| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
ABSTRACT
In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and cholesterol side chain cleavage (P450scc), 17
-hydroxylase/lyase (P450c17), and aromatase (P450arom) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E2) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450c17, P450scc, and P450arom were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E2 and T titers. Alternatively 3ß-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.
estradiol, follicle, follicular development, gene regulation, seasonal reproduction, testosterone
INTRODUCTION
It has been well established that reproductive steroid hormones (estrogens, androgens, and progestins) play an important role in the onset of puberty, development of gametes, expression of sexually dimorphic characteristics, and evoking reproductive behaviors in all vertebrates studied to date. The secretion of steroid hormones is in turn primarily controlled by the pituitary gonadotropins (FSH and LH). In the study of bony fishes (teleosts), there is a plethora of studies [1, 2] describing the seasonal and biological significance of the changing titers of plasma testosterone (T) and estradiol-17ß (E2); however, it is becoming increasingly important to understand the cellular and molecular regulatory mechanisms by which these changing titers are mediated.
In mammals it has been shown that the rapid and acute changes in steroidogenesis (nonenzymatic regulation) are mediated by the mobilization of cholesterol via the steroidogenic acute regulatory protein (StAR [3]) and potentially the peripheral-type benzodiazepine receptor (PBR [4]). On the other hand, the cyclic (long-term) changes in the type and quantity of secreted steroids in mammals are the result of changes in gene expression of steroidogenic enzymes that are regulated by a suite of factors via a complex process [5–8]. Unfortunately, these types of studies with teleosts are rare, because the genes or gene products encoding the steroidogenic enzymes have only been isolated from a few species of fish [9–20]. These reports include limited studies of expression of selected genes at specific stages of follicular development; however, in this communication, we present the first comprehensive study of the simultaneous changes in the gene expression (transcript abundance) of four key steroidogenic enzymes in the ovary of a fish species as it correlates with changing steroid titers and gonadal development.
The model selected for this study is the channel catfish, Ictalurus punctatus that exhibits an annual breeding pattern characterized by an extended period of oocyte growth (8–10 mo) culminating in a single spawning event [21, 22]. The synchronous development of follicles in this species facilitates the correlation of the expression of steroidogenic genes with gametogenesis, thereby avoiding the complexity of gamete interaction in model vertebrates in which gametes develop and mature asynchronously. In lower vertebrates, ovarian steroids are major endocrine agents controlling ooctye development and maturation. The catfish ovary produces a number of steroid products (Fig. 1 [23]), but this study will focus on the synthesis of sex steroids. Figure 1 illustrates the catalytic roles and critical positions of the four enzymes examined in this study: 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and cholesterol side chain cleavage enzyme (P450scc), 17-hydroxylase/lyase (P450c17), and aromatase (P450arom). The changes in plasma steroid titers and in vitro ovarian steroidogenesis have been previously determined throughout a reproductive cycle of the channel catfish [22–25]. The present study extends these findings by examining the seasonally dependent changes in the expression of steroidogenic genes. Analyses were performed on ovaries of a maturing but reproductively naive population of catfish using the method of real-time quantitative reverse transcription-polymerase chain reaction (rtqRT-PCR). These data were corroborated by a similar study using a reproductively experienced population of fish from which the samples were analyzed by ribonuclease protection assay (RPA).
|
MATERIALS AND METHODS
Chemicals
The SYBR Green Core Reagents Kit was used for the rtqRT-PCR analysis of transcript abundance (PE Applied Biosystems, Foster City, CA). Authentic steroids were purchased from Steraloids Inc. (Wilton, NH) and Sigma Chemical Company (St. Louis, MO). The [-32P]UTP (29.6 Tbq/mmol) was obtained from New England Nuclear (DuPont, Boston, MA); [1,2,6,7-3H]T (specific activity of 3.8 TBq/mmol) and [2,4,6,7,16,17-3H]E2 (specific activity of 5.6 TBq/mmol) were from Amersham Pharmacia Biotech Inc. (Piscataway, NJ) and were used without additional purification. Antisera against each of T and E2 were gifts from Dr. Peter Thomas (University of Texas Marine Sciences Institute, Port Aransas, TX). Leibovitz-15 (L-15) tissue culture media was purchased from Sigma Chemical Company. Oligonucleotide primers (described in Table 1) were synthesized by the BioAnalytical Services Laboratory at the Center of Marine Biotechnology or the Biopolymer Laboratory at the University of Maryland School of Medicine, Baltimore. All other chemicals were of the highest quality available.
