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GENERAL AND COMPARATIVE ENDOCRINOLOGY 87, 451-460 (1992)

Sex Steroid Levels in the Testes, Ovaries, and Pyloric Caeca during Gametogenesis in the Sea Star Aster& vulgaris

GENE A. HINES AND STEPHEN A. WATTS

Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294

AND

STACIA A. SQWER AND CHARLES W. WALKER

Department of Zoology, University of New Hampshire, Durham, New Hampshire 03824

Accepted February 18, 1992

The concentrations of progesterone, testosterone, and estradiol were determined via radioimmunoassay in testes, ovaries, and pyloric caeca of the sea star Asten’as vulgaris during one complete and two partial gametogenic cycles. These compounds were found in all tissues examined and were present in quantities similar to those reported previously in other echinoderms and in vertebrates. Testes and ovaries exhibited annual growth cycles during which testicular and ovarian mass increased up to lOOfold as gametes were produced and stored until spawning. Pyloric caecal mass varied during the annual reproductive season; however, no seasonal trends were apparent. In the testes, sex steroid levels were highest at the onset of spermatogenesis. Transient increases in the levels of estradiol coincided with spermatogonial mitotic proliferation. Transient increases in the levels of testosterone and progesterone in the testes coincided with spermatogenic column formation and with spermiogenesis, respectively. In the ovaries, estradiol and testosterone levels were highest at the onset of oogenesis while progesterone levels did not change significantly throughout the annual gametogenic cycle. Male and female pyloric caeca exhibited similar seasonal variations in levels of sex steroids as compared with the gonads. It is hypothesized that transient increases in the levels of sex steroids during gametogenesis may serve as endogenous modulators of reproduction. Q 1992 Academic PISS, IK.

Early evidence of steroid-like compounds in echinoderms was provided in 1937 when Donahue and Jennings injected ovarian extracts of the echinoid Lytechinus variegatus into ovariectomized rats. Sub- stantial epithelial growth in uterus and va- gina was noted suggesting the presence of mitogenic estrogens in the extract. Similar results were obtained with ovarian extracts from several other species of echinoderms including Echinometra sp., Stichopus mo- bius (Donahue, 1940), Aster& forbesi, Ar- bacia punctulata, and Strongylocehtrotus droebachiensis (Hagerman et al., 1956). Botticelli et crl. (1960, 1961), using similar bioassays, identified estradiol-like and pro-

gesterone-like compounds in ovaries of Pi- saster ochraceus and Strongylocentrotus franciscanus. Later, Dieleman and Scho- enmakers (1979) developed radioimmunoassay procedures to measure progesterone and estrone levels in ovaries of the asteroid Aster& rubens.

Several investigations in vitro demonstrated that gonads and pyloric caeca of asteroids were capable of steroid synthesis. Schoenmakers (1979) and Schoenmakers and Voogt (1980, 1981) showed, respectively, that tissues of Asterias rubens could convert cholesterol to C2i steroids, p~oges- terone to C,, steroids, and androstenedione to other androgens. In female A. rz&elses

451 0016-6480192 $4.00 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.

452 HINES ET AL.

(Schoenmakers et al., 1977) and in male As- terias vulgaris (Walker, 1980), cells with potential steroid synthesizing capabilities were identified. Tissue-specific and sex- specific differences in androgen metabolism were demonstrated in gametic and somatic tissues of A. vulgaris (Hines et al., in press). The qualitative and quantitative differences in steroid metabolism in asteroids suggest important physiological functions for these hormones,

Schoenmakers and Dieleman (1981) determined the levels of progesterone and estrone in female Aster& rubens throughout the annual gametogenic cycle. Seasonal patterns of progesterone and estrone were related to stages of oogenesis and indicated an antagonistic relationship in the regulation of vitellogenesis. Similarly, Voogt and Dieleman (1984) reported varying levels of esterone and progesterone during the annual gametogenic cycle in male A. rubens and suggested that these steroids were involved with spermatogenesis. The levels of estrone, estradiol, and progesterone were measured in ovaries, testes, and pyloric caeca of the asteroid Sclerasterias rnollis during the annual gametogenic cycle under natural and photoperiod-manipulated conditions (Xu, 1990). The presence of these steroids in different levels during the annual cycle suggested an involvement with gametogenesis.

Investigations have shown that the physiological functions of sex steroids in echinoderms are similar to those recognized for vertebrates (reviewed by Voogt et al., 1985; Shirai and Walker, 1988). Most studies have provided information on steroids in female echinoderms and few have docu- mented steroid profiles over the entire gametogenic cycle. The present study reports steroid levels in male asteroids and pro- vides comparative information on the levels of progesterone and estradiol, and the first report of testosterone, in male and female gonads and pyloric caeca during the gametogenic cycle. We have also used a new,

more reliable, method for extraction of steroids from echinoderm tissues providing a more accurate determination of steroid levels.

