Kiyomi Matsumiya

Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukemia

More than 70% of patients survive childhood leukemia, but chemotherapy and radiation therapy cause irreversible impairment of spermatogenesis. Although autotransplantation of germ cells holds promise for restoring fertility, contamination by leukemic cells may induce relapse. In this study, we isolated germ cells from leukemic mice by FACS sorting. The cell population in the high forward-scatter and low side-scatter regions of dissociated testicular cells from leukemic mice were analyzed by staining for MHC class I heavy chain (H-2K b / H-2D b ) and for CD45. Cells that did not stain positively for H-2K b /H-2D b and CD45 were sorted as the germ cell-enriched fraction. The sorted germ cell-enriched fractions were transplanted into the testes of recipient mice exposed to alkylating agents. Transplanted germ cells colonized, and recipient mice survived. Normal progeny were produced by intracytoplasmic injection of sperm obtained from recipient testes. When unsorted germ cells from leukemic mice were transplanted into recipient testes, all recipient mice developed leukemia. The successful birth of offspring from recipient mice without transmission of leukemia to the recipients indicates the potential ofautotransplantation of germ cells sorted by FACS to treat infertility secondary to anticancer treatment for childhood leukemia.

Early induction of apoptosis in androgen‐independent prostate cancer cell line by FTY720 requires caspase‐3 activation

We previously reported that FTY720, a metabolite from Isaria sinclairii, induced some cancer cells to undergo apoptosis, and that FTY720‐induced apoptosis was not related to Fas‐antigens. In this study we investigated whether FTY720 was able to induce apoptosis in an androgen‐independent prostate cancer cell line, DU145, which is not only resistant to androgen‐withdrawal‐induced apoptosis but also Fas‐ and TNF‐α‐mediated apoptosis.

Mouse germ cell-less as an essential component for nuclear integrity.

ABSTRACT A mouse homologue of the Drosophila melanogaster germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2β (lamina-associated polypeptide 2β). To elucidate the role of mgcl-1, we analyzed two mutant mouse lines that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that were probably due to impaired nuclear envelope integrity were observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities were observed in testis in which the highest expression of mgcl-1 was detected. Fertility was significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations showed insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, were aberrant. Considering that the first abnormality during the process of spermatogenesis was abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling.

Anticarcinogenic effect of FTY720 in human prostate carcinoma DU145 cells: Modulation of mitogenic signaling, FAK, cell‐cycle entry and apoptosis

Despite the high frequency of prostate cancer, therapeutic options for advanced disease are limited to chemotherapy, radiation or hormonal therapy and eventually fail in all patients. Therefore, alternative approaches need to be developed. We previously reported that FTY720, a metabolite from Isaria sinclarii, is a unique antitumor agent for an androgen‐independent prostate cancer cell line and requires caspase‐3 activation in apoptosis. In our study, we have evaluated the effect of FTY720 on a family of mitogen‐activated protein kinases (MAPKs), focal adhesion kinase (FAK), mitochondrial transmembrane potential, caspase‐9 and caspase‐8 and analyzed the expression of some cell‐cycle regulator proteins in DU145 cells in order to understand the various antitumor effects of FTY720. Apoptosis was quantified by phosphatidylserine exposure. Activation of MAPKs, cleavage of caspase‐9 and caspase‐8, status of cyclin‐dependent kinases (CDKs) and Cip1/p21, a cyclin‐dependent kinase inhibitor, were evaluated by Western blot analysis, in addition to FAK and phospho‐FAK immunoprecipitation and cell‐cycle analysis by FACScan. We found that in DU145 cells, 40 μM FTY720 caused activation of p38 MAPK and the upstream kinase MKK3/MKK6 but not SAPK/JNK. Mitochondrial transmembrane potential, FAK and ERK1/2 were reduced while caspase‐9 and caspase‐8 were cleaved. The p38‐specific inhibitor had no effect on apoptosis induced by FTY720, whereas z‐VAD.FMK, a broad‐spectrum caspase inhibitor, did not inhibit the p38 MAPK activation. An amount of 20 μM FTY720 resulted in G1 arrest and a decrease of CDK2 as well as CDK4, whereas it induced Cip1/p21. FTY720 may exert anticarcinogenic effects against prostate cancer cells possibly involving modulation of mitogenic signaling, cell‐cycle regulators, induction of G1 arrest and apoptotic death in DU145 cells.

