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To investigate the effect of 1800 MHz electromagnetic radiation (EMR) on apoptosis, we exposed NIH/3T3 cells at 1800 MHz with a specific absorption rate (SAR) of 2 W/kg intermittently for 12, 24, 36, and 48 h. After exposure, Cell Counting Kit-8 (CCK-8) and flow cytometry were used to detect cell viability and apoptosis, the expression of p53, a molecule with the key role in apoptosis, was measured by real-time qPCR, western blot, and immunofluorescence, and images of the structure of the mitochondria, directly reflecting apoptosis, were captured by electron microscopy. The results showed that the viability of cells in the 12, 36, and 48 h exposure groups significantly decreased compared with the sham groups, after 48 h of exposure, the percentage of late apoptotic cells in the exposure group was significantly higher. Real-time qPCR results showed that p53 mRNA in the 48 h exposure group was 1.4-fold of that in the sham group, significant differences of p53 protein fluorescence expression were observed between the exposure groups and the sham groups after 24 h and 48 h. The mitochondrial swelling and vesicular morphology were found in the electron microscopy images after 48 h exposure. These findings demonstrated 1800 MHz, SAR 2 W/kg EMR for 48 h may cause apoptosis in NIH/3T3 cells and that this apoptosis might be attributed to mitochondrial damage and upregulation of p53 expression.

Scutellaria altissima L. is a common traditional Chinese medicine used to treat inflammation in some countries. Scutellarin, an active major flavone glycoside isolated from the traditional Chinese medicine Scutellaria altissima L., has been shown to offer various beneficial biochemical effects on cerebrovascular diseases and inflammation. However, the antiproliferative effects of Scutellarin in prostate cancer and the underlying mechanism are not fully elucidated. In the present study, we aimed to ascertain whether Scutellarin inhibits cancer cell growth and to further explore the molecular mechanism. Scutellarin enhanced the sensitivity of prostate cancer cells to cisplatin. MTT assays revealed that cell viability was significantly decreased in the prostate cancer cells treated with Scutellarin. Flow cytometric analysis indicated that Scutellarin suppressed cell proliferation by promoting G2/M arrest and inducing apoptosis. We employed western blotting to delineate the underlying mechanisms involved in the G2/M arrest and apoptosis. Comet assay and γH2AX immunocytochemistry were used to detect levels of DNA damage in PC3 cells exposed to Scutellarin and/or cisplatin. Our data revealed that Scutellarin significantly induced prostate cancer cell apoptosis by activating the caspase cascade. An increase in the Bax/Bcl-2 ratio, depolarization of mitochondrial membrane potential and cell cycle arrest at G2/M phase were accompanied by the apoptosis induction. Additionally, Scutellarin altered the protein expression of cell cycle and apoptosis regulatory genes by downregulating Cdc2, cyclin B1 and Bcl-2 and upregulating caspase-3, caspase-9 and Bax in prostate cancer cells. Furthermore, Scutellarin sensitized PC3 cells to cisplastin treatment in a dose-dependent manner. Taken together, our data confirmed the cytotoxicity of Scutellarin against prostate cancer PC3 cells and provide new findings in regards to Scutellarin sensitizing prostate cancer cells to chemotherapy. Our findings suggest that Scutellarin has potential to be used as a novel antineoplastic therapeutic candidate for prostate cancer patients.

The aim of the present study was to evaluate the efficacy of paclitaxel combined with curcumin (CUR) against drug resistance in ovarian cancer cells. PLGA-phospholipid-PEG nanoparticles were prepared using the nano precipitation method. The size and morphology of the nanoparticles were determined using a transmission electron microscope and particle size analyzer. The encapsulation efficiency of nanoparticles was determined using the ultrafiltration centrifugation method. The dialysis method was used to study the release of PLGA-phospholipid-PEG nanoparticles. ADM was used to induce the A2780 cell line (human ovarian cancer cell line) to establish the model of the multidrug-resistant (MDR) cell line, and the protein activity of P-glycoprotein (P-gp) in the A2780 cell line and A2780/ADM resistant cell line was determined using western blot analysis. The results showed that, the prepared nanoparticles were uniform in size, with a size of approximately 100 nm, and round in shape. Additionally, the nanoparticles had a more gentle and slow release than the free drug release. The results of the protein trace printing experiment showed that the P-gp content of the drug-resistant cell line was significantly reduced by the CUR nanoparticles. In conclusion, PLGA-phospholipid nanoparticles containing taxol and CUR have improved solubility and stability together with a slow release effect. In addition, CUR was able to overcome the MDR of tumor cells by elevating the paclitaxel concentration in the tumor cells to improve the antitumor activity of this combination.

