Carnitine/organic cation transporter OCTN2-mediated transport of carnitine in primary-cultured epididymal epithelial cells

Carnitine is essential for the acquisition of motility and maturation of spermatozoa in the epididymis, and is accumulated in epididymal fluid. In this study, carnitine transport into primary-cultured rat epididymal epithelial cells was characterized to clarify the nature of the transporter molecules involved. Uptake of carnitine by primary-cultured epididymal epithelial cells was time, Na(+) and concentration dependent. Kinetic analysis of carnitine uptake by the cells revealed the involvement of high- and low-affinity transport systems with Km values of 21 microM and 2.2 mM respectively. The uptake of carnitine by the cells was significantly reduced by inhibitors of carnitine/organic cation transporter (OCTN2), such as carnitine analogues and cationic compounds. In RT-PCR analysis, OCTN2 expression was detected. These results demonstrated that the high-affinity carnitine transporter OCTN2, which is localized at the basolateral membrane of epididymal epithelial cells, mediates carnitine supply into those cells from the systemic circulation as the first step of permeation from blood to spermatozoa.     

OCTN2-mediated transport of carnitine in isolated Sertoli cells

Carnitine is extensively accumulated in epididymis. Carnitine is also accumulated in testis at higher concentration than in the plasma and is used in spite of the presence of the blood-testis barrier. In this study, we examined the characteristics of carnitine transport in primary-cultured rat Sertoli cells, which constitute a part of the blood-testis barrier. Uptake of [3H]carnitine (11.4 nM) from the basal side of Sertoli cells was Na+-dependent and was significantly decreased in the presence of 10 microM (48.0 +/- 7.4% of control) or 100 microM unlabeled carnitine (14.6 +/- 5.7% of control). Furthermore, the uptake was significantly inhibited in the presence of 100 microM acetyl-L-carnitine, 100 microM gamma-butyrobetaine or 500 microM quinidine. In RT-PCR analysis, the high-affinity carnitine transporter OCTN2 was detected in rat whole testis tissue and primary-cultured Sertoli cells. In contrast, the low-affinity carnitine transporter ATB(0,+) was detected in rat whole testis tissue, but not in primary cultured Sertoli cells. These results demonstrate that OCTN2 mediates carnitine supply to Sertoli cells from the circulation.     

Nucleosides are efficiently absorbed by Na(+)-dependent transport across the intestinal brush border membrane in veal calves

In previous work, a comparatively high capacity for Na(+)-dependent transport of nucleosides across the intestinal brush border membrane (BBM) was observed in dairy cows, which might be related to digestion of the large amount of nucleic acids present in ruminal microorganisms in the ruminant small intestine. If this were the case, the capacity for Na(+)-dependent intestinal nucleoside transport should be much lower in veal calves, in which only small amounts of nucleic acids, nucleotides, and nucleosides reach the small intestine via the milk replacer. To test this hypothesis, we investigated Na(+)-dependent transport of 3H-labeled thymidine and guanosine across the BBM using BBM vesicles (BBMV) isolated from the small intestine of veal calves. In the presence of a transmembrane Na+ gradient both substrates were transported against a concentration gradient. Inhibitory studies showed that thymidine and guanosine are transported by two different transporters with overlapping substrate specificity, one accepting predominantly pyrimidine nucleosides (N2) and one accepting particularly purine nucleosides (N1). Nucleoside transport was inhibited by glucose along the whole small intestine. Maximal transport rates similar to those in dairy cows were obtained for the proximal, mid-, and distal small intestine. These findings suggest that the high absorptive capacity for nucleosides is a genetically fixed property in the bovine small intestine, which is already present in the preruminant state of veal calves. It may contribute to the high digestibility of nucleic acids observed by others in veal calves receiving milk replacer supplemented with RNA. Its main function may be the efficient absorption of nucleosides resulting from the digestion of nucleic acids associated with desquamated enterocytes. Due to the limited de novo synthesis of nucleotides in enterocytes intracellular uptake of nucleosides across the BBM may contribute to nucleic acid synthesis in enterocytes and thus may have a trophic effect on the intestinal epithelium.     

Characteristics of transport systems of L-alanine in mouse mammary gland and their regulation by lactogenic hormones: evidence for two broad spectrum systems.