MATERIALS AND METHODS

Chemicals and supplies. Optima grade methylene chloride and reagent grade sodium chloride, sodium phosphate, potassium chloride, calcium chloride, and magnesium chloride were purchased from Fisher Sci- entific (Fair Lawn, NJ). Glass distilled Baker Resi- Analyzed methanol and Baker Analyzed hydrochloric acid were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ). Water for homogenization, solu- tions, and washes was double-distilled. Sep-Pak C,, cartridges with octadecyl-silane bonded reversed- phase packing were purchased from Waters Associ- ates (Milford, MA). Sterile Leur Lok plastic syringes (10 cc) were purchased from Beckton Dickinson and Co. (Rutherford, NJ).

Animals. Specimens of adult Asterias vulgaris (approximately 15 cm arm length) were purchased from Ocean Resources (Portland Harbor, ME). Animals were collected when possible (weather permitting) by SCUBA from Casco Bay, Portland, beginning in No- vember 1987 until January 1990 and sent via overnight express to the laboratory at the University of Alabama at Birmingham where tissues were excised. Steroid levels were not determined in the summer when individuals contained minimal gonads with insufficient tissue mass for analyses. Total animal and organ wet weights were measured to calculate tissue indices. Wet mounts of gonads were viewed by microscopy to determine sex and reproductive state. Parasitized (Wicklow et al., 1985) or otherwise unhealthy animals were not used. For each individual, four 0.1-g subsam- ples of testes or ovaries and of pyloric caeca were frozen in liquid nitrogen and stored at - 80” until anal- ysis.

Seawater temperatures were measured from a thermistor mounted in an iron frame at 25 feet below mean low water levels in Boothbay Harbor, Maine. Data were provided courtesy of Daniel Smith of the Environmental Monitoring Project (State of Maine, Department of Marine Resources, West Boothbay Harbor, ME 04575).

Steroid extraction. Steroid extraction and processing were performed according to Hines et al. (1990). Frozen tissues were thawed and each sample was homogenized on ice in 500 ~1 of water for ca. 5 set using a Wheaton glass and Teflon homogenizer. Homoge- nates were then pulse-sonicated on ice for 10 set using a Branson Sonitier Cell Disruptor 350 with micro tip (output level = 6, duty cycle = 40%). Next, 400 p,l of prewarmed (40”) HCl was added to each homogenate, vortexed, and incubated for 15 mm at 40”. After incu-

STEROIDS IN Asterius vulgaris 453

bation, 1.25 ml of 0.07 M Na,HPO, buffer (pH = 7.4) of sea star extracts were 2.09% (n = 6) and 17.2% (n was added to each homogenate. = S), respectively.

Three methylene chloride (MC) extractions were performed on each homogenized sample to extract steroids. MC (7 ml) was added to each sample, vortexed thoroughly, and centrifuged in a Beckman TJ-6 table- top centrifuge at 2500 rpm for 10 min. The lower MC phase (from each of three extractions) was removed by Pasteur pipet, pooled, and dried under nitrogen gas at 40”. Extracted steroids were resuspended in 500 p,l methanol and vortexed and 4.5 ml buffer (as above) was added to prepare samples for Sep-Pak processing.

Sep-Pak cartridges were primed with three sequen- tial washes of 10 ml methanol, 10 ml water, and 5 ml 0.15 M NaCl at a flow rate of 14 ml/min. Steroid samples were then applied to primed cartridges and washed sequentially with 5 ml 0.15 M NaCl, 10 ml water, and 5 ml methanol. The methanol wash eluted steroids and the eluant was dried under nitrogen gas at 40”. Steroids were then resuspended in phosphate- buffered saline (final concentration 0.136 M NaCl, 2.68 mM KCl, 8.11 mM Na*HPO,, 1.47 mM KH,PO,, 0.68 mM CaCl,, 0.492 mM MgCl,): 100 p.1 for estradiol and progesterone and 50 l.~l for testosterone estimations via radioimmunoassay (RIA).

Testosterone concentrations were determined similarly. The antiserum used was highly specific and cross-reactivity was ~5% with dihydrotestosterone. The lowest detectable concentration of testosterone was 0.04 @ml or 0.04 nglg tissue. Antibody binding ranged between 32.0 and 39.8%. The intra- and interassay coefficients of variation for testosterone RIA of sea star extracts were 2.16% (n = 6) and 32.7% (n = 4), respectively.

For estradiol determinations, steroids competed with tracer of high specific activity of antibody in fixed volumes of antiserum. Addition of a second highly specific antiserum precipitated steroids that were bound by the first antibody. Cross-reactivity was ~1.5% with cw-estradiol. The lowest detectable concentration of estradiol was 10 pgJm1 or 10 pglg tissue. Antibody binding ranged between 41.7 and 48.3%. The intra- and interassay coefficients of variation for estradiol RIA of sea star extracts were 2.89% (n = 6) and

11.7% (n = 5), respectively.

