Glucocorticoids play a physiologic role in the adult male reproductive functions, modulating gonadal steroid synthesis and spermatogenesis, through the glucocorticoid receptor (GR). The expression of GR has been described in several key testicular cell types, including somatic cells and early germ cell populations. Nothing is known on GR in human spermatozoa. Herein, we explored the GR expression and its possible role in normal and testicular varicocele semen samples from volunteer donors. After semen parameter evaluation by macro- and microscopic analysis, samples were centrifuged; then spermatozoa and culture media were recovered for further investigations. By western blotting and immunofluorescence analyses we evidenced for the first time in spermatozoa the presence of GR-D3 isoform which was reduced in sperm from varicocele patients. By treating sperm with the synthetic glucocorticoid dexamethasone (DEXA), we found that survival, motility, capacitation, and acrosome reaction were increased in both healthy and varicocele samples. GR involvement in mediating DEXA effects, was confirmed by using the GR inhibitor mifepristone (M2F). Worthy, we also discovered that sperm secretes different cortisol amounts depending on its physio-pathological status, suggesting a defence mechanism to escape the immune system attach in the female genital tract thus maintaining the immune-privilege as in the testis. Collectively, our data suggests a role for glucocorticoids in determining semen quality and function, as well as in participating on sperm immune defensive mechanisms. The novelty of this study may be beneficial and needs to take into account in artificial insemination/drug discovery aimed to enhancing sperm quality.
Sa, S.J.;Wee, M.S.;Oh, J.Y.;Cheong, H.T.;Park, S.B.;Yang, B.K.;Kim, C.I.;Park, C.K.
Korean Journal of Animal Reproduction
/
v.25
no.4
/
pp.327-337
/
2001
This study investigated the effects of superoxide dismutase (SOD) on lipid peroxidation and fertilizing ability in vitro of boar spermatozoa frozen-thawed. The percentages of motile sperm were highest when SOD of 10 units/$m\ell$ was added to washing medium for spermatozoa. However, the rates of motile sperm were not significantly different in different concentrations of SOD. On the other hand, the motile rates of sperm washed with SOD were lower in sperm inculbated for 120 min than 30 min regardless of the different concentrations of SOD. The percentage of spermatozoa that reached acrosome reaction were increased with incubation periods prolonged. No significant differences, however, were observed in acrosome reaction rates between sperm incubated with and without SOD supplementation for 0, 60 and 120 min. When oocyies inseminated with different concentrations of SOD, the penetration rates were significantly (P<0.05) higher in medium with 1 unit/$m\ell$ than 0, 10 and 100 units/$m\ell$ of SOD. However, the proportions of polyspermit oocytes were significantly (P<0.05) lower in medium with 10 and 100 units/$m\ell$ than 0 unit/$m\ell$ of SOD. In another experiment, the sperm suspension were also treated with different concentrations of SOD and were assayed far sulfhydryl(-SH) group content. In the groups treated with 100 units/$m\ell$ of SOD, sperm-SH group were higher than another groups. However, sperm-SH group content were not siginificantly different in spermatozoa treated with different concentrations of SOD. Under the same conditions, the lipid peroxidation of sperm was evaluated on the basis of malondialdehyde production. The addition of SOD to sperm suspension decreased the formation or malondialdehyde. However, there were not significantly different in sperm treated with different concentrations of SOD. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. The sperm binding to zona pellucida were gradually increased with SOD concentrations added. The number of spermatozoa binded to zona pellucida were significantly (P<0.05) higher in medium with 100 units/$m\ell$ than 0 units/$m\ell$ of SOD. These findings suggested that SOD cause an enhancement penetrarion ability and sperm zona binding in boar spermatozoa frozen-thawed.
