Importance of calcium for the binding of oviductal fluid proteins to the membranes of bovine spermatozoa

Our laboratory has recently shown that in vitro-cultured oviductal cells secrete sperm motility maintaining factor(s). Since the binding of oviductal proteins to spermatozoa (SPZ) has been demonstrated in many species, the motility factor was postulated to bind the membranes of SPZ. Therefore, the current study was performed to evaluate which proteins from in vivo oviductal secretions bind to sperm membranes, to characterize binding conditions, and to evaluate the effect of this binding on sperm survival. Bovine oviducts were dissected, and oviductal cells and fluid were collected by pressing the oviductal tube with a glass slide. This mixture was incubated in Tris-EDTA buffer at 37°C for 30 min, and the cells were washed twice by centrifugation. The supernatant containing oviductal fluid proteins (OFP) was reserved, filtered, frozen (for later motility tests), or lyophilized and labeled with ¹²⁵|. Frozen-thawed SPZ were incubated either immediately, following capacitation, ionophore-induced acrosome reaction, death by heating, or flagellar removal with labeled OFP for 30 min. The resulting pellet after three washes was dissolved in SDS and submitted to 10% SDS-PAGE. An autoradiogram showed that 72, 66, 39, 38, and 36 kDa proteins bind strongly to the five types of SPZ used, and that this binding is very specific, since unlabeled OFP inhibited binding while serum proteins did not. Furthermore, for 39, 38, and 36 kDa proteins, the presence of calcium in the incubation medium was essential for dose-dependent binding, whereas magnesium was not. Preincubation of SPZ for 30 min at 37°C with oviductal fluid, followed by one wash and 6 hr of incubation in control media, showed that the percentage of motile SPZ is significantly higher (52 ± 6%) compared with SPZ not preincubated with oviductal fluid (24 ± 6%: P < 0.01). In summary, a limited number of proteins from oviductal secretions bind to the surface of bovine SPZ only in the presence of calcium, and this binding appears to be important for subsequent sperm viability. © 1996 Wiley-Liss, Inc.     

Systemic pilocarpine increases deposition of and decreases responsiveness to inhaled carbachol in sheep.

The purpose of this study was to determine whether excessive airway secretions could serve as a barrier function against inhaled particulate matter. To increase airway secretions, six conscious sheep were treated with pilocarpine (0.8 mg/kg i.v.). Pilocarpine increased pulmonary resistance (RL) and total aerosol deposition within five breaths (AD5) as determined by the rebreathing of an inert monodisperse aerosol. When RL had returned to baseline, AD5 remained elevated [21 +/- 2% (SE), P < 0.05] and tracheal secretions were increased (237 +/- 77%, P < 0.05) above the values before pilocarpine administration. A carbachol aerosol dose-response curve was carried out at this time and compared with a control carbachol dose-response curve by calculating the dose of carbachol required to increase RL by 400% (PD400). Mean PD400 was increased postpilocarpine by 53 +/- 18 (P < 0.05) and 85 +/- 25% (P < 0.05) when normalized for increased aerosol deposition. Thus, pilocarpine decreased airway responsiveness to inhaled carbachol despite increasing aerosol deposition. The pilocarpine-induced airway hyporesponsiveness to inhaled carbachol is consistent with the hypothesis that excessive secretions have a protective role in the airways.     

P物质对兔离体输卵管峡部平滑肌活动的影响

本文在离体状态下观察P物质对不同性激素状态兔输卵管峡部平滑肌活动的影响。动物分成动情组、间情组、去卵巢组。实验结果:(1)P物质对间情兔输卵管峡部平滑肌的收缩有明显的抑制作用。(2)P物质对动情兔和去卵巢兔输卵管峡部平滑肌的收缩没有影响。结果表明P物质对不同性激素状态下的兔输卵管峡部平滑肌的作用不一致。     

Effect of steroids and oviductal cells, from the different parts of the oviduct, on the incidence of monospermy in porcine in vitro fertilization

A high incidence of polyspermy occurs in porcine in vitro fertilization. It is also known that in vivo, the oviductal cells and their secretions play an important role in fertilization and early development. Vesicles from oviductal cells from different parts of the oviduct (isthmus or ampulla) pretreated with estradiol or progesterone or ethanol were used to assess their role in the fertilization process. Oviductal cells were co-cultured with 0.5 million motile sperm/ml for 30 min. A 10-microl sample (spermatozoa bound with the cells) was added to 40-microl droplets of fertilization medium containing 5 oocytes. After 15 to 18 h, oocytes were examined for penetration and monospermy. The results show a lower penetration rate with oviductal cells than that of the control. The use of oviductal cells from the isthmus treated with estradiol significantly decreased the percentage of polyspermy compared with that of ampulla treated with the estradiol or with the control. When the isthmus cells were treated with progesterone, an increase in the incidence of polyspermy was observed. Therefore, it is possible to use oviductal cells to increase the incidence of monospermy in porcine in vitro fertilization; moreover, estradiol increases the proportion of monospermy when added to isthmus-derived oviductal cells.     

DHEA attenuates catecholamine secretion from bovine adrenal chromaffin cells

Dehydroepiandrosterone (DHEA) is a putative anti-stress agent and stress is associated with the secretion of catecholamine from the adrenal gland, but the effects of DHEA on catecholamine secretion are not fully understood. Using bovine chromaffin cells, we found that DHEA inhibited catecholamine secretion and cytosolic Ca²⁺ ([Ca²⁺]_i) rise coupled with nicotinic acetylcholine receptor (nAChR) without exerting an effect on³H-nicotine binding. In the case of high K⁺ stimulation, DHEA effectively suppressed secretion without affecting [Ca²⁺]₁ rise. Trifluoperazine (TFP), a calmodulin inhibitor, was capable of counteracting the inhibition of DHEA on high K⁺-induced secretions. In permeabilized cells, DHEA suppressed the Ca²⁺-induced secretion. These results suggest that DHEA (a) acts as a channel blocker that suppresses Ca²⁺ influx and subsequent secretions associated with nAChR, or (b) affects the intracellular secretion machinery to suppress high K⁺-induced secretions without affecting the high K⁺-induced [Ca²⁺]_i rise.     

Postnatal development of the oviduct of intact and ovariectomized rabbits

The length of the oviduct, the thickness of its wall, and the height of its mucosal epithelium and cilia were measured in (a) 0-, 2-, 4- and six-month-old rabbits, (b) rabbits ovariectomized at birth and (c) ovariectomized, estrogen-treated rabbits. The length and external diameter of the oviduct increased progressively until four months of age, after which their rates of increase declined. The thickness of the oviductal wall at the uterotubal junction was twice as large as that of the isthmus at two months of age and six times as large at four and six months of age. The height of the mucosal epithelium in the fimbriae was less than that in other oviductal segments at birth, but exceeded that in other segments at six months of age. Ciliated cells and motile cilia were absent 24 hours after birth; they were first observed two months after birth. The cilia of fimbriae were shorter than cilia elsewhere in the oviduct. Neonatal ovariectomy retarded the development of the oviduct and the mesotubarium and caused pyknosis of ciliated and non-ciliated cells of the oviductal mucosa. Cells with scarcely motile cilia were present five and one-half months after neonatal ovariectomy.     

In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release

The mammalian oviduct plays a key role in sperm storage, capacitation, and selection. Specific oviduct secretions and/or binding to oviductal cells are thought to be responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm-oviductal cell adhesion and release in the bovine species. Distribution and acrosomal status of sperm bound to in vitro-cultured ampullary and isthmic cell monolayers were followed until the time of sperm release by means of fluorescence labeling techniques. In order to understand whether release is due to surface changes of sperm or oviductal cells, double incubation experiments with unlabeled and Hoechst-labeled sperm have been performed. Main findings demonstrate that (1) only acrosome-intact sperm bind specific bovine oviductal epithelial cells; (2) acrosomes of bound sperm are preserved intact over time; and (3) release of unreacted sperm is likely to be due to changes of the sperm surface, probably triggered by capacitation. These findings support the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.     

The Anticonvulant Effect of Cooling in Comparison to α-Lipoic Acid: A Neurochemical Study

Brain cooling has pronounced effects on seizures and epileptic activity. The aim of the present study is to evaluate the anticonvulsant effect of brain cooling on the oxidative stress and changes in Na⁺, K⁺-ATPase and acetylcholinesterase (AchE) activities during status epilepticus induced by pilocarpine in the hippocampus of adult male rat in comparison with α-lipoic acid. Rats were divided into four groups: control, rats treated with pilocarpine for induction of status epilepticus, rats treated for 3 consecutive days with α-lipoic acid before pilocarpine and rats subjected to whole body cooling for 30 min before pilocarpine. The present findings indicated that pilocarine-induced status epilepticus was accompanied by a state of oxidative stress as clear from the significant increase in lipid peroxidation (MDA) and superoxide dismutase (SOD) and significant decrease in reduced glutathione and nitric oxide (NO) levels and the activities of catalase, AchE and Na⁺, K⁺-ATPase. Pretreatment with α-lipoic acid ameliorated the state of oxidative stress and restored AchE to nearly control activity. However, Na⁺, K⁺-ATPase activity showed a significant decrease. Rats exposed to cooling for 30 min before the induction of status epilepticus revealed significant increases in MDA and NO levels and SOD activity. AchE returned to control value while the significant decrease in Na⁺, K⁺-ATPase persisted. The present data suggest that cooling may have an anticonvulsant effect which may be mediated by the elevated NO level. However, brain cooling may have drastic unwanted insults such as oxidative stress and the decrease in Na⁺, K⁺-ATPase activity.     

Mucus-glycoproteins (mucins) of the cat trachea: characterisation and control of secretion

Glycoproteins produced by the tracheae of anaesthetized cats were radiolabelled biosynthetically by a pulse administration of Na2 35SO4 and [3H]glucose into the tracheal lumen. Subsequently, radiolabelled secretions were washed from the tracheal lumen. Repeated doses of pilocarpine and then ammonia vapour were given to stimulate secretion. Pilocarpine-stimulated glycoproteins, which came mainly from the submucosal glands, were particularly enriched with 35S. Ammonia-stimulated secretions, which probably came mostly from the microvillous border of the surface epithelium, contained mainly 3H radioactivity but little 35S. Two negatively-charged glycoproteins of different molecular size were identified in the secretions: the larger component was excluded on Sepharose CL-4B and it had a higher 3H 35S ratio than the smaller component which was retarded on Sepharose CL-4B. The relative amount of the smaller component decreased progressively with repeated pilocarpine stimulation and it was not detected in secretions induced by ammonia. Pilocarpine stimulation caused little alteration in carbohydrate composition of the secreted glycoproteins. In response to ammonia, glycoproteins were secreted with a high sialic acid content but quantitatively they represented a small amount of material compared with that induced by pilocarpine. These findings suggest that tracheal glycoproteins from different epithelial-cell sources have distinctive chemical compositions and that their secretions may be independently regulated. The 35S-rich high-molecular-weight glycoproteins from the submucosal glands were of the mucin-type but those derived from the microvillus border may represent a different class of airway glycoproteins from typical epithelial mucins.     

Differentiated structure and function of primary cultures of monkey oviductal epithelium

Summary We have established well-differentiated, polarized cultures of monkey oviductal epithelium. Oviductal epithelial cells were isolated by protease digestion and plated on collagen-coated, porous cell culture inserts. About 5 d after plating, cells developed detectable transepithelial electrical resistance of up to 2000 Ω.cm² (an index of tight junction formation) and transepithelial voltages of up to 20 mV (an index of vectorial transepithelial ion transport). Measurements of short-circuit current in Ussing chambers indicated that active secretion of Cl was the major transepithelial active ion transport process, and that this was stimulated by elevation of either cAMP or Ca. Furthermore, estimates of the volume of mucosal liquid were consistent with Cl secretion mediating fluid secretion. Various microscopical methods showed that the cultures were densely ciliated and contained mature secretory cells. Transport across the oviductal epithelium determines the composition of the oviductal fluid, and the study of the relevant transport processes will be greatly enhanced by well-differentiated cultures of oviductal epithelium of the kind established here.     

Analysis of gene expression in mouse 2-cell embryos using fluorescein differential display: comparison of culture environments

The effect of the oviductal environment on gene expression in 2-cell mouse embryos was examined with mRNA differential display. Embryos used for experiments were cultured in modified Whitten medium with or without oviductal tissue until late 2-cell stage. The results of sequencing indicated that the genes for ATP synthase (ATPase 6), S:-adenosylmethionine decarboxylase (S:-AMDC) and nuclear autoantigenic sperm protein (NASP) were differentially expressed in embryos cultured in the oviductal environment (nonblocking culture condition). The ATPase 6 gene is encoded by mitochondrial DNA and is essential for the production of ATP. This indicates that the expression of ATP synthesis-related genes at the 2-cell stage may be required to maintain normal development in vitro. S:-Adenosylmethionine decarboxylase decarboxylates adenosylmethionine, which is a substrate of DNA methylation. The expression of S:-AMDC may be responsible for the low level of methylation of preimplantation development. As NASP is a histone-binding protein that is thought to be testis and sperm specific, its function in embryos remains unclear. On the other hand, the Tcl1 gene and a novel gene, the c-1 gene, were strongly expressed in embryos cultured without oviductal tissue (blocking culture condition). The expression patterns of these genes are quite similar. However, the detailed functions of these genes in embryos remain to be determined.     

Acetylcholine Formation in Mouse Brain and Effect of Cholinergic Drugs

TREATMENT of mice with arecoline or oxotremorine produces tremors accompanied by a transient increase in the brain level of acetylcholine lasting for 20 to 30 min¹. We reported that administration of pilocarpine, whose peripheral cholinomimetic effects resemble those of arecoline, also markedly increases the level of brain acetylcholine in rats^2,3. By contrast with other cholinomimetics, however, the pilocarpine-induced increase in acetylcholine lasts for several hours and is not accompanied by tremors. These findings suggest that arecoline and pilocarpine increase brain acetylcholine levels by different mechanisms. This study compares the effects of arecoline and pilocarpine administration on the conversion of choline-³H to acetylcholine-³H to elucidate the mechanism by which these drugs raise the brain acetylcholine level.     

Biodistribution of bis[β-(N,N,N-trimethylamino)ethyl]selenide-75Se diiodide,a potential articular cartilage imaging agent

In an effort to develop a specific radiodiagnostic agent for articular cartilage imaging, we have investigated the biodistribution of bis[β-(N,N,N-trimethylamino)ethyl]selenide-⁷⁵Se diiodide (⁷⁵Se BISTAES) in rabbits. At an intravenous dose of 5 mg/kg, the greatest localization of the compound occurred in articular cartilage 15 min after injection. The compound was excreted rapidly in the urine. The results suggest that ⁷⁵Se BISTAES has potential clinical use as an articular cartilage imaging agent.     

Mechanism Underlying the Effect of Mecamylamine on Nicotinic Acetylcholine Receptors of Rat Sympathetic Ganglion Neurons

On neurons of the superior cervical ganglion of 3-week-old rats, we studied the mechanism underlying the blocking effect of mecamylamine on transmembrane currents evoked by iontophoretic application of acetylcholine (ACh currents); these currents were recorded with the use of a patch-clamp technique in the whole-cell configuration. The IC₅₀ of the above agent equaled (2.7 ± 0.3) · 10^-10 M. The blocking effect of mecamylamine on ACh current did not depend on the membrane potential and decreased with rise in the concentration of the drug. Thus, a competitive blocking mechanism mostly underlies the above phenomenon.     

Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH

The effect of pH (from 4.8 to 9.8) on the production of pilosine and pilocarpine and on their partition between cell and medium was studied in two lineages (P and PP) of Pilocarpus microphyllus cell suspension cultures. Highest mass accumulation was observed at high pHs and both lineages produced pilocarpine while only lineage PP produced pilosine. Both alkaloids were released in the medium but higher accumulation occurred in the cells. The highest production of pilocarpine was at pH 8.8–9.8 in both cell lineages. Other imidazole alkaloids were also identified in both lineages. At all pHs tested, the pH in the media cultures tended to stabilize around 6 after 10–15 days of cultivation. NO₃ ⁻ and NH₄ ⁺ variation in the media might partially explain the pH stabilization.     

Antiserum inhibition of rabbit spermatozoal adherence to ova

The in vitro effects of different normal and immune sera, rabbit oviductal fluid, and culture media from incubations of female reproductive tissues on the adherence of rabbit spermatozoa to rabbit and rat ova is described. There were no apparent differences in results due to species of ova. Adherence of spermatozoa to ova was not affected by treatment with normal sera. Antisera against materials that contained spermatozoa such as semen epididymal spermatozoa and testis completely inhibited the spermatozoa from attaching to ova and caused considerable agglutination of sperm cells in the incubation slides. When the agglutinating and immobilizing activities of rabbit and goat antisera were removed by papain treatment the antiadherent effect of the antisera was unaffected. Oviductal secretions and concentrated media from the culture of reproductive tissue from female rabbits isoimmunized with semen inhibited adherence of sperm.     

MEDIATION BY β-ADRENERGIC RECEPTORS OF EFFECT OF NOREPINEPHRINE ON PINEAL SYNTHESIS OF [14C]SEROTONIN AND [14C]MELATONIN

Rat pineal organs maintained in organ culture converted [¹⁴C]tryptophan to [¹⁴C]serotonin and [¹⁴C]melatonin. The synthesis of both indoles was stimulated by the presence of norepinephrine or dibutyryl adenosine 3′,5′-monophosphate. This effect of norepinephrine could be blocked by the α-adrenergic blocking drug, propranolol, but was not modified by the a-adrenergic blocking agent, phenoxybenzamine. Neither blocking agent modified the pineal response to dibutyryl adenosine 3′,5′-monophosphate. Unlike dibutyryl adenosine 3′,5′-monophosphate, the naturally occurring adenosine phosphates did not stimulate synthesis of [¹⁴C]melatonin in vitro.     

Effect of vaginally administered 15(S)15-methyl-PGF2alpha on egg transport and fertility in rabbits.

The effects of vaginal suppositories containing 1.0 mg of 15[S]15-methy-PGF2alpha on oviductal motility, egg transport, and fertility were determined in rabbits. Suppository treatment caused a significant increase (P less than 0.02) in the amplitude of oviductal contractions, and a decrease in the frequency of contractractions (P less than 0.04). Altered oviductal motility persisted for an average of 2 hr after treatment. Treatment with 1, 2, or 3 suppositories at various times after ovulation caused a significant reduction in the number of eggs located in the oviducts (P less than 0.025). There was, however, a great deal of variation in egg recovery in treated animals (range 0 to 100%). Treatment of mated rabbits during the time of tubal egg transport caused a significant reduction in the number of Day-12 implants (P less than 0.05). The percentage of corpora lutea represented by Day-12 implants was similar to egg recovery rates in animals similarly treated. The treatment had no effect on fetal survival from Day 12 to 28 of pregnancy. The decrease in fertility caused by these vaginal suppositories is presumably due to the stimulatory effect on oviductal motility which accelerates tubal transport of the embryos into the uterus. Embryos that arrive in the uterus prematurely probably do not implant and degenerate or are expelled.     

Genetic deletion of the neuronal glutamate transporter,EAAC1, results in decreased neuronal death after pilocarpine-induced status epilepticus

Excitatory amino acid carrier 1 (EAAC1 also called EAAT3) is a Na⁺-dependent glutamate transporter expressed by both glutamatergic and GABAergic neurons. It provides precursors for the syntheses of glutathione and GABA and contributes to the clearance of synaptically released glutamate. Mice deleted of EAAC1 are more susceptible to neurodegeneration in models of ischemia, Parkinson’s disease, and aging. Antisense knock-down of EAAC1 causes an absence seizure-like phenotype. Additionally, EAAC1 expression increases after chemonvulsant-induced seizures in rodent models and in tissue specimens from patients with refractory epilepsy. The goal of the present study was to determine if the absence of EAAC1 affects the sensitivity of mice to seizure-induced cell death. A chemoconvulsant dose of pilocarpine was administered to EAAC1^−/− mice and to wild-type controls. Although EAAC1^−/− mice experienced increased latency to seizure onset, no significant differences in behavioral seizure severity or mortality were observed. We examined EAAC1 immunofluorescence 24 h after pilocarpine administration and confirmed that pilocarpine causes an increase in EAAC1 protein. Forty-eight hours after induction of seizures, cell death was measured in hippocampus and in cortex using Fluoro-Jade C. Surprisingly, there was ∼2-fold more cell death in area CA1 of wild-type mice than in the corresponding regions of the EAAC1^−/− mice. Together, these studies indicate that absence of EAAC1 results in either a decrease in pilocarpine-induced seizures that is not detectable by behavioral criteria (surprising, since EAAC1 provides glutamate for GABA synthesis), or that the absence of EAAC1 results in less pilocarpine/seizure-induced cell death, possible explanations as discussed.     

Autoradiography of lymph lodes with 99mTc-dextran in rabbits

^99mTc-dextran (D) was further evaluated in the present study as a lymphoscintigraphic agent compared to radiocolloids. It was injected intra-dermally into the web space between the second and third toes in both hind feet of two rabbits. ^99mTc-nanocolloid (NC) was injected subcutaneously in both hind feet of two other rabbits. Popliteal lymph nodes were taken out and frozen in liquid nitrogen after the animals were sacrificed at 2 h post-injection. Three frozen sections in 10μm thickness were prepared from each node for autoradiographic studies. The lymph node slices were exposed for 18 h using Ilford G 5 emulsion. The obtained autoradiographs showed that the distribution of ^99mTc-D radioactivity within lymph nodes was more uniform indicating better tissue penetration compared to ^99mTc-NC which remained mostly in the lymph canaliculi.