• Journal of Animal Science and Technology
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• v.64 no.2
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• pp.235-246
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• 2022

In this study, we used more reliable experimental materials and methods to detect the effects of osteopontin (OPN) on boar sperm in vitro capacitation, acrosome reaction, and fertilization efficiency. We reorganized and obtained the OPN protein of the porcine source. Immunofluorescence and Western blot show the localization and expression of the OPN protein before and after sperm capacitation. To determine whether OPN can affect sperm during sperm capacitation, we examined cyclic adenosine monophosphate (cAMP) concentrations after sperm capacitation, and the results showed that OPN significantly increased the cAMP concentration in sperm (p < 0.05). Flow cytometry showed that 0.1 ㎍/mL OPN-treated sperm had better acrosome reaction ability. In vitro fertilization (IVF) showed that 0.1 ㎍/mL OPN significantly increased the rate of embryo division. In conclusion, this study found that 0.1 ㎍/mL porcine OPN protein can significantly improve porcine capacitated sperm motility, cAMP concentration after capacitation sperm, acrosome reaction ability, and embryo division during IVF and provides new clues to explore the mechanism of OPN's function on sperm.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.285-291
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• 2022

In 1951, Colin Russell Austin and Min Chueh Chang identified "capacitation", a special process involving ejaculated spermatozoa in the female reproductive tract. Capacitation is a phenomenon that occurs in vivo, but almost all knowledge of capacitation has been obtained from in vitro studies. Therefore, numerous trials have been performed to establish in vitro capacitation methods for various studies on reproduction. Although a series of studies have been conducted to develop an optimal protocol for inducing capacitation, most have focused on identifying the appropriate chemical compounds to induce the capacitation of boar spermatozoa in vitro. Therefore, the purpose of this study was to identify the optimal incubation time for inducing capacitation in vitro. Duroc semen was incubated for various periods (60, 90, and 120 min) to induce capacitation. Sperm function (sperm motility, motion kinematic parameters, and capacitation status) was evaluated. The results showed that total sperm motility, rapid sperm motility, progressive sperm motility, curvilinear velocity, and average path velocity significantly decreased in a time-dependent manner. However, the capacitation status did not show any significant changes. Taken together, these results indicate that an incubation time of more than 60 min suppresses sperm motility and motion kinematic parameters. Therefore, we suggest that 60 min may be the best incubation time to induce capacitation without negative effects on sperm motility and motion kinematics in boar spermatozoa in vitro.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.287-296
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• 2011

Mammalian fertilization is a complex cascade process consisting of sperm migration through the female reproductive tract, physiological changes to sperm such as sperm capacitation and acrosome reaction, and sperm-egg interaction in the oviduct in vivo. On the other hand, in vitro fertilization (IVF) is a process by which egg cells are fertilized by sperm outside the body: in vitro. IVF has been used for a variety of purposes in reproductive biotechnology for human and animals. The discovery of sperm capacitation in 1951 promoted the development of IVF technology. In the initial stage of IVF, sperm capacitation in preincubation medium was shown to be essential to fuse with eggs. Besides, sperms should detour some of the in vivo regulations for IVF. This review introduces a general mammalian fertilization process, including sperm capacitation, removal of cumulus matrix, acrosome reaction, and sperm-egg fusion and focuses on the roles of key biochemical molecules, signal mechanisms, and genes involved during IVF and novel results of sperm-oocyte interaction elucidated in various gene-knockout mice models.

• Journal of Embryo Transfer
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• v.7 no.1
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• pp.21-30
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• 1992

