Fluorescence in situ hybridization analysis of sex-chromosome mosaicism in azoospermic men.

A retrospective study was carried out to determine the prevalence of sex-chromosome mosaicism among azoospermic men and to evaluate the feasibility of using fluorescence in situ hybridization (FISH) technique to assess mosaicism and the origin of marker chromosomes. Nine hundred eighty patients with azoospermia who were referred to a male infertility clinic at a university hospital were karyotyped by G-banding using peripheral blood lymphocyte (PBL) metaphase spreads. When sex chromosome mosaic karyotype was detected, FISH analyses using sex chromosome-specific probes were performed. Seventeen of 980 patients showed evidence of sex chromosomal mosaic karyotype or mosaicism with marker chromosomes by G-banding studies of PBLs. Ten patients showed mosaicism in the number of sex chromosomes and 7 showed mosaicism with marker chromosomes. All 17 patients agreed to undergo FISH analysis. FISH confirmed mosaicism in 88.2% of these patients (15 of 17). Low-frequency mosaicism showing a frequency of less than 10% by G-banding was proved to be nonmosaicism by FISH. Marker chromosomes detected in 7 patients were proved to be derived from the Y chromosome by FISH analyses. From these data the prevalence of sex chromosome mosaicism confirmed by FISH analysis is 1.5% (15 of 980 patients). FISH analysis should be applied when mosaicism shows a frequency of less than 10% by the G-banding technique. Also, FISH analysis is indicated when a marker chromosome is detected by G-banding.

Serum inhibin pro alphaC in infertile men.

Specific assays have been developed for bioactive inhibin dimers, inhibin A and B, and inhibin alpha-subunit precursor pro alphaC. To better understand the role of serum inhibin pro alphaC in infertile men, the authors measured these forms of inhibin in sera from 39 infertile men and analyzed inhibin relationships with serum gonadotropins, testosterone, and estradiol. All subjects had oligozoospermia. Inhibin A levels were undetectable in all subjects. Inhibin B concentrations were 117 +/- 59 pg/mL. Inhibit B concentrations correlated negatively with serum FSH (r = .584, p < .0001) and positively with sperm count (p < .01) and bilateral testicular volume (r = .607, p < .0001). The concentration of pro alphaC was 556 +/- 236 pg/mL (normal range, 446 +/- 28). Inhibin pro alphaC showed no correlation with serum FSH, LH, testosterone, sperm concentration, and bilateral testicular volume. In addition, inhibin pro alphaC was not correlated with inhibin B. Pro alphaC is unlikely to be a useful marker for spermatogenesis in infertile men compared with inhibin B.

Inhibition of steroidogenesis in Leydig cells by exogenous nitric oxide occurs independently of steroidogenic acute regulatory protein (star) mRNA.

Nitric oxide (NO) plays multiple roles in the reproductive system. The authors studied the effect of NO on LH-stimulated steroidogenesis in primary cultures of rat Leydig cells, particularly seeking a link between inhibition of steroidogenesis and changes in expression of steroidogenic acute regulatory protein (StAR). Sodium nitroprusside (SNP), an NO generator, did not alter basal testosterone, but dose-dependently reduced testosterone production in the Leydig cells stimulated by LH (100 ng/mL) at 3 h after addition of SNP. Induction of StAR mRNA transcripts could be detected as early as 1 h after the addition of LH, but no effect was detected of SNP on LH induction of StAR mRNA. StAR, then, is not affected in the inhibition by NO of LH-stimulated steroidogenesis in Leydig cells.

Sertoli cell number in testes of azoospermic men: trends in biopsy specimens.

The general male population has shown a long-term decline in sperm count. Induced hormonal imbalance can produce adverse reproductive effects in men, especially decreases in sperm count. One suggested cause for a decreased sperm count is a reduction in the number of Sertoli cells. The authors assessed numbers of Sertoli cells in testicular biopsy specimens obtained from azoospermic men between 1989 and 1999. The number of Sertoli cells per tubular area or circumference did not vary by year of biopsy among the tissue samples during this 10-year period. Patient age, taken as a measure of exposure duration to hypothesized abnormal hormonal environment, did not affect the number of Sertoli cells. Since no reduction in the number of Sertoli cells was observed, other pathogenic factors may be the cause of long-term decreases in sperm count.