The incidence of ovarian cancer in women has been on the increase in recent years. The aim of the present study was to examine the effects of taxol on the expression of ovarian cancer-associated gene forkhead box transcription factor M1 (FoxM1) and its therapeutic effects for ovarian cancer. The expression of FoxM1 gene was examined in patients with or without ovarian cancer. RNA and protein levels of FoxM1 gene of ovarian cancer patients were detected at different time periods (1, 3, 6, 8, 12 and 24 months) after treatment with taxol. The results showed that the mRNA level of FoxM1 gene in patients with ovarian cancer was significantly higher than that in normal women (P

The present study was planned to investigate miR-143 expression during stomach cancer. The study explored the relationship between miR-143 expression and clinicopathological characteristics including proliferation, migration and apoptosis of stomach cancer cells. Sixty-three samples from each of stomach cancer tissue and surrounding tissue were obtained. Total RNA was extracted. The expression levels of miR-143 from stomach cancer tissue as well as from surrounding tissue were measured by semi-quantitative PCR. The effects of miR-143 overexpression on the migration of stomach cancer cells were examined by Transwell assay. The effects of miR-143 overexpression on the apoptosis of stomach cancer cells were examined by flow cytometer. The expression level of miR-143 was significantly decreased in stomach cancer tissues in comparison to surrounding tissues (P

Aim To examine the possible effect of heat treatment on expression of heat shock proteins (Hsps) 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods Western blot analysis, real-time polymerase chain reaction (PCR), and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43 degrees treatment of Sertoli cells isolated from pubertal monkey testes. Results Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hsp105 was expressed in cytoplasm of untreated Sertoli cells. Both Hsp105 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase (ERK) 1/2 inhibitor (U0126) or phosphoinositide kinase-3 (PI3K) inhibitor (LY294002) could partially block the response of Hsp105 and Hsp70 induced by heat treatment. Conclusion These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.

Male contraception has focused, to a great extent, on approaches that induce azoospermia or severe oligospermia through accelerated germ cell apoptosis. Understanding the specific steps in the germ cell apoptotic pathways that are affected by male contraceptives will allow more specific targeting in future contraceptive development. In this study, we have used a nonhuman primate model to characterize the key apoptotic pathway(s) in germ cell death after mild testicular hyperthermia, hormonal deprivation, or combined interventions. Groups of 8 adult (7- to 10-year-old) cynomolgus monkeys (Macaca fascicularis) received one of the following treatments: 1) two empty silastic implants; 2) two 5.5-cm testosterone (T) implants; 3) daily exposure of testes to heat (43 degrees C for 30 min) for 2 consecutive days; and 4) two T implants plus testicular heat exposure for two consecutive days. Testicular biopsies were performed before and at Days 3, 8, and 28 of treatment. Treatment with T, heat, or both led to sustained activation of both mitogen-activated protein kinase (MAPK) 1/3 and MAPK14. Activation of MAPK1/3 and MAPK14 were accompanied by an increase in B-cell leukemia/lymphoma (BCL) 2 levels in both cytosolic and mitochondrial fractions of testicular lysates (BAX levels remained unaffected) and cytochrome c and DIABLO release from mitochondria. These treatments also resulted in inactivation of BCL2 through phosphorylation at serine 70, thereby favoring the death pathway. We conclude that the serine phosphorylation of BCL2 and activation of the MAPK14-mediated mitochondria-dependent pathway are critical for male germ cell death in monkeys.

To investigate the possible role of testicular orphan receptors (TR) TR2, TR3, and TR4 in the process of germ cell apoptosis in the heat-treated testis of monkey, we have examined the spatiotemporal expression of the 3 TR mRNAs in relation to p53 mRNA levels in the monkey testis by in situ hybridization and reverse transcription polymerase chain reaction techniques. The results showed that TR2 mRNA was confined to spermatocytes; TR4 and TR3 mRNAs were expressed in both spermatocytes and spermatids. The heat treatment did not change TR2 mRNA level but significantly reduced TR4 mRNA expression in spermatocytes on days 3 and 8 after the heat treatment. TR3 mRNA expression was affected by the heat treatment in a time-dependent manner, with the lowest level on day 30 after the heat shock. Low to moderate signal for p53 mRNA was detected in spermatocytes before treatment, which increased dramatically on days 3, 8, and 30 after the heat shock. The coincident expression of the testicular TR3 and p53 mRNA, spatially and time dependently, implied that the decrease in TR3 expression in the heat-treated testis might be closely related to the p53 signal pathway, whereas the temporal decrease in TR4 production in the testis at the early stage indicated that this orphan receptor might be also involved in germ cell apoptosis. The data suggest that TR3, TR4, and p53 could be important regulators of germ cell apoptosis induced by the heat treatment, whereas TR2 might not be a key regulator in this process.

Stem cell factor (SCF) is essential for the development of primordial follicles. One of its functions is to prevent oocytes from apoptosis. However, the underlying mechanism remains largely unknown. By using cultured ovaries that are rich in primordial follicles, the anti-apoptotic action of SCF and the potential signal transduction pathways were investigated. The apoptosis was evaluated by means of in situ 3′-end labeling. The expressions of proteins were analyzed with immunohistochemistry and Western blot. The data showed that SCF significantly prevented oocytes from apoptosis in the cultured organs. Addition of a specific pharmacological inhibitor of PI3K abolished the anti-apoptotic action of SCF while that of a MEK inhibitor did not. The phosphorylation of two mitogen activated protein kinases (MAPKs) (p42 and p44) and AKT, the respective substrates of MEK and PI3K, were enhanced by SCF treatment. Not surprisingly, the MAPK activation occurred only in theca cells. The expressions of apoptosis-related gene products, the Bcl-2 family proteins, in response to SCF treatment were also investigated. While SCF up-regulated the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, it did the opposite to the pro-apoptotic factor Bax. The PI3K inhibitor reversed the regulation of SCF on Bcl-xL and Bax but not on Bcl-2. Therefore, it seemed that SCF initiated an anti-apoptotic signal starting from its membrane receptor c-kit to Bcl-2 family members through PI3K/AKT and other signaling cascades in the oocytes of primordial follicles. Mol. Reprod. Dev. 70: 82–90, 2004. © 2004 Wiley-Liss, Inc.