It has been reported that plasma membrane activity of the spermatozoa may be susceptible to be influenced by extracellular osmolality and such membranous changes involve infracellular molecular changes, special regard to the structure of membranous lipids, and the accompanying ion-channel of which are closely related with their fluidity of $Ca^{2+}$ and HCO$^{-}_{3}$. It is of common recognition that a certain kind of sterol acceptor player an important to induce lipid fluctuation of the sperm plasma membrane which have been influenced by BSA administration and came in effect to outflow of cholesterol from the spermatozoa and resulted in changes of ionic fluidity to facilitate adenylyl cyclase, and to induce protein tyrosine phosphorylation by increase of cAMP and activation of PKA. Thus it seems likely that an augmentation of the acrosomal reaction is closely related with protein tyrosine phosphorylation. The following experimental results were obtained in the present study; Under the high osmolality conditions, the spermatozoa motility declined significantly and the structural change of the plasma membrane diminished to confirm that the response degrees to the osmolality depended upon the water transfer volume through the plasma membrane and the changes of cellular volume. Those experimental results suggest that a physiological parameter such as low temperature condition played an important role for presentation of spermatozoa and that inducement of spermatozoa activation for reinforcement of protein tyrosine phosphorylation. On the other hand, it seemed likely that the BSA administration as one of sterol accepters might represent a key role also under the high osmolality condition and their result also suggests that osmolality change, special regard to high osmolality condition may play an important role also in the processes of signal transmission.
Objective: Present investigation was aimed to study the Single Nucleotide Variants of the luteinizing hormone beta ($LH{\beta}$) gene and to analyze their association with the semen quality (fresh and post-thawed frozen semen) and luteinizing hormone (LH) concentrations in Murrah buffalo bulls. Methods: Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and Sanger sequencing method is used to study genetic variability in $LH{\beta}$ gene. LH assay was carried out using enzyme-linked immunosorbent assay method. A fixed general linear model was used to analyze association of single nucleotide polymorphism (SNP) of $LH{\beta}$ gene with semen quality in 109 and LH concentrations in 80 Murrah bulls. Results: $LH{\beta}$ gene was found to be polymorphic. Total six SNPs were identified in $LH{\beta}$ gene g C356090A, g C356113T, g A356701G, g G355869A, g G356330C, and g G356606T. Single Stranded Conformational Polymorphism variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on sperm concentration (million/mL), percent mass motility, acrosome integrity and membrane integrity in fresh and frozen semen whereas significant (p<0.05) effect was observed on percent live spermatozoa. SSCP variants of pattern 2 of exon 1+pattern 2 of exon 2+pattern 1 of exon 3 had highly significant (p<0.01) effect on luteinizing hormone concentrations too. Conclusion: The observed association between SSCP variants of $LH{\beta}$ gene with semen quality parameters and LH concentrations indicated the possibilities of using $LH{\beta}$ as a candidate gene for identification of markers for semen quality traits and LH concentrations in Murrah buffaloes.
Park, C. K.;J. Y. Ann;Kim, I. C.;Lee, J. H.;B. K. Yang;Kim, C. I.;H. T. Cheong
Korean Journal of Animal Reproduction
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v.25
no.4
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pp.317-325
/
2001
This study investigated the effect of ferrous sulfate (Fe$^{2+}$) and/or ascorbic acid (Asc) on fertilizing ability in vitro of frozen-thawed boar spermatozoa. Using chlortetracycline (CTC) fluorescence, the spermatozoa was treated in preincubation medium with control, Fe$^{2+}$(1 mM), Asc (0.5 mM) and Fe$^{2+}$Asc to assessed for acrosome reaction, and the oocyte penetration test to determine whether the Fe$^{2+}$ and/or Asc can promote the penetration ability in vitro. When frozen-thawed spermatozoa was washed with preincubation medium, there were significantly (P < 0.05) more acrosome-reacted in medium with Fe$^{2+}$Asc (38%) than control (27%). The penetration rates were also significantly (P < 0.05) higher in medium with Fe$^{2+}$Asc (76%) than control (55%). Next, the lipid peroxidation of sperm was evaluated on the basis of malondialdehyde production following same treatments. The addition of Fe$^{2+}$Asc to sperm suspension increases the formation of malondialdehyde. However, there were not significantly different under the all conditions. The sperm suspension were also treated with control, Fe$^{2+}$, Asc and Fe$^{2+}$/Asc and assayed for sulfhydry1(-SH) group content. In the Fe$^{2+}$/Asc group, sperm-SH group were higher than another groups. In spermatozoa treated with Fe$^{2+}$ and/or Asc, however, no changes in sperm -SH-groups were detected when compared to controls. In another experiment, the activity of sperm binding to zona pellucida was evaluated through binding to salt-stored porcine oocytes. In control and Asc treatment groups, sperm binding to zona pellucida were significantly (P < 0.05) higher than in medium with Fe$^{2+}$. On the other hand, there is not a significant increase in binding to zona pellucida with spermatozoa treated by Fe$^{2+}$/Asc. In summary, the present study suggests that Fe$^{2+}$/Asc causes an enhancement in fertilizing ability that is associated with penetration rate increased without change of spermatozoa binding capacity to homologous zona pellucida.o homologous zona pellucida.
