Observations on the spawning behavior of artificially matured Japanese eels Anguilla japonica in captivity

Spawning behavior of artificially matured Japanese eels Anguillo japonica in captivity was investigated using a DVD Video image system. Following a routine hormone treatment technique for this fish, female eels were artificially matured by weekly intramuscular injections of salmon pituitary extracts (SPE) at a dosage of 40 mg kg(-1) BW for a total of 7-11 doses to induce ovarian maturation, while male eels received weekly intramuscular injections of human chorionic gonadotropin (HCG) at a dosage of 1000 IU kg(-1) BW for a total of 6-11 doses at 18 degrees C to induce testicular maturation in a separate aquarium. In this experiment, three pairs of such hormone-treated matured eels were acclimatized in seawater in 1.5 m(3) experimental aquaria with or without shelters at 20 degrees C for 24 h. Twenty four hours after the acclimatization terminated, the females received SPE injections to boost maturation and ovulation. Twenty four hours following these injections, the females received injections of HCG (1000 IU per fish, HCG injection) and 17 alpha-hydroxyprogesterone (2 mg per fish) to induce ovulation, while males were given HCG injections (1000 IU per fish, HCG injection) to induce spermiation. Video taping started after the 24 h acclimatization terminated and last for a total of 96 h. Before the HCG injections, both sexes were inactive, staying on the bottom or in shelters if available. Following these HCG injections, they became active and frequently left the bottom swimming in the water column. During the 24 h following HCG injections, activity accounted for 67% and 45% of the total activity in no shelter treatment for females and males, respectively, in comparison with 77% and 78% in shelter treatment. Activity was significantly more pronounced during this phase than during other phases for each sex in either shelter treatment. Egg release and sperm ejection occurred in the water column around the time eels' activity reached peaks. Eels either returned into the shelters or stayed motionlessly on the bottom of the aquaria after egg release and sperm ejection. Eight out of nine (89%) females in no shelter treatment spontaneously released eggs with a total of 11 batches 14-18 h following HCG injections, in contrast with four out of nine (44%) females releasing eggs for 4 batches 16-20 h in shelter treatment. Males arrived at activity peaks 11-13 h following HCG injections in no shelter treatment, 2-4 h ahead of the females (14-16 h), in comparison with 8-11 h in shelter treatment with 5-6 h ahead of the females (14-17 h). Courtship behavior indicative of spawning such as pairing, chasing and touching bodies was not observed in the eels in this study. However, on many occasions, eels of both sexes (male-female or female-female) were found to "cruise together" in water column for a short time period or frequently come together prior to releasing eggs and ejecting sperm, suggesting the possibility of group mating in artificially matured Japanese eels. (c) 2007 Elsevier B.V. All rights reserved.

Dynamic changes in ovarian follicles measured by ultrasonography during gonadotropin stimulation in rhesus monkeys

The objective was to study dynamic changes of ovaries in rhesus macaques stimulated by gonadotropins to identify an indicator for predicting ovarian response to stimulation. Twenty-one cycling monkeys were given 36 IU/d recombinant human follicle-stimulat

Ovarian response to gonadotropin stimulation in juvenile rhesus monkeys

The objective of this study was to investigate juvenile rhesus monkeys responding to various gonadotropin regimen Stimulations. Thirty-two prepubertal rhesus monkeys were randomly allocated into five groups for ovarian stimulation as follows: Groups I, II

Changes of urinary steroid conjugates and gonadotropin excretion in the menstrual cycle and pregnancy in the Yunnan snub-nosed monkey (Rhinopithecus bieti)

The Yunnan snub-nosed monkey (Rhinopithecus bieti) is one of the most endangered species in the world, and it is endemic to China. According to our knowledge, there was no information on reproduction for this species. The present study was designed to und

Identification and characterization of a novel splice variant of gonadotropin alpha subunit in the common carp Cyprinus carpio

In this study, an alternative splicing transcript GtH-alpha 291 was identified by RT-PCR, which is 291 nt and exists not only in the pituitary but also in the ovary in common carp Cyprinus carpio. The analysis of GtH-alpha 291 amino acid sequence by the SignalP server predicted that the 'missing segment' might characterize as a signal peptide. In the secretion experiment, GtH-alpha 357 subunit could be secreted out of HeLa cells while GtH-alpha 291 could not, which confirmed the prediction. Co-immunoprecipitation assay proved that GtH-alpha 291 subunit is able to interact with both FSH-beta and LH-beta as GtH-alpha 357 does. This is the first report concerning an alternative splicing transcript of a GtH alpha subunit. Further studies are necessary to elucidate the specific role of this variant in the regulation of gonadal development and sexual maturation. (c) 2007 The Authors.

