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A defect in the structure of the obese gene is responsible for development of obesity in the ob/ob mouse. The product of expression of the gene is the protein hormone leptin. Leptin causes weight loss in ob/ob and normal mice, it is secreted by adipocytes, and it is an important controller of the size of fat stores by inhibiting appetite. The ob/ob mouse is infertile and has a pattern of gonadotropin secretion similar to that of prepubertal animals. Consequently, we hypothesized that leptin might play a role in the control of gonadotropin secretion and initiated studies on its possible acute effects on hypothalamic-pituitary function. After a preincubation period, hemi-anterior pituitaries of adult male rats were incubated with leptin for 3 hr. Leptin produced a dose-related increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release, which reached peaks with 10(-9) and 10(-11) M leptin, respectively. Gonadotropin release decreased at higher concentrations of leptin to values indistinguishable from that of control pituitaries. On the other hand, prolactin secretion was greatly increased in a dose-related manner but only with leptin concentrations (10(-7)-10(-5) M). Incubation with leptin of median eminence-arcuate nuclear explants from the same animals produced significant increases in LH-releasing hormone (LHRH) release only at the lowest concentrations tested (10(-12)-10(-10) M). As the leptin concentration was increased, LHRH release decreased and was significantly less than control release at the highest concentration tested (10(-6) M). To determine if leptin can also release gonadotropins in vivo, ovariectomized females bearing implanted third ventricle cannulae were injected with 10 microgram of estradiol benzoate s.c., followed 72 hr later by microinjection into the third ventricle of leptin (0.6 nmol in 5 microliter) or an equal volume of diluent

Background The evaluation of prepubertal gonadal Leydig cells secretion requires gonadotropin stimulation. Urinary hCG (human chorionic gonadotropin) is currently unavailable in many countries, however, recombinant hCG (rhCG) can be used. Our aim was to evaluate rhCG-stimulated testicular hormones in a group of patients with cryptorchidism. Methods We evaluated 31 prepubertal boys (age range, 0.75–9.0 years) presenting with unilateral ( n  = 24) or bilateral ( n  = 7) cryptorchidism. Patients with other genital abnormalities, previous use of hCG or testosterone or previous surgeries were excluded. Blood samples were obtained at baseline and 7 days after a single subcutaneous dose of rhCG (Ovidrel® 250 mcg) to measure the testosterone, DHT (dihydrotestosterone), AMH (anti-Mullerian hormone), and inhibin B levels. Results rhCG stimulation significantly increased testosterone levels from 10 ng/dl to 247.8 ± 135.8 ng/dl, increased DHT levels from 4.6 ± 0.8 to 32.3 ± 18.0 ng/dl, and increased the T/DHT ratio from 2.2 ± 0.4 to 8.0 ± 3.5. There was also a significant increase in inhibin B (from 105.8 ± 65.2 to 132.4 ± 56.1 pg/ml; p  < 0.05) and AMH levels (from 109.4 ± 52.6 to 152.9 ± 65.2 ng/ml; p  < 0.01) after the rhCG stimulation. Conclusions In this cohort, hormonal responses can be elicited after the rhCG stimulation test, suggesting that rhCG is a promising stimulation test to replace the urinary hCG test during the evaluation of gonadal Leydig cells function. The clinical applicability and adequate performance of rhCG testing must be investigated in future studies.

