Lunar synchronization of in vitro steroidogenesis in ovaries of the golden rabbitfish, Siganus guttatus (Bloch)

To assess the relationship between lunar cycle and steroidogenesis in the ovaries of the golden rabbitfish, Siganus guttatus, the intact follicles of oocytes were incubated in vitro with human chorionic gonadotropin (hCG) and seven steroid hormones, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), 17alpha-hydroxyprogesterone (17alpha-OHP), progesterone (P), cortisol, estradiol-17beta (E2) and testosterone, during the two lunar phases, the new moon (1 week before spawning) and the first lunar quarter (just before spawning). Around the new moon, germinal vesicle breakdown (GVBD) could not be induced by addition of hCG or any steroid hormones. Around the first lunar quarter, GVBD was induced by addition of hCG, DHP, 20beta-S, 17alpha-OHP, P, and cortisol. DHP was the most potent steroid hormone. When the intact follicles of oocytes were incubated with hCG in both lunar phases, the production of E2 and DHP measured by enzyme immunoassay decreased and increased significantly from the new moon to the first lunar quarter, respectively. These results suggest that the ovarian follicles produce E2 around the new moon and DHP around the first lunar quarter and that the production/conversion of the steroid hormones is under the influence of gonadotropin(s). The synchronous increase in ovarian activity supports the hypothesis that lunar periodicity is a major factor for the ovarian development of S. guttatus.     

Variation with semilunar periodicity of plasma steroid hormone production in the mudskipper Boleophthalmus pectinirostris

Variation in the production of the plasma steroid hormones E(2), 17alpha-OHP and T in females and T and 11-KT in males, was investigated in the mudskipper Boleophthalmus pectinirostris during the spawning season. Females with oocytes at the vitellogenic stage (GSI 5.97-6.86%) and mature males with GSI of 0.255-0.288% were collected at intervals of 3-4 days within the two complete semilunar cycles from May 31 to June 30, 2006. The results showed that variations in the levels of plasma steroid hormones were synchronized obviously with semilunar periodicity in both females and males. Each steroid hormone level exhibited two cycles, each cycle with a peak. In females, the first peaks in plasma E(2), 17alpha-OHP and T levels were observed 3 days after the first lunar quarter, and the second ones, 4 days after the last lunar quarter. In males, the first peaks of plasma T and 11-KT levels occurred 3 days after the first lunar quarter, and the second ones, at the last lunar quarter. The fact that, in the present study, changes in the levels of plasma steroid hormones were synchronized with semilunar periodicity, although the fish were at the same stages of gonadal development, suggests that variation of plasma steroid hormones is basically regulated by biological rhythms (Zeitgebers), and that tidal movement (with its semilunar periodicity) is the major environmental factor stimulating steroid hormone production in B. pectinirostris.     

Profiles of plasma sex steroids and gonadotropin in coho salmon, Oncorhynchus kisutch, during final maturation

The plasma concentrations of estradiol-17 alpha (E2), 17 mu-hydroxy-20 beta-dihydroprogesterone (DHP), progesterone, 11-ketotestosterone (11-KT), testosterone (T), and gonadotropin (GtH) were measured in adult coho salmon (Oncorhynchus kisutch) concomitantly with advancing maturation of the gonads during the spawning season. Concentrations of E2 were higher in females with eggs showing central (premigrating) germinal vesicles, migrating germinal vesicles, or peripheral germinal vesicles than in fish in which the eggs had undergone germinal vesicle breakdown or ovulation. Conversely, plasma levels of DHP were low at central, migrating, and peripheral germinal vesicle stages and then increased dramatically at germinal vesicle breakdown and ovulation. Levels of 11-KT, T, and GtH generally increased with advancing maturity of the eggs. When time rather than degree of maturation was used as a variable, plasma E2 was high in females first returning from the ocean but then dropped off precipitously in fish sampled at the end of the run. Plasma DHP followed the opposite pattern, being low in fish sampled early in the run and high in fish sampled late in the run. No easily discernible patterns emerged from the profiles of the other steroids and GtH, although some significant variation in the concentrations occurred. Even though sampling was initiated at the time when the salmon first returned from the ocean, most of the hormones were not variable in males when viewed according to sampling date or predominant cell type in the testes, which indicates that the males were extremely close to final maturity during the sampling period. Plasma levels of DHP were higher in males that were producing milt than in any other group of males. Concentrations of 11-KT were higher than those of T in all males, but no patterns in the levels of either steroid emerged.     