Apoptosis occurs spontaneously during spermatogenesis. However, little is known about its regulation in primate. Using an experimental cryptorchidism model in rhesus monkey, we have investigated the relationship between apoptosis and the Bcl-2 family members, Bcl-2 and Bax. Apoptotic cells were identified by in situ end labeling of fragmented DNA. The expressions of Bcl-2 and Bax in the testes during the heat stress-induced testicular germ cell apoptosis were detected by immunohistochemistry and Western blot techniques. The results showed that the apoptotic signals increased after heat treatment and the most susceptible cell types were spermatocytes and spermatids. A redistribution of Bax from the cytoplasmic to nuclear localization in some germ cells was observed. However, its total expression levels in the cells remained unchanged in the cryptorchid testes as determined by Western blot analysis; on the other hand, Bcl-2 levels increased significantly in response to heat stress. The subcellular redistribution of Bax and the increase in Bcl-2 expression in the cryptorchid testis suggest an involvement of Bcl-2 family members in heat stress-induced germ-cell apoptosis.

Hsp70-2 functions as a molecular chaperone that assists other proteins in their folding, transport and assembly into complexes, and is postulated to be linked to the mechanisms that inhibit apoptosis. Here we have determined the association between Hsp70-2 gene and germ cell apoptosis induced by a high dose of testosterone undecanoate (TU). In this study, in situ analysis of cell DNA fragmentation and expression of Hsp70-2 in TU-treated monkey testes were compared with the normal testes. The TUNEL analysis data showed that a large number of germ cell apoptosis occurred in the testes on Day 30 after TU injection. Therefore, we speculate that spermatogenesis failure in TU-treated monkey testis may be a result of the germ cell apoptosis induced by a high dose of TU. As compared with that of normal testes, however, the level of Hsp70-2 mRNA was only slightly decreased while that of Hsp70-2 protein was almost unchanged in the testes from Day 7 to day 30 at the early stage of the germ cell apoptosis after TU treatment, but the levels of both Hsp70-2 mRNA and protein dropped dramatically on Day 60 when a large number of germ cells had undergone apoptosis and were depleted. Therefore, it is suggested that the Hsp70-2 may be not a molecule to prevent germ cell apoptosis induced by injection of TU in the testes at the early stage.

We investigated the possible role of Hsp70-2 in germ cell apoptosis induced by heat stress in monkey unilateral cryptorchid testis. The study focused on in situ analysis of the testicular cell DNA fragmentation and on the possible relationship between Hsp70-2 expression and germ cell apoptosis. The TUNEL result showed that most of the germ cells were labeled in the cryptorchid testis on d 5 after induction of cryptorchidism; that with most of the apoptotic germ cells depleted, only a few germ cells were labeled on d 10; and that almost no apoptotic signal was observed in the cryptorchid testis on d 15 and thereafter. This indicates that the increasing germ cell degeneration in cryptorchid testis may take the form of apoptosis. Using in situ hybridization, immunohistochemistry, and Northern blot, we examined the changes of Hsp70-2 expression in the monkey cryptorchid testis. The level of Hsp70-2 mRNA decreased slightly, while the expression of HSP70-2 protein was almost unchanged at the early stage of germ cell apoptosis in the cryptorchid testis on d 5 and dropped dramatically along with the loss of apoptotic germ cells in the cryptorchid testis on d 10 after operation. It is therefore suggested that Hsp70-2 might not take part in inhibiting the apoptosis of germ cells at the early stage during operation-induced cryptorchid testis, and that Hsp70-2 gene does not belong to the immediate early related gene responsible for germ cell apoptosis induced by heat stress.

Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by usingin situ hybridization andin situ 3′-end labeling of DNA fragments (TUNEL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle, (ii) In the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter, (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA probably plays an important role in spermatogenesis and germ cell apoptosis.

