The ontogeny of nuclear estrogen receptor isoform expression and the effect of 17beta-estradiol in embryonic rainbow trout (Oncorhynchus mykiss).

Ligand bound nuclear estrogen receptor (ER) acts as a transcription factor regulating the expression of estrogen dependent genes. There are four nuclear ER isoforms in rainbow trout (Oncorhynchus mykiss). The objective of this study was to measure whole body mRNA levels of the two ERalpha isoforms (alpha1/alpha2) and the two ERbeta isoforms (beta1/beta2) in male and female embryos from 50 to 600 degree-days (DD; days post-fertilizationxwater temperature) and in embryos exposed to vehicle or 17beta-estradiol (E2) for 2h at 230, 240 and 250 DD. All four isoforms were detected at every time point in both sexes. Sexual dimorphism was rarely observed; at 50 DD the level of ERalpha2 mRNA was significantly greater in males than in females and at 100 DD the level of ERbeta1 mRNA was significantly greater in females than in males (p<0.05). Expression profiles of the two ERalpha isoforms were slightly different from one another, whereas the ERbeta isoforms exhibited similar expression patterns. The effect of E2 was not different between male and female embryos. The level of ERalpha1 mRNA increased significantly at 240 DD; a similar but not statistically significant trend was observed at 230 and 250 DD. Despite the critical role of estrogen during sex differentiation in rainbow trout, the receptivity to this hormone as measured by the response in mRNA levels of ER appears to be largely the same between males and females and ERalpha1 is the only E2 responsive isoform.

Regulation of hepatic estrogen receptor isoform mRNA expression in rainbow trout (Oncorhynchus mykiss).

The complete nuclear estrogen receptor family in rainbow trout consists of two subtypes (ERalpha and ERbeta) each of which consists of two isoforms (alpha1/alpha2 and beta1/beta2). Transcription rate and mRNA stability of ERalpha1 is affected by 17beta-estradiol (E2) but no information on estrogen regulation exists for the other isoforms. The objective of this study was to compare the mRNA expression patterns of the four ER isoforms in the liver of male trout and in immortalized trout hepatocyte lines (RTH-149 and SOB-15) treated with E2 or 17alpha-ethynylestradiol (EE2) using quantitative RT-PCR. To determine the in vivo dose-response, isogenic male trout were injected intra-peritoneally with 0, 1.5, 15 or 150 microg E2 or an equimolar amount of EE2 and the liver sampled 24 h later. Treatment with either E2 or EE2 significantly (p<0.05) increased the level of ERalpha1 mRNA at all doses tested compared to vehicle, while the response of mRNAs for the other three isoforms did not change. The in vitro dose-response was tested by treating both cell lines with 0, 0.1, 1.0 or 10.0 microM E2 for 48 h. In RTH-149 cells, ERalpha1, ERalpha2 and ERbeta2 mRNAs were significantly higher in cells incubated with 10 microM E2 as compared to cells treated with only vehicle (p<0.05). In SOB-15 cells, ERalpha2 and ERbeta1 mRNAs were significantly higher in cells incubated with 1.0 microM E2 as compared to cells incubated with only vehicle (p<0.05). These results support the conclusion that the mRNAs for the four ER isoforms respond differentially to estrogen regulation.

Aneuploid sperm formation in rainbow trout exposed to the environmental estrogen 17{alpha}-ethynylestradiol.

