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The prolactin-induced germ cell apoptosis in the seminiferous tubules is through an extrinsic caspase pathway
RESULTS: No differences were observed in age, abstinence, volume, sperm morphology, progressive motility, and vitality (P>0.10). Controls presented higher sperm concentration (x106/mL) (108.1; 87.9) than the study group (78.5; 52.6) (0 < 0.0001). Leukocytospermia lead to a decrease in DNA integrity, while semen processing lead to a decrease in DNA fragmentation and to an increase in mitochondrial activity (table 1). No mixed efffects (processing x leukocytospermia) could be observed. TABLE. Sperm mitochondrial activity and DNA integrity before and after seminal processing in samples with and without leukocytospermia
Pre-processing
Post-processing
RESULTS: The presence of c-fos mRNA was detected by the predicted PCR bands and confirmed by restriction enzyme digestion of the PCR product. Both forms of c-Fos proteins were immunoreactive, mainly in germ cells (spermatogonia, spermatocytes and spermatids) and Sertoli cells. ERbeta was primarily present in somatic cells (Leydig, Sertoli and myofibrillar cells). A summary of c-Fos, phosphorylated c-Fos and ERbeta distribution in human testis is shown in Table 1. CONCLUSIONS: This pattern of ER expression suggests two putative pathways for estrogen action over spermatogenesis, either directly involving Sertoli cells, where estrogens could alter gene expression and/or the transcriptional activity of c-Fos protein, or indirectly through paracrine signaling from ER-positive interstitial cells. Supported by: CNPq and CAPES, Brazil.
Control Group Study Group Control Group Study Group Comet I (%) Comet II (%)a Comet III (%)b Comet IV (%)c DAB I (%)d DAB II (%)e DAB III (%)f DAB IV (%)g
30; 16.5 55.2; 16.5 11.1; 5.6 3.8; 2.9 3.1; 4.9 78.3; 8.2 11.4; 6.2 7.3; 5.3
38; 17.4 46.3; 14.9 11.4; 6.4 4.4; 2.7 2.1; 3.4 81.5; 8.9 10.1; 4.8 6.3; 5.3
33.7; 22.5 59.6; 22.2 5.2; 5.9 1.6; 3 5.4; 8 90; 8.3 2.8; 2.7 1.8; 1.9
39.4; 20.5 50.5; 18.3 7.7; 6.6 2.4; 3.3 4.1; 6.1 89.7; 8.1 3.4; 3.5 2.8; 2.8
a effect of leukocytospermia (P¼0.034); b,c,d,e,f,g Effect of semen processing (P<0.0001).
CONCLUSIONS: While semen processing improves DNA integrity and mitochondrial activity independent of the presence of leukocytes in semen, samples with leukocytospermia present more DNA fragmentation. Supported by: None.
MALE REPRODUCTIVE ENDOCRINOLOGY P-836 EXPRESSION OF THE PROTO-ONCOGENE c-fos AND THE IMMUNOLOCALIZATION OF c-Fos, PHOSPHORYLATED c-Fos AND ERbeta PROTEINS IN THE HUMAN TESTIS. F. C. Araujo, A. B. Reis, C. A. Oliveira, F. M. Reis. Department of Physiology, UFMG, Belo Horizonte, Brazil; Department of Morphology, UFMG, Belo Horizonte, Brazil; Department of Obstetrics and Gynecology, UFMG, Belo Horizonte, Brazil. OBJECTIVE: The testes are glands with endocrine and exocrine functions, the latter being characterized by sperm formation, or spermatogenesis. This is under the control of a complex system involving endocrine and paracrine signalling. In view of an interrelationship between the expression of c-Fos and estrogen receptor beta (ERbeta) proteins, this investigation evaluated the expression of the proto-oncogene c-fos and the immunolocalization of c-Fos, phosphorylated c-Fos and ERbeta proteins in the human testis. DESIGN: A immunohistochemical and molecular study on human testicular tissue samples. TABLE 1. Summary of immunolocalization of c-Fos, phosphorylated c-Fos and ERbeta proteins in the human testis
Cell Type
c-Fos
Phosphorylated c-Fos
ER-beta
Leydig cell Myoid cell Sertoli cell Spermatogonia Spermatocyte Type A spermatid Type B spermatid Type C spermatid Type D spermatid
þþ þþþ
þþ þþ þþ
þþþ þþþ þþþ
, negative; þ, weak; þþ, moderate; þþþ, strong. MATERIALS AND METHODS: Testis tissue was obtained from 12 men undergoing orchiectomy as a treatment for prostate cancer. These patients had received no hormonal, chemo or radiation therapy before the operations. Tissues were stained by immunohistochemistry using the avidin-biotinperoxidase method, and c-fos mRNA expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR).
