Photoperiodic regulation of pituitary thyroid-stimulating hormone and brain deiodinase in Atlantic salmon.

Seasonal timing is important for many critical life history events of vertebrates, and photoperiod is often used as a reliable seasonal cue. In mammals and birds, it has been established that a photoperiod-driven seasonal clock resides in the brain and pituitary, and is driven by increased levels of pituitary thyroid stimulating hormone (TSH) and brain type 2 iodothyronine deiodinase (DIO2), which leads to local increases in triiodothyronine (T3). In order to determine if a similar mechanism occurs in fish, we conducted photoperiod manipulations in anadromous (migratory) Atlantic salmon (Salmo salar) that use photoperiod to time the preparatory development of salinity tolerance which accompanies downstream migration in spring. Changing daylength from short days (light:dark (LD) 10:14) to long days (LD 16:8) for 20 days increased gill Na+/K+-ATPase (NKA) activity, gill NKAα1b abundance and plasma growth hormone (GH) levels that normally accompany increased salinity tolerance of salmon in spring. Long-day exposure resulted in five-fold increases in pituitary tshßb mRNA levels after 10 days and were sustained for at least 20 days. tshßb mRNA levels in the saccus vasculosus were low and not influenced by photoperiod. Increased daylength resulted in significant increases in dio2b mRNA levels in the hypothalamus and midbrain/optic tectum regions of the brain. The results are consistent with the presence of a photoperiod-driven seasonal clock in fish which involves pituitary TSH, brain DIO2 and the subsequent production of T3, supporting the hypothesis that this is a common feature of photoperiodic regulation of seasonality in vertebrates.

Assessment of the timing and degree of smolt development in southern populations of masu salmon Oncorhynchus masou.

The present study assessed whether non-anadromous masu salmon Oncorhynchus masou in Miyazaki, southern Japan, smoltify, and if so, at what time of the year. Yearling O. masou of Miyazaki and an anadromous population from Hokkaido, northern Japan, were reared in hatcheries in their respective regions and sampled monthly from February to June to examine the spring smoltification period. The Hokkaido population showed a peak of gill Na+ -K+ -ATPase (NKA) activity in May, which was accompanied with an increase in mRNA levels of the seawater (SW)-type NKA alpha subunit, nkaα1b. Increases in gill NKA activity and nkaa1b levels were not seen in Miyazaki populations. Transferring yearling Miyazaki population to 70% SW (salinity of 23) in mid-April resulted in an increased serum osmolality over 4 days. These results suggest that they do not smoltify in their second spring. Next, profiles of gill NKA activity and its subunit mRNA levels in under-yearling Miyazaki population in the autumn were examined. Two phenotypes differing in body color during this period were categorized as parr and smolt-like fish. Smolt-like fish had higher gill NKA activity than parr in December while there was no significant difference in gill nkaα1b levels. Smolt-like fish acclimated to 70% SW better than parr as judged by lower serum osmolality. However, serum osmolality in smolt-like fish did not return to the basal level 7 days after transfer to 70% SW, suggesting that their hypo-osmoregulatory ability was not fully developed to a level comparable to anadromous populations of this species. The present study suggests that, if O. masou in Miyazaki go though a smoltification process, it occurs in its first autumn instead of the second spring and is less pronounced compared with anadromous populations.

Relationships between plasma concentrations of sex steroid hormones and gonadal development in the brown hagfish, Paramyxine atami.

The relationship between sex steroid hormone profiles in plasma and gonadal function in hagfish is poorly understood. In the present study, plasma concentrations of estradiol, testosterone, and progesterone were examined with respect to gonadal development, sexual differences, and possible function of atretic follicles in the brown hagfish, Paramyxine atami, using a time-resolved fluoroimmunoassay. Plasma concentrations of these three hormones were low in juveniles of both sexes. In females, plasma estradiol showed a significant correlation with ovarian development, with the highest concentrations in late vitellogenic adults. Plasma testosterone and progesterone also increased significantly in non-vitellogenic adult females; however, plasma testosterone showed no significant differences among adult females at different ovarian developments, while plasma progesterone was significantly lower in late vitellogenic adults than it was in non-vitellogenic adults. Vitellogenic females that possessed atretic follicles showed significantly lower concentrations of all three hormones than females that only possessed normal follicles. In males, no significant differences were found in plasma estradiol or testosterone levels among groups of different developmental stages of the testis, while plasma progesterone showed a clear inverse relationship with testicular development. Thus, differences were found in plasma sex steroid hormone profiles between male and female P. atami. Moreover, plasma estradiol showed a significant correlation with ovarian development, which suggests that estradiol is involved in the regulation of ovarian development. The present study also revealed that steroid hormone production was strongly suppressed in females that possessed atretic follicles in their ovaries.

