C.S. Teng

Gossypol-induced apoptotic DNA fragmentation correlates with inhibited protein kinase C activity in spermatocytes

After exposure of cultured rat spermatocytes to gossypol acetic acid for five hours, DNA fragmentation in a ladder pattern was found in the medium and supernatants of cell lysates. The concentrations of gossypol used for the induction of apoptosis ranged from 100 microM to 300 microM. Within this dose range, gossypol was also found to be effective at inhibiting protein kinase C (PKC) activity. This inhibitory effect was demonstrated by measuring the PKC residing in cytosolic and particulate fractions. However, the gossypol-induced inhibition of PKC activity was protected by phorbol 12,13-dibutyrate (PDBu), an activator of PKC. Furthermore, the presence of PDBu prevented gossypol-induced DNA fragmentation. These results suggest that spermatocyte apoptosis induced by gossypol is correlated with the reduction of PKC activity, and that maintenance of PKC basal activity is essential for protecting the spermatocyte from apoptosis.

Protooncogenes as mediators of apoptosis

Apoptosis has been well established as a vital biological phenomenon that is important in the maintenance of cellular homeostasis. Three major protooncogene families and their encoded proteins function as mediators of apoptosis in various cell types and are the subject of this chapter. Protooncogenic proteins such as c-Myc/Max, c-Fos/c-Jun, and Bcl-2/Bax utilize a synergetic effect to enhance their roles in the pro- or antiapoptotic action. These family members activate and repress the expression of their target genes, control cell cycle progression, and execute programmed cell death. Repression or overproduction of these protooncogenic proteins induces apoptosis, which may vary as a result of either cell type specificity or the nature of the apoptotic stimuli. The proapoptotic and antiapoptotic proteins exert their effects in the membrane of cellular organelles. Here they generate cell-type-specific signals that activate the caspase family of proteases and their regulators for the execution of apoptosis.

Biphasic c-Myc Protein Expression During Gossypol-Induced Apoptosis in Rat Spermatocytes

The presence of protooncogene products such as c-Myc proteins in rat spermatocytes has been quantitatively detected by Western immunoblot and a computer-controlled Spotdenso-program with an IS-1000 digital imaging system. Cellular levels of c-Myc proteins in response to gossypol were measured in spermatocytes during the process of gossypol-induced apoptosis. Within 0.5 to 2 h of the addition of gossypol, levels of c-Myc proteins fall dramatically and remain at a low level for the next several hours. The reduction in c-Myc proteins occurs 4.5-6 h before the apoptosis of spermatocytes in the presence of gossypol. Between 3 and 5 h after exposure to gossypol, the c-Myc protein content returns to preexposure (or higher) levels. In addition, the increase in c-Myc proteins occurs 1.5-4 h before the apoptotic death of spermatocytes. An identical pattern of c-Myc protein response to gossypol was also found in total testicular tissue in vitro. These results suggest that spermatocyte apoptosis induced by gossypol is correlated with biphasic c-Myc protein expression. This article present some hypothetical models with which to explain c-Myc protein-mediated apoptosis.

The effects of gossypol on nuclear protein in rat testes. I. Reduction in the content of basic proteins in the spermiogenic cells

In rat testes, after 45 days of gossypol treatment, the number of round spermatids (RS) reduced from 9.8 x 10(6) to 6.2 x 10(6) per testis. The number of elongating spermatids (ES) reduced from 6.4 x 10(6) to 3.1 x 10(6) per testis. Total nuclear basic proteins (TNBP, i.e., somatic type of histones H1, H2A,B, H3, and H4, plus testis-specific proteins TP1,2,3, and sperm-specific S1 proteins) were extracted from RS and ES. Gossypol has no detectable effect on the concentration of TNBP in RS. However, significant effect was found in ES, where TNBP was reduced from 8.7 micrograms to 6.8 micrograms/10(6) cells. A two-stage polyacrylamide gel has been used to separate the TNBP and the individual proteins were quantified by a Beckman Model DU-7 computer-monitored spectrophotometer. In the RS cells, the content of the TNBP was not sensitive to gossypol. However, in the ES cells, the content of histones H1, H2A, and H2B were reduced by gossypol treatment (with an inhibition of 50%, 58%, and 31%, respectively). The inhibition on H3, TP1, TP3 and S1 was insignificant by gossypol.