The characteristics of the transport systems of L-alanine in lactating mouse mammary gland and their regulation by lactogenic hormones have been studied. L-alanine uptake was mediated by three Na(+)-dependent and one Na(+)-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na(+)-dependent uptake exhibited the usual characteristics of system A. Cl- dependency has been established for system A. The other two Na(+)-dependent systems, which we have named BCl(-)-dependent and BCl(-)-independent, are described for the first time. These are systems with broad specificity and were distinguished on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. The Na(+)-independent route was identified as system L, which operates independent of Cl-. The A, L and BCl(-)-independent transport systems were upregulated in pregnant mouse mammary tissue cultured in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin). Insulin alone also upregulated systems A and L to some extent in pregnant mouse mammary tissue. BCl(-)-dependent activity was not detected in pregnant mouse mammary tissue and was not induced by lactogenic hormones in vitro.     

Rice sodium-insensitive potassium transporter, OsHAK5, confers increased salt tolerance in tobacco BY2 cells

Potassium ion (K(+)) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na(+)) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K(+) uptake activity of the cells, while keeping Na(+) out during salt stress. Here, in search for Na(+)-insensitive K(+) transporter for this purpose, bacterial expression system was used to characterize two K(+) transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K(+) transporter families in bacteria, fungi and plants. When expressed in an Escherichia coli K(+) transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K(+)-limiting conditions by significantly increasing the K(+) content of the cells. Under the condition with a large amount of extracellular Na(+), we found that OsHAK5 functions as a Na(+)-insensitive K(+) transporter, while OsHAK2 is sensitive to extracellular Na(+) and exhibits higher Na(+) over K(+) transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K(+) but not Na(+) in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na(+) insensitive K(+) transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells.     

Channels for water efflux and influx involved in volume regulation of murine spermatozoa

The nature of the membrane channels mediating water transport in murine spermatozoa adjusting to anisotonic conditions was investigated. The volume of spermatozoa subjected to physiologically relevant hypotonic conditions either simultaneously, or after isotonic pre-incubation, with putative water transport inhibitors was monitored. Experiments in which quinine prevented osmolyte efflux, and thus regulatory volume decrease (RVD), revealed whether water influx or efflux was being inhibited. There was no evidence that sodium-dependent solute transporters or facilitative glucose transporters were involved in water transport during RVD of murine spermatozoa since phloretin, cytochalasin B and phloridzin had no effect on volume regulation. However, there was evidence that Hg(2+)- and Ag(+)-sensitive channels were involved in water transport and the possibility that they include aquaporin 8 is discussed. Toxic effects of these heavy metals were ruled out by evidence that mitochondrial poisons had no such effect on volume regulation.     

Expression of ion transport-associated proteins in human efferent and epididymal ducts

Appropriate intraluminal microenvironment in the epididymis is essential for maturation of sperm. To clarify whether the anion transporters SLC26A2, SLC26A6, SLC26A7, and SLC26A8 might participate in generating this proper intraluminal milieu, we studied the localization of these proteins in the human efferent and the epididymal ducts by immunohistochemistry. In addition, immunohistochemistry of several SLC26-interacting proteins was performed: the Na(+)/H(+) exchanger 3 (NHE3), the Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR), the proton pump V-ATPase, their regulator Na(+)/H(+) exchanger regulating factor 1 (NHERF-1), and carbonic anhydrase II (CAII). Our results show that SLC26A6, CFTR, NHE3, and NHERF-1 are co-expressed on the apical side of the nonciliated cells, and SLC26A2 appears in the cilia of the ciliated cells in the human efferent ducts. In the epididymal ducts, SLC26A6, CFTR, NHERF-1, CAII, and V-ATPase (B and E subunits) were co-localized to the apical mitochondria rich cells, while SLC26A7 was expressed in a subgroup of basal cells. SLC26A8 was not found in the structures studied. This is the first study describing the localization of SLC26A2, A6 and A7, and NHERF-1 in the efferent and the epididymal ducts. Immunolocalization of human CFTR, NHE3, CAII, and V-ATPase in these structures differs partly from previous reports from rodents. Our findings suggest roles for these proteins in male fertility, either independently or through interaction and reciprocal regulation with co-localized proteins shown to affect fertility, when disrupted.     

