Immunocytochemical localization of luteinizing hormone-releasing hormone in the brain of the goldfish Carassius auratus

Using an indirect immunofluorescence technique, LH-RH was localized in the brain of the goldfish (Carassius auratus). Immunoreactive cell bodies were inconsistantly found in two hypothalamic nuclei: the anteroventral nucleus preopticus periventricularis (npp) and the posterior nucleus lateralis tuberis (nltp). Numerous immunoreactive fibers were observed in hypothalamic as well as in the extrahypothalamic parts of the brain and in the proximal pars distalis of the pituitary gland. Our results are discussed in relation to the neuroendocrine control of the pituitary gonadotropic function in teleosts.     

Effects of pinealectomy and melatonin treatment on liver and plasma metabolites in the goldfish, Carassius auratus

The effects of pineal removal on daily variations and levels of some liver and plasma metabolites were examined in the goldfish, Carassius auratus. Pinealectomy abolishes the liver glycogen daily variation and decreases hepatic glycogen levels in goldfish maintained on specific photoperiod regimes during spring and summer. Neither the daily variation nor the levels of hepatic glycogen are affected by pinealectomy during the fall. Pinealectomy alters the plasma glucose daily variation and increases the levels of this metabolite in fishes maintained on long photoperiods during the summer. During the fall the daily variation of plasma glucose is abolished by pinealectomy and blood sugar levels are decreased. The diurnal fluctuation of plasma lipid is altered by pinealectomy during the summer. Plasma lipid levels are elevated in pinealectomized fishes during winter, spring, and summer. The daily variation of liver lipid levels is altered by pinealectomy during the autumn. Melatonin treatment increases liver glycogen reserves in goldfish maintained on a long, but not a short, photoperiod during the spring. Plasma glucose levels are also elevated by melatonin treatment. Therefore, the pineal organ participates in regulating levels and daily variations of some metabolites. The influence of the pineal on metabolites varies with season and day length.     

Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus

Gonadotrophin-inhibitory hormone (GnIH) plays an important role in regulating of reproduction in teleosts. To clarify the mode of action of GnIH on the synthesis of gonadotropin releasing hormone (GnRH) and gonadotrophin (GtH), three GnIHR cDNAs were cloned from the goldfish brain. In situ hybridization results showed that GnIHRs were localized to the hypothalamus and pituitary. In the hypothalamus, GnIHRs were found in the NPP, NPO and NLT, whereas sGnRH neurons were reported to be located, and potentially regulated by GnIH. In the pituitary, only two GnIHRs were observed and they were localized to the PI instead of the adenohypophysis where GtH-expressing cells are localized, suggesting indirect regulation of GtH by GnIH. In vivo, intraperitoneal (i.p.) injections of synthetic goldfish GnIH-II peptide and GnIH-III peptide significantly decreased sGnRH and FSHβ mRNA levels. Only GnIH-II decreased LHβ mRNA levels significantly. In vitro, both GnIH-II and GnIH-III showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LHβ and FSHβ synthesis was observed when GnIH-III was applied to primary pituitary cells in culture. Thus, GnIH could contribute to the regulation of gonadotropin in the brain and pituitary in teleosts.     

Involvement of gonadotrophin and steroid hormones in spermiation in the amago salmon, Oncorhynchus rhodurus, and goldfish, Carassius auratus