Antisense for gonadotropin-releasing hormone reduces gonadotropin synthesis and gonadal development in transgenic common carp (Cyprinus carpio)

Generating transgenic fish with desirable traits (e.g., rapid growth, larger size, etc.) for commercial use has been hampered by concerns for biosafety and competition if these fish are released into the environment. These obstacles may be overcome by producing transgenic fish that are sterile, possibly by inhibiting hormones related to reproduction. In vertebrates, synthesis and release of gonadotropin (GtH) and other reproductive hormones is mediated by gonadotropin-releasing hormone (GnRH). Recently two cDNA sequences encoding salmon-type GnRH (sGnRH) decapeptides were cloned from common carp (Cyprinus carpio). This study analyzed the expression of these two genes using real-time polymerase chain reaction (RT-PCR) in different tissues carp at varying developmental stages. Transcripts of both genes were detected in ovary and testis in mature and regressed, but not in juvenile carp. To evaluate the effects of sGnRH inhibition, the recombinant gene CAsGnRHpc-antisense, expressing antisense sGnRH RNA driven by a carp beta-actin promoter, was constructed. Blocking sGnRH expression using antisense sGnRH significantly decreased GtH in the blood of male transgenic carp. Furthermore, some antisense transgenic fish had no gonadal development and were completely sterile. These data demonstrate that sGnRH is important for GtH synthesis and development of reproductive organs in carp. Also, the antisense sGnRH strategy may prove effective in generating sterile transgenic fish, eliminating environmental concerns these fish may raise. (c) 2007 Published by Elsevier B.V.

Molecular and expression characterization of three gonadotropin subunits common alpha, FSH beta and LH beta in groupers

A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSH beta and LH beta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSH beta and LH beta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSH beta and LH beta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSH beta and LH beta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSH beta and LH beta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSH beta cells mainly distributed in the middle area of PPD, while the LH beta cells distributed more widely, including in the area similar to the FSH beta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSH beta and LH beta cells. Double immunofluoresence localization further demonstrated FSH beta and LH beta expression in distinct cells in the PPD area, although the FSH beta and LH beta cells were detected in the identical area of PPD. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Cryopreservation of rhesus macaque (Macaca mulatta) spermatozoa and their functional assessment by in vitro fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris-egg yolk freezing media. TEST and TTE. which have: been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa From four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization or oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes. 82 +/- 13% were fertilized and 63 +/- 22 and 39 +/- 21% of the resulting zygotes developed into morulae and blastocysts. respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species. (C) 2001 Academic Press.

THE POSSIBLE INVOLVEMENT OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR IN LUTEOLYSIS OF RHESUS-MONKEY

Changes of plasminogen activators (PA) during different stages of development of the corpus luteum, and their possible physiological role in luteolysis were studied in rhesus monkeys. It was demonstrated for the first time that monkey corpus luteal cells not only produce PA, but that the function of the corpus luteum is also closely related to the activity of this enzyme system. Generally, the life span for a corpus luteum in monkey is approximately 14-16 days, its demise beginning thereafter. In the present study, we found that urokinase in the corpus luteum is higher on day 5 and day 10 after human chorionic gonadotrophin injection, while the tissue type (t) PA is mainly produced on day 13 when luteolysis may take place. Progesterone production remained high on day 5 and day 10 and decreased dramatically from day 13, indicating the important role of tPA but not urokinase (u) PA in suppressing luteal function. When purified tPA (but not uPA) monoclonal antibody was added to luteal cell culture to neutralize endogenously produced tPA activity, progesterone production in the cells was increased significantly. Interestingly, prolactin alone was capable of increasing PA production by luteal cells; prolactin together with luteinizing hormone, however, had a synergistic luteotrophic effect.

Ovulation in the tree shrew (Tupaia belangeri) induced by gonadotrophins

The induction of ovulation by exogenous gonadotrophins is an important approach for recovering oocytes used for studies on the reproductive biology of some mammals. In the present study, pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadot

黄鳝cGnRH-Ⅱ的cDNA克隆与序列分析

促性腺激素释放激素(Gonadotropin-releasing hormone,GnRH)是一个保守的十肽神经家族激素,在脊椎动物的性腺发育和繁殖功能的维持方面起着重要的调控作用。本文通过运用RACE和RT-PCR方法,从黄鳝脑组织中克隆得到cGnRH-ⅡcDNA全序列,其核苷酸序列长度为617bp。该cDNA编码的cGnRH-Ⅱ的前体氨基酸序列结构组成与其他物种的cGnRH-Ⅱ前体结构一致,其推导的蛋白前体长度为83个氨基酸,包括一个信号肽、cGnRH-Ⅱ十肽和一个由蛋白水解位点(Gly-Lys-Ar

Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSH beta (AsFSH beta) and LH beta (AsLH beta) subunit cDNAs are 345, 363, 387 and 414 bp in length, and encode mature peptides of 115, 121, 129 and 138 aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSH beta and AsLH beta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSH beta and AsLH beta) in mature sturgeon pituitaries, but only AsFSH beta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSH beta was expressed in 4-year-old individuals, whereas in females, AsFSH beta was just expressed in 5-year-old individuals. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

A perspective on fish gonad manipulation for biotechnical applications

The gonad is an essential organ for generating sperm and ova in vertebrates. This review describes several pilot studies on gonad gene manipulation and development in fish. With antisense RNA techniques, we suppressed the gonad development, and thus the fertility, of an antisense gonadotropin-releasing hormone (sGnRH) transgenic common carp. Then, using a tissue-specific exogenous gene excision strategy with sexual compensation, we knocked out the gonad-specific transgene. Under the control of the rainbow trout protamine promoter, the transgenic fish expressed the reporter gene eGFP specifically in the spermary. These results indicate that the fish gonad is a new model organ that can improve contemporary biotechnology experiments. Herein we discuss the potential of fish gonad manipulation for resolving important biosafety problems regarding transgenic fish generation and producing the new transgenic animal bioreactor.

Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.