Determination of steroids. Concentrations of progesterone, testosterone, and estradiol extracted from sea star tissues were determined by RIA. Parallel dis- placement curves were obtained with serial dilutions of sea star extracts and steroid standards. Previous identification of sex steroids extracted from sea star tissues was determined via RIA after separation by LH 20 columns. Native and modified steroid products were further separated and identified by thin layer chromatography in several chromatographic systems. Furthermore, these steroids synthesized in vitro from radiolabeled precursors could be recrystallized to con- stam specific activity (Hines et al., in press).

Standard curves were generated from kit-supplied calibrants ranging O-40 rig/ml, O-16 ngiml, and O-5120 pgiml for progesterone, testosterone, and estradiol, respectively. An ICN Micromedic Systems (courtesy of Jim Edmonson, Department of Obstetrics and Gyne- cology, University of Alabama at Birmingham) com- puter program reduced data to log-logit representa- tions and unknown (samples) and control steroid concentrations were extrapolated from these curves,

Progesterone and testosterone concentrations were determined by COAT-A-COUNT solid-phase ‘251-RIA kits purchased from Diagnostic Products Corp. (Los Angeles, CA). Estradiol concentrations were determined by liquid-phase lz51-RIA kits purchased from PANTEX (Santa Monica, CA). Intra- and interassay controls CIBA Ligand A, B, and C were purchased from Corning Diagnostics Corp. (Irvine, CA). Intra- and interassay controls of pooled Asterias vulgaris pyloric caeca steroid extracts were also used.

Calculations. For each animal collection data (total = 16), four samples each of gonads and pyloric caeca of 4-10 individuals of each sex were assayed for progesterone, testosterone, and estradiol content. Mean steroid levels (2 standard error of the mean) are pre- sented as steroid concentration per gram fresh weight of tissue. ANOVA (P < 0.05) was used to test for significant differences among means at different collection periods.

RESULTS

Steroid Levels in the Testes

For progesterone determinations, steroids (standards, controls, and unknown samples) competed with tracer of high specific activity for antibody bound to tubes. The antiserum used was highly specific and cross-reactivity was <2.5% with 11-deoxycortisol. The lowest detectable concentration of progesterone was 0.05 rig/ml or ‘0.05 rig/g tissue. Antibody binding ranged between 33.9 and 37.3%. The intra- and interassay coefficients of variation for progesterone RIA

Mean testicular indices determined from November 1987 through January 1990 showed annual patterns of variation indicating two distinct gametogenic cycles and a partial third cycle (Fig. 1). Testicular indices increased during the fall, continued to increase during the winter, and peaked in the early spring. Testicular indices remained low during the summer. Wet mount microscopic observations of testes showed

454 HINES ET AL.

FIG. 1. Mean (+-SEM, n = 4-10 individuals) sex steroid levels and indices of the testes of A. vulgaris

during consecutive annual gametogenic cycles. Ste- roid levels are expressed per gram fresh tissue wt. Testis index = (testes wet wt/total animal wet wt) x 100%.

changes in germinal cell populations similar to those described previously (Walker, 1974, 1980; Watts et al., 1990a).

In the testes, mean progesterone levels ranged from 0.3 to 6.1 rig/g during the study (Fig. 1). Considerable annual variation was found but major peaks in the levels of progesterone were seen in November, during spermatogonial proliferation, and in April, just prior to spawning, of the 1988-1989 season. Progesterone levels were also relatively high in September of the 1989-1990 season just prior to spermatogonial proliferation.

Mean testosterone levels ranged from 0.14 to 5.5 rig/g during the study (Fig. 1). A peak of testosterone was noted during April of the 1988-1989 season just prior to spawning. Testosterone levels were relatively higher during December and January of the 1989-1990 season during growth of spermatogenic columns.

Mean estradiol levels in the testes ranged

from 11 to 460 pg/g during the study (Fig. 1). Significantly higher levels of estradiol were seen in November and October of the 1987-1988 and 1988-1989 seasons, respectively, during spermatogonial proliferation. Little variation was seen in estradiol levels during the 1989-1990 gametogenic season. The estradiollprogesterone ratios were highest (>0.4) immediately prior to the increases in testicular mass during the 1987- 1988 and 1988-1989 annual gametogenic cycles, but did not increase substantially during the 1989-1990 cycle (Fig. 3A).