The present study evaluated whether an exogenous antioxidants, taurine and $\alpha$-tocopherol, could, when added to the freezing extender, improve the post-thaw sperm characteristics, function, the level of reactive oxygen species (ROS) generation, and the level of lipid peroxidation (LPO) in frozen-thawed porcine semen. CASA (computer-aided sperm analysis), HOST (hypoos-motic swelling test), chemiluminescence using luminol and lucigenin and the detection of malondialdehyde for LPO was performed in frozen-thawed porcine sper-matozoa. The results obtained in these studies are as follows. While no beneficial effects of taurine and $\alpha$-tocopherol supplementation were visible in motility, viability, acrosome reaction, tail swelling patterns, and the generation of $O^{2-}$ of frozen-thawed porcine sper-matozoa, $H_{2}O_{2}$ was decreased by all treatments except taurine 50mM treatment. In conclusion the taurine and $\alpha$-tocopherol treatments during freezing reduced generation of reactive oxygen species and production of malondialdehyde in frozen-thawed porcine semen, and the ROS savangers may minimize various damages of spermatozoa during freezing.
The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and embryo cleavage in vitro in pig. The semen of miniature pig collected by gloved-hand method pre-warmed ($37^{\circ}C$) in thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with cleavage rate of embryo after IVF using separated and un-separated sperm by percoll. The result, viability of separated sperm was significantly(p<0.05) higher($83.6{\pm}$2.0 vs $59.0{\pm}4.4%$) than un-separated sperm. The results of CTC analysis showed the percentage of F- and B-patterned separated sperm was higher in separated that than un-separated sperm. On the contrary, the percentage of AR-patterned form unseparaed sperm was significantly(p<0.05) higher($13.6{\pm}0.8$ vs $8.1{\pm}0.6%$) than separated sperm. Also, abnormality of un-separated sperm was significantly(p<0.05) higher($2.2{\pm}0.4$ vs $16.8{\pm}2.8%$) than separated sperm. However, the cleavage rates of embryo using separated sperm by percoll and un-separated sperm had not significantly difference on 2 cell stage(9.25 vs 11.88%), 4 cell stage(26.76 vs 24.51%) and >4 cell stage(63.99 vs 63.61%) at 48h of IVF. Therefore, the sperm separated by percoll method showed improvement in sperm quality than un-separated sperm in miniature pig.
