PS1 Expression is Downregulated by Gonadal Steroids in Adult Mouse Brain

Mutations in presenilin (PS) 1 and PS2 genes are associated with early onset (≤65 years) of Alzheimer’s disease (AD). PS1 is involved in γ-secretase mediated cleavage of β-amyloid precursor protein (APP), but its regulation is poorly understood. Sex steroids influence APP cleavage pathways resulting in reduced burden of both intra- and extra-cellular nonamyloidogenic products. As gonadal hormones are implicated in AD and their levels change with age, we have analyzed the effect of 17β-estradiol and testosterone on PS1 expression in the cerebral cortex of adult and old AKR mice of both sexes. Northern and Western-blot analysis revealed that PS1 mRNA and protein expression followed similar pattern of regulation. PS1 expression was downregulated by 17β-estradiol and testosterone in the cerebral cortex of females and adult male, but upregulated in old male mice. Such sex-dependent regulation of PS1 expression during aging by gonadal steroids might account for the PS-related brain functions.     

Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis

Summary. Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17β-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17β-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 ± 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17β-estradiol. The enzyme’s K_m was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17β-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17β-estradiol that they produce (autocrine effect).     

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.     

Effects of Estradiol and IGF-1 on the Sodium Calcium Exchanger in Rat Cultured Cortical Neurons

The Na⁺/Ca²⁺ exchanger (NCX) is an important bidirectional transporter of calcium in neurons and has been shown to be involved in neuroprotection. Calcium can activate a number of cascades that can result in apoptosis and cell death, and NCX is a key factor in regulating the cytoplasmic concentration of this ion. 17-β-estradiol and insulin-like growth factor 1 (IGF-1) are known neuroprotective hormones with interacting mechanisms and effects on intracellular calcium; however, their relationship with the NCX has not been explored. In this article, the effects of these two hormones on neuronal NCX were tested using the whole-cell patch clamp technique on rat primary culture neurons. Both 17-β-estradiol and IGF-1 produced an increase in the NCX-mediated inward current and a decrease in the NCX-mediated outward current. However, the IGF-1 effect was lower than that of 17-β-estradiol, and the effect of both agents together was greater than the sum of each agent alone. Neither of the agents affected the pattern of regulation by extracellular or intrapipette calcium. Inhibitors of the IGF-1 and 17-β-estradiol receptors and inhibitors of the main signaling pathways failed to change the observed effects, indicating that these actions were not mediated by the classical receptors of these hormones. These effects on the NCX could be a mechanism explaining the neuroprotective actions of 17-β-estradiol and IGF-1, and these findings could help researchers to understand the role of the NCX in neuroprotection.     

Estrogen-like properties of brominated analogs of bisphenol A in the MCF-7 human breast cancer cell line

Tetrabromobisphenol A (TeBBPA) is a four-meta-brominated variant of bisphenol A (BPA) and is one of the most commonly used brominated flame retardants worldwide. We compared the estrogenic potency of TeBBPA, BPA and the brominated analogs mono- (MBBPA), di- (DBBPA), and tribromobisphenol A (TrBBPA) in the estrogen-dependent human breast cancer cell line MCF-7. All of the compounds competed with 17β-estradiol for binding to the estrogen receptor, although the affinity of the test chemicals to the estrogen receptor was much lower than that of 17β-estradiol. TrBBPA and TeBBPA showed a considerably lower access to the estrogen receptors within intact MCF-7 cells incubated in 100% serum compared to incubation in serum-free medium, indicating a strong binding to serum proteins. BPA, MBBPA, and DBBPA showed only a slightly reduced access to the receptors. All of the test compounds induced proliferation in MCF-7 cells, the potential decreasing with increasing number of bromo-substitutions. TeBBPA did not induce maximal cell growth, indicating cytotoxic effects at high concentrations. BPA and the brominated analogs, except TeBBPA, induced progesterone receptor and pS2 to the same extent as 17β-estradiol, although at much higher concentrations. Our studies demonstrate that compared to 17β-estradiol, BPA and the brominated analogs have much lower estrogenic potencies for all of the endpoints tested, TeBBPA being the least estrogenic compound. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gonadal stage-dependent effects of gonadal steroids on gonadotropin II secretion in the Atlantic croaker (Micropogonias undulatus).