Steroid Levels in the Ovaries

Ovarian indices (Fig. 2) showed annual and seasonal patterns similar to testicular indices (Fig. l), although mean ovarian indices were greater than testicular indices on corresponding dates. Steroids in the ovaries were also present in similar quantities

FIG. 2. Mean (?SEM, n = 4-10 individuals) sex steroid levels and indices of the ovaries of A. vulgaris during consecutive annual gametogenic cycles. Ste- roid levels are expressed per gram fresh tissue wt. Ovary index = (ovaries wet wtkotal animal wet wt) x 100%.

STEROIDS IN Asterias vulgaris 455

1987 1988 1989 1990

1 s x B

0

1987 19Ea 1989 1990

FIG. 3. Ratios of the levels of estradiol (E) and progesterone (P) in the testes (A) and ovaries (B) of A. vulgaris during consecutive annual gametogenic cycles (solid lines). Dotted lines represent mean (kSEM, n = 4-10 individuals) gonad indices (gonad index = (gonad wet wtitotal animal wet wt) X 100%.

and exhibited similar seasonal variations when compared to the testes.

Mean progesterone levels in ovaries ranged from 0.5 to 2.3 rig/g during the study (Fig. 2). Considerable annual variation in the levels of progesterone occurred and no seasonal trends were apparent. Progester- one levels were consistently higher during the 1988-1989 gametogenic season when compared to the levels found in previous and subsequent gametogenic seasons.

Mean testosterone levels in ovaries ranged from 0.3 to 3.3 nglg during the study (Fig. 2). Increases in the levels of testosterone were seen in November, during oocyte growth, and in March, during early oogenesis,, of the 1988-1989 season. After the ini- tial increase of testosterone in November of the 1989-1990 season testosterone levels remained relatively high through January.

Mean estradiol levels in ovaries ranged from 4 to’ 360 pg/g throughout the study

(Fig. 2). There was a sharp and distinct peak in estradiol which coincided with a peak of testosterone in November of 1989. There is a higher peak of testosterone in March for which there is not correlating peak in estradiol. The estradiol/ progesterone ratios in the ovaries were highest (>-0.2) at the onset of ovarian growth (Fig. 3B).

Steroid Levels in the Pyloric Caeca

Male and female pyloric caeca indices were higher than and exhibited greater vari- ability than gonad indices (Figs. 4 and 5, respectively). No seasonal or annual patterns were apparent as were exhibited by the gonads. Similar levels of and variations in levels of sex steroids were present in the pyloric caeca when compared to the gonads. Some transient increases in steroid levels in the pyloric caeca coincided with similar transient increases in steroid levels

27

23

19

15

11

1987 1988 1989 1990

FIG. 4. Mean (-+SEM, n = 4-10 individuals) sex steroid levels and indices of the pylaric caeca of male A. vulgaris during consecutive annual gametcgenic cycles. Steroid levels are expressed per gram f&h tissue wt. Pyloric caecum index = (pyloric caeca wet wti total animal wet wt) X 100%.

456 HINES ET AL.

FIG. 5. Mean (?SEM, n = 4-10 individuals) sex steroid levels and indices of the pyloric caeca of female A. vulgaris during consecutive annual gametoge- nit cycles. Steroid levels are expressed per gram fresh tissue wt. Pyloric caecum index = (pyloric caeca wet wt/total animal wet wt) X 100%.

in the gonads during periods of gametoge- nit activity.

Annual Cycle of Seawater Temperatures

Seawater temperatures ranged from <O” to 15” during this study and an annual pattern of changing seawater temperatures was evident for each gametogenic cycle (Fig. 6). Generally, temperatures decreased during the fall and winter months and increased during the spring and summer months. Temperatures were lowest in Jan- uary, March, and December of the 1987- 1988, 1988-1989, and 1989-1990 gametoge- nit seasons, respectively. Small (-2’) transient increases in temperatures occurred during 4 and 6 weeks after respective minimum temperatures were recorded in the 1987-1988 and 1989-1990 gametogenic seasons. Similar transient changes in temperatures occurred prior to the minimum temperature recording in the 1988-1989 game-

togenic season. Temperatures were highest in September of 1988 and 1989. The time differential between maximal and minimal temperature recordings during the 1988- 1989 gametogenic season was approximately twice that compared to the 1989- 1990 gametogenic season.

DISCUSSION

Annual and seasonal variations in tissue indices and steroid concentrations were observed in testes, ovaries, and pyloric caeca of the sea star Asterias vulgaris. Other asteroids have shown similar variations in tissue indices and biochemical parameters, coinciding with changes in biotic or physi- cal factors (Lawrence and Lane, 1982). Changes in the timing or magnitude of tissue indices are often reflected by the under- lying changes in biochemical processes (Watts and Lawrence, 1990). Unseasonable fluctuations in seawater temperatures during the onset of the 1989-1990 gametogenic cycle may have partially accounted for observed annual variations in steroid levels. Other investigations with echinoderms collected from the same area noted decreased or delayed reproductive effort during 1989- 1990 (David Quinby, personal communica- tion). Temperature has been shown to influence gonadal steroidogenesis in teleost fish (Manning and Kime, 1984) and reptiles

’ 4 ,-. I

N D J F’M’A’M’J’J’A’S’O’N’D’J’F’M’A’M’J’J’A’S’O’N’D’J’F’M’A’ 1987 1988 1989 1990

FIG. 6. Average daily seawater temperatures at 25 ft. below mean low water at Boothbay Harbor, Maine, during the collection period. Each data point repre- sents the daily average of values recorded every 15 min. Data courtesy of Daniel Smith, Maine Depart- ment of Marine Resources.