Environmental contaminants that mimic native estrogens (i.e., environmental estrogens) are known to significantly impact a wide range of vertebrate species and have been implicated as a source for increasing human male reproductive deficiencies and diseases. Despite the widespread occurrence of environmental estrogens and recognized detrimental effects on male vertebrate reproduction, no specific mechanism has been determined indicating how reduced fertility and/or fecundity is achieved. Previous studies show that male rainbow trout, Oncorhynchus mykiss, exposed to the environmental estrogen 17alpha-ethynylestradiol (EE2) before gamete formation and fertilization produce progeny with significantly reduced embryonic survival. To determine whether this observed decrease results from sperm chromosome alterations during spermatogenesis, male rainbow trout were exposed to 10 ng of EE2/l for 50 days. After exposure, semen was collected and sperm aneuploidy levels analyzed with two chromosome markers by fluorescent in situ hybridization. In vitro fertilizations were also conducted by using control and exposed sperm crossed to eggs from an unexposed female for offspring analysis. Evaluations for nucleolar organizer region number and karyotype were performed on developing embryos to determine whether sperm aneuploidy translated into embryonic aneuploidy. Results conclusively show increased aneuploid sperm formation due to EE2 exposure. Additionally, embryonic cells from propagated progeny of individuals possessing elevated sperm aneuploidy display high levels of embryonic aneuploidy. This study concludes that EE2 exposure in sexually developing male rainbow trout increases levels of aneuploid sperm, providing a mechanism for decreased embryonic survival and ultimately diminished reproductive success in EE2 exposed males.

Initiation of motility by steelhead (Oncorhynchus mykiss) sperm: membrane ion exchangers and pH sensitivity.

Initiation of motility in salmonid sperm is sensitive to the pH of the extracellular medium, however, the basis of this sensitivity is not clear. Sperm incubated in an immobilization buffer (SI) at low pH ( approximately 7.1-7.2) become motile when diluted with activating medium (AM) at high ( approximately 8.5) but not low pH. Based on this observation, various agents were tested to determine whether the onset of steelhead sperm motility upon activation with high pH AM, following incubation with low pH SI, could be blocked by inhibiting membrane exchangers postulated to be important in intracellular pH (pHi) regulation. Amiloride (inhibitor of proton:sodium exchange), SITS and DIDS (inhibitors of anion exchange) and bafilomycin A 1 (inhibitor of H(+)-ATPase activity) were not effective in this experimental design. However, regardless of SI pH, DIDS was effective in blocking motility as was replacing chloride with thiocyanate or including the chloride channel blocker, niflumic acid, in SI suggesting that chloride efflux plays a key role in motility initiation. Nonetheless, the results of this study suggest that the rapid onset of sperm motility with activation at high pH following incubation at low pH is probably not based on rapid adjustment of pHi via membrane exchangers/transporters but rather due to an effect of pH on motility-associated processes at the extracellular surface of the sperm.

Oxygen consumption in unfertilized salmonid eggs: an indicator of egg quality?

A negative correlation between oxygen consumption and fertility was observed in both steelhead and chinook salmon eggs. However, this relationship was attributed to bacterial growth. Elimination of samples with bacterial growth resulted in no significant relationship between the rate of oxygen consumption (VO2) and fertility. VO2 of unfertilized eggs of both steelhead and chinook salmon was measured over a storage period of up to 24 days (d). Despite declines in fertility during storage, VO2 did not significantly change throughout storage. The average respiration rate for steelhead eggs was 3.4 nmol O2 per egg per h, and was 4.3 nmol O2 per egg per h for chinook salmon eggs. Treatment of chinook salmon eggs with uncouplers of mitochondrial respiration, 2,4-dinitrophenol (2,4-DNP) and carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone (FCCP), resulted in an increase in VO2 to 12.9 and 11.5 nmol O2 per egg per h, respectively. Treatment with the putative uncoupler, clove oil, resulted in no change in VO2, while KCN, an inhibitor of oxidative phosphorylation, reduced oxygen consumption to zero. Copper caused an increase in oxygen consumption, even in the absence of eggs, suggesting a need for caution in interpreting changes in respiration rates as a result of metal exposure. Thus, unfertilized salmonid eggs demonstrated submaximal VO2, which was not correlated with fertility.

Adenosine triphosphate levels in steelhead (Oncorhynchus mykiss) eggs: an examination of turnover, localization and role.

