Origin of a sperm motility inhibitor from boar seminal plasma

We have investigated the origin of the sperm motility inhibitor (SPMI) from boar seminal plasma. SPMI was measured by its capacity to inhibit the motility of demembranated spermatozoa and by an enzyme-linked immunosorbant assay (ELISA). Among the various reproductive and now reproductive tissues and fluids tested, only the seminal vesicle fluid and seminal plasma contained significant amounts of SPMI biological activity and SPMI antigen. Like other seminal vesicle fluid proteins, SPMI is diluted 6- to 8-fold upon ejaculation. By immunohistochemical detection at the light microscope with antibodies obtained from rabbits immunized with SPMI purified from boar seminal plasma, SPMI was found in the cytosol and/or on the plasma membrane bordering the lumen of the seminal vesicles. At the electron microscope level, SPMI appeared to be present only on the surface of the secretory cells. The data indicate that SPMI originates from a single tissue, the seminal vesicle, and suggest that only the mature form present on the luminal surface of the gland can react with the antibody generated from rabbits immunized with the secreted form of SPMI. © 1993 Wiley-Liss, Inc.     

Drosophila sperm motility in the reproductive tract

Motile cilia and flagella exhibit many waveforms as outputs of dynein activation sequences on the highly conserved axoneme. Motility change of sperm in the reproductive tract is difficult to study and remains an important area of investigation. Sperm typically execute a sinusoidal waveform. Increased viscosity in the medium induces somewhat unusual arc-line and helical waveforms in some sperm. However, whether the latter two waveforms occur in vivo is not known. Using green fluorescence protein imaging, we show that Drosophila sperm in the uterus move in circular foci via arc-line waves, predominantly in a tail-leading orientation. From the uterus, a small fraction of the sperm enters the seminal receptacle (SR) in parallel formations. After sperm storage and coincident with fertilization of the egg, the sperm exit the SR via head-leading helical waves. Consistent with the observed bidirectional movements, the sperm show the ability to propagate both base-to-tip and tip-to-base flagellar waves. Numerous studies have shown that sperm motility is regulated by intraflagellar calcium concentrations; in particular, the Pkd2 calcium channel has been shown to affect sperm storage. Our analyses here suggest that Pkd2 is required for the sperm to adopt the correct waveform and movement orientation during SR entry. A working model for the sperm's SR entry movement is proposed.     

Measurement of boar sperm motility by the trans-membrane migration method

The conventional microscopic methods for evaluating sperm motility of domestic animals are mostly inadequate due to their subjectivity and lack of precision. Recently, a trans-membrane migration method, originally developed for the examination of human sperm motility, has substantially overcome these problems. This study investigated the applicability of the method to boar sperm motility measurement. The apparatus used was simple and consisted only of syringe plungers, poriferous membranes, and modified multi-well culture plates. It measured the proportion of sperm in the semen that moved across the membrane after incubation at 37 degrees C for 3 hr. The sperm motility as measured by this method correlated well with that measured by direct microscopic examinations. The measurement was more reliable using an 8-microns instead of a 5-microns pore-size membrane. The method was found to work equally well for the sperm motility measurement of the semen with a sperm concentration between 1.5 x 10(8)/ml and 6.0 x 10(8)/ml. The results indicate that this method is a simple, objective, quantitative, and reproducible design for the measurement of boar sperm motility.     

The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (HSP90)

Sperm motility is an important parameter for fertility. The molecular mechanisms of mammalian sperm motility are still largely undefined. Our previous observations suggested that heat shock protein 90 (HSP90) may be associated with porcine sperm motility. The aim of the present study was to further characterize the plausible novel function of HSP90 on sperm motility. Semen from normal, sexually mature boars with sperm motility higher than 80% was used. An HSP90-specific inhibitor, geldanamycin (GA), was added to diluted semen at 0.5, 1.0, 2.5 or 5.0 microg/mL and the semen was then incubated at 37 degrees C for 15, 30, 45 or 60 min. Sperm motility was determined by using computer-assisted semen analyzer at the end of incubation. The results indicated that GA significantly reduced sperm motility in a dose and time dependent manner. Moreover, incubation of semen with 5.0 microg/mL GA for 15 min completely stopped sperm motility. To test the reversibility of the GA effect on sperm motility, GA was removed after 30 min incubation and was replaced with fresh extender alone or with extender plus 5 mM caffeine, then incubated for another 15, 30, 45 or 60 min. The results showed that simply removing GA did not reverse the inhibitory effect on sperm motility, while adding caffeine partially reversed this inhibitory effect. However, the effect of 2.5 or 5.0 microg/mL GA was not reversed by caffeine. Considering the specificity of GA targeting to HSP90, the above observations suggested that HSP90 may play a crucial role in regulating porcine sperm motility.     