Significance of serum inhibin B concentration for evaluating improvement in spermatogenesis after varicocelectomy.

BACKGROUND: The study aim was to clarify the relationship of serum inhibin B concentrations to recovery of spermatogenic function after varicocelectomy, both as a predictor of improvement in the seminogram and as a means of monitoring hormonal function after surgery. METHODS: Fifty-two varicocele patients, including five with normal sperm concentrations, were studied. Changes in the seminogram, serum hormone concentrations and serum inhibin B were evaluated in the 47 oligozoospermic patients after surgery. Preoperative inhibin B concentrations correlated significantly with serum concentrations of FSH (r = 0.598, P < 0.0001) and testosterone (r = 0.380, P < 0.02). Inhibin B concentrations also correlated significantly with sperm concentration (r = 0.351, P < 0.02) and total testicular volume (r = 0.578, P < 0.0001). No significant correlation was seen between inhibin B and the Johnsen score. Preoperative concentrations of inhibin B were higher in patients who increased their sperm concentration after surgery (responders) than in those without improved concentrations (non-responders). No significant difference was observed between pre- and postoperative inhibin B concentrations in responders or non-responders. However, 15 of 25 (60%) patients with increased inhibin B showed improvement of the seminogram, while only five of 22 (23%) patients with no change or a decrease in inhibin B had any improvement (P < 0.05). CONCLUSIONS: Preoperative serum inhibin B concentration could not reliably predict a response to varicocelectomy. However, a change in serum inhibin B concentration after varicocelectomy might be helpful to evaluate the improvement of testicular function after varicocelectomy.

[Clinical laboratory data and reference intervals standardized in Fukuoka].

In Fukuoka whose population is approximately five million inhabitants, surveys on the accuracy of laboratory data have been performed by the Fukuoka Prefecture Medical Association for the last 30 years. We have been attempting to evaluate the data for routine use since 1988, and it has become possible to share laboratory data between all institutions in Fukuoka prefectures. As a result, reference intervals for 23 clinical chemistry analytes were established in 1995, to which were added in 1996 five serum protein constituents that have been utilized for clinical examinations. Methods for documentations and monitorings the data obtained in the prefecture were also established, standardization of the above analytes extended to 97% of the institutions in the prefecture. Results for 14 of the 23 clinical chemistry analytes have become highly reliable and clinically useful as differences between institutions in terms of results have narrowed. Standardization of other analytes is now in progress.

Development of follicular dendritic cells: a study using short-term bone marrow cell grafting in SCID mice.

To evaluate the cellular origin of follicular dendritic cells (FDC) in lymphoid follicles (LFs), severe combined immunodeficient (SCID) mice (H-2d) were grafted with 5-bromo-2'-deoxyuridine (BrdU)-incorporated bone marrow cells from CB-17 mice (H-2d) and with non-BrdU-incorporated bone marrow cells from C3H mice (H-2k) and Wistar rats (RT1u). This procedure was followed by antigenic stimulation with horseradish peroxidase and related immune complex (mouse peroxidase anti-peroxidase) administration. Secondary LFs in the lymph nodes and spleen of the reconstructed SCID mice were examined morphologically and immunocytochemically. LFs reconstructed with CB-17 mouse bone marrow cells contained FDCs capable of trapping and/or retaining mouse peroxidase anti-peroxidase as immune complexes. Secondary LFs contained BrdU-incorporated germinal center lymphocytes but not non-lymphoid stromal cells. A cell grafting study in SCID mice using bone marrow cells from C3H mice and Wistar rats demonstrated that FDCs in reconstructed LFs exhibited a marker specific for the recipient but not for the donor. These data indicate that functionally active FDCs occur de novo in reconstructed LFs in SCID mice, and do not support the view that FDCs originate from bone marrow cells in short-term reconstructed LFs.

The distribution, immune complex trapping ability and morphology of follicular dendritic cells in popliteal lymph nodes of aged rats.