Involvement of gonadal steroids in the control of gonadotropin II (GTH II) (homologous to LH) secretion was investigated in the Atlantic croaker (Micropogonias undulatus) using gonadectomy (Gx) and steroid replacement paradigms. Gonadectomy in males and females during the late gonadal recrudescence phase elicited significant increases in the gonadotropin response to stimulation by an LHRH analog (LHRHa), without altering basal GTH II secretion. Slow-release silicone elastomer implants of testosterone or estradiol significantly inhibited LHRHa-induced GTH II secretion in gonad-intact and Gx males, and in Gx females, whereas 5alpha-dihydrotestosterone, a nonaromatizable androgen, was ineffective. Pretreatment of fish with an aromatase inhibitor, 1,4, 6-androstatrien-3,17-dione, 2 days before the administration of testosterone implants, completely blocked the negative effect of testosterone on LHRHa-induced GTH II secretion in males, but only partially restored it in females. This suggests that the negative feedback of testosterone in males is primarily mediated by its conversion to estradiol at the level of the hypothalamus and/or pituitary gland, while in females the androgen may also exert a direct inhibitory effect on GTH II secretion, probably mediated via an androgen receptor. In addition, estradiol and testosterone exerted positive effects on basal and LHRHa-induced GTH II secretion during the early-recrudescence phase of the gonadal cycle. The steroids switched to a negative effect on LHRHa-induced GTH II secretion once the fish had fully developed gonads, possibly as a mechanism that prevents a precocious surge in GTH II secretion and final gamete maturation until gametogenesis is complete and the environmental conditions are appropriate for spawning.     

Effects of constant short and long photoperiod regimes on the spawning performance and sex steroid levels of female and male sea bass

Under constant short photoperiod, the spawning time of 2-year-old sea bass Dicentrarchus labrax was advanced as compared to controls, whereas spawnings were delayed under constant long photoperiod. High plasma levels of 17β-oestradiol (E_2/) and testosterone (T) in females were coincident with the appearance of vitellogenic oocytes in the ovary, while high levels of 11-ketotestosterone (11-KT) and T in males were coincident with the presence of spermiating males. Although plasma levels of E₂ in females and 11-KT in males were low during the remainder of the cycle, levels of T were always >1 ng ml⁻¹ in both sexes, suggesting that T could play an important role during the initial stages of gonadal development. The profiles of E₂ and T in females and 11-KT and T in males exposed to constant short days were similar to those in the control group, but fish which were maintained under constant long photoperiods showed a bimodal pattern of these steroids. The results obtained from fish exposed to constant photoperiod regimes provide further evidence that an endogenous process could be operating to control the reproduction of sea bass.     

Stress-induced effects on feeding behavior and growth performance of the sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>): a self-feeding approach

Repetitive aquaculture-related protocols may act as cyclic stressors that induce chronic stress in cultured fish. The sea bass is particularly sensitive to stressful conditions and the mere presence of humans will disturb feeding behavior. In this paper, we study whether chronic stress induced by repetition of acute stress protocols affects long-term feeding behavior and growth performance in sea bass and whether exogenous cortisol may induce stress-like changes in these parameters. We demonstrate that both chronic stress and dietary cortisol decrease food intake and have a negative effect on feed conversion efficiency, severely impairing sea bass performance. Both experimental approaches induced changes in the daily feeding activity by lengthening the active feeding periods. Fish subjected to a cyclic stressor modify their daily feeding pattern in an attempt to avoid interference with the time of the stressor. The delay in feeding when fish are acutely and repeatedly stressed could be of substantial adaptive importance.     