This study investigates the role of caspase 2 in apoptotic signaling of nonhuman primate male germ cells triggered by mild testicular hyperthermia, testosterone (T(e)) implants, or by combined interventions. Mean incidence of germ cell apoptosis increased significantly by Day 3 in the heat (H(e)) alone group and by Day 8 in the Te alone group but peaked at Day 3 in H(e) + T(e) group. We found activation of caspase 2 in both germ cells and Sertoli cells after induction of apoptosis. Most notably, active caspase 2 immunoreactivity was detected only in those germ cells susceptible to apoptosis compared with controls, where little or no such staining is detected. To further explore the role of caspase 2 in regulating male germ cell death, we next evaluated the efficacy of caspase 2 inhibition in preventing or attenuating heat-induced germ cell apoptosis in rats. Caspase 2 inhibition significantly (P0.05) prevented such heat-induced germ cell apoptosis. The protection offered by the caspase 2 inhibitor occurred upstream of mitochondria, involving suppression of mitogen-activated protein kinase (MAPK) 14 activation and inducible nitric oxide synthase (NOS2) induction and, in turn, suppression of cytochrome c-mediated death pathway. Together, our results show that caspase 2 is activated in male germ cells undergoing apoptosis in nonhuman primates after heat stress, hormonal deprivation, or after combined interventions. Blockade of caspase 2 activation prevents heat-induced germ cell apoptosis in rats by suppressing the MAPK14- and NO-mediated intrinsic pathway signaling.

This study investigates the possible involvement of nitric oxide synthase (NOS) in activating germ cell death in monkeys after mild testicular hyperthermia and/or hormonal deprivation. Groups of 8 adult male monkeys received 1 of the following treatments for 12 weeks: 1) 2 empty Silastic implants, 2) 2 testosterone (T) implants, 3) daily exposure of testes to heat (43 degrees C for 30 minutes) for 2 consecutive days, or 4) 2 T implants plus testicular heat exposure. Testicular biopsies were performed before and on days 3, 8, 28, and 84 of the treatment. In control monkey testes, endothelial NOS (eNOS) was observed mainly in Sertoli cells and spermatogonia. No obvious alteration in eNOS levels was detected in any of the treatment group as assessed by Western blotting. Induction of inducible NOS (iNOS) in testes of the 3 treated groups was detected by immunoblotting as early as day 3 after treatment compared with that of controls. Immunocytochemistry further revealed a small increase in iNOS expression in both germ cells and Sertoli cells after T treatment. However, treatment of heat or heat in combination with T markedly induced iNOS expression in germ cells. These data suggest that iNOS, but not eNOS, may be involved in monkey testicular germ cell death after heat and/or T treatment.

To investigate the molecular mechanism of cell apoptosis induced by tyrosine protein kinase inhibitor, genistein, in human salivary adenoid cystic carcinoma cell line SACC-83.SACC-83 cells cultured in vitro were treated with genistein, the expressions of bax, bcl-2 and survivin proteins were detected with Western blotting, and the results were quantitatively analyzed by FluorChem V2.0 software, statistical analysis was performed with analysis of variance using SPSS11.5 software package.With the increase of concentration of genistein and the elongation of time, the expression of bax protein was significantly increased, but the expression of bcl-2 and survivin proteins was significantly decreased. Treated with 220 micromol/L genistein for 3 days, the expression level of bax protein was 3.43 folds of the control group (P0.01),but the expression level of bcl-2 and survivin proteins was respectively 85%(P0.05) and 35%(P0.01) of the control group.The cell apoptosis induced by genistein in human salivary adenoid cystic carcinoma cell line SACC-83 may be associated with the upregulation of bax protein expression,and the downregulations of bcl-2 and survivin proteins expression.

Male contraception has focused, to a great extent, on approaches that induce azoospermia or severe oligospermia through accelerated germ cell apoptosis. Understanding the specific steps in the germ cell apoptotic pathways that are affected by male contraceptives will allow more specific targeting in future contraceptive development. In this study, we have used a nonhuman primate model to characterize the key apoptotic pathway(s) in germ cell death after mild testicular hyperthermia, hormonal deprivation, or combined interventions. Groups of 8 adult (7- to 10-year-old) cynomolgus monkeys (Macaca fascicularis) received one of the following treatments: 1) two empty silastic implants; 2) two 5.5-cm testosterone (T) implants; 3) daily exposure of testes to heat (43 degrees C for 30 min) for 2 consecutive days; and 4) two T implants plus testicular heat exposure for two consecutive days. Testicular biopsies were performed before and at Days 3, 8, and 28 of treatment. Treatment with T, heat, or both led to sustained activation of both mitogen-activated protein kinase (MAPK) 1/3 and MAPK14. Activation of MAPK1/3 and MAPK14 were accompanied by an increase in B-cell leukemia/lymphoma (BCL) 2 levels in both cytosolic and mitochondrial fractions of testicular lysates (BAX levels remained unaffected) and cytochrome c and DIABLO release from mitochondria. These treatments also resulted in inactivation of BCL2 through phosphorylation at serine 70, thereby favoring the death pathway. We conclude that the serine phosphorylation of BCL2 and activation of the MAPK14-mediated mitochondria-dependent pathway are critical for male germ cell death in monkeys.

To investigate the anti-proliferation effect of tyrosine protein kinase inhibitor, Genistein, on human salivary adenoid cystic carcinoma cell line SACC-83, and its effect on Survivin expression.SACC-83 cells were treated with different concentration Genistein for different time, cell survival rate was calculated with MTT assay, apoptosis was detected with flow cytometry, the expression of Survivin was quantitatively analyzed by Western blotting and FluorChem V2.0 software.When treated with Genistein of certain concentration for certain time, SACC-83 cell growth was significantly inhibited. With the increase of concentration and elongation of acting time, the inhibitory effects increase. Treated with 220 micromol/L Genistein for 72 hours, SACC-83 cell growth was significantly inhibited, cell apoptosis was induced (P0.01), and the expression of Survivin decreased.Genistein inhibits the growth of human salivary adenoid cystic carcinoma cell line SACC-83, and induces cell apoptosis; the decrease of Survivin expression may be one of the mechanisms of Genistein inducing apoptosis.

