Yoshihiro Endo

Calyculin A, protein phosphatase inhibitor, enhances capacitation of human sperm*

OBJECTIVE To examine the effects of protein phosphatase inhibitors on capacitation and protein phosphorylation in human sperm. DESIGN The chlortetracycline (CTC) fluorescence assay was used to monitor capacitated sperm treated with or without protein phosphatase inhibitors. Capacitation was confirmed by the ability of sperm to undergo the acrosome reaction in response to Ca++ ionophore A23187 or mouse zonae pellucidae. 32P-labeled sperm phosphoproteins were analyzed by one-dimensional gel electrophoresis to detect the effects of protein phosphatase inhibitors on protein phosphorylation. RESULTS The treatment of sperm with calyculin A resulted in the following: [1] the rapid appearance of the clear perimeter pattern, specifically, distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece, in a dose-dependent manner in the 1 to 100 nM range; [2] an accelerated ability to undergo the acrosome reaction; and [3] an enhanced phosphorylation of sperm phosphoproteins in a dose-related fashion in the 1 to 100 nM range. A similar stimulatory effect was observed only with a 100-fold higher concentrations of okadaic acid, another protein phosphatase inhibitor. CONCLUSION Our results strongly suggest that protein phosphorylation and dephosphorylation may be involved in the regulation of human sperm capacitation.

Effect of epidermal growth factor on human sperm capacitation

OBJECTIVE To examine the effect of epidermal growth factor (EGF) on human sperm capacitation and the involvement of protein phosphorylation in the regulation of the EGF action. DESIGN Human sperm were capacitated in modified Krebs-Ringers bicarbonate medium of Biggers, Whitten, and Whittingham in the presence of EGF and various agents known to phosphorylate the EGF receptor. The chlortetracycline fluorescence assay was used to monitor capacitated sperm. Capacitation was confirmed by the ability of sperm to undergo the acrosome reaction in response to solubilized mouse zonae pellucidae (ZP). RESULTS In 15 minutes, the appearance of the clear perimeter pattern increased significantly in the sperm treated with 100 ng/mL EGF compared with the controls. In EGF-treated sperm, the percent clear perimeter pattern remained stable for 3 hours without affecting the acrosome reaction pattern and the motility. Epidermal growth factor stimulated the appearance of the clear perimeter pattern at concentrations > 100 pg/mL. The stimulation by EGF was attenuated by the treatment with genistein, 12-O-tetradecanoylphorbol-13-acetate, or thapsigargin. In sperm that were incubated in the presence of 100 ng/mL EGF for 30 minutes and further induced the acrosome reaction by mouse ZP, the percent acrosome reaction pattern increased significantly compared with the controls. CONCLUSION Epidermal growth factor stimulates human sperm capacitation by activating the tyrosine kinase of the EGF receptor which is regulated by multisite phosphorylation.

Effects of modulators of protein kinases and phosphatases on mouse sperm capacitation

We examined effects of modulators of protein kinases and phosphatases on the kinetics of mouse sperm capacitation. The chlortetracycline fluorescence assay was used to monitor the process of capacitation (in terms of the appearance of the B pattern). The treatment of sperm with dibutyryl cyclic AMP (cAMP) or dibutyryl cGMP resulted in a higher percentage B pattern at various times during capacitation compared with the control. The addition of 100 µM H8 inhibited the cyclic nucleotide-dependent stimulation of capacitation. Tumor promotors, 12-O-tetradecanoyl phorbol 13-acetate (TPA; a stimulator of protein kinase C) and okadaic acid (an inhibitor of protein phosphatases 1 and 2A), induced a rapid appearance of the B pattern (15 min after addition) and maintained a percentage B pattern similar to that of the control in the later period of capacitation. An inhibitor of protein kinase C, staurosporine, inhibited the TPA-dependent acceleration of capacitation. Furthermore, the addition of genistein, an inhibitor of protein tyrosine kinases, resulted in a strong inhibition of capacitation. All agents tested did not affect sperm motility. These data suggest that protein phosphorylation and dephosphorylation may play regulatory roles in mediating mouse sperm capacitation.

Protein phosphorylation regulates the mouse sperm acrosome reaction induced by the zona pellucida.

Recently, the ligand-receptor signal transduction mechanism has been implicated in mediating the zona pellucida (ZP)-induced acrosome reaction. Little is known about the role of protein phosphorylation in this specific event. We examine whether modification of protein phosphorylation and dephosphorylation affects the kinetics of the acid-solubilized ZP-induced acrosome reaction of mouse sperm. Mouse epididymal sperm were incubated in modified Krebs-Ringer bicarbonate medium for a period of 90 to 120 min and then treated with 2 acidsolubilized ZP/µl for an additional 60 min. The chlortetracycline fluorescence assay was used to monitor the acrosome reaction. Capacitated sperm were inhibited from undergoing acid-solubilized ZP-induced acrosome reaction in the presence of an inhibitor of cyclic nucleotide-dependent protein kinase, H8; activators of the Ca2+-and phospholipid-dependent protein kinase (protein kinase C); an inhibitor of phosphatases 1 and 2A, okadaic acid; or an inhibitor of protein tyrosine kinases, genistein. The addition of inhibitors of protein kinase C, such as staurosporine, H7, and protein kinase C [19–36] pseudosubstrate, inhibited the phorbol ester-dependent inhibition of the acid-solubilized ZP-induced acrosome reaction. The present study suggests that protein phosphorylation and dephosphorylation play a regulatory role in the process of the ZP-induced acrosome reaction.