How histopathology can contribute to an understanding of defense mechanisms against cryptococci.

Invasive fungal infections, particularly those considered opportunistic, have become a common and significant complication of procedures performed in advanced contemporary medicine. Among such infections, cryptococcosis, which is usually caused by infection with Cryptococcus neoformans and Cryptococcus gattii, is particularly problematic because this fungal infection occurs in immunocompromised and apparently immunocompetent individuals. It has been largely accepted that Cryptococcus species are recognized by cellular receptors and that Th1-type immune responses play an important role in defense mechanisms against the yeast. However, the interaction between the yeast and host tissue varies depending on the characteristics of the yeast and the immune status of the host. To gain a better understanding of the pathophysiology of cryptococcosis, we wish to emphasize the usefulness of histopathological examinations, because it allowed more detailed information of an extremely complex interaction between the causative yeasts and tissue response. In the present review, we describe the pathophysiology of cryptococcosis as largely revealed in our previous histopathological investigations of the experimental infection.

Effects of estradiol or testosterone treatment on expression of gonadotropin subunit mRNAs and proteins in the pituitary of juvenile brown hagfish, Paramyxine atami.

A single functional gonadotropin (GTH) comprising two subunits, α and ß, was recently identified in the pituitary of brown hagfish (Paramyxine atami). Little is known about the feedback mechanisms that regulate these GTH subunits by sex steroids in the hagfish. The present study was designed to examine feedback effects of estradiol and testosterone on mRNA expression and protein expression of both GTHα and GTHß subunits in the pituitary of the juvenile P. atami. Intraperitoneal administration of estradiol over the course of 27days resulted in substantial accumulation of immunoreactive (ir)-GTHα and ir-GTHß in the adenohypophysis, but testosterone treatments over 27days had no effects on ir-GTHα or ir-GTHß. Estradiol treatment for 1, 2, 4 or 14days had no effect on GTHα mRNA levels. In contrast, after 2days of estradiol treatment, GTHß mRNA levels had increased significantly from baseline, while these levels were not affected after treatment over 1, 4, or 14days. After 14days of testosterone treatment, both GTHα and GTHß mRNA levels had decreased significantly from baseline levels. These results indicate that estradiol acted primarily to suppress the secretion of GTH, and hence resulted in the accumulations of ir-GTHα and ir-GTHß in the pituitary. On the other hand, testosterone appeared to suppress both the synthesis and the secretion of GTH. Thus, estradiol and testosterone probably differ in their effects on the regulation of pituitary GTH synthesis and secretion in juvenile hagfish.

Evolutionary origin of a functional gonadotropin in the pituitary of the most primitive vertebrate, hagfish.

Hagfish, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Hagfish have long been the enigma of vertebrate evolution not only because of their evolutionary position, but also because of our lack of knowledge on fundamental processes. Key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Here, the presence and identity of a functional glycoprotein hormone (GPH) have been elucidated from the brown hagfish Paramyxine atami. The hagfish GPH consists of two subunits, alpha and beta, which are synthesized and colocalized in the same cells of the adenohypophysis. The cellular and transcriptional activities of hagfish GPHalpha and -beta were significantly correlated with the developmental stages of the gonad. The purified native GPH induced the release of gonadal sex steroids in vitro. From our phylogenetic analysis, we propose that ancestral glycoprotein alpha-subunit 2 (GPA2) and beta-subunit 5 (GPB5) gave rise to GPHalpha and GPHbeta of the vertebrate glycoprotein hormone family, respectively. The identified hagfish GPHalpha and -beta subunits appear to be the typical gnathostome GPHalpha and -beta subunits based on the sequence and phylogenetic analyses. We hypothesize that the identity of a single functional GPH of the hagfish, hagfish GTH, provides critical evidence for the existence of a pituitary-gonadal system in the earliest divergent vertebrate that likely evolved from an ancestral, prevertebrate exclusively neuroendocrine mechanism by gradual emergence of a previously undescribed control level, the pituitary, which is not found in the Protochordates.