c-Fos protein expression in apoptotic rat spermatocytes induced by gossypol

Proto-oncogene products such as c-fos protein with a molecular weight of 62 kDa have been identified in rat spermatocytes. In this study, cellular levels of c-fos proteins in spermatocyte, either with or without gossypol exposure, were quantitatively detected by Western immunoblot and a computer-controlled Spot-denso-program with an IS-1000 Digital Imaging System. Within 0.5-3.5 h (an average of 2 h) of the addition of gossypol, levels of c-fos proteins fell dramatically. The reduction in c-fos proteins occurred 6 h before the apoptosis of spermatocytes in the presence of gossypol. Four hours after exposure to gossypol, the c-fos protein content was overexpressed. The period of c-fos up-regulation lasted for approximately 8 h. The increase in c-fos protein coincided with a high rate of apoptotic cell death. Morphologic structure of the dying cell was revealed by electron microscopy. These results suggest that spermatocyte apoptosis induced by gossypol correlates with biphasic c-fos protein-mediated apoptosis.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment. V. Inhibition and recovery of microtubular dynein

After oral administration with gossypol acetic acid for various times, young male rats developed a low content of microtubular (or cytoplasmic) dynein in the spermatogenic cells, e.g., spermatids and primary spermatocytes. The content of dynein in the cells was measured by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-dynein antibody. The results were expressed as ng dynein/10(6) cells and compared with those of the control rats. After gossypol treatment for 8, 12, and 19 weeks, the content of dynein in spermatids was reduced by 61%, 70%, and 68%, respectively; whereas, the amount of dynein in primary spermatocytes was reduced by 37%, 44%, and 31%, respectively. The microtubular dynein associated with spermatids was more vulnerable to gossypol than that of the primary spermatocytes. Immunofluorescent staining technique confirmed the finding that the control cells have more dynein than that of the drug-treated cells. Eight weeks after the withdrawal of the drug treatment, the content of dynein in spermatids and primary spermatocytes was fully recovered. The possible effects of this change in conjunction with the function of microtubules during spermatogenesis and sperm motility are discussed.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment: I. The activity for DNA synthesis

Mature male rats were treated with gossypol for various lengths of time after which the treatment was stopped to allow the arrested spermatogonia to revive. Fifteen days after withdrawal of the drug treatment, the arrested spermatogonia entered into the meiotic division and then reached the pachytene stage. The meiotic cells derived from the arrested spermatogonia were the first generation of the differentiated germ cells after long-term gossypol treatment. Centrifugal elutriation technique was used to isolate the repopulating pachytene spermatocytes (RPS) or the control pachytene spermatocytes (CPS) from the rat testis with or without receiving gossypol pretreatment. In vitro culture condition for RPS and CPS was established for the synthesis of DNA by the measurement of 3H-thymidine incorporation. The activity for DNA synthesis in RPS was studied and compared to that of the CPS. It was concluded that after gossypol treatment for various times, the incorporation of 3H-thymidine into the cellular thymidine pool of RPS was not affected. However, the activity for DNA synthesis in RPS was significantly lower than that in the CPS. The synthetic activity for DNA was reduced by 14%, 26%, 42%, 40%, and 40% in the RPS for the gossypol pretreatment of 3, 4, 6, 7, and 8 weeks, respectively.