S384
Abstracts
P-837 THE PROLACTIN-INDUCED GERM CELL APOPTOSIS IN THE SEMINIFEROUS TUBULES IS THROUGH AN EXTRINSIC CASPASE PATHWAY. W. J. Huang, P. S. Wang, J.-Y. Yeh, L. S. Chang. Urology, National Yang-Ming University, School of Medicine, Taipei, Taiwan; Physiology, National Yang-Ming University, School of Medicine, Taipei, Taiwan; Medical Education and Research, Taipei Veterans General Hospital, Taipei, Taiwan; Urology, Taipei Veterans General Hospital, Taipei, Taiwan. OBJECTIVE: Hyperprolactinemia (hyperPRL) is associated with impaired reproductive function in the mammals. In the testis, the germ cell apoptosis is significantly more frequent in hyperPRL rats than in the control normal rats. And in in vitro study, the percentage of apoptotic germ cells occurs proportionally to the amount of PRL added in the incubation medium. This study was to investigate the underlying mechanism to induce germ cell apoptosis by PRL. DESIGN: Animal model of hyperPRL of male rats. MATERIALS AND METHODS: The hyperPRL status was produced by allografting of 2 anterior pituitary glands (þAP) to the subrenal capsule, while the control rats were grafted with similar amount of cerebral cortex (þCX). Six weeks after grafting, the serum PRL levels reached to a plateau. The testes from 12 rats (6 from þAP and 6 from þCX) were then removed and sections of the tissue were studied with TUNEL assay. The apoptotic germ cells were scored under fluorescent microscopy. The rest of the testes were prepared for protein extraction. Western blotting for caspase 3, 8 and 9 were performed for samples from both groups. RESULTS: The TUNEL studies of the seminiferous tubules demonstrated significantly increase in apoptotic germ cells in the þAP groups (20.7 2.2 vs. 2.9 1.0 per tubule, P<0.0001). The Western blotting studies showed an equal density in caspase 3 for both groups, but the density of caspase 8 was significantly stronger then the control (relative density 1.37 0.14 vs. 0.79 0.07, P¼0.007). Interestingly, there was almost no blotting density for caspase 9. CONCLUSIONS: We concluded that the effect of PRL to induce germ cell apoptosis is very evident in status after AP-grafting. The underlying mechanism to start apoptosis cascade might be via a caspase 8 rather than a caspase 9 related pathway. Supported by: National Science Council Taiwan; Taipei Veterans General Hospital. P-838 PROLACTIN INDUCED SPERMATOGENIC CELL APOPTOSIS PRESENTS DIFFERENTLY AT VARIOUS HORMONE ENVIRONMENTS. W. J. Huang, P. S. Wang, J.-Y. Yeh, L. S. Chang. Urology, National Yang-Ming University, School of Medicine, Taipei, Taiwan; Physiology, National Yang-Ming University, School of Medicine, Taipei, Taiwan; Medical Education and Research, Taipei Veterans General Hospital, Taipei, Taiwan; Urology, Taipei Veterans General Hospital, Taipei, Taiwan. OBJECTIVE: The effect of prolactin (PRL) on spermatogenesis has been observed to induce apoptosis in germ cells, mostly the spermatogonia. However the spermatogenesis is a process regulated by an orchestra of hormones. It is difficult to treat patients with spermatogenesis failure effectively with gonadotropins alone. The purpose of this study is to investigate the roles of testosterone and follicle stimulating hormone (FSH) in PRL-induced spermatogenic cell apoptosis. DESIGN: In vitro study using male hyperPRL rats animal modle. MATERIALS AND METHODS: The hyperprolactinemia (hyperPRL) status was induced by allografting anterior pituitay (AP) glands to subrenal space in the Sprague-Dawley rats (n ¼ 6 in each group). Six weeks after the grafting, the testicular tissue was retrieved and the seminiferous tubules
Vol. 88, Suppl 1, September 2007
Pre-processing
Post-processing
RESULTS: The presence of c-fos mRNA was detected by the predicted PCR bands and confirmed by restriction enzyme digestion of the PCR product. Both forms of c-Fos proteins were immunoreactive, mainly in germ cells (spermatogonia, spermatocytes and spermatids) and Sertoli cells. ERbeta was primarily present in somatic cells (Leydig, Sertoli and myofibrillar cells). A summary of c-Fos, phosphorylated c-Fos and ERbeta distribution in human testis is shown in Table 1. CONCLUSIONS: This pattern of ER expression suggests two putative pathways for estrogen action over spermatogenesis, either directly involving Sertoli cells, where estrogens could alter gene expression and/or the transcriptional activity of c-Fos protein, or indirectly through paracrine signaling from ER-positive interstitial cells. Supported by: CNPq and CAPES, Brazil.