To investigate the germ cell apoptosis under body temperature in testis, we analyzed the gene expression patterns on day 1, day 4, day 7, day 14, day 28 and normal control adult mouse testis after experimental cryptorchidism (EC) using Affymetrix MOE430A microarray. Our data showed that EC led to the oxidative stress and gene expression fluctuation in the first 28 days, both of which were highly coincident in timing. Cryptorchid testis showed more effective antioxidative capability in the first 4 days, and suddenly lowered the capability from day 5 on, then gradually restored the antioxidation from day 10 to day 14, and turned to worse on day 28 again. The extensive high gene expression on day 4 after EC and the up-rising of oxidative stress level on day 5 and the abrupt down-regulation of the gene on day 7 were closely related. From the chip data, we have found that the high level of reactive oxidative species (ROS) was not only related to the dysfunction or abnormality of the direct origin of ROS generation, but also related to the abnormality of the more upstream physiological events in energy metabolism, lipid metabolism. The selective regulation of metabolic substrate transporter in different cell population implied the existence of various regulation of the selective signal pathways among different cell populations by EC.

Context: The context of the study was to examine whether combined testosterone (T) and heat (H) treatment have additive or synergistic effects on suppression of spermatogenesis.Objective: The objective of the study was to determine whether T+H induces a greater suppression of spermatogenesis than either treatment alone in monkeys.Design: The study was a randomized, placebo-controlled study.Setting: The study was conducted at a primate center in China.Participants: The study population was comprised of 32 adult cynomolgus monkeys.Interventions: Groups of eight adult monkeys were treated for 12 wk with: 1) two empty implants (C); 2) two T implants (T); 3) daily testicular heat exposure (43 C for 30 min) for 2 consecutive days (H); or 4) two T implants plus testicular heat exposure (T+H). Treatment was followed by an 8-wk recovery period.Main Outcome Measures: Measures included sperm counts and germ cell apoptosis.Results: Serum T levels were elevated in both the T and T+H groups during treatment but not in the C or H group. Sperm counts were transiently suppressed after heat to 16.4% of baseline at 4 wk and then returned to pretreatment levels. Sperm counts were suppressed slowly after T treatment to nadir of 6.4% of pretreatment levels at 12 wk. T+H rapidly suppressed sperm output as early as 4 wk to 3.9% of pretreatment levels that was maintained throughout treatment. The decreased sperm counts were due to increased germ cell apoptosis in all treatment groups. Sperm counts recovered to the pretreatment levels in all groups by 8 wk after treatment.Conclusion: This proof-of-concept study demonstrates that transient testicular warming enhances and hastens the effect of T implant on the suppression of spermatogenesis in monkeys.

To confirm that transient increase in temperature of the testis (43C for 30 minutes once daily for 2 consecutive days) could induce apoptosis of germ cells in non-human primates and to investigate the possible roles of Hsp105 and Hsp60 in regulation of germ cell loss, we conducted the study on eight cynomolgus monkeys. The sperm concentration on day 28 after heat shock decreased to 8.4% of pretreatment levels and recovered to baseline on day 144. Using the TUNEL assay, increased numbers of apoptotic spermatocytes and round spermatids were detected on days 3, 8, and 30 post heat treatment. Hsp105 and Hsp60 mRNA and protein levels were analyzed using in situ hybridization, RT-PCR, immunohistochemical and Western blot methods. Hsp105 was confined to nuclei of spermatids before treatment, decreased dramatically with the loss of spermatids on days 3, 8, and 30, before returning to baseline levels on days 84 and 144. The expression of Hsp60 was high on days 3, 8, 30 and was only detected in Sertoli cells and spermatogonia. These results suggested that exposure of the testis to heat resulted in selective, but reversible damage to the seminiferous epithelium via increased germ cell apoptosis. Temporal changes in the expression pattern of Hsp105 and Hsp60 in relation to germ cell death suggests they may be involved in key processes in regulation of germ cell apoptosis.

To investigate the anti-proliferation effect of tyrosine protein kinase inhibitor, genistein (4'-5,7-trihydroxyisoflavone), on human salivary adenoid cystic carcinoma cell line SACC-83 in vitro, and its effect on cell cycle.SACC-83 cells cultured in vitro were treated with genistein,cell survival rate was calculated with MTT assay, phase contrast microscope was used to observe the status of cell growth and the morphological changes in each group, and cell cycle was detected with flow cytometry. Apoptosis was detected with Annexin V/PI staining. The results were statistically analyzed by SPSS11.5 software.When treated with genistein of certain concentration for certain time, SACC-83 cell growths were significantly inhibited. With the increase of concentration of genistein and elongation of acting time, the inhibitory effects increased. Cells treated with genistein showed changes in morphology, cells' volume was decreased ,and suspended cells increased. Treated with 220 micromol/L genistein for 72 hours, SACC-83 cell growths were significantly inhibited, cell cycle was arrested in G2/M phase, and cell apoptosis was induced(P0.01).Genistein inhibits growth of human salivary adenoid cystic carcinoma cell line SACC-83, arrests cell cycle in G2/M phase, and induces cell apoptosis; these suggest that tyrosine protein kinase has important effect on the neoplasia and development of salivary adenoid cystic carcinoma.