Identification and functional analysis of bull (Bos taurus) cauda epididymal fluid proteome

Despite recent advances in bull epididymal fluid proteome research, significant numbers of proteins secreted to epididymal lumen remain unidentified. The objective of this study was to expand the number of identified cauda epididymal fluid proteins in bulls and to contextualize them in a broader view of their mutual interactions and involvement in biological processes and pathways, to fully elucidate the ways in which epididymal fluid proteins are involved in storage and maturation of spermatozoa in epididymis. We collected postmortem cauda epididymal fluid from 6 mature Holstein Friesian bulls. We performed the identification of proteins using 2-dimensional electrophoresis coupled with MALDI mass spectrometry. Analysis of functionality and pathway involvement of identified proteins was performed using Ingenuity Pathway Analysis software. We identified a total of 189 epididymal fluid proteins, out of which 100 were newly identified in bull epididymal fluid. We have combined our data with 2 previously performed bull epididymal fluid proteome identifications, yielding 280 proteins total, and analyzed it. The main canonical pathways involving epididymal proteins were glycolysis, gluconeogenesis, protein ubiquitination pathway, nuclear factor-erythroid 2-related factor 2-mediated oxidative stress response, and farnesoid X receptor/retinoid X receptor activation. The main biological functions potentially performed by epididymal fluid proteins included carbohydrate metabolism, cellular growth and proliferation, cell death and survival, and small molecule biochemistry. Overall, our results have pointed out multiple novel pathways in bull epididymal fluid that might take part in various aspects of maturation and protection processes of epididymal spermatozoa.     

Carnitine-dependent metabolic activities in Saccharomyces cerevisiae: three carnitine acetyltransferases are essential in a carnitine-dependent strain

L-carnitine is required for the transfer of activated acyl-groups across intracellular membranes in eukaryotic organisms. In Saccharomyces cerevisiae, peroxisomal membranes are impermeable to acetyl-CoA, which is produced in the peroxisome when cells are grown on fatty acids as carbon source. In a reversible reaction catalysed by carnitine acetyltransferases (CATs), activated acetyl groups are transferred to carnitine to form acetylcarnitine which can be shuttled across membranes. Here we describe a mutant selection strategy that specifically selects for mutants affected in carnitine-dependent metabolic activities. Complementation of three of these mutants resulted in the cloning of three CAT encoding genes: CAT2, coding for the carnitine acetyltransferase associated with the peroxisomes and the mitochondria; YAT1, coding for the carnitine acetyltransferase, which is presumably associated with the outer mitochondrial membrane, and YER024w (YAT2), which encodes a third, previously unidentified carnitine acetyltransferase. The data also show that (a) L-carnitine and all three CATs are essential for growth on non-fermentable carbon sources in a strain with a disrupted CIT2 gene; (b) Yat2p contributes significantly to total CAT activity when cells are grown on ethanol; and that (c) the carnitine-dependent transfer of activated acetyl groups plays a more important role in cellular processes than previously realised.     

Partial characterization, sperm association and significance of N- and O-linked glycoproteins in epididymal fluid of rhesus monkeys (Macaca mulatta)

The present study investigated regional modifications of glycosylation status, sperm association and functional significance of N- and O-linked glycoproteins in epididymal luminal fluid of the rhesus monkey (Macaca mulatta). The predominant glycoproteins of the epididymal luminal fluid that increase in the extent of glycosylation or unmasking of exposed epitopes in a region-specific, maturation-dependent manner, included those of 150, 116, 68, 64, 58 (N- and O-linked) and 170 kDa (O-linked). The higher expression of 40 (N-linked), 38 (N- and O-linked) and 60, 56 and 33 kDa (O-linked) glycoproteins in the proximal caput epididymal fluid was followed by alteration or reorganization of 60, 38 and 33 kDa (O-linked) glycoproteins in the distal segments of the epididymis. The association of epididymal fluid glycoproteins with maturing spermatozoa was identified by generating polyclonal antiserum against monkey caudal sperm membrane in female albino rabbits. The antiserum crossreacted strongly with 58 and 33 kDa epididymal fluid glycoproteins of monkeys and also reacted with 116, 68, 58, 56 and 33 kDa glycoproteins from Triton X-100 extracts of human spermatozoa, indicating the presence of antigenically related components in both species. The functional significance of epididymal fluid glycoproteins in sperm functions was investigated by raising antiserum against a heavily glycosylated 58 kDa glycoprotein (MEF1) of caudal epididymal fluid, which crossreacted with the Triton X-100 extracts of epididymal spermatozoa of monkey and ejaculated human spermatozoa on immunoblots. In an in vitro micro-sperm agglutination assay, anti-MEF1 serum agglutinated both rat caudal epididymal spermatozoa and human spermatozoa. MEF1 seemed to be involved in fertilization as demonstrated by inhibition of fertility (100%) in female albino rabbits and rats immunized with this protein. A sperm-agglutinating 58 kDa glycoprotein of rhesus monkey epididymis with functional significance in fertility was identified, thus indicating that it is a potential candidate for contraceptive vaccine development.     

