro. Our results showed that epithelial cell monolayers (10, 14-day old) create a suitable microenvironment for the survival of proximal caput sperm and the maintenance of sperm motility over a 72 h period. Moreover, corpus epididymal tubule fragments in culture (1, 4-day old) are capable of promoting the migration of the cytoplasmic droplet along the sperm tail after 24h of co-incubation.     

Androgenic control of antagglutinin secretion in the boar epididymal epithelium

Summary Antagglutinin, a specific protein synthesized by the boar epididymis, was secreted by the principal cells of the initial segment, the caput and the corpus, but was not detectable in the caudal cells. Castration completely abolished the synthesis and secretion of antagglutinin in all epididymal cells. Androgen replacement suggests that the epithelial cells from different segments have differential regulatory mechanisms. The proximal zone appeared refractory to exogenous testosterone; the median zone was a typical androgen-dependent region; and the caudal cells, where an unusual secretion of antagglutinin was detected, revealed still a different reaction pattern. It is postulated that these latter cells depend not solely on androgen but also or exclusively on other factors. Our results, which demonstrate a primary role of the Golgi complex in the secretory process in the epididymal cells, also suggest that the apical smooth endoplasmic reticulum may be implicated in the intracellular transport of glycoproteins to the cell surface.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Identification of anti-agglutinin for spermatozoa in epididymal boar plasma

The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs–Ringer bicarbonate at 37°C (5% CO₂ in air). In the samples washed three or five times and then incubated for 3–5 h, higher rates (72–79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2–5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. © 1994 Wiley-Liss, Inc.     

Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines

Several antibiotics were examined for their potential to eliminate mycoplasmas from contaminated cell cultures. Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma hyorhinis and Mycoplasma orale were effectively eliminated from experimentally contaminated mouse fibroblasts and mink epithelial cells by the use of the antibiotics minocycline and tiamutin. An elimination procedure was established, which involved the consecutive treatment of the cultures over a period of 3 weeks, followed by cell cloning. This procedure was effective when applied to cell lines which had been contaminated with unidentified and partially non-cultivable strains of mycoplasmas.     

The establishment of cell suspension cultures ofGladiolus that regenerate plants

Summary Inflorencence stalks from greenhouse-grownGladiolus plants of the cultivars ‘Blue Isle’ and ‘Hunting Song’ cultured on a Murashige and Skoog basal salts medium supplemented with 53.6 μM 1-napthaleneacetic acid formed a compact, not friable type of callus that regenerated plantlets. Cormel slices and intact plantlets of three cultivars (‘Peter Pears’, ‘Rosa Supreme’, ‘Jenny Lee’) propagated through tissue culture formed a friable type of callus when cultured on Murashige and Skoog basal salts medium supplemented with 2,4-dichlorophenoxyacetic acid. This friable callus readily formed a cell suspension when the callus was placed in a liquid medium. Plants were regenerated from two-month-old suspension cell cultures of the commercial cultivar ‘Peter Pears’ after the suspension cells had been cultured on solid medium.     

CD52 mRNA is modulated by androgens and temperature in epididymal cell cultures

Cultured rat epididymal tissue explants formed >90% pure, adherent growing epithelial cell monolayers. Despite their flattened and apparently androgen receptor-negative phenotype, these cells for a short period kept characteristics of the epididymal duct epithelium, i.e., expression of the tissue-specific marker CD52 and responsiveness of its mRNA toward temperature elevation and androgen withdrawal. When cells were grown on permeable supports at 33 degrees C, androgen supplementation or withdrawal specifically modulated the levels as well as the length of the CD52 mRNA. Elevation of the culture temperature to a quasi abdominal milieu of 37 degrees C selectively reduced the CD52 mRNA levels under all culture conditions. This reduction was not affected by the presence of androgens and was not accompanied by changes in length, suggesting that the modulation of CD52 mRNA in epididymal cells by androgens and by temperature is synergic, but may involve different molecular mechanisms. CD52 mRNA levels, however, were not stable in the primary cultures but decreased rapidly to undetectable levels after 4-5 days at all culture conditions. GAPDH mRNA levels, on the other hand, were stable throughout the culture period.     

Changes in the lipid content of boar sperm plasma membranes during epididymal maturation

Plasma membranes of boar sperm from caput, corpus and cauda of the epididymis were purified by differential- and sucrose-density equilibrium centrifugation and were found to yield a single band at a density of 1.13 g/cm3. This fraction was enriched in acid and alkaline phosphatase, 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas it contained minimal amounts of hyaluronidase and N-acetylglucosaminidase and no succinic acid dehydrogenase activities. The plasma membrane of caput, corpus and cauda sperm had the same phospholipid/protein and cholesterol/phospholipid ratios but yielded different amounts of protein and individual lipid classes. Several changes in the plasma membrane were observed during transit of sperm through the epididymis. Within the phospholipid class a decrease in the percentage of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol was detected accompanied by an increase in amount of phosphatidylcholine, sphingomyelin and polyphosphoinositides. In the other lipid classes there was a decrease in the amount of free fatty acid and the major glycolipid. The amount of cholesterol decreased, while the amount of desmosterol and cholesterol sulfate increased. There was an increase in the amount of diacylglycerol. In addition, the changes in the fatty acid composition of the total membrane lipid and each phospholipid were determined. The above changes in the lipid composition of the plasma membrane during epididymal maturation may help to explain the decreased resistance to cold shock and changes in membrane fluidity of sperm during transit in the epididymis.     