The interaction of living boar sperm and sperm plasma membrane vesicles with the porcine zona pellucida

Enzymological, morphological, and immunological methods were used to characterize further the interaction of noncapacitated boar spermatozoa with the porcine zona pellucida. Transmission electron microscopy showed that sperm usually bind to the zona over the head region of the cell. Only the plasma membrane is involved in this binding. Bound sperm will undergo the acrosome reaction when treated with calcium and the ionophore A23187. The ability of intact sperm to bind to porcine eggs in vitro and the ability of sperm plasma membrane vesicles to absorb univalent antibody to the sperm binding site for the zona were used to determine the effects of various physical, chemical, and enzymological treatments on the sperm binding sites. These sites were resistant to a number of enzymes including proteases and polysaccharidases, but were inactivated by heat and trichloroacetic acid. Binding sites on the zona were inactivated by extracts from small quantities of sperm. Binding was also blocked by Fab antibody to whole zonae absorbed to other swine tissue and by similarly absorbed Fab antibody to sperm plasma membranes. These data provide further support for the presence of zona recognition sites on the plasma membrane of noncapacitated boar sperm. The binding sites on the sperm plasma membrane do not appear to be peripheral membrane proteins nor major constituents of a surface glycocalyx.     

Investigation of wild boar (Sus scrofa) for porcine reproductive and respiratory syndrome in some territories of Russia

Samples of blood sera and internal organs were collected from 90 shot wild boars (Sus scrofa) in five regions of Russian Federation. Blood sera were tested for antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) using enzyme-linked immunosorbent assay (ELISA). In addition, samples of internal organs (lungs, lymph nodes, spleen) were tested by polymerase chain reaction (nested PCR) for PRRSV antigen. The result of our investigation showed that all samples were negative. However, PRRSV is widespread in domestic swine throughout Russia including the examined regions. Since the results show the absence of PRRSV infection in wild boars in the five examined regions of Russia, wild boars seem not to play any role in the epidemiology of PRRSV in Russia.     

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.     

Activity of angiotensin-converting enzyme (ACE) in reproductive tissues of the stallion and effects of angiotensin II on sperm motility

A testis-specific isoform of angiotensin-converting enzyme (ACE) has been identified in a number of mammalian species. The purpose of this study was to characterize the activity of ACE in equine spermatozoa, seminal plasma, and testis. Activity of ACE was determined in seminal plasma, ejaculated and epididymal spermatozoa from mature stallions as well as from pre- and postpubertal testis. The effect of addition of angiotensin II on equine sperm motility was also evaluated.The activity of ACE in detergent extracted sperm plasma membrane was approximately 13-fold higher than that detected in seminal plasma (93.7 mU/mg versus 7.0 mU/mg protein, respectively). Activity of ACE in equine testis was significantly higher in postpubertal than in prepubertal males (3.0 mU/mg versus 0.4 mU/mg protein, respectively), and ACE activity was reduced (P<0.001) in a dose-dependent fashion by the addition of captopril.The effect of angiotensin II on sperm motility was evaluated by computer-assisted semen analysis in sperm incubated with angiotensin II (0, 1, 10, 100 nM) at 38.5 degrees C. There was no significant effect of angiotensin II on the percent motile sperm; however, there was a significant main effect of angiotensin II (P<0.01) on the kinematic parameters beat cross frequency (BCF), average path velocity (VAP), and curvilinear velocity (VCL), respectively. In addition, there were significant stallionxconcentration interactions for amplitude lateral movement (ALH), BCF, linearity (LIN), straightness (STR), and VCL.This study demonstrates that ACE activity is present in sperm membrane from ejaculated and epididymal spermatozoa and in postpubertal testis. Further studies are required to determine the role of this testis-specific enzyme.     

Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study

Owing to the quick genetic turnover of the pig industry, most AI-boar sires live 2-3 yr, a period during which for 1-2 yr their semen is extended and used in liquid form for AI. Despite showing low cryosurvival, affecting fertility after AI, boar semen is frozen for easiness of transport overseas and reposition of valuable genetics. For the latter, semen is stored in liquid nitrogen (LN₂, cryostorage) for many years, a controversial practice. Here we studied how length of cryostorage could affect sperm quality. Straws (0.5 mL) frozen using the same cryopreservation protocol at one specific location from AI- sires of proven fertility were stored in LN₂ for up to 8 yr. Post-thaw sperm quality was evaluated after 2, 4 or 8 yr of cryostorage, always compared to early thawing (15 d after freezing). Sperm motility and kinematics were evaluated post-thaw using CASA and sperm viability was cytometrically evaluated using specific fluorophores. Sperm viability was not affected by length of cryostorage, but total and progressive sperm motility were lower (p < 0.01) in sperm samples cryostored for 4 or 8 yr compared to those thawed 15 d after freezing. Cryostorage time affected sperm kinetics, but with greater intensity in the samples cryostored for 4 yr (p < 0.001) than in those for 2 yr (p < 0.01). The fact that the major phenotypic characteristic of boar spermatozoa, motility, is constrained by time of cryostorage should be considered when building cryobanks of pig semen. Attention should be placed on the finding that >2 yr of cryostorage time can be particularly detrimental for the post-thaw motility of some sires, which might require increasing sperm numbers for AI.     

Ultrastructure of sperm and male reproductive system in Lineus viridis (Heteronemertea, Nemertea)

Nemerteans possess serially arranged gonads that lie between the midgut pouches. In both sexes the gonads are lined with an epithelium. During maturity, they gain contact to the exterior by a ciliated duct, which is generally assumed to be a derivative of the gonad. Gonad lining and sperm ultrastructure are little known in heteronemerteans, a group of nemerteans belonging to the Anopla, one of the two large nemertean subgroups. Reproduction biology in heteronemertean Lineus viridis allows predicting a modified sperm type, so-called introsperm for this taxon. Nothing is known on the fate of the testes at the end of the reproductive period of this perennial species. In order to test the predictions and to broaden the data base, males of L. viridis were collected at different times of the year. Histological and ultrastructural data show that the gonad wall is lined with different aciliated endothelial cells and germ cells, while the gonoduct is formed by densely ciliated cells. The testes are completely filled with sperm cells during maturity; there is no hint at ongoing spermiogenesis at this time. The sperm consists of head, midpiece and tail. Externally, head and midpiece cannot be discriminated. The acrosome is cup-shaped and lies anterior to the nucleus which contains 6–8 lateral ridges. Three long mitochondria mark the midpiece. They line the posterior section of the nucleus and extend up to the level of the ciliary basal structures. The sperm morphology corroborates the predictions derived from the mode of reproduction. At the end of the reproductive period the male gonads change cellular composition, while the gonoduct degenerates. Provided that both sexes show the same growth rate, male offspring acquire sexual maturity earlier than female offspring, since L. viridis males are always smaller than the females. In contrast to the males, females keep their gonads and gonoducts during most time of the year. Since large males were never found within the studied population, these data indicate that L. viridis might be a consecutive hermaphrodite.     

Porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 infections in wild boar (Sus scrofa) in southwestern Germany

Samples were collected from 203 wild boars (Sus scrofa) hunted in Baden-Wurtemburg, Germany from November-January 2008 and 2009. Samples from the lung and tonsil were analyzed by quantitative polymerase chain reaction (qPCR) for porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European type) and type 2 (American type). A qPCR to detect porcine circovirus type 2 (PCV2)-specific genome was performed on tissue homogenates including lung, tonsils, and inguinal lymph nodes. Serum samples were tested for antibodies against PRRSV and PCV2 by enzyme-linked immunosorbent assay (ELISA). No PRRSV was detected in any of the 203 samples and one sample had detectable antibodies against PRRSV. We detected PCV2 in organ materials from 103 wild boars with a prevalence of 50.7%. The number of wild boars positive for PCV2 by PCR varied according to the population density of wild boars among woodlands. More positive samples were detected in woodlands with a high density of wild boars. We found no correlation between the number of PCV2-positive wild boars and the density of domestic pigs in the surrounding area. The number of wild boars positive for antibodies against PCV2 by the INGEZIM Circovirus IgG/IgM test kit was low (53 sera positive for IgG- and three sera positive for IgM-antibodies) in comparison to the higher positive results from the INGEZIM CIRCO IgG test kit (102 positive and 12 inconclusive results).     

Direct contact between boar spermatozoa and porcine oviductal epithelial cell (OEC) cultures is needed for optimal sperm survival in vitro

Oviductal epithelial cell (OEC) co-culture prolongs sperm viability and motility in vitro in a number of species including humans and horses. This study has sought to determine the effects of homologous OEC co-culture on boar sperm function. To determine whether the effects on spermatozoa were specifically caused by co-culture with or by OEC secretions, or by both factors together, a number of co-culture and cell-conditioned medium (CM) experiments were conducted. Firstly, Percoll-washed spermatozoa were co-cultured with OECs and pig kidney epithelial (LLC-PK1) cells, and in medium without cells. Secondly, Percoll-washed spermatozoa were incubated with CM derived from both OECs and LLC-PK1 cells and in unconditioned medium. A number of sperm function parameters were assessed after 5, 30, 60, 90, 120, and 180 min, and 24h of co-culturing or incubation with CM. Of all the sperm function parameters investigated, the percentage (%) viability data yielded the most interesting results. OECs (mean+/-S.E.M.; 31.2+/-1.10) were better than LLC-PK1 cells (24.3+/-0.93) at prolonging the viability of unbound spermatozoa after 24h of co-culturing (P<0.05). Also after 24h, the viability of spermatozoa bound to the OECs (77.6+/-1.83) was significantly higher than in the case of the LLC-PK1 cells (53.5+/-1.43; P<0.001). Other sperm function parameters, e.g., capacitation and motility, were also influenced by OEC co-culturing and incubation with CM, although to a lesser degree. In conclusion, porcine homologous OEC co-culture and CM incubation specifically affect sperm function. However, we propose that it is OEC co-culturing, rather than OEC-CM, that has the greater influence.     

Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation

We investigated the effect of supplementing extended boar semen with different amounts of hyaluronan (HA) prior to freezing on post-thaw sperm characteristics. Using a split sample design, the effect of HA at a final concentration of 500 or 1000 microg/ml semen on post-thaw motility parameters, and membrane lipid architecture status assessed by merocyanine-540/YOPRO-1 and flow cytometry were evaluated. HA-supplementation improved motility parameters (P < 0.05 to P < 0.001) and decreased the percentage of hyperactivated spermatozoa (P < 0.05). HA-supplemented samples had more spermatozoa showing high lipid membrane stability as assessed with merocyanine-540. In conclusion, HA appeared to preserve post-thaw spermatozoa viability in vitro and maintained membrane stability after cryopreservation.     

Lactose-egg yolk diluent supplemented with N-acetyl-D-glucosamine affect acrosome morphology and motility of frozen-thawed boar sperm

These experiments were carried out to investigate the effect of N-acetyl-D-glucosamine, and to obtain additional information about the effect of orvus es paste (OEP) and egg yolk concentration in the freezing of boar sperm in the maxi-straw. The highest post-thaw acrosomes of normal apical ridge (NAR) and motility were obtained with 0.025 or 0.05% N-acetyl-D-glucosamine concentration in the first diluent. However, there were no effects of N-acetyl-D-glucosamine among the diluents with or without N-acetyl-D-glucosamine at the second dilution. The N-acetyl-D-glucosamine in the first and second diluents was added at room temperatures (20-23 degrees C) and 5 degrees C, respectively. It is suggested that the temperature of N-acetyl-D-glucosamine addition is important for the effect of boar sperm protection during freezing and thawing. When the 0.05% N-acetyl-D-glucosamine was supplemented in the first diluent, the optimum final OEP content was 0.5%. The optimum content of egg yolk in the diluent with 0.05% N-acetyl-D-glucosamine concentration was 20% and egg yolk was one of the main cryoprotective agents. In conclusion, we found out that the diluent with 0.025 or 0.05% soluble N-acetyl-D-glucosamine in the first diluent, 0.5% final orvus es paste concentration and 20% egg yolk concentration significantly enhanced NAR acrosomes and motility of boar sperm after freezing and thawing.     