|
Animals and Tissue Preparation
Two populations of the channel catfish (I. punctatus) were investigated in the present study. The northern (Maryland) population of fish was composed of reproductively inexperienced fish (naive) presumably entering their first reproductive year, whereas the southern (Louisiana) population was comprised of experienced breeders. All animals were treated in a manner as approved by appropriate Institutional Animal Use and Care Committees. Up to seven naive breeders were captured directly from ponds of a local commercial producer (Bowling Catfish Farms, Charles City, MD) at 4-wk intervals, except in the winter months when few fish were available. The fish were sacrificed in the field and plasma samples from the caudal artery of each fish were recovered by centrifugation and storage at -80°C until analysis by RIA. The ovaries were dissected immediately after bleeding, weighed, portions were taken for analyses not described here, and the remainder was flash frozen in liquid nitrogen. The tissues were stored at -80°C until analysis by rtqRT-PCR (described below). The ovary weight is expressed as a percentage of the fish weight (sans ovary; gonadosomatic index, GSI) and is used as a reflection of the seasonal changes in ovarian development. Local air temperature data were obtained from the Baltimore weather station of the National Climatic Data Center.
The experienced breeders were obtained from the Aquaculture Center of the Louisiana State University Agricultural Farm (Baton Rouge, LA). These animals were treated similar to the naive breeders except that the remaining ovarian tissue was prepared as described previously [23] in order to harvest deyolked ovarian follicles. The tissues were stored at -80°C until analyzed by RPA (described below). Seasonal changes in sex steroid titers and gonadal growth of this population have been published elsewhere [24].
Isolation of Total RNA
Total RNA was isolated from 50 mg of intact ovarian tissue fragments (naive breeders) or from 80–100 mg of deyolked ovarian follicular envelopes (experienced breeders) [23] using Trizol Reagent (Life Technologies, Gaithersburg, MD) and the FastPrep RNA Isolation System (Bio 101, La Jolla, CA) according to the manufacturer's instructions. The quantity and quality of RNA were determined by UV absorbance at 230, 260, and 280 nm wavelength.
Real-Time Quantitative RT-PCR
Transcript abundance of 3ß-HSD, P450c17, P450arom, P450scc, and 18S rRNA (internal control) was quantified by rtqRT-PCR of total RNA isolated from the ovaries of naive breeders. Described briefly, 400 ng of total RNA was reverse transcribed in a 20-µl reaction volume using random hexamer primers and Moloney-murine leukemia virus reverse transcriptase (Life Technologies). The sequences of the four full-length cDNAs encoding the catfish steroidogenic enzymes have been posted in GenBank (accession numbers S75715, AF063835, AF063836, and AF063837) while a full description of the P450arom has been published [11]. Gene-specific primers (Table 1) for PCR amplification were designed according to the requirements set forth by Primer Express software and the developer of the ABI Prism Sequence Detector (see below). In short, the primer sets were designed to generate amplicons of 100–150 base pairs (bp) in length, and the 3' ends of all primers were A/T-rich. The cDNA corresponding to 5 ng of reverse-transcribed RNA served as templates for each of duplicate 25-µl PCR reactions using SYBR Green Core Reagents. The PCR amplifications and fluorescence detection were performed with the ABI Prism Sequence Detector 7700 under the manufacturer's universal thermal cycling conditions. Each gene, including 18S, was amplified in a separate reaction. The computations and descriptions of a modification of the procedure, called TaqMan technology, have been published elsewhere [26–28]. Transcript abundances of the steroidogenic genes were normalized to those of 18S and reported as fold change in abundance relative to the values obtained in the July of the first year of sampling.
Ribonuclease Protection Assay
This procedure was used to measure the transcript abundance of the four steroidogenic enzymes and actin (internal control) in the ovaries of the experienced breeders. The procedure and its validation have been reported before [24]. Described briefly, up to 25 µg of total RNA was hybridized with an excess of each of the 32P-labeled specific antisense riboprobes, the protected riboprobes were resolved on 7 M urea denaturing-PAGE and the gel was exposed to a storage phosphor screen. The signals from the protected riboprobes were visualized with a Storm 840 Laser scanner (Molecular Dynamics, Sunnyvale, CA) and quantified digitally (ImageQuant, Molecular Dynamics). Hybridization of each gene, including ß-actin, was performed separately. The analyses of all samples were conducted in single RPA analyses and the assay was conducted twice. Transcript abundance of the steroidogenic genes was determined after the background was subtracted and normalized to the abundance of the ß-actin transcript. The data are reported as fold change in abundance relative to the values obtained in July of the first year of sampling.