THE EFFECTS OF GOSSYPOL ON NUCLEAR PROTEIN IN RAT TESTES. II. THE SYNTHESIS OF HISTONES AND TESTIS-SPECIFIC PROTEINS AFTER GOSSYPOL TREATMENT

This communication is to report the effects of gossypol on the synthesis of chromosomal basic proteins in the spermiogenic cells. A double-labelling experiment has been performed. The control rats received 14C-arginine, whereas the gossypol-treated rats received a 3H-arginine injection. An equal volume of the tissues (from control and gossypol-treated) were combined, and the total nuclear basic proteins (TNBP) from round spermatids (RS) and elongating spermatids (ES) were extracted for electrophoretic separation. By studying the ratio of 3H/14C, it was indicated that the synthesis of H1, H3, H2B plus H2A and TP1 in the RS was reduced by gossypol. The amount of inhibition of these proteins were 48%, 19%, 27%, and 11%, respectively. In the ES, the synthesis of H1, H3, H2B plus H2A, H4 plus TP2, TP3, and TP1 was reduced by gossypol, the reduction was 33%, 22%, 26%, 14%, 33%, and 8%, respectively. The synthesis of S1 protein was not inhibited by gossypol in both RS and ES.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment: II. The activity for the synthesis of cellular and nuclear basic proteins

The synthesis of cellular and nuclear basic proteins in the first generation of the repopulated pachytene spermatocyte (RPS) and the control pachytene spermatocyte (CPS) was studied by the incorporation of 3H-arginine into proteins. After pretreatment with gossypol for various lengths of time, the synthetic activity for cellular protein in the RPS was slightly inhibited. In comparison to CPS, on the other hand, the inhibition ranged from 12% to 26% (for gossypol pretreatment from 3 to 12 weeks, respectively). However, the synthetic activity for nuclear basic protein in the RPS was drastically reduced in comparison to that of the CPS. The reduction ranged from 46% to 61% in RPS after receiving gossypol pretreatment for 3 to 12 weeks, respectively. Gel electrophoretic separation of the basic protein extracted from the pachytene cell indicated that the major basic proteins are nucleosomal linker and core proteins, i.e., histone H1, H2A,B, H3, and H4, and with a lesser amount of sperm-specific BP and X1 proteins. After gossypol treatment in the RPS, the synthesis of sperm-specific proteins (BP and X1) and core histones (H2A and H4) became drastically reduced. Finally, the effect of gossypol on the ratio of nuclear basic protein to DNA in RPS and the direct correlation of this ratio to nucleosomal spacing and chromatin structure are discussed.

Differential activation of MAPK during Müllerian duct growth and apoptosis: JNK and p38 stimulation by DES blocks tissue death

Abstract. To investigate the involvement of the mitogen-activated protein kinases (MAPKs) in c-Jun-mediated Müllerian duct (MD) differentiation, Western immunoblot with antibodies against c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK1 and 2), p38, phosphorylated JNK (p-JNK), and p-ERK were used to investigate these kinases in the left and right MDs (LMD and RMD, respectively) of female chicks. The content of these kinases in the LMD and RMD of various stages of embryos was detected by measuring their density in autoradiograms by a Spot-denso-program with Alpha Ease software. In the LMD, the growing embryonic sex tract, the amount of JNK increased from the 8th to 10th embryonic day and reached its highest at the 12th to 18th day. The content of ERK1, ERK2, and p38 remained at the same level throughout development. In the RMD, the apoptotic embryonic sex tract, the level of these four MAPKs showed a linear increase from the 8th to 10th day and then declined at the 12th day. Before the RMD entered the apoptotic stages (10th day of incubation), MAPKs were overexpressed. The findings following the application of p-MAPK antibodies, e.g., p-JNK and p-ERK, mirrored the result showing that differential activation of MAPKs existed in the LMD and RMD. When the RMD entered the apoptotic stages (13th to 18th day of incubation), the reduction in JNK activity was higher than that of the other three kinases. The apoptotic death of RMD was prevented by in vivo diethylstilbestrol treatment, which restored the level of JNK, p-JNK, and p38. No stimulatory effect was found for ERK and p-ERK.