Immunohistochemical localization of gonadotropin releasing hormones in the brain and pituitary gland of the Nile perch,Lates niloticus (Teleostei,Centropomidae)

In the present study we investigated the distribution of gonadotropin-releasing hormones (GnRH) in the brain of Lates niloticus and their association with different pituitary cell types using immunohistochemical techniques. We found immunoreactive (ir) chicken GnRH-II (cGnRH-II) and mammalian GnRH (mGnRH) as the main components of the GnRH-ir system within the brain of the Nile perch. The results indicate that mGnRH and cGnRH are localized in different neurons: mGnRH-ir perikaria were observed in the preoptic region particularly in the organum vasculosum laminae terminalis (OVLT) and in the nucleus lateralis tuberis pars posterior (NLTP) of the mediobasal hypothalamus. These cell bodies are located along a continuum of ir-fibers that could be traced from the olfactory nerve to the pituitary. mGnRH-ir fibers were detected in many parts of the brain (olfactory bulbs, ventral telencephalon, hypothalamus, and mesencephalon) and in the pituitary. cGnRH-ir cell bodies are restricted to the optic tract, but few scattered fibers could be detected in different parts of the brain. The pituitary exhibited very few cGnRH-II ir fibers, contrasting with an extensive mGnRH innervation. Moreover, mGnRH-ir fibers were targeting the three areas of the pituitary gland: rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI). Double immunolabeling studies showed GnRH-ir fibers in close proximity with prolactin (PRL)- and adrenocorticotropic hormone (ACTH)-producing cells in the RPD, growth hormone (GH)-producing cells in the PPD, gonadotropins (GTHs)-producing cells in the PPD in the external border of the PI, and with somatolactin (SL)- and alpha-melanocyte stimulating hormone (alpha-MSH)-producing cells in the PI. Our results showed direct morphological evidence for a close association of GnRH-ir fibers with the different adenohypophysial cell types. These results suggest a multiple role of GnRH in the regulation of various pituitary hormones' release.     

Immunocytochemical study of growth hormone, prolactin, and thyroid-stimulating hormone in the adenohypophysis of the sea lamprey, Petromyzon marinus L., during its upstream migration

Immunoreactivity to growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) was found in the adenohypophysis of the sea lamprey, Petromyzon marinus L., during its upstream migration, by immunoperoxidase techniques with antisera to mammalian pituitary hormones. Cross-reactivity to GH and PRL was found in two different cell populations in the proximal pars distalis. Specific immunostaining for GH and PRL was absent in other parts of the lamprey pituitary. Immunoreactive TSH cells were located only in the rostral pars distalis, and corresponded in shape and size to the large basophils in this region of the lamprey pituitary. These results suggest that mammalian-like GH, PRL, and TSH are produced in the pituitary of the lamprey.     

Gonadotropin release from the pars distalis of goldfish, Carassius auratus, transplanted beside the brain or into the brain ventricles: additional evidence for gonadotropin-release-inhibitory factor

The pars distalis of the pituitary was transplanted from one goldfish to another, of matched gonadal condition and body size, either beside the brain ("juxta" location) or into the brain ventricle in the preoptic region or under the optic tectum. Recipients of a pars distalis had significantly higher serum gonadotropin (GtH) levels than sham-operated controls without transplants; following removal of the transplants, serum GtH levels decreased to levels found in controls within 24 hr. This demonstrates that the pars distalis releases GtH spontaneously when removed from its normal connections with the hypothalamus providing evidence for tonic inhibition of GtH release by a GtH-release-inhibitory factor (GRIF). The juxta-transplanted pars distalis of sexually recrudescing and mature (= completed recrudescence) females released more GtH than the pars distalis of sexually regressed females. The juxta-transplanted pars distalis of sexually mature males released more GtH than the pars distalis of males in early stages of testicular recrudescence. These results demonstrate a seasonal variation in the ability to release GtH independent of GRIF. This may be due to the greater content of GtH in the pituitary, and a greater ability to synthesize GtH by the pituitary in sexually mature and recrudescing goldfish compared to sexually regressed fish. In sexually recrudescing and mature males and females, transplantation of the pars distalis into the brain ventricle in either the preoptic region or ventral to the optic tectum resulted in significantly lower serum GtH levels in recipients than transplantation beside the brain. This demonstrates the presence of some factor in the brain that inhibits GtH release, supporting the idea of GRIF. In sexually regressed female goldfish, GtH release from the transplanted pars distalis was not influenced by location in the brain ventricles versus beside the brain. This suggests a seasonal variation in GRIF activity in the brain, with reduced activity in sexually regressed females coincident with less ability to release GtH by the pars distalis.     