This study was aimed to develope the new method for bovine sperm capacitation through testing and combinating factors relating to sperm capacitation. For verifying the efficiency of this method on inducing capacitation, in vitro fertilization was carried out. The results obtained were as follows: When pHs of HBS /PR for sperm-washing were 5.99, 6.38, 6.78, 7.10, 7.40, 7.69, 8.15, 8.45 and 8.83, variances between light absorhance differences obtained from sperm pre-washing solution and post-washing solution(VADs) were 0.000, 0.001, -0.001, -0.005, -0.005, -0.021,-0.017,-0.016,and-0.036, respectively. There were significant decreases of VADs in pH 7.69 and pH 8.83. When sperm were firstly, secondly, and thirdly washed with HBS /PR, VADs were -0. 024, - 0.006, and - 0.004, respectively. There was significant decrease of VAD in 1st sperm-washing. When washed-sperm were secondly washed with HBS /PR supplemented with no (con-trol), heparin, CSA, PC12, and BSA, VADs were 0.009, -0.024, -0.008, -0.009, and 0.014, respectively. When the sperm were thirdly washed with HBS /PR with no(control), heparin, CSA, PC12 and BSA, VADs were 0.020, -0.007, 0.005, 0.006, and 0.019, respectively. Only heparin treatment showed the negative values of VADs in 2nd and 3rd sperm-washing. Of oocytes cultured with sperm which were repeatedly washed with heparin and high pH, 52% (57/110) were cleaved over 2 cell stage. However, percent of oocytes parthenogenetically divided was 5%(2 /42).

• The Korean Journal of Zoology
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• v.38 no.4
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• pp.514-522
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• 1995

In order to examine the interaction of albumin with the sperm during capacitation in mouse, proteins of cauda epididymal sperm were extracted under various conditions and analyzed with SDS-PAGE. Sperm surface labeling patterms were also examined using fluorochroin~conjugated wheat germ agglutinin (WGA) and bovine serum albumin (BSA). Albumin was detached from the sperm surface during the incubation and seemed to be constituted the major protein components of the conditioned media in which sperm incubated for 90 mm. Detachment of albumin from the sperm was not affected by the Ca2+ in the medium. WGA-FITC labeling confirmed that Triton X-100 permeabilired plasma membrane overlaying the apical segment of sperm head and detached plasma membrane associated proteins having negatively charged glycoconjugates. BSA-FITC labeling of epididymal sperm occurred on the apical segment of periacrosoinal region and postacrosomal region of the head. BSA-FITC labeling was not observed in periacrosoinal region of the sperm treated with Ca2+-ionophore ~3187 (10 MM)~ whereas the postacrosome region of acrosome-reacted sperm was still labeled after the AR. These results suggest that albumin bound to the surface of epididymal sperm is detached during the capacitation process, and it might be involved In physiological change of sperm plasma membrane accompanying the capacitation.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.6
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• pp.748-756
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• 2000

Effects of xanthine (X), xanthine oxidase (XO), and catalase (C), $H_2O_2$, and carbohydrates on sperm capacitation, acrosome reaction, and fertilizing ability in vitro were examined. Glucose alone, but not fructose, supported the maximum rate of sperm capacitation and acrosome reaction. However, in the combination of X, XO, and C (XXOC) or $H_2O_2$, fructose alone also supported maximum capacitation, acrosome reaction, and fertilization. Either insufficient or excessive amounts of $H_2O_2$ decreased sperm capacitation and the acrosome reaction. In order to understand how glucose generates $H_2O_2$ or other reactive oxygen species in sperm cells, 6-aminonicotinamide, an inhibitor of the pentose-phosphate pathway (PPP), and apocynin, an inhibitor of NADPH oxidase, were added to sperm suspensions in glucose-containing medium. Results appeared that sperm capacitation, acrosome reaction, and fertilization were consequently inhibited by either one of these compounds. These inhibitory effects were nullified by addition of XXOC. These results support the hypothesis that glucose, in addition to being a substrate for glycolysis, facilitates sperm capacitation and the acrosome reaction by generating reactive oxygen species through G-6-P dehydrogenase and NADPH oxidase.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.169-174
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• 2013

