Dynamics of excretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate, and of the glucuronides of testosterone and 17β-oestradiol, by urine of reproductively mature male and female rainbow trout (Oncorhynchus myklss)

Levels of sulphated 17α20β-dihydroxy-4-pregnen-3-one (17,20 β -P; the oocyte maturation inducing steroid) in blood plasmas of sexually mature male and female rainbow trout Oncorhynchus mykiss , were very low in comparison to those of the free steroid. However, relatively large amounts were found in urine of both sexes.
Catheters were inserted into the urinary bladders of unovulated and ovulated females and of ripe-running males, and the fish then placed in spawning channels. Three-hourly urine samples were collected between 09.00 and 18.00 hours and then a 15-h sample between 18.00 and 09.00 hours the next morning. Measurements were made of 17,20 β -P-sulphale, testosterone glucuronide (T-G) and 17 β -oestradiol glucuronide (E2-G). In females, the highest rates of excretion of E2-G, T-G and 17,20 β -P-sulphate were found in unovulated, ovulating and ovulated females, respectively. The rates of excretion of 17,20 β -P-sulphate, T-G and E2-G in ovulated females were unaffected by the presence of a male. id males, however, there was a sharp increase in the rate of excretion of 17,20 β -P-suiphate and T-G in fish which were paired with an ovutated (nesting) female. A similar increase was found in males injected with male trout pituitary extract.     

Female rainbow trout urine contains a pheromone which causes a rapid rise in plasma 17,20β-dihydroxy-4-pregnen-3-one levels and milt amounts in males

Previous studies have shown that spermiating male rainbow trout respond to the presence of female urine in the water with significant increases in plasma levels of gonadotrophin II, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and testosterone. The present results show that males only need a single brief exposure to female urine in order to respond; levels of 17,20β-P rise significantly within 1 h of exposure, and peak between 3 and 4 h. Also, milt amounts increase significantly following exposure of males to female urine. Levels of 17,20β-P are also related positively to the amount of female urine to which the males are exposed. Furthermore, when live females are placed, out of physical and visual contact, in the same tank as males, levels of 17,20β-P rise in the same way as in males which are exposed to female urine. However, if females are fitted with urinary catheters (which drain the urine outside the tank), males respond more slowly. These results indicate that urine is the main source of the male 'priming' pheromone.     

Plasma levels of C18-, C19- and C21-steroids in captive and feral female sea bass

Morphometric analysis of the gonads of sea bass Dicentrarchus labrax revealed that captive fish matured 1 month later than feral fish, but levels of gonadal steroids were identical in both groups at the same stage of sexual development. 17β-oestradiol (E₂) (up to 3 ng ml-1) and testosterone (T) (up to 4 ng ml-1) were highest during the gametogenetic period while 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) (free and sulphated) were maximal during the spawning period. Free 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was very low and did not change (c. 0·5 ng ml⁻¹) while 17,20β-P-sulphate increased during the spawning period in both groups (up to 2 ng ml⁻¹). In contrast cortisol levels were higher in captive fish and increased during the spawning period (up to 100 ng ml⁻¹). These results suggest that captivity delays vitellogenesis and spawning in sea bass without affecting the final levels of the gonadal steroids and further indicates a role for cortisol in the latter period. The increased levels during the spawning period suggests a pheromonal role for 17,20β-P-sulphate and 17,20β,21-P-conjugates and the involvement of 17,20β,21-P in final ooccyte maturation.     

Stimulatory and inhibitory effects of testosterone on testes in Atlantic salmon male parr

Immature 1-year-old Atlantic salmon Salmo salar parr were implanted with Silastic capsules of different sizes filled with testosterone (T). Testosterone had both positive and negative effects on testicular weights, spermatogenesis and steroidogenesis. The positive effects: higher incidence of males with enlarged gonads, spermiation, and high plasma levels of 11-ketotestosterone (11-KT) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), were most pronounced in males treated with small T capsules. The negative effects: suppression of gonadal development and depressed plasma levels of 11-KT and 17,20β-P compared with mature controls, were most evident in fish treated with large T capsules.     