A homolog of mammalian PRL-releasing peptide (fish arginyl-phenylalanyl-amide peptide) is a major hypothalamic peptide of PRL release in teleost fish

Two PRL-releasing peptides (PrRP20 and PrRP31) were recently identified from mammalian hypothalamus by an orphan receptor strategy, and a C-terminal RF (arginyl-phenylalamyl-) amide peptide (RFa), structurally related to mammalian PrRP, was also identified from the brain of the Japanese crucian carp (C-RFa) by an intestine-contracting assay. However, to date there have been no reported studies that have examined the PRL-releasing effects of RFa in fish. In the present study we determined the cDNA, primary structure, and function of a homolog of the mammalian PrRP20 in the chum salmon, Oncorhynchus keta. An RFa cDNA encoding a preprohormone of 155 amino acids was cloned from the hypothalamus of chum salmon by 3'- and 5'-rapid amplification of cDNA ends. A native RFa was purified from an acid extract of salmon hypothalami by a Sep-Pak C(18) cartridge, affinity chromatography using anti-synthetic C-RFa, and reverse phase HPLC on an ODS-120T column. The salmon RFa proved to be identical with C-RFa on the basis of elution position on reverse phase HPLC. Immunocytochemical staining in rainbow trout, Oncorhynchus mykiss, revealed that C-RFa-immunoreactive cell bodies were located in the posterior part of hypothalamus and C-RFa-immunoreactive fibers were abundant from the hypothalamus to the ventral telencephalon. A small number of immunoreactive fibers were projected to the pituitary and terminated close to the PRL cells in the rostral pars distalis and to the somatolactin (SL) cells in the pars intermedia. The hypophysiotropic effects of the fish homolog were determined on the release of PRL, SL, and GH from the pituitary of the rainbow trout. Plasma PRL and SL levels were increased at 3 and 9 h, respectively, after ip injection of the synthetic C-RFa into the rainbow trout at doses of 50 and 500 ng/g body weight. In contrast, plasma GH levels were decreased after 1 h at 500 ng/g body weight. Perifusion of the trout pituitaries with synthetic C-RFa at concentrations of 10 pM to 100 nM demonstrated maximum PRL release at 100 pM and maximum SL release at 10 and 100 nM. However, GH release was not affected. These data are the first to demonstrate that a homolog of mammalian PrRP (fish RFa) is a major hypothalamic peptide of PRL release in teleost fish.     

Ontogeny of adenohypophyseal cells in the pituitary of the American shad (Alosa sapidissima)