Effects of intraperitoneal injections of chum salmon gonadotrophin (SGA) and various steroid hormones (17α,20β-dihydroxy-4-pregnen-3-one; 17α,20β-diOHprog, 17α-hydroxy-progesterone, testosterone, 11-ketotestosterone) on the induction of in vivo spermiation were examined in nonspermiating amago salmon (Oncorhynchus rhodurus) and goldfish (Carassius auratus). A single injection of SGA to amago salmon and two successive injections to goldfish induced strong spermiation responses. Similarly two successive injections of 17α, 20β-diOHprog caused precocious spermiation in both species; however, the response to 17α,20β-diOHprog was of lesser magnitude than that to SGA. The spermiation response of goldfish to 17α-hydroxyprogesterone was similar to that of 17α,20β-diOHprog. Neither testosterone nor 11-ketotestosterone were effective in inducing spermiation in amago salmon, but these steroids were found to be slightly effective in goldfish. Effects of a single injection of SGA on changes in serum levels of 17α,20β-diOHprog and 11-ketotestosterone were also examined in nonspermiating amago salmon. Serum levels of 17α,20β-diOHprog dramatically increased after treatment and peaked on Day 2 and thereafter declined quickly. Similarly, 11-ketotestosterone peaked on Day 2, but the levels remained high throughout the experimental period. Considered together, these findings are consistent with the suggestion that 17α,20β-diOHprog is involved in the process of spermiation in teleosts. It is further suggested that this hormone is a testicular steroidal mediator of gonadotrophin-induced spermiation in amago salmon.     

Effects of hypothalamic lesions on serum growth hormone levels and growth rates in goldfish, Carassius auratus

Serum growth hormone (GH) levels and growth rates of goldfish were measured concurrently following discrete electrothermic lesions of a number of hypothalamic nuclei. Following lesions of the nucleus preopticus periventricularis (NPP), significant increases in serum GH levels were evident at 4 weeks postlesioning in comparison to both sham-operated and normal control groups. In addition, the NPP-lesioned fish showed significant increases in both total body weight and standard length increments compared to control groups. Lesions in other hypothalamic areas including the nucleus anterior tuberis, nucleus lateralis tuberis and the nucleus recessus lateralis had no consistent effect on growth rates or serum GH levels. The present results suggest that the NPP is concerned with the inhibition of GH secretory function in the goldfish.     

Effects of monosodium l-glutamate on pituitary innervation in goldfish, Carassius auratus

Monosodium l-glutamate was injected intraperitoneally into goldfish at a dosage of 2.5 mg/g body wt. The first noticeable effect in the pituitary, 30 min postinjection, was a marked swelling of pituicytes in the neurohypophysis (NH). Eighteen hours postinjection a large number of degenerating type B fibers were found in the NH of the proximal pars distalis (PPD), while all type A fibers, and type B fibers in other parts of the pituitary, remained unaffected. Because glutamate causes a lesion in the nucleus lateral tuberis (NLT), from the anterior margin of the pituitary stalk through to the posterior end of the nucleus, and a minor lesion in the nucleus preopticus periventricularis, it is likely that the NLT is the origin of the degenerative type B fibers in the proximal NH. The significance of these results is discussed in relation to the neuroendocrine regulation of the pituitary functions in goldfish.     

Microelectrophoretic identification of pituitary prolactin in Carassius auratus

A microelectrophoretic polyacrylamide slab gel system has been adapted to analyze prolactin levels in individual goldfish pituitaries. The putative prolactin (R_f = 0.40) was identified within the gels by use of what is known about hypothalamic, dopaminergic control of secretion of this hormone, the effect of osmotic pressure on prolactin secretion, and the effect of administration of ergot drugs in fish. The putative goldfish prolactin was also shown to cross-react with anti-ovine and anti-pollack prolactin in two systems.     

Neurohypophysial hormonal control of cortisol secretion in the teleost Carassius auratus

Intravenous injections of arginine vasopressin (AVP), arginine vasotocin (AVT), and isotocin (IST) produced dose-dependent increments in circulating levels of plasma cortisol in goldfish in which endogenous ACTH secretion was suppressed by prior administration of dexamethasone. Injections of met⁵-enkephalin (ENK) or saline had no significant effect on plasma cortisol in dexamethasone-blocked fish. These results demonstrate that AVT and IST stimulate cortisol secretion in the goldfish and suggest that in teleosts AVT and IST possess corticotrophin-releasing factor (CRF) activity.     