Identification and characterization of ERp29 in rat spermatozoa during epididymal transit

The mammalian epididymis is able to create sequential changes in the composition of luminal fluid throughout its length, wherein spermatozoa undergo morphological, biochemical, and physiological modifications. Subsequently, spermatozoa acquire the ability for fertilization upon reaching the epididymal cauda. In this study, protein variations in Sprague-Dawley rat spermatozoa along the caput and caudal regions of epididymis were investigated by high-resolution two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry. From total protein spots on the 2DE maps, 43 spots were shown to be significantly modified as sperm traverse the epididymis, and seven unambiguous proteins were identified from them. Finally, using indirect immunofluorescence, we demonstrated that localization of one of these seven proteins, the endoplasmic reticulum protein (ERp29) precursor, which was first reported in mammalian spermatozoa, was apparently up-regulated as the sperm underwent epididymal maturation and expressed mainly on caudal sperm. Western blot analysis also revealed that ERp29 precursor, from both whole spermatozoa and membrane proteins, increased significantly as the sperm underwent epididymal maturation. Furthermore, the results from immunofluorescence-stained epididymal frozen sections demonstrated that ERp29 was localized in cytoplasm of epididymal epithelia, and the fluorescence intensity was significantly higher in the caudal epididymis than in the caput. These clues indicated that the ERp29 precursor, perhaps related to secretory protein synthesis and absorbed by spermatozoa, may play a vital role in sperm maturation during the epididymal transit, particularly, in the sperm/organelle membrane.     

Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs

This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the 'Entrepelado' and 'Lampi?o' breeds. The samples were suspended in a commercial extender and refrigerated to 17 degrees C for transport to the laboratory (step A), where they were further extended with a lactose-egg yolk-based extender and chilled to 5 degrees C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS-PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the 'Entrepelado' breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing-thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.     

Post-translational modifications in glycosylation status during epididymal passage and significance in fertility of a 33 kDa glycoprotein (MEF3) of rhesus monkey (Macaca mulatta)

The present study reports data on post-translational modifications in the glycosylation status during epididymal passage and significance in fertility of a 33 kDa glycoprotein of rhesus monkey (Macaca mulatta), designated as MEF3 (monkey epididymal fluid protein 3). MEF3 exhibited strong affinity for N-linked alpha-D-mannose groups and O-linked N-Ac-galactosamine linkages in epididymal fluids and exhibited moderate affinity for N-Ac-glucosaminylated (wheat germ agglutinin), fucosylated (Tetragonolotus purpurea), and N-Ac-galactosamine (peanut agglutinin) residues on more mature corpus and caudal spermatozoa in a maturation-dependent manner on Western blots probed with specific biotinylated lectins. Polyclonal antiserum raised against affinity-purified MEF3 from caudal epididymal fluid (CEF) cross-reacted specifically with CEF and caudal sperm membrane of macaque and with Triton X-100 extract of ejaculated human spermatozoa, suggesting the existence of antigenically related components in both species. The tangled agglutination caused by anti-33 kDa serum of human spermatozoa, along with localization of MEF3 on entire sperm surface of epididymal and testicular sperm of monkey and human spermatozoa, suggest the significance of MEF3 in sperm function. The 100% inhibition of fertility of immunized female rabbits with this protein in vivo and inhibition of human sperm penetration in zona-free hamster eggs in vitro suggests the functional significance of MEF3 in fertility. Together, these results clearly indicate that MEF3 has potential significance as a target for antibodies that inhibit sperm function and fertility.     

