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Our recent studies demonstrated that experimental autoimmune orchitis (EAO) model was produced in C3H/He mice with high incidence by two subcutaneous injections of viable syngeneic testicular germ cells (TC) without the use of any adjuvants or immunopotentiators. In this study the developmental patterns of autoantigenicity of TC during postnatal period were investigated by examining the orchitogenic activity of TC, the lymphostimulatory activities of TC (including the TC-induced in vitro lymphocyte proliferative response and the cytokine release from sensitized spleen cells (SPC) in response to TC) and the immunohistochemical localization of target autoantigens in the testes of mice at various weeks of age. Delayed-type hypersensitivity-inducing capacity and anti-TC antibody-eliciting capacity were initially observed in mice that were immunized with TC of 4-week old (w.o.) mice. The TC from 6-w.o. mice had the capability of inducing EAO (orchitogenicity) for the first time. A significant stimulation of in vitro lymphocyte proliferative response, as well as of interleukin (IL) 5 and IL-6 production by sensitized SPC were detectable when TC of mice 3-w.o. or more than were employed as stimulant. IL-2 and interferon gamma production were detected with TC of 4-w.o. mice. Immunohistochemical staining reaction with anti-TC antisera was primarily localized at the acrosomal portion of spermatids and spermatozoa in the seminiferous tubules, being already detected in spermatids of as early as 3-w.o. mice. Thus, from these data it is suggested that the appearance of the lymphostimulatory activities of TC consistently precedes that of the orchitogenic activity and that relatively mature germ cells such as spermatids and spermatozoa developing in the testes during the postnatal weeks may be responsible for the induction of disease and relevant immune responses in our EAO system.

A rare case of bilateral spermatocele developed 17 years after vasectomy was presented. A forty-seven year old man visited to our hospital with a chief complaint of swelling of bilateral intrascrotal contents. Sonographic findings demonstrated multilocular lesions of the bilateral intrascrotal contents. Operative procedure revealed bilateral cysts originating from the caput of the epididymis. Bilateral epididymectomy were performed. It was diagnosed as spermatocele because of demonstration of spermatozoa in the cystic fluid. We concluded that bilateral spermatocele has been induced by prolonged increased pressure of the intraepididymal duct following vasectomy 17 years ago.

The sexual dysfunction and infertility after treatment of bilateral germ cell tumors (GCT) becomes the serious problem. Therefore andrological aspects as well as cancer curability should be considered in planning of bilateral GCT treatment. Here we report 3 cases of metachronous bilateral GCT treated with different regimens, and discuss from the viewpoint of preservation of sexual function. Case presentations: (1) A 38-year-old man underwent right-sided orchitectomy for a right testicular tumor at the age of 26 years. Pathological diagnosis was seminoma and clinical stage was T1N0M0S2. 12 years later, contralateral testicular tumor developed. Left-sided orchitectomy was performed. Pathological diagnosis was seminoma and clinical stage was T1N0M0S2. He has been followed up for 4 years after the second operation without any evidence of tumor recurrence. Endocrinological examination show low testosterone level, and high LH and FSH levels. Erection and ejaculation are impossible but he does not request androgen replacement therapy. (2) A 21-year-old man underwent right-sided orchitectomy for a right testicular seminoma at the age of 20 years (T1 N0M0S0). 1 year later, contralateral seminoma (T1N0M0S0) developed and left-sided organ-preserving operation was performed. Histologic specimens showed seminoma and intratubular malignant germ cells (ITMGC) in surrounding seminiferous tubules. 2 cycles of BEP was added after the operation. He has been followed up for 5 years without any evidence of tumor recurrence. Endocrinological examination shows normal levels of testosterone and LH, but FSH is slightly high. Erection and ejaculation are possible. (3) A 36-year-old man underwent right-sided orchitectomy for a right testicular embryonal carcinoma at the age of 30 years. Clinical T1N0M0S1 was confirmed. 6 years later, he noticed the induration at his left testis. The result of fine needle aspiration cytology was embryonal carcinoma. At first, organ-preserving operation after chemotherapy was planned. However, he refused the operation considering the possibility of erectile dysfunction and infertility. As a result, he received only chemotherapy (3 cycles of BEP), and has been free of the disease for 11 years after chemotherapy. The level of testosterone, LH, and FSH are all normal. Erection and ejaculation are possible.