Radioimmunoassay of E2 and T
Steroids were extracted from 100-µl aliquots of plasma with 3 ml methylene chloride. The organic phase was evaporated and the residue reconstituted in 500 µl PBS containing BSA. The efficiency of steroid recovery was determined by spiking the plasma samples with 400 cpm of tritiated steroid. The steroid extracts were incubated overnight at 4°C with appropriate antiserum and tritiated steroid (4000 cpm), after which the antisera-bound steroid was separated from the free fraction with dextran-coated charcoal. After centrifugation, the radioactivity within an aliquot of the supernatant was determined by liquid scintillation spectrometry (Beckman LS 6000LC, Beckman, San Ramon, CA), and steroid concentrations were determined from a standard curve as calculated by the Prizm sofware program (GraphPad, San Diego, CA).
The T antiserum was used at a dilution of 1:7500 and cross-reacted with 5-dihydrotestosterone (14%), 11-ketotestosterone (39%), and 11ß-hydroxytestosterone (2%). The E2 antiserum was used at a dilution of 1:3000 and cross-reacted with estriol (0.08%), cortisol (0.02%), androstenedione (0.06%), dehydroepiandrosterone (0.12%), T (0.02%), and progesterone (0.02%). All other steroids that were tested cross-reacted at less than 0.002%.
Statistics
Transcript abundance and steroid titer data were log-transformed and significant differences in the means were tested by ANOVA. Where differences existed (P < 0.05), the differing monthly groups were identified by Tukey's multiple comparison test.
RESULTS
Seasonal Changes in GSI and Plasma Steroid Titers
The GSI was lowest during the summer months following spawning and the onset of gonadal recrudescence was evident by October (Fig. 2). Beginning in October the GSI gradually increased during the cooler months of the year (temperature indicated by dotted line), although this increase was not statistically significant. The most rapid rate of ovarian growth occurred after April, which coincided with increasing temperatures. The peak GSI in June immediately preceded the time of spawning (early July), which is 4–6 wk later than that seen in southern populations of the same species [22–25].
|
Significant titers of T and E2 were detectable throughout the year, even during ovarian regression (July to September; Fig. 2). The E2 titers, which remained relatively constant during gonadal regression (from July to September), exhibited a rise at the onset of ovarian recrudescence (October) and quickly returned to the levels seen during the period of gonadal regression. A second and steady increase in E2 titers occurred during early vitellogenic growth and reached the highest level late in the vitellogenic growth period. Upon completion of vitellogenic growth, E2 titers decreased sharply through the period of spawning (July) to the low levels associated with gonadal regression (August).
The overall plasma titers of T were much lower than that of E2 and the T titers seen in experienced catfish broodstock [22–25]. In this population of fish, due to large animal-to-animal variations, none of the monthly groups was statistically different from the others. Nevertheless it was apparent that the T titers were elevated only during the later half of vitellogenic growth, remained elevated during postvitellogenesis when the E2 titers began to drop (June) and returned to the low titers associated with regression (August). An initial elevation in T titers, which is associated with the onset of recrudescence in mature broodstock [24], was noticeably absent in this population (Fig. 2).
Validation of rtqRT-PCR
Efficiency of amplification of the target genes and the internal control (18S) was verified to be statistically equal according to the procedure of the manufacturer [27]. In addition, the specificity of the primers used was verified by electrophoretic analysis of randomly selected rtqPCR reactions. In all cases, single amplicons were detected for each of the five gene products. Negative controls in which RNA was included in place of cDNA produced no signals confirming the lack of interference by genomic DNA. Multiple PCR experiments were performed and the interassay variability was monitored by measuring aliquots of a standard sample in every assay. The coefficient of interassay variability was 6.04%.