(Licht, 1972; Pieau et al., 1982), and may with the relative inactivity of the testes dur- have similarly affected steroidogenic pro- ing this aspermatogenic ‘period support the cesses in Asterias vulgaris. Therefore, we hypothesis that transient increases in ste- will discuss the 1988-1989 gametogenic cy- roid concentrations may regulate spermato- cle as representing the “typical” cycle. genie events in A. vulgaris.

Transient, seasonal increases in steroid levels observed in the present study coincided with specific transitions in germinal cell populations. The beginning of a new spermatogenic cycle in Asterias vulgaris was characterized by an increase in the testicular index (Lowe, 1978; Walker, 1980; Smith and Walker, 1986; Watts et al., 1990a) and by changing levels of testicular sex steroids. Estradiol increased in the fall concomitantly with mitotic proliferation of previously quiescent spermatogonia. Tes- tosterone increased approximately 1 month after the apparent proliferative response of spermatogonia to estradiol. These increases in testosterone coincided with the beginning of differentiation of spermatogonia into columns of primary spermatocytes. Approximately 1 month after the increases in testosterone, an increase in progesterone occurred and was coincident with the onset of spermiogenesis. During the winter and early spring all steroid concentrations remained low in the testes while testicular mass continued to increase as spermatozoa differentiated and were stored in the testicular lumen. These data suggest that increased concentrations of steroids, which may have been important for the initiation of spermatogonial proliferation, differentiation, and spermiogenesis, may not be re- quired for maintenance of spermiogenesis. Later in the spring increases in the concentrations of steroids in the testes of A. vulgaris (most notably progesterone and testosterone) occurred just prior to spawning. The increased concentrations of steroids may be important for the preparation of testes for spawning as most spermatozoa are fully differentiated at that time. In the late spring spawning occurred, all steroids decreased, and the testes returned to an inac- tive state. The low,level of steroids together

The levels of testicular sex steroids in two other sea stars also change during the annual gametogenic cycle. Similar to Aste- rias vulgaris, increases in the levels of estrone and progesterone, determined by RIA, marked the beginning of a new gametogenic cycle in male S. mollis (Xu, 19901, In A. rubens (Voogt and Dieleman, 19841, highest levels of progesterone and estrone, also determined by RIA, occurred after the onset of testicular growth. However, a significant increase in testicular growth en- sued, suggesting that these hormones were important for gametogenesis.

The function of sex steroids in the testes of sea stars may be comparable with that of vertebrates where testicular sex steroids are associated primarily with the regulation of spermatogenesis (Mainwaring et ail., 1988). In particular, increases in the levels of testosterone in testes of Asterias vulgaris coincide with the beginning of testicular growth, indicating a potential regulatory (or stimulatory) function similar to the testes of vertebrates (Mainwaring et al., 1988). Holmes et al. (1986) demonstrated that Ser- toli cells in the rat secrete a potent mitogen which is regulated by testosterone. Since somatic cells are present in testes of A. vd- garis, and are in many ways similar to Ser- toli cells of vertebrates (Walker, 19881, increased levels of testosterone at the begjn- ning of spermatogenesis may similarly influence germinal cell proliferation in the sea star.

The onset of gametogenesis in female As- terias vulgaris occurred in the fall ‘with an increase in ovarian indices accompanied by increased levels of sex steroids. In: partieu- lar, increases in the levels of estradioi l~o- incided with the apparent proliferation of oogonia and early stages of vitellogenesis. Testosterone levels increased also at t,his

458 HINES ET AL.

time and again during the late growth/ partial maturation phase of oocyte develop- ment. Progesterone levels did not exhibit a seasonal pattern. In addition, an increase in the levels of sex steroids immediately prior to spawning in females was less apparent than in male A. vulgar-is.

Levels of ovarian sex steroids determined by RIA changed seasonally also in Aster& rubens (Schoenmakers and Diele- man, 1981) and Sclerasterias mollis (Xu and Barker, 1990). Both species exhibited relatively high steroid levels prior to vitellogenesis. Later, with the onset of oogenesis, estrogen levels increased and were suggested to be important for vitellogenesis in these two species. These authors suggested that the inverse relationship which existed between progesterone levels and ovarian size in A. rubens and S. mollis may be important for oogenesis.