Influence of light-dark and lunar cycles on the ocular melatonin rhythms in the seagrass rabbitfish, a lunar-synchronized spawner

The aim of this study was to investigate the effects of light-dark (LD) cycles and lunar phases on ocular melatonin rhythms in the seagrass rabbitfish, Siganus canaliculatus, a lunar-synchronized spawner. Under a natural 24-hr LD (12.00:12.00) cycle, ocular melatonin levels were low during daylight hours. The levels significantly elevated to peak during the mid-dark phase at 24.00 hr and then declined sharply in the early morning around 06.00 hr. These rhythms disappeared under either constant light (LL) or constant dark (DD) conditions. Melatonin levels remained low in LL compared with those in DD condition. These results suggest that ocular melatonin rhythms in the seagrass rabbitfish are suppressed in the presence of light. When fish were exposed to natural moon phases, ocular melatonin concentrations were higher around the new moon than both the first quarter and full moon phases. Exposure to experimental new moon conditions caused a significant increase in melatonin levels while those of the fish exposed to experimental full moon conditions were decreased. These results suggest that the seagrass rabbitfish perceives moonlight through the eye and that moonlight directly causes melatonin suppression.     

Steroid profiles during spawning in male common carp

The profiles of testosterone (T) 11-ketotestosterone (11-KT), 17 alpha-hydroxy-4-pregnen-3-one (17-P), and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) were determined in groups of spermiating male common carp (Cyprinus carpio) which were or were not mated with females and subjected to an increase in water temperature, a treatment which usually induces females to spawn. The steroid levels were correlated with changes in gonadotropin (GtH). All males mated with ovulatory females showed a GtH surge and spawned. Two distinct classes of these spawned males were differentiated based on their initial androgen levels and hormone profiles. Androgen levels were initially elevated in the "high" males. In these fish there was a very rapid shift in the steroidogenic pathway from androgen to progestogen production coincident with the GtH surge. In the "low" males, 17-P levels rose significantly in response to the GtH surge, but there was no steroidogenic shift. In the males mated with nonovulatory females, the levels of 17-P, 17,20 beta-P, T, and 11-KT all increased coincident with a small rise in GtH; there was no steroidogenic shift. In the unmated males, the steroid profiles were similar to those in the nonspawned fish, except that T and 11-KT levels were significantly lower in comparison. In all groups, 17-P rose higher than 17,20 beta-P, and 11-KT was generally the predominant androgen. A model is proposed which explains the steroidogenic shift in the "high" males. This involves the inhibition of androgen biosynthesis by 17,20 beta-P or by a related progestogen.     

Gonadotropin surge during spawning in male goldfish

Changes in plasma hormone levels during spawning were studied in male goldfish. Blood samples were analyzed for gonadotropin (GtH), testosterone, 11-ketotestosterone (11-KT), and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOH-P) in males, and for GtH in females. Spawning was induced by raising water temperature from 12 to 20 degrees in sexually mature goldfish kept in pairs. Plasma GtH levels in males showed a marked increase (GtH surge) which was synchronous with the preovulatory surge in females, and peaked at the onset of spawning. Plasma levels of testosterone and 17 alpha, 20 beta-diOH-P increased almost at the same time as the GtH surge in males, whereas 11-KT levels remained low during the spawning. The small increase in GtH in males could be induced in the absence of females by raising the water temperature, but the levels were significantly enhanced by the presence of ovulatory females. A large amount of milt was observed during spawning, and the amount was correlated with plasma levels of GtH. Increased levels of GtH may be involved in milt production. We can propose that the synchronous GtH surge in both sexes causes ovulation and milt production to occur at the same time, favoring a higher rate of fertilization of the eggs.     

