Activin stimulates goldfish FSH biosynthesis by enhancing FSHβ promoter activity

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play critical roles in controlling vertebrate gonadal development and function. Activin, a dimeric growth factor initially identified in the gonads, is important in the differential regulation of the two gonadotropins in mammals. Using goldfish as a model, we have demonstrated that activin stimulates FSHβ but suppresses LHβ expression. The present study demonstrated that the 5′-flanking region of goldfish FSHβ gene is functional in the mouse gonadotrope cell line, LβT2 cells. Similar to its effect on the cultured pituitary cells, activin stimulated FSHβ promoter activity in the LβT2 cells and the effect could be blocked by its binding protein follistatin. Follistatin also significantly suppressed the basal FSHβ promoter activity, suggesting secretion of endogenous activin by the LβT2 cells. Further characterization of the cis-regulatory elements responsible for activin stimulation is now under way in our laboratory.     

Production of channel catfish (Ictalurus punctatus) recombinant gonadotropins using the S2 Drosophila cell line system

The S2 cell system was utilized for the production of recombinant luteinizing hormone (LH) and follicle stimulating hormone (FSH) of the channel catfish (Ictalurus punctatus) as C-terminal His-tagged proteins. When expressed individually, the common α-subunit was secreted in abundance but both β-LH and β-FSH were poorly expressed. However, co-expression of the α-subunit with each of the β subunits using a duel promoter vector resulted in the abundant secretion of LH and FSH α/β heterodimers. These recombinant gonadotropins (GtH) were able to stimulate estradiol secretion in an ovarian follicle bioassay and activate recombinant gonadotropin receptors.     

Regulation of LH synthesis and release by GnRH and gonadal steroids in masu salmon

The roles of salmon GnRH (sGnRH) and gonadal steroid hormones in regulation of LH synthesis and release were examined in primary pituitary cell cultures of masu salmon (Oncorhynchus masou). Pituitaries were taken from fish at four reproductive stages: in March (initiation of sexual maturation); May (early maturation); July (pre-spawning); and September (spawning period). Amounts of LHβ subunit mRNA in the pituitary cells were determined by real-time PCR, and LH levels in the medium were determined by RIA. sGnRH and gonadal steroids including estradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11-KT) were added to the cultures to examine their direct effects on LH response. sGnRH had no significant effect on LHβ mRNA levels at any stages, although a stimulatory trend was noted in March. In contrast, E2 and T considerably increased LHβ subunit mRNA levels in March and May during initial stages of maturation, and the effects were less pronounced in July and September. On the other hand, sGnRH stimulated LH release at all stages in the males and the effects were most prominent in July and September. E2 and T also stimulated LH release in July and September, but their effects were weaker than that of sGnRH. The present results indicate that sGnRH and gonadal steroids directly regulate LH synthesis and release in masu salmon pituitary cells: sGnRH mainly stimulates LH release in the late stage of sexual maturation; whereas, E2 and T are effective in stimulating LH synthesis at earlier stages of maturation.     

Activin and its receptors in the goldfish

Activin (_AA, _AB and _BB) is a dimeric protein that belongs to the transforming growth factor- (TGF-) superfamily of growth factors and is involved in the regulation of many physiological and developmental processes. Recently, we have demonstrated that porcine activin stimulated goldfish gonadotropin-II (GTH-II) and growth hormone (GH) secretion from dispersed pituitary cells in static culture and pituitary fragments in perifusion. The action of activin in the goldfish is unique in that it has an acute stimulatory effect on the secretion of GTH-II and GH, whereas in mammals activin usually exhibits long-term stimulatory actions on FSH secretion. The action mechanism of activin is different from that of gonadotropin-releasing hormone (GnRH). Using domain-specific antibodies against mammalian activin subunits, we subsequently demonstrated the existence of immunoreactive activin subunits (_A and _B) in the goldfish ovary, testis, pituitary and brain, suggesting endocrine, paracrine and autocrine roles for activin in the regulation of goldfish reproduction. Both activin _A and _B subunits have been cloned from goldfish genome by polymerase chain reaction (PCR). Using the PCR fragments as probes, we have cloned a full length cDNA coding for activin _B subunit from the goldfish ovary. Both activin _A and _B subunits show high homology to those of other vertebrates with the _B subunit much more conserved (93 and 98% identity with human and zebrafish _B subunit, respectively). The identity of the cloned _B subunit was further confirmed by expression in the Chinese hamster ovary (CHO) cells and detection of the specific activity of activin in the culture medium. The messenger RNA of activin _B subunit is expressed in a variety of goldfish tissues including ovary, testis, brain, pituitary, kidney and liver, suggesting a wide range of physiological roles for activin in the goldfish. We have also cloned a full length cDNA coding for the activin Type IIB receptor from the goldfish ovary, suggesting that activin may have paracrine or autocrine actions on the ovarian functions. The identity of the cloned receptor was confirmed by specific binding of¹²⁵ I-activin on COS-1 cells transfected with the cloned Type IIB receptor.     