Calcium-induced quiescence of sperm motility in the bluegill (Lepomis macrochirus)

Before dilution in hypoosmotic media sperm of freshwater fish are maintained quiescent by a range of factors including osmolality, K+ and pH, and the onset of motility is generally associated with an increase in cytoplasmic Ca2+. In contrast, Ca2+ in conjunction with osmolality was found to inhibit motility of intact bluegill sperm. Consistent with seminal plasma composition, 0.16 mmol/L Ca2+ and greater, in conjunction with an osmotic concentration of 290 mOsm/kg, inhibited the onset of bluegill sperm motility; sperm diluted in saline at 290 mOsm/kg without Ca2+ became motile. Cations Mn2+ and Sr2+, in conjunction with osmolality, had an inhibitory effect on initiation of sperm motility similar to that of Ca2+. Sperm motility was inhibited by Ca2+ channel blockers nimodipine and nifedipine, the mitochondrial Ca2+ uniporter inhibitor ruthenium red and the calmodulin inhibitors W-7 and trifluoperazine dihydrochloride. These results provide evidence that elevated cytoplasmic Ca2+ inhibits sperm motility and yet low levels permit or promote motility. This study demonstrates a unique inhibitory action of Ca2+ on the motility of intact fish sperm at physiologically relevant levels.     

Seroprevalence of six reproductive pathogens in European wild boar (Sus scrofa) from Spain: the effect on wild boar female reproductive performance

We studied the seroprevalence of six reproductive pathogens in Spanish hunter-harvested wild boar females. The sample was representative of the hunting harvest in the studied hunting estates. Mean antibody prevalences were: 60.6+/-0.06% for Aujeszky's disease virus (ADV), 56.6+/-0.09% for porcine parvovirus (PPV), 51.8+/-0.06% for porcine circovirus type 2 (PCV2), 29.7+/-0.09% for Brucella spp. and 36.3+/-0.1% for Toxoplasma gondii. We did not detect antibodies against porcine reproductive and respiratory syndrome virus (PRRSv). ADV seroprevalence was associated with PPV and PCV2 seroprevalence in Spanish wild boar females. Ovulation rate in the studied wild boar females was 4.41+/-0.16 (n=120), mean litter size was 3.91+/-0.16 (n=82) and the partial resorption index 0.92+/-0.17 (n=66). Ovulation rate and litter size were statistically associated with age. T. gondii seroprevalence was negatively related to ovulation rate and partial resorption index. Wild boars from managed fenced estates had antibodies against more pathogens than wild boars from open estates. Potential relations between management of wild boar populations and exposure of individuals to different reproductive pathogens are discussed.     

Zinc ion-dependent protein in boar semen. II. Effects on sperm motility and antibacterial properties

New functions of a zinc ion-dependent protein isolated from boar seminal plasma are presented. The results of studies suggest that plasma proteins in boar semen are arranged specifically and control the motility of the spermatozoa. The zinc ion-dependent protein seems to be a factor inactivating the plasmatic inhibitor of spermatozoa motility. The protein exhibited a regulating activity in the pH range 7.3–8.2, and at a quantitative protein ratio of 13:1, in favour of the inhibitor.This protein also inhibited the growth of bacteria, especially Gram-positive species. At a concentration of 4 mg/ml of the medium, the protein or its fractions totally inhibited the growth of bacteria of the genus Micrococcus after 6 h of incubation. As regards other strains of bacteria, total inhibition of growth was observed after 24–48 h. Antibacterial activity of the protein did not change after a short period of heating at 100°C, nor after freezing/thawing.