• Asian-Australasian Journal of Animal Sciences
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• v.15 no.7
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• pp.949-956
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• 2002

There are several physiological and pharmacological evidences indicating that opening of voltage dependent $Ca^{2+}$ channels play a critical role in induction of acrosome reaction in mammalian sperm. We determined the intracellular free $Ca^{2+}$ concentration in ejaculated goat sperm using a fluorescent, $Ca^{2+}$-specific probe, Fura2/AM, after the suspension of sperm in KRB medium, capable of sustaining capacitation and the acrosome reaction. We used nifedipine, D-600 and diltiazem, the $Ca^{2+}$ channel antagonists belonging to the classes of dihydropyridines, phenylalkylamines and benzothiazepines, to investigate the possibility that L-type voltage gated $Ca^{2+}$ channels play a role in the progesterone-stimulated exocytotic response. Progesterone promoted a rise in intracellular $Ca^{2+}$ in goat sperm and addition of nifedipine (100 nM) just prior to progesterone induction, significantly inhibited both intracellular $Ca^{2+}$ rise and exocytosis suggesting that $Ca^{2+}$ channels are involved in the process. However, the intracellular $Ca^{2+}$ increase during the process of capacitation was not affected with the addition of nifedipine suggesting a role of focal channel for $Ca^{2+}$ during capacitation. Studies using monensin and nigericin, two monovalent cation ionophores showed that an influx of $Na^+$ also may play a role in the opening of $Ca^{2+}$ channels. These results strongly suggests that the entry of $Ca^{2+}$ channels with characteristics similar to those of L-type, voltage-sensitive $Ca^{2+}$ channels found in cardiac and skeletal muscle, is a crucial step in the sequence of events leading to progesterone induced acrosome reaction in goat sperm.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.41-48
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• 2013

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

• Applied Microscopy
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• v.30 no.1
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• pp.21-44
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• 2000

This study was carried out to investigate sperm storage, and the fate of spermatozoa in the female reproductive tract during hibernation in Korean greater horseshoe bat, Rhinolophus ferrumequinum korai. (1) Numerous sperm occurring in uterine lumen and glands were engulfed, and disappeared by the polymorphouclear leucocytes during the hibernation. (2) The stored sperm present in caudal isthmus of oviduct only, the heads of sperm toward the oviductal epithelial cells. Therefore, the projected sperm during the mating season are only alive in the caudal isthmus of oviduct in the long hibernation. The present result suggests that the caudal isthmus of oviduct may play an important role as the principal storage site in capacitation of sperm. (3) In March, the sperm do not occur in the caudal isthmus of oviduct. It suggests that the stored sperm in the caudal isthmus of oviduct should migrate to the ampulla of the site of fertilization to meet ovum in the period of ovulation. The results of this experiment consider that prolonged sperm storage, fate of sperm and sperm migration in the long hibernation have a kind of mechanism for the fertilization.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 1998.07a
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• pp.41-42
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• 1998

The changes in calcium binding protein(CBP) of mouse epididymal sperm during their post-testicular differentiation were analyzed by two-dimensional SDS-PAGE. According to dpididymal maturation, capacitation and acrosome reaction of spermatozoa, both quantitative and qualitative changes of CBPs in the epididymal sperm was detected. It suggested that the development of fertilizing ability of epididymal sperm was closely related to the changes in the CBPs profiles of sperm during epidiyaml transit.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.2
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• pp.181-186
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• 2009

In our previous report, we demonstrated that the tyrosine phosphorylation of sperm proteins (TPSP) of guinea pig was associated with capacitation and hyperactivation (CAHA), and $Ca^{2+}$ and ${HCO_3}^-$ were required for the initiation of CAHA and increasing the TPSP. The aim of this study was to further investigate the mechanism underlying the above events. The results showed that addition of cAMP agonists, dibutyryl-cAMP (db-cAMP) and isobutyl-methylxantine (IBMX), to ${HCO_3}^-$ -free medium significantly increased CAHA to the normal level (when sperm were incubated in TALP). Although addition of the cAMP agonists to $Ca^{2+}$-free medium increased CAHA, the percentages of hyperactivated and capacitated sperm were still significantly lower than the normal level. Compared with ${HCO_3}^-$ -free or $Ca^{2+}$-free medium, TPSP was increased when db-cAMP and IBMX were added in the media. H-89, a specific inhibitor of protein kinase A (PKA), inhibited CAHA in a dose-dependent manner and totally blocked TPSP. These results confirm a previous observation that $Ca^{2+}$ and ${HCO_3}^-$ regulated CAHA and TPSP in a cAMP/PKA pathway, and support an interation between TPSP and CAHA of sperm. Besides the cAMP/PKA pathway, $Ca^{2+}$ might have also played a role in regulating CAHA by other pathways since the normal level of CAHA did not recover by adding cAMP agonists in the media.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.41-46
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• 2010