The distribution and ontogeny of adenohypophyseal cells have been studied in the pituitary gland of embryos, larvae, and juveniles of the clupeid American shad (Alosa sapidissima) using immunocytochemical techniques. In juvenile specimens, adenohypophysis was composed of rostral pars distalis (RPD), formed by cavities lined by prolactin (PRL), adrenocorticotropic hormone (ACTH), and gonadotropic hormone (GTH) cells; proximal pars distalis (PPD), containing growth hormone (GH), GTH, and putative thyroid stimulating hormone (TSH) cells; and pars intermedia (PI) with somatolactin (SL) and melanophore stimulating hormone (MSH) cells. At 3 days post-fertilization (3 days pre-hatching) the pituitary of embryos consisted of an oval mass of cells, close to the ventral margin of the diencephalon, divided in rostral and caudal regions. At this time PRL and ACTH cells appeared in the rostral region of the adenohypophysis, while SL cells were observed in the caudal region where MSH cells showed reactivity 1 day before hatching. At variance, GH cells showed a weak immunoreactivity in the rostral portion at hatching that increased 2 days latter. GTH cells also showed weak immunoreactivity in the rostral region of the adenohypophysis at hatching time. Two days later GTH cells were located in the rostral and central regions of the adenohypophysis. At hatching, the neurohypophysis was very small and no nerve processes were seen to penetrate the adenohypophysis tissue. After hatching, the pituitary gland elongated and in 7 days old larvae, the RPD showed a small lumen surrounded by a palisade of PRL, ACTH, and GHT cells; the PPD showed GH and GTH cells while the PI contained SL and MSH cells. The adenohypophysis and neural lobe increased in size with development and, in 42 days old larvae, they were similar to those of juvenile specimens.     

Immunocytochemical identification of the prolactin- and growth hormone-secreting cells in the teleost pituitary with antisera to tilapia prolactin and growth hormone

Antisera raised to highly purified tilapia (Sarotherodon mossambicus) prolactin (PRL) and growth hormone (GH) were used to locate PRL and GH cells in the adenohypophysis of seven species of teleosts by immunoenzymological methods using horseradish peroxidase. The only pituitary cells in the tilapia that immunologically reacted with anti-tilapia PRL were the PRL cells located in the rostral pars distalis (RPD). In the pituitary glands of two species of salmonids and two species of marine fishes, the tilapia PRL antibodies bound to both PRL and GH cells; no prolactin-immunoreactive cells were identified in the goldfish and eel pituitaries. Antiserum to tilapia GH reacted specifically with the GH cells located in the proximal pars distalis of all teleosts examined in this study, but not with any presumed PRL cells in the RPD. These results indicate that tilapia PRL antisera are specific for tilapia PRL cells but not necessarily for these cells in other teleost species. Tilapia GH antisera showed no species specificity among the teleosts tested.     

Seasonal changes of responses to gonadotropin-releasing hormone analog in expression of growth hormone/prolactin/somatolactin genes in the pituitary of masu salmon

Gonadotropin-releasing hormone (GnRH) is considered to stimulate secretion of growth hormone (GH), prolactin (PRL), and somatolactin (SL) at particular stages of growth and sexual maturation in teleost fishes. We therefore examined seasonal variation in the pituitary levels of GH/PRL/SL mRNAs, and tried to clarify seasonal changes of responses to GnRH in expression of GH/PRL/SL genes, in the pituitaries of growing and maturing masu salmon (Oncorhynchus masou). Pituitary samples were monthly collected one week after implantation with GnRH analog (GnRHa). The levels of mRNAs encoding GH, PRL, and SL precursors in single pituitaries were determined by a real-time polymerase chain reaction method. The fork lengths and body weights of control and GnRHa-implanted fish of both sexes gradually increased and peaked out in September of 2-year-old (2+) when fish spawned. GnRHa implantation did not stimulate somatic growth, nor elevate gonadosomatic index (GSI) of 1+ and 2+ males, whereas it significantly increased GSI of 2+ females in late August to early September. The GnRHa-implanted 1+ males had higher levels of GH and PRL mRNAs in July, and SL mRNA from June to August than the control males. The levels of GH, PRL, and SL mRNAs in the control and GnRHa-implanted 1+ females, however, did not show any significant changes. Afterward, the PRL mRNA levels elevated in the control 2+ fish of both sexes in spring. GnRHa elevated the GH mRNA levels in both males and females in 2+ winter, and the PRL mRNA levels in females in early spring. Regardless of sex and GnRHa-implantation, the SL mRNA levels increased during sexual maturation. In growing and maturing masu salmon, expression of genes encoding GH, PRL, and SL in the pituitary is thus sensitive to GnRH in particular seasons probably in relation to physiological roles of the hormones.     