Male priming pheromone is present in bile, as well as urine, of female rainbow trout

The bile of sexually mature female rainbow trout Oncorhynchus mykiss has pheromonal activity which causes a significant increase in concentrations of 17,20β-dihydroxy-4-pregnen-3-one in the plasma of males. Bile from male trout is inactive. The activity in the female bile binds to octadecylsilane and can be eluted with methanol. The synthetic bile acids, taurocholic acid and taurolithocholic acid, are inactive.     

Lack of species-specific primer effects of odours from female Atlantic salmon, Salmo salar, and brown trout, Salmo trutta

We exposed, in two successive spawning seasons, individually placed precocious male Atlantic salmon ( Salmo salar ) and brown trout ( Salmo trutta ) parr to odour stimuli (ovarian fluid and urine mix) from ovulated conspecific or heterospecific anadromous females. Atlantic salmon parr had significantly higher plasma concentrations of the hormones 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), 11-ketotestosterone (11-KT) and testosterone (T) after exposure to odours from conspecific females or from brown trout females compared to parr exposed to a control solution (0.9% NaCl). We did not observe any significant differences between the hormone levels in salmon parr exposed to the two female odours. The salmon parr exposed to conspecific odours had significantly higher volumes of strippable milt compared to the controls, but we did not find any significant differences when comparing the effect of the two female odours. Brown trout parr had significantly higher plasma 17,20β-P levels following exposure to heterospecific female odours compared to control males, but there was no significant difference between males exposed to the different female odours. We did not observe any significant differences in plasma levels of T and 11-KT and in milt volumes between exposed and control trout. Taken together, the results from both tested species indicate that the potency of heterospecific stimuli in stimulating increased plasma sex steroid hormone levels in male parr was as strong as stimuli from conspecific females. The results are discussed in connection to observed hybridisation between the two sympatric species.     

The role of F-series prostaglandins as reproductive priming pheromones in the brown trout

Electrophysiological studies demonstrated that the olfactory epithelium of mature male brown trout Salmo trutta parr was acutely sensitive to F-series prostaglandins (PGFs) PGF_1α and PGF_2α, with detection threshold concentrations of 10⁻¹¹ M. The olfactory epithelium was also sensitive to the PGF metabolite 15-ketoPGF_2α (threshold 10⁻⁸ m), but did not detect a further metabolite, 13,14,-dihydro-15-ketoPGF_2α Immature brown trout did not detect any of the prostaglandins tested. Exposure of mature male brown trout parr to waterborne PGF_1α and PGF_2α (concentration 10⁻⁸ m), resulted in significant increases in levels of expressible milt and the plasma concentrations of 17,20β-dihydroxy-4-pregnen-3-one, testosterone and 11-ketotestosterone. The olfactory epithelium of both immature and mature male brown trout parr was sensitive to the urine and ovarian fluid from ovulated female brown trout. Exposure of mature male brown trout parr to ovarian fluid resulted in an increase in the levels of plasma 17,20β-dihydroxy-4-pregnen-3-one whilst exposure to urine increased the levels of expressible milt. In addition, PGF_2α was found to be present within both the urine and ovarian fluid of mature female brown trout. It is suggested that the F-series prostaglandins have a role as priming pheromones in male brown trout.     

Seasonal changes in serum steroid hormones in a protandrous teleost, the sobaity (Sparidentex hasta Valenciennes)

Serum concentrations of 11-ketotestosterone, 11β-hydroxytestosterone, testosterone, testoster-one glucuronide, oestradiol and 17,20β -dihydroxy-4-pregnen-3-one (17,20β -P) were measured in the sobaity at monthly intervals through their second breeding season. Concentrations of the 11-oxygenated androgens in the males and of oestradiol in the females peaked during the spawning season in January-February, while maximum levels of testosterone were found in the summer when these steroids were low. Testosterone glucuronide showed two peaks, one in the post-spawning period as oestradiol and the 11-oxygenated androgens were falling and the other coincident with the summer peak of testosterone. 17,20β -P was detectable in only one male and one female fish in February. Serum concentrations of 11-oxygenated androgens are more reliable than those of oestradiol for determining the sex of sobaity, and may also be used as indicators of the occurrence of sex reversal. The seasonal pattern of serum steroids correlated well with the changes of sexual status of the gonads during regression and recrudescence observed histologically and suggests that oestradiol may be involved in the sex inversion of this species.     

17α,20β,21-Trihydroxy-4-pregnen-3-one: the probable maturation-inducing steroid of the Lusitanian toadfish

17,20β,21-Trihydroxy-4-pregnen-3-one (17,20β,21-P) was identified as the major metabolite of incubations of Lusitanian toadfish Halobatrachus didactylus ovarian follicles with [³H]-17hydroxyprogesterone. The potency of several steroids in inducing germinal vesicle breakdown of follicle-enclosed oocytes of Lusitanian toadfish was systematically examined by using an in vitro germinal vesicle breakdown (GVBD) bioassay. 17,20β-Dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-P, two confirmed maturation-inducing steroids (MIS) in teleosts, were the most potent in inducing GVBD with ED₅₀s ranging between 9 and 271 nM. Structure-activity relationships followed similar patterns to what has been observed in similar bioassays, i.e. a vital requirement for 17- and 20β-hydroxyl groups in C21 steroids and a reduction in activity of 14 and 5–6%, respectively, for 5-pregnene and 5β-pregnanes compared to 4-pregnenes. Corticosteroids, testosterone and 17β-oestradiol were ineffective. Folliculated oocytes stimulated by pituitary homogenate produced 17,20β,21-P from endogenous substrates in amounts one order of magnitude higher than 17,20β-P. These results strongly support the hypothesis that 17,20β,21-P is the likely MIS in this species.     

Plasma levels of gonadotropin and 17α,20β-dihydroxy-4-pregnen-3-one in relation to spawning behaviour of rainbow trout, Oncorhynchus mykiss(Walbaum)

Manipulation of the opportunity to spawn was used to investigate the relationship between endocrine events, egg viability and spawning behaviour in female rainbow trout. Females were prevented from spawning by isolating them from males and gravel for up to 21 days after ovula- tion. Blood samples were taken before pairing with a male, at the onset of nesting activity, and at the completion of spawning. Plasma hormone levels of gonadotropin (GtH) and 17α,20β-dihydroxy-4-pregnen-3-one (17,2OP) were measured by specific radioimmunoassays. There were no qualitative or quantitative differences in the spawning behaviour of females paired on the day of ovulation or 7. 14, or 21 days after ovulation. There was a general decrease in the viability of eggs with increasing retention times. In females paired on the day of ovulation, or after 7 or 14 days, GtH levels increased with the onset of nesting behaviour and declined as fish reached the post-spawning condition. By day 21, GtH levels before pairing were significantly higher than prepairing levels in the other three treatment groups, and did not increase at the onset of nesting, or decrease in post-spawning fish. Plasma 17,20P remained high in prepairing and nesting samples of all four groups and declined to low levels in fish in post-spawning condition. In females paired on the day of ovulation there was a significant increase in 17,20P from the prepairing to the nesting stage. These results suggest that 17,20P plays a key role in the synchronization of behavioural and maturational events at the time of spawning.     

Effects of capture and recovery on plasma levels of cortisol, lactate and gonadal steroids in a natural population of rainbow trout

Rainbow trout were captured by angling from a run of spawning fish on the Tongariro River in northern New Zealand, to examine the effects of catch and release angling on stress and reproductive parameters. Fish were blood sampled immediately after capture at playing times of <5 or 15 min, or after 1 or 24 h of recovery in stream enclosures. Plasma samples were assayed for cortisol (F), lactate, testosterone (T), 17β-oestradiol (E₂), and 17a,20β-dihydroxy-4-pregnen-3-one (17,20βP). Plasma F levels were similar to those of hatchery stocks of rainbow trout, at capture, and became significantly elevated 1 h after capture. Plasma F was still clevated in some fish 24 h after capture. Plasma lactate levels began to increase 15 min after capture, were further elevated 1 h after capture, and had returned to normal 24 h after capture. We proposed that metabolic recovery had occurred but that some animals were still experiencing some degree of stress, possibly in response to holding conditions in the river. Both plasma T and E₂ were depressed 24 h after capture, whereas there was no change in plasma 17,20βP. This is consistent with other findings showing that acute stress is associated with depression of plasma levels of T and E₂. There was no mortality as a result of capture or any of the handling protocols. We conclude that catch and release angling will result in negligible mortality, but may have an inhibitory effect on some reproductive processes.     

Stress-induced changes in concentrations of plasma sex steroids in black bream

Cortisol levels of black bream Acanthopagrus butcheri at capture did not change with time of day, gonadal stage or season and were 1·9±0·2 and 2·8±0·4 ng ml⁻¹ for male and female fish, respectively. Confinement resulted in significantly elevated cortisol levels at all time periods; however, levels after 24 h of confinement were significantly lower than peak cortisol levels (15 min for males and 1 h for females). Confinement stress resulted in reduced levels of 17β-oestradiol (E2) and testosterone (T) within 1 h in sexually mature females. In mature males, suppression of T and 11-ketotestosterone (11KT) occurred after 30 min and 6 h of confinement, respectively. The relationship between confinement stress and levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20β P) was more complex, with levels in males being elevated after 15 min and 24 h and suppressed after 6 h of confinement. In contrast, 17, 20β P levels in females were elevated after 1 h of confinement. In regressed females, plasma E₂ and T concentrations were low at capture and were not affected by confinement stress whereas plasma 17, 20β P was elevated within 1 h. This study indicates that stress exerts a rapid inhibitory effect on gonadal steroidogenesis.     

Reproductive priming in mature male Atlantic salmon parr exposed to the sound of redd cutting

The sounds of a wild female Atlantic salmon cutting a redd were associated with significant increases in the levels of plasma 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) and of expressible milt in mature male parr, comparable with levels of the steroid and milt produced in parr exposed to the priming pheromone, prostaglandin F_2a. Hence auditory cues may have a significant role in synchronising reproductive physiology in Atlantic salmon.     

Pheromone of male blue gourami and its effect on vitellogcnesis, steroidogenesis and gonadotropin cells in pituitary of the female

Female Trichogaster trichopterus were exposed to aquarium water in which males had built nests. Gonadotropin cells in the pituitary gland, and exovitellogenesis and steroidogenesis in the ovary were studied. In females in which the percentage of oocytes in vitellogenesis (%V) was low initially, it rose significantly in comparison with an unexposed control group. In females in which the %V was higher initially, it increased further, and in addition a significant percentage of oocytes reached maturation. Thin layer chromatography, using the precursors ³H-pregnenolone and ¹⁴progesterone, revealed high yields of the steroids 17β-estradiol (E₂), 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P), 5β-pregane,3α,17α,20β-triol (5β-P-triol) and 11-ketotestosterone (11KT) in both experimental groups. Significant differences were found in E₂, 17,20-P and 5β-P-triol between the test and control groups. The immunoresponse of GtH-producing cells in the pituitary of the females maintained in nest water was lower than in the control group, suggesting that the GtH was secreted from the cells, which would explain the vitellogenic and steroidogenic changes found in the ovary.     

Shift in Steroidogenesis in the Ovarian Follicles of the Goldfish (Carassius auratus) during Gonadotropin-Induced Oocyte Maturation

Both partially purified chum salmon gonadotropin and 17α-hydroxyprogesterone stimulated in vitro production of testosterone by postvitellogenic follicles of goldfish ( Carassius auratus ). Chum salmon gonadotropin further enhanced the conversion of exogenously supplied 17α-hydroxyprogesterone to 17α, 20β-dihydroxy-4-pregnen-3-one. The increased medium concentrations of 17α, 20β-dihydroxy-4-pregnen-3-one were associated with the induction of final oocyte maturation.
The capacity of postvitellogenic follicles to produce steroids in response to exogenous 17α-hydroxyprogesterone was examined in females at various stages of final oocyte maturation following the administration of human chorionic gonadotropin in vivo combined with elevation of holding temperature. The maximum production of testosterone in response to 17α-hydroxyprogesterone was obtained in follicles from initial controls. In contrast, 17α 20β-diOHprog production was very low in initial controls and markedly increased during oocyte maturation (3–6 hr following injection), followed by a significant decrease in follicles collected at 15 hr. Estradiol-17β production by the follicles was very low at any stages of gonadotropin-induced oocyte maturation. These results suggest that gonadotropin-induced shift in the biosynthetic pathway in the follicle from the secretion of predominantly testosterone to 17α, 20β-dihydroxy-4-pregnen-3-one secretion is a prerequisite step for the induction of oocyte maturation in goldfish.     

Prolonged-release gonadotrophin-releasing hormone analogue implants enhance oocyte final maturation and ovulation, and increase plasma concentrations of sulphated C21 steroids in North Sea plaice

Reproductively mature female plaice were implanted with or without 50 μg of gonadotrophin-releasing hormone analogue (GnRHa), suspended in either coconut oil or methacrylate resin. The weight of the GnRHa-treated fish increased significantly (due to hydration of the oocytes) and reached a peak between 10 and 14 days. The fish produced several batches of eggs, which were consistently bigger than those produced by control fish. Plasma concentrations of free 17β-oestradiol and glucuronidated testosterone rose briefly (4 days) in response to the GnRHa, but then fell continuously till the end of the experiment (20 days). Plasma concentrations of sulphated 5β-pregnane-3α,17,20β-triol and 5β-pregnane-3β,17,20β-triol (which are putative metabolites of 17,20β-dihydroxy-4-pregnen-3-one, the oocyte maturation-inducing steroid) increased significantly at 4 days and reached a peak between 12 and 16 days. Concentrations were still very elevated on day 20. Plasma concentrations of sulphated 3α,17,21-trihydroxy-5β-pregnan-20-one showed a slight increase on day 4 but did not change thereafter. There was a highly significant difference in the amounts of GnRHa released into the bloodstream by the two methods of administration on day 4. However, this was not matched by significant differences in the concentrations of any of the steroids.     

Hormonal regulation of the European sea bass reproductive cycle: an individualized female approach

Fifteen tagged female sea bass Dicentrarchus labrax were sampled weekly from September to April and plasma vitellogenin (VTG), testosterone (T), 17β-estradiol (E₂), and two potential maturation inducing steroids (MISs): 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) and 17,20β,21-trihydroxy-4-pregnen-3-one (20βS) assayed. An oocyte sample was obtained via intraovarian cannulation at each sampling time from every female and the stage of development of the most advanced clutch of oocytes determined and related to VTG and hormone plasma levels for each female. The mean number of ovulations per female was 1·75+0·25 when those females that did not present ovulations were excluded and up to 4 ovulations detected in some females. The highest plasma levels of T ( c. 6 ng ml^-1) were observed during postvitellogenesis and the beginning of maturation while maximum plasma levels of E2 (>5 ng ml^-1) were obtained during late vitellogenesis. VTG plasma levels increased throughout vitellogenesis peaking ( c. 2·5 mg ml^-1) at postvittelogenesis. For the first time significant changes of plasma progestogens were detected in European sea bass during the sexual cycle. The highest plasma level of 17,20βP ( c. 1·1 ng ml^-1) was observed during postvitellogenesis while the highest level of 20βS ( c. 1·4 ng ml^-1) coincided with final maturation. These results suggest that 17,20βP and 20βS play a role in the early and final maturation, respectively, in the European sea bass.     

The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

There is a rapid shift in the steroidogenic pathway from androgen to progestogen production in spawning male common carp, Cyprinus carpio. Experiments were conducted to determine the mechanism regulating this shift using in vitro cultures of testicular fragments and isolated sperm of spermiating male carp. The levels of 11-ketotestosterone (11-KT) continually increased for 48 h with or without gonadotropin (GtH) stimulation, suggesting that 11-KT is the principal androgen produced by carp testes. Ovine prolactin (oPRL) enhanced GtH-stimulated 11-KT production, but by itself had no effect. Gonadotropin, carp pituitary extract, and pregnenolone all enhanced the production of 11-KT, testosterone (T), and 17 alpha-hydroxyprogesterone (17-P) in a dose-dependent manner. No 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was detected in response to any of these agents; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) was not measured. Both 17,20 beta-P and 17,20 alpha-P inhibited 11-KT production in a dose-dependent manner in the presence of either GtH, 17-P, or T. Isolated sperm and testicular fragment preparations both produced 17,20 beta-P and approximately tenfold more 17,20 alpha-P when incubated with 17-P. Only testicular fragments, however, produced 11-KT. We conclude that androgen synthesis occurs only within somatic cells of common carp testes. GtH, and perhaps PRL, stimulates the production of steroid precursors that, under normal physiological conditions, are metabolized to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid metabolism by ovarian follicles of the sea bass Dicentrarchus labrax

Ovarian samples from fear sea bass, Dicentrarchus labrax, were collected for the in vitro incubations during the spawning period. Follicles with fully developed vitellogenic oocytes showing central germinal vesicle (stage I follicles) and follicles with oocytes showing initial germinal vesicle migration (stage II follicles) were treated with either (1) 20 μg sea bass hypophysis plus 50 ng 17-hydroxyprogesterone (17-P), (2) 20 μg hypophysis alone, (3) 50 ng 17-P alone and (4) media alone. Structure-activity experiments used stage II follicles treated with several dosages (0.1, 1.0 and 10.0 ng/ml) of either 17-P, 17,20β-P, or 17,20β,21-P. Free and conjugated (sulfates and glucuronides) levels of the established teleost oocyte maturation inducing steroids (MIS), i.e. 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) were measured in the incubation media by high performance liquid chromatography. Our results show that the synthesis of free and conjugated 17,20β-P is constant (0.1–0.2 ng/ml) in all incubates. In contrast, the synthesis of free and conjugated 17,20β,21-P is higher in incubates containing stage II follicles (up to 5 ng/ml) than in those having stage I follicles (up to 3 ng/ml; P<0.01). Structure-activity data reveal that 17-P is not effective at inducing in vitro germinal vesicle breakdown whereas both 17,20β-P and 17,20β,21-P are equally potent and highly effective. These results demonstrate that 17,20β-P and 17,20β,21-P are synthesized in vitro by follicles of sea bass and that sulfation is the main route for the metabolism of the C₂₁-steroids in riper follicles. The highest levels of 17,20β,21-P, found in incubates containing stage II follicles, points at 17,20β,21-P, rather than 17,20β-P, as the most probable MIS in sea bass, nonetheless, this hypothesis requires further confirmation.     

Plasma concentrations of ovarian steroids in relation to oocyte final maturation and ovulation in female plaice sampled at sea

Blood plasma concentrations of free 17 β -oestradiol, free testosterone and glucuronidated testosterone were strongly positively related to the percentage of vitellogenic oocytes remaining in the ovaries of plaice Pleuronectes platessa caught at sea–being at their highest in pre-spawning (stage IV) females (i.e. those in which the oocytes were close to fully grown, but had not yet entered the stage of final maturation). In contrast, the concentrations of free and sulphated 17,20 β -P, 3α aL ,17, 20 β -P-5 β , and 3 α ,17,21-P-5 β were at their lowest in stage IV females. Free 17,20 β -P (the putative maturation-inducing steroid) became only slightly elevated (less than twofold) during spawning (i.e. in stage V and VI females with hydrated and/or ovulated eggs). Sulphated 17,20 β -P and 3 α ,17,21-P-5 β became slightly more elevated (three- to fourfold). However, sulphated 3 α , 17,20 β -P-5 β concentrations increased 30-fold and were at their highest in fish in which only 40% of vitellogenic oocytes remained in the ovaries. Sulphated 17,20 β -P, 3 α , 17, 20 β -P-5 β and 3 α ,17,21-P-5 β concentrations were significantly positively related to hyaline oocyte batch size; and sulphated 17,20 β -P and sulphated 3 α , 17,20 β -P-5 β were significantly negatively related to the degree of hydration of the hyaline oocytes. None of the steroid concentrations, however, was related to the time of capture. More ovulated females were found in the afternoon than at any other time of the day.