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The female European mole (Talpa europaea) presents a vivid paradox in relation to our contemporary understanding of mammalian sexual differentiation. These animals are exceptional among female mammals in that they possess bilateral ovotestes. The ovotestis contains a morphologically normal ovarian component that develops during the spring breeding season and a histologically defined testicular region, the interstitial gland, which enlarges during autumn when the ovarian component decreases in size. In correlation with this unusual gonadal situation, the female mole displays a penile clitoris traversed by a urethral canal. Although the histology of the ovotestis is well documented and has recently been extended to an additional three species of the genus Talpa, there have been no clear indications of the physiological function, particularly androgen production, of the ovotestis in these female moles. This paper presents the first clear evidence of seasonal variation in plasma testosterone concentrations, which parallel the growth and regression of the "testicular" interstitial gland, in T. europaea. Plasma androstenedione did not show significant seasonal variation, but plasma testosterone (1.06 +/- 0.2 ng/ml) and gonadal testosterone concentration (1.57 +/- 0.65 microgram/mg protein) in females in autumn were significantly higher (p < 0.02) than plasma (0.4 +/- 0.2 ng/ml) and gonadal (0.24 +/- 0.21 microgram/mg) concentrations in pregnant or immediately postpartum females in spring. Our data also reveal selective metabolic production of testosterone from radiolabeled steroid precursors (progesterone and androstenedione) by these ovarian interstitial tissues and male testes; estradiol is produced by ovarian tissue but not interstitial gland or testis.

We tested whether 1) glucose availability is a signal for initiation of torpor in male hamsters and 2) glucoprivation can override the inhibitory effects of androgens on daily torpor. Male hamsters maintained at ambient temperatures of 8-16 degrees C were injected with 2-deoxy-D-glucose (2DG), a glucose analogue that interferes with cellular glucose oxidation. 2DG (2,000-2,500 mg/kg body mass) induced torpor within 1 h of treatment in normal adult males in reproductive condition and in those bearing testosterone (T)-filled capsules that produced supraphysiological blood T concentrations; body temperatures were reduced from 37 to 25 degrees C for several hours. Latency to torpor was increased and duration of torpor was decreased in the T-treated hamsters. Food intake decreased substantially both on the day of torpor and on the succeeding day. Glucoprivation appears to counteract the potent inhibitory effect of androgens on torpor and induces a hypometabolic state that results in overall energy savings.

17 alpha-Hydroxylase Deficiency (17 alpha-OHDS) is a rare defect of steroid biosynthesis, characterized by the inability to synthesize cortisol, androgens or estrogens, by the complete absence of follicular maturation, hypergonadotropic hypogonadism, primary amenorrhea and hypertension. Since the ovaries of such patients contain numerous primordial follicles, we hypothesized that the absence of spontaneous follicular maturation could be due to a lack of aromatizable substrate. To provide this substrate, testosterone was administered either by intra-ovarian injection or by vaginal administration. Ovarian stimulation was performed with human urinary gonadotropins. Follicular maturation and ovulation could be induced by this treatment, as determined from ultrasonography, the analysis of LH, estradiol and progesterone serum levels and the aspiration of oocytes from the mature follicles. Fertilization of these oocytes in vitro, however, did not succeed. We conclude that follicular maturation can be induced in 17 alpha-OHDS by gonadotropins when testosterone is provided as an aromatizable substrate and that estrogens are a necessary component of follicular maturation.

The effects of experimental conditions--steroid concentration, temperature, treatment duration, tadpole density, and delivery mode--on testosterone (T) metabolic rates were examined in larval anurans. T uptake into tadpoles was measured by scintillation counting of [3H]T and metabolism of T was studied by thin layer chromatography in two anuran species, the African clawed frog (Xenopus laevis) and the western toad (Bufo boreas). T was extensively metabolized by both species, but the effects of each experimental condition on T metabolism varied between species. In the study of delivery mode, tadpoles injected with T dissolved in saline metabolized T faster than animals in groups where the steroid was added directly to the water. Animals injected with T dissolved in oil metabolized T the slowest, but T was short-lived in all cases. The current study demonstrates that concentration, temperature, treatment duration, tadpole density, and delivery mode may all affect steroid metabolic rates, but these effects vary between species in unpredictable ways. Since steroid metabolism may either activate or deactivate steroids, these factors are important in understanding the potency of steroids in tadpoles. A review of the literature dealing with steroid effects in anuran larvae illustrates these problems.