Immune system function declines with age, and lymph nodes involute. The aims of the study were to describe the distribution of follicular dendritic cells (FDCs) in the lymphoid follicles of aged rats, and to determine whether these cells have reduced ability to trap immune complexes (ICs). Popliteal lymph nodes of rats aged 24-28 months were immunostained for S-100 protein as a marker of FDCs. Some rats were pretreated with peroxidase-anti-peroxidase complex (PAP) as an IC. FDCs were densely distributed in lymphoid follicles, which contained many primary follicles and a few secondary follicles. FDCs in primary follicles stained weakly for S-100 protein, and showed weak trapping, while those in secondary follicles stained strongly for both S-100 protein and trapping. Ultrastructurally, in involuted lymphoid follicles FDCs were atrophic. We conclude that FDCs in aged rats are densely distributed in involuted follicles and show reduced trapping ability and atrophic morphology. This seems to reflect the long life of FDCs and the reduced numbers of lymphoid cells in these follicles. We suggest that FDCs in aged rats may show some of their normal functions if fully developed germinal centers are induced, and may not play an important role in the process of involution of the follicles.

Ultrastructural localization of S-100 protein in rat popliteal lymph nodes, and very slight proliferative activity of follicular dendritic cells.

The purposes of this study were to examine the tissue distribution of S-100 protein in rat lymph nodes at the ultrastructural level with respect to the relationship between follicular dendritic cells (FDCs) and antigen transporting cells (ATCs), and to determine whether FDCs increase after secondary stimulation with sheep red blood cells (SRBCs). We examined the ultrastructural localization of S-100 protein in rat popliteal lymph nodes, and the density of S-100 protein-positive FDCs in lymphoid follicles, after secondary stimulation with SRBCs, on paraffin wax sections. We found S-100 protein expression in FDCs in all regions of lymphoid follicles, although FDCs in the central portion of lymphoid follicles showed stronger reactions than FDCs in the periphery. S-100 recognized ATCs weakly. At the border between the subsinus layer and the lymphoid follicles, ATCs were very close to FDcs. There were only two mitotic S-100 protein-positive cells in the lymphoid follicles of all specimens. The density of S-100 protein-positive FDCs in the lymphoid follicles in secondary stimulated rats was significantly lower than in primary stimulated rats. We suggest that S-100 protein expression reflects FDC development and supports a close relationship between FDCs and ATCs. FDCs may have only very slight proliferative activity through the FDC density in the lymphoid follicles decreased after secondary stimulation.

A change in the localization of the region trapping immune complexes in rat popliteal lymph nodes during development of germinal centers, with regard to the distribution of follicular dendritic cells.

A study was conducted to clarify changes in the relationship between the region of immune complex (IC) trapping by follicular dendritic cells (FDCs) and the distribution of FDC during reaction of germinal centers (GCs), and to examine the relationship between the tridimensional shape of the IC-trapping regions and their two-dimensional shape. Five-week-old rats were given footpad injections of sheep red blood cells, and then their popliteal lymph nodes were excised between days 0 and 42, 24 h after injection of peroxidase-antiperoxidase complex (PAP) as an IC. The specimens were immunostained for PAP trapping on serial paraffin sections, and for S-100 protein as a marker of FDCs. It was found that during the GC reaction, PAP trapping became weak and then disappeared on the basal side of developing GCs where S-100 protein-positive FDCs were still present. All of the 1933 lymph follicles examined were found to trap PAP. Whereas the tridimensional shapes of the trapping regions showed similar patterns according to the development of lymph follicles, their two-dimensional shapes varied. We suggest that FDCs in primary follicles may differentiate into FDCs in the light zone and FDCs in the dark zone in secondary follicles. To evaluate each of the compartments of a lymph follicle more accurately, investigators should pay attention to the tridimensional shape of the compartment.

The density and distribution of follicular dendritic cells in rat popliteal lymph nodes: a decrease in their density and a change in their distribution after stimulation.

