Phospholipid hydroperoxide glutathione peroxidase (PHGPx) was intensely expressed in mitochondria in the midpiece of human spermatozoa by immunostaining with anti-PHGPx monoclonal antibodies. The PHGPx not only reduced phospholipid hydroperoxide but also scavenged hydrogen peroxide in human spermatozoa. We found a dramatic decrease in the level of expression of PHGPx in the spermatozoa of some infertile males by immunoblotting with anti-PHGPx monoclonal antibodies. These individuals accounted for about 10% of the group of 73 infertile males that we examined. All seven patients with PHGPx-defective spermatozoa were classified as suffering from oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Males with PHGPx-defective spermatozoa accounted for 26% of the 27 infertile males with oligoasthenozoospermia. No defects in expression of PHGPx in spermatozoa were observed in 31 fertile volunteers. After a 3-h incubation, the relative number of motile spermatozoa with low-level expression of PHGPx was significantly lower than that of spermatozoa with normal expression of PHGPx. The PHGPx-defective spermatozoa failed to incorporate rhodamine 123, revealing a loss of mitochondrial membrane potential. Ultrastructual analysis of mitochondria by electron microscopy demonstrated that the morphology of mitochondria in PHGPx-defective spermatozoa was abnormal. The results suggest that failure of the expression of mitochondrial PHGPx in spermatozoa might be one of the causes of oligoasthenozoospermia in infertile men.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674 -683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a CreloxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.A frequent cause of male infertility is defective sperm function, which is the main problem for close to a quarter of couples who attend infertility clinics (1-4). Considerable efforts are now focused on the identifying ultrastructural and/or molecular defects in the spermatozoa or seminal plasma to develop solutions to various types of male infertility.Phospholipid hydroperoxide glutathione peroxidase (GPx4) 2 is an intracellular selenoprotein that directly reduces peroxidized phospholipids produced in cell membranes (5). The GPx4 gene has a complex intron/exon structure (6, 7). Three different transcripts of GPx4 exist, differing in their 5Ј extension and coding for a cytosolic protein (non-mitochondrial GPx4), a mitochondrial protein (mitochondrial GPx4), and a nuclear protein (nucleolar GPx4), respectively (6, 7). After cleavage of the N-terminal mitochondrial import sequence of mitochondrial GPx4, the mature protein becomes identical to the 20-kDa non-mitochondrial GPx4 (8, 9). Nuclear GPx4 was recently identified as a sperm nucleus-specific 34-kDa selenoprotein (called snGPx, for sperm nucleus-specific glutathione peroxidase) (10). It is formed by use of an alternative promoter and start codon localized in the first intron of the GPx4 gene (7, 10, 11). We previously reported that 34-kDa GPx4 localized in nucleoli in several cell lines by using an N-terminal nucleolar import signal (11). We call hereafter nuclear GPx4 nucleolar GPx4, because non-mitochondrial 20-kDa GPx4 exists both in cytosol and in the nucleus (12). Expression of three types of GPx4 is induced significantly in testis during spermatogenesis, especiall...

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) is a neuropeptide related to vasoactive intestinal peptide-secretin-glucagon which stimulates adenylate cyclase in cultured rat pituitary cells and stimulates LH and FSH release in vitro and in vivo. Because the cAMP-protein kinase-A pathway regulates the gonadotropin subunit messenger RNAs (mRNAs) and modulates GnRH-stimulated gonadotropin secretion in vitro, we examined the effects of PACAP38 on gonadotropin secretion and subunit mRNA levels. Anterior pituitary cells were prepared from 7-week-old male rats castrated at 5 weeks of age. In monolayer cultures stimulated with GnRH, 0.1-10 nM PACAP38 decreased (P < 0.05) the EC50 for GnRH dose-dependently without affecting the maximum LH secretory response. Cells were next stimulated with 1-min pulses of 2.5 nM GnRH every hour for 9 h in the absence or presence of 10 nM PACAP38, which was perifused continuously. The amplitude of GnRH-induced LH, FSH, and alpha-subunit secretory episodes from PACAP38-treated cells rose (P < 0.01) gradually to 233 +/- 54%, 197 +/- 44%, and 378 +/- 104%, respectively (mean +/- SEM; n = 5 experiments), of the value for control cells lacking PACAP38. This enhancement was sustained for at least 3 h after PACAP38 was removed from the perifusion medium. With PACAP treatment, interpulse secretion of LH and alpha-subunit increased gradually (P < 0.01) to 174 +/- 21% and 212 +/- 64% of the value for chambers stimulated with GnRH alone (control), respectively, whereas interpulse secretion of FSH declined (P < 0.001) to 75 +/- 7% of the control value. In contrast to the gradual effect of PACAP38 to enhance GnRH-induced hormone secretion, PACAP38 alone produced a transient burst of gonadotropin secretion. At the completion of the perifusions, total RNA was extracted and gonadotropin subunit mRNA levels were determined by Northern analysis. GnRH increased (P < 0.01) FSH beta mRNA to 438 +/- 52% of the level in cells stimulated with medium alone (control). Adding PACAP38 to the perifusion medium partially blocked (P < 0.01) the effect of GnRH (178 +/- 20% of the control value), and PACAP38 alone reduced (P < 0.01) FSH beta mRNA levels to 31 +/- 3% of the control value. By contrast, alpha-subunit mRNA levels were increased by both PACAP38 (143 +/- 4% of the control value; P < 0.01) and GnRH (121 +/- 2% of the control value; P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