Alterations in pituitary GnRH and dopamine receptors associated with the seasonal variation and regulation of gonadotropin release in the goldfish (Carassius auratus)

Seasonal variations in the serum concentrations of gonadotropin (GtH) and the serum GtH response to intraperitoneal injection of domperidone, a specific dopamine receptor antagonist, were examined in goldfish. In addition, the effects of in vivo treatment of goldfish with a superactive analog of salmon gonadotropin-releasing hormone (sGnRH-A) and domperidone on the binding parameters of pituitary GnRH and dopamine receptors were investigated in goldfish. Serum concentrations of GtH and the maximum GtH response to domperidone increased in correlation with advancing gonadal maturation; values increased from those in sexually regressed fish in January to maximal levels observed in fish in late stages of gonadal recrudescence in March, followed by a decrease with gonadal regression. At all stages, injection of domperidone increased serum concentrations of GtH in a dose-related manner; however, the ED50 of domperidone did not vary significantly over the course of the reproductive cycle. Multiple injections of sGnRH-A caused a progressively increasing and more prolonged serum GtH response; as well, multiple sGnRH-A treatment significantly potentiated the serum GtH response to domperidone without altering the ED50 of domperidone. sGnRH-A treatment caused a significant increase in the number of dopamine/neuroleptic receptors in the goldfish pars distalis, accompanied by a nonsignificant increase in dopamine/neuroleptic receptors in the neurointermediate lobe, and significantly increased the number of high-affinity GnRH receptors in the goldfish pituitary. Treatment with domperidone also significantly increased pituitary high-affinity GnRH receptor numbers. Receptor affinities were not significantly altered by either sGnRH-A or domperidone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogenesis of the three parts of the fetal rat adenohypophysis. A detailed immunohistochemical analysis

The sequential changes in the histological pattern of anterior pituitary cytodifferentiation of the rat are described. The first labeled cells were ACTH positive and were detected in the pars tuberalis on postconceptual day 13. On day 14 ACTH cells also appeared in the ventral periphery of the pars distalis. On fetal day 15 the pars tuberalis anlage was characterized by numerous well-stained ACTH cells and by some weakly labeled FSH-beta, LH-beta, TSH-beta, GH and PRL cells while the pars distalis showed only ACTH positivity. On day 16 of gestation the ACTH cells were equally distributed throughout the whole pars distalis, while LH-beta, FSH-beta, TSH-beta, PRL and GH immunoreactive cells were localized either in the ventral region of the pars distalis only or were evenly distributed throughout the pars distalis. The present immunocytochemical data suggest that in the pars distalis the hypophyseal cell differentiation follows a clear rostrocaudal, ventrodorsal direction and that the time sequence of the functional differentiation of the adenohypophysis is pars tuberalis, pars distalis and pars intermedia.     

Chronological appearance of the different hypophysial hormones in the pituitary of sea bass larvae (Dicentrarchus labrax) during their early development: an immunocytochemical demonstration

Antisera raised against chum salmon prolactin (PRL), rainbow trout growth hormone (GH), mammalian adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) were used to study the chronological appearance of immunoreactivity for PRL, GH, ACTH, TSH, LH, and melanocyte-stimulating hormone (MSH) in the pituitary of sea bass larvae (Dicentrarchus labrax) during the first 26 days after hatching. The anti-ACTH gives positive immunostaining in the ACTH cells as well as in the MSH cells; however, the two cell types can easily be distinguished by their different localization in the pituitary: ACTH in the rostral pars distalis, MSH in the pars intermedia. The first day after hatching cells immunoreactive for TSH, GH and ACTH could already be noticed, ACTH reacted strong in the pars intermedia but very weak in the rostral pars distalis. Cells immunopositive for PRL became visible between Days 9 and 15. With anti-LH, no positive reaction could be obtained during the first 26 days after hatching.     