Immunocytochemical localization of nuclear protamine in boar spermatozoa during epididymal transit

Protamine was specifically demonstrated in boar spermatozoa collected from the rete testis, caput, corpus and cauda epididymidis and the ejaculate by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera and an indirect post-embedding immunogold technique. Spermatozoa from all collection sites stained after incubation although with different degrees of labelling. Controls were negative. Labelling increased from the rete testis towards the epididymal corpus, where it was most intense, decreasing sharply thereafter. The weakest binding of the assayed antibodies was obtained in the ejaculated spermatozoa but it could be reversed by in-vitro induction of chromatin decondensation with sodium dodecyl sulphate and the metal-chelating EDTA. The finding of a significant decrease in the immunolabelling detected from the corpus epididymidis onwards indicates a critical point for the interaction between DNA and the protamines in boar spermatozoa during the epididymal maturation.     

Proteomic analysis of differentially expressed proteins in fungal elicitor-treated Arabidopsis cell cultures

Slow progress has been made in discovering plant genes governing the interaction of plant pathogens and their hosts using classical genetic approaches. Extensive studies employing DNA microarray techniques to identify global changes in gene expression during pathogen-host interaction have greatly enhanced discovery of genetic components regulating the plant defence response to pathogen attack. In this study, a complementary approach was used to identify changes in protein abundance during interaction of Arabidopsis cell cultures with a pathogen-derived elicitor. The soluble protein fractions were analysed by two-dimensional difference gel electrophoresis and proteins differentially expressed in response to treatment with fungal elicitor were identified via matrix-assisted laser desorption ionization-time of flight mass spectrometry. Elicitor responsive proteins included molecular chaperones, oxidative stress defence proteins, mitochondrial proteins, and enzymes of a diverse number of metabolic pathways. The findings, in combination with currently available microarray data, will form the basis of a filter to identify pivotal genes whose role in pathogen defence systems will require confirmation using gene knockout mutants.     

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here “the chaperome,” which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10 % of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.     

Density and distribution of anionic sites on boar ejaculated and epididymal spermatozoa

Current knowledge implies that spermatozoa successively acquire negative surface charges as they migrate through the epididymis. Until recently, however, techniques used were not amenable to statistical analysis. In the present study, a novel approach allowing numerical assessment of negative charge labelling was used in order to determine the density and distribution of anionic sites on ejaculated and maturing spermatozoa collected from six regions of the boar epididymis. Labelling was assessed quantitatively for the three morphologically distinct membrane domains on the sperm head. Statistical analysis revealed that labelling density was highest on efferent duct spermatozoa, declined up to the proximal corpus and then increased again. Densities of anionic sites on distal corpus, proximal cauda and ejaculated sperm cells were similar but significantly below the values obtained for efferent duct spermatozoa. All three sperm membrane domains underwent parallel changes. However, the overall density of negative charges on the postacrosomal segment was significantly higher as compared to the acrosomal plasma membrane. These alterations reflect sperm surface modifications through removal and addition of anionic groups. Since charge interactions are considered to play a pivotal role in sperm-egg interactions, these processes should be viewed as an integral part of sperm maturation.     

A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Successful establishment of primary small airway cell cultures in human lung transplantation

Background

Although both animal and human studies suggested the association between placenta growth factor (PlGF) and chronic obstructive pulmonary disease (COPD), especially lung emphysema, the role of PlGF in the pathogenesis of emphysema remains to be clarified. This study hypothesizes that blocking PlGF prevents the development of emphysema.

Methods

Pulmonary emphysema was induced in PlGF knock-out (KO) and wild type (WT) mice by intra-tracheal instillation of porcine pancreatic elastase (PPE). A group of KO mice was then treated with exogenous PlGF and WT mice with neutralizing anti-VEGFR1 antibody. Tumor necrosis factor alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), and VEGF were quantified. Apoptosis measurement and immuno-histochemical staining for VEGF R1 and R2 were performed in emphysematous lung tissues.

Results

After 4 weeks of PPE instillation, lung airspaces enlarged more significantly in WT than in KO mice. The levels of TNF-α and MMP-9, but not VEGF, increased in the lungs of WT compared with those of KO mice. There was also increased in apoptosis of alveolar septal cells in WT mice. Instillation of exogenous PlGF in KO mice restored the emphysematous changes. The expression of both VEGF R1 and R2 decreased in the emphysematous lungs.

Conclusion

In this animal model, pulmonary emphysema is prevented by depleting PlGF. When exogenous PlGF is administered to PlGF KO mice, emphysema re-develops, implying that PlGF contributes to the pathogenesis of emphysema.     

Biochemical studies on proacrosin and acrosin from epididymal boar spermatozoa: in vitro translation of boar testicular proacrosin mRNA

An inactive form of acrosin was extracted from epididymal boar spermatozoa utilizing acid pH conditions. When subjected to activation in alkaline environment, this form turns into an enzymatically active species, which exhibits close-related electrophoretic characteristics. Both the precursor and the activated species, when incubated in the presence of thermolysin, give rise to two fastly moving acrosin molecular forms. In order to establish the nature of the true acrosin zymogen, we isolated poly(A+)-RNA from boar testicles, performed its translation in vitro in the presence of [35S]-methionine and reticulocyte lysate, immunoprecipitated the translation products with anti-boar acrosin antibody, and analyzed them by SDS-polyacrylamide gel electrophoresis and autoradiography. A single translation product of molecular weight 55,000 was detected. It is concluded that the polypeptide chain of the boar zymogen is of 55,000; increases in molecular weight are due to post-translational modifications, like glycosylation.