Experimental evidence for the influence of cumulus-oocyte-complexes on sperm release from the porcine oviductal sperm reservoir

In the pig, a temporal relationship is suggested between sperm release from the sperm reservoir (SR) and ovulation, but the mechanism(s) is still under discussion. In two experiments, the influence of transferred ova on the release of SR-spermatozoa at ovulation and the effect of supplementation with non-sulfated glycosaminoglycan hyaluronan (HA) on embryo development and the number of accessory spermatozoa, respectively, were examined. PMSG/hCG primed ovectomized gilts that had previously received endoscopic low-dose insemination into the cranial uterine horn were used as an experimental model. After salpingectomy, tubal segments (ampulla, cranial, and caudal isthmus) were flushed and sperm numbers or respective accessory spermatozoa were counted. In Experiment 1, the distribution of the sperm population was altered in the presence of cumulus-oocyte-complexes (COCs). A higher proportion of spermatozoa was found after transfer of COCs into one oviduct in the ampulla and cranial isthmus segments compared with the controls (17.5 vs. 4.9%, p<0.05). In Experiment 2, the quality of the transferred ova and treatment influenced the presence of accessory spermatozoa. Transfer of COCs together with HA increased (p<0.05) the number of accessory spermatozoa compared with the other treatment groups and was similar to those in the "undisturbed" controls. No modifications were obtained regarding mean blastomere numbers (2.6 +/- 0.2 to 3.1 +/- 0.2). In summary, this study was demonstrated that cumulus-oocyte-complexes may be involved in triggering sperm release from the pig oviductal SR and that HA might be related to sperm release.     

Prolactin signaling in porcine adrenocortical cells: involvement of protein kinases

Prolactin (PRL) was found to have a stimulatory effect on adrenal steroidogenesis in vivo and in vitro in several species including pigs. PRL signal transduction pathways, however, in adrenocortical cells are poorly recognized. Therefore, the goal of this paper is to ascertain the involvement of protein kinase C (PKC) and tyrosine kinases in PRL signaling in porcine adrenal cortex. Adrenals were harvested from locally slaughtered mature gilts. Cortical cells were dispersed by sequential treatment with collagenase. The cells were seeded into 24-well culture plates at a density of 3×10⁵/mL. Cells were incubated with or without PRL (500 ng/mL), ACTH (5 nM—a positive control), tyrosine kinase inhibitor—genistein (1; 2.5 or 5 μM), PKC inhibitor—sphingosine (20–1000 nM) and PKC activators—diacylglycerol (DiC8; 10–100 μM) and phorbol ester (PMA; 1–1000 nM). All incubations were performed for 8 h (95% air and 5% CO₂, 37°C). PRL and ACTH (P<0.05) increased cortisol and androstenedione (A₄) secretion. DiC8 and PMA mimicked the stimulatory effect of PRL. Sphingosine (P<0.05) suppressed basal and PRL-stimulated steroid secretion. Genistein inhibited (P<0.05) PRL-stimulated cortisol secretion and enhanced (P<0.05) basal and PRL-stimulated A₄ secretion. Moreover, PKC activation was assessed by measuring the specific association of [³H]phorbol dibutyrate ([³H]PDBu) with adrenocortical cells after treatment with PRL or ionomycin (a positive control). PRL (within 2–3 min) and ionomycin (within 2–5 min) increased (P<0.05) specific binding of [³H]PDBu to the porcine adrenocortical cells. In addition, PRL did not augment the cortisol and A₄ secretion by PKC-deficient adrenocortical cells. In conclusion, presented results support the hypothesis that PKC and tyrosine kinases are involved in PRL signaling in adrenocortical cells in pigs. Moreover, activation of PKC is associated with the increased secretion of cortisol and A₄.     