Effects of fasting, medium glucose, and amino acid concentrations on prolactin and growth hormone release, in vitro, from the pituitary of the tilapia Oreochromis mossambicus.

Previous investigations have shown that the release of PRL and GH from the tilapia pituitary is directly sensitive to osmotic pressure and a variety of endocrine and neuroendocrine factors. The present studies were aimed at determining whether the spontaneous release of PRL and GH, in vitro, is: (1) sensitive to the nutritional status of the fish, and (2) responsive to variations in the D-glucose and total amino acid content of the incubation medium. In the first series of experiments, male fish (50 to 60 g) were divided into two groups. One group was fed twice daily for 2 weeks while the second received no food. A nearly homogeneous mass of PRL-secreting cells was dissected from the rostral pars distalis (RPD) and incubated for 18 to 20 hr in either hyposmotic (300 mOsmolal) or hyperosmotic (355 mOsmolal) medium. Similarly, a mass of GH-secreting cells was dissected from the proximal pars distalis (PPD) and incubated for 18 to 20 hr in isosmotic (320 mOsmolal) medium. Fasting was found to alter the total amount of PRL and GH in the culture well (tissue + medium) at the end of the incubations, decreasing PRL and increasing GH. Fasting was also found to both reduce spontaneous PRL release in vitro and suppress its stimulation by reduced osmotic pressure (P less than 0.01). By contrast, fasting resulted in a substantial increase in spontaneous GH release from the PPD in vitro (P less than 0.01). In the second series of experiments, GH release was found to increase as the D-glucose concentration of the medium decreased (P less than 0.01), while prolactin release was unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of gonadotropin-releasing hormone receptors in cultured rat pituitary cells

The properties of gonadotropin-releasing hormone (GnRH) receptors were analyzed in isolated pituitary cells prepared by enzymatic dispersion with trypsin or collagenase-hyaluronidase. The initial impairment of LH responses to GnRH in isolated cells prepared by both methods was reversed during culture for 2 days in multiwell vessels. However, specific binding sites for GnRH, assayed by equilibration with [125I]iodi0[D-Ser(t-BU)6]des-Gly10-GnRH N-ethylamide (GnRH-A) were demonstrable in collagenase-dispersed cells, both initially and after 2 days in culture. Cellular uptake of GnRH-A was temperature dependent, with rapid and saturable binding to a limited number of specific receptor sites with high affinity for the labeled analog (Ka = 4.0 +/- 0.8 X 10(9) M-1). These sites showed common binding specificity for GnRH-A and the native GnRH peptide, with significantly lower affinity for the natural peptide (Ka = 2.3 X 10(7) M-1). Other protein and peptide hormones, including ovine LH, ovine PRL, hCG, TRH, somatostatin, and angiotensin II (up to 10(-6) M) did not inhibit binding of GnRH-A to its receptors. Cellular binding of GnRH-A was followed by increased cGMP production and LH release within 10 min. The analog was 50 times more potent than native GnRH in stimulating LH release. The Kact values derived for GnRH and GnRH-A were 0.5 and 0.01 nM, respectively, considerably lower than the Kd values of 50 and 0.25 nM derived from receptor binding analysis. These results indicate that GnRH receptors can be identified in isolated pituitary cells, in which peptide binding is followed by increased cGMP production and LH release. The impaired LH responses in acutely dispersed pituitary cells are not due to the loss of receptor sites but to a reversible postreceptor defect. Occupancy of about 20% of the GnRH-binding sites elicits a near-maximal LH response, indicating the nonlinearity of GnRH-receptor coupling to secretory responses in the cultured gonadotroph.     