Control Group Study Group Control Group Study Group Comet I (%) Comet II (%)a Comet III (%)b Comet IV (%)c DAB I (%)d DAB II (%)e DAB III (%)f DAB IV (%)g
30; 16.5 55.2; 16.5 11.1; 5.6 3.8; 2.9 3.1; 4.9 78.3; 8.2 11.4; 6.2 7.3; 5.3
38; 17.4 46.3; 14.9 11.4; 6.4 4.4; 2.7 2.1; 3.4 81.5; 8.9 10.1; 4.8 6.3; 5.3
33.7; 22.5 59.6; 22.2 5.2; 5.9 1.6; 3 5.4; 8 90; 8.3 2.8; 2.7 1.8; 1.9
39.4; 20.5 50.5; 18.3 7.7; 6.6 2.4; 3.3 4.1; 6.1 89.7; 8.1 3.4; 3.5 2.8; 2.8
a effect of leukocytospermia (P¼0.034); b,c,d,e,f,g Effect of semen processing (P<0.0001).
CONCLUSIONS: While semen processing improves DNA integrity and mitochondrial activity independent of the presence of leukocytes in semen, samples with leukocytospermia present more DNA fragmentation. Supported by: None.
MALE REPRODUCTIVE ENDOCRINOLOGY P-836 EXPRESSION OF THE PROTO-ONCOGENE c-fos AND THE IMMUNOLOCALIZATION OF c-Fos, PHOSPHORYLATED c-Fos AND ERbeta PROTEINS IN THE HUMAN TESTIS. F. C. Araujo, A. B. Reis, C. A. Oliveira, F. M. Reis. Department of Physiology, UFMG, Belo Horizonte, Brazil; Department of Morphology, UFMG, Belo Horizonte, Brazil; Department of Obstetrics and Gynecology, UFMG, Belo Horizonte, Brazil. OBJECTIVE: The testes are glands with endocrine and exocrine functions, the latter being characterized by sperm formation, or spermatogenesis. This is under the control of a complex system involving endocrine and paracrine signalling. In view of an interrelationship between the expression of c-Fos and estrogen receptor beta (ERbeta) proteins, this investigation evaluated the expression of the proto-oncogene c-fos and the immunolocalization of c-Fos, phosphorylated c-Fos and ERbeta proteins in the human testis. DESIGN: A immunohistochemical and molecular study on human testicular tissue samples. TABLE 1. Summary of immunolocalization of c-Fos, phosphorylated c-Fos and ERbeta proteins in the human testis
Cell Type
c-Fos
Phosphorylated c-Fos
ER-beta
Leydig cell Myoid cell Sertoli cell Spermatogonia Spermatocyte Type A spermatid Type B spermatid Type C spermatid Type D spermatid
þþ þþþ
þþ þþ þþ
þþþ þþþ þþþ
, negative; þ, weak; þþ, moderate; þþþ, strong. MATERIALS AND METHODS: Testis tissue was obtained from 12 men undergoing orchiectomy as a treatment for prostate cancer. These patients had received no hormonal, chemo or radiation therapy before the operations. Tissues were stained by immunohistochemistry using the avidin-biotinperoxidase method, and c-fos mRNA expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR).