Seasonal Changes in the Expression of the Steroidogenic Enzymes
The abundance of the mRNA encoding the four steroidogenic enzymes was determined by rtqRT-PCR in whole ovarian follicles of the naive breeders. These data were normalized to the abundance of 18S RNA (Fig. 3) and presented as a value relative to the transcript abundance during the period of regression (July) of Year 1. It is evident from these data that each steroidogenic enzyme is expressed throughout the year and the expression of the changed seasonally, whereas the expression of 3ß-HSD was relatively stable.
|
There were two periods during the year when the expression of the cytochrome P450s were elevated. The first period of induced expression occurred at the time of onset of ovarian recrudescence (October) at which time the transcripts of P450arom increased about 20-fold while those of P450scc and P450c17 rose three fold. The second period of elevated expression was recorded during the months of mid-vitellogenesis (January to May/June). It is important to note that the elevations in the (Fig. 3) corresponded to similar changes in the E2 titers (Fig. 2). On the other hand, the expression of 3ß-HSD seems to be the least variable throughout the year; however, there was a minor increase in the RNA of this enzyme in March corresponding to the peak abundance of transcripts for the other enzymes.
In the population of experienced breeders, the transcript abundance of the enzymes was measured in deyolked follicles by RPA and the data were normalized to ß-actin. This set of samples was too small to subject to statistical analysis; however, it is apparent that the pattern of transcript expression was very similar to those of the naive breeders in that the abundance of the transcripts for the steroidogenic cytochrome P450s showed a seasonally dependant variation while the abundance of the 3ß-HSD transcript showed negligible variation. These data also suggest that the experienced breeders may respond differently than the naive breeders at the onset of recrudescence. Around the time of the onset of recrudescence, a small increase in the expression of P450c17 preceded a 2.5-fold increase in the expression of P450arom (Fig. 4). There was no apparent change in the abundance of the P450scc transcript at this time, but expression was greatly increased during early vitellogenic growth. The latitudinal differences in the temperature-dependant periods of spawning is evident by the 6- to 8-wk shift in the peak GSI of the two populations (shown by the dotted line in Figs. 3 and 4).
|
DISCUSSION
The purpose of this study was to determine systematically, for the first time, the expression profiles of four key steroidogenic enzymes simultaneously in the ovary of a lower vertebrate, the channel catfish, throughout an annual reproductive cycle. These experiments were conducted with presumably virgin catfish entering their first year of reproductive activity, and these data were supported by a smaller study using experienced breeders and a different analytical method. The overall conclusion is that the abundance of the mRNAs encoding the three steroidogenic cytochrome P450s (particularly P450arom; Figs. 3 and 4) were well correlated with E2 titers (Fig. 2 and [24], respectively). This positive correlation suggests that gene transcription is, the primary mechanism for regulating steroidogenesis and that downstream processes (e.g., translation of the P450 transcript) contribute a more subtle influence on the seasonal steriod titers. Transcriptional regulation of the three P450s appears to be similar, but not identical, to each other and yet differs considerably from the regulation of 3ß-HSD.
Even though all four enzymes occupy critical positions within the steroidogenic pathway (Fig. 1), it seems that only the three cytochrome P450s exhibit significant seasonal variation in the expression of mRNA and thereby suggesting that the steroid-dependent events of ovarian development (e.g., onset of recrudescence, vitellogenic growth, and oocyte maturation) are largely mediated by the regulation of these genes. The expression of 3ß-HSD appears to be independent of seasonal influences. The use of transcript abundance as a measure of gene expression, as in the present study, is widely accepted even though there is evidence in mammals that post-transcriptional regulation can influence expression in some specific situations [29].
The presence of transcripts for all four steroidogenic enzymes within the ovarian follicle (Figs. 3 and 4), together with the significant plasma titers of sex steroids (Fig. 2 [24]) demonstrate that the entire steroidogenic pathway is intact in the catfish ovary throughout the year. This differs from the data obtained from other seasonally spawning, temperate fish species where the expression of steroidogenic enzymes are low or undetectable during the regressed phase when sex steroid titers are very low [9, 10, 15, 17]. Northern blot analysis utilized by these studies of trout, medaka, and tilapia failed to detect the presence of the mRNAs encoding specific steroidogenic enzymes in pre- or postvitellogenic ovaries. Similar results are evident in mammals where certain steroidogenic genes are poorly expressed during specific stages of folliculogenesis [6, 7]. The detection of steroidogenic transcripts in a fish ovary at all times of the year, even during the ovarian regression, is unique to this study, however, this may be a consequence of the highly sensitive methods of analysis (rtqRT-PCR and RPA) utilized in this report.