Gonadotropin-releasing hormone in Cyprinidae as in other vertebrates functions as a brain signal which stimulates the secretion of luteinizing hormone from the pituitary gland. Two forms of gonadotropin-releasing hormone have been identified in cyprinids, chicken gonadotropin-releasing hormone II and salmon gonadotropin-releasing hormone. Hypophysiotropic functions are fulfilled mainly by salmon gonadotropin-releasing hormone. The only known factor having an inhibitory effect on LH secretion in the family Cyprinidae is dopamine. Most cyprinids reared under controlled conditions exhibit signs of reproductive dysfunction, which is manifested in the failure to undergo final oocyte maturation and ovulation. In captivity a disruption of endogenous gonadotropin-releasing hormone stimulation occurs and sequentially that of luteinizing hormone. In addition to the methods based on the application of exogenous gonadotropins, the usage of a method functioning on the basis of hypothalamic control of final oocyte maturation and ovulation has become popular. The replacement of natural gonadotropin-releasing hormones with chemically synthesized gonadotropin-releasing hormone analogues characterized by amino acid substitutions at positions sensitive to enzymatic degradation has resulted in a centuple increase in the effectiveness of luteinizing hormone secretion induction. Combining gonadotropin-releasing hormone analogues with dopamine inhibitory factors have made it possible to develop an extremely effective agent which is necessary for the successful artificial reproduction of cyprinids.

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.

We previously demonstrated the biological activities of single-chain recombinant gonadotropins (scGTHs) of goldfish Carrassius auratus follicle-stimulating hormone (scFSH) and luteinizing hormone (scLH), produced by a baculovirus-silkworm larvae system, by using in vivo bioassays with some fishes including Japanese eel Anguilla japonica. Among the bioassays, we succeeded in induction of spermatogenesis of sexually immature male Japanese eels by both scFSH and scLH, especially resulting in the occurrence of spermatozoa in scLH-administered males. However, those recombinant hormones did not induce enlargement of testes. In order to further confirm the potency of recombinant GTHs for use in aquaculture species, we administered scFSH and scLH to males of Japanese eel at higher dosage and frequency (eight times with 2-5 days interval) than those of the previous study (five or six times with 7 days intervals), including combination of scFSH and scLH administration (scFSH-scLH). Gonadosomatic indices (GSI) of scLH- and scFSH-scLH-administered males were larger than those of initial control males and of control males that were injected with saline. Enlargement of testes was also confirmed by measurement of testicular lobe size in scFSH-, scLH-, and scFSH-scLH-administered males. By histological observation, occurrence of spermatozoa was confirmed in scLH- and scFSH-scLH-administered eels. Although milt production was not induced, higher dosage and frequency of scGTH administration was effective in promoting testicular development of immature eels. Thus, single-chain fish GTHs produced by the baculovirus-silkworm larvae system could be a useful tool for promotion of gonadal maturation in aquaculture fishes.

:  Cultured immature female Japanese eels acclimated to sea water at either 10 or 20°C were treated weekly with salmon gonadotropin fraction (sGTH) in order to investigate the effects of water temperature on artificial induction of ovarian maturation. In eels maintained at 20°C, ovulation was induced in 11 of 18 fish during the experimental period of 13 weeks, whereas at 10°C all fish showed a low gonadosomatic index (GSI) at the end of the experiment. Plasma vitellogenin levels were higher in eels kept at 20°C than in eels at 10°C throughout the experiment. However, no significant differences were observed in the plasma testosterone and estradiol‐17β levels between groups. Eels pretreated with sGTHs at 10°C for 13 weeks were separated to two different temperature (10 and 20°C) groups, and received the same weekly sGTH injections. In eels transferred to 20°C, plasma vitellogenin levels, GSI and oocyte diameter were increased, but these values were maintained at low levels in eels that remained at 10°C. These results clearly indicate that water temperature is an important factor regulating vitellogenesis in the Japanese eel.

