Masahiko Maegawa

A repertoire of cytokines in human seminal plasma.

The pathophysiological significance of seminal cytokines in sperm function is still controversial. We determined the repertoire of cytokines in seminal plasma obtained from men with or without abnormalities in semen and assessed the pathophysiological significance of seminal cytokines. After conventional analysis of semen samples obtained from 86 men, levels of seminal cytokines (interleukin [IL]-1alpha, IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-alpha [TNF-alpha], interferon-gamma, granulocyte colony-stimulating factor [G-CSF], macrophage CFS [M-CSF]) and granulocyte elastase were measured by an enzyme-linked immunosorbent assay. Leukocytospermia was defined as seminal plasma, which has > or =1000 ng/ml granulocyte elastase. Leukocytospermia was found in nine of 62 of the subjects in the normozoospermic group but in none of the 24 subjects showing abnormal sperm parameters (azoospermia, n=5; oligozoospermia, n=4; asthenozoospermia, n=15). The IL-8 level in the leukocytospermic group was significantly higher than those in the normal and oligozoospermic groups. IL-1alpha and TNF-alpha levels in the leukocytospermic group were significantly higher than those in the normal and asthenozoospermic groups. Although the G-CSF level in the leukocytospermic group was significantly higher than that in the normal group, high levels of M-CSF were detected in all groups. The IL-8 level was strongly correlated with IL-1alpha (r=0.935, P<0.0001) and G-CSF (r=0.916, P<0.0001) levels. Cytokines detected in seminal plasma are associated with the pathogenesis of leukocytospermia but not with the pathogenesis of asthenozoospermia and oligozoospermia.

Essential Role of IκB Kinase α in Thymic Organogenesis Required for the Establishment of Self-Tolerance

IκB kinase (IKK) α exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-κB activation pathway. The in vivo function of IKKα, however, still remains elusive. Because a natural strain of mice with mutant NF-κB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKα axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKα-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKα-dependent manner was associated with defective processing of NF-κB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKα in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.

Immunohistochemical study on the development of CRF-containing neurons in the hypothalamus of the rat

SummaryAppearance of immunoreactive corticotropin-releasing factor (CRF)-containing neurons was studied in developing hypothalamus of the rat by use of antisera against rat- and ovine CRF. These neurons were first recognized in the lateral and paraventricular nuclei on days 15.5 and 16.5 of gestation, respectively, when antiserum against rat CRF was employed. Antiserum against ovine CRF revealed the cells two days later exclusively in the latter nucleus. In both nuclei, the neurons increased in number with development. The neurons in the paraventricular nucleus appeared to project their immunoreactive processes to the median eminence via the periventricular and lateral pathways. In the median eminence, the immunoreaction with antiserum to rat CRF was first recognized in its anterior portion in the form of dots on day 16.5 of gestation but as beaded fibers in the external layer on day 17.5; these structures increased in amount with development in rostro-caudal direction. Although antiserum to ovine CRF was less potent in immunostainability than antiserum to rat CRF, it also revealed the beaded fibers in the median eminence on day 17.5 of gestation. Since evidence is available that the paraventricular nucleus is involved in corticotropin release, it is concluded that, in rats, the hypothalamic regulatory mechanism controlling the release of corticotropin initially appears on days 16.5–17.5 of gestation.

Differential immunolabeling for electron microscopy of diverse peptidergic neurons.

We describe a simple and reliable method for differential immunolabeling of pre- and post-synaptic signal peptides at the ultrastructural level. Hypothalamic tissues of rats, including the suprachiasmatic nucleus, were cut on a Vibratome. Visualization of the immunolabeling of somatostatin (SRIH) and vasoactive intestinal polypeptide (VIP) was performed with avidin-biotin-peroxidase-diaminobenzidine (DAB). The end product of the DAB to VIP was further silver-intensified in a physical processing using silver nitrate, and the silver grains were finally substituted for gold. DAB-labeled SRIH fibers synapse on gold-labeled VIP perikarya and dendrites in the suprachiasmatic nucleus.

CRF-containing neurons of the rat hypothalamus.

SummaryThe immunoreactive CRF-neurons of the rat hypothalamus have been examined immunohistochemically employing anti-rat CRF serum. These neurons are confined to the paraventricular nucleus, dorsomedial-lateral hypothalamic area, and suprachiasmatic nucleus, and are, respectively, also immunoreactive to anti-Met-enk, -alpha-MSH, and -VIP sera. Intraventricular administration of colchicine (50 μg/5 μl/rat) induces a dramatic enhancement of the immunostainability of the cell somata, and also accelerates the development of immunoreactivity of other stored peptides, especially in the paraventricular nucleus.The CRF-neurons respond to adrenalectomy by showing increased immunoreactivity and an increase in the number of cell bodies; in the dorsomedial-lateral area and suprachiasmatic nucleus, there is also an enhanced immunoreactivity for alpha-MSH and VIP, respectively. CRF-cells in the paraventricular nucleus become markedly hypertrophied, but do not show any enhanced immunoreactivity for Met-enk. Since the axons of the paraventricular neurons run to the median eminence, it is probable that they are involved with the endocrine control of hypophysial ACTH release. It is concluded that the CRF-containing neurons in rat hypothalamus consist of three types which are functionally and morphologically different.

Postmenopausal changes in production of type 1 and type 2 cytokines and the effects of hormone replacement therapy.

ObjectiveAn appropriate defense against infective agents or malignant cells is attributed to the exquisitely balanced T helper 1 type (cellular) and T helper 2 type (humoral) immune reactions. We investigated the effect of hormone replacement therapy (HRT) on postmenopausal changes in the production of interferon (IFN)-&ggr; and interleukin (IL)-10, a type 1 and a type 2 cytokine, respectively. DesignBoth cytokines were measured by ELISA in the supernatant of lipopolysaccharide-stimulated whole blood cells from 72 untreated and 44 HRT-treated women. Thirteen women were examined before and during HRT. ResultsThe production of IFN-&ggr; in women in their 40s and in postmenopausal women was significantly higher compared with that of younger women. However, IFN-&ggr; fell to the lowest level in the late postmenopausal stage, whereas the production of IL-10 increased gradually with age and in parallel with the postmenopausal period. Thus, in women in the mid-and late postmenopausal period, excessive production of type 2 cytokine (IL-10) compared with type 1 cytokine (IFN-&ggr;) occurred. The IFN-&ggr; levels of women on HRT were significantly lower than those of untreated women in the early and mid-postmenopausal stages, and IL-10 levels of women on HRT were significantly lower than those of untreated women in the mid-and late postmenopausal stages. HRT induced a significant decrease in the production of IL-10 and tended to lower the level of IFN-&ggr;. ConclusionsProduction of IL-10 is augmented in postmenopausal women. HRT probably prevents postmenopausal women from an aberration of the immune system by improving the balance of type 1 and type 2 immune reactions.

Hypothalamic thyrotropin-releasing hormone (TRH)-containing neurons involved in the hypothalamic-hypophysial-thyroid axis. Light microscopic immunohistochemistry

The localization of neurons containing immunoreactive thyrotropin-releasing hormone (TRH) was examined in the hypothalamus of intact, propylthiouracil (PTU)-treated, and colchicine-treated adult rats. In intact animals, immunoreactive TRH neurons were occasionally found in the paraventricular and dorsomedial nuclei, and in the anterior and lateral hypothalamic areas. In PTU-treated animals, the cellular appearance of the hypothalamus with the exception of the paraventricular nucleus was almost similar to that of intact animals. In the paraventricular nucleus, only the cells localized in the periventricular and medial parvocellular subdivisions significantly increased in number and became hypertrophic in comparison with intact animals. The distribution of immunoreactive fibers in the hypothalamus was almost equal among the 3 animal groups with the exception of that in the median eminence, in which the fibers were most densely concentrated in intact animals, and most sparse in PTU-treated rats. The fibers projecting into the median eminence were distinguished into the periventricular and lateral pathways, which are derived from the neurons in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus, respectively. Thus, among immunoreactive TRH neurons in the hypothalamus, only those in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus may be involved in the hypothalamic-hypophysial-thyroid axis.

Transient Increase in the Levels of T-Helper 1 Cytokines in Postmenopausal Women and the Effects of Hormone Replacement Therapy

The aim of this study was to determine, at least in part, T-cell function in postmenopausal women and the effects of hormone replacement therapy (HRT). Levels of T-helper 1 (Th1) cytokines (IL-2 and IFN-γ) and T-helper 2 (Th2) cytokines (IL-4 and IL-10) produced by phytohemagglutinin-stimulated whole blood cells from 72 untreated and 44 HRT-treated women were measured by ELISA. Thirteen of the 44 HRT-treated women were examined before and during HRT. The production of IL-2 increased gradually with advance of the postmenopausal period. The levels of IL-2 in women in the early (≤10 years) and mid (>10 and <30 years) postmenopausal stages were significantly higher than those in women in their second, third and fourth decades. The level in women in the late (≧30 years) postmenopausal stage, however, was significantly lower than those in women in the early and mid postmenopausal stages. The level of IFN-γ was highest in women in the mid postmenopausal stage. On the other hand, the levels of Th2 cytokines did not change with age or after menopause until the mid postmenopausal period but were significantly lower in women in the late postmenopausal stage. IFN-γ levels in women on HRT were significantly lower than those in untreated postmenopausal women at all postmenopausal stages. HRT induced a significant decrease in the production of IL-2 and IL-4. In conclusion, production of Th1 cytokines is augmented in women after menopause. HRT prevents this increase, thereby improving the aberration of Th1/Th2 balance that is implicated in an inadequate immune response and pathological conditions.

High implantation rate and consequently high pregnancy rate by in vitro fertilization-embryo transfer treatment in infertile women with antisperm antibody

OBJECTIVE To examine the effect of antisperm immunity on postfertilization steps, such as implantation of embryos and fetal growth in IVF-ET treatment of women with sperm-immobilizing antibodies. DESIGN Retrospective analysis of clinical laboratory data. SETTING The IVF-ET program of the Department of Obstetrics and Gynecology. The University of Tokushima, School of Medicine. PATIENTS Eighteen women with sperm-immobilizing antibodies and 122 infertile patients with nonimmune etiology as controls. Infertile couples due to a male factor and with unknown etiology were excluded. INTERVENTIONS All patients received the same IVF-ET program with GnRH agonist. MAIN OUTCOME MEASURES Rates of fertilization and cleavage, implantation rate per embryo transferred and pregnancy rate (PR) in both test and comparison groups. RESULTS The rate of fertilization in the antisperm group (61.3%) was significantly lower than that in the comparison group (76.8%). But the implantation rate per embryo transferred (23.5%) and consequently the modified PR per oocyte recovery procedure (34.4%) in immunologically infertile women were significantly higher than those in the comparison group (7.9% and 17.8%, respectively). CONCLUSIONS Although sperm-immobilizing antibodies prevent sperm-egg interaction, they do not seem to have any adverse effects on achievement of pregnancy. Moreover, the existence of antisperm immunity in woman with antisperm antibodies is suggested to be favorable for successful pregnancy by the IVF-ET procedure.

Immunohistochemical evidence of serotoninergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus

SummaryBy applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptie membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.