In all echinoderms investigated, transient peaks in gonadal steroid levels during the annual gametogenic cycle suggest an involvement with the regulation of gametogenesis. Peak levels of different steroids were closely related temporally, and often coincided with specific gametogenic events. Examination of the ratios of specific steroids during the annual gametoge- nit cycle showed significant changes in es- troneiprogesterone in gonads of Asterias rubens (Voogt et al., 1985) and estradiol/ progesterone in Asterias vulgaris. In breast cancer cells, estrogens have been reported to stimulate the synthesis of proteins necessary for cell proliferation (Dickson et al., 1984; Vignon et al., 1982), among these was the progesterone receptor (Katzenellenbo- gen et al., 1984; Horwitz et al., 1978). Se- quential, transient increases in steroid levels during the annual gametogenic cycle of echinoderms (as evidenced by steroid ratios) may thus be important mechanisms for initiating certain gametogenic events.

Progesterone, testosterone, and estradiol were present in similar levels and exhibited similar seasonal variations in the pyloric

caeca as in the gonads of Asterias vulgaris. This indicates a commonality between the two tissue types that may reflect a common steroid biosynthetic capacity or require- ment. The pyloric caeca of Asterias rubens (Schoenmakers and Dieleman, 1981; Voogt and Dieleman, 1984) and Sclerusterius mollis (Xu, 1990; Xu and Barker, 1990) exhibited seasonal variations of sex steroids during the annual gametogenic cycle. Signifi- cant changes in the levels of progesterone and estrone in the pyloric caeca of A. rubens coincided with the beginning of a new gametogenic cycle and were suggested to be involved with nutrient allocation to support gonadal growth (Schoenmakers and Dieleman, 1981; Voogt and Dieleman, 1984; Voogt et al., 1985). However, the physiological significance of steroids in the pyloric caeca of A. vulgaris remains ob- scure.

Extrinsic factors such as temperature or photoperiod may ultimately regulate the annual gametogenic cycle of echinoderms (Giese and Pearse, 1974). Bouland and Jan- goux (1988) suggested that environmental factors are necessary for the initiation of, rather than the maintenance of, gametogenesis in Asterias rubens. Watts et al. (1990b) proposed that seasonal fluctuations in field temperatures regulate testicular growth of Aster& vulgaris by influencing the activity and kinetics of metabolic enzymes. In addition, the onset of the gametogenic cycle in A. vulgaris can be manipulated under experimental photoperiodic conditions, indicating that photoperiod may serve as a gating mechanism for the initiation of gametogenesis (Pearse and Walker, 1986) as seen in other echinoderms (Pearse and Eernise, 1982; Pearse et al., 1986; Bay- Schmith and Pearse, 1987; McClintock and Watts, 1990).

The seasonal variations in steroid levels observed in this study suggest that gametogenesis may be regulated hormonally: espe- cially mitotic and/or meiotic transitions among germinal cell populations. This hy-


pothesis is supported by data from many investigations which indicate physiologi- cally relevant roles for steroids in echinoderms. However, the ultimate control cues which may initiate changes in steroid levels, thus initiating physiological responses, remain unknown. In addition, the seasonal changes in the levels of progesterone, testosterone, and estradiol in Asterius vulgaris suggest an evolutionarily conserved hor- monal regulation of reproduction among many different phyla. Although a relationship between transient increases in sex steroid levels and gametogenic events in the sea star A. vulgar-is seems apparent, future work on the physiological role of these hormones and their potential interactions with extrinsic and intrinsic factors is needed.

ACKNOWLEDGMENTS

We appreciate the technical assistance of Carl Mas- sey, Kara Lee, and Dory Curtis. This study was supported by grants from the NSF (DCB-8711425); from NATO (0413/86); from the Hubbard Endowment of UNH to C. W.W. and from the Alabama Academy of Science to G.A.H.

REFERENCES

Bay-Schmith, E., and Pearse, J. S. (1987). Effect of fixed daylengths on the photoperiodic regulation of gametogenesis in the sea urchin Strongylocen- trotus purpuratus. Int. J. Invert. Repro. Dev. II,

287-294. Botticelli, C. R., Hisaw, F. J., Jr., and Wotiz, H. H.

(1960). Estradiol-17B and progesterone in ovaries of the starfish Pisaster ochraceus. Proc. Sot. Exp. Biol. 103, 875-877.

Botticelli, C. R., Hisaw, F. J., Jr., and Wotiz, H. H. (1961). Estrogens and progesterone in the sea urchin (Strongyiocentrotus fransiscanus) and pecten (Pecten hericius). Proc. Sot. Exp. Biol.

