Testis transplantation in male rainbow trout (Oncorhynchus mykiss)

The objective of the present study was to establish a procedure for the transplantation of an intact testis from one male rainbow trout (Oncorhynchus mykiss) to another individual and evaluate the reproductive function of the transplanted testis at sexual maturity. Isogenic (cloned) male rainbow trout were produced by crossing a completely homozygous male (YY) with a homozygous female (XX) to eliminate any problem of tissue rejection. Transplantation was performed on four pairs of sexually immature animals (n = 8); each served both as a donor and recipient. The left testis was removed by making a ventral midline incision to expose the body cavity and gonads. The left testis was disconnected at the anterior and posterior points of attachment and transferred to the recipient fish where it was placed in position adjacent to the pyloric cecae. The right testis was left intact. After 4 wk, the fish were injected (i.p.) twice weekly for 8 or 9 wk with salmon pituitary extract (1.5 mg/kg) to induce precocious sexual maturation. A similar number of untreated fish were maintained as controls. Following this treatment, all the fish were killed, and the right (intact) and left (transplanted) testes were removed, weighed, and sampled for sperm. Although the mean weights of the left, transplanted testes were significantly (P: < 0.05) smaller than the intact testes (transplants = 1.2 g; intact = 3.9 g), transplanted testes were present in all animals, had increased in mass, and were sexually mature containing sperm. The mean fertility, as measured by the proportion of eggs completing first cleavage, of sperm derived from transplanted testes (92%) was no different from the sperm obtained from intact testes (84%). Similarly, there was no difference in the number of embryos attaining the eyed stage of development, after 18 days of incubation, that were derived from transplanted (84%) or intact testes (85%).     

On remembrance of the abdominal pores in rainbow trout, Salmo gairdneri Richardson, and some other salmonid spp.

Direct connections (abdominal pores) between the peritoneal cavity and the external environment of elasmobranch fishes and some teleost species including the Salmonidae were recognized almost 100 years ago but over this period their existence in these teleost species has been omitted from anatomical texts. In this report, the abdominal pores of rainbow trout, Salmo gairdneri , Atlantic salmon, Salmo salar , and the cisco, Coregonus artedii , were examined in relationship to ip injected particulate material. Both carbon particles and suspensions of the bacterial kidney disease organism were found to be extruded within masses of macrophages and as free particles through the abdominal pores in fish injected ip 72 h previously. The pores were patent in male and female, mature and immature fish. The role and significance of the abdominal pores in rainbow trout in the clearance from the body of material, including material likely to be used for vaccinating fish, is discussed.     

Expression of HSP70 and CYP1A protein in ovary and liver of juvenile rainbow trout exposed to beta-naphthoflavone

Cytochrome P4501A (CYP1A) and the 70-kDa stress protein (HSP70) were determined using Western blotting in the ovary and liver of juvenile female rainbow trout (Oncorhynchus mykiss) exposed for 4 days to beta-naphthoflavone (betaNF) following a single intraperitoneal injection. Ovarian CYP1A protein was observed in both control and betaNF-exposed fish, indicating constitutive and inducible expression of CYP1A in immature trout ovaries. CYP1A protein levels determined using densitometry were 14- and 46-fold greater in betaNF-exposed trout compared to controls in the liver and ovary, respectively. Hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity, a specific catalytic marker of CYP1A, was also induced 38-fold above controls following betaNF exposure. Hepatic HSP70 protein expression was significantly higher in whole cell homogenates, but not in cytosolic fractions, collected from betaNF-exposed fish in comparison to control fish. There was no difference in ovarian HSP70 levels determined in whole cell homogenates between control and betaNF-exposed fish. The observation that unlike liver, ovarian HSP70 expression remained unchanged following induction of CYP1A protein may be related to the sensitivity of the teleost ovary to environmental toxicants that act as aryl hydrocarbon receptor agonists.     

Gonadal regeneration in masculinized female or steroid-treated rainbow trout (Oncorhynchus mykiss)

In salmonids, the development of an indifferent gonad into a testis or an ovary is normally determined chromosomally but can be reversed or changed by the administration of exogenous steroids during specific times in embryonic development. Because the gonads of sexually mature rainbow trout (RBT) are capable of regeneration following surgical removal and since regeneration of some tissue involves dedifferentiation, the objective of this experiment was to determine if the phenotypic sex of RBT gonads could be reversed during the regenerative process. In experiment 1, male RBT were surgically gonadectomized (Gx) or left intact and subsequently treated with estradiol-17beta, a steroid that feminizes male RBT embryos. All Gx males regenerated testicular tissue regardless of treatment. Likewise, the gonads of sham-operated, intact fish treated with exogenous estrogen showed no evidence of sex-reversal. In experiment 2, testes from masculinized females (XX genotype; male phenotype) were surgically removed. In all cases, only testicular tissue was regenerated in the masculinized females. Taken together, these results are consistent with the conclusion that gonads of salmonid fishes are not susceptible to sex-reversing stimuli during the regenerative process and that gonadal regeneration in salmonids is a result of cellular proliferation of the remaining gonadal remnant.     