To determine whether follicular dendritic cells (FDCs) increase after stimulation, and also to confirm the widely accepted finding that the density of FDCs in the light zone is higher than that in the dark zone, we examined the density of FDCs in lymph follicles of rat popliteal lymph nodes. Rats aged five weeks were stimulated by injection of sheep erythrocytes, and then examined 10 days later. Unstimulated rats were also examined. After embedding in paraffin, the removed lymph nodes were immunostained with anti-S-100 protein antibody as a marker of rat FDCs. The density of FDCs was determined by measuring the area of the lymph follicle and counting the number of S-100 protein-positive cells within it. The density of FDCs in the lymph follicles of stimulated rats was found to be significantly lower than in 5-week-old and unstimulated rats. The density of FDCs in the light zone was similar to that in the dark zone when germinal center bordering cells (GCBCs), distributed at the border between the dark zone and the adjacent corona, were counted as FDCs. We conclude that the density of FDCs in lymph follicles decreases after stimulation, and suggest that FDCs have no or only very slight proliferative activity under normal conditions. Investigators may need to consider GCBCs in order to understand how FDCs differentiate.

Immunohistochemical analysis of dendritic/Langerhans cells in thyroid carcinomas.

Recent immunohistochemical investigations of thyroid carcinomas have revealed that dense infiltration by dendritic cells (DCs) is correlated with a favorable prognosis. The present study was done to clarify the frequency and characteristics of DC infiltration in thyroid carcinomas, and also cytokines associated with DC maturation and migration. Compared with follicular carcinomas, papillary carcinomas contained significantly higher numbers of DCs, interleukin (IL)-1 alpha- and tumor necrosis factor (TNF)-alpha-positive cells, and cells positive for two TNF-alpha receptors (p60 and p80). The centers of cancer nodules had large numbers of CD1a- and CD1c-positive DCs suggesting that they were Langerhans cells, whereas the periphery of cancer nodules and inflamed surrounding thyroid tissues had numerous CD1b-, L-M2- and X-12-positive DCs suggesting that they were interdigitating cells, as well as many CD1a- and CD1c-positive DCs. Neoplastic cells of papillary carcinomas were more frequently reactive with antibodies against IL-1 alpha and TNF-alpha than those of follicular carcinomas, and a good correlation between their immunoreactivity and the frequency of DCs was found. These data suggest that cytokines such as IL-1 alpha and TNF-alpha released from carcinoma cells and cells in the cancer stroma may regulate the infiltration and maturation of dendritic/Langerhans cells, and that this process may be better preserved in papillary than in follicular carcinomas.

Protection of thyroid cancer cells by complement-regulatory factors.

BACKGROUND: Clinical and experimental studies have suggested that complement activation may play a role in tumor cytotoxicity. Little information is available concerning the presence of complement activation and the localization of complement-regulatory factors in cells or tissues of malignant tumors. The aim of the present study was to examine, using immunohistochemistry and immunoelectron microscopy, whether the complement system is activated in tissues of thyroid carcinoma and whether thyroid carcinoma cells are protected from cell lysis by in situ complement activation. METHODS: Fresh tissues were obtained by thyroidectomy from 15 patients with papillary carcinomas, 7 with follicular carcinomas, and 5 with follicular adenomas. In addition, five specimens of histologically normal thyroid tissue and five specimens of chronically inflamed tissue adjacent to thyroid neoplasms were studied. Immunohistochemical and immunoelectron microscopic localization of complement components, C3d and C5b-9, and the complement-regulatory factors, such as s-protein, decay-accelerating factor (CD55), membrane cofactor protein (CD46), complement receptor types 1 (CD35) and 2 (CD21), and protectin (CD59), were examined in these tissues. RESULTS: The staining patterns of C3d, C5b-9, and s-protein were positive and homogeneous in the nonneoplastic and most neoplastic thyroid tissues. Immunoelectron microscopy showed these antigens were localized mainly on the subepithelial and vascular basement membranes and attached to the cell surface of thyroid follicular cells. Decay-accelerating factor (CD55) was present homogeneously on the basement membranes, on the basal cell border of the thyroid follicular cells, and often on the luminal surface of carcinoma cells. Both membrane cofactor protein (CD46) and protectin (CD59) were expressed strongly on the cell surface of almost all benign and malignant thyroid follicular cells. Membrane cofactor protein was expressed on both the basal and lateral membrane, showing cell-to-cell interaction, but rarely on the luminal surface, whereas protectin was expressed strongly on the luminal surface and often on the basal cell border but rarely on the lateral membrane. Neither complement receptor type 1 (CD35) nor complement receptor type 2 (CD21) was expressed on any thyroid follicular cells. CONCLUSIONS: The present study confirmed the presence of complement activation with subsequent deposition of C3d and C5b-9 complexes in thyroid carcinomas. It also indicated that thyroid carcinoma cells are protected from cell lysis because of complement activation in multiple phases by complete coverage of the entire cell membrane surface with complement-regulatory factors. These findings were similar to those found in nonneoplastic thyroid follicular cells.