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of actin-related protein 2/3 (Arp2/3) complex in actin polymerization is well appreciated, the underlying mechanism(s) and its relationship with capacitation are poorly understood. Therefore, to evaluate the potential role of Arp2/3 complex on capacitation, bovine spermatozoa were incubated with multiple doses (1, 10 and $100{\mu}M$) of CK-636, an inhibitor of Arp2/3 complex with heparin. The cellular localization of the Arp2/3 complex in spermatozoa was identified by immunohistochemistry, whereas western blot was also applied to detect the protein tyrosine phosphorylation of sperm proteins. Additionally, sperm motility and kinematic parameters were evaluated using a computer-assisted sperm analysis system. CK-636 resulted in significant changes in the ratio of Arp2/3 complex localization between acrosome and equatorial region of the spermatozoa. Short-term exposure of spermatozoa to $100{\mu}M$ of CK-636 significantly decreased sperm motility, however a non-detectable effect on protein tyrosine phosphorylation was observed during capacitation. On the basis of these results, we propose that Arp2/3 complex is associated with morphological changes during capacitation and compromised sperm motility.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.2
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• pp.181-189
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• 2008

The aim of this study was to evaluate the effects of $Ca^{2+}$, $HCO_3{^-}$ and BSA on the in vitro capacitation-associated protein tyrosine phosphorylation, hyperactivation and acrosome reaction in guinea pig sperm. Caudal epididymal sperm were incubated in four different groups: modified TALP (Tyrode's albumin lactate pyruvate) or TALP without one of the medium constituents ($Ca^{2+}$, $HCO_3{^-}$ and BSA). After incubation for the required time (0 h, 0.5 h, 1 h, 3 h, 5 h, and 7 h), sperm were removed for further experiment. The capacitation effect was assessed by CTC (Chlortetracycline) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7 h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum. Moreover, an absence of $Ca^{2+}$ or $HCO_3{^-}$ inhibited in vitro hyperactivation and acrosome reaction and decreased the phosphorylation of the proteins throughout the period of in vitro capacitation. However, an absence of BSA could not influence these processes if substituted by polyvinyl alcohol (PVA) in the medium.

• Korean Journal of Animal Reproduction
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• v.14 no.4
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• pp.303-308
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• 1990

To investigate the effect of heparin on sperm capacitation. The acrosome reaction of bovine sperm which were incubated in mTALP containing heparin and the in vitro development of bovine follicular oocytes which were cocultured with heparin-treated sperm were evaluated, and the resutls were as follows : 1. When bovine fresh sperm were incubated in mTALP solution containing 0, 5, 10 and 25$\mu\textrm{g}$/ml heparin for 15 to 840 minutes, there was no significant difference between motilies of heparin-treated sperm and untreated sperm, but the acrosome-reaction rate of heparin-treated sperm was significantly higher than that of untreated sperm. Moreover the acrosome reaction rate of was sperm treated with heparin was significantly increased after incubating for 15 minutes. 2. When fresh sperm were incubated in mTALP solution containing 10$\mu\textrm{g}$/ml heparn for 840 minutes, the motility of sper incubated for 840 minutes was lower than those of sperm incubated for 0, 15, 60 and 120 minutes, but the acrosome-reaction rate of sperm incubated for 840 minutes was higher than those of the others. 3. When frozen-sperm were incubated in mTALP solution containing 10$\mu\textrm{g}$/ml heparin for 120 minutes, the acrosome reaction rate of sperm was significantly increased after incubating for 15 minutes. 4. When fresh sperm treated with heparin were cocultured with bovine follicular oocytes, 16.7 to 23.7% of the oocytes were developed to 2-8 cell stage.

• Clinical and Experimental Reproductive Medicine
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• v.18 no.1
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• pp.23-34
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• 1991

In order to study the sperm-egg interaction during fertilization process in mouse, the effects of sperm concentration, the duration of capacitation and insemination, the stages of maturation and development of eggs, and sperm extracts and BSA on sperm binding to egg were examined. Sperm-egg binding was increased depending on sperm concentration within the range of $10^3-10^6$ sperm/ml. It showed the most numbers of sperm-egg binding at 60min from the beginning of preincubation(capacitation) and insemination, respectively. During sperm capacitation, sperm-egg binding inhibitor was released from sperm into the incubation medium. Sperm extracts containing trypsin-like enzyme which is secreted through the acrosome reaction increased the binding. BSA in the culture medium showed a positive effect on the binding. It is suggested that physicochemical alterations of zona pellucida in the process of maturation and fertilization of eggs leaded to inhibition of sperm-egg binding.