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm,Bombyx mori

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.     

Fine structure of the corpus luteum of the autografted ovary in the rat

Summary The rat ovary has been transplanted successfully to subcutaneous tissue areas by several investigators. Light microscopy has revealed that corpora lutea in ovarian autografts are formed by luteinization of intact follicles and contain entrapped ova. In the present study, corpora lutea from autografted ovaries in castrate rats were obtained at metestrus and examined electron microscopically to determine whether their cellular morphology correlated with the normal progesterone levels in these animals. Cellular features usually accepted as regressive were apparent. The findings suggest either structural luteolysis is occurring before functional luteolysis or that the adrenal has increased steroidogenic activity in the castrate with ovarian autografts to account for the normal progesterone levels.Supported in part by USPHS Grant T01-DE00241-04The authors wish to thank J. Canale and Y. Tablada for technical, G. Kerimian for photographic, and M.A. Anderson for secretarial assistance     

Fish gonadotropin(s). III. Evidence for more than one gonadotropin in chum salmon pituitary glands.

Two proteins with gonadotropin activity have been isolated from a highly purified chum salmon (Oncorhynchus keta) gonadotropin preparation (G-75 Fraction II) by chromatography on DEAE Bio Gel A. These gonadotropins exhibited distinct behaviour in polyacrylamide gel electrophoresis, chromatography on Sephadex G-75 superfine, and ratios of cAMP stimulation in immature rainbow trout ovaries and testes. Rechromatography of G-75 Fraction II on Sephadex G-75 superfine gave a symmetrical protein peak with a coincident cAMP activity profile. Repeated freezing and thawing elicited a shift in the cAMP activity profile toward the trailing edge of the protein peak. Data are discussed in terms of two gonadotropin molecules which respond differently to phase changes. Charge polymorphism was exhibited by isoelectric focusing in polyacrylamide gels of one of the DEAE fractions. Five UV absorbing bands were observed which stimulated cAMP production in immature rainbow trout gonads. Three of these bands increased adenyl cyclase activity in trout ovaries and testes. One of the bands stimulated cAMP production primarily in trout testes and the other stimulated trout ovaries, providing evidence for two gonadotropins, each of which is sex specific.     

Distribution of cortisol among its free and protein-bound fractions in rainbow trout (Oncorhynchus mykiss): evidence of control by sexual maturation.

1. Total cortisol concentrations did not differ in sexually matured female, male, and immature rainbow trout. 2. The per cent cortisol bound to a corticosteroid binding protein was greater in mature female fish (48.2%) than in mature male (16.0%) and immature fish (19.5%). 3. The mature female fish exhibited a lower percentage of free cortisol (21.8%) compared to mature males (44.8%) and immature fish (43.2%). 4. Parallel aspects of the teleostean cortisol binding-protein and the mammalian counterpart are compared and commented upon.     

The effect of LH on cyclic AMP and progesterone in rat ovaries in vivo.

LH injected into the abdominal vein of immature female rats increased ovarian cyclec AMP levels within 1 min, and the effect lasted at least 2 hr. In PMS stimulated rats, a maximal effect on cyclic AMP levels was obtained with 5 mug of LH 10 min after injection. Significant effects were obtained with as little as 0.1 mug. The minimum effective dose required to increase ovary cyclic AMP levels in proestrus rats was 0.05 mug of LH. While 0.01 mug did not affect cyclic AMP levels, an increase in ovary progesterone was found. In other experiments, the response of cyclis AMP to LH in vivo was greater in immature ovaries than in those from animals pretreated with PMS and hCG. Small increases in cyclic AMP levels were also seen with FSH in PMS and PMS + hCG treated animals and with prostaglandin E2 and prolactin in PMS treated rats. These results parallel, to some extent, previous results found in vitro, and show that very small doses of LH can stimulate ovary cyclic AMP increases in vivo.     