Gonadotropin-like and adrenocorticotropin-like cells in the pituitary gland of hagfish, Paramyxine atami; immunohistochemistry in combination with lectin histochemistry.

The pituitary system of the hagfish remains an enigma. The present study has aimed to detect possible adenohypophysial hormones in the pituitary gland of the brown hagfish, Paramyxine atami, by means of immunohistochemistry in combination with lectin histochemistry. Rabbit antisera raised against ovine luteinizing hormone (LH)beta, proopiomelanocortin (POMC)-related peptides, and the growth hormone/prolactin family of tetrapod and fish species were used, and 25 kinds of lectins were tested. Three different types of adenohypophysial cells were revealed in the pituitary of brown hagfish. The first was stained with both anti-ovine LH beta and several D-mannose-binding lectins, such as Lens culinaris agglutinin and Pisum sativum agglutinin. This cell type predominated in the adenohypophysis in adults with developing gonads and thus appeared to be involved in the regulation of gonadal functions. The second was negative for anti-ovine LH beta but was stained with several N-acetylglucosamine-binding lectins, such as wheat germ agglutinin and Lycopersicon esculentum lectin. This cell type exhibited a weak positive reaction with anti-lamprey adrenocorticotropin (ACTH) and thus appeared to be related to POMC-like cells. The second cell type was found in the adenohypophysis regardless of the developmental state of the gonads. The third cell type was negative for both antisera and lectins. Since this cell type was numerous in juveniles and adults without developing gonads, most cells of this type were probably undifferentiated. These findings suggest that GTH and ACTH are major adenohypophysial hormones in the hagfish.

Immunohistochemical detection of gonadotropin-like material in the pituitary of brown hagfish (Paramyxine atami) correlated with their gonadal functions and effect of estrogen treatment.

Since hagfish are members of the most primitive group of living vertebrates, studies on their reproduction are indispensable for understanding phylogenetic aspects of vertebrate reproductive system. Nevertheless, our knowledge of the reproductive physiology of the hagfish, especially of the pituitary-gonadal axis, is almost completely lacking. In the present study, the relationship between the amount of immunoreactive gonadotropin (GTH)-like material in the pituitary gland and gonadal conditions was examined in the brown hagfish, Paramyxine atami. First, pituitary sections were stained immunohistochemically with anti-ovine LHbeta, and the degrees of the accumulation of GTH-like material were compared among three different groups of gonadal conditions; juveniles and adults with and without developing gonads. Immunoreactive GTH-like material was heavily accumulated in adults with developing gonads, whereas it was not or only weakly accumulated in juveniles or adults without developing gonads. Thus, there was a strong positive correlation between the amount of GTH-like material and gonadal conditions. Second, effect of estradiol benzoate on GTH-like material was examined using three groups of juvenile hagfish: initial control, sham control, and experimental animals. Experimental animals received estradiol benzoate resolved in sesame oil intraperitoneally every third day for 1 month, whereas sham control animals received the same doses of sesame oil. GTH-like material was heavily or moderately accumulated in most estrogen-treated animals, whereas it was not or weakly accumulated in initial or sham control animals. Thus, estrogen treatment in juvenile hagfish resulted in the large increase in the amount of GTH-like material. From these results, it is suggested the presence of not only GTH but also the hypophysial-gonadal feedback system in the hagfish.

Identification of sea lamprey GTHbeta-like cDNA and its evolutionary implications.