Analysis of cytoplasmic factors in developmental cleavage of mouse embryo

One-cell embryos from certain mouse strains were found incapable of developing beyond the 2-cell stage in vitro (2-cell block), but a microinjection of EDTA effectively overcame this block. When 2-cell arrested embryos were fused with embryos that had developed to the late 2-cell stage in vivo, the fusants developed beyond the 2-cell stage. Microinjection of cytoplasm of in vivo 2-cell embryos into 1-cell embryos also obviated the 2-cell block. Analyses of 35S-labeled embryos by 2-dimensional polyacrylamide gel electrophoresis indicated changes in synthetic protein patterns possibly related to this block.

Effect of Epidermal Growth Factor on Mouse Sperm Acrosome Reaction Induced by Zona Pellucida

PROBLEM: The effect of epidermal growth factor (EGF) on the acid‐solubilized zona pellucida (ZP)‐induced acrosome reaction was investigated in mouse sperm.

Protein kinase C activity and protein phosphorylation in mouse eggs

The treatment of mouse eggs with phorbol esters and diacylglycerol inhibits sperm peretration and results in biochemical modification of the zona pellucida. In this report, we have demonstrated the presence of protein kinase C (PKC) activity in mouse eggs as determined by 12-O-tetradecanoyl phorbol-13-acetate (TPA) dependent in vivo and in vitro protein phosphorylation in mouse eggs. When mouse eggs were radiolabeled with [32P]phosphate and treated with TPA, two specific proteins, 70 and 20 kDa, were phosphorylated. The 70-kDa protein was also phosphorylated in vitro by endogenous PKC. In addition, we have shown that exogenous PKC induced the in vitro phosphorylation of 70-, 55-, and 20-kDa proteins in egg extract. The 70-kDa protein was also phosphorylated in vitro after treatment of the cytosol fraction of mouse eggs with TPA, suggesting that this protein might be a specific substrate for PKC and that it is located in the cytosol. These results demonstrate that mouse eggs contain PKC activity and suggest that PKC-catalyzed protein phosphorylation of specific proteins might be involved in the regulation of egg-induced modification of the zona pellucida.

Demonstration of maturation-promoting and -inhibiting activities in mouse oocytes.

The microinjection of cytoplasm from mature mouse oocytes into immature mouse oocytes induced the resumption of meiosis in the presence of 100 μg/ml dibutyryl cyclic AMP or 100 μM 3-isobutyl-1-methylxanthine. The volume of cytoplasm injected was critical to bringing about this maturation-promoting activity in mouse oocytes, and 20 pl of cytoplasm seems to be required to overcome the inhibitory effect of cyclic AMP on oocyte maturation. Furthermore, it was demonstrated that the microinjection of cytoplasm from immature mouse oocytes into immature mouse oocytes augmented the inhibitory effect of cyclic AMP on the resumption of meiosis. These results suggest that the appearance and disappearance of maturation-promoting and- inhibiting activities appear to be dependent on the meiotic stages in the mouse oocytes.

Effects of modulators of protein kinase C on human sperm capacitation**Supported by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, (project 63640510 to S.S.) Tokyo, Japan; and by the World Health Organization Special Program of Research, Development and Research Training in Human Reproduction, (project 87088 to S.S.) Geneva, Switzerland.

OBJECTIVE To examine the effects of stimulators or inhibitors of protein kinase C on capacitation and protein phosphorylation in human sperm. DESIGN Capacitated sperm treated with or without modulators of protein kinase C were monitored by the chlortetracycline fluorescence assay. Capacitation was confirmed by the ability of sperm to undergo the acrosomal reaction in response to mouse zonae pellucidae. 32P-labeled sperm phosphoproteins were analyzed by one-dimensional gel electrophoresis to detect the effect of protein kinase C stimulator, 12-O-tetradecanoyl-phorbol-13-acetate, on protein phosphorylation. RESULTS The treatment of sperm with protein kinase C stimulators resulted in the following: [1] the rapid appearance of the clear perimeter pattern, featuring distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece; [2] an accelerated ability to undergo the acrosomal reaction; and [3] an enhanced phosphorylation of 57.5-kd sperm phosphoprotein. Furthermore, these stimulatory effects were inhibited by protein kinase C inhibitors. CONCLUSION Protein phosphorylation mediated by protein kinase C may be involved in the regulation of human sperm capacitation.

Gametes- and Embryo-Oviduct Interactions

Although the time of ovulation in women can now be gauged with accuracy by means of echo sonography of the ovarian structure, there is as yet no comparable means of judging precisely when the embryos have passed from the fallopian tube to the uterus. It is worth reminding that what is occurring in the oviduct is still not clearly defined. The extent to which the post-ovulatory environment influences gametes maturation, fertilization and pre-implantation development is largely unknown. The oviductal environments, however, may have key roles in functional regulations of gametes and embryos (Figure 1).