Chromosomal variants among 1790 infertile men

Abstract Background: The largest cytogenetic survey involving infertile men was undertaken to clarify whether chromosomal abnormalities, including autosomal abnormalities, affect semen qualities.

Prostate stromal cell-derived hepatocyte growth factor induces invasion of prostate cancer cell line DU145 through tumor-stromal interaction

In prostate cancer, several growth factors derived from stromal cells regulate tumor cell growth. Hepatocyte growth factor (HGF) possesses biological activities that promote cancer proliferation and invasion through tumor‐stromal interaction. We examined how prostate stromal cell‐derived HGF affects invasion of prostate cancer cells through this interaction.

Testosterone Suppresses Spermatogenesis in Juvenile Spermatogonial Depletion (jsd ) Mice

Abstract Male juvenile spermatogonial depletion (jsd/jsd) mice are sterile because of a failure of spermatogonial differentiation. We have previously reported the recovery of spermatogonial differentiation by suppressing the levels of gonadotropins and testosterone with Nal-Glu, a GnRH antagonist. To determine whether suppression of testosterone or the gonadotropins was responsible for spermatogenic recovery, we examined the effect of supplementation of LH or FSH along with Nal-Glu treatment. Systemic administration of flutamide, an androgen receptor antagonist, was also examined. LH supplementation elevated both serum and intratesticular testosterone levels and suppressed the recovery of spermatogonial differentiation in a dose-dependent manner. Supplementation with FSH did not affect either testosterone levels or spermatogonial differentiation. Furthermore, the mice treated with flutamide showed some recovery of spermatogonial differentiation. The overall findings revealed that testosterone action mediated by androgen receptors suppressed the spermatogonial differentiation in jsd/jsd mice and suggested that spermatogonial differentiation in the jsd mutant is highly sensitive to testosterone suppression.

Clinical Practice Manual for Late-onset Hypogonadism Syndrome

With the aging of the population, the quality of life (QOL) of middleaged and elderly men has come into question and it has been taken up from an interdisciplinary standpoint in recent years. Partial androgen deficiency of the aging male (PADAM) or lateonset hypogonadism (LOH) is a syndrome consisting of symptoms caused by partial deficiency of androgens, but the time of onset varies and the epidemiological status is unclear. Therefore, in Japan to date, this syndrome has been considered as a general phenomenon associated with aging, the medical authorities have not reacted and patients are not being treated. In Western countries however, this phenomenon has attracted attention in relation to geriatrics and reproductive endocrinology since the 1980s. In 1998, the International Society for the Study of the Aging Male (ISSAM) was founded to conduct basic and clinical research, to provide postgraduate education and to engage in publicity activities for the enlightenment of the public. The social background is characterized by the appearance of a very rapidly aging society with longer average life spans. The importance of improving the health of the elderly and preventive medicine as government policy has increased. Improving the health of the elderly not only promotes self-reliance of the elderly but also increases the work force. A high QOL is also possible. The main topic for healthcare in the 21st century is how to maintain the QOL of the elderly. In women, hormone replacement therapy (HRT) is widely applied internationally, but specific healthcare for elderly men appears to be limited to the widespread use of phosphodiesterase type 5 (PDE5) inhibitors to treat erectile dysfunction (ED). Although the delay in healthcare policies for elderly men is not a direct reason, a large gap has appeared between the average life spans of men and women in recent years and in Japan, men have a shorter life span than women by about seven years. In response to this sense of crisis, the World Health Organization (WHO) issued the Geneva Manifesto in 1997 and ‘healthy aging for men’ became an international movement. ISSAM was established in 1998 with the goal of ‘aging male research on gender specific issues in male health’. The first meeting of the society in Asia was held in Malaysia in 2001 and this topic was adopted on an international level from an early stage. The reason appeared to be strong economic and social concern that Asian countries with a current pyramid-type population distribution will become aging societies with a lower birth rate than in developing countries. Japan has already become an aging society with a low birth rate. In the national census (summary) in 2005, the elderly population of 65 years and older accounted for about 21% of the total population, the highest in the developed world. In Japan, scientific research on the aging male started at about the same time as in the rest of Asia, and the Japanese Society for the Study of the Aging Male (JSSAM) was founded in November 2001 with Yoshiaki Kumamoto, professor emeritus of Sapporo Medical University, and Hajime Nawata, professor of Kyushu University as representative facilitators. The goal of this society is ‘undertaking basic, clinical and social research and surveys on policies for the diagnosis, treatment and prevention of male-specific medical problems, and contributing widely to men’s health by development, promotion and spread of proper healthcare’. As mentioned above, the concept of research on the aging male is being promoted as ‘healthy aging for men’ but almost no actual treatment for such patients has been performed. When the JSSAM was established , so-called ‘male climacteric symptoms’ or ‘male menopause’ was popular in the media, and when such treatment was started , many patients mainly with a chief complaint of climacteric symptoms appeared in medical practice. These patients included many with psychiatric problems such as depression and considerable confusion arose in clinics and hospitals. Based on this background , the Subcommittee on Endocrinology, Reproductive Function and Sexual Function of the Japanese Urological Association asked the Scientific Committee to prepare a clinical practice guideline, and a working group was organized to prepare the guideline by a collaborative team from the Japanese Urological Association and JSSAM after a review by the Board of Directors. In this clinical practice manual, the term ‘late–onset hypogonadism (abbreviation: LOH)’ syndrome was adopted as the term that best expresses this condition medically. In order to recommend standard procedures for diagnosis, treatment, prevention and monitoring of adverse reactions due to androgen replacement therapy (ART) and post-treatment assessments, a literature survey of clinical papers was performed , but since treatment of LOH Syndrome has just started , almost all papers had a low recommendation rank. Therefore, the name was changed to ‘Clinical Practice Manual for Treatment of Late-onset Hypogonadism (LOH) Syndrome’ (‘Manual’ hereinafter) instead of the initially planned ‘clinical practice guideline’. Care of LOH Syndrome is in its initial stages and such treatment requires careful consideration. Since many men visiting medical institutions at present complain of ‘climacteric symptoms’, measures must be taken to have this disease recognized in the mental health field. In the future, it will be necessary to establish evidence for treatment of LOH Syndrome from the broad perspective of promotion of ‘healthy aging for men.’ This ‘Manual’ is the first edition aimed at gathering evidence through future diagnosis and treatment of LOH and it is hoped that it will serve as a reference for routine medical practice. Correspondence: Eitetsu Koh MD, Department of Urology, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa 920-8641, Japan. Email: kohei@med.kanazawa-u.ac.jp This is an English translation of text originally published in Japanese in (LOH ) , 2007, Jihou

REGULATION OF INVASIVE POTENTIAL OF HUMAN PROSTATE CANCER CELL LINES BY HEPATOCYTE GROWTH FACTOR

Background: The growth and progression of prostate cancer depends on the stromal‐epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro.

Ejaculated spermatozoa in patients with non-mosaic Klinefelter's syndrome.

Background : Non‐mosaic Klinefelter patients are generally azoospermic and there is no therapy to improve the spermatogenesis. Some patients have a few spermatozoa in their ejaculates, which can be used for intracytoplasmic sperm injection (ICSI), but only a few cases resulting in a successful birth have been reported.