Purpose: Vasectomy may result in damage to spermatogenesis. There are several explanations for this damage, including an increase of pressure in the seminiferous tubules and an autoimmune reaction. Recently, vasectomy has been reported to induce germ cell death by apoptosis. However, the exact mechanism of this vasectomy-induced germ cell apoptosis is unclear. To elucidate this mechanism, we designed a vasectomized rat model and examined the testiscular alterations and apoptotic degeneration biochemically and microhistopathologically. Particularly, we analyzed the expression of nitric oxide synthase (NOS) and nuclear factor kappa B (NF kappa B), which plays a critical role in the induction of the iNOS gene, in the testis after vasectomy to gain insight into the association between germ cell apoptosis and these factors., Materials and Methods: The testes of 40 Wistar rats (10-weeks old) were studied at 1, 2, 5 and 10 weeks after unilateral (left) vasectomy. Wistar rats weighting 290 to 310 g were divided into 2 groups and subjected to underwent either unilateral vasectomy or sham surgery under ether anesthesia. Bilateral testes were carefully observed biochemically and histopathologically. Apoptosis was detected by an in situ end-labeling technique (detection of cellular DNA fragmentation) and electron microscopy. Neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) protein was detected by Western blotting and immunohistochemical studies using each NOS monoclonal antibody. To confirm the co-localization of cellular DNA fragmentation in germ cells and each NOS, each set of consecutive testis sections (one stained for cellular DNA fragmentation and the others for each NOS) were examined. Expression of NF kappa B proteins was examined immunohistochemically using a NF kappa B p65 polyclonal antibody., Results: At 5 and 10 weeks after vasectomy, the vasectomized left testis was significantly lighter than the unvasectomized right testis and sham-operated testis. At that time, the seminiferous tubules of vasectomized testes were highly damaged, presenting narrow tubular diameter, disorder of cellular arrangement, depletion of the germ cells, and local interstitial fibrosis. Vasectomized testes demonstrated a significantly increased number of apoptotic germ cells per cross-sectional area compared with sham-operated testes at 5 and 10 weeks after operation (p < 0.01). Electron microscopy revealed apoptotic germ cells each with a darkly stained nucleus. Western blotting and immunohistochemical studies demonstrated that iNOS proteins were more strongly expressed on vasectomized testes as time passed after vasectomy. Examination of consecutive sections from the vasectomized testis revealed that visibly apoptotic germ cells that exhibited positive staining for cellular DNA fragmentation were also intensely stained for eNOS and iNOS. NF kappa B p65 proteins were more strongly expressed in the nucleus of germ cells in the vasectomized testis than in the sham-operated testis., Conclusions: We found that vasectomy results in damage to spermatogenesis in adult rats, that may induce germ cell apoptosis, and that iNOS and NF kappa B may play a critical role in the germ cell apoptosis after vasectomy.

Purpose: Controversy exists on how to diagnose the vanishing testis and the degree of investigation required. In this series, we reviewed anatomical and histological findings in vanishing testes and investigated the effectiveness of diagnostic laparoscopy and imaging studies., Materials and Methods: Between 1974 and March 1999, 107 boys with nonpalpable testis underwent surgery. Of the total, 52 had spermatic vessels, vas deferens, and/or nubbin, and as a result the diagnosis of vanishing testis was made., Results: The affected side of vanishing testis was left 41, right 9 and bilateral 2.35 nubbins were found and the lengths of 24 nubbins were 5 mm or less. Histological examinations were performed in 43 cases including 27 nubbins. From that total, 31 had vas deferens and 11 had epididymis. Only two nubbins had seminiferous tubules but they included no germ cells. The two nubbins were greater than 5 mm long. Laparoscopic surgery was undertaken in 12 separate cases of the vanishing testis and as a result hypoplastic spermatic vessels were present in 7 of the 12 cases., Conclusion: The incidence of viable testicular tissue in vanishing testes was 4.7% in our series and it ranges from 0-16% in other series. We submit that one can diagnose the inguinal vanishing testis with preoperative imaging and laparoscopy, and that the nubbin seldom contains testicular tissue. Our results do not support the necessity to remove nubbins.

Background and Purpose: Chinese herbal medicine, "Hochuekkito" is widely used for male infertility in Japan. There have been many reports concerning its clinical usefulness but very few reports of in vitro experiments studying the mechanism of its effects. In addition to stimulating germ cells, we analyzed its direct effects on sperm using computer assisted semen analyzer (CASA)., Materials and Methods: Motile sperm were prepared using swim up technique from semen collected from ten healthy volunteers. Sperm movements (motility, velocity, linearity) were analyzed by CASA after adding either serum containing anti-sperm antibody (ASA) or normal serum with or without Hochuekkito., Results: Two hours after adding serum with ASA, the decrease of sperm motility was significantly reduced from 25.1% (92.8%-->67.7%) to 12.5% (92.9%-->80.6%) by adding Hochuekkito. No significant difference in velocity and linearity was observed between two groups. By adding normal serum, any of three parameters differed significantly with or without Hochuekkito., Conclusion: Protective effects of Hochuekkito on sperm was suggested. Although normal sperm with ASA was used in this report, since the sperm of infertile patients are said to be more fragile, this results imply that direct protective effect is one of the mechanism of Hochuekkito for male infertility.