The purpose of the trial was to evaluate follicular growth and endometrial folding in mares after human chorionic gonadotropin (hCG) treatment in comparison with untreated mares during oestrus. In addition, the influence of follicle size at the time of hCG treatment was evaluated. HCG (3000 IU) was administered intravenously in 17 mares bearing dominant follicles 35-40 mm in diameter (Group A) and in 13 mares with larger follicles (Group B). Ten mares with follicles larger or equal to 35 mm were untreated (Group C). Ultrasonographical examination of the mares continued in 6 h intervals until ovulation. The growth of the dominant follicle was faster in Group A than in Groups B and C (1.3 vs. 0.3 and 0.7 mm/6h, P less than 0.05) but diameters of the preovulatory follicles were similar - 44, 48 and 44 mm in Groups A, B and C, respectively. Similarly, the reduction of endometrial folding (on a three point scale) during observation was higher in Group A than in B and C (2.1 vs. 1.2 and 1.8, A:B: P less than 0.05) but endometrial folding values in the term before ovulation were not different (0.6, 0.9 and 0.6 in Groups A, B and C). A positive correlation between the speed of follicular growth and reduction of endometrial folding was found (rs - 0.479, P = 0.003). Irregularity in follicle shape (the difference between the longest axis and its perpendicular axis) at the beginning of observation (3.3, 4.0 and 3.2 mm) was lower than before ovulation (7.4, 10.4 and 9.2 mm) in all groups (P less than 0.01). The interval from the beginning of observation until ovulation was significantly shorter in Groups A and B versus C (37 and 31 vs. 103 h, P less than 0.01). The results show that the growth of dominant follicles after hCG is influenced by the size of the follicles at the time of treatment and correlates with the reduction in endometrial folding as well as irregularity of follicle shape. Nevertheless, hCG treatment does not influence the size and shape of preovulatory follicles or endometrial folding immediately before ovulation.

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a λgt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.

:  Manchurian trout Brachymystax lenok is an endangered fish species in East Asia including the Korean peninsula. To establish a method for artificial propagation and to improve our understanding of reproduction in the species, cloning of the pituitary gonadotropin (GTH) subunit cDNAs encoding the Manchurian trout gonadotropin common α (mtGTHα), follicle‐stimulating hormone β (mtFSHβ), and luteinizing hormone β (mtLHβ) by polymerase chain reaction, were attempted. The open reading frames of the mtGTHα, mtFSHβ, and mtLHβ encoded 114, 137, and 142 amino acid residues, respectively. The mature peptides showed strikingly high sequence identities to those of salmonid species GTHα2, FSHβ, and LHβ subunits (92–100%). In an examination of tissue distribution of the mtGTHα, mtFSHβ, and mtLHβ, it was revealed that all of the three subunit genes are specifically expressed in the pituitary by reverse transcription–polymerase chain reaction analysis. Next, single‐chain recombinant FSH and LH constructs using the mtGTHα, mtFSHβ, and mtLHβ cDNAs were produced. In a transient transfection of the FSH and LH constructs into Chinese hamster ovary (CHO‐K1) cells followed by Western blot analysis, the tethered FSH in cell lysates or LH secreted into a medium was detected from 48 to 72 h after transfection. The present study provides a possibility of bioactive GTH production for studies on the reproductive physiology of GTH in the Manchurian trout.

:  Specific antibodies against follicle‐stimulating hormone β subunit (FSHβ), prolactin (PRL), and somatolactin (SL) of the Japanese eel Anguilla japonica were produced. These antibodies, as well as antibodies against luteinizing hormone β subunit (LHβ) and growth hormone (GH) produced previously, were used to examine changes in the production of pituitary hormones in female eels during maturation induced by salmon pituitary homogenate (SPH) injection. Immunohistochemical observations showed a decrease in FSH production after SPH injection, suggesting that SPH inhibits FSH production. In contrast, LH production increased markedly with maturation. The number of GH producing cells decreased gradually during maturation, possibly because of inhibition by exogenous GH present in the SPH and/or endogenous insulin‐like growth factor‐I produced by the stimulation of salmon GH. Although changes in the number of PRL producing cells with maturation were not evident, the number of SL producing cells showed a peak at the late vitellogenic stages, and thereafter decreased to the migratory nucleus stage. These results suggest that GH and SL are involved in sexual maturation in SPH injected eels, as in other fishes.