It has already been demonstrated that the Notch signaling system is essential for gametogenesis in the adult germ line of Caenorhabditis elegans. However, the role of the Notch signaling system in mammalian spermatogenesis has not been well investigated. Recently, it has been revealed that this signaling system is expressed in the mammalian testis by showing coexpression of Jagged 2 and its receptor, Notch 1, is consistent with Notch 1 being a cognate receptor for Jagged 2 in the mammalian testis. Therefore, we investigated expressions of messenger RNAs of Notch 1 and Jagged 2 in the testicular tissues of developing Sprague-Dawley rats by reverse transcription-polymerase chain reaction and Northern blot analysis, expressions of their proteins in the testicular tissues of developing rats, fertile human controls and infertile human patients with maturation arrest by immunohistochemistry, and effects of antibodies to this system by culturing rat testicular tissues with these antibodies. Transcripts of Notch 1 and Jagged 2 in the rat testis were positive throughout the examined period; these intensities became higher at day 13 after birth, coincidence with the formation of spermatocytes, and peaked at day 19 after birth. Expressions of Notch 1 and Jagged 2 were recognized at first in the perinuclear regions of spermatocytes in the rat testis as a round structure at day 19 after birth and thereafter in further differentiated germ cells as meiosis proceeded. In the adult rat testis, positive staining was present as a round structure in spermatocytes, as a typical horseshoe-shaped structure in round spermatids, and as a covering structure spreading around the nucleus of elongated spermatids, but not in spermatozoa. Notch 1 was recognized in the vacuole of the Golgi complex of primary spermatocytes and the acrosome of elongated spermatids with electron microscopy. When rat testicular tissues were cultured with anti-Notch 1 or anti-Jagged 2 antibody, round and elongated spermatids decreased after 5 and 7 days of culture, respectively, and disappeared at around 9 and 12 days of culture, respectively, with shrinkage of the diameter of seminiferous tubules. Spermatocytes, however, increased after 11 days of culture. Expressions of both proteins have been detected in the testicular tissues of human fertile controls as in the rat testicular tissues. However, Notch 1 expression has not been detected in testicular tissues of 11 patients with maturation arrest, whereas Jagged 2 expression has been recognized in all of them. In conclusion, the results presented in this study offer the possibility that Notch 1/Jagged 2 signaling system plays an important role for male germ cells to differentiate or at least to survive in the rat testis and fails to express in the testis of spermatogenic maturation arrest patients.

CD169+ macrophages are suggested to play a pivotal role in establishing anti‐tumor immunity. They capture dead tumor cell‐associated antigens and transfer their information to lymphocsytes, including CD8+ T cells, which is important for successful tumor suppression. This study aimed to determine the prognostic significance of CD169+ macrophages residing in the tumor‐draining lymph nodes from cases of bladder cancer. In this retrospective study, 44 bladder cancer patients who received radical cystectomy were examined. The abundance of CD169+ macrophages in the regional lymph nodes and the number of CD8+ T cells in the primary tumor were investigated by immunohistochemistry. A CD169 score was calculated based on the intensity of CD169 staining and the proportion of CD169+ macrophages, and the scores were compared to the patients’ clinicopathological parameters. A high CD169 score was significantly associated with low T stage and with a high number of CD8+ T cells infiltrating into the tumor. The group with high CD169 expression had significantly longer cancer‐specific survival than the group with low CD169 expression (5‐year cancer‐specific survival rate: 83.3% vs 31.3%). In a multivariate analysis, the CD169 score was identified as a strong and independent favorable prognostic factor for cancer‐specific survival. Our findings suggest that CD169+ macrophages in the lymph nodes enhance anti‐tumor immunity by expanding CD8+ T cells in bladder cancer. The CD169 score may serve as a novel marker for the evaluation of bladder cancer prognosis.

No abstract

Background : To assess the feasibility of laparoscope-guided minilaparotomy (endoscopic minilaparotomy) for renal cell carcinoma in patients on chronic dialysis. Methods : Endoscopic retroperitoneal minilaparotomy using a 30 ° telescope was carried out through single skin incision (5-8 cm) in eight patients with renal cell carcinoma who were on chronic dialysis. Outcomes of the operations were compared to those in eight patients on chronic dialysis with renal cell carcinoma who underwent standard translumbar radical nephrectomy.Results : Resection of the tumor was successfully completed without complication and the postoperative course was uneventful in both of the treatment groups. No significant difference in mean operative time or mean blood loss was observed between the treatment groups. Wound pain was minimal and analgesics were generally not required in the minilaparotomy group. The endoscopic laparotomy group resumed full diet and began walking earlier than the group that underwent standard radical nephrectomy. Conclusions : Endoscopic minilaparotomy seems to be a valuable alternative treatment for renal cell carcinoma in patients on chronic dialysis.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide expressed in the central nervous system and peripheral organs. Previous studies revealed the role and distribution of PACAP in the rodent testis, however, its presence in the human testis and in testicular germ cell tumors is not known. We used RT-PCR and immunohistological observations to investigate whether human testicular tissue and testicular germ cell tumors contain PACAP. The mRNAs for PACAP and its receptors were detected in total RNA extracted from human testes. PACAP immunoreactivity was observed in spermatogonia and spermatids from normal testes. In contrast, diffuse PACAP immunopositivity was observed in seminoma tumor cells, while only faint immunoreactivity was observed in embryonal carcinoma cells. Our data suggest that PACAP may play a role in human spermatogenesis and in testicular germ cell tumor development.