The porcine sperm reservoir in relation to the function of hyaluronan

The oviduct plays a role in successful animal reproduction not only in spermatozoa and ova transport to the fertilization site but also by affording a microenvironment for fertilization and early embryonic development. The sperm reservoir (SR) is restricted in the uterotubal junction (UTJ) and caudal isthmus. Billions of porcine spermatozoa are distributed to the female reproductive tract during/after insemination, and small amounts of them are stored for about 36–40 hours in the SR, which maintains sperm viability in the pre-ovulation period through its surface epithelium and production of fluid. The SR regulates the release of spermatozoa so that only a small population moves towards the fertilization site (ampulla) to decrease polyspermy. This review attempts to provide information about the structure and function of the porcine SR, its intraluminal content (hyaluronan, HA), and the influences of HA on porcine spermatozoa in vivo. In pigs, the spermatozoa are stored in a mucous-like fluid within the UTJ and caudal isthmus in the pre-ovulation period. The oviduct fluid contains sulfated glycosaminoglycans (GAGs) and non-sulfated GAGs, i.e., HA. It is interesting to note that HA is synthesized by hyaluronan synthase-3 (HAS-3), and its receptor, CD44, is found in the epithelium of the porcine SR site. Additionally, sperm capacitation does not occur in vivo in the SR during the pre- and peri-ovulation periods, but spermatozoa in the SR will attempt to capacitate if exposed to bicarbonate. However, capacitation in the SR will rise in the post-ovulation period, indicating the role of HA in modulating sperm capacitation after ovulation. All data support the understanding that the porcine SR ensures the viability of fertile spermatozoa and maintains the non-capacitated status during the pre-ovulation period. This basic knowledge about the SR is believed to be useful to advance sperm preparation procedures for in vitro fertilization (IVF) and improve the preservation process of porcine semen.     

Purification and characterization of an angiotensin I‐converting enzyme inhibitory peptide derived from porcine troponin C

To search for a novel angiotensin I‐converting enzyme (ACE) inhibitory peptide, porcine skeletal troponin was hydrolyzed with pepsin. This hydrolysate showed ACE inhibitory activity, and was applied to various kinds of chromatography to separate an active peptide. Analysis using a protein sequencer identified this peptide as RMLGQTPTK (9mer). This sequence was estimated to occur at the 44–52 position of troponin C, and its 50% inhibitory protein concentration (IC₅₀) was 34 µM. RMLGQTP (7mer), a partial peptide of 9mer, showed activity with an IC₅₀ of 503 µM. RP‐HPLC analysis of a reaction mixture of 9mer and ACE showed that 9mer was slowly hydrolyzed by ACE. On the other hand, 7mer was rapidly hydrolyzed by ACE. Activity of 9mer was reduced as its hydrolysis by ACE proceeded. To estimate the resistance of 9mer to digestive proteases after oral administration, it was reacted with pepsin, α‐chymotrypsin, or trypsin. In each of these reaction mixtures, a significant amount of 9mer remained as a substrate after digestion. Remaining ACE inhibitory activity was close to that of 9mer. These results suggest that 9mer might not be digested after oral administration, because of its relatively high resistance to digestive proteases. Therefore, 9mer might be expected to work well in vivo as an ACE inhibitor.     

Acetylcholine-induced contractions in the porcine internal mammary artery: possible role of muscarinic receptors

The effect of acetylcholine on the isolated, non-precontracted, porcine internal mammary artery (IMA) was investigated. Acetylcholine induced concentration-dependent contractions of non-precontracted IMA rings with denuded endothelium (pEC50 = 5.80 +/- 0.04) and was without effect on arterial segments with intact endothelium. The muscarinic receptor antagonists atropine, pirenzepine, methoctramine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) antagonized the response to acetylcholine. The constrained pA2 values were 10.14, 7.74, 7.34 and 10.5, respectively. It is concluded that acetylcholine induces concentration-dependent contractions of porcine internal mammary artery rings on basal tone and that this contractile effect is probably due to direct cholinergic stimulation of smooth muscle cells, maybe including activation of muscarinic M1 receptors.     

Plasma pyruvate kinase activity vs creatine kinase activity as an indicator of the porcine stress syndrome

Plasma pyruvate kinase (PK) and creatine kinase (CK) activities were increased significantly (P less than 0.001 and P less than 0.05, respectively) in homozygote halothane-reacting pigs (nn), compared with those activities in homozygote nonreacting pigs (NN). Pyruvate kinase activity was less variable within groups than was CK activity, allowing more effective discrimination between nn and NN geno-types. The PK and CK activities in plasma increase with age in halothane-reacting pigs and the nonreacting pigs. Enzyme activities in heterozygote (Nn) nonreacting pigs did not differ significantly (P greater than 0.05) from enzyme activities of homozygote (NN) nonreacting pigs. Although PK was better than CK in identifying stress-susceptible pigs, age-related effects and the failure to identify heterozygotes may restrict the use of plasma PK activity as a diagnostic test for the stress syndrome.     