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution I: 11% Lactose or Triladyl + egg yolk; solution II: solution I + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CIC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p<0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.201-209
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• 2000

Objective: This study has been carried out to evaluation the effect of fertilization promoting peptide (FPP) on the kinematic parameters, capacitation and acrosome reaction of the frozen-thawed human spermatozoa. Methods: After FPP treatment, we examined kinematic parameters, capacitation and acrosome reaction, using the methods of computer-aided sperm analysis (CASA) and chlortetracycline (CTC) fluorescence analysis. Results: We have obtained the evidence that FPP can promote the capacitation and inhibit the spontaneous acrosome reaction of frozen-thawed human spermatozoa in vitro. FPP ($25{\sim}100$ nM) induced a significant increase in the proportion of B-pattem capacitated spermatozoa, and a significant decrease in the proportion of F-pattem uncapacitated ones without significant stimulation of acrosomal exocytosis. In the kinematic parameters treatment, FPP treated groups maint3ined higher LIN, BCF and STR than those of control. The VAP, VSL, VCL and ALH were not different. Therefore it is suggested that FPP in human seminal plasma may play a positive role in promoting human sperm function.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.2
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• pp.178-188
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• 2013

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.225-231
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• 1996

This study was conducted to investigate the effects of preincubation time, concentration and exposure time of sperm on in vitro fertilization of porcine follicular oocytes rnatured in in vitro. The results obtained are as follows ; 1. Effect of preincuhation time for porcine sperm capacitation on in vitro fertilization in medium with heparin was investigated. Normal fertilization rate was highest in 15 min(26.4%). However, there were no significant differences among preincuhation times of 5~90 min, 2. Normal fertilization rates of sperm concentrations were 17.0~26.5%, and normal fertilization rate from l$\times$ l05cell /ml concentration was also higher than those of other sperm concentration. 3. Normal fertilization rates of sperm exposure time of 4, 8, 12, 16 and 20 hours were 6.1, 20.8, 27.8, 25.0 and 26.7%, respectively. Normal fertilization rate from sperm exposure time of 12 hours was also higher than that of other sperm exposure times.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.142-148
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• 2020

The research work was undertaken to determine an effective fertilization medium, sperm separation method and sperm capacitating agent for optimum in vitro fertilization (IVF) rates of indigenous zebu cow oocytes. In experiment 1, tissue culture medium (TCM 199), Tyrode's albumin lactate pyruvate (TALP) and Brackett and Oliphant (BO) medium were used as basic medium for IVF of oocytes of indigenous zebu cows. In experiment 2, three sperm separation methods namely centrifugation, swim up and percoll gradient methods were used for separation of motile and viable spermatozoa for IVF. In experiment 3, for capacitation of spermatozoa, IVF medium supplemented with the heparin, mixture of penicillamine, hypotaurine and epinephrine (PHE) or the combination of heparin with PHE were used for fertilization. In vitro culture (IVC) of presumptive zygotes was done in modified synthetic oviduct fluid (mSOF) medium using standard procedure 24 h after sperm-oocytes co-culture. The cleavage rate was determined to evaluate the efficacy of fertilization medium, sperm separation method and sperm capacitating agent 24 h after IVC. The cleavage rate was higher in oocytes fertilized in TALP (63.3%) than in TCM 199 (47.5%) (p < 0.05). The cleavage rate was higher in oocytes fertilized by spermatozoa separated by percoll gradient method (62.3%) than by centrifugation (51.6%) (p < 0.05). The cleavage rate of oocytes was higher when insemination was done with spermatozoa capacitated in TALP supplemented with heparin and PHE (61.3%) compared to control (40.9%) (p < 0.05). In conclusions, TALP based medium and percoll gradient sperm separation followed by capacitation with combination of heparin and PHE are suitable for IVF of indigenous zebu cow oocytes in Bangladesh.