The dynamics of energy production and utilization in fish eggs before and shortly after fertilization may be critical for embryo survival. Therefore, the current study examined the turnover of adenosine triphosphate (ATP) as well as examined the possible role and localization of ATP in unfertilized steelhead (Oncorhynchus mykiss) eggs and early embryos. The mean ATP level in unfertilized steelhead eggs was 1.92+/-0.10 (mean+/-S.E.M., n=17) nmol ATP per egg. Exposure of the unfertilized egg to 10 degrees C water (water activation) and fertilization resulted in comparable and substantial decreases (approx. 20-50%) in egg ATP levels within 3 min. This suggests that the energy expended at fertilization is used in response to water activation rather than fertilization per se. Unfertilized eggs maintained in ovarian fluid for 9 days at 10 degrees C under air showed a progressive decline of fertility that reached zero after 6 days. In contrast, no significant changes were seen in ATP levels throughout this 9 days period. Thus, fertility does not positively correlate with egg ATP levels in stored eggs. In the unfertilized egg, the ATP stored in the yolk accounted for approximately 1.5% of the total egg ATP. After fertilization, the concentration of ATP in the yolk increased approximately seven-fold, with the yolk and blastoderm each now accounting for approximately 20% of the total remaining ATP. Finally, to estimate the changes in oxidative metabolism following fertilization, the cyanide (KCN)-sensitive decline in total ATP was determined for unfertilized eggs and 1 day embryos. In the presence of KCN, ATP levels declined to approximately 50% within 24 h in both unfertilized eggs as well as embryos; the rates of ATP decline were not different. Therefore, there was not a discernible increase in ATP generation by oxidative phosphorylation at the time of fertilization.

Surgical transplantation of sexually immature ovaries in rainbow trout (Oncorhynchus mykiss).

Transplantation of testes between isogenic rainbow trout males has been recently demonstrated. The objective of the present investigation was to determine if ovaries detached from the body wall and removed from the abdominal cavity would reestablish themselves when autografted to an ectopic site. In the first experiment, eleven sexually immature, female rainbow trout were laparotomized midventrally, and the right ovary was removed and transplanted to the abdominal cavity and positioned along the pyloric cecae on the right side. In the second experiment, the ovary was autografted in four animals as in experiment 1 or was transferred to and allografted in four other sexually immature female trout. The animals were examined three months following surgery. At the termination of experiment 1, the autografted ovaries were present in 73% of the animals; the transplanted ovaries were smaller in size than the intact control ovaries. Histological examination did not reveal any necrotic tissue in these transplanted gonads, and oocyte development was not different between the transplanted and the intact ovary within animal. Transplanted ovaries allografted from another female were not found. Taken together, these data support the conclusion that rainbow trout ovaries detached from the body wall can reestablish their blood supply and maintain ovarian development and that female trout appear to reject gonadal tissue from other individuals of their species.

Carbon dioxide and pH affect sperm motility of white sturgeon (Acipenser transmontanus).

Maintenance of sperm at pH values less than approximately 7.5 inhibited the onset of motility when sperm were subsequently diluted with water; maintenance at pH values above approximately 8.2 was associated with maximal motility upon dilution with water. Within 5 approximately min of exposure to low pH buffer (pH 6.9), there was a 50% decline in sperm motility upon dilution with water suggesting that exposure to low pH interferes with motility within a time frame that may affect fertilization. In most instances, maintenance of sperm under CO(2) at a pressure of 4-5 kPa almost completely blocked their capacity for motility. Furthermore, exposing semen to increasing partial pressures of CO(2) up to about 1 kPa resulted in a marked decrease in semen pH. These observations are consistent with the findings that the buffering capacity of semen is particularly low at physiological pH, and that this low buffering capacity corresponds to the highest pH sensitivity of the capacity for sperm motility. The low seminal buffering capacity may represent a physiological adaptation in the control of sperm function. It may also represent a vulnerability to environmental hypercapnia or metabolic acidosis.

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.

Does CO2 enhance short-term storage success of Chinook salmon (Oncorhynchus tshawytscha) milt?