人参多糖体外抗猪生殖与呼吸综合征病毒活性

猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是严重危害养猪业的传染病之一,也是重点防控的疾病之一。人参多糖(ginseng polysaccharides complex,GPS)具有抗病毒及增强免疫的作用。为了寻找有效预防和治疗PPRS的药物,本试验以Marc-145细胞为模型,采用细胞病变抑制试验和MTT法测定细胞活力,观察GPS在体外抗猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的作用,并通过改变药物作用方式初步探讨GPS的抗病毒机制。试验分为6组,分别为50,100,200和400 mg/L GPS组、病毒对照组及正常细胞对照组。在GPS对PRRSV感染Marc-145细胞的阻断和抑制作用中,各质量浓度GPS均显著提高感染PRRSV后Marc-145细胞的存活率(P<0.05),GPS处理组Marc-145细胞中的PRRSV量均显著低于病毒对照组(P<0.05)。而且,GPS可以促进Marc-145细胞中IL-6和IFN-γ的mRNA表达,也能增强细胞上清中IL-6和IFN-γ的活性。本试验研究表明GPS在体外可有效阻断或抑制PRRSV对Marc-145细胞的感染,提示GPS可作为预防和治疗PRRS的候选药物。     

Immunology of the porcine respiratory disease complex.

PRDC is a multifactorial respiratory syndrome that includes several respiratory pathogens. As can be observed in this article, although the pathogenesis of some of the respiratory pathogens of pigs is fairly well defined, the host response and the immune response necessary to control the pathogen often remain unclear. As our ability to evaluate the porcine immune system and its ability to respond to disease improves, the knowledge of how each of these respiratory pathogens alter and evade the immune system will increase. The pathogens most commonly isolated from pigs with clinical signs of PRDC either infect the cells of the immune system or induce significant immunopathology. Thus, PRRSV and M. hyopneumoniae, the two most common pathogens associated with PRDC, alter the ability of the respiratory immune system to respond to their presence and the presence of other pathogens. By changing the respiratory immune system, these two common pathogens increase the susceptibility to the many other pathogens associated with PRDC. As we learn more about the pathogens of the respiratory system, their interactions with each other, and the mechanisms by which they modulate the immune system, our ability to develop effective control measures will improve.     

Neutralizing activity in the gastrointestinal contents of piglets vaccinated with an ethylenimine-inactivated porcine enterovirus.

The T80 strain of porcine enterovirus was rapidly and completely inactiviated by ethylenimine in a reaction which appeared to follow first order kinetics. The virus was effectively concentrated 35- to 88-fold, with recovery rates of 23 t0 53%, by adsorption to the polyelectrolyte PE60. Multiple doses of adjuvanted, PE60-concentrated, ethylenimine-inactivated T80 virus given by both the oral and subcutaneous routes induced the appearance of significant levels of virus neutralizing activity in the gastrointestinal tract of piglets vaccinated at four weeks of age. This activity, found predominantly in large intestine, was present 14 days after administration of the first dose of vaccine and significant levels of activity were still detectable six weeks later. Titres of serum virus neutralizing activity were higher and more persistent than in piglets which received live or formaldehyde-inactivated T80 virus by the oral or intramuscular routes.     

Trichomonas gallinae: a possible contact-dependent mechanism in the hemolytic activity

The in vitro hemolytic activity of Trichomonas gallinae was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of six adult animals of different species (rabbit, rat, chicken, horse, bovine, and sheep). Results showed that T. gallinae lysed all human erythrocytes groups, as well as rabbit, rat, chicken, horse, bovine and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. gallinae, or with killed organisms. The scanning electron microscopy (SEM) showed that the erythrocytes adhered to the parasite surface and were phagocytosed. These observations suggest that the contact between T. gallinae and erythrocytes may be an important mechanism in the injury caused to the erythrocytes. The hemolytic activity of T. gallinae may be an efficient means of obtaining nutrients for the parasite and allow the investigation of the mechanism used by T. gallinae to damage cellular membranes.     

Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach

Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.     

Selenium stimulates estradiol production in bovine granulosa cells: possible involvement of nitric oxide

Reduction in fertility is well known to be possibly related to selenium deficiencies, even if target organ for selenium action is, at present, unclear. The present study was aimed to examine whether selenium directly influences granulosa cells. Bovine granulosa cells from different size follicles were used to investigate the effect of selenium (5 ng/ml), with or without bovine follicle-stimulating hormone (bFSH) (100 ng/ml), on proliferation and steroidogenesis. In addition, we sought to determine if selenium modulates the production of nitric oxide, which is known to play an important role in ovarian activity. Our data demonstrate that selenium significantly (P < 0.001) stimulates the proliferation of the cells from small follicles; moreover, it further potentiates the stimulatory effect of the gonadotropin in the same cells. Furthermore, selenium significantly (P < 0.01) augments E2 output by cells from both kinds of follicles. bFSH increases E2 production (P < 0.01) by cells from large follicles, whereas it exerts a stimulatory (P < 0.01) effect only in the presence of selenium in the cells from the small ones. The production of nitric oxide is significantly increased (P < 0.001) by bFSH, but only in cells from small follicles. Selenium inhibits (P < 0.001) nitric oxide production in cells from both kinds of follicles and significantly decreases (P < 0.001) bFSH-induced nitric oxide production in cells from the small ones. We conclude that selenium acts on granulosa cells by modulating their proliferation and E2 synthesis; moreover, its effect could be mediated, at least in part, through an inhibition of nitric oxide.     

Endothelium-dependent relaxation of canine uterine artery in response to acetylcholine: the possible involvement of alternative pathways

The effect of acetylcholine on the isolated, pre-contracted, uterine artery of non-pregnant dog was investigated. Acetylcholine-induced concentration-dependent relaxation of isolated canine uterine artery with endothelium (pEC50 = 6.48 +/-0.01, n = 37) and was without effect on arterial segments denuded of endothelium. Indomethacin, 4-aminopyridine (10-5 m) and pre-contraction with K+-rich Krebs-Ringer bicarbonate solution had no effect on acetylcholine-induced relaxation. NG-nitro-l-arginine (l-NOARG) (10-5 m) inhibited relaxation evoked by acetylcholine. Indomethacin applied with l-NOARG led to further inhibition of acetylcholine-induced relaxation. In the presence of both l-NOARG and indomethacin, 4-aminopiridine did not provoke further inhibition of acetylcholine-induced relaxation of canine uterine artery. It is concluded that the acetylcholine-induced relaxation of canine uterine artery is probably mediated by endothelial production of nitric oxide (NO). However, if NO-synthase is inhibited, acetylcholine-induced vasorelaxation may be, in part, mediated through activation of cyclooxygenase pathway.     

Changes in sperm ultrastructure of bulls with decreased sperm activity and freezability]

A decrease of sperm freezability occurred at the K. breeding station, and this situation lasted longer than a year. Out of the 2550 ejaculates taken from 42 breeding bulls within 12 months, 685, i.e. 26.7%, were unfit for use immediately after sperm collection, mostly owing to a low activity of spermatozoa and pathological forms of their motility, and another 469 ejaculates, i. e. 18.3%, were unfit for use after sperm freezing; on the whole, 1154 (i. e. 45.2%) ejaculates had to be excluded. It was revealed by the vital-lethal primuline test that the spermatozoa died quickly after collection. The findings obtained during an electron-microscopic examination of the spermatozoa at the beginning of the process included visible changes in the ultrastructure of the flagellum, particularly its middle piece (deformed shape, incomplete set of axial filaments, vacuolization of the flagellum, abnormal arrangement of the mitochondrial spiral), numerous abnormities of the external cytoplasmic membrane and invagination, vacuolization, and abnormal density of nucleoplasm. The primary changes on the flagella and in the nucleus give evidence that the testicular tissues were altered. The etiological factors behind these processes are believed to include a reduction in the resistance of bulls due to long-lasting consumption of feeds contaminated with the fungus Aspergillus fumigatus, insufficient movement and bad zoo-hygienic practices, all this combined with the secondary action of the infectious germs of Mycoplasma bovigenitalium, which were revealed by cultivation tests in 50% of the ejaculates of the bulls; a positive antibody titre was demonstrated in all bulls.     