S384
Abstracts
P-837 THE PROLACTIN-INDUCED GERM CELL APOPTOSIS IN THE SEMINIFEROUS TUBULES IS THROUGH AN EXTRINSIC CASPASE PATHWAY. W. J. Huang, P. S. Wang, J.-Y. Yeh, L. S. Chang. Urology, National Yang-Ming University, School of Medicine, Taipei, Taiwan; Physiology, National Yang-Ming University, School of Medicine, Taipei, Taiwan; Medical Education and Research, Taipei Veterans General Hospital, Taipei, Taiwan; Urology, Taipei Veterans General Hospital, Taipei, Taiwan. OBJECTIVE: Hyperprolactinemia (hyperPRL) is associated with impaired reproductive function in the mammals. In the testis, the germ cell apoptosis is significantly more frequent in hyperPRL rats than in the control normal rats. And in in vitro study, the percentage of apoptotic germ cells occurs proportionally to the amount of PRL added in the incubation medium. This study was to investigate the underlying mechanism to induce germ cell apoptosis by PRL. DESIGN: Animal model of hyperPRL of male rats. MATERIALS AND METHODS: The hyperPRL status was produced by allografting of 2 anterior pituitary glands (þAP) to the subrenal capsule, while the control rats were grafted with similar amount of cerebral cortex (þCX). Six weeks after grafting, the serum PRL levels reached to a plateau. The testes from 12 rats (6 from þAP and 6 from þCX) were then removed and sections of the tissue were studied with TUNEL assay. The apoptotic germ cells were scored under fluorescent microscopy. The rest of the testes were prepared for protein extraction. Western blotting for caspase 3, 8 and 9 were performed for samples from both groups. RESULTS: The TUNEL studies of the seminiferous tubules demonstrated significantly increase in apoptotic germ cells in the þAP groups (20.7 2.2 vs. 2.9 1.0 per tubule, P<0.0001). The Western blotting studies showed an equal density in caspase 3 for both groups, but the density of caspase 8 was significantly stronger then the control (relative density 1.37 0.14 vs. 0.79 0.07, P¼0.007). Interestingly, there was almost no blotting density for caspase 9. CONCLUSIONS: We concluded that the effect of PRL to induce germ cell apoptosis is very evident in status after AP-grafting. The underlying mechanism to start apoptosis cascade might be via a caspase 8 rather than a caspase 9 related pathway. Supported by: National Science Council Taiwan; Taipei Veterans General Hospital. P-838 PROLACTIN INDUCED SPERMATOGENIC CELL APOPTOSIS PRESENTS DIFFERENTLY AT VARIOUS HORMONE ENVIRONMENTS. W. J. Huang, P. S. Wang, J.-Y. Yeh, L. S. Chang. Urology, National Yang-Ming University, School of Medicine, Taipei, Taiwan; Physiology, National Yang-Ming University, School of Medicine, Taipei, Taiwan; Medical Education and Research, Taipei Veterans General Hospital, Taipei, Taiwan; Urology, Taipei Veterans General Hospital, Taipei, Taiwan. OBJECTIVE: The effect of prolactin (PRL) on spermatogenesis has been observed to induce apoptosis in germ cells, mostly the spermatogonia. However the spermatogenesis is a process regulated by an orchestra of hormones. It is difficult to treat patients with spermatogenesis failure effectively with gonadotropins alone. The purpose of this study is to investigate the roles of testosterone and follicle stimulating hormone (FSH) in PRL-induced spermatogenic cell apoptosis. DESIGN: In vitro study using male hyperPRL rats animal modle. MATERIALS AND METHODS: The hyperprolactinemia (hyperPRL) status was induced by allografting anterior pituitay (AP) glands to subrenal space in the Sprague-Dawley rats (n ¼ 6 in each group). Six weeks after the grafting, the testicular tissue was retrieved and the seminiferous tubules
Vol. 88, Suppl 1, September 2007