Active intestinal absorption of nucleosides by Na+-dependent transport across the brush border membrane in cows

Transport of 3H-labeled nucleosides across the bovine intestinal brush border membrane (BBM) was characterized with BBM vesicles (BBMV) isolated from mid-jejunum of cows because large amounts of nucleic acids are digested in the small intestine of ruminants. Two Na+-dependent electrogenic nucleoside transporters with overlapping substrate specificity were shown to be present in the jejunal BBM, one for pyrimidine nucleosides and one for purine nucleosides. As indicated by inhibitory studies, thymidine seemed to be a specific substrate for the pyrimidine nucleoside transporter, while this applied to guanosine and deoxyguanosine for the purine nucleoside transporter. Uridine and adenosine appear to have an affinity to both transporters. This also applies to deoxyadenosine and deoxyuridine. Nucleobases (uracil, hypoxanthine) did not affect transport of nucleosides. The kinetic constants (Km and Vmax) for Na-dependent thymidine and guanosine transport were 29 and 24 micromol/L and 78 and 51 pmol (mg protein)(-1) s(-1), respectively. These values are much higher than those reported for monogastric species.     

Sequential development of flagellar defects in spermatids and epididymal spermatozoa of selenium-deficient rats

In this study cauda epididymal spermatozoa of rats maintained on a selenium-deficient diet for 5 and 7 months exhibited an array of flagellar defects. Spermatids and spermatozoa were analyzed by light and electron microscopy to define the appearance of flagellar abnormalities during spermiogenesis and post-testicular sperm development. Late spermatids of selenium-deficient rats displayed normal structural organization of the flagellar plasma membrane, axoneme, outer dense fibers, fibrous sheath and annulus, but they exhibited a premature termination of the mitochondrial sheath. A comparison of late spermatids and caput epididymal spermatozoa revealed that a late step in flagellar differentiation was the structural remodeling of the annulus and its accompanying fusion with both the fibrous sheath and the mitochondrial sheath. In selenium-deficient animals, however, the annulus failed to fuse with the mitochondrial sheath, generating an apparent weak point in the flagellum. After epididymal passage, cauda epididymal spermatozoa of selenium-deficient animals also exhibited extensive flagellar disorganization resulting from the apparent sliding and extrusion of specific outer dense fiber-doublet microtubule complexes from the proximal and the distal ends of the mitochondrial sheath and the accompanying loss of the midpiece plasma membrane. Only fiber complex number 4 was extruded proximally, whereas fibers 4, 5, 6 and 7 were extruded from the mitochondrial sheath-deficient posterior midpiece. Axonemal fibers 8, 9, 1, 2 and 3 retained their normal geometric relationships. These data suggest that the known loss of male fertility in selenium deficiency results from the sequential development of sperm defects expressed during both spermiogenesis and maturation in the epididymis.     

Presence, processing, and localization of mouse ADAM15 during sperm maturation and the role of its disintegrin domain during sperm-egg binding

Successful fertilization requires gametes to complete several stages, beginning with maturation and transport along the male and female reproductive tracts and ending with the interaction between the sperm and the egg. This last step involves sperm-egg adhesion and membrane fusion. ADAMs (disintegrin and metalloprotease domain proteins) are a family of membrane-anchored glycoproteins that are thought to play diverse roles in cell-cell adhesion through their interaction with integrins. This study analyzes the presence, location, processing, and possible role of ADAM15 in mouse sperm. The presence of ADAM15 in mouse spermatozoa was detected by Western blotting, which revealed that ADAM15 is post-translationally processed, during epididymal sperm maturation and the acrosome reaction. The 35 kDa antigen present in the acrosome-reacted sperm is the last proteolytic product of the 110/75 kDa ADAM15 found in non-capacitated sperm. This 35 kDa protein contains the disintegrin domain. By indirect immunofluorescence, ADAM15 was identified in the acrosomal region and along the flagellum of mouse spermatozoa. In acrosome-reacted sperm, ADAM15 was lost from the acrosomal region, but remained diffusely distributed throughout the head and flagellum. Furthermore, the ADAM15 disintegrin domain (RPPTDDCDLPEF) partially inhibited fusion and almost completely inhibited sperm-oolemma adhesion. In conclusion, our data indicate that ADAM15 is present in the testis and in spermatozoa from the caput, corpus, and cauda epididymis, as well as in non-capacitated and acrosome-reacted gametes. Results also indicate that ADAM15 is processed during epididymal maturation and acrosome reaction and that it may play a role during sperm-egg binding.     