Continued in vitro growth of fibroblast-like cells (RBCF-1) derived from the caudal fin of the fish, Carassius auratus

Fibroblast-like cells derived from primary culture of the caudal fin of a 2-year old goldfish were used to find out whether or not fish cells have a limited in vitro lifespan during the observation period of 22 months. The cells cultivated at 37°C (RBCF-1 line) attained exponential growth from the beginning of culture and reached to 200 population doubling number (PDN) at the 310th day. The growth rate of the cells cultured at 30°C (RBCF-2 line) was almost the same as that of RBCF-1 cells except for a temporal decline between the 80 and 160th day of culture. The plating efficiency of RBCF-1 cells, as determined by colony formation, increased with passage and reached around 20% after 183 PDN. Subcutaneous injection of RBCF-1 cells into nude mice failed to develop any tumour so far as examined up to 5 months after injection. Feulgen-DNA cytofluorometry simultaneously combined with tritiated thymidine autoradiography revealed that, in both RBCF-1 and RBCF-2 lines, unlabelled cells which escaped from 48 hours' labelling gradually diminished from each population as PDNs increased and also that labelling indices for each line were 83 and 90% at 113 and 91 PDNs, respectively. The distribution of DNA content per cell was almost the same irrespective of the line and PDN. The modal number of RBCF-1 chromosomes was 100 at both 62 and more than 200 PDNs, and no significant change in the distribution of chromosome number could be found. The saturation cell density of RBCF-1 cells was almost identical at 20, 30 and 37°C, while extremely high saturation density was observed at 27°C. These results were discussed from a viewpoint of ageing characteristics of fish cells in culture.     

Effect of catecholamines and synthetic mammalian hypothalamic hormones on the adenylyl cyclase activity of the pituitary of the teleost, Carassius auratus

The catecholamines norepinephrine and epinephrine, at concentrations as low as 10^?6M, were found to increase adenylyl cyclase activity of the pars distalis of the teleost fish, Carassius auratus. Dopamine increased the pars distalis enzyme activity at 10^?4M only. Except for an inhibition by norepinephrine at 10^?3M the catecholamines were without effect on the adenylyl cyclase activity of the neuro-intermediate lobe. Melatonin, serotonin and γ-aminobutyric acid, at concentrations from 10^?6 to 10^?3M, were without effect on the activity of the adenylyl cyclase of either lobe.The synthetic mammalian hypothalamic hormones, thyrotropin-releasing hormone (TRH) and gonadotrophin-releasing hormone (GnRH), were without effect on the pars distalis enzyme system at concentrations from 2–200 ng/ml.     

Corpus allatum activity in vitro during the reproductive cycle of the viviparous cockroach, Diploptera punctata (eschscholtz)

Isolated pairs of corpora allata (CA) from the viviparous cockroach, Diploptera punctata have been shown to synthesize and release C₁₆JH at a linear rate for at least 5 hr. No storage of C₁₆JH has been observed at any time during the first oviposition cycle. It is suggested that rate limitation in JH biosynthesis does not occur at the terminal enzymic stage because the immediate precursor, methyl farnesoate, does not accumulate at any level of CA activity. It is concluded that the short-term incubation procedures employed represent an accurate assessment of CA activity in vivo.The synthesis and release of C₁₆JH by CA has been followed during the first oviposition cycle. High rates of release of JH were observed during rapid oocyte growth—CA became highly active over a 24 hr period as the oocytes were entering vitellogenesis. An analysis of CA activity relative to oocyte length revealed that the release rate of C₁₆JH was highest when oocyte length was in the range 1.0–1.6 mm. However, it was not possible to ascertain if vitellogenesis was initiated before or after the increase in the JH release rate. C₁₆JH synthesis of 40 pmol hr^?1 is the highest mean rate yet reported for an insect.     