To study the apoptosis and its mechanism at the fetal-maternal interface of early gestation, localization of apoptotic cells in the implantation sites of the rhesus monkey on day 17, 19, 28 and 34 of pregnancy were first examine by using the TUNEL technique. The expression of Ki67, a molecular marker of proliferating cells, and two apoptotic proteins, B cell lymphoma/leukaemia-2 (Bcl-2) and P53, were then studied by immunohistochemistry. Apoptotic nuclei were observed mainly in the syncytiotrophoblast. Ki67 was confined almost exclusively to cytotrophoblasts. The localization of Bcl-2 protein follows that of the apoptotic nuclei and its expression level increased as the development of the placenta progressed on. P53 was detected to some extent in cytotrophoblasts and syncytiotrophoblast covering the basal feet of the anchoring villi during the late stage of placentation. Based on these observations, it might be suggested that Bcl-2 could be possible to play an interesting role in limiting degree of nuclear degradation and sustaining cell suvival in the multi-nucleated syncytiotrophoblast cells during early pregnancy, and P53 could also be essential in regulating the trophoblastic homeostasis by controlling its proliferation or apoptosis.

We have established the well-defined cycling, pseudo-pregnant and pregnant rhesus monkey models, and used these to analyze expression of the common molecules specifically related to angiogenesis, apoptosis or proteolysis, such as vascular endothelial growth factor (VEGF) and its receptors KDR, flt-1, flt-4 and flk-1, basic fibroblast growth factor (bFGF) and its receptors Flg, transforming growth factor-alpha and beta1 (TGF-a/beta1), and TGF-beta1 receptor type I (TbetaR-I) and type II (TbetaR-II), as well as steroidogenic acute regulatory protein (StAR), tissue type plasminogen activator/urokinase plasminogen activator/plasminogen activator inhibitor type 1 (tPA/uPA/PAI-1) and matrix matalloproteinase type 1, -3/tissue inhibitor matalloproteinase type 1, -2, -3 (MMP-1, -3/TIMP-1, -2, -3), Fas/FasL, BcL-2/Bax, in the corpus luteum (CL), in the functional layer of the endometrium and in the materno-fetal boundary of the implantation site. We have demonstrated that: expression of these molecules in the monkey CL, endometrium and materno-fetal boundary of the implantation site is correlated well with CL functional and vascular development and with the processes involved in the establishment of the implantation window as well as with the early stages of placentation. A coordinated increase in tPA and its inhibitor PAI-1 expression in the monkey and rat CL may be instrumental in initiating luteal regression in both species, and correlated well with the timing of the closure of the implantation window, whereas high uPA activity in the CL is important for the early formation of the CL and for maintaining its function which is closely correlated to the period of establishment of the implantation window. Apoptosis, proteolysis and angiogenesis occur in the CL and in the endometrium during the time of establishment of the implantation window, as well as in the materno-fetal boundary of the implantation site at the early stages of placentation. It seems that these processes occur in these tissues in a coordinated and time- and cell-dependent manner, and are reliant on each other. Based on these observations, we have designed experiments to test the actions of some related available compounds on mouse implantation, used alone or in combination. The preliminary data showed that the compounds which could effectively affect apoptosis, angiogenesis or proteolysis in the implantation site were capable of effectively inhibiting implantation by acting on the endometrium and/or on the CL. Furthermore, the combined use of these compounds produced an obvious additive effect on inhibiting implantation. This finding suggested this may be a good approach for developing an anti-implantation agent.

Primate placentation involves a series of cell proliferation, immigration and apoptosis which account for the progressive tissue remodelling at the implantation site. p53 is an important proto-oncogene involved in the regulation of cell-cycle and apoptosis. To study the effect of RU486 on apoptosis and expression of p53 at the fetal-maternal interface, the location of apoptotic cells and expression of p53 were examined using in situ 3'-end labeling method, immunohistochemistry and Western blot assay at the fetal-maternal interface of normal and RU486 treated rhesus monkey. Western blot analysis showed the specificity of the anti-human antibody used with the monkey tissue. In the placental villi, the apoptotic nuclei were observed mainly in the syncytiotrophoblast and part of the cytotrophoblast within the cell column; p53 protein was detected mainly in the cytotrophoblast. In the endometrium, positive signals for apoptosis and p53 were detected in some stromal cells. After two days of mifepristone treatment, the apoptotic cells increased significantly in both placental villi and endometrium. In the villi, the increased apoptotic nuclei were mainly localized to the cytotrophoblast. At the same time, p53-positive nuclei also increased in both villous cytotrophoblast cells and endometrial stromal cells, after the treatment of RU486. These results suggest that apoptosis and expression of p53 are essential in regulating trophoblastic homeostasis by controlling its proliferation in normal placenta, whereas the up-regulation of p53 protein may play an important role in apoptosis that happens at the fetal-maternal interface induced by RU486.