Influence of testosterone and/or luteinizing hormone releasing hormone analogue on precocious sexual development in the juvenile rainbow trout

The influence of testosterone, luteinizing hormone releasing hormone (LHRH) agonist and combinations of these hormones on gonadotropic hormone (GtH) levels in the sexually immature trout was investigated. Both the steroid and releasing hormone preparations, testosterone in Silastic capsules and cholesterol-pelleted LHRH-A, were formulated for sustained release and long-term biological action following a single hormone implantation. Marked increases in pituitary GtH followed testosterone and/or testosterone and LHRH analogue treatment combined, but the low pituitary GtH level in controls remained unchanged after LHRH analogue administration alone. Plasma GtH titers increased with time after testosterone treatment, indicating a positive steroid feedback effect by androgen on GtH in the juvenile rainbow trout. When combined with testosterone treatment, LHRH analogue augmented plasma GtH levels compared to fish receiving testosterone treatment alone. In males the elevated plasma GtH levels were associated with testes stimulation and onset of spermatogenesis; in females, however, no significant stimulation of the ovaries was observed. It can be concluded from these studies that the testosterone stimulus is sufficient to induce onset of sexual development in immature males but not females. Whereas LHRH analogue releases GtH from the testosterone-primed trout pituitary, LHRH treatment alone under these conditions fails to stimulate the juvenile trout reproductive system.     

Detection of VHSV IVb within the gonads of Great Lakes fish using in situ hybridization

Viral haemorrhagic septicaemia virus (VHSV) genotype IVb was recently detected as the cause of numerous mortality events in Great Lakes fish. In situ hybridization was used to examine the gonads from 13 fish, including freshwater drum Aplodinotus grunniens and muskellunge Esox masquinongy that were infected naturally, as well as rainbow trout Oncorhynchus mykiss and fathead minnows Pimphales promelas, which were experimentally infected. Although the ovaries and testes of fish infected by VHSV IVb had few lesions, viral RNA was present in the ovaries of the rainbow trout and fathead minnow and was abundant in the gonads of muskellunge and in the ovaries of freshwater drum. Viral RNA was present mainly surrounding yolk vacuoles/granules or adjacent to the germinal vesicle, with lesser amounts found within the germinal vesicle, in the mesovarium and/or tunica albuginea and blood vessels of the ovary. Viral RNA was also found in and surrounding primary and secondary spermatocytes of the muskellunge.     

虹鳟spindlin基因克隆及不同倍性的表达分析

spindlin基因是减数分裂纺锤体相关因子,为了研究spindlin基因在二倍体和三倍体雌性虹鳟减数分裂过程中出现的差异,通过cDNA末端快速扩增(RACE)技术获得spindlin基因cDNA 4529 bp(GenBank登录号:MN378564),其中3′非编码区(UTR)和5′非编码区(UTR)分别长3662 bp和141 bp,开放阅读框(ORF)长726 bp,编码241个氨基酸,该蛋白质序列的相对分子量为28.3 kD,理论等电点值为5.94,无跨膜结构。同源性分析表明,虹鳟(Oncorhynchus mykiss)与银大马哈鱼(Oncorhynchus kisutch)同源最高,高达99.59%。系统发育进化树显示,虹鳟与大鳞大马哈鱼(Oncorhynchus tshawytscha)和红点鲑(Salvelinus alpinus),聚为一支。实时荧光定量(RT-PCR)结果显示, spindlin基因在二倍体雌性虹鳟卵巢、肾、肝、脾、肌、鳃、心、眼、肠和鳍组织中均有表达,其中,在卵巢中的表达量极显著高于其他组织(P<0.01)。对于二倍体雌性虹鳟,在受精后24...     

Vascularization of ovaries from golden hamsters following implantation into the chick chorioallantoic membrane

In order to study the angiogenic activity, entire ovaries of immature or adult golden hamsters in different functional stages were implanted into the chick chorioallantoic membrane (CAM). Ovaries or other organs (heart, lung, liver, spleen, kidney, adrenal gland, testis) were applied during days 9-14 of incubation. Histological serial sections and ultrastructural studies displayed vascularization from the host to the ovary. Vascularization was marked for unstimulated immature ovaries compared to superstimulated immature ovaries or adult ovaries obtained during the period of proestrus-estrus. When no vascularization developed, grafted ovaries of immature or adult animals induced a mild erosive reaction of the CAM. In contrast, few other implanted organs obtained from immature animals caused a response with vascularization which was absent in grafted organs from adult hamsters. The erosive reaction of the CAM was rare, but the ulcerative response accompanied by infiltrating eosinophils was frequently seen both in immature and adult animals. These results may indicate that angiogenetic activity of the ovary is not the sole cause for vascularization of grafts. Other factors such as tissue death or a low histocompatibility barrier are likely to be involved.     

Effects of acute 17alpha-methyltestosterone,acute 17beta-estradiol,and chronic 17alpha-methyltestosterone on dopamine,norepinephrine and serotonin levels in the pituitary,hypothalamus and telencephalon of rainbow trout (Oncorhynchus mykiss)

This study investigated: (a) the effects of acute 17alpha-methyltestosterone (MT) or 17beta-estradiol (E(2)) administration on norepinephrine (NE), dopamine (DA), serotonin (5-HT), 3,4, dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) contents in the hypothalamus, telencephalon and pituitary of previtellogenic female rainbow trout Oncorhynchus mykiss, and (b) the effects of chronic MT administration on the levels of these neurotransmitters in these brain regions in immature male rainbow trout. The acute administration of MT induced a significant decrease in pituitary levels of DOPAC as well as in the DOPAC/DA ratio. On the other hand, the acute administration of E(2) induced an increase in pituitary 5-HT levels as well as a decrease in the 5-HIAA/5-HT ratio. In a second experiment, 20 mg MT per kilogram body weight was implanted for 10, 20 or 40 days into sexually immature male rainbow trout. Implanted rainbow trout showed increased testosterone and decreased E(2) levels. In the pituitary, MT induced long-term decreases in NE, DA, DOPAC and 5-HT levels, as well as in the DOPAC/DA ratio. Hypothalamic and telencephalic DA, NE and 5-HT levels were not affected by MT implantation. However, 5-HIAA levels and the 5-HIAA/5-HT ratio were reduced by MT implantation in both brain regions. These results show that chronic treatment with MT exerts both long-term and region-specific effects on NE, DA, and 5-HT contents and metabolism, and thus that this androgen could inhibit pituitary catecholamine and 5-HT synthesis. A possible role for testosterone in the control of pituitary dopaminergic activity and gonadotropin II release is also discussed.     

Expression of bradykinin B2 receptor in the mouse ovary

The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mare's serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B(2) receptor mRNA was constitutively expressed. Bradykinin B(2) receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B(2) receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B(2) receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B(2) receptor mRNA level. The present data indicate that the level of B(2) receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.     

Some blood parameters of the rainbow trout (Salmo gairdneri Richardson)

Some of the blood constituents of male, female and immature rainbow trout of the Shasta variety, maintained in known environmental and dietetic conditions, were examined and the results statistically treated. The parameters for male and female trout were similar, only the erythrocyte count exhibiting a significant difference. Large but expected differences were evident between the parameters for mature and immature fish.     

Response of serum carotenoid levels to dietary astaxanthin and canthaxanthin in immature rainbow trout Oncorhynchus mykiss

Rainbow trout were fed a diet supplemented with astaxanthin (89 mg/kg) or canthaxanthin (116 mg/kg) in two different experiments: experiment 1 was designed to measure the kinetics of the appearance and disappearance of carotenoids in the serum; experiment 2 was undertaken to establish the serum dose-response to synthetic astaxanthin and canthaxanthin for immature rainbow trout. The serum carotenoid concentrations of immature rainbow trout increased when fish were fed carotenoid supplemented feed and then reached a plateau after 1 day of intake for astaxanthin and after 2 days for canthaxanthin. Circulating astaxanthin represented a value 2.3 times that of canthaxanthin. After dietary supplementation was discontinued, the serum carotenoid concentrations decreased within 3 days for both carotenoids. The average decreasing slopes for the two carotenoid pigments were parallel, indicating a similarity in the rate of which astaxanthin and canthaxanthin are utilized by rainbow trout. The serum dose-response of trout that received dietary keto-carotenoids increased with increasing pigment levels. The hypothesis that absorption of dietary carotenoids in 12.5–200 mg/kg range of concentration across the gut wall may be by passive diffusion is proposed.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,4,7,8-pentachlorodibenzofuran reduces growth and disrupts reproductive parameters in female rats

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) are widespread environmental pollutants. TCDD is well known for its adverse effects on female reproduction when administered acutely to immature or adult rats. It is also known that fetal/neonatal exposure to this compound alters reproductive parameters. It is unknown whether exposure to PCDF causes similar adverse effects in offspring. The objectives of the study were to investigate the effects of in utero and lactational (IUL) exposure to TCDD and PCDF on subsequent growth, estrous cycles, and ovulation. Additionally a gonadotropin-primed immature rat model was used to investigate possible direct effects on the ovary after IUL exposure to TCDD (2.5 microg/kg) by evaluating 1) ovarian morphometrics and 2) serum estradiol concentrations. Body weights were reduced in animals with IUL exposure to TCDD and PCDF relative to those in controls at 10 days of age (P < 0.05 for each), and this difference was maintained until termination of the experiment at 125-165 days of age (P < 0.05). Exposure to TCDD or PCDF also disrupted regular estrous cycles and inhibited ovulation rate. On Day 23 (before eCG stimulation), ovaries from animals exposed to TCDD contained the same number of primordial, primary, secondary, preantral, and antral follicles as ovaries from control animals. On Day 25 (48 h after eCG stimulation), ovaries from TCDD-exposed rats had significantly fewer large preovulatory follicles when compared with ovaries from controls. The numbers of smaller follicles (both antral and small antral) were not different. Serum estradiol was significantly lower in TCDD-exposed animals 48 h after eCG stimulation.