Successful short-term storage of salmonid milt depends on numerous factors, including temperature, fluid volume, and gaseous environment, with storage at low temperatures under an atmosphere of 100% O2 being the most common method. Salmonid sperm maintained in a storage environment with elevated carbon dioxide (CO2) levels, such as the approximately 4% CO2 in exhaled air, are not motile when activated. While these modest levels of CO2 inhibit sperm motility, the effect is reversible within hours after exposure to a CO2-free oxygenated environment. Therefore, the effect of CO2 (as a component gas in the storage environment) on chinook salmon (Oncorhynchus tshawytscha) sperm motility and viability was examined. The hypothesis of the current investigation was that CO2-exposure with subsequent CO2 removal would be beneficial during short-term chinook salmon milt storage. Milt samples were collected from mature (adult) and precocious (jack) male chinook salmon and stored under various CO2 and O2 levels at 3 to 4 degrees C for up to 14 days. Milt samples were then removed from the incubation environments and maintained under CO2-free humidified air with continuous mixing for 4 h at 10 degrees C before analysis of motility. The resultant motility of samples incubated under 3.5% or less CO2 was not different than controls during the 14 d incubation period; motility of samples stored under higher CO2 tensions were significantly lower. The motility of samples incubated under 3.5% CO2 reached the maximum recovered motility after 2 h exposure to CO2-free humidified air, while the motility of sperm incubated under 13.4% CO2 levels recovered no motility even after 6 h exposure to CO2-free humidified air. The motility of samples incubated under normoxia was significantly greater than that of samples incubated under hyperoxia (approximately 90% O2) at both 7 and 14 d, regardless of the CO2 level. Sperm viability was relatively unaltered by any of the incubation conditions examined. The results of this investigation suggest that there is no apparent advantage to storage of chinook salmon sperm in the presence of CO2 and that storage under hyperoxia negatively affects sperm function compared to storage under normoxia.

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%).

Gonadal morphology of female diploid gynogenetic and triploid rainbow trout.

Chromosome sets of fishes can be manipulated; this practice includes the production of triploid and gynogenetic salmonids. Such chromosomal modifications often result in abnormal ovarian development. In rainbow trout (RBT), triploid females have string-like gonads lacking significant developing oocytes and are suggested to be sterile due to the odd set of chromosomes disrupting oogenesis. Aberrant ovarian development is reported to occur in about 30% of gynogenetic females. It has been suggested that gynogenetic fish are more prone to expressing developmental abnormalities due to either increased homozygosity or to incomplete inactivation of the paternal chromatin. This investigation was done to compare the ovarian morphology of female triploid and induced gynogenetic diploid RBT. The objective was to determine whether the presence of supernumerary chromosomal fragments, potentially generated during the process of sperm genome inactivation, would result in abnormal gonadal development in gynogens comparable to that observed in triploid females. Gonadal morphology was observed and karyotypical analysis was completed on 21 gynogenetic fish. In 90% of the fish examined, the presence of chromosomal fragments was positively correlated with irregular ovarian development. The atypical gonadal morphology observed in the gynogens resembled triploid RBT ovarian morphology. The results of this investigation support the hypothesis that disruption of the normal diploid chromosomal complement alters germ cell development in gynogenetic female RBT due to the unbalanced nature of the genome. J. Exp. Zool. 286:505-512, 2000.

Gonadal sex reversal in triploid rainbow trout (Oncorhynchus mykiss).

Sex determination in salmonids is primarily governed by sex chromosomes; however, phenotypic expression and successful development of the gonads may be influenced by additional factors. Exposure to exogenous steroids during the critical period of gonadal differentiation will reverse the expected phenotypic sex of both female and male trout. Triploidy, a viable condition in rainbow trout (RBT), alters the degree of gonadal development in a gender-specific manner. Males produce testes with similar morphology and function as diploid fish, but females produce underdeveloped ovaries devoid of growing oocytes. One possible explanation for this observed gender difference is that the timing of meiotic initiation may influence ovarian/testicular development in triploid RBT. To determine whether the early entrance of germ cells into meiosis results in the lack of ovarian development in triploid females, the objective of this study was to sex-reverse genotypic triploid female RBT (XXX) into phenotypic males and genotypic triploid male RBT (XXY) into phenotypic females. Male fish were exposed to estradiol-17beta (E(2)) and females were exposed to the non-aromatizable androgen 17alpha-methyldihydrotestosterone (MDHT). Over 90% of the male fish treated with exogenous E(2) developed gonadal structures indistinguishable from the gonads of triploid females. Triploid female RBT treated with MDHT developed testes; however, not all fish treated with this androgen were completely sex reversed. The results of this investigation are consistent with the hypothesis that the failure of ovarian development in triploid RBT is due to the early onset of meiosis and does not appear to be due to genotypic sex. J. Exp. Zool. 284:466-472, 1999.