To investigate the possible effect of nitric oxide on receptivity and apoptosis of mouse endometrium and the possible pathway.Female pregnant mice were treated with either molsidomine, a generator of nitric oxide (NO), or N-omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. The pregnancy rates of each group were calculated; 3'-end-labeling was used to detect DNA fragmention of apoptotic cells; immunohistochemistry, in situ hybridization, and Western blot were applied respectively to estimate expression levels of Fas/FasL proteins and mRNA.The pregnancy rate in the drug treated group was reduced in a dose-dependent manner; apoptosis, Fas protein and mRNA levels in the endometrium of drug treated mice were correlatively decreased during the peri-implantation period.The decreased pregnant rate in mice by abnormal levels of nitric oxide may be brought about by inhibiting the normally occurrence of apoptosis in the receptive endometrium.

Previous studies on azoospermia or oligozoospermia induced by heat stress or high doses of testosterone mainly focused on germ cell apoptosis; no data regarding their possible effect on spermatogonia mitosis are available. We have established unilateral cryptorchid and testosterone undecanoate (TU)-treated monkey models and examined expression of P16INK4a in the testis to look at its possible role in azoospermia or oligozoospermia induced by the heat stress or the TU treatment. The results showed that both heat stress and TU were capable of inducing expression of P16INK4a mainly in spermatogonia and other types of germ cells as well as Sertoli cells at the later stage of germ cell apoptosis, namely on Day 10 after operation or on Day 60 after TU injection. It is, therefore, suggested for the first time that P16INK4a protein may inhibit the spermatogonia mitosis in the testis at the later stage of the germ cell apoptosis, resulting in arrest of spermatogenesis.

The exact role of apoptosis that occurs in human placenta during the early stage of pregnancy remains unknown because of the difficulty in obtaining the intact implantation site. In this study, we used rhesus monkey as an animal model to examine apoptosis occurring in the implantation site at various stages of early pregnancy. It was shown that Fas and FasL mRNA and protein were localized in both the chorionic villi and glandular epithelium from day 15 to day 30 of pregnancy. Fas and FasL protein were also expressed in the epithelial plaque on day 15 of pregnancy. In situ 3'-end-labeling results showed that glandular epithelial cells underwent extensive apoptosis with obvious morphological degradation during the early stage of pregnancy. It was found that the cells that were 3'-end-labeled in the chorionic villi and anchoring villi were mainly localized in cytotrophoblast and cytotrophoblast column. It is therefore suggested that in primates apoptosis, which may be involved in the regulation of proliferation of trophoblast villi and degradation of epithelial plaque, as well as remodeling of the glands in the maternal decidua, may play an important role during the early stage of implantation and placentation.

Objective In earlier studies, we have shown the involvement of the mitochondria-dependent intrinsic pathway for induction of male germ cell apoptosis in monkeys after transient testicular warming or administration of exogenous testosterone. The JNK (c-Jun NH2-terminal kinase) signaling pathway has been implicated in the activation of apoptosis in various cell systems by stimulating the intrinsic pathway, but its role in testicular germ cell death is unclear. The goal of this study was to define the role of JNK in male germ cell apoptosis in monkeys after mild testicular hyperthermia or deprivation of intratesticular T or the combination of both interventions. Study Design Groups of eight adult cynomolgus monkeys received one of the following treatments: (1) two empty Silastic implants (C); (2) two 5.5 cm T-implants (T); (3) daily exposure of testes to heat (43°C for 30 minutes) for 2 consecutive days (H); and (4) two T-implants plus exposure of the testes to heat for 2 consecutive days (T + H). Testicular biopsies were performed before and at 3, 8, and 28 days during treatment. Results Activation of JNK, as evidenced by increase in phospho-c-Jun in testis lysates, was detected in all treatment groups on day 3. Compared with controls, where no staining was detected, a strong phospho-c-Jun staining was detected in the nuclei of apoptotic germ cells in all treatment groups and in the Sertoli cell nuclei at day 8 in H and H + T groups. To further define the role of JNK in apoptotic signal transduction, we examined the expression of JNK1, JNK2, and JNK3 in testes after these interventions. In the control testes, the expression of JNKs was localized in the Sertoli cell cytoplasm. Costaining for JNK2 and -3 and for TUNEL shows expression of both of these isoforms only in those germ cells undergoing apoptosis when compared with controls where these proteins were detected in cytoplasm. In contrast, JNK1 was detected in the Sertoli cell nuclei at day 8 in H and H + T groups. Conclusion Our results indicate that (1) the JNK pathway may play a role in male germ cell apoptosis in monkeys; (2) JNK isoforms could have preferential effects on testis function; and (3) Sertoli cells participate in germ cell apoptosis triggered by heat stress via JNK signaling.