Naltrexone attenuates stress-induced suppression of LH secretion in the pituitary gland in the Cichlid fish Oreochromis mossambicus: evidence for the opioidergic mediation of reproductive stress response

Opioid peptide β-endorphin (β-EP) plays a modulatory role in vertebrate reproduction. However, the role of opioid peptides in reproductive stress response is least understood in fishes. The aim of the present study was to determine the effect of different doses of β-EP on luteinizing hormone (LH) secretion in normal and the opioid receptor antagonist naltrexone (NALT) in stressed female tilapia Oreochromis mossambicus. Administration of 4 μg β-EP, but not 0.5 or 1.5 μg β-EP, daily for 22 days caused suppression of LH-secreting cells at the proximal pars distalis of the pituitary gland, concomitant with a significant reduction in the mean GSI and HSI in 4 μg β-EP-treated fish compared to controls. On the other hand, exposure of the fish to mild acute stressors for 22 days caused changes in the LH-secreting cells similar to that of high dose of β-EP, whereas administration of NALT attenuated these effects. Taken together, the results indicate that increased concentration of β-EP as may occur during stressful conditions can cause suppression of LH secretion, leading to the inhibition of spawning, and that treatment of NALT attenuates the stress-induced inhibition of LH secretion in fish.     

Time course of final oocyte maturation and ovulation in chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis> induced by hCG and GnRHa

Abstract:   The question of whether the ovulation and spawning time in chub mackerel Scomber japonicus is entrained by a circadian rhythm was raised by our previous experiments. Further questions were also raised about whether the time course of human chorionic gonadotropin (hCG)-induced final oocyte maturation (FOM) and ovulation reflected the natural time course induced by endogeneous pituitary gonadotropin (GtH). To address these questions, hCG and gonadotropin-releasing hormone analog (GnRHa) were administered at two 'opposite' times, 14:00 and 02:00 hours, and the time courses of FOM and ovulation were compared. When hCG was injected, ovulation occurred 33 h post-injection in both groups, regardless of the timing of the hCG injection. The timing of ovulation in chub mackerel depends on the timing of hCG injection, but apparently not on circadian rhythms. When GnRHa was injected, ovulation began at 36 h post-injection of GnRHa, regardless of the timing of injection. These results indicate that the time course of FOM and ovulation in the chub mackerel followed a similar pattern whether stimulated by hCG injection or spontaneous luteinizing hormone (LH) surge because GnRHa induces the secretion of endogenous GtH (primarily LH) from the fish pituitary. Thus, it is concluded that the time course of hCG-induced FOM and ovulation in chub mackerel follows the natural time course induced by endogenous pituitary LH.     

Gonadotropins and their paracrine signaling network in the zebrafish ovary

Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play fundamental roles in vertebrate ovarian development and function. However, there has been an increasing body of evidence that the actions of FSH and LH are mediated or modulated by a variety of locally produced peptide or protein factors, which form an intimate regulatory network within and between the ovarian follicles. In the past few years, a variety of growth factors have been identified and characterized in the zebrafish ovary including activin and epidermal growth factor (EGF), which are important components of the intraovarian communication network. To understand how this local network interacts with the gonadotropins from the pituitary, we have recently cloned and characterized all the subunits of zebrafish FSH and LH from the pituitary as well as their receptors (FSHR and LHR) from the ovary. Using the Chinese hamster ovary (CHO) cells as the bioreactor, we have produced recombinant zebrafish FSH and LH with biological activities. With the recombinant hormones available, the functions of zebrafish FSH and LH in the ovary and their interactions with the local factors will be an important issue to address in the future. This review briefly summarizes some recent work from our laboratory and others on both gonadotropins and their potential intraovarian signaling factors in the zebrafish.     