Rainbow trout chimeras produced by injection of blastomeres into recipient blastulae.

In mammals, the ability to successfully introduce isolated cells into a recipient embryo and to document their development has been an important experimental advance in determining the developmental potential of cells. These techniques are also useful in reestablishing the genome of embryonic cells into a germ line. The objective of the present study was to determine whether blastomeres isolated from rainbow trout (Oncorhynchus mykiss) will incorporate and continue to develop when injected into recipient embryos. In the first experiment, donor cells, previously labeled by injecting fluorescein isothiocyanate-dextran into the zygote, were isolated from blastulae; approximately 1000 of these cells were microinjected into each unlabeled recipient embryo of the same developmental stage. Following subsequent development through gastrulation, microscopic examination revealed that 19 of 114 injected embryos (17%) contained fluorescent cells. These labeled cells were present at numerous sites within embryos, and the pattern of distribution of these cells varied among embryos. In experiment two, blastomeres from normal diploid embryos were injected into triploid blastulae. The injected embryos were incubated until hatching and then sacrificed; cells from these embryos were dispersed and treated with 4',6-diamidino-2-phenylindole. The proportion of diploid cells, as determined by flow cytometry, varied from 2.0% to 12%. From these results we conclude that blastomeres isolated from rainbow trout blastulae will incorporate and continue to develop following injection into recipient embryos.

Electrical stimulation of the starfish's radial nerve in vitro induces the release of a gonadotrophin.

In starfish a neuropeptide responsible for the induction of ovulation and the reinitiation of meiotic maturation in fully grown oocytes appears to be released from the radial nerves at the time of spawning. The objectives of this investigation were to determine if the radial nerve would release this gonadotrophin when electrically stimulated in vitro and to locate other possible sources of this factor. Electrical stimulation of radial nerves isolated from the starfish, Pycnopodia helianthoides, resulted in the release of the gonadotrophin. Significant amounts of this neuropeptide were detected neither in other tissues of the starfish nor in mammalian nervous tissue.

Hydrostatic pressure inhibition of hormone-induced resumption of meiotic maturation in isolated oocytes.

Prior to ovulation, fully grown oocytes of both the amphibian (Rana pipiens) and the starfish (Piaster ochraceus), like those of many other organisms, are arrested in late prophase I of meiosis. Reinitiation of meiotic maturation in oocytes from either of these organisms is hormonally induced. Although the meiosis-inducing substance (MIS) for each organism is chemically dissimilar (a steroid in the frog and a purine in the starfish) induction of oocyte maturation in both biological systems appears to be initiated by the interaction of the MIS with the plasma membrane of the oocyte. The objective of this investigation was to determine if elevated hydrostatic pressure affected hormonal induction of oocyte maturation and to compare the effect of pressure on oocytes stimulated with different meiosis-inducing substances. In isolated oocytes from either the frog or the starfish, increasing ambient pressure reduced the percentage of oocytes which matured, and a stepwise increase in pressure resulted in a corresponding shift in the dose response curves of hormone-induced, oocyte maturation to the right. In experiments using only starfish oocytes, this inhibitory effect of pressure was found to be reversible and limited to the initial period of the maturation event. Taken together, these data suggest that elevated ambient pressure inhibits an early cellular event in the hormonal induction of meiotic maturation which is common to both amphibian and starfish oocytes.