Objective Caspase 2 is an initiator caspase whose activation has been found to promote apoptosis through mitochondria-dependent intrinsic pathway signaling in various cell systems, including the oocyte. Previously, we have shown that the intrinsic pathway is the key pathway for male germ cell apoptosis in rodents, monkeys, and men. The present study investigates if germ cell apoptosis induced by mild testicular hyperthermia or deprivation of intratesticular testosterone (T) or after combined interventions involves activation of caspase 2. Study Design Groups of eight adult cynomolgus monkeys received one of the following treatments: (1) two empty silastic implants (C); (2) two 5.5 cm-T implants (T); (3) daily exposure of testes to heat (43°C for 30 minutes) for 2 consecutive days (H); and (4) two T-implants plus exposure of the testes to heat for 2 consecutive days (H + T). Testicular biopsies were performed before and at 3, 8, and 28 days during treatment. Results Mean incidence of germ cell apoptosis increased significantly by d 3 in the H-alone group and by d 8 in the T-alone group but peaked at d 3 in the H + T group. Maximum activation of caspase 2 in respective treatment groups, as evidenced by immunocytochemistry and immunoblotting using an active caspase 2 antibody, coincided with the increased incidence of apoptosis. In control testes, we detected moderate immunostaining for active caspase 2 in Sertoli cells with little or no expression of germ cells. In contrast, we found a strong staining for active caspase 2 in apoptotic germ cells and in the Sertoli cells Co-staining for TUNEL and active caspase 2 further confirmed activation of caspase 2 only in those germ cells undergoing apoptosis. Conclusion Caspase 2 is activated in male germ cell apoptosis in nonhuman primates after heat stress, hormonal deprivation, or combined interventions. Future studies aimed at determining the expression of inhibitor of apoptosis proteins in testis will be needed to determine why Sertoli cells are not dying in spite of enhanced expression of caspase 2.

Experimental cryptorchidism is a common model for examining the expression and function of heat-sensitive spermatogenesis-related genes in testis. Previous studies have shown that germ cells in cryptorchid testis die mainly in an apoptotic way. The molecular mechanism, however, is still unclear. We have established unilateral cryptorchid monkey model (Cynomolgus Macaque) to identify possible molecules involved in the germ cell apoptosis. The degree of germ cell apoptosis, the morphology of the cryptorchid testis, and the changes in the serum concentration of FSH, LH and testosterone after cryptorchid surgery were analyzed. Sertoli cell marker molecule vimentin, the orphan receptor LRH-1, as well as the mitochondria-related protein HSP60 and Bcl-2 were examined. Our results showed that the weight of the cryptorchid testis decreased in a time-dependent manner started from day 7 after the surgery, while the weight of the scrotal testis had no obvious change. HE staining showed that from day 5, some germ cells were detached from the epithelium. A massive degeneration of the seminiferous epithelium characteristic of epithelial structural disorganization and the formation of multinucleated giant cells as well as vacuoles was observed on day 10 and 15. The cryptorchidism induced a marked germ cell apoptosis on day 3 after the operation, reaching a peak level on day 7.

Objective To determine whether the mitochondria-dependent pathway was involved in testicular germ cell death in monkeys after mild testicular hyperthermia or deprivation of intratesticular T or the combination of both interventions. Study Design Groups of 8 adult monkeys received one of the following treatments: (1) two empty Silastic implants; (2) two 5.5 cm T implants; (3) daily exposure of testes to heat (43°C) for 2 consecutive days; and (4) two T implants plus heat exposure for 2 consecutive days. Testicular biopsies were performed before and at 3, 8, and 30 days during treatment. Results Translocation of both cytochrome c and DIABLO from mitochondria to cytosol was detected in all treatment groups compared to controls. This event was accompanied by an increase in Bcl-2 levels in both cytosolic and mitochondrial fraction of testicular lysates, whereas BAX levels remained unaffected. Conclusion These results indicate that the mitochondria-dependent pathway plays an important role for male germ cell death in monkeys. Timed testes biopsies will be used for transcriptional and translational assessment of androgen-responsive genes.

To study the apoptosis and its mechanism at the fetal-maternal interface of early gestation, localization of apoptotic cells in the implantation sites of the rhesus monkey on day 17, 19, 28 and 34 of pregnancy were first examine by using the TUNEL technique. The expression of Ki67, a molecular marker of proliferating cells, and two apoptotic proteins, B cell lymphoma/leukaemia-2 (Bcl-2) and P53, were then studied by immunohistochemistry. Apoptotic nuclei were observed mainly in the syncytiotrophoblast. Ki67 was confined almost exclusively to cytotrophoblasts. The localization of Bcl-2 protein follows that of the apoptotic nuclei and its expression level increased as the development of the placenta progressed on. P53 was detected to some extent in cytotrophoblasts and syncytiotrophoblast covering the basal feet of the anchoring villi during the late stage of placentation. Based on these observations, it might be suggested that Bcl-2 could be possible to play an interesting role in limiting degree of nuclear degradation and sustaining cell suvival in the multi-nucleated syncytiotrophoblast cells during early pregnancy, and P53 could also be essential in regulating the trophoblastic homeostasis by controlling its proliferation or apoptosis.