Cresyl Violet Stains Mast Cells Selectively: Its Application to Counterstaining in Immunohistochemistry.

The thiazine dye toluidine blue (TB) is well known to stain mast cells and hyaline cartilage metachromatically, and thus is mostly often used for their identification. However, TB is not suitable for counterstaining in immunohistochemistry, because of its high-background staining in the cytoplasm of other cell species and in extracellular structures. To expand the knowledge about dyestuffs staining mast cells in consideration with their usage in immunohistochemistry, we determined the stainability of several thiazines and oxazines, which are structurally related compounds to TB, using sections of mast cell-containing tissues. We found that all azine dyes used metachromatically stained mast cells and cartilage. Among these dyes, an oxazines cresyl violet (CV) stained mast cells with lower background, suggesting that those are useful for detecting mast cells and for counterstaining in immunohistochemistry. To ascertain its utility, CV was used in immunostaining of bHSDs in sections from adult rat ovary. Immunopositive signals reflected by DAB development in brown were clearly detected even after CV staining. We conclude that, similar to thiazines, oxazines stain mast cells metachromatically, and that of these, CV is more useful as a counterstain in immunohistochemistry than TB.

The Stage- and Cell Type-Specific Localization of Fragile X Mental Retardation Protein in Rat Ovaries.

Premutations of the fragile X mental retardation 1 (FMR1) gene are associated with increased risk of primary ovarian insufficiency. Here we examined the localization of the Fmr1 gene protein product, fragile X mental retardation protein (FMRP), in rat ovaries at different stages, including fetus, neonate, and old age. In ovaries dissected from 19 days postcoitum embryos, the germ cells were divided into 2 types: one with decondensed chromatin in the nucleus was FMRP positive in the cytoplasm, but the other with strongly condensed chromatin in the nucleus was FMRP negative in the cytoplasm. The FMRP was predominantly localized to the cytoplasm of oocytes in growing ovarian follicles. Levels of FMRP in oocytes from elderly (9 or 14 months of age) ovaries were lower than in those from younger ovaries. These results suggest that FMRP is associated with the activation of oogenesis and oocyte function. Especially, FMRP is likely to be implicated in germline development during oogenesis.

Effects of 5α-dihydrotestosterone and 17ß-estradiol on the mouse ovarian follicle development and oocyte maturation.

We have previously reported that androstenedione induces abnormalities of follicle development and oocyte maturation in the mouse ovary. In granulosa cells of the ovarian follicle, androstenedione is aromatized to 17ß-estradiol (E2). To determine whether the androgen or estrogen acts directly on the follicle to induce the above-mentioned abnormalities, we compared the effects of a non-aromatizable androgen, 5α-dihydrotestosterone (DHT), with those of E2 on murine follicular development and oocyte maturation in a single follicle culture system. The high dose (10(-6) M) of DHT prompted normal follicular development, and there was no effect on oocyte meiotic maturation after stimulation with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). In contrast, culture with the high dose (10(-6) M) of E2 delayed follicular growth and also suppressed proliferation of granulosa cells and antrum formation. Furthermore, culture with E2 delayed or inhibited oocyte meiotic maturation, such as chromosome alignment on the metaphase plate and extrusion of the first polar body, after addition of hCG and EGF. In conclusion, these findings demonstrate that E2, but not DHT, induces abnormalities of follicular development, which leads to delay or inhibition of oocyte meiotic maturation.

The rat oocyte synthesises melatonin.

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine originally identified in the pineal gland, where it is synthesised enzymatically from serotonin (5-hydroxytryptamine) by the sequential action of arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT; also known as hydroxyindole O-methyltransferase). Melatonin directly affects ovarian functions and previous studies have suggested that melatonin is synthesised in the ovary. In the present study, we examined whether AANAT and ASMT are expressed in the adult rat ovary. Reverse transcription-polymerase chain reaction analyses demonstrated that both AANAT and ASMT mRNAs are expressed in the ovary. Western blotting for AANAT protein showed that the ovary, like the pineal gland, contains this enzymatic protein with a molecular mass of 24kDa. Immunohistochemistry revealed that the AANAT protein is localised to the oocyte, corpus luteum and medulla, including mast cells. AANAT protein was found in oocytes at all stages of follicular development, and its levels in oocytes increased progressively throughout follicular development. Furthermore, isolated oocytes metabolised exogenous serotonin to melatonin. These findings demonstrate that melatonin is synthesised from serotonin in oocytes. Melatonin synthesised in the oocyte may be implicated in its own growth or maturation, for example, by acting as a calmodulin antagonist or an antioxidant.