Objective: Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the zona pellucida (ZP) of several mammalian species including pigs. Treatment of oocytes with various lectins blocks sperm binding to the ZP in various mammalian species. This study was undertaken to examine the distribution of sugar residues in the ZP of pig oocytes matured in vitro and the ability of spermatozoa to bind to ZP and in vitro penetration in oocytes treated with fluorescein isothiocyanate (FITC)-labelled lectins. Materials and Methods: The lectins of Banderiaea simplicifolia (BS-II, bind to $\beta$-D-N-acetylglucosamine), Canavalin ensiformis (Con A, bind to $\alpha$-D-Mannose), Lens culinaris (LCA, bind to a-D-Mannose), Ricinus communis (RCA-I, bind to $\beta$-D-Galactose) and Ulex europaeus (UEA-I, bind to $\alpha$-L-Fucose) were examined for spermatozoa penetration, binding capacity to ZP and distribution of lectins. Results: The penetration rates were significantry (p<0.05) higher in control oocytes (63%) than those treated with all lectins, but penetration rates ($40{\sim}49%$) were simililar in group treated with lectins. The incidence of monospermy was similar in oocytes untreated and UEA-I, but it was higher in oocytes treated with BS-II, Con A, RCA-I and LCA. The porcine oocytes cultured for 48 h in TC-199 medium were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescein illumination, higher (p<0.001) proportions of oocytes showed fluorescein of zona pellucida after treatment with Con A (93%), LCA (93%) and RCA-I (100%) than BS-II (37%) and UEA-I (50%). All of the oocytes treated with RCA-I exhibited strong fluorescein in the outer region of the zona pellucida while those treated with LCA exhibited strong fluorescein throughout the zona pellucida. BS-II bounded mainly to the outer region and UEA-I bounded mainly to the inner region of the zona pellucida, with either strong or weak fluorescein. At 120 min after insemination in vitro, fewer spermatozoa were bound to the zona pellucida of the oocytes treated with BS-II, Con-A and RCA-I. Of the lectins, Con A most inhibited sperm binding. Conclusions: These results suggest that $\beta$-D-Galactose residues in the porcine zona pellucida may act as primary sperm receptors and inducers of the sperm acrosome reaction and these sugar residues may be involved in the block to polyspermy.
Ji, D.Y.;Kim, C.K.;Lee, J.H.;Park, S.J.;Ryu, L.S.;Ryu, J.W.;Lee, J.H.;Jeong, Y.C.;Pang, M.G.
Journal of Animal Science and Technology
/
v.47
no.6
/
pp.925-936
/
2005
The present study evaluated whether pentoxifylline added to the freezing extender could improve the sperm characteristics and function in canine frozen semen. Also the conception rate following AI with frozen-thawed semen was investigated. The beneficial effects of pentoxifylline supplementation were visible in motility, viability, acrosome reaction, and tail swelling patterns. Especially, highest sperm viability and function were obtained in the forzen semen supplemented with 1mM pentoxifylline. The follicle size measured by ultrasonography was 6.48 mm, 11.52 mm and 8.9 mm on 11, 13 and 15 days after the onset of natural estrus, respectively and ovulation occurred on 13 and 15 days. The pregnancy rates in bitches inseminated with frozen semen on natural and induced estrus were 71.4% and 75.0%, respectively. There was no significant difference between the pregnancy rates in bitches inseminated with frozen semen following natural and induced estrus, but the litter size was slightly increased in natural cycle.
This study was undertaken to evaluate the effects of $\beta$-mercaptoethanol ($\beta$-ME) on lipid peroxidation and fertilization ability in vitro by xanthine (X) - xanthine oxidase (XO) system in boar spermatozoa frozen-thawed. The boar spermatozoa were treated with X and/or XO, and the spermatozoa viability were measured by the eosin-nigrosin stain method. In control group, level of vitality in boar spermatozoa were higher than in medium with X, XO and X+XO groups. No significant differences, however, were observed under the all conditions. The percentage of spermatozoa that reached acrosome reaction were significantly (P<0.05) higher in sperm treated without that than with $\beta$-ME under the all conditions. On the other hand, when spermatozoa were inseminated in medium with X and/or XO, the penetration rates in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. The lipid peroxidation of sperm was evaluated on the basis of malondialdehyde (MDA) production. The MDA were higher in sperm treated without that than with $\beta$-ME under the above all conditions. However, significant differences were not observed between medium with and without $\beta$-ME. Sperm-SH group were higher detected in medium with that than without $\beta$-ME under the all conditions. The activity of sperm binding to Bona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were significantly (P<0.05) higher than in medium with X+XO groups. The sperm binding in all conditions were higher in medium with that than without $\beta$-ME. However, significant differences were not observed between medium with and without $\beta$-ME. These results suggest that addition of $\beta$-ME in X-XO system may play a positive role in improving of fertilization ability in vitro.
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