Dynamics in the membrane organization of the mammalian sperm cell and functionality in fertilization

The capacitation process of sperm cells involves complex changes in the composition and orientation of molecules at the surface of the sperm cell. Here we focus on the lipid architecture in the sperm plasma membrane and demonstrate that the sperm plasma membrane is not static but is an extremely dynamic structure. Advanced fluoroscopic techniques enabled continuous monitoring of lipid organization in living cells and extremely rapid lipid movements were observed. The orientation of lipids in the sperm plasma membrane changed under capacitative treatments, was found to be sensitive for temperature and also changed upon binding of sperm cells to the zona pellucida. The changes in membrane properties coincided with an activation of protein kinases resulting in tyrosine phosphorylation of specific plasma membrane proteins. The detected membrane changes relate to intrinsic membrane properties such as fluidity, permeability, adhesiveness and fusibility. We think that these results may provide a physiological basis for new assays, able to discriminate between functional and non-physiological sperm cells.     

Dynamics in the membrane organization of the mammalian sperm cell and functionality in fertilization

Summary

The capacitation process of sperm cells involves complex changes in the composition and orientation of molecules at the surface of the sperm cell. Here we focus on the lipid architecture in the sperm plasma membrane and demonstrate that the sperm plasma membrane is not static but is an extremely dynamic structure. Advanced fluoroscopic techniques enabled continuous monitoring of lipid organization in living cells and extremely rapid lipid movements were observed. The orientation of lipids in the sperm plasma membrane changed under capacitative treatments, was found to be sensitive for temperature and also changed upon binding of sperm cells to the zona pellucida. The changes in membrane properties coincided with an activation of protein kinases resulting in tyrosine phosphorylation of specific plasma membrane proteins. The detected membrane changes relate to intrinsic membrane properties such as fluidity, permeability, adhesiveness and fusibility. We think that these results may provide a physiological basis for new assays, able to discriminate between functional and non‐physiological sperm cells.     

Xylazine-induced mydriasis: possible involvement of a central postsynaptic regulation of parasympathetic tone

Intravenous injection of xylazine (0.01 – 1 mg/kg) produced a dose-dependent mydriasis associated with a depression of tonic ciliary nerve activity in anesthetized cats. Xylazine-induced mydriasis was apparent in the sympathectomized iris but was absent in the parasympathectomized, physostigmine-treated iris. Epinephrine (30 μg/kg, i.v.) produced a slighdy greater mydriasis in the sympathectomized iris than in the parasympathectomized, physostigmine-treated iris. The α₂-adrenergic blocking agent, yohimbine (0.5 mg/kg, i.v.) antagonized the pupillary dilation and reversed the depression of ciliary nerve activity induced by xylazine administration.
In rats pretreated with reserpine (7.5 mg/kg, s.c., 20 h) and α-methyl-p-tyrosine (250 mg/kg, i.p., 5 h), intravenous injection of xylazine (0.01 – 1 mg/kg) resulted in mydriasis of similar magnitude as control animals. However, xylazine induced bradycardia in the control group but not in die pretreated animals.
The results suggest that pupillary dilation produced by i.v. xylazine is primarily die result of a central inhibition of parasympathetic tone to the iris. It also appears that xylazine produces this effect via postsynaptic α₂-adrenergic mechanisms, while it produces bradycardia through a presynaptic α₂-adrenergic mechanism.     

Cryodamage of plasma membrane and acrosome region in chicken sperm

Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen‐thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil⁺ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non‐permeating cryoprotectants: trehalose (KEG‐TRE) or glycine (KEG‐GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo‐PRO‐1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG‐GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome–sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo‐PRO‐1) was noted in the KEG‐GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.