Both 17 beta-estradiol (E2) and testosterone (T) were shown to inhibit in vitro pituitary LH secretion in the turtle Trachemys scripta. Since T was approximately 500 times less potent than E2, and 5 alpha-dihydrotestosterone was even less active than T, the inhibitory action of T may result from its aromatization to estrogen. We utilized both in vivo and in vitro approaches to elucidate the roles of T and estrogen in the negative feedback of pituitary LH secretion. Gonadectomy of adult (vitellogenic) females significantly elevated plasma LH. Adult females treated with fadrozole (an aromatase inhibitor) with or without daily injections of keoxifene (an antiestrogen) also showed an increase in plasma LH to a level comparable to that observed in gonadectomized females, whereas plasma LH levels of juvenile females treated with fadrozole remained undetectable. In vitro LH secretion in response to GnRH in juvenile females was significantly inhibited by 48-h exposure to 50 ng/ml T or 100 pg/ml E2. Both fadrozole (200 microM) and keoxifene (200 nM) significantly blocked this T-induced inhibition of LH secretion, demonstrating that T lacks intrinsic inhibitory activity. Confirmation of the inhibition of aromatase activity by fadrozole comes from metabolic studies of 1 beta-[3H]androstenedione using turtle brain, ovary, and pituitary. In vitro, fadrozole altered the metabolism of 1 beta-[3H]androstenedione and inhibited aromatase activities in these tissues. These results indicate that the inhibitory effect of T is largely mediated through its aromatization to estrogen, and that estrogen is primarily responsible for the suppressed LH secretion in vitellogenic adult turtles.

The effects of temperature and steroids [testosterone (T), estradiol (E2), and corticosterone (B)] on premetamorphic growth and development were investigated in the toad (Bufo boreas). The effects of steroids were both temperature and age dependent. In the first experiment, steroids (1.1-1.4 microM) were administered by dissolving them in the water beginning 1 day after hatching at 22 degrees C or 27 degrees C. At 22 degrees C, B inhibited growth (P < 0.001) but had no significant effect on development. Forelegs never emerged in B-treated animals and all died before complete tail resorption. Discontinuation of B treatment allowed normal growth and metamorphosis, but the resulting post-metamorphic animals were significantly shorter (snout-vent length, P < 0.001) than after other treatments. At 22 degrees C, T and E2 had no effect on larval growth and development or size at metamorphosis (P > 0.05), but T induced early foreleg emergence (FLE) (P < 0.005). At 27 degrees C, B was fatal after 2 weeks of treatment, and T and E2 inhibited growth (P < 0.001) and development (P < 0.001), but did not affect time to FLE. In a second experiment at 27 degrees C, treatment with 1.1 microM B starting 15 days after hatching induced early metamorphic events (P < 0.001), such as tail resorption and emergence of the left foreleg (but not the right), but jaw and head restructuring failed to occur. All B-treated animals died before complete tail resorption. In a third experiment, 0.275 and 1.11 microM B, starting at day 43 (stage 43), induced early FLE (P < 0.05) and decreased snout-vent length at tail resorption (P < 0.005) without a dose effect. A higher dose of B (4.44 microM) decreased snout-vent length at tail resorption and time to FLE (P < 0.05) but did not affect body weight at metamorphosis (P > 0.05). Animals in this experiment survived to complete tail resorption and transformed normally. The actions of B in these experiments closely resemble those observed with administration of thyroid hormones, suggesting that steroids may interact with endogenous thyroid hormones.

Female spotted hyenas exhibit male-like genitalia and dominance over males. Hyena ovarian tissues incubated in vitro produced large quantities of the steroid hormone precursor androstenedione. The activity of aromatase, which converts androstenedione to estrogen, was one-twentieth as great in hyena versus human placental homogenates. In comparison, the activity of 17 beta-hydroxysteroid dehydrogenase, which converts androstenedione to testosterone, was equal in the two homogenates. The limited aromatase activity may allow the hyena placenta to convert high circulating concentrations of androstenedione to testosterone, which results in virilization of the fetal external genitalia and possibly destruction of fetal ovarian follicles. Androstenedione production by residual ovarian stromal cells during reproductive life accounts for the epigenetic transmission of virilization in female spotted hyenas.