Progesterone and estrone levels in the ovaries,pyloric ceca,and perivisceral fluid during the annual reproductive cycle of starfish, Asterias rubens

The levels of progesterone and of estrone in the ovaries and pyloric ceca have been determined with specific radioimmunoassay techniques throughout the annual reproductive cycle of Asterias rubens. The progesterone level in the ovaries is high during stage 1, decreases during stage 2, and remains low in stages 3 and 4. In the pyloric ceca the progesterone level is high during stages 1 and 2, low in stage 3, and increases to the high level during stage 4, i.e., before spawning. Variations in the progesterone level in the ovaries related to the actual stage of the annual reproductive cycle, the presence of steroid-synthesizing cells, and biochemical data suggest that progesterone may have a certain function during the growth of oocytes and is possibly primarily synthesized in the pyloric ceca. The estrone level in the ovaries is low, but at the end of stage 1 (i.e., at the start of vitellogenesis) the ovaries contain more estrone. In the pyloric ceca the estrone level remains low throughout the annual reproductive cycle. The relation between estrone levels and protein contents is discussed and lead to the hypothesis that estrone or other estrogens affect the biosynthesis of proteins and possibly also their incorporation in the oocytes of Asterias rubens.     

Molecular characterization of sea bass gonadotropin subunits (alpha,FSHbeta, and LHbeta) and their expression during the reproductive cycle

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass (Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain-pituitary cDNA library. The nucleotide sequences of the common alpha, the FSHbeta, and the LHbeta subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHbeta is more similar to higher vertebrate FSHbeta's (35-37%) than to LHbeta's (26-30%), whereas sea bass LHbeta is more similar to LHbeta's (40-53%) than to FSHbeta's (26-41%). Phylogenetic analysis of fish GTH sequences grouped the beta subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.     

The two pools of pituitary gonadotropin: regulation during the menstrual cycle.

Information on the relative activity and on the functional relationships between the acutely releasable (1st) and reserve (2nd) pools of pituitary LH during the course of the normal menstrual cycle was obtained via a 4 h LRF infusion (0.2 mug/min X 4 h). This was immediately followed by 3 pulses of LRF (10 mug at 2 h intervals) to assess further the size of the acutely releasable pool after activation of the reserve pool by the infusion. Our observations indicate that two functional pools of LH are present in all phases of the menstrual cycle and that comparative pool size or activity is influenced profoundly by ovarian steroid feedback as well as by the pattern of input of hypothalamic LRF. From the early to the late follicular phase, in synchrony with the rising levels of E2, the size of the 2nd pool is preferentially augmented. A small increase in the 1st pool activity is not apparent until the late follicular phase when a 5-fold increase in the size of the 2nd pool is also attained. During the mid-luteal phase and in association with relatively high progesterone (P) and E2, th large 2nd pool is maintained as in the late follicular phase but the 1st pool is strikingly smaller. Activation of the 2nd pool of LH by the LRF infusion (priming) increases the acutely releasable LH (1st pool) in all three phases of the cycle, as evidenced by an enhanced response to the 1st but not subsequent pulses of LRF at the end of the infusion as compared with non-infused controls. This priming effect is likely a reflection of activation or "shifting" of LH from the larger 2nd pool to the smaller 1st pool. It is found that this priming effect is greatest during the mid-luteal phase as compared to other phases of the cycle. During the days of mid-cycle LH surge, a dramatic reversal of the relative activity of the two pools in observed and this is manifested by an enormous increase in the activity of the 1st relative to the 2nd pool. In contrast to other phases of the cycle, the release of LH from the 2nd pool is not sustained and this premature decline in LH release despite continuous LRF infusion appears to be due to pituitary depletion of LH as evidenced by the failure of the pituitary response to pulses of LRF immediately following the infusion...     

The relationship between plasma steroid hormone concentrations and the reproductive cycle in the Northern Pacific rattlesnake, Crotalus oreganus

