W. M. van Dijk

Cloning and Spatiotemporal Expression of the Follicle-Stimulating Hormone {beta} Subunit Complementary DNA in the African Catfish (Clarias gariepinus)

Abstract The gene and cDNA encoding a putative follicle-stimulating hormone β subunit (cfFSHβ) from African catfish (Clarias gariepinus) were cloned. Similar to other FSHβ genes, the cfFSHβ gene consisted of three exons interrupted by two introns. The cfFSHβ cDNA coded for a mature protein of 115 amino acids. The 12 cysteines that are required for the typical tertiary folding of glycoprotein hormone β subunits were positionally conserved in cfFSHβ. The cfFSHβ mRNA expression was exclusively detected in the pituitary and was detectable before pubertal development was initiated. The cfFSHβ transcript levels increased in particular during early stages of puberty and reached constantly high levels after the first appearance of spermatids in the testis. The cfFSHβ mRNA-positive cells were localized in the proximal pars distalis. Castration of mature males caused elevated cfFSHβ mRNA levels that were decreased by steroid replacement. Previous work indicated that the African catfish is an interesting model to study the regulation of gonadal functions because cfLH is able to activate both the catfish luteinizing hormone receptor (cfLH-R) and follicle-stimulating hormone receptor (cfFSH-R). Because cfFSH purification has failed so far, ongoing studies are directed toward the production of recombinant cfFSH. After all, the developmental and hormonal regulation of cfFSHβ transcript levels opens the possibility for physiologically relevant actions of the putative cfFSH, next to the presumptive bifunctionally acting cfLH.

Steroid hormones stimulate gonadotrophs in juvenile male African catfish (Clarias gariepinus).

Abstract In juvenile African catfish (Clarias gariepinus), the pituitary LH content strongly increased after the beginning of spermatogonial proliferation. We hypothesized that a signal of testicular origin is involved in stimulating the gonadotrophs. We investigated the effects of castration and sex steroid treatment on gonadotrophs in juvenile males by quantifying LH production and release and LH subunit transcript levels and by examining gonadotroph morphology and proliferation. Castration reduced but did not abolish the maturation-associated elevation in pituitary LH content. Treatment with testosterone but not with 11-ketotestosterone, an otherwise potent androgen in fish, reversed the castration-induced decrease of pituitary LH levels. An increased pituitary LH content was accompanied by an increased number of cytologically mature gonadotrophs. However, no evidence was found for gonadotroph proliferation, so that quiescent gonadotrophs may have become activated. Although 11-ketotestosterone treatments had no effect in castrated males, this androgen attenuated gonadotroph activation in intact males. Because androgen production in juvenile catfish is downregulated by treatment with 11-ketotestosterone, its inhibitory effects on gonadotrophs in gonad-intact males may be due to suppression of Leydig cell testosterone production, which appears to be a limiting factor for the activation of catfish gonadotrophs. Aromatizable androgens may have opposite effects on fish (stimulatory) and mammalian (inhibitory) gonadotrophs.

Inhibitory and Stimulatory Interactions Between Endogenous Gonadotropin-Releasing Hormones in the African Catfish (Clarias gariepinus)

Abstract In the brain of all vertebrate classes, chicken (c) GnRH-II ([His5,Trp7,Tyr8]GnRH, cGnRH-II) is expressed in the mesencephalon. In addition, at least one other form of GnRH is expressed in the preoptical area/hypothalamus. In the human pituitary stalk and the mouse median eminence, cGnRH-II is present together with mammalian GnRH. Similarly, in the pituitary of several teleost fish (e.g., goldfish and eel, but not salmon or trout), a teleost GnRH is found together with cGnRH-II. These GnRHs are not colocalized in the same cells. Hence, these GnRH peptides may differentially regulate gonadotropin secretion and, in addition, may exert their effects simultaneously. The current study therefore investigated the effects of combinations of the two forms of GnRH present in the African catfish (Clarias gariepinus) pituitary—cGnRH-II and catfish GnRH ([His5,Asn8]GnRH, cfGnRH)—on the cytosolic free calcium concentration ([Ca2+]i) in single, Fura-2-loaded catfish gonadotrophs, as well as their effects on both in vitro and in vivo LH secretion. Both inhibitory and stimulatory effects of combinations of cfGnRH and cGnRH-II on [Ca2+]i were observed, which were mirrored by their effects on both in vitro and in vivo LH secretion. The following pattern became apparent. The effect of intermediate or maximal effective cfGnRH doses was inhibited by the simultaneous presence of subthreshold or borderline effective cGnRH-II doses. Conversely, subthreshold or borderline effective concentrations of cfGnRH enhanced the effects of intermediate and maximal concentrations of cGnRH-II. In addition, combinations of cfGnRH and cGnRH-II concentrations that were equally active when tested separately showed an additive effect. The observed interactions between the two GnRHs may be of particular physiological relevance in the control of seasonal LH levels in the African catfish, as well as in other teleost species. Moreover, the occurrence of mutual inhibitory and stimulatory interactions between endogenous GnRHs may be a widespread aspect of GnRH action in vertebrates.

Role of calcium ions in action of gonadotropin-releasing hormone on gonadotropin secretion in the African catfish, Clarias gariepinus

The aim of the present study was to establish the role of calcium ions in the mechanism of action of gonadotropin-releasing hormone (GnRH) in stimulating gonadotropin (GTH) release in the African catfish, Clarias gariepinus. For that purpose, GTH release from pituitary fragments was monitored in a perifusion system. GTH release, induced by the GnRH analog Buserelin, was strongly diminished in the absence of Ca2+, as well as in the presence of the Ca2+ channel antagonist nifedipine. In addition, the Ca2+ ionophore A23187 stimulated GTH secretion in the absence of GnRH. These results indicate that calcium ions play an intermediate role in the mechanism of action of GnRH in the African catfish.