Progestin is an essential factor for the initiation of the meiosis in spermatogenetic cells of the eel

Meiosis is an indispensable process of sexual reproduction. However, detailed information on the regulatory mechanisms that initiate meiosis is not available. Progestins are important steroids regulating final maturation in male and female vertebrates. In male teleosts, it is known that progestin induces spermiation and sperm maturation. However, a role for progestin in early spermatogenesis or meiosis has not yet been described. In this study, we examined the functions of progestin on the initiation of meiosis in male Japanese eel. A natural progestin in teleost fish 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) and its receptors were present in the testis at an early stage of spermatogenesis. By using an eel testicular culture system, DHP was shown to induce DNA replication in spermatogonia. Although 11-ketotestosterone, a known initiator of spermatogenesis, also stimulated DNA synthesis in spermatogonia, antibodies against DHP prevented DNA replication when added during the period in which meiosis was initiated. DHP treatment also induced the expression of meiosis-specific markers, such as DmcI and Spo11. Furthermore, Spo11 expression and synaptonemal complexes, specific features of the meiotic prophase, were detected in testicular fragments cultured with DHP in some germ cells that showed morphological characteristics of undifferentiated spermatogonia. We conclude that DHP, a progestin, is an essential factor for the initiation of meiosis.     

Kinetic studies on ovarian C-17,20-lyase activity: effect of luteinizing hormone surge

It has been shown that 3 h after the preovulatory gonadotropin surge, an abrupt decrease occurs in follicular C-17,20-lyase (lyase) activity which causes a decrease in C19-steroid production. To determine the reason for the reduced lyase activity, we used rats that were induced to ovulate by means of PMSG administration. In these rats, a 54% decrease in lyase activity occurred at the peak of the LH surge. When the gonadotropin surge and ovulation were blocked by pentobarbitone the decrease was prevented. Administration of LH to the pentobarbitone-blocked rats reduced lyase activity to well below the level reached after the endogenous gonadotropin surge. In cycling proestrous rats as well, human CG (hCG) decreased the lyase activity, as measured in isolated follicles 3 h after hCG administration. Out of three potential inhibitory steroids for lyase activity; progesterone, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnen-20-one, and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, only the last compound inhibited competitively ovarian lyase activity. The inhibition constant (Ki) value was 29 microM. In order to explore which of the two activities of the lyase complex is regulated by the gonadotropin, a double label double substrate experiment was conducted using [14C]progesterone with [3H]17 alpha-hydroxyprogesterone (17 alpha-OHP). With this assay procedure, we could determine in the same experiment the site of stimulation, the preferred substrate, and the amount of conversion. The conversion of progesterone to 17 alpha-OHP, as well as the conversion to androstenedione were significantly inhibited throughout the reaction by the gonadotropin. Thus, the changes in ovarian lyase after hCG mimic those of 17 alpha-hydroxylase. The labeling pattern of androstenedione showed that the ovarian lyase complex catalyzed the conversion preferentially from progesterone. Whereas the 3H/14C ratio in androstenedione varied between 0.29 to 0.76, the ratios in the 17 alpha-OHP were from 5 to 22. Thus, the exogenous 17 alpha-OHP did not equilibrate with the product formed from progesterone. The effect of the hCG was to decrease the preference of progesterone over 17 alpha-OHP as substrate. It is concluded that: the LH of the surge inhibits both the 17 alpha-hydroxylase and lyase activities. The ovarian lyase complex shows a preference for progesterone as a substrate rather than 17 alpha-OHP. 17 alpha-OHP is not an obligatory intermediate in androstenedione production in ovarian tissue. hCG affects the ovarian lyase complex by shifting the relative preference of substrates towards 17 alpha-OHP.     

Endocrine changes associated with spawning behavior and social stimuli in a wild population of rainbow trout (Salmo gairdneri). I. Males

Rainbow trout (Salmo gairdneri) were collected from a natural spawning population at Pennask Lake, B.C. Blood samples were taken from male trout at different stages of spawning and assayed by radioimmunoassay for gonadotropin (GtH), testosterone (T). 11-ketotestosterone (11-KT), and 17 alpha-hydroxy-20 beta-dihydroprogesterone (17,20-P). Testosterone and 11-KT were detected in the plasma of males in spawning condition: 11-KT was at a higher concentration than T and declined to undetectable levels in postspawning fish. There were striking increases in plasma GtH and 17,20-P in males actively courting nest building females. The increase in 17,20-P was evidently not dependent upon an elevation of the pituitary hormone: 17,20-P remained low in males placed with sexually inactive, unovulated females even though GtH levels rose above levels measured in males isolated from females. The significance of the social control of hormone levels is not clear. There is some evidence that 17,20-P and/or GtH may cause an increase in quantity of the milt, and thus the endocrine response to a sexually active female may serve to synchronize maximum male fertility with oviposition readiness in the female.     

Effects of sex steroids on the smoltification of masu salmon, Oncorhynchus masou

Testosterone (T; 25-micrograms/g diet), estradiol-17 beta (E2; 2.5-, 25-micrograms/g diet), and 5 alpha-dihydrotestosterone (DHT; 2.5-, 25-micrograms/g diet) were administered to yearling masu salmon from February 3 through May 8. T and E2 inhibited the natural smoltification in spring and stimulated gonadotropin (GtH) accumulation in the pituitary, but DHT had no effect. T (25-micrograms/g diet) was administered to underyearling masu salmon from September 30 through December 14. At the end of the T treatment, fish body color darkened and pituitary GtH content increased in the T-treated group. On May 8 in the next spring, however, plasma T levels and pituitary GtH contents already decreased and smoltification occurred in both control and T-treated groups. On October 29, underyearling masu salmon reared under the short daylength (8 L:16 D) for 4 months were switched to a long daylength (16 L:8 D) and simultaneously administered with T (25-micrograms/g diet) or 11-ketotestosterone (11-KT; 25-micrograms/g diet). On December 17, the proportion of smolts was 52.8% in the control, whereas T and 11-KT administration inhibited smoltification under the artificially increased daylength. Pituitary GtH content significantly increased both in the T-treated and 11-KT-treated groups, though it was much lower in the latter. These results indicate that the springtime smoltification which is induced by increasing daylength is inhibited by biologically active sex steroids such as E2, T, and 11-KT.     

Endocrine changes during natural spawning in the white sucker, Catostomus commersoni. II. Steroid hormones

Blood samples were taken from white suckers (Catostomus commersoni) during their annual spring spawning migration and analyzed by radioimmunoassay for gonadotropin (GtH), estradiol-17 beta (E2), testosterone (T), 11-ketotestosterone (11-KT), 17 alpha-hydroxyprogesterone (17-P), 17 alpha-hydroxy-20 beta-dihydroprogesterone (17, 20-P), androstenedione (A), and cortisol. GtH, 17-P, and 17,20-P levels were low in prespawning fish of both sexes, rose to their highest levels in ovulated females and spawning males, and then fell to low levels in spent fish. In females, E2, T, and A levels were high in prespawning fish and declined significantly at ovulation, dropping to lowest levels in spent fish. In males, 11-KT, T, and A levels were highest in prespawning fish, and lowest in spent fish. Cortisol levels were highest in spermiating males and ovulated females. Plasma profiles of the sex steroids in the white sucker are very similar to those observed in the rainbow trout (Salmo gairdneri).     

Testicular steroidogenic response to human chorionic gonadotropin of fifteen male transsexuals on chronic estrogen treatment

Fifteen transsexuals were prepared for surgery with estrogen treatment. The response to human chorionic gonadotropin (hCG) in the untreated state was similar to that of normal subjects when testosterone (T), estradiol-17 beta (E2), 17 alpha-hydroxyprogesterone (17 alpha-OHP), progesterone (P), 4-androstenedione (delta 4A), and dehydroepiandrosterone (DHA) were used as indices. Following estrogen therapy, plasma T, 17 alpha-OHP, and DHA levels were markedly reduced whereas delta 4A and P were not. In spite of the suppressive effects of estrogen, a good response to hCG was noted in such subjects in plasma levels of T, 17 alpha-OHP, and, to a lesser extent, delta 4A even after estrogen administration for 24 months. The high rates of 17 alpha-OHP to T induced by estrogen treatment is restored to normal by the administration of hCG.     

Gonadotropin therapy in men with isolated hypogonadotropic hypogonadism: the response to human chorionic gonadotropin is predicted by initial testicular size

This study was designed to determine whether exogenous hCG alone can complete spermiogenesis in men with isolated hypogonadotropic hypogonadism (IHH). hCG was administered to 22 men with IHH until maximal testicular growth was achieved. Their mean testicular volume increased from 5.5 +/- 1.1 (+/- SE) mL (pretreatment) to 10.8 +/- 1.6 mL (maximum) during treatment (P less than 10(-6)). The maximum mean testicular volume was highly positively correlated with initial volume (r = 0.84; P less than 10(-6)). All men attained normal serum testosterone levels, and 7 of 22 men achieved supraphysiological serum estradiol levels. During hCG treatment, 14 of the 22 men had sperm appear in their semen. Six of 11 men with complete gonadotropin deficiency, defined as an initial mean testicular volume less than 4 mL, became sperm positive during hCG treatment. In contrast, 9 of 11 men with partial gonadotropin deficiency (initial mean testicular volume of 4 mL or more) produced sperm during treatment (P less than 0.001). Sperm concentration was highly positively correlated with both pretreatment (r = 0.65; P less than 0.01) and final testicular volume (r = 0.73; P less than 0.0001). Of 13 men attempting to impregnate their partners, 7 were successful in initiating conception; a total of 8 pregnancies ensued. The sperm concentration at the time of conception was less than 10 million/mL in all but 1 man. Our study demonstrates that hCG, in the absence of exogenous FSH, can complete spermiogenesis in men with partial gonadotropin deficiency. The response to hCG in men with IHH is predicted by the initial testicular volume.     

Two-year comparison of testicular responses to pulsatile gonadotropin-releasing hormone and exogenous gonadotropins from the inception of therapy in men with isolated hypogonadotropic hypogonadism

Men with the complete form of isolated hypogonadotropic hypogonadism (initial mean testes volume less than 4 mL) require 2 or more yr of exogenous gonadotropin therapy combining hCG and human menopausal gonadotropin (hMG) to achieve maximal, but subnormal, testis size and sperm output. To test whether pulsatile GnRH therapy, which more closely mimics normal hormonal stimulation, would accelerate or further augment testicular growth, hasten the onset of sperm production, and/or increase sperm output more than occurs during conventional exogenous gonadotropin therapy, we administered either hCG/hMG or GnRH from the inception of therapy to 2 comparable groups of men with complete IHH (initial testicular volume, less than 4 mL) and compared their testicular responses during the first 2 hr of therapy. Five men were treated with pulsatile GnRH in doses of 143-714 ng/kg every 2 h, sc, while 11 other men received hCG (2000 IU) and hMG (75 IU FSH and 75 IU LH) im 3 times/week. In the GnRH-treated men, the mean plasma total and free testosterone levels during therapy rose to within the normal range, but were significantly lower (P less than 0.01 and P less than 0.02, respectively) than those in the hCG/hMG-treated men. The mean plasma estradiol concentrations during therapy were within the high normal range and were similar in the two groups. The mean plasma FSH levels achieved in the GnRH-treated men were significantly (P less than 0.01) and 1.3- to 3.2-fold higher than those in the hCG/hMG-treated men. The mean testicular size achieved in the GnRH-treated men was not significantly different from that in the hCG/hMG-treated men (P = 0.08); the mean testicular volumes after 2 yr were 4.8- and 4.3-fold the pretreatment values in the GnRH and hCG/hMG groups, respectively. After 12 months of therapy, sperm production had occurred in one man in the GnRH group and in no subject in the hCG/hMG group. After 24 months, two men in the GnRH group and eight men in the hCG/hMG group produced sperm. Thus, 40% of the GnRH-treated men and 80% of the hCG/hMG-treated men (P = NS) produced sperm after 2 yr of therapy. The sperm concentrations in all men were below 5 million/mL and were comparable in the two groups (P = NS). These results suggest that pulsatile sc GnRH therapy for the first 2 yr does not accelerate or enhance testicular growth, hasten the onset of sperm production, or increase sperm output significantly compared to hCG/hMG.     

Seasonal changes in serum gonadotropin, testosterone, 11-ketotestosterone, and estradiol-17 beta levels and their relation to tumor burden in gonadal tumor-bearing carp x goldfish hybrids in the Great Lakes

The hybrids of carp (Cyprinus carpio) and goldfish (Carassius auratus) collected from the lower Great Lakes between 1978 and 1981 exhibited a high frequency of gonadal neoplasms and apparent sterility, conditions which were rare in the parental species. A pronounced hyperplasia of gonadotropic cells in hybrids resulted in the accumulation of large stores of biologically active gonadotropin (GtH) in the pituitary gland. Serum GtH levels were generally higher in hybrids than in carp collected from the same areas, but seasonal trends were similar for both species. Among hybrids, tumored individuals had higher serum GtH levels than nontumored individuals. Serum levels and seasonal trends for testosterone (T), 11- and ketotestosterone (11-KT), and estradiol-17 beta (E2) were, for the most part, similar when carp and nontumored hybrids of the same gender were compared. Serum levels of T and 11-KT (but not E2) were generally higher in tumored hybrids when compared to nontumored individuals. High serum steroid levels were related to a specific tumor type, possibly of Sertoli cell origin. Although the etiology of the gonadal tumors is not yet known, the serum levels of gonadal steroids in hybrids make it unlikely that a lack of steroidal feedback is responsible for the gonadotrop hyperplasia.     

Male hypogonadotropic hypogonadism: factors influencing response to human chorionic gonadotropin and human menopausal gonadotropin, including prior exogenous androgens

Although testosterone (T) therapy is sufficient for maturation and maintenance of secondary sex characteristics in hypogonadal men, gonadotropins are required for stimulation of spermatogenesis. Thirteen men with hypogonadotropic hypogonadism received treatment with hCG, followed in 12 by the addition of human menopausal gonadotropin (hMG). All initially had undetectable serum LH and FSH and low T levels and were azoospermic with small testes. During therapy, all achieved normal male levels of T. Twelve of 13 had marked and continuous increase in testicular volume. Three men had sperm in the ejaculate with hCG treatment alone. All but 1 patient developed sperm in their seminal fluid during combined hCG and hMG therapy. Two men achieved three pregnancies, and 2 more had semen that produced hamster oocyte penetration assays in the fertile range during the protocol period. Four of 5 who achieved sperm densities greater than 1 million/ml while receiving combined therapy maintained or increased sperm production while receiving continued hCG therapy after hMG was withdrawn. We examined the response to gonadotropin therapy of men who had received previous T therapy and those who had not. There were no differences in rapidity or degree of response, as assessed by rise in serum T, increase in testis volume, or maximal sperm density achieved. Multiple pituitary deficits and cryptorchidism were negative prognostic factors. In summary, the prognosis for successful stimulation of spermatogenesis in men with hypogonadotropic hypogonadism treated with hCG/hMG is good and not adversely affected by prior androgen treatment. Despite undetectable serum FSH levels, hCG treatment was sufficient to both initiate and maintain spermatogenesis in some patients.     

Endocrine profiles in the males of a twice-annually spawning strain of rainbow trout, Salmo gairdneri

The male reproductive cycles of a twice-annually spawning strain of rainbow trout were studied by monitoring the plasma gonadotropin (GtH) and steroid hormone levels in individual fish for more than a year using thirty-five 2.5-year-old mature males. Twenty-five males survived the whole experimental period and were divided into four groups according to the amount of milt and endocrine profiles. In the summer breeding season, milt amount was negligible in Group I and small in Group II with low plasma testosterone, 11-ketotestosterone (11-KT), and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOH-P) levels, whereas both groups showed a large amount of milt and a distinct increase in steroid levels in the winter breeding season (November-January). Group III expelled a large amount of milt in both the summer and winter breeding seasons, and plasma testosterone, 11-KT, and 17 alpha,20 beta-diOH-P showed a clear peak in each breeding season. In Group IV, milt was expelled from December to July, and plasma steroid levels remained high until June before declining to the basal levels; however, these fish failed to mature in the ensuing winter breeding season. Plasma GtH levels in Groups III and IV were significantly higher than those in Groups I and II in the summer breeding season. These results clearly indicate that fish in Group III are twice-annual spawners.