106, 887-889. Bouland, C., and Jangoux, M. (1988). Investigation of

the gonadal cycle of the asteroid Asterias rubens under static conditions. In “Echinoderm Biology, Proceedings of the Sixth International Echino- derm Conference” (R. D. Burke, R. D., P. V. Mladenov, P. Lanbert and R. L. Parsley, Eds.), pp. 169-175. A. A. Balkema, Rotterdam, The Netherlands.

Dickson, R. B., McManaway, M. E., and Lippman, M. E. (1984). Estrogen-induced factors of breast

cancer cells partially replace estrogen to promote tumor growth. Science 232, 1.540-1543.

Dieleman, S. J., and Schoenmakers, H. J. N. (1979). Radioimmunoassays to determine the presence of progesterone and estrone in the starfish Asterias

rubens. Gen. Comp. Endocrinol. 39, 534-542. Donahue, K. J. (1940). Occurrence of estrogens in the

ovaries of certain marine invertebrates. Endocri- nology 27, 149-1.52.

Donahue, J. K., and Jennings, D. E. (1937). The occurrence of estrogenic substances in the ovaries of echinoderms. Endocrinology 21, 875-877.

Giese, A. C., and Pearse, J. S. (1974). In “Reproduc- tion of Marine Invertebrates,” pp. 20-31. Aca- demic Press, New York.

Hagerman, D. D., Wellington, F. M., and Villee, C. A. (1956). Estrogens in marine invertebrates. Biol. Bull. 112, 180-183.

Hines, G. A., Watts, S. A., Sower, S. A., and Walker, C. W. (1990). Sex steroid extraction from echinoderm tissues. J. Liquid Chromatogr. 13(12), 2489-2498.

Hines, G. A., Watts, S. A., Voogt, P. A., and Walker, C. W. Androgen metabolism in somatic and germinal tissues of the sea star Asterias vulgaris.

Camp. Biochem. Physiol.. in press. Holmes, S. D., Spotts, G., and Smith, R. 6. (1986).

Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF) binding to its receptor. J. Biol. Chem. 261(9), 4076-4080.

Horwitz, K. B., Koseki, Y., and McGuire, W. L. (1978). Estrogen control of progesterone receptor in human breast cancer: role of estradiol and an- tiestrogen. Endocrinology 103, 1742-1751.

Katzenellenbogen, B. S., Norman, M. J., Eckert, R. L., Peltz, S. W., and Mangel, W. F. (1984.). Bioactivities, estrogen receptor interactions, and plasminogen activator-inducing activities of ta- moxifen, and hydroxytamoxifen isomers in MCF-7 human breast cancer cells. Cancer Res.

44, 112-119. Lawrence, J. M., and Lane, J. M. (1982). The utiliza-

tion of nutrients by post-metamorphic echinoderms. In “Echinoderm Nutrition” (M. Jangoux and J. M. Lawrence, Eds.), pp. 331-371. A. A. Balkema Press, Rotterdam, The Netherlands.

Licht , P. (1972). Environmental physiology of reptilian breeding cycles: Role of temperature. b;en. Comp. Endocrinol. (Suppl.) 3, 477-488.

Lowe, E. F. (1978). “Relationships between Bio- chemical and Caloric Composition and Reprodnc- tive Cycle in Asterias vulgaris (Echinodermata: asteroidea) from the Gulf of Maine.” Ph.D. dis- sertation, Univ. Maine Orono. 107 pp.

Mainwaring, W. I. P., Haining, S. A., and Harper, B. (1988). The functions of testosterone and its me- tabolites. In “Hormones and Their Actions Part

460 HINES ET AL.

1” (B. A. Cooke, R. J. B. King, and H. J. van der Molen, Eds.), pp. 169-196. Elsevier Science, New York.

Manning, N. J., and Kime, D. E. (1984). Temperature regulation of ovarian steroid production in the common carp, Cyprinus cnrpio L., in vivo and in

vitro. Gen. Comp. Endocrinol. 56, 376388.

McClintock, J. B., and Watts, S. A. (1990). The ef- fects of photoperiod on gametogenesis in the trop- ical sea urchin Eucidaris tribuloides (Lamarck) (Echinodermata: echinoidea) J. Exp. Mar. Biol. Ecol. 139, 175-184.

Pearse, J. S., and Eemisse, D. J. (1982). Photoperi- odic regulation of gametogenesis and gonadal growth in the sea star Pisaster ochraceus. Mar. Biol. 67, 121-125.

Pearse, J. S., and Walker, C. W. (1986). Photoperi- odic regulation of gametogenesis in a North At- lantic sea star, Asterias vulgaris. Int. J. Invert. Repro. Dev. 9, 71-77.

Pearse, J. S., Pearse, V. B., and Davis, K. K. (1986). Photoperiodic regulation of gametogenesis and growth in the sea urchin Strongylocentrotus purpuratus. J. Exp. Zool. 237, 107-118.