Development of a homologous radioimmunoassay for Mediterranean yellowtail (Seriola dumerilii, Risso 1810) LH

Abstract Purified Mediterranean (M.) yellowtail luteinizing hormone-like gonadotropin (MyLH) and its β-subunit (MyLHβ) served to develop a radioimmunoassay (RIA) for MyLH. The rabbit antisera against MyLH and MyLHβ used for this purpose were tested on pituitary sections by immunocytochemistry. Anti-MyLHβ specifically detected a single type of cells, which were located at the periphery of the proximal pars distalis (PPD) and surrounding the pars intermedia (PI). Anti-MyLH, however, also recognized two other cell types, thyrotropin β-immunoreactive (ir) cells and putative follicle-stimulating hormone-like (MyFSH)-producing cells. Labeling of the two latter cell types was prevented by preabsorption of anti-MyLH with M. yellowtail pituitary glycoprotein α-subunit. The standard curve for the RIA was generated using purified MyLH, 125 I-labeled MyLHβ and anti-MyLHβ at a dilution of 1:70,000, which resulted in the binding of 30% of the tracer added. The standard curve ranged from 0.25 to 50 ng/ml. The midrange of the assay (ED50) was obtained with 5.48–7.87 ng LH/ml. The variation between assays was less than 15%. An average cross-reactivity of FSH in the LH RIA of 8.4% was found. Serial dilutions of M. yellowtail pituitary extracts displaced radiolabelled MyLHβ parallel to the MyLH standard. Application of the LH RIA to blood samples and pituitary cell culture medium provided physiological validation of the assay. Significant increases in LH levels were recorded after salmon GnRH treatments in vivo and in vitro. Serum LH levels from wild fish sampled at the spawning season were significantly higher than those from captive fish sampled in the same period.

Linking the morphology of fluvial fan systems to aquifer stratigraphy in the Sutlej‐Yamuna plain of northwest India

The Indo-Gangetic foreland basin has some of the highest rates of groundwater extraction in the world, focused in the states of Punjab and Haryana in northwest India. Any assessment of the effects of extraction on groundwater variation requires understanding of the geometry and sedimentary architecture of the alluvial aquifers, which in turn are set by their geomorphic and depositional setting. To assess the overall architecture of the aquifer system, we used satellite imagery and digital elevation models to map the geomorphology of the Sutlej and Yamuna fan systems, while aquifer geometry was assessed using 243 wells that extend to ∼200 m depth. Aquifers formed by sandy channel bodies in the subsurface of the Sutlej and Yamuna fans have a median thickness of 7 and 6 m, respectively, and follow heavy-tailed thickness distributions. These distributions, along with evidence of persistence in aquifer fractions as determined from compensation analysis, indicate persistent reoccupation of channel positions and suggest that the major aquifers consist of stacked, multistoried channel bodies. The percentage of aquifer material in individual boreholes decreases down fan, although the exponent on the aquifer body thickness distribution remains similar, indicating that the total number of aquifer bodies decreases down fan but that individual bodies do not thin appreciably, particularly on the Yamuna fan. The interfan area and the fan marginal zone have thinner aquifers and a lower proportion of aquifer material, even in proximal locations. We conclude that geomorphic setting provides a first-order control on the thickness, geometry, and stacking pattern of aquifer bodies across this critical region.

Gonadotropin-Releasing Hormone Does Not Directly Stimulate Luteinizing Hormone Biosynthesis in Male African Catfish

Abstract Besides gonadotropin release, GnRH stimulates gonadotropin subunit gene transcription and translation in gonadotrophs. In the African catfish, Clarias gariepinus, chicken GnRH-II (cGnRH-II: [His5,Trp7,Tyr8]-GnRH) and catfish GnRH (cfGnRH: [His5,Asn8]-GnRH) are two endogenous forms of GnRH. Studying their effects on LH subunit steady-state mRNA levels, LH de novo synthesis, and LH release in primary pituitary cell cultures of adult males, we found that cGnRH-II hardly influenced the steady-state levels of LH subunit mRNAs or LH de novo synthesis, although it stimulated LH release. Although cfGnRH stimulated LH secretion as well, high concentrations—although apparently still within the physiologic range—reduced LH transcript levels and de novo synthesis in primary pituitary cell cultures. In vivo experiments demonstrated a biphasic response of LH subunit transcript levels after a single GnRH injection: a decrease after 2 h was followed by an increase at 8 h. When the testes were removed before GnRH treatment, however, LH transcript levels remained depressed at 8 h after GnRH injection, indicating that the secondary increase in LH transcript levels depends on the presence of the testes. We conclude that the up-regulation of LH production subsequent to GnRH stimulation in adult male African catfish is mediated by factors originating from the testis. Previous work suggests that aromatizable androgens may play an important role in this context. Under the present experimental conditions, however, GnRHs had no, or an inhibitory, direct effect on LH production in catfish gonadotrophs.

Sertoli cell proliferation and FSH signalling in African catfish, Clarias gariepinus

Sertoli cell proliferation occurs mainly during the phase of rapid spermatogonial proliferation, allowing the cyst-forming Sertoli cells to form an increasingly large space for housing the growing germ cell clone. There is no information in fish on the regulation of Sertoli cell proliferation; follicle-stimulating hormone (FSH) stimulates Sertoli cell proliferation in mammals. Increasing or decreasing FSH and FSH receptor expression experimentally in male African catfish was associated with respective changes in Sertoli cell proliferation or testis growth, suggesting that also in fish, one role of FSH may be to regulate Sertoli cell numbers.