We describe the reproductive cycle of Northern Pacific rattlesnakes (Crotalus oreganus) by quantifying steroid hormone concentrations and observing reproductive behaviors in free-ranging individuals. Additionally, we examined reproductive tissues from museum specimens. Plasma steroid hormone concentrations were quantified for both male and female snakes throughout the active season (March-October). We measured testosterone (T), 5α-dihydrotestosterone (DHT), and corticosterone (B) concentrations in both sexes and 17β-estradiol (E2) and progesterone (P) in females only. We observed reproductive behaviors (e.g., consortship, courtship, and copulation) in the field and measured testis and follicle size in male and female snakes from museum collections to relate steroid hormone concentrations to the timing of reproductive events. Our study revealed that C. oreganus in central California exhibits a bimodal pattern of breeding, with most mating behavior occurring in the spring and some incidences of mating behavior observed in late summer/fall. Each breeding period corresponded with elevated androgen (T or DHT) levels in males. Testes were regressed in the spring when the majority of reproductive behavior was observed in this population, and they reached peak volume in August and September during spermatogenesis. Although we did not detect seasonal variation in female hormone concentrations, some females had high E2 in the spring and fall, coincident with mating and with increased follicle size (indicating vitellogenesis) in museum specimens. Females with high E2 concentrations also had high T and DHT concentrations. Corticosterone concentrations in males and females were not related either to time of year or to concentrations of any other hormones quantified. Progesterone concentrations in females also did not vary seasonally, but this likely reflected sampling bias as females tended to be underground, and thus unobtainable, in summer months when P would be expected to be elevated during gestation. In females, P was positively correlated with T and DHT, and E2 was positively correlated with T.     

Serum concentrations of relaxin, chorionic gonadotropin, estradiol-17 beta, and progesterone during the reproductive cycle of the chimpanzee (Pan troglodytes).

Levels of serum relaxin were measured by a specific RIA and correlated with serum patterns of estradiol-17 beta, progesterone, LH, or cCG during a single menstrual cycle in each of 10 female chimpanzees, and throughout 24 pregnancies in 21 chimpanzees. Significant concentrations of relaxin, higher than those reported for the human being, were detected in serum of nonpregnant chimpanzees during the late luteal phase of the menstrual cycle. During pregnancy in the chimpanzee, serum relaxin concentrations, exceeding levels found during the luteal phase, were highest during the first third of gestation, and declined thereafter. Although the absolute concentrations were higher, the patterns of relaxin secretion throughout the reproductive cycle in chimpanzees was qualitatively very similar to that observed in other primates, including the human being. The chimpanzee should thus provide a useful model for examining the role of relaxin in human reproduction.     

Ovulatory surge of gonadotropin in the goldfish, Carassius auratus.

The periovulatory pattern of circulating gonadotropin is described for the first time in a poikilothermic vertebrate. In sexually mature female goldfish (Carassius auratus) maintained on a 16L:8D photoperiod, ovulation occurs during the latter part of the dark phase, approximately 20 hr after the fish are warmed from 13 to 21°. Serum gonadotropin levels increase gradually during the first half of the light phase, and by the latter part begin to increase rapidly, reaching the peak of the gonadotropin surge by the onset of the dark phase. Gonadotropin levels remain high through the period of ovulation, and decrease markedly by the onset of the following light phase. The findings indicate that the goldfish provides a valuable model for the study of ovulation in teleosts.     

In vitro uptake and metabolism of testosterone by goldfish,Carassius auratus,mesenteric adipose tissue

Mesenteric adipose tissue was removed from adult goldfish, Carassius auratus, and incubated in vitro with (1alpha,2alpha)-3[H]testosterone (T). Total radioactivity in the medium decreased and tissue total radioactivity increased in a time-dependent fashion between 1 and 6 h of incubation with maximum uptake occurring between 4 and 6 h. After ether extraction and thin layer chromatography the amount of radioactivity comigrating with authentic T standard decreased over time in both medium and tissue samples. Radioactivity in the aqueous fraction remaining after ether extraction increased in a time-dependent manner, suggesting the presence of water-soluble conjugated steroids. Acid hydrolysis of the aqueous fraction yielded radioactivity that principally comigrated with T standard following a second ether extraction and TLC, along with a small, unidentified peak of poorly migrating radioactivity. Aromatization of T was assessed after charcoal stripping samples and measuring radioactivity remaining in the form of 3H(2)O. Small but significant amounts of 3H(2)O were present in both incubation medium and tissue samples. Incubation with the aromatase inhibitor 4-androsten-4-ol-3,17-dione acetate (ATD) decreased the formation of 3H(2)O in a dose-dependent manner. These results suggest that goldfish mesenteric adipose tissue is capable of converting T to several metabolites including water-soluble conjugates and estrogen.