Follicular dendritic cell and ICCOSOMES in germinal center reactions.

The development and destination of immune complex coated bodies (ICCOSOMES) was investigated at the ultrastructural level in the germinal centers (GC) of murine lymph nodes after secondary-immunization with horseradish peroxidase. Immediately after a booster injection, large amounts of immune complex (IC) were trapped on the surface of the cytoplasmic extensions of follicular dendritic cells (FDC). Cytoplasmic extensions, consisting mainly of FDC processes with associated IC, formed complex lamellar labyrinth-like structures (LBS). We describe here morphological changes of LBS. Lamellar or stationary LBS before the injection sequentially changed into distorted, filiform, glomerated and transitional LBS. Around day 1, IC-coated cytoplasmic extensions developed into glomerated LBS (G-LBS) which were invaginated into their own cytoplasmic ends, which swelled up to several microns in diameter. Around day 2, tufts of the glomerated LBS were unlaced and dispersed as (ICCOSOMES) in the interstitium. Some ICCOSOMES were endocytosed by germinal center cells (GCCs) and transported to the Golgi apparatus and perinuclear space. These ICCOSOME-bearing GCCs were B220-positive and some produced specific antibody (spAb) in the perinuclear space indicating they are in the B cell lineage. ICCOSOME-ingesting GCCs are thought to be able to process the antigen and present it to T cells. This may be important in the development of plasma cells and memory cells which developed from the GC B cells.

The development of porcine zona pellucida using monoclonal antibodies: I. Immunochemistry and light microscopy.

We established three monoclonal antibodies (Mabs) against the zonae pellucidae (ZP) of porcine oocytes, named STA-1, STA-2, and STA-3, and eventually we determined that they all reacted with the isolated ZP. Based on Western blotting without 2-mercaptoethanol (2-ME), STA-1 reacted with the 80,000-110,000 Mr component, STA-2 with the 42,000-63,000 Mr component, and STA-3 with the 40,000-80,000 Mr component of ZP. We immunohistochemically specified the components of porcine ZP reactive with the three Mabs during the course of follicular development. Each Mab reacted with both the ZP and the interfollicular cell space (IFCS). One ZP component, reactive with STA-2 and STA-3, was first produced in the primordial follicle and was not found at the cumulus follicle stage, which corresponds to the stage of large antral follicles more than 5 mm in diameter. Another ZP component, reactive with STA-1, was not produced until the secondary follicle stage, and was never found at the antral follicle stage. These results suggest that each ZP component is produced and secreted at a specific stage or stages of folliculogenesis.

The development of porcine zona pellucida using monoclonal antibodies: II. Electron microscopy.

Following our previous study on the immunohistochemistry of porcine zonae pellucidae (ZP), we undertook the present study to localize the components of the ZP with immunoelectron microscopy, using three types of anti-porcine-ZP monoclonal antibodies (Mabs), named STA-1, STA-2, and STA-3. Some organelles of the oocyte were seen to react with STA-2 and STA-3 prior to ZP formation. As soon as a follicle began to mature, STA-2 and STA-3 reacted with the perinuclear space and the endoplasmic reticular membrane of the oocyte. The follicle first reacted with STA-1 at the secondary follicle stage. At this stage, the positive reaction involved the follicular cell layer as well as the oocyte and ZP. Positive reaction was scattered within and limited to the interfollicular cell space and was never found in the cytoplasm of follicular cells. At the antral follicle stage, the oocyte was surrounded by a thick, electron-dense ZP. A strong reaction was observed in the outer layer, but no significant reaction occurred in the inner layer. The convex and ragged outer margin of the ZP was characterized by the strongest reaction.