Background: The analysis of cell kinetics of germ cells have been implicated as important clue to investigate the etiology of infertile patients. We examined the germ cell kinetics of varicocele testis using proliferating cell nuclear antigen (PCNA) immunostaing by image cytometry (CAS 2000), which is very accurate and useful method to evaluate the staining intensity., Patients and Methods: Testicular biopsy specimen obtained from 28 cases of male infertility with left varicocele and 5 normal controls were quantitatively examined concerning PCNA expression. Positive rate of PCNA (PR) was expressed as the percent of nuclear area positively stained with PCNA monoclonal antibody compared with total nuclear area analyzed in more than 20 seminiferous tubules. A hematoxylin and eosin-stained sections, which were fixed in Bouin's solution were used to examine Johnsen's mean score by photomicroscopy., Results: PR of right and left testes of varicocele patients were 11.3 +/- 4.8% and 11.3 +/- 4.1%, respectively. PR of normal testes was 21.7 +/- 3.1%. PR of each side were reduced similarly and the difference of PR between right and left testes of varicocele patients were not significant. Bilateral PR was 11.4 +/- 4.1%. Bilateral PR was significant lower than that of normal testes (p < 0.001). Concerning varicocele grade, sperm concentration of seminogram, Johnsen's mean score and the improvement of seminogram, no significant correlations between PR and those were observed., Conclusion: PR evaluated by image cytometry is very useful biological parameter to assess the germ cell kinetics. In varicocele patients, not only left side but also right side of testes were deteriorated in the cell kinetics and the decrease in DNA synthesis is one of the causes that have deleterious effects on spermatogenesis.

It is hard to differentiate leucocytes from immature germ cells among many round cells in ejaculated semen. Recently monoclonal antibodies (MoAbs) which recognize surface antigen of various subtypes of leucocytes are available. In this study we applied a panel of MoAbs for leucocyte subpopulations (granulocytes, T-lymphocytes, B-lymphocytes, Monocytes/Macrophages and pan-leucocytes). Semen smears from 64 mild oligozoospermic patients and 5 fertile volunteers were stained by immunohistological technique (avidin-biotin-immunoperoxidase method). We found highly varying total leucocyte numbers ranging from 1200 to 2.2 x 10(8) per ejaculate. When comparing semen from infertile patients with those from fertile donors, statistically significant higher medians of total leucocytes, total granulocytes and monocytes/macrophages were seen in infertile group. We compared a total number of leucocytes obtained from conventional Papanicolaou staining with that from immunohistochemical staining. Both were almost identical but the former gave a little bit higher value, because of the difficulty in differentiating leucocytes from immature germ cell by Papanicolaou staining. Moreover we performed an experiment to evaluate the applicability of flow-cytometry (FCM) to count leucocytes in semen. Cells in the semen smears were stained with MoAbs recognizing the surface antigen shared by every leucocyte, and percentage of the leucocytes which were thus stained with MoAbs was compared with that determined by FCM. Results from FCM constantly gave lower value than these from immunostaining. This is probably because FCM could not differentiate leucocytes from contaminated non cell substances (e.g. cell debris). From the present study it is not adequate to use FCM for the detection of leucocytes in semen as applied in haematology. Further techniques to remove non-cellular substances should be established for its application in semen analysis.

Using the antibody for glutathione S-transferase (GST) purified from human kidney, normal testes and experimental cryptorchid testes from newborn to 20-week-old rats were immunohistochemically stained by the peroxidase antiperoxidase (PAP) method. The cryptorchidism was surgically created at 1 week of age. The localization of GST was particularly examined by light microscopy, and the amount of Leydig cells was measured by a stereological method. 1. Leydig cells in the normal and cryptorchid testes showed strong GST activity at all ages. The amount of these cells in normal testes increased from 4 to 8 weeks of age and then slightly decreased, whereas in cryptorchid testes it was significantly larger than in the normal testes at 20 weeks of age, indicating hyperplasia of Leydig cells. 2. In the normal and cryptorchid testes, degenerating primary spermatocytes with GST activity appeared in the seminiferous tubules at 2 to 4 weeks of age. In the cryptorchid testis, degenerating germ cells with GST activity were also found in the regressing seminiferous tubules after 4 weeks of age. It is possible that GST acts as a detoxification system in the degenerating germ cells. 3. The PAP staining of GST in the rat testes is considered to be useful method for evaluating metabolic function of the spermatogenic cells and the distribution and amount of Leydig cells. 4. Experimental cryptorchidism showed that germ cells become sensitive after 4 weeks of age.