Removal of the olfactory bulbs lengthened the period of circannual rhythms (CARs) of body mass and plasma testosterone (T) concentrations in male golden-mantled ground squirrels, but did not otherwise alter their expression. The period of the CAR was approximately 2 months longer in bulbectomized than in sham-operated animals. Peak values of body mass and T were unaffected by bulbectomy. All neurologically intact, but only 50% of bulbectomized, squirrels displayed normal hibernation patterns. We conclude that the olfactory bulbs are not the site of essential circannual oscillators. Circannual reproductive and hibernation cycles of ground squirrels are less subject to modulation by the olfactory bulbs than are the corresponding rhythms of several non-circannual hamster species.

Female Siberian hamsters maintained in a winter photoperiod (8 h light/day) ceased to undergo daily torpor during infusion of prolactin (PRL) from osmotic minipumps; winter torpor was reinstated within 3 days of discontinuation of treatment. By contrast, PRL infusion was ineffective in suppressing daily torpor elicited by restricting food intake in female hamsters housed in a summer photoperiod (16 h light/day). Summer daily torpor was, however, completely inhibited in long-day gonadectomized male hamsters treated with testosterone (T). We suggest that the hyperprolactinemia, which in previous studies is characteristic of hamsters that sustain ablation of the suprachiasmatic nucleus, is incompatible with winter torpor. Summer torpor may be controlled by a mechanism less responsive to variations in plasma PRL concentration. Both winter and summer torpor are inhibited by exogenous T; it remains uncertain, however, whether sustained decreases in endogenous T secretion are as essential for the expression of summer as they are for winter torpor.

This report is concerned with hormone concentrations accompanying sexual maturation in a highly 'masculinized' female mammal, the spotted hyaena, Crocuta crocuta. Plasma concentrations of testosterone, androstenedione and oestrogen were determined by radioimmunoassay in a longitudinal study of 12 female and eight male hyaenas 2.5-62.5 months old. Concentrations of testosterone were significantly higher in males than in females after 26.5 months of age, but earlier measurements did not differ between sexes. Mean testosterone concentrations in adult female hyaenas (0.4-0.5 ng ml-1) were similar to those in several other female mammals that do not display a 'masculine' profile, but mean concentrations of androstenedione (2.5-5.5 ng ml-1) in female hyaenas were significantly higher than in males (1.0-2.0 ng ml-1), at most ages. Oestrogen could not be detected (less than 0.03 ng ml-1) in females until about 14 months of age and then increased (to approximately 0.13 ng ml-1) between 18 and 30 months; oestrogen remained undetectable in males. This rise in oestrogen in females corresponded to nipple enlargement and to changes in the size and elasticity of the urogenital meatus, permitting copulation and parturition through the clitoris. Gonadectomy (two males and four females) at 4-7 months resulted in nondetectable concentrations of testosterone and oestrogen and a marked attenuation in androstenedione (to approximately 0.39 ng ml-1), indicating that the gonads are the major source of these three steroids. Gonadectomy also eliminated sex differences in weight, nipple development and elasticity of the urogenital meatus.

At different phases of the hibernation season, castrated male golden-mantled ground squirrels were implanted with capsules that either were filled with testosterone (T) or left empty (blank). Blank-treated animals hibernated normally when housed at 5 degrees C. Entry into hibernation was prevented in the majority of squirrels treated with T several days before the initial cold challenge. T concentrations that inhibited torpor (greater than 1.2 ng/ml) were comparable with those of intact males at the end of the hibernation season. In some squirrels, moderate T concentrations were compatible with hibernation, but torpor bout duration was shorter than normal. The inhibitory effect of T on hibernation did not appear to require aromatization of T to estradiol. We suggest that a steroid-independent mechanism triggers arousal from hibernation and that T-dependent processes determine whether hibernation is resumed at the end of an arousal period.