Pieau, C., Mignot, Th-M., Dorizzi, M., and Guichard, A. (1982). Gonadal steroid levels in the turtle Emys orbicularis L: A preliminary study in em- bryos, hatchlings, and young as a function of the incubation temperature of eggs. Gen. Comp. En- docrinol. 47, 392-398.

Schoenmakers, H. J. N. (1979). In vitro biosynthesis of steroids from cholesterol by the ovaries and pyloric caeca of the starfish Asterias rubens. Camp. Biochem. Physiol. 63B, 179-184.

Schoenmakers, H. J. N., Colenbrander, P. H. J. M., and Peute, J. (1977). Ultrastructural evidence for the existence of steroid synthesizing cells in the ovary of the starfish, Asterias rubens (Echinoder- mata). Cell Tissue Res. 182, 275-279.

Schoenmakers, H. J. N., and Dieleman, S. J. (1981). Progesterone and estrone levels in the ovaries, pyloric caeca and perivisceral fluid during the annual reproductive cycle of the starfish Asterias

rubens. Gen. Camp. Endocrinol. 43, 63-70.

Schoenmakers, H. J. N., and Voogt, P. A. (1980). In

vitro biosynthesis of steroids from progesterone by the ovaries and pyloric caeca of the starfish As- terias rubens. Gen. Camp. Endocrinol. 41,408-416.

Schoenmakers, H. J. N., and Voogt, P. A. (1981). In vitro biosynthesis of steroids from androstenedione by the ovaries and pyloric caeca of the starfish Asterias rubens. Gen. Camp. Endocrinol. 45, 242-248.

Shirai, H., and Walker, C. W. (1988). Chemical control of asexual and sexual reproduction in echinoderms. In “Endocrinology of Selected Inverte- brate Types,” pp. 4.53-476. A.R. Liss, New York.

Smith, F. F., and Walker, C. W. (1986). Biochemical changes in the composition of the testes during spermatogenesis in the sea star Asterias vulgaris.

J. Exp. Zool. 237, 351-364. Vignon, R., Chambon, D., Derocq, D., and Rochefort,

H. (1982). Estrogens stimulate cell proliferation and induce secretory proteins in a human breast cancer cell. J. Clin. Endocrinol. Metub. 55, 276-283.

Voogt, P. A., and Dieleman, S. J. (1984). Progester- one and estrone levels in the gonads and pyloric caeca of the male sea star Asterias rubens: A com- parison with the corresponding levels in the female sea star. Camp. Biochem. Physiol. 79A, 635- 639.

Voogt, P. A., Broertjes, J. J. S., and Oudejans, R. C. H. M. (1985). Vitellogenesis in sea stars: Physiological and metabolic implications. Camp.

Biochem. Physiol. 80(2), 141-147. Walker, C. W. (1974). Studies on the reproductive

systems of sea stars. I. The morphology and his- tology of the gonad of Asterias vulgaris. Biol.

Bull. 147, 661-667.

Walker, C. W. (1980). Spermatogenic columns, somatic cells, and the microenvironment of germinal cells in the testes of asteroids. J. Morphol. 166,

81-107.

Watts, S. A., and Lawrence, J. M. (1990). The effect of reproductive state, temperature, and salinity on DNA and RNA levels and activities of metabolic enzymes of the pyloric caeca in the sea star Luidia

clathrata (Say). Physiol. Zool. 63, 1196-1215. Watts, S. A., D., Roy, J., and Walker, C. W. (1990b).

Omithine decarboxylase exhibits negative ther- mal modulation in the sea star Asterias vulgaris: Potential regulatory role during temperature- dependent testicular growth. Biol. Bull. 179, 159-

162.

Watts, S. A., Hines, G. A., Lee, K. Jaffurs, D., Roy, J., Smith, F. F., and Walker, C. W. (199Oa). Sea- sonal patterns of omithine decarboxylase activity and levels of polyamines in relation to the cytol- ogy of germinal cells during spermatogenesis in the sea star Asterias vulgaris. Tissue Cell 22(4),

43.5447. Wicklow, B. J., Borror, A., and Walker, C. W. (1985).

Spermatophagy: A unique symbiosis between Or-

chitophyru (Ciliophora) and Asterias (Echinoder- mata). Trans. Amer. Micro Sot. 104, 109.

Xu, R. A. (1990). “Studies on the Reproductive Biol- ogy of the Forcipulate Starfish Sclerasterias mol-

lis (Echinodermata: Asteroidea).” Ph.D. disserta- tion, Univ. Otago, New Zealand, 144 pp.

Xu, R. A., and Barker, M. F. (1990). Annual changes in the steroid levels in the ovaries and the pyloric caeca of Sclerasterias mollis (Echinodermata: As- teroidea) during the reproductive cycle. Camp. Biochem. Physiol. 95A(l), 127-133.

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