In vitro biosynthesis of 1,4-β-galactan attached to rhamnogalacturonan I

 The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[¹⁴C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [¹⁴C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-β-galactanase released 65–70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [¹⁴C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-β-galactanase. Thus, the majority of the ¹⁴C-labelled product was 1,4-β-galactan. Compounds released by the endo-1,4-β-galactanase treatment were mainly [¹⁴C]galactose and [¹⁴C]galactobiose, indicating that the synthesized 1,4-β-galactan was longer than a trimer. In vitro synthesis of 1,4-β-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn²⁺. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [¹⁴C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-β-galactanase, radioactivity was not only found as [¹⁴C]galactose or [¹⁴C]galactobiose but also as larger fragments. The larger fragments were likely the [¹⁴C]galactose or [¹⁴C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with β-galactosidase together with endo-1,4-β-galactanase digested all radioactivity to the fraction eluting as [¹⁴C]galactose. The data indicate that the majority of the [¹⁴C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-β-galactan sidechains in the endogenous pectic rhamnogalacturonan I. Received: 24 June 1999 / Accepted: 30 August 1999     

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.     

Steroid hormones modulate prolactin binding by cultured porcine granulosa cells.

We have studied the effects of the gonadal steroids — testosterone, 17β-estradiol, progesterone, and 5α-dihydrotestosterone on the prolactin-binding activity of porcine granulosa cells maintained in monolayer culture. Testosterone, estradiol, and progesterone all significantly enhanced prolactin binding (55%, 107%, and 112% above control, respectively). In contrast, the non-aromatizable androgen, 5α-dihydrotestosterone, caused an insignificant suppression of prolactin binding. The anti-androgen, cyproterone acetate, did not influence prolactin binding when used alone, and did not inhibit the effects of testosterone. These data suggest that the stimulatory effects of testosterone may require aromatization to estradiol.     

Effects of dopaminergic system activation on feeding behavior and growth performance of the sea bass (Dicentrarchus labrax): A self-feeding approach

Dopamine is synthesized from l-dopa and subsequently processed into norepinephrine and epinephrine. Any excess neurotransmitter can be taken up again by the neurons to be broken down enzymatically into DOPAC. The effect of dopamine on mammalian food intake is controversial. Mice unable to synthesize central dopamine die of starvation. However, studies have also shown that central injection of dopamine inhibits food intake. The effect of dopaminergic system in the fish feeding behavior has been scarcely explored. We report that the inclusion of l-dopa in the diets results in the activation of sea bass central dopaminergic system but also in the significant increase of the hypothalamic serotonin levels. Dietary l-dopa induces a decrease of food intake and feed conversion efficiency that drives a decline of all growth parameters tested. No behavioral effects were observed after l-dopa treatment. l-dopa treatment stimulated central expression of NPY and CRF. It suggests that CRF might mediate l-dopa effects on food intake but also that CRF neurons lie downstream of NPY neurons in the hierarchical forebrain system, thus controlling energy balance. Unexpectedly, dietary administration of haloperidol, a D₂-receptor antagonist, cannot block dopamine effects but also induces a decline of the food intake. This decrease seems to be a side effect of haloperidol treatment since fish exhibited a decreased locomotor activity. We conclude that oral l-dopa inhibits sea bass food intake and growth. Mechanism could also involve an increase of hypothalamic serotoninergic tone.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Comparison of the gonadal development and plasma levels of sex steroid hormones in diploid and triploid sea bass, Dicentrarchus labrax L