Gonadotropin-releasing hormone neuronal development during the sensitive period of temperature sex determination in the pejerrey fish,Odontesthes bonariensis

The development of gonadotropin-releasing hormone (GnRH) neurons was studied in relation to the sensitive period of thermolabile sex determination in the pejerrey Odontesthes bonariensis, an atherinid fish from South America. Fish were raised from hatching at three different temperatures: 17 degrees C (100% females), 24 degrees C (70% females), and 29 degrees C (100% males). Three groups of immunoreactive GnRH (ir-GnRH) neurons were identified at the terminal nerve ganglion (TNG), the midbrain tegmentum (MT), and the preoptic area (POA). Immunoreactive GnRH (ir-GnRH) neurons were identified in the TNG at hatching (day 0) and in the MT at day 3 at all the experimental temperatures. In the POA ir-GnRH neurons were identified in the nucleus preopticus periventricularis simultaneously with the first appearance of ir-GnRH fibers in the pituitary on days 11, 14, and 17 for larvae kept at 29, 24, and 17 degrees C, respectively. The number of ir-GnRH neurons in the TNG did not show any statistical difference between temperatures. The number of ir-GnRH neurons in the MT increased in number during the experiment for larvae kept at 17 and 24 degrees C but decreased between days 17 and 31 in larvae kept at 29 degrees C. The number of ir-GnRH neurons in the POA increased during development with a peak at day 28 for all temperatures studied and the magnitude of this peak showed a correlation with incubation temperature. These results reinforce the notion that the hypothalamus-pituitary-gonadal axis is active during sex determination in pejerrey suggesting a possible role of the central nervous system and GnRH in this process. It is also suggested that GnRH neurons located in the preoptic area might be the physiological transducers of temperature during the temperature sensitive period in this species.     

Immunocytochemical identification of GtH1 and GtH2 cells during the temperature-sensitive period for sex determination in pejerrey, Odontesthes bonariensis.

The ontogeny of gonadotropin 1 (GtH1) and 2 (GtH2) cells and its possible link to gonadal sex differentiation were studied in pejerrey, Odontesthes bonariensis, by immunocytochemistry using anti-chum salmon beta-GtH1 and beta-GtH2 antisera. In adults, GtH1 cells were found in the proximalis pars distalls (PPD) close to the neurohypophysis, whereas GtH2 cells were identified surrounding GtH1 cells, at the external layer of the PPD and in the pars intermedia. Essentially the same distribution was observed in larvae. In pejerrey, the phenotypic sex is governed by the temperature during the critical period of sex determination (temperature-dependent sex determination, TSD). Female proportions vary gradually from 100% at 15-19 degrees to 0% at 29 degrees, and the critical time of TSD has been estimated to be 3-5, 2-4, and 1-4 weeks after hatching at 17, 19, and 27 degrees, respectively. Thus, the expression of both GtHs was examined weekly in larvae reared from hatching to week 11, at 17, 24, and 29 degrees. The proportion of females at 17, 24, and 29 degrees was, 100%, 78%, and 0%, respectively. Histological ovarian differentiation was noticed at 7 and 4 weeks at 17 and 24 degrees, whereas testicular differentiation occurred at 7 and 4 weeks at 24 and 29 degrees. GtH1 cells were first observed at week 3 at 29 degrees and at week 4 at 17 and 24 degrees. These cells increased in number until week 4 and then decreased, disappearing after week 6 at all temperature regimes. GtH2 cells appeared at week 2 at 24 and 29 degrees and at week 3 at 17 degrees. GtH2 cell number increased until week 3 at 29 degrees and until week 4 at 17 and 24 degrees and then temporarily decreased, thereafter increasing again. These results strongly suggest that GtH1 and GtH2 are expressed by different cells. The fact that GtH1 and GtH2 cells appear just before histological gonadal differentiation at all temperatures, together with the peak of GtH1 and GtH2 cell number during the temperature-sensitive period, suggests that GtHs are related to sex differentiation or TSD in O. bonariensis.     