Mechanism of glycine transport in mouse mammary tissue

The mechanism of glycine transport in lactating mouse mammary gland was investigated. Three Na+-dependent systems of glycine transport, distinguished on the basis of their ionic requirement and sensitivity to 2-(methylamino)isobutyric acid (MeAIB), were A (Na+-dependent, MeAIB-sensitive); (Na++Cl-)-dependent, MeAIB-insensitive; and Na+-dependent, Cl--independent, MeAIB-insensitive. These systems were further distinguished on the basis of inhibition analysis and sensitivity to pH of the extracellular medium and preloading mammary tissue with amino acids. The uptake of glycine via the A system (Km 0.53 mM) was inhibited by preloading mammary tissue with alanine, while glycine uptake mediated by the (Na++Cl-)-dependent, MeAIB-insensitive system (Km 0.47 mM) was downregulated by preloading mammary tissue with all amino acids (alanine, sarcosine and histidine) tested. Treatment of mammary tissue with N-ethylmaleimide inhibited the uptake of glycine via both these systems. Decreasing the pH of the extracellular medium inhibited the uptake of glycine via the A system but not the (Na++Cl-)-dependent, MeAIB-insensitive system. On the basis of ionic requirement, system A appears to comprise two components, one dependent on Na+ plus Cl- and the other on Na+ alone. Insulin upregulated the A system-mediated uptake of glycine in pregnant mouse mammary tissue cultured in vitro, while the (Na++Cl-)-dependent, MeAIB-insensitive system remained unaffected.     

Differential modulation of bovine epididymal activity by oxytocin and noradrenaline

Passage of spermatozoa through the epididymis and emission of sperm during ejaculation are based on spontaneous and induced contractions of epididymal peritubular muscle layers. This study deals with the ejaculation-relevant factors noradrenaline (NA) and oxytocin (OT) and their contractile effects in the course of the bovine epididymal duct. Muscle tension recording revealed excitatory effects of NA in all duct regions. A peculiarity was found in a duct section between the mid-cauda and ductus deferens, where the responsiveness to NA was particularly faint in comparison with the adjacent regions. NA-induced contraction was primarily mediated by postjunctional alpha(2)-adrenoceptors (ADRA) in the caput and corpus regions, and by alpha(1)-ADRA in the cauda region. Contrary to NA, OT exerted regionally varying effects. The peptide induced contraction in intact and epithelium-denuded caput as well as in epithelium-denuded corpus segments but had a relaxant net effect in intact corpus and proximal cauda segments. Within the mid-cauda, OT evoked strong contraction, which progressively decreased distally. Receptor specificity of the epididymal OT effects was verified using the selective OT receptor (OTR) agonist [Thr(4),Gly(7)]OT and vasopressin. OTR immunoreactivity was detected in the epididymal peritubular muscle wall and epithelial principal cells. RT-PCR analysis confirmed the presence of OTR in all duct regions. In summary, different contractile responses to OT and NA occur in the course of the epididymal duct, possibly preventing excessive sperm transport through the corpus and serving orthograde emission of sperm during ejaculation.     

Regulation of in vitro metabolism of palmitate by carnitine and propionate in liver from dairy cows.

Regulation of in vitro palmitate metabolism by carnitine and propionate was investigated in liver obtained by biopsy from fasted nonlactating cows and from cows during early lactation. Liver slices from nonlactating cows during a 7-d fast esterified less palmitate than those from the same cows before fasting. Carnitine added in vitro increased hepatic oxidation and decreased esterification of palmitate in fed cows, but effects of carnitine were less during fasting. Propionate added in vitro decreased oxidation of palmitate; the effect was greater during fasting. In liver slices from cows during early lactation, carnitine increased oxidation and total utilization of palmitate and decreased palmitate esterification. Addition of tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I, prevented the carnitine-induced changes in palmitate metabolism. Substantial carnitine-independent oxidation of palmitate was observed in the presence of tetradecylglycidic acid. Tetradecylglycidic acid decreased esterification of palmitate to triglycerides but increased esterification to diglycerides. Effects of tetradecylglycidic acid and either propionate or pyruvate on palmitate oxidation were additive, indicating that propionate and pyruvate affect palmitate oxidation at sites other than carnitine palmitoyltransferase I. No interactions were detected between carnitine and propionate, but both compounds were potent regulators of palmitate metabolism in liver slices from cows during early lactation.