A short-lived elevation in the transcript levels of the three cytochrome P450s in September/October seems to be associated with the onset of ovarian recrudescence (Figs. 3 and 4). These data suggest that the onset of recrudescence is a photoperiod-dependent process because the catfish from both latitudes responded simultaneously. P450arom was the most responsive steroidogenic gene to the hormonal signals associated with recrudescence (presumably FSH) because P450arom expression increased the most in both populations. This pronounced change in P450arom expression is likely to be directly responsible for the elevation of E2 titers (Fig. 2 [22, 24]) which has been shown to be important for initiation of follicular growth [30]. The pivotal role of FSH in the process of recrudescence would presumably be reflected in its plasma titers but these determinations cannot be conducted at this time because this hormone has not been isolated from any catfish species. However, the importance of FSH to this process is suggested by the timely increase in ovarian expression of the catfish FSH receptor (in preparation).
An elevation in the abundance of the P450 transcripts was sustained throughout the spring months and closely mirrored the elevation in E2 titers (Fig. 2 for the naive breeders and [24] for the experienced breeders). The sharp decrease in the abundance of transcripts of the cytochrome P450s during the rapid vitellogenic growth of the experienced breeders (Fig. 4) was predicted by the decrease in E2 titers that often occurs at this time in many species (Fig. 3 [22, 25]). Presumably, lower titers of E2 are sufficient to maintain hepatic vitellogenesis once it has been induced [1, 30]. A marked shift in the steroidogenic pathway at the time of the transition from the estrogen-mediated growth phase to the progestin-mediated maturation phase has been widely described [30], however, little is known about the changes in the expression and the regulation of the genes encoding steroidogenic enzymes responsible for the shift. Our recent findings on the seasonal expression of the catfish gonadotropin receptors (FSH-R and LH-R) are consistent with the shift in steroidogenesis at the onset of recrudescence and oocyte maturation (in preparation).
As the terminal and rate-limiting enzyme in the synthesis of E2, P450arom is important in the female reproductive physiology and hence has been studied intensively in a variety of vertebrates. It is also the steroidogenic enzyme cloned in most species of teleosts [19]. In some of these species, northern blot analyses have demonstrated abundant P450arom transcripts in mid- to late-vitellogenic ovaries and undetectable levels in postvitellogenic ovaries [10, 15, 17]. However, the present study has revealed a short-lived elevation in the transcript level at the onset of recrudescence. The ovarian P450arom transcript levels clearly reflect the E2 titers at different phases of the ovarian cycle, including the onset of recrudescence. Similarly, a high correlation between the ovarian levels of P450arom transcripts and the plasma levels of E2 has also been demonstrated in rat ovaries under various reproductive physiological states [8, 31]. These observations indicate that the ovarian P450arom transcripts were readily translated into active protein and that the ovary is the predominant source of the circulatory E2 levels in catfish. Some teleosts express P450arom in the brain, at levels higher than in the ovary [19]. In one such species, the goldfish, the brain P450arom is the product of a second P450 gene [32] and such duality is believed to be related to the polyploid nature of its genome. The diploid nature of the catfish genome (suggesting a single copy of the P450arom gene) and the high correlation of the ovarian P450arom with E2 titers suggest that it is unlikely brain P450arom contributed significantly to circulating E2 titers.
Both P450scc and P450c17 are critical enzymes for the synthesis of sex steroids. The P450scc catalyzes the first and rate-limiting step in steroidogenesis and both the hydroxylase and lyase activities of P450c17 are required for the synthesis of T. Thus the increases in the transcripts of these two cytochromes at the onset of recrudescence and during vitellogenic growth are consistent with the rises in the steroid titers in both catfish populations (Fig. 2 [24]). However, other studies of fish [9, 33] did not detect the mRNA of these enzymes until mid-vitellogenesis, even though an elevation in steroid titers was clearly evident. These differences are certainly due to the increased sensitivity of the analyses used in this study compared to the Northern blot analyses.
Studies using other fish species have shown that mRNA abundances of P450arom sharply decreased while P450scc and P450c17 mRNA were elevated during the period of final oocyte maturation and ovulation [9, 30, 33]. Presumably, elevated P450scc and P450c17 were required for the synthesis of the maturation-inducing steroid (MIS). In this study with the channel catfish, the transcripts for all three cytochrome P450s decreased simultaneously as they approached spawning period but none of the females examined were in the process of final oocyte maturation. Even though the present data corroborate the very small amounts of 17,20ß-dihydroxy-4-pregnen-3-one (the MIS in catfish) produced by spawning catfish [22], the pattern of expression of these steroidogenic genes during final oocyte maturation are likely to be different from the pattern seen in prematurational or postspawning fish.