Androstenedione induces abnormalities in morphology and function of developing oocytes, which impairs oocyte meiotic competence.

OBJECTIVE: To obtain insight into the effects of androstenedione on ovarian folliculogenesis and oogenesis. DESIGN: Experimental study. SETTING: St. Marianna University School of Medicine. ANIMAL(S): Prepubertal (14-day-old) BDF1 female mice. INTERVENTION(S): Early secondary follicles were isolated from the ovaries and were cultured individually in vitro with or without androstenedione (10(-11) to 10(-5) M) for 12 days. Thereafter, the follicles were treated with hCG and epidermal growth factor (EGF). MAIN OUTCOME MEASURE(S): Diameters and morphology of follicles and oocytes; E(2) and P secretion; and chromatin configuration and expression of growth differentiation factor 9 (GDF9) in oocytes were examined. RESULT(S): Early secondary follicles developed to the preovulatory stage. Androstenedione treatments increased the follicle diameters, reduced survival rates of follicles, and promoted the formation of follicles with abnormal morphology, including misshapen oocyte. The secretion of E(2) and P was significantly higher in androstenedione-exposed follicles. Androstenedione prevented the alteration in chromatin configuration and reduced oocyte GDF9 expression. When follicles cultured with androstenedione were treated with hCG and EGF, the first polar body exclusion, chromosome alignment on metaphase plate, and spindle assembly were inhibited in the oocytes. CONCLUSION(S): These results demonstrate that excess androgen induces abnormalities in the morphology and function of developing oocytes, which impairs oocyte meiotic competence.

Secondary solid tumors after allogeneic hematopoietic SCT in Japan.

To evaluate the incidence and risk factors for secondary solid tumors in Japan after allogeneic hematopoietic SCT (allo-HSCT), 2062 patients who had received allo-HSCT between 1984 and 2005 were retrospectively analyzed. Twenty-eight patients who developed 30 solid tumors were identified a median of 5.6 years after transplantation. The risk for developing tumors was 2.16-fold higher than that of the age- and sex-adjusted general population. The cumulative incidence of solid tumors at 10 years after allo-HSCT was 2.4%. The risk was significantly higher for tumors of the skin, oral cavity and esophagus (standard incidental ratio 40.23, 35.25 and 10.73, respectively). No increase in gastric, colon or lung cancer, despite being the most prevalent neoplasm in the Japanese, was observed. In multivariate analysis, occurrence of chronic GVHD and malignant lymphoma as a primary disease was associated with a higher risk for developing solid tumors. Eighteen patients are still alive, and their 5-year probability of survival since diagnosis of solid tumors is 59.7%. Our data suggest that the incidence and risk factors of secondary solid tumors in Japanese allo-HSCT recipients are comparable to those reported in Western countries and emphasize that the early detection of solid tumors has a crucial role in improving OS.

Number of CGG repeats in the FMR1 gene of Japanese patients with primary ovarian insufficiency.

OBJECTIVE: To define the number of CGG repeats in the FMR1 gene of Japanese patients with primary ovarian insufficiency (POI) and normal controls. DESIGN: Retrospective, controlled cohort study. SETTING: Outpatient department of an academic tertiary center. PATIENT(S): One hundred twenty-eight consecutive Japanese patients with sporadic, nonsyndromic POI and 98 controls with normal menstruation. INTERVENTION(S): Deoxyribonucleic acid was obtained from the plasma of each subject. MAIN OUTCOME MEASURE(S): Differences in the distribution of CGG repeat numbers between patients with POI and controls. RESULT(S): Six alleles in the intermediate range and two in the premutation range were found in five and two patients with POI, respectively, but none were identified in normal controls. The prevalence of FMR1 premutation among Japanese POI patients was 1.56% (2 of 128). The prevalence of having >36 CGG repeats in the FMR1 gene was significantly higher in patients with POI than in controls, and age at the onset of amenorrhea was significantly lower in patients with >38 repeats. CONCLUSION(S): More than 36 CGG repeats in the FMR1 might intensify the etiology of POI, at least up to the premutation range.