The goal of this study was to compare the reproductive physiology of triploid and diploid European sea bass (Dicentrarchus labrax L.). Gonads of diploid and triploid fish (males and females) were examined both microscopically and macroscopically, together with the plasma levels of the major sex steroids produced (testosterone and estradiol-17beta) when fish were adults. Prior to sexual maturation, the gonadosomatic index (GSI) of triploid males was similar to that of diploids. However, the GSI in 4-year-old adult triploid males was 1.8 times lower than that of diploids (P < 0.05). All diploid males exhibited normal gonadal development. In contrast, in triploid males spermatogenesis was impaired during late meiosis, affecting severely spermiogenesis. This was achieved by an increasing imbalance in the amount of DNA present in daughter cells of the same type as spermatogenesis progressed, as demonstrated by abnormal cell sizes, culminating in inviable spermatids. Thus, no spermiating triploid fish were observed during 4 years, which included three full consecutive maturation cycles. Furthermore, the germ cells from triploids were significantly larger than those from diploids (P < 0.001). Seasonal profiles of plasma levels of testosterone in 4-year-old males were essentially similar in both ploidies. On the other hand, triploid females had rudimentary ovaries containing oogonia and primary oocytes that were arrested during meiotic prophase I, while diploid females exhibited all stages of ovarian development. Diploid females showed levels of testosterone and estradiol-17beta significantly higher than those of triploids (P < 0.05), in which no endocrine signs of maturation were observed at all. Regarding sex ratios, triploids had 10% more females than diploids (P < 0.05) but in both ploidies males predominated, as is usually found in this species under culture conditions. These results show that triploidy blocked the initial phases of meiosis in females and the latter ones in males, resulting in the absence of or reduced gonadal development, respectively. In conclusion, we provide an explanation for the lack of gonadal development in triploid male fish, and, to the best of our knowledge, we report for the first time a case in which induced triploidy completely blocks meiosis in both sexes, thus conferring functional sterility in the sea bass.     

Reproductive periodicity and steroid hormone profiles in the sex-changing coral-reef fish, <Emphasis Type="Italic">Plectropomus leopardus</Emphasis>

The reproductive biology of coral trout, Plectropomus leopardus, from the Great Barrier Reef (Australia) was investigated by correlating gonadal condition with plasma levels of gonadal steroids. Female fish were found to be regressed from mid-summer to early spring, after which rapid and cyclical increases in gonado-somatic index (I _G), maximum oocyte diameter (MOD) and plasma concentrations of estradiol-17β and testosterone were detected. Male fish, in contrast, commenced recrudescence slightly earlier in winter and responded with less dramatic increases in both I _G and plasma concentrations of testosterone and 11-ketotestosterone. The mode of oocyte development was multiple group-synchronous, and cyclical fluctuations in reproductive parameters (I _G, MOD and gonadal steroid concentrations) were synchronized with new-moon lunar phases. It is likely, therefore, that individual P. leopardus have the capacity to spawn on multiple occasions, with lunar periodicity. However, evidence suggests that early bouts of reproduction may be more important in terms of reproductive investment than subsequent bouts later in the same season. It is concluded that patterns of gametogenesis and steroidogenesis in P. leopardus are similar to the patterns displayed by other tropical groupers, suggesting that management regimes and propagation protocols developed for these fishes may also be appropriate for use with P. leopardus.     

The effect of physiological concentrations of sex hormones,insulin, and glucagon on growth of breast and prostate cells supplemented with unmodified human serum

The majority of cell culture studies have assessed the effect of hormones on cancer cell growth using media supplemented with charcoal-treated fetal bovine serum (CTS). We aimed to determine whether using a system more reflective of the human condition by changing the charcoal-treated serum to an untreated pooled human serum (PHS) resulted in the same hormone responses in breast and prostate cell lines. MCF-7 breast cancer, MCF-10A non-transformed breast, and LNCaP prostate cancer cell lines supplemented with PHS were treated with high and low physiological concentrations of six hormones (17β-estradiol, dehydroepiandosterone (DHEA), dihydrotestosterone (DHT), testosterone, insulin, and glucagon). Cell growth was measured after 72 h of incubation. All hormones stimulated growth of MCF-7 cells (p < 0.05). MCF-10A cell growth was inhibited by DHEA, DHT, and testosterone (p < 0.05), unaffected by 17β-estradiol and glucagon, and stimulated by insulin (p < 0.05). LNCaP cell growth was stimulated by the highest concentration of DHEA and DHT (p < 0.05) and inhibited by the highest concentration of 17β-estradiol (p < 0.05), while insulin and testosterone, had no effect. Overall, PHS lowered the magnitude of the effect of hormones on cell growth in comparison to CTS. Due to the presence of all serum constituents, our model represents a more appropriate physiological environment for determining the effect of hormones on cancer cell growth. Further studies are required to determine the mechanisms by which added hormones interact with the constituents of untreated human serum.     