Developmental ontogeny of prolactin and its receptor in fish

Prolactin (PRL) is a member of a family of structurally similar proteins which includes growth hormone (GH) and somatolactin (SL) in teleost fish. The genes encoding these proteins are expressed principally in the pituitary gland and sequence analysis reveals they share considerable similarity. GH, PRL, and SL bring about their physiological action by binding to specific receptors localised in the membrane of cells in target tissue. The PRL receptor (PRLR) and GH receptor (GHR) have been identified in a number of teleosts but the SL receptor remains to be characterised. On hormone binding, receptors dimerise, and signal transduction occurs via the JAK/STAT signalling pathway. The principal action of PRL in fish is freshwater osmoregulation, although it has also been implicated in reproduction, behaviour, growth, and immunoregulation. The role of PRL in early development and metamorphosis is well established, respectively, in mammals and amphibians, although its role in fish is not so well known. Studies have shown that PRL mRNA and protein are restricted to the developing pituitary gland in fish embryos and larvae. PRLR mRNA and protein is also present in fish embryos and has a widespread tissue distribution in larvae. The levels of PRLR and PRL mRNA vary throughout embryonic and early larval development. The potential role of PRL in fish embryos and larvae is considered in relation to their physiological status.     

Perspectives on fish gonadotropins and their receptors

Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common α subunit non-covalently associated with a hormone-specific β subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders.Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper.The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review.In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone–receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.     

Preparation and use of specific antibodies to the beta-I and beta-II subunits of gonadotropic hormone from Fundulus heteroclitus pituitary

Fundulus heteroclitus naturally spawns with a semilunar periodicity throughout most of the year in its southernmost habitat, an activity that can be maintained in the laboratory. The alpha and two beta subunits comprising F. heteroclitus gonadotropic hormones (GtHs) I and II have been sequenced, and antibodies have been raised against unique peptides found in each of the two beta subunits. On immunoblots of pituitary proteins, each antibody recognizes a single band with a molecular mass of 16-17 kDa, somewhat larger than the deduced sizes (11-13 kDa) of the unglycosylated subunits. Each antibody also recognizes a different subset of pituitary cells in the central (GtH I) and peripheral (GtH II) proximal pars distalis, regions that display the typical tinctorial properties of gonadotrops. The distribution and distinct separation of cells containing GtH beta subunits I and II thus differ from those found for previously described teleost species, most of which are salmonids that engage in a single spawning episode during the year. The availability of these antibodies thus makes F. heteroclitus an inexpensive, easily manipulated model system for studies on the hormonal regulation of fractional spawning common to a large class of commercially important species other than salmonids.     

Isolation and characterization of carp prolactin

Prolactin (PRL) was extracted with acid-acetone from common carp (Cyprinus carpio) pituitary glands and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography (HPLC) on TSK-gel TMS 250 with a yield of 0.7 mg/g wet tissue. At each stage of purification, fractions were monitored by HPLC on TSK-gel ODS 120T and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Carp PRL was almost equipotent with ovine PRL in retaining plasma Na concentrations in the hypophysectomized killifish, Fundulus heteroclitus. Immunocytochemistry at both the light and electron microscopic levels revealed that carp PRL antiserum specifically stained cells in the goldfish rostral pars distalis. No cross reaction with putative growth hormone (GH) cells in the proximal pars distalis was observed. The specificity of the carp PRL antiserum was confirmed by immunoblot studies. Although immunostaining of both carp and salmon PRL was observed, there was no cross reaction to GHs from these species. Carp PRL had a sole N-terminal residue of valine, a molecular weight of 23 kDa in SDS-PAGE, and an isoelectric point of 7.3 by gel electrofocusing. Based on these results, together with the knowledge of physicochemical properties of salmon and tilapia PRLs, we propose a standard procedure for isolation of fish PRLs.