We have identified the first and perhaps only gonadotropin beta-like protein by cDNA cloning in sea lamprey, a member of the oldest lineage of vertebrates, the agnathans. Two pituitary gonadotropins (GTHs: follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) have been identified in representative species of all classes of vertebrates except the agnathans. The present study was undertaken to identify GTH in sea lamprey, Petromyzon marinus, to gain a further understanding of the origin and evolution of reproductive pituitary hormones and their respective genes in vertebrates. Sea lamprey preGTHbeta-like cDNA was cloned from a plasmid cDNA library using an expressed sequence tag analysis. The preGTHbeta-like cDNA encoded 150 amino acids, in which the GTHbeta-like protein consisted of 134 amino acid residues. Sea lamprey GTHbeta-like protein contained 12 Cys residues and two N-glycosylation sites at homologous positions to those of FSHbeta and LHbeta. The region of the molecule that has been proposed to control receptor binding specificity (i.e., the region between the 10th and 12th Cys residues) suggests that the proposed heterodimer would be more like a FSH than a LH. Sea lamprey GTHbeta-like protein-producing cells were identified immunocytochemically in the ventral part of the proximal pars distalis of pituitary using antiserum prepared against a synthetic peptide of preGTHbeta-like protein (52-68). Intraperitoneal administration of sea lamprey GnRH-I and -III at 100 microg/g body weight (twice at a 24h interval) increased expression of GTHbeta-like protein in the pituitary of adult female sea lamprey during the final maturational period. Thus, these results are the first to demonstrate the presence of a single GTH-like system in lampreys. Because the sea lamprey GTHbeta-like protein is a clear out-group compared to those of the LH and FSH family based on phylogenic analysis, we propose that an ancestral glycoprotein hormone gave rise to only one GTH in lampreys and to the glycoprotein hormone family that gave rise to LH, FSH, and TSH during the early evolution of gnathostomes.

Development of the adenohypophysis in the lamprey: evolution of epigenetic patterning programs in organogenesis.

In gnathostomes, the adenohypophysis, a component of the hypothalamo-hypophysial complex, is believed to develop through hierarchically organized epigenetic interactions based primarily on the topographical relationships between tissues. From a comparison of developmental processes and gene expression patterns of pituitary-related genes between the agnathan species, lampreys and gnathostomes, we speculate on the evolutionary pathway of the vertebrate adenohypophysis. In the lamprey, this is derived from the nasohypophysial placode (NHP) that develops anterior to the oral ectoderm. The NHP can be identified by the expression of LjPitxA, before actual histogenesis, but it is initially distant from the future hypothalamic region. Subsequently, the NHP expresses both LjFgf8/17 and LjBmp2/4a gene transcripts, and grows caudally to establish a de novo contact with the hypothalamic region by the mid-pharyngula stage. Later, the NHP gives rise to both the adenohypophysis and an unpaired nasal organ. Thus, the topographical relationship between the NHP and the hypothalamic region is established secondarily in the lamprey, unlike gnathostomes in which the equivalent relationship appears early in development. Comparing the developmental pattern of the amphioxus homologue of the adenohypophysis, we hypothesize that a modification of the regulation of the growth factor encoding gene lies behind the evolutionary changes recognized as heterochrony and heterotopy, which leads to the gnathostome hypophysial developmental pattern.

Effect of lactoferrin feeding on the host antifungal response in guinea-pigs infected or immunised with Trichophyton mentagrophytes.

Earlier studies revealed that oral administration of lactoferrin (LF), a multi-functional milk protein, facilitated curing of dermatophytosis in guinea-pigs and man by an unknown mechanism. The present study aimed to assess the effect of feeding bovine LF on the host antifungal defence systems in guinea-pigs infected or immunised with Trichophyton mentagrophytes, a dermatophytosis-causing fungus. The unbound iron-binding capacity (UIBC) of the plasma of individual animals varied, and plasma with higher UIBC inhibited growth of T. mentagrophytes in vitro. However, LF administration did not enhance plasma UIBC or the anti-T. mentagrophytes activity of plasma in infected or uninfected animals. Phagocytic activity and reactive oxygen (RO) production of blood neutrophil polymorphonuclear leucocytes (PMNLs) were estimated by flow cytometry. LF administration caused no significant effects on phagocytic activity or RO production of neutrophil PMNLs in infected or uninfected animals. The functions of mononuclear cells (MNC) from the spleen were investigated in guinea-pigs immunised with heat-killed T. mentagrophytes conidia. The MNC were cultured with concanavalin A or inactivated T. mentagrophytes. In the bromo-deoxyuridine incorporation assay, the stimulation index was higher for MNC derived from LF-treated animals than for those from control animals. The culture supernates of MNC enhanced the ability of macrophages to kill T. mentagrophytes conidia. Furthermore, stronger augmentation was observed with the culture supernate from LF-treated animals than with that from control animals. In conclusion, LF feeding may potentiate the host antifungal defence systems by modulating MNC function rather than plasma antifungal activity or peripheral blood neutrophil PMNL activity.

A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient.

A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis-complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell-free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P-450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 X g pellet (P-120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative-staining method suggests that most of the vesicular vacuoles were recovered in the P-120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P-450.