Impact of long‐term dietary exposure to lead on some reproductive parameters of a female Common carp (Cyprinus carpio L.)

During a long‐term 3‐year dietary exposure of mature carp females to lead, its bioaccumulation in the brain, changes in the neurohormonal activity at the level of the hypothalamus and the pituitary gland as well as the growth and maturation of ovaries were analysed. Moreover, an analysis of the effectiveness of hormone‐stimulated spawning during two subsequent spawning seasons was carried out. The results of the analyses show that chronic exposure of maturing and mature carp females to lead in feed results in its accumulation in the brain (1.365 μg g^?1). This impairs the endocrine activity of the hypothalamus, which is manifested by, among others, an increased secretion of dopamine and impaired spontaneous secretion of luteinizing hormone (LH). The observed decrease in the concentration of gonadotropin in blood inhibits vitellogenesis, which is manifested by a lower degree of the maturity of oocytes, lower fecundity (as measured by egg number, egg weight, egg maturity and egg survival) and lower gonadosomatic index values. The final negative impact of lead is the impairment of reproductive functions, as manifested by a smaller number of spawning females and in their lower fecundity during spawning.     

越冬池溶解氧含量的无线监测与控制系统初步设计

为确保鱼类安全越冬,避免因越冬池缺氧而大量死亡,设计了越冬池溶解氧含量无线监测和增氧机控制系统。该系统运行安全可靠,能够保证鱼类越冬生产的安全性,提高鱼类越冬成活率。     

越冬期间黄颡鱼、大鳍组织器官含脂量变化的研究

通过对嘉陵江北碚江段黄颡鱼，大鳍Hu越冬期间其胃，肠，肝胰脏，肌肉含脂量变化的研究。表明：1．黄颡鱼，大鳍Hu内脏各器官（胃,肠，肝胰脏）含脂量以越立中期为最低，肌肉则相反，表现出越立饥饿期间，该鱼主要利用内脏脂肪作用为主要能量来源而不用大量肌肉脂肪；2，越冬期间，黄颡锺，大鳍Hu内脏各器官（胃，肠，肝胰脏）含脂量与含水量的变化有负相关关系存在。     

不同交配,越冬方式对河蟹育苗的影响

不同交配、越冬方式对河蟹育苗产量有着显著的影响，三年的生产实践表明：秋季室外土池交配的抱卵率、抱卵量明显高于水泥交配；秋季交配优于春季交配；室外土池自然越冬抱卵卵蟹的胚胎发育优于室外塑料大棚保温越冬的抱卵蟹。适宜的秋，春交配有利于河蟹多苗育苗，延长生长期，增加产量。     

鲈生物学及池塘养殖技术

报道了鲈繁殖、食性、摄食、生长等习笥及鲈池塘养殖技术，在山东越冬的３种方式、鲈的主要病害及防治。     

Androgen secretion activity of recombinant follicle-stimulating hormone of Japanese eel, Anguilla japonica in immature and maturing eel testes

The androgen secretion activities of recombinant Japanese eel follicle-stimulating hormone (rjeFSH) were investigated in immature and maturing eel testes. The rjeFSH stimulated testosterone (T) and 11-ketotestosterone (11-KT) secretion in immature testis but not in maturing testis. This result suggests that eel FSH plays an important role through the sex steroid secretion in immature testis rather than in maturing testis.     

Physiological roles of FSH and LH in red seabream, Pagrus major

The duality of gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), has been confirmed in most teleost species, but very little is known about their biological functions. To elucidate the physiological roles of FSH and LH in fish reproduction, the expression profiles of GTH subunit genes during gonadal development were analyzed in both male and female red seabream. Furthermore, in vitro studies were carried out to examine the effects of GTHs on steroid hormone production and cytochrome P450 aromatase (P450arom) expression in red seabream gonads. In both sexes, LHβ mRNA was maintained at high levels from the early gametogenesis until spawning season, and declined with gonadal regression. Interestingly, FSHβ mRNA levels in males increased in parallel with testicular development, whereas those in female were remained low throughout oocyte development. From in vitro studies using purified red seabream FSH and LH, both GTHs had a similar potency in stimulating 11-ketotestosterone production by testicular slices, while the biological activity of FSH was much lower than that of LH in stimulating production of estradiol-17β by vitellogenic follicles. Moreover, expression of P450arom mRNA was induced by LH, but not FSH, in ovarian follicles in vitro. FSH was also ineffective in inducing maturational competence and final oocyte maturation. These results suggest that, unlike salmonids, FSH may play an important role during gametogenesis in male, but not female, red seabream, whereas LH may be involved in regulation of both early and late gametogenesis in both sexes.     

Spermatogenesis and its endocrine regulation

Three major phases compose spermatogenesis: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and spermiogenesis, the restructuring of spermatids into flagellated spermatozoa. The process is fuelled by stem cells that, when dividing, either self-renew or produce spermatogonia that are committed to proliferation, meiosis, and spermiogenesis. During all phases, germ cells are in close contact with and require the structural and functional support of Sertoli cells. In contrast to germ cells, these somatic cells express receptors for sex steroids and follicle-stimulating hormone (FSH), the most important hormones that regulate spermatogenesis. A typical Sertoli cell response to an endocrine stimulus would be to change the release of a growth factor that would then mediate the hormone's effect to the germ cells. Recent studies in the Japanese eel have shown, for example, that in the absence of gonadotropin Sertoli cells produce a growth factor (an orthologue of anti-Müllerian hormone) that restricts stem cell divisions to the self-renewal pathway; also estrogens stimulate stem cell renewal divisions but not spermatogonial proliferation. Gonadotropin or 11-ketotestosterone (11-KT) stimulation, however, induces spermatogonial proliferation, which is in part mimicked by another Sertoli cell-derived growth factor (activin B). Since FSH (besides luteinizing hormone, LH) stimulates steroidogenesis in fish, and since FSH is the only gonadotropin detected in the plasma of sexually immature salmonids, increased FSH signalling may be sufficient to initiate spermatogenesis by activating both Sertoli cell functions and 11-KT production. Another important androgen is testosterone (T), which seems to act via feedback mechanisms that can compromise FSH-dependent signalling or steroidogenesis. The testicular production of T and 11-KT therefore needs to be balanced adequately. Further research is required to elucidate in what way(s) 11-KT stimulates later stages of development, such as entry into meiosis and spermiogenesis. At this period, LH becomes increasingly important for the regulation of androgen production. Results from mammalian models suggest that during the later phases, the control of germ cell apoptosis via Sertoli cell factors is an important regulatory mechanism. In many species, sperm cells cannot fertilize eggs until having passed a maturation process known as capacitation, which includes the acquisition of motility. Progestins that are produced under the influence of LH appear to play an important role in this context, which involves the control of the composition of the seminal plasma (e.g., pH values). This revised version was published online in July 2006 with corrections to the Cover Date.     

雄烯二酮和甲基睾酮诱导雌性日本鳗鲡性腺发育的反馈调节作用

采用间隔15d多次埋植雄激素雄烯二酮(4-androstene-3,17-dine,ADSD)或甲基睾酮(17α-methyl-testosterone,MT)诱导雌性日本鳗鲡(Anguilla japonica)性腺发育成熟;并着重研究埋植雄激素后,雌鳗血清中促性腺激素(GtH)及脑和垂体中哺乳类促性腺激素释放激素(mGnRH)含量的动态变化.一次或多次埋植ADSD后,1～5d的血清GtH含量上升,然后下降;并且,血清GtH含量上升的幅度随埋植次数的增加而增加;对于MT处理组,只在埋植7次后第15天测得血清GtH水平显著高于对照组,但仍显著低于ADSD处理组.这表明埋植ADSD和MT可促进GtH的分泌,但两者促进GtH分泌的作用存在显著差异.埋植ADSD 1d后,脑和垂体中mGnRH含量明显增加,第2天后和对照组无明显差别,表明ADSD促进了mGnRH的合成,并可能有一定程度的释放.研究表明雄激素对性腺未发育成熟的雌性日本鳗鲡在脑和垂体两个水平存在正反馈调节作用.     

Effects of gonadotropin-releasing hormone agonist and testosterone on gonadotropin subunit gene expression in female red seabream (Pagrus major)

We examined the effects of gonadotropin-releasing hormone agonist (GnRHa) and testosterone (T) on the level of gonadotropin subunit mRNAs in the pituitary of ovariectomized or intact female red seabream. Ovariectomy induced increase of seabream (sb) GnRH, glycoprotein (GP) α and luteinizing hormone (LH) β mRNA levels. GnRHa treatment also stimulated GPα and LHβ mRNA levels. T treatment reduced GPα and LHβ mRNA expression probably via negative feedback action on sbGnRH. Both GnRHa and T treatment had no effect on follicle-stimulating hormone (FSH) β mRNA levels. These results suggest that the regulatory mechanisms of GPα and LHβ gene expression differ from those of FSHβ gene.     

Complementary use of natural and artificial wetlands by waterbirds wintering in Doñana,south‐west Spain

• 1. The Doñana wetland complex (SW Spain) holds more wintering waterfowl than any other wetland in Europe.
• 2. This study focused on the use made by 12 common waterbirds (eight ducks and four waders) of the natural seasonal marshes in Doñana National Park (DNP) and the adjacent Veta la Palma (VLP) fish ponds created in the early 1990s. Data used were from aerial and terrestrial surveys collected between October and February during six consecutive winters from 1998/99 to 2003/04. Changes in distribution of each bird taxon were related to changes in the extent of flooded marshes within DNP. Up to 295 000 ducks were counted in VLP during dry periods, and up to 770 000 in DNP when it was flooded.
• 3. The timing and extent of flooding in DNP was highly variable, but there was a consistent pattern in which ducks concentrated in VLP during dry months and winters but redistributed to DNP as more of it was flooded. This refuge effect was also strong for black‐tailed godwits Limosa limosa, but much less so for other waders. Waders feed mainly on invertebrates, and invertebrate biomass in VLP was found to be higher than in DNP. Ducks feed mainly on seeds and plant material, which are more abundant in DNP when flooded.
• 4. When water levels in DNP were stable over the course of a winter, or controlled for in multivariate models, the numbers of ducks at VLP declined over time, probably due to reduced availability of plant foods. In contrast, numbers of waders at VLP were more stable, and their invertebrate prey became more abundant over time, at least in the winter 2003/4.
• 5. In this extremely important wetland complex, the value of natural and artificial wetlands for wintering waterbirds are complementary, providing suitable habitat for different species and for different conditions in a highly variable Mediterranean environment.

Copyright © 2009 John Wiley & Sons, Ltd.     

Winter mortality of young-of-the-year pikeperch (Stizostedion lucioperca)

Abstract— The wintering success of young-of-the-year pikeperches ( Stizostedion lucioperca ) was examined in the laboratory during the winter of 1993-1994. From September to November half the fishes were offered artificial fishfood; the others received plankton with river water. Pikeperch did not eat the fishfood properly, and poorer condition of the smallest fish fed artificially was reflected in a lower amount of lipid in the whole fish at the beginning of the experiment in November. The autumnal diet played an important role in determining the wintering success even when the effect of size was removed. In contrast, feeding during the experiment did not affect survival. However, feeding fishes had more lipid at the end of the experiment in April than starved ones and their protein content also increased during the winter.     

Influence of estradiol-17β, testosterone, and 11-ketotestosterone on testicular development, serum steroid hormone, and gonadotropin secretion in male red sea bream Pagrus major

ABSTRACT:   In order to investigate the influence of estrogen and androgen on reproductive activities of male teleosts, male red sea bream were implanted with silicone capsules containing estradiol-17β (E2), testosterone (T) or 11-ketotestosterone (11-KT) in immature and early spermatogenic stages. One month after implantation of either E2 or T, the gonadosomatic index decreased in accordance with testicular regression in both stages. Implantation of E2 decreased circulating 11-KT levels but did not affect gonadotropin (GTH) subunits, follicle stimulating hormone-β (FSHβ), luteinizing hormone-β (LHβ), α glycoprotein subunit (αGSU) gene expression, and serum LH levels in both stages. Alternatively, T decreased serum 11-KT and LH levels, and FSHβ and LHβ mRNA levels in the early spermatogenic stage but not in the immature stage. These results suggest E2 may directly inhibit testicular development through the suppression of 11-KT production. Meanwhile, T may decrease serum 11-KT levels through the suppression of FSH and LH secretion, resulting to inhibition of testicular development in the early spermatogenic stage. Treatment with 11-KT did not affect the testis in either stage, whereas 11-KT increased LHβ and αGSU mRNA levels in immature, and decreased FSHβ mRNA levels in the early spermatogenic stage. These results suggest that 11-KT may have different effects on GTH subunit gene expression in each reproductive stage.