The small temporal changes in the expression of mRNA for 3ß-HSD are similar to that seen in the rat [7] and suggest that this gene is not a key site for the regulation of steroidogenesis. Although 3ß-HSD is critical for the synthesis of the 
4 steroids (e.g., T and MIS), it does not appear to be rate-limiting, and thus the minor increase in the transcript of this enzyme recorded at midvitellogenesis could be sufficient to support the enhanced rate of steroidogenesis during this period. In the rainbow trout ovary, this enzyme shows a greater increase in late vitellogenic follicles presumably to facilitate the surge in the MIS [12].
This study utilized two populations of catfish that differ in their reproductive experience and latitudinal habitat; however, the seasonal pattern of transcript expression in the ovarian follicles of the steroidogenic enzymes seem to have a similar, but not identical, relationship to steroid titer and ovarian physiology. Even though the data from the experienced breeders are not sufficient in number to stand on their own, this data set is a valuable supplement. Although a direct comparison of the transcript abundances of the two populations of catfish is not appropriate due to the procedural difference in analyses, it is reassuring that ovarian follicles from both populations that were analyzed by distinctly different methods and normalized to different controls (18S and ß-actin) displayed similar patterns of seasonal changes in transcript abundance of the four steroidogenic enzymes. It will require further study to determine if the differences in the pattern of transcript abundances seen in the two populations and the muted steroid titers of the naive population were a consequence of a prepubertal dummy run (a nonovulatory cycle) and what was the molecular mechanism that manifested these differences.
The similar expression patterns of the three catfish P450 genes strongly suggests that they have a common regulatory mechanism even though similar studies in mammals show these genes are differentially regulated [6, 7]. Follicle-stimulating hormone and LH are likely to be the primary regulators [30] of these three genes; however, it is important to understand how other hormones (GH, growth factors, GnRH, etc.) contribute, what are the signal transduction mechanisms utilized by the activated receptors of these hormones, and how do these signals interact at the gene level within the growing follicle.
ACKNOWLEDGMENTS
The authors thank Ms. Carla Berard and Ms. Jennifer Childress for their technical assistance, the Bowling Catfish Farm and Dr. Terrence Tiersch for the catfish, and Dr. Peter Thomas for the gift of antisera.
FOOTNOTES
1 This research was supported by grants from the United States Department of Agriculture (Enhancing Reproductive Efficiency, grant 94-37203-0761) and Wallenburg Foundation awarded to J.M.T. A portion of this study has been published in the Proceedings of the 6th International Symposium on Reproductive Physiology of Fish, Bergen, Norway, 4–9 July 1999. 
2 Correspondence: John M. Trant, Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street, Baltimore, MD 21202. FAX: 410 234 8896; trant{at}umbi.umd.edu
Accepted: July 20, 2000.
Received: April 12, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  N. Zmora, Y. Kazeto, R S. Kumar, R. W Schulz, and J. M Trant Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions J. Endocrinol., August 1, 2007; 194(2): 407 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P M. Lokman, K. A N George, S. L Divers, M. Algie, and G. Young 11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro Reproduction, May 1, 2007; 133(5): 955 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M Kusakabe, I Nakamura, J Evans, P Swanson, and G Young Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes. J. Endocrinol., June 1, 2006; 189(3): 541 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y Kazeto and J M Trant Molecular biology of channel catfish brain cytochrome P450 aromatase (CYP19A2): cloning, preovulatory induction of gene expression, hormonal gene regulation and analysis of promoter region J. Mol. Endocrinol., December 1, 2005; 35(3): 571 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Gen, K. Okuzawa, N. Kumakura, S. Yamaguchi, and H. Kagawa Correlation Between Messenger RNA Expression of Cytochrome P450 Aromatase and Its Enzyme Activity During Oocyte Development in the Red Seabream (Pagrus major) Biol Reprod, October 1, 2001; 65(4): 1186 - 1194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Kumar, S. Ijiri, and J. M. Trant Molecular Biology of the Channel Catfish Gonadotropin Receptors: 2. Complementary DNA Cloning, Functional Expression, and Seasonal Gene Expression of the Follicle-Stimulating Hormone Receptor Biol Reprod, September 1, 2001; 65(3): 710 - 717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sampath Kumar, S. Ijiri, and J. M. Trant Molecular Biology of Channel Catfish Gonadotropin Receptors: 1. Cloning of a Functional Luteinizing Hormone Receptor and Preovulatory Induction of Gene Expression Biol Reprod, March 1, 2001; 64(3): 1010 - 1018. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
,17-pregnenolone, presumptively 16