Male squirrels, born approximately June 1 and kept under constant conditions of photoperiod (14 h of light per day) and temperature (23 +/- 2 degrees C) from birth, manifested circannual rhythms in plasma testosterone (T) and luteinizing hormone (LH) levels. Blood samples were collected once per month for 22 months and hormone levels determined by radioimmunoassay. Testosterone levels were low or undetectable ( less than or equal to 0.05 ng/ml) between October and January of the first year, and began increasing in February and peaked during April and May (approx. 4.5 ng/ml). By August of the second year T had declined to low levels which were maintained through January (0.05 ng/ml). The cycle then repeated, with T titers elevated during April and May of the second year. LH levels were undetectable (less than or equal to ng/ml) throughout most of the first 8 months of life; LH peaks were reached by individual animals between March and July. There was a close correspondence between seasonal peaks and troughs of testosterone and those in LH. The circannual pattern of plasma T and LH also was documented for a group of field-trapped males (less than or equal to 11 months old) maintained under similar conditions and bled at monthly intervals for 14 consecutive months. Rhythms of plasma T and LH persisted with a period of 11 to 12 months under constant conditions and thus qualify as circannual cycles.

Steroid hormone concentrations change in response to social or environmental stimuli in many vertebrates. To test this phenomenon in an amphibian, we examined plasma androgen (A) and corticosterone (B) concentrations in male marine toads (Bufo marinus), a tropical species exhibiting intermale competition, amplectic clasping of females, and bouts of breeding behavior following rains. When males clasped females for 0, 1, 2, or 3 hr, plasma A concentrations were significantly and positively correlated with hours spent in amplexus. In field-sampled males, plasma A concentrations were higher in amplexing males than in single males. Among single males those found closer to breeding ponds had higher A concentrations than those more distant. These data support the hypothesis that sexual stimuli enhance plasma A concentrations in this amphibian. In 3-hr experimental tests and field-sampled males, B concentrations were higher in amplexing than in single males. Unlike some amphibians, short-term elevations of B apparently are not associated with decreased reproductive function. However, as in other amphibians in which high B concentrations are associated with stress-induced inhibition of reproduction, after 48-72 hr in captivity male toads showed high B concentrations and low plasma androgen concentrations. The bursts of sexual activities exhibited by B. marinus following heavy rains were associated with no changes in A concentration and with increased B concentration.

Plasma levels of testosterone (T), thyroxine (T4) and follicle-stimulating hormone (FSH) were measured in a field population of the painted turtle, Chrysemys picta, in Michigan. All three hormones showed pronounced seasonality; plasma luteinizing hormone (LH) remained nondetectable. Plasma T and FSH concentrations were highly correlated and exhibited biphasic cycles with peaks in spring and fall, whereas T4 showed a single peak in summer, coincident with the nadir in T. Hormone levels were minimal on the day that animals emerged from hibernation at the end of March; T and FSH then increased rapidly (in ca. 1 week after emergence) to a transient peak that persisted for only ca. 2 weeks. Plasma T and FSH rose again in September before the onset of hibernation and shortly after the late summer peak in spermatogenetic recrudescence. Plasma T4 increased more gradually after emergence in spring and did not peak for 1-2 months, coincident with the nadir in T. The profile in plasma FSH is consistent with the view that this gonadotropin may regulate androgen secretion but it did not show the expected relationship to the spermatogenetic cycle. Separate studies indicated that the turtles are sensitive to capture and handling; hormone levels (especially androgen) fall precipitously within a day of capture. These "stress" effects may account for discrepancies with previous studies of plasma T in this species.

Ovaries from the leopard frog, Rana pipiens, and bullfrog, R. catesbeiana, were used to study potential direct extrapituitary effects of gonadotropin-releasing hormone (GnRH). GnRH alone did not alter steroid secretion from ovaries in either species. Ovarian fragments containing mature follicles from R. pipiens were incubated in several submaximal doses of homologous pituitary homogenate or purified bullfrog luteinizing hormone (LH) together with 1000 ng/ml GnRH. The addition of GnRH failed to alter testosterone (T) or progesterone secretion or germinal vesicle breakