[PKA-regulated phosphorylation status of S149 and S321 sites of CDC25B inhibits mitosis of fertilized mouse eggs].

To further test whether protein kinase A (PKA) can affect the mitotic cell cycle, one-cell stage mouse embryos at S phase (22 h after hCG injection) were incubated in M16 medium containing various concentrations of H-89, a PKA inhibitor. With increasing concentrations of H-89 (0-50 µmol/L), the G(2) phase of eggs was decreased and the cleavage rate was accelerated. A concentration of 40 µmol/L H-89 led to all of the mouse eggs entering the M phase of mitosis. Furthermore, to study the role of PKA in regulating the phosphorylation status of S149 and S321 sites of cell division cycle 25B (CDC25B) on one-cell stage fertilized mouse eggs, pBSK-CDC25B-WT, pBSK-CDC25B-S149A, pBSK-CDC25B-S321A and pBSK-CDC25B-S149A/S321A were transcribed into mRNAs in vitro, then mRNAs were microinjected into S phase of mouse fertilized eggs and cultured in M16 medium pretreated with H-89. Then, the cleavage of fertilized eggs, maturation promoting factor (MPF) activity and phosphorylation status of CDC2-Tyr15 were observed. In the presence of 40 µmol/L H-89, the cleavage rate of fertilized eggs in CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups was significantly higher than that in the control groups, and the peak of MPF activity appeared in the CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups earlier than that in the control groups. CDC2-Tyr15 phosphorylation state was consistent with MPF activity. In conclusion, the present study suggests that PKA regulates the early development of mouse embryos by phosphorylation of S149 and S321 of CDC25B, which plays an important role in the regulation of G(2)/M transition in the mitotic cell cycle of fertilized mouse eggs.

Protein kinase B/Akt may regulate G2/M transition in the fertilized mouse egg by changing the localization of p21(Cip1/WAF1).

Protein kinase B (PKB, also called Akt) is known as a serine/threonine protein kinase. Some studies indicate that the Akt signalling pathway strongly promotes G2/M transition in mammalian cell cycle progression, but the mechanism remains to be clarified, especially in the fertilized mouse egg. Here, we report that the expression of Akt at both the protein and mRNA level was highest in G2 phase, accompanied by a peak of Akt activity. In addition, the subcellular localization of p21(Cip1/WAF1) has been proposed to be critical in the cell cycle. Hence, we detected the expression and localization of p21(Cip1/WAF1) after injecting fertilized mouse eggs with Akt mRNA. In one-cell stage fertilized embryos microinjected with mRNA coding for a constitutively active myristoylated Akt (myr-Akt), p21(Cip1/WAF1) was retained in the cytoplasm. Microinjection of mRNA of kinase-deficient Akt(Akt-KD) resulted in nuclear localization of p21(Cip1/WAF1) . Meanwhile, microinjection of different types of Akt mRNA affected the phosphorylation status of p21(Cip1/WAF1) . However, there was no obvious difference in the protein expression of p21(Cip1/WAF1) . Therefore, Akt controls the cell cycle by changing the subcellular localization of p21(Cip1/WAF1) , most likely by affecting the phosphorylation status of p21(Cip1/WAF1) .

[Recombinant human testis sperm binding protein increases sperm motility parameters].

OBJECTIVE: To investigate the effects of recombinant human testis sperm binding protein (TSBP) on human sperm motility parameters in vitro. METHODS: Sperm specimens obtained from 22 healthy fertile men were prepared by the Percoll gradient-centrifugation technique. The sperm suspension was incubated with recombinant His6-TSBP at the concentration of 0.01 mg/ml or 0.1 mg/ml at 37 degrees C for 1 or 3 hours in vitro. The combination of the recombinant protein and sperm membrane was determined by Western blot, and the sperm motility parameters were analyzed by computer-aided sperm analysis (CASA). The same procedure was performed for 12 asthenospermia patients. RESULTS: In the 22 healthy volunteers, the percentage of forward motile sperm was increased after incubated with 0.1 mg/ml recombinant protein for 1 h (P < 0.05), both forward motile sperm percentage and motility were increased after incubated with recombinant protein at the same concentration for 3 h (P < 0.05), but no effect was observed after incubation with 0.01 mg/ml recombinant protein. In the 12 asthenospermia patients, the forward motile sperm percentage was increased after incubated with 0.1 mg/ml recombinant protein for 3 h (P < 0.05), but no statistically significant difference was observed in sperm motility. CONCLUSION: Recombinant His6-TSBP at the concentration of 0.1 mg/ml can increase sperm motility in healthy fertile men and the forward motile sperm percentage in both healthy fertile men and asthenospermia patients in vitro.

Characterization of p70 S6 kinase 1 in early development of mouse embryos.

The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes mTORC1 and mTORC2. p70 S6 Kinase 1 (S6K1) is characterized as downstream effector of mTOR. Until recently, the connection between S6K1 and mTORC1 /mTORC2 during the early development of mouse embryos has not been well elucidated. Here, the expression level of total S6K1 and its phosphorylation at Thr389 was determined in four phases of one-cell embryos. S6K1 was active throughout the cell cycle especially with higher activity in G2 and M phases. Rapamycin decreased the activity of M-phase promoting factor (MPF) and delayed the first mitotic cleavage. Down-regulating mTOR and raptor reduced S6K1 phosphorylation at Thr389 in one-cell embryos. Furthermore, rapamycin and microinjection of raptor shRNA decreased the immunofluorescent staining of Thr389 phospho-S6K1. It is proposed that mTORC1 may be involved in the control of MPF by regulating S6K1 during the early development of mouse embryos.

Involvement of the p110 alpha isoform of PI3K in early development of mouse embryos.

Class I of phosphoinositide 3-kinases (PI3Ks) is characterized as a group of intracellular signal proteins possessing both protein and lipid kinase activities. Recent studies implicate class I of PI3Ks acts as indispensable mediators in early development of mouse embryos, but the molecular mechanisms are poorly defined. In this paper, mouse one-cell embryos were used to investigate a possible contribution of the catalytic subunit of PI3K, p110 alpha, to cell cycle progression. The expression level of p110 alpha was determined in four phases of one-cell embryos. Silencing of p110 alpha by microinjection of p110 alpha shRNA into one-cell embryos resulted in a G2/M arrest and prevented the activation of Akt and M-phase promoting factor (MPF). Further, microinjection of the synthesized mRNA coding for a constitutively active p110 alpha into one-cell embryos induced cell cleavage more effectively than microinjection of wild-type p110 alpha mRNA, whereas microinjection of mRNA of kinase-deficient p110 alpha delayed the first mitotic cleavage. Taken together, this study demonstrates that p110 alpha is significant for G2/M transition of mouse one-cell embryos and further emphasizes the importance of Akt in PI3K pathway.

Protein kinase A modulates Cdc25B activity during meiotic resumption of mouse oocytes.

Protein kinase A (PKA) play a critical role in maintaining the meiotic arrest. However, the steps downstream of PKA remain largely unknown. In this study, we investigated the regulation of meiotic resumption by PKA/Cdc25B pathway in mouse oocytes. Injection of mRNA coding for Cdc25b-S321A had a more potent maturation-inducing ability than Cdc25b-WT. When co-injected with PKA inhibitor, Cdc25B-WT had similar activities with Cdc25B-S321A. Meanwhile, the phosphorylation of Cdc25B-S321 was detected in germinal vesicle (GV) oocytes by Western blotting with a phospho-Ser321-specific antibody and the band disappeared when oocytes reenter into the meiotic cell cycle. Furthermore, Cdc25B-WT translocated to the nucleus shortly before GV breakdown (GVBD), whereas phosphorylated Cdc25B-S321 expressed exclusively in the cytoplasm and the signal could not be detected in GVBD oocytes. Taken together, these data indicate that Cdc25B-Serine321 is the potential PKA target and Cdc25B subcellular localization determines its function during the process of maintaining GV arrest in mouse oocytes.

Effect of LHRH-PE40 on target cells via LHRH receptors.

OBJECTIVE: To detect the effect and cytotoxicity of luteinizing hormone-releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) on target cells using LHRH receptors (LHRHR). METHODS: The affinity of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of target cells were measured by enzyme linked immunosorbent assay. Morphological observations with light microscope were used to analyze its receptor pathway, with Spiegelmer, and cytotoxicity. IC(50) values of LHRH-PE40, which caused 50% inhibition of tumor cell growth were evaluated by MTT assay. The target cells were exposed to LHRH-PE40 and its cytotoxicity was analyzed by scanning and transmission electron microscopies, agarose gel electrophoresis, and flow cytometry. RESULTS: LHRH-PE40 killed target cells by LHRHR pathway. The morphological changes in these cells showed decreased cell size, cytoplasmic membrane blebbing, and chromatin condensation and margination. At a certain concentration and time point, HeLa cells were also induced to undergo programmed cell death. CONCLUSION: LHRH-PE40 induced target cells apoptosis via LHRHR.

[The effect of protein kinase B on the expression and location of p21 in early development of mouse fertilized eggs].

To investigate the effect of Protein kinase B on the expression and location of p21 in mouse early development. Immunopreciptation technology was used to detect the localization of p21 and Western blotting was used to analyze the expression of p21 after microinjecting mRNA of WT-PKB, myt-PKB and PKB-KD to mouse eggs. There was no obvious difference between the three kinds of mRNA in the expression of p21. But the cell localization altered. The p21 retain in cytoplasm after microjecting myt-PKB. In mouse fertilized egg PKB/Akt controls the cell cycle by changing the cell localization of p21.

Polo-like kinase 1 may regulate G2/M transition of mouse fertilized eggs by means of inhibiting the phosphorylation of Tyr 15 of Cdc2.

Polo-like kinase 1(Plk1) has been reported to be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during mammalian early embryonic mitosis. In the present study, we examined the expression of Plk1 at protein and mRNA level in mouse fertilized eggs by Western blot and RT-PCR. We also examined the kinase activity of Plk1. At various developmental phases of mouse one-cell stage embryos, both the protein and the mRNA of Plk1 were uniformly distributed; but the kinase activity of Plk1 increased at G2/M phase and decreased at the end of M phase. At the meantime, the phosphorylation of Tyr 15 of Cdc2 was inhibited at M phase. To investigate its function in mammalian fertilized eggs further, we used specific short hairpin RNAs (shRNA) and scytonemin, the putative inhibitor of Plk1 to suppress the activity of Plk1 in mouse fertilized eggs. Upon blockage of the activation of with Plk1 shRNA and scytonemin in mouse one-cell stage embryos, the cleavage rate decreased and the phosphorylation level of Tyr 15 of Cdc2 increased. These results imply that the Plk1 may regulate cell cycle progression of mouse fertilized eggs by means of inhibiting the phosphorylation of Tyr 15 of Cdc2.

Expression and activity of mTOR and its substrates in different cell cycle phases and in oral squamous cell carcinomas of different malignant grade.

In order to study the relationship between mTOR (mammalian target of rapamycin) and tumorigenesis, we investigated the expression and activity of mTOR, and its substrates, alpha1, alpha2, beta1 and beta2 isoforms of p70S6 kinase (p70S6K) and eukaryotic initiation factor 4E binding protein-1 (4EBP-1) in oral squamous carcinoma and Hela cells using RT-PCR, immunohistochemistry, statistical analysis and Western blotting. The result of Western blots showed that in poorly differentiated oral squamous carcinoma, the expression level of mTOR and p70S6k increased in M phase, while that of 4EBP-1 decreased. The results of RT-PCR and immunohistochemistry assay are the same as that of Western blot. In Hela cells, the RT-PCR results showed that the level of mTOR mRNA did not change during the cell cycle. In M phase, the expression of alpha1, alpha2, beta1 and beta2 isoforms of p70S6K increased noticeably, while the expression of 4EBP-1 decreased. The immunoblot results in Hela cells were consistent with the RT-PCR results. Furthermore, the activity assays in Hela cells suggested that,in phase G2 and M, the activity of mTOR was maintained at a higher level than in any other phase, while 4EBP-1 decreased in phase M. These results may help in further investigations of the important role of mTOR in cell cycle and tumorigenesis.

Effects of PKC zeta on early genome transcription activation in mouse 1-cell stage fertilized eggs.

Effects of PKC zeta on the activation of embryonic transcription in 1-cell stage fertilized mouse eggs were explored. The effects of PKC antagonist calphostin C and PKC zeta specific inhibitor on the activation of embryonic early transcription were observed by Western blotting and cell immunofluorescence. PKC activity increased gradually from G1 phase to late G2 phase in mouse 1-cell stage fertilized eggs, and reached a maximum in G2 stage. Calphostin C inhibited PKC activity by about 47% in 1-cell stage fertilized eggs. Calphostin C inhibited early transcription in 1-cell stage fertilized eggs (p < 0.01). PKC zeta-Thr410 in G2 were about 27% and 110% higher than those in G1 phase of 1-cell stage fertilized eggs and MII oocytes, respectively. PKC zeta specific inhibitor can also inhibit early transcription in 1-cell stage fertilized eggs (p < 0.05). The results suggest that PKC zeta participates in early transcription activation in mouse 1-cell stage fertilized eggs.

[Fusion expression and affinity purification of a human novel gene tsbp in eukaryotic cells].

AIM: To construct an eukaryotic expressing vector of the human novel gene testis sperm binding protein (tsbp), and to express fusion protein and purify the recombinant protein by affinity chromatography. METHODS: The novel gene tsbp was amplified by PCR from the prokaryotic expressing plasmid pGEX-5X-1/tsbp and an eukaryotic expressing vector pcDNA3.1/myc-His(-)B/tsbp was constructed after DNA recombination. After transfecting HEK293 cells with this recombinant vector via liposome mediation, the expression of the fusion protein was detected by RT-PCR, immunofluorescence and Western blot. Fusion protein His6-tsbp was purified from the cell lysis by immobilized metal affinity chromatography (IMAC) and the efficiency of purification was detected by SDS-PAGE and Western blot. RESULTS: DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pcDNA3.1/myc-His(-)B/tsbp had been constructed successfully. After the recombinant plasmid being transfected into HEK293 cells, RT-PCR verified the expression of tsbp mRNA. The result of immunofluorescence assay was positive and the fusion protein could be detected by Western blot of transfected HEK293 cells. The purified fusion protein could also be detected by SDS-PAGE and Western blot. CONCLUSION: The novel gene tsbp was successfully cloned, expressed and purified in the form of His6 fusion protein, which is helpful for further study of the function of this testis sperm binding protein.

Expression and immunohistochemical localization of Cdc2 and P70S6K in different stages of mouse germ cells.

In order to determine the function and possible relationship between Cdc2 and P(70)S6K, Western blot analysis and immunohistochemistry analysis were used to study the expression and kinase activity of Cdc2 and P(70)S6K in male mouse germ cells. With the maturation of germ cells in the testis, the expression of Cdc2 and P(70)S6K was relatively constant. However, the kinase activity of P(70)S6K was increased and the phosphorylation of Tyr15 residue of Cdc2 was enhanced, which suggests that the kinase activity of Cdc2 is decreasing. Immunohistochemistry analysis also showed that there was a P(70)S6K transfer from nucleus to cytoplasm during spermatogenesis. During spermatogenesis, cell division of the germ cell in male mouse is decelerated; nevertheless, cell growth is enhanced. Cdc2 and P(70)S6K are involved in these two processes. It could be an alternative mechanism to prepare for future fertilization that Cdc2 is able to maintain a subtle balance between the production and growth of male germ cells by regulating P(70)S6K.

[Chromosomal localization and inhibitory effect on TPK activition of a human novel gene hbrp].

The purpose of present study is to determine the location of the novel gene hbrp which is related to bovine seminal plasma (BSP) proteins in the human chromosome by the FISH (fluorescent in situ hybridization) technique, and to investigate the relation of HBRP to TPK( tyrosine protein kinase)by genetic engineering. The result is that the novel gene hbrp was successfully localized on human chromosome 19q1.3; HBRP protein obviously inhibited the activity of PKC. It is concluded that the structure of BSP contained two tandemly arranged fibronectin type II(Fn2)--domains which were related to the function of binding protein BSP. Amino acid sequence of HBRP protein with four tandemly arranged Fn2-domains showed that the protein might be binding protein functionly relating to BSP protein. We have concluded that HBRP protein obviously inhibited the activity of TPK. The location of the novel gene on human chromosome 19q1.3 is very important to study the other biological functions of the protein encoded by gene hbrp.

[The expression of mTOR and its substrates in oral squamous cell carcinoma].

OBJECTIVE: The aim of this study was to observe the expression of mTOR (mammalian target of rapamycin) and its substrates in oral squamous cell carcinoma. METHODS: mTOR and its substrates alpha1, alpha2, beta1, beta2 isoforms of p70 S6 kinase (p70S6k) and 4EBP1 were examined by means of RT-PCR, Western-blot test. RESULTS: The result of RT-PCR showed that in poorly differentiated tissue, the expression level of mTOR and its substrates alpha1, alpha2, beta1, beta2 isoforms of p70S6k increased obviously, while that of 4EBP1 decreased, while that in well differentiated tissue was second to it, the normal oral tissue was the last. The expression of Western-blot was the same as the RT-PCR. CONCLUSION: The expression of mTOR and its substrates differs in different types of oral squamous cell carcinoma. The result suggests that mTOR, p70S6K and 4EBP1 might play important roles in oral squamous cell carcinoma. It may be an important target protein to treat tumor in the future.

Effects of protein kinase C on M-phase promoting factor in early development of fertilized mouse eggs.

The mechanism of development of mouse fertilized eggs from the one-cell stage to the two-cell stage remains unclear to date. In the present study, we have evaluated protein kinase C (PKC) and M-phase promoting factor (MPF) kinase activity in fertilized mouse eggs treated with a PKC modulator. PKC and MPF activity have similar activity. The two subunits of MPF, p34(cdc2) and cyclin B, were shown to be included in the substrates phosphorylated by PKC in fertilized mouse eggs, while PKC modulator affected the electrophoretic mobility shift of cdc2 and cdc25C by dephosphorylation and phosphorylation. These results clearly indicate that PKC may affect the progression of the cell cycle through post-translational modification of MPF activity.

[Gene localization and tissue expression of a novel human reproduction-related gene].

Gene localization is significant in elucidating the interaction between genes,gene and diseases. Using radiation hybrid (RH) technique,we cloned and localized a novel gene,designated human BSP-related protein (HBRP) on 19q13.2 - 13.3, in line with its localization in data bank of overlapping fragment of human genome through bioinformatics method. It is suggested RH is rapid, precise,simple and powerful in gene localization. In addition, we detected the expression and distribution of HBRP in human tissues by RT-PCR. The results showed HBRP was highly expressed in intestine, kidney, liver, spleen, stomach and pancreas, whereas lowly in brain, lung, muscle and heart.