Biological Activity of Sea Bass (Dicentrarchus labrax L.) Recombinant Interleukin-1β

Biological activities of a putative mature sea bass interleukin-1β peptide, produced as a recombinant protein (rIL-1β) in Escherichia coli, have been investigated. The rIL-1β contains a 6-histidine tag at the N-terminus, and protein purification has been achieved through this tag by affinity chromatography. Biological activities have been investigated both at the cellular and gene expression levels. In in vitro assays sea bass rIL-1β induced the proliferation of murine D10.G4.1 cells and increased yeast phagocytosis by sea bass head kidney leukocytes. The purified cytokine was also tested in a lymphocyte-activation factor assay, where it induced the proliferation of sea bass thymocytes. Finally, in an in vivo assay, rIL-1β administered intraperitoneally increased expression levels of the IL-1β gene and activated macrophages to produce a cyclooxygenase 2 homologue (COX-2) gene in the head kidney.     

Annual cycles of growth and reproduction in hatchery–reared Florida largemouth bass, Micropterus salmoides floridanus, raised on forage or pelleted diets

Annual cycles of growth and reproduction of hatchery–reared Florida largemouth bass, Micropterus salmoides floridanus , were investigated. Animals were raised on either forage (goldfish, Carassius auratus ) or a pelleted salmon feed. Male and female year–class 1 largemouth bass were sampled throughout one complete yearly cycle (January–December). A biphasic growth cycle was observed in both forage–fed and pellet–fed fish. No increase in body length or weight was observed until approximately midway through the spawning period (May), after which fish grew at a consistent rate for the remainder of the study. The reproductive cycle of forage–fed fish was characterized by a rapid increase in gonadosomatic index (GS1) between January and April, followed by a prolonged spawning period (April–July) during which GSI progressively declined. Fully regressed gonads were observed in September and October, and a resumption of gonadal recrudescence was observed between October and December. Visceral adipose deposits (expressed as mesenteric fat index; MFI) were resorbed during gonadal growth and the initial stages of the spawning period, and restored during the post–spawning phase. Fish raised on pelleted feed had growth and reproductive cycles that parallelled those of forage–fed fish, but several significant differences were observed between the two diet groups. During the growth phase of the cycle, pellet–fed largemouth bass grew significantly faster than forage–fed largemouth bass, and had significantly larger MFIs than forage–fed largemouth bass at all times of the year. Pellet–fed fish also had significantly larger GSIs than forage–fed fish. These data indicate that diet composition may be an important determinant of growth and reproductive function in this species.     

Testicular development and serum levels of gonadal steroids during the annual reproductive cycle of captive Japanese sardine

Gonad and blood samples were taken throughout the year from captive males of the Japanese sardine,Sardinops melanostictus, and changes in serum levels of gonadal steroids were examined in relation to the annual gonadal cycle. On the basis of testicular histology, the annual gonadal cycle was divisible into four periods: immature (July–September), spermatogenesis (October–December), spermiation (January–April), and post-spawning (May–June). The pattern of seasonal changes in the gonadosomatic index (GSI) was inversely correlated with that of water temperature, and reflected the degree of testicular maturity. The serum testosterone level was relatively low during spermatogenesis (2.2–2.5 ng/ml), rose markedly around the time of spermiation (7.7–24.6 ng/ml), and became low after spawning and during immature periods (0.6–0.7 ng/ml). The serum 17α,20β-dihydroxy-4-pregnen-3-one level was high in males with spermatogenic or spermiating testes (0.6–1.0 ng/ml), but became low (0.2 ng/ ml during the post-spawning period and was undetectable in immature fish. Although 11-ketotestosterone was detectable in some fish, the values obtained were thought to reflect cross-reactivity of the antiserum employed with testosterone. These findings are discussed in relation to male reproduction of the Japanese sardine and steroidal regulation of spermatogenesis and spermiation in other teleosts.