Reproductive biology and endocrine regulation of final oocyte maturation of captive white bass

The ovarian development, and plasma levels of gonadotropin II (GtH II) and sex-steroid hormones at the end of vitellogenesis were examined in captive white bass Morone chrysops. The changes in plasma hormone levels and oocyte morphology associated with gonadotropinreleasing hormone agonist (GnRHa)-induced final oocyte maturation (FOM) were studied. Although plasma 17β-oestradiol (E₂) and oocyte diameter increased, there were no changes in GtH II, testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) or 17,20β,21-dihydroxy-4-pregnen-3-one (17,20β,21-P) in non-hormone-treated females, and no FOM was observed. Treatment with a sustained-release GnRHa delivery system (GnRHa implant) induced two FOM cycles separated by about 24 h, with the release of approximately equal numbers of eggs in each spawn. Plasma GtH II levels were elevated significantly throughout FOM, reaching a maximum of 9·07 ± 1·55 ng ml^?1 in ovulated fish. Both plasma E₂ and T increased soon after the GnRHa treatment, but E₂ declined in fish undergoing germinal vesicle (GV) migration. Plasma T increased further during FOM (7·55 ± 2·87 ng ml^?1), but declined precipitously at ovulation. A surge in plasma 17,20β-P and 17,20β,21-P (4·11 ± 0·97 ng ml^?1 and 3·10 ± 0·77 ng ml^?1, respectively) was observed in females undergoing GV breakdown (GVBD). Based on the involvement of different sex-steroid hormones, FOM was separated into two stages. Early FOM included lipid-droplet coalescence and GV migration, and was associated with elevations in plasma GtH II and T. Late FOM included GVBD and yolk-globule coalescence, and was associated with elevations in plasma GtH II, 17,20β-P and 17,20β,21-P. The results of this study point to the absence of a surge in plasma GtH II as the missing link in the reproductive axis responsible for the failure of captive white bass to undergo FOM at the end of vitellogenesis. Sustained elevation of plasma GtH II via treatment with a GnRHa implant induced two consecutive spawns with an overall egg production two- to eightfold higher than previously obtained from captive broodstocks, and similar to annual egg production Values reported for wild fish.     

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.     

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.     

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.     

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.     

Amino acid cues emanating from Pacific salmon eggs and ovarian fluid

The eggs of salmonid fishes are an important food source for many aquatic predators that detect eggs using olfaction. Moreover, chemicals from eggs and ovarian fluid aid sperm cells in detecting and locating eggs for fertilization, and ovarian fluid is attractive to conspecific males. Thus chemicals from eggs and ovarian fluid may facilitate reproduction but may also attract egg predators. The authors sampled mature females of three Pacific salmon species – Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch) and sockeye (Oncorhynchus nerka) – and determined the proportional representation of amino acids, potent fish odorants, from their eggs and ovarian fluid (Chinook and coho salmon only). They then tested juvenile coho salmon, an egg predator, for responses to ovarian fluid and egg odours using the electro-olfactogram (EOG) recording technique. The amino acid compositions of the salmon species were significantly and positively correlated with each other, and the interspecific differences were comparable to those between individuals of the same species. The egg water samples were, on average, dominated by lysine, alanine and glutamine (12.6%, 12.4% and 10.9%, respectively). The ovarian fluid samples were dominated by lysine (20.5%), followed by threonine (9.7%), glycine (9.2%) and arginine (8.8%). EOG recordings demonstrated the ability of juvenile coho salmon to detect the chemical traces of eggs and ovarian fluid. It is concluded that salmon eggs are a potent source of odours for potential predators but likely not highly differentiated among salmon species.     

Gonadal development and sex steroids in a female Arctic charr broodstock

Gonadal development and plasma levels of sex steroids were investigated in female Arctic charr at 3-week intervals over a 12-month period. Circulating levels of oestradiol-17β (E₂), testosterone (T) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) were measured by radioimmunoassay, and gonadal status assessed through histological examination and measurement of gonadosomatic index (GSI) and frequency distribution of oocyte size-classes. Gonadal recrudescence during March-July was characterized by modest but insignificant increases in plasma levels of E₂ (2–4 ng ml^?1) and T (2–5 ng ml^?1) and recruitment of oocytes into yolk accumulation. Only a small and insignificant rise in GSI and no apparent increase in oocyte diameter occurred during this period, indicating that the rate of yolk formation and oocyte growth was low. Following transformation from stage V (peripheral yolk granule stage) to stage VI (yolk granule migration stage) in late July, the vitellogenic oocytes entered a phase of rapid growth which resulted in a marked rise in GSI until ovulation commenced in late September. Gonadal growth during this period was accompanied by increases in plasma levels of E₂ and T which peaked at 11 ± 1 (mid-August) and 71 ± 5ng ml^?1 (late September), respectively. The levels of both steroids dropped rapidly during final maturation and ovulation, followed by a surge in plasma levels of 17,20β-P which peaked at an average of 74 ± 17 ng ml^?1 in early October. All three steroids returned to basal levels within a month after ovulation, and all steroids, except E₂, remained low until March of the following year. A slight increase in E₂ detected in February and March during the second season may have been associated with recruitment into vitellogenesis of a new generation of oocytes. It is suggested that the abrupt increase in vitellogenesis in late July may reflect a condition-dependent decision to proceed with maturation, once the energy reserves have been repleted during spring-early summer.     

The effect of four C21-steroids on oocyte maturation in Siberian sturgeon (<Emphasis Type="Italic">Acipenser baeri</Emphasis> Brandt)

The effect of four C21-steroids, progesterone (P₄), 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), 17,20β,21-trihydroxy-4-pregnen-3-one (20βS) and 11-desoxycortisol (S), on in vitro oocyte maturation in Siberian sturgeon (Acipenser baeri Brandt) females was demonstrated using short-term (5 and 30 min) exposures of ovarian follicles to steroid solutions followed by incubation in steroid-free medium. The study aimed to find out which of the four candidates for a maturationinducing steroid (P₄, 17,20βP, 20βS or S) induces a fastest germinal vesicle breakdown (GVBD) in oocytes of Siberian sturgeon. Dissolution of the oocyte nucleus or GVBD was taken as a criterion of oocyte maturation. Dose-response profiles of hormone activities as well as effects of the hormones under short-term exposures of follicles to their equal doses were compared. P₄ was found to be a most active GVBD inducer compared to other C21-steroids, S was the second in its activity, whereas 17,20βP and 20βS were less efficient. A comparison of the present and previously obtained data on the dynamics of C21-steroids in vivo and their effect on ovarian follicles in vitro indicates an important role of the above hormones, particularly P₄ and 20βS, in the regulation of the final stage of oocyte maturation in sturgeons.     

Ovarian development and serum sex steroid and vitellogenin profiles in the female cultured sturgeon hybrid, the bester

Changes in serum levels of estradiol-17/J (E₂), testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and vitellogenin (VTG), in cultured adult female bester were examined in relation to ovarian development during a 1-year sampling period. Considerable variations in oocyte development were found among fish. Oocytes at previtellogenic stage (≥0–6mm in diameter) generally started to develop concomitantly with the degeneration of the first batch of oocytes. In vitellogenic individuals, ovaries were comprised of more advanced oocytes with diameter ranging from 0–6 to 2–6 mm and in the post-vitellogenic class, oocytes attained their largest size (>2–6mm) while the germinal vesicle was migrating towards the animal pole. Oocytes with a migrating nucleus were maintained during the winter period and massive degeneration started in April–May without germinal vesicle breakdown (GVBD) or ovulation occurring. Seasonal changes in E₂, T and VTG levels were well correlated with the advancement of oogenesis. Their levels increased during vitellogenesis, whereas in the post-vitellogenic (migratory nucleus) stage the levels of E₂ declined from 2–4 ng ml ^?1± to 1–2 ng ml ^?1 and VTG from 4–10 mg ml ^?1 to 0.–0.5 mg ml ^?1 while T levels remained high (50–60 ng ml ^?1). In contrast, serum levels of 17,20?-P were constantly low (less than 0.2 ng ml^?L) throughout the reproductive cycle. These results indicate that the time appropriate for induction of artificial reproduction would be from October–November to April–May when the oocytes are in the late Stages of the development.     

Electrophoretic patterns of yolk proteins throughout ovarian development and their relationship to vitellogenin in the rainbow trout,Oncorhynchus mykiss

• 1.1. Electrophoretic patterns of yolk proteins were investigated throughout ovarian development and their relationship to vitellogenin determined in a pulse-chase experiment with ³H-vitellogenin.
• 2.2. Using a radioimmunoassay for vitellogenin, vitellogenin/yolk protein products of vitellogenin were detected in follicles throughout ovarian development and in ovulated eggs.
• 3.3. The majority of yolk proteins in follicles measuring less than 1.0 mm in diameter appeared to be derived from sources other than vitellogenin. In contrast, in the larger follicles all of the major yolk proteins detected were derived from vitellogenin.
• 4.4. Pulse-chase with ³H-vitellogenin revealed that all of the major yolk proteins in 3.0 mm follicles were derived from vitellogenin. The major peptides eluted with molecular masses of 110 and 30 kDa under non-reducing conditions (these are very likely to represent lipovitellin 1 and lipovitellin 2), and 88, 22, 16, and 12 kDa under reducing conditions.
• 5.5. There were no apparent differences in the major yolk proteins in ovulated eggs compared to those in vitellogenic follicles, indicating that no extensive proteolysis of these proteins had occurred during maturation and/or ovulation.

     

Effects of indomethacin and prostaglandins on the spawning behaviour of female goldfish

In goldfish, injection of ovulated eggs (from donor females) through the ovipore and into the ovarian lumen of females with vitellogenic oocytes induces spawning behaviour within several hours. Intraperitoneal (i.p.) injection of indomethacin (IM), 10 μg/g, either 10 h prior to, or coincident with, injection of ovulated eggs, completely inhibits the onset of spawning behaviour. IM injection similarly terminates ongoing spawning behaviour induced by egg injection. PGF_2α (5 μg/g; i.p. injection) restores spawning behaviour of egg-injected, IM-blocked fish; at the same dosage, PGE₂ is marginally effective and PGE₁ is without effect. As PGF_2α and PGE₂ also induce spawning behaviour in females which have not been injected with ovulated eggs, it is suggested that distension of the oviduct following ovulation or egg injection results in the release of PG which then acts in some way to induce spawning behaviour. The ability of PG to induce spawning behaviour is eliminated by hypophysectomy and restored by treatment with salmon gonadotropin: no steroid treatment was effective in restoring PG-induced spawning in fish which had been hypophysectomized for 3–4 months. The possible mode of action of PG in inducing spawning behaviour in female goldfish is discussed.     

Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: Role of cyclic AMP and protein kinases

The transduction of the serotonin (5-HT) signal in Fundulus heteroclitusovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) was investigated. Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10⁻⁴ M 5-HT. Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT. Serotonin was found to slightly increase the levels of follicular 3′,5′-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10⁻⁴ M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20βP in IBMX-free culture media in the presence or absence of 10⁻⁴ M 5-HT. The exposure of follicles to 17,20βP alone produced a small and transient reduction in cAMP (40%) within 1–3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20βP, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20βP-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5-HT may inhibit 17,20βP-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT. Mol. Reprod. Dev. 49:333–341, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of the onset of vitellogenesis by abnormal retention of mature eggs in the stick insect,Carausius morosus

Mature eggs stored in the lateral oviducts are unusual in Carausius morosus. When egg laying is experimentally prevented, ovarian production is inhibited by the accumulation of mature eggs within the lateral oviducts. Such storage affects only the very small terminal oocytes and not the vitellogenic follicles, which attain to term and are ovulated. The fact that unilateral retention of eggs affects only the corresponding ovary strongly suggests that the mature eggs themselves inhibit ovarian production in the stick insect without the involvement of an endocrine organ. It is likely that this mode of action, different from that suggested for the housefly or Rhodnius prolixus, is correlated with the independence of ovarian development from the corpus allatum hormone in the stick insect.     

Cyclical Changes in the Ovary of the Hagfish Eptatretus burgeri (Cyclostomata)

Abstract The development of eggs and the degeneration of ovulated follicles in Eptatretus burgeri was studied over a period of one year. It was found that small resting eggs with a diameter of about 1.5 mm continue to grow in November. By the spawning season in October of the following year, the follicles attain a length of about 24 mm. Only one fertilized egg containing an embryo of 7.5 mm length was obtained. The ratio of males to females was found to vary throughout the year. The annual increase of body length in correlation to ovarian development in the hagfish is calculated to be 4 to 5 cm.     

GCMS-indentified steroids and steroid glucoronides in gonads and holding water of Trichogaster trichopterus (Anabantidae,Pallas 1770)

• 1.1. Analysis by gas chromatography—mass spectrometry was performed to identify steroids and steroid glucoronides in gonads of the tropical fish Trichogaster trichopterus and in the water in which the fish were maintained.
• 2.2. Full mass spectra of estradiol-17β (E₂), testosterone, 17α-hydroxyprogesterone, cholesterol, stigmasterol, 4β-methylcholesterol, estrone, 17α,20β-hydroxy-4-pregnen-3-one (17,20-P) and sitosterol were obtained.
• 3.3. The above steroids were detected in both female and male gonads, with the exception of estrone, which was detected only in the male, and 17,20-P, which was detected only in the female.
• 4.4. All steroids except 17,20-P were detected in the water in which the fish were maintained.

     

Synthesis of 17,20β,21‐trihydroxypregn‐4‐en‐3‐one by ovaries of reproductively mature Atlantic cod Gadus morhua

Atlantic cod Gadus morhua ovaries were incubated in vitro with tritiated 17‐hydroxypregn‐4‐ene‐3,20‐dione (17‐P) to determine whether 17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) or 17,20β, 21‐trihydroxypregn‐4‐en‐3‐one (17,20β,21‐P), or both, were more likely to be the steroid responsible for inducing oocyte final maturation (i.e. resumption of meiosis). Only 17,20β,21‐P was produced, in addition to 11‐deoxycortisol (17,21‐P), which is intermediate between 17‐P and 17,20β,21‐P. Also, the 5β‐reduced forms of 17‐P, 17,21‐P and 17,20β,21‐P were all found. Some sulphation of 21‐hydroxylated steroids was demonstrated. The ability of female G. morhua to make 17,20β,21‐P but not 17,20β‐P was confirmed by radioimmunoassay of plasma samples from spawning fish. Although small amounts of 17,20β‐P immunoreactivity were detected in a few plasma samples, this was shown, by thin‐layer chromatography, to be mostly due to cross‐reaction with other unidentified compounds. The evidence strongly suggests that 17,20β,21‐P is more likely than 17,20β‐P to be the maturation‐inducing steroid in G. morhua.     

The impact of antiandrogen 2-hydroxyflutamide on the expression of steroidogenic enzymes in cultured porcine ovarian follicles

We used our model system for agonism and antagonism of the androgen receptor (AR), in which the porcine ovarian follicles were exposed on the excessive concentration of an AR agonist- testosterone (T) or an AR antagonist- 2-hydroxyflutamide (2-Hf) to: (1) analyze the spatiotemporal expression of ovarian 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase/c17,20-lyase (P450c17) and cytochrome P450 aromatase (P450arom); (2) to determine the contribution of AR-mediated action during steroidogenesis and (3) to establish some correlations between the onset and expression pattern of the investigated proteins. Whole follicles (6–8 mm in diameter) isolated from mature porcine ovaries have been incubated (for 24 h) in an organ culture system in the presence of T (10^?7 M), 2-Hf (1.7 × 10^?4 M) or both T and 2-hydroxyflutamide (T+2-Hf, at the same concentrations as when added separately). Thereafter, sections obtained from cultured follicles were processed for main steroidogenic enzymes detection by immunohistochemistry. Moreover, expression of their mRNA and protein was determined by real-time PCR and Western blot analysis. Progesterone, androgens and estradiol concentrations in the culture media were measured by radioimmunoassays (RIA). Our results demonstrated that 2-Hf can influence the steroidogenic activity of porcine follicles in vitro through the blockade of AR. It was shown that follicular 2-Hf treatment brought about dramatic decline in the production of the investigated steroids. What is more the addition of 2-Hf separately caused a negative effect on 3β-HSD and P450c17 mRNA and protein expression by ovarian follicles, while it was without effect on P450arom mRNA level. Quite opposite effect was observed in case of the simultaneous addition of 2-Hf and T. It caused high increase, in both P450arom mRNA and its protein. What was interesting, addition T+2-Hf evoked 3β-HSD and P450c17 increase on mRNA level, but decreased their protein expression. This was against our expectations but the reason for that finding remains undiscovered, intriguing and worth reporting. These results suggest that alike, steroidogenic enzymes activity and their expression is associated with the presence of androgens and AR in the porcine ovary.     

The tempo of reproduction in Hyphessobrycon pulchripinnis(Characidae), with a discussion on the biology of ‘multiple spawning’ in fishes

Synopsis We describe the short-term patterns of egg production and release in the lemon tetra, Hyphessobrycon pulchripinnis (Characidae) as observed over a six month aquarium study and then use our results and those of others to both describe general patterns and derive comparative predictions. Female lemon tetras ovulated about once every four days; differences among individuals were small and inconsistent. As in other species, the probability of ovulation depended strongly on time since last ovulation, indicative of an ovarian cycle; furthermore we found no obvious long-term patterns of ovulation within females and batch fecundity was independent of the length of the two previous interovulation intervals and of the one following. Each batch of ovulated eggs was released over an average of 23.1 spawning acts, beginning as soon as the lights went on in the morning. The number of spawning acts increased with ovulation fecundity but decreased with the number of other females spawning in the tank on that day. The mass of eggs produced every four days was greater than that of the remaining ovary, and the mass of eggs that could be produced in a season was greater than that of the female. This latter observation highlights the most important consequence of repeated reproduction within a season - increased reproductive output - and leads to predictions associating it with less seasonal environments (e.g. low latitudes), as well as with small ovaries and small body size. The significance of releasing a batch of eggs over many spawning acts remains unknown.     

Mating‐induced ovulation in loggerhead sea turtles,Caretta caretta

Mating‐induced ovulation is common in mammals, but has been rarely described in other taxa. Observations of several mature female loggerhead sea turtles, Caretta caretta, held in captivity seemed to indicate that ovulation did not occur in the absence of a male. This study was designed to determine whether this was an effect of captivity or an effect of the absence of a male. Two mature female loggerheads were followed over six annual reproductive cycles. Ultrasound exams were performed approximately every 2 weeks to follow the development of follicles in the ovaries. During the first two seasons, no male was present, in the next two seasons, a mature male was present without mating, and in the final two seasons a mature male was present, mating with one or both females. When no male was present or when present without mating, ovarian follicles developed to full size, but ovulation did not occur and the follicles gradually began to decrease in diameter and undergo changes evident on ultrasound. In the fifth season, only one of the females mated, dropping two eggs after 7 days, and continued to oviposit throughout the following months (total 275 eggs). The unmated female did not ovulate, showing the same pattern as earlier seasons. In the final season, both females mated and ovulated, dropping eggs for the next four months (total 539 eggs). The following year, no males were present and neither female ovulated. This study provides clear evidence that ovulation in loggerhead sea turtles is induced by mating. Zoo Biol 27:213–225, 2008. © 2008 Wiley‐Liss, Inc.     

Steroids involved with final oocyte maturation in the winter flounder.

A number of androgens and progestogens including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20-P) were examined in female winter flounder as possible maturation inducing steroids (MIS). During final oocyte maturation serum levels of testosterone (T) and 17 beta-hydroxy-5 beta-androsten-3-one (5 beta-T) peaking at over 200 ng/ml and pregnenolone (PE) at 40 ng/ml were the predominant steroids found from each major group. High levels of T and 5 beta-T were correlated with oocyte stages characterized by germinal vesicle migration. Of the PEs measured, maximum serum levels of PE, 3 beta,17 alpha-hydroxy-5-pregnen-20-one (17-PE) and 3 beta,17 alpha, 20 beta-dihydroxy-5-pregnene (17,20-PE) were found during later oocytes stages associated with germinal vesicle breakdown. Levels of 17,20-P, an established MIS in most fish, were almost non-detectable (less than 0.1 ng/ml serum) in females throughout all stages of final oocyte maturation. Incubations of ovarian follicles in vitro with physiological concentrations of T and 5 beta-T indicated that these steroids could induce all stages of final oocyte maturation. Similar in vitro incubations showed that 17-PE and 17,20-PE were only effective on germinal vesicle breakdown. The principal conclusions are that T, 5 beta-T and the PEs can be considered as MISs in winter flounder and the PE pathway predominates during the final stages of oocyte maturation in winter flounder in contrast to progesterones which predominate in other fish species, mostly salmonids, studies to date.