Gonadotropins up-regulate the expression of enolase 2, but not enolase 1, in the rat ovary.

It has been demonstrated that the glycolytic enzymes, enolase 1 (ENO1) and enolase 2 (ENO2), are expressed in the rat ovary. In the present study, we found that mRNA levels of ovarian ENO2 but not ENO1 in normal cycling adult female rats changed significantly during the estrous cycle: ovarian ENO2 mRNA levels at metestrus were lower than those at estrus. Single injection of human CG (hCG) or equine CG (eCG) into immature (3 week old) rats up-regulated ovarian expression of ENO2. hCG mainly increased ENO2 expression in oocytes and theca cells of preantral and antral follicles, and eCG did in theca cells of these follicles. In contrast, hCG and eCG did not affect the expression of ENO1, which was mainly expressed in granulosa cells. These results suggest that endogenous gonadotropins up-regulate expression of ENO2 in oocytes and theca cells of preantral and antral follicles, which would activate glycolysis in these cells. It is also suggested that the activated glycolysis is necessary for ovarian functions such as follicle growth and maturation, and hormone production.

Exogenous androstenedione induces formation of follicular cysts and premature luteinization of granulosa cells in the ovary.

OBJECTIVE: To investigate the effects of androstenedione on ovarian follicle development. DESIGN: Experimental study. SETTING: University research laboratory. ANIMAL(S): Female Wistar-Imamichi rats and BDF1 mice. INTERVENTION(S): Rats were injected with androstenedione. Ovarian follicles of mice were cultured in the presence of androstenedione. MAIN OUTCOME MEASURE(S): Ovarian morphology; ovarian cell types undergoing apoptosis; ovarian expression of cytochrome P450 aromatase (P450arom), cytochrome P450 side-chain cleavage (P450scc), and cyclin-dependent kinase inhibitor p27(kip1); serum levels of T, E(2), and P in rats; and ultrastructure of granulosa cells from cultured follicles of mice. RESULT(S): In androstenedione-treated rat ovaries, follicular cysts were formed, and apoptotic cells were found in the inner part of granulosa cell layers of antral follicles. Androstenedione administration down-regulated expression of P450arom but up-regulated expression of P450scc and p27(Kip1) in the granulosa cells of antral follicles. Serum T levels were significantly increased in androstenedione-treated rats. In mouse follicles exposed to androstenedione, the granulosa cells contained abundant lipid droplets and mitochondria with complex tubular cristae. CONCLUSION(S): Excess androgen enhances apoptosis in the inner part of granulosa cell layers of antral follicles, resulting in the formation of follicular cysts. It is also demonstrated that androgen stimulates premature luteinization of granulosa cells.

Human chorionic gonadotropin induces nestin expression in endothelial cells of the ovary via vascular endothelial growth factor signaling.

The intermediate filament protein nestin was originally found to be expressed in neuronal progenitor cells, but recent studies have shown that other cell types, including endocrine and vascular endothelial cells, express nestin. In the present study, we examined the expression and localization of nestin in the ovaries of developing, peripubertal, and adult rats. RT-PCR and Western blot analyses revealed that nestin mRNA and proteins were expressed in adult rat ovaries. Immunohistochemical analyses using adult rat ovaries showed that nestin was mainly localized to capillary endothelial cells of theca interna in follicles with more than two layers of granulosa cells and that its expression increased with follicle growth. Ontogenetically, ovarian nestin expression started at the peripubertal period when the first gonadotropin surge occurs. To test the possibility that gonadotropins induce nestin expression, prepubertal (postnatal d 21) rats were sc injected with equine chorionic gonadotropin (eCG) and/or human chorionic gonadotropin (hCG). A single injection of hCG, but not eCG, was sufficient to induce nestin expression in follicles, mainly in capillary endothelial cells of theca interna. Furthermore, pretreatment with an inhibitor of vascular endothelial growth factor receptor prevented the induction of the nestin expression by hCG. These findings demonstrate that the endogenous LH surge induces nestin expression in capillary endothelial cells of theca interna via the vascular endothelial growth factor signaling pathway. Nestin may be involved in angiogenesis in growing follicles, which is followed by follicle maturation and subsequent ovulation.

Expression and cellular localization of melatonin-synthesizing enzymes in the rat lens.

Melatonin (N-acetyl-5-methoxytryptamine) prevents oxidative stress-induced cataract development, and previous studies have suggested that the ocular lens synthesizes melatonin. In the present study, we examined whether two key enzymes in melatonin biosynthesis, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), are expressed in the lens of adult male rats. Reverse transcriptase-polymerase chain reaction analyses demonstrated that both AANAT and HIOMT mRNAs are expressed in the lens. Western blotting for AANAT protein showed that the lens, like the pineal gland, contains this enzyme protein with a molecular mass of 24 kDa. Immunohistochemistry revealed that AANAT protein is localized to the lens cortical fiber cells. Serotonin, which is a substrate for AANAT and a melatonin precursor, was also found in this region. These findings demonstrate that the lens expresses AANAT and HIOMT, and suggest that the cortical fiber cells are the main melatonin-synthesizing sites in the lens. Locally synthesized melatonin in the lens cortical fiber cells may protect the lens itself from cataract development.

Expression of luteinizing hormone/chorionic gonadotropin receptor in the rat pineal gland.

Luteinizing hormone (LH) influences the secretion of melatonin (N-acetyl-5-methoxytryptamine) from the pineal gland. The present study examined the possible presence of LH/chorionic gonadotropin (CG) receptor in the pineal gland of adult female rats. Reverse transcriptase-polymerase chain reaction analyses demonstrated that LH/CG receptor mRNA is expressed in the pineal gland. Western blotting showed that the pineal gland, like the ovary, contains an 80 kDa receptor protein. Immunohistochemistry revealed that LH/CG receptor, arylalkylamine N-acetyltransferase (a regulatory enzyme in melatonin biosynthesis) and serotonin (a melatonin precursor) are localized primarily to the same cells of the pineal gland. We further found that the levels of pineal LH/CG receptor protein in normal cycling female rats change significantly during the estrous cycle, being lowest at early metestrus. These results demonstrate that LH/CG receptor is expressed in the pineal gland, primarily in melatonin-synthesizing cells, namely pinealocytes. Furthermore, it is suggested that LH influences pineal melatonin secretion through binding to this receptor. In addition, LH/CG receptor levels in the pineal gland are regulated during the estrous cycle under normal physiological conditions.

alpha1-Adrenergic receptor in rat ovary: presence and localization.

Noradrenaline modulates ovarian steroidogenesis, stimulates ovulation, and probably promotes follicular development in the ovary. It has been suggested that these effects of noradrenaline are mediated by alpha- and/or beta-adrenergic receptors (ARs) in the ovary. The purpose of the present study was to examine whether alpha(1)-AR is present in the rat ovary. In Western blotting, antibody against alpha(1)-ARs recognized a major protein in the ovary of adult (10-week-old) rats with a molecular weight of 80 kDa, which is similar to that of the alpha(1B)-AR subtype. Immunohistochemistry using this antibody showed that alpha(1)-AR was detected at various sites in the ovary, including large antral follicle, germinal epithelium at the circumference of large antral follicle, corpus luteum, and interstitial tissue. These results confirm that the ovary contains alpha(1)-AR (probably alpha(1B)-subtype), and suggest that this receptor mediates some of the activities of noradrenaline in the regulation of ovarian functions. Furthermore, we found that alpha(1)-AR is present in oocyte of large antral follicle, suggesting that noradrenaline acts on oocyte via this receptor.

Removal of both antennae influences the courtship and aggressive behaviors in male crickets.

To test whether insect antennae are necessary for eliciting courtship and aggression toward appropriate partners, we antennectomized adult male crickets (Gryllus bimaculatus) and observed their behavior toward other antennectomized males and intact females. At 7 days after removal of both antennae, pairs of antennectomized males were placed together; 80% displayed courtship behavior, generating courtship song by rubbing their forewings together, toward other antennectomized males, and 20% displayed aggressive behavior. Only 45% courted intact females. No intact males courted antennectomized males, and 80% displayed aggressive behavior. All intact males courted females. The results for males with one antenna removed were essentially the same as for intact males. These findings indicate that a high proportion of male crickets with both antennae removed court other males and fail to display male-male aggression, demonstrating that removal of antennae from male crickets induces male-male courtship and that an antenna is necessary for the expression of male-male aggression. Moreover, brain serotonin (5-hydroxytryptamine; 5-HT) levels in male crickets were significantly reduced at 7 days after removal of antennae. The reduction of 5-HT was detected primarily in the central body of the brain. Thus, 5-HT in the central body of the male cricket brain may be involved in the behavioral changes.

Melatonin binding sites in estrogen receptor-positive cells derived from human endometrial cancer.

Our previous work showed that melatonin (N-acetyl-5-methoxytryptamine) inhibits proliferation of the human endometrial cancer cell line, Ishikawa, which is estrogen receptor-positive. The aim of the present study was to determine whether Ishikawa cells possess membrane melatonin receptors. Binding of the radioligand 2-[125I]-iodomelatonin to membrane preparations obtained from Ishikawa cells was detectable, saturable and stable. Scatchard analysis revealed that the dissociation constant (Kd) of the binding sites was 179.0 pm (similar to that of the MT2 [Mel1b] melatonin receptor subtype), and that the concentration (Bmax) of the binding sites was 12.9 fmol/mg protein. Luzindole, a selective MT2 melatonin receptor antagonist, significantly suppressed binding of 2-[125I]-iodomelatonin at all concentrations tested (10(-8) to 10(-4) m). These results suggest that the MT2 melatonin receptor subtype is present in the membranes of Ishikawa cells, and that the antiproliferative effect of melatonin on Ishikawa cells is mediated via the MT2 receptor. This may have implications for the use of melatonin in endometrial cancer therapy.

The effect of female wings on male courtship behavior in the cricket Gryllus bimaculatus.

Sexual dimorphism exists in the shape and the structure of the forewings of the cricket (Gryllus bimaculatus). However, the functional significance of the wings in the G. bimaculatus female has been unclear. In common blue butterflies (Polyommatus icarus), wings in females have been suggested as being important for attracting males. To test whether female crickets need wings for conspecific males to recognize them and initiate mating behavior, we removed all wings from females and observed the behavior of males towards them. Most males (87.5%) showed mating behavior towards the wingless females: they produced courtship song and transferred spermatophores to the wingless females. Similarly, 88.5% of the males showed mating behavior towards intact females. When males were placed with both a wingless female and an intact female, no significant difference was detected in male mate choice. The findings demonstrate that the wing of the G. bimaculatus female is not necessary for female recognition by conspecific males and the initiation of male mating behavior, and that it is not important in male mate choice.

Immunohistochemical study of HNK-1 (Leu-7) antigen in prostate cancer and its clinical significance.

Anti-HNK-1 monoclonal antibody (MAb) was reactive with non-cancerous and cancerous prostatic epithelia, as well as natural killer cells, myelinated nerves and cells from APUD systems. The expression of HNK-1 antigen on prostate cancer was investigated immunohistochemically by avidinbiotin-peroxidase complex (ABC) method with anti-HNK-1 MAb to clarify the relationship between anti-HNK-1 immunostaining of the cancerous tissue and the tumor differentiation, and that between the former and the survival rate of patients. Of 52 patients with prostate cancer, 49 were reactive with anti-HNK-1 MAb, the positive rate being 94%. The well differentiated cancer showed the highest percentage of positive cancer cells and the strongest staining, while the poorly differentiated cancer had the lowest percentage of positive cancer cells and the weakest staining. HNK-1 antigen was highly expressed on prostate cancer, and the better differentiated the cancer, the more HNK-1 antigen expressed. We also analyzed the total 5-year survival rate of the 52 patients, and the average 5-year survival rate and non-progression rate of 32 patients in stage D2 who had received only endocrinotherapy. Significantly higher survival rate and non-progression rate were observed in the group with more than two-thirds positive cancer cells as compared with the group with less than two-thirds positive cancer cells. The results suggest that the expression of HNK-1 antigen on prostate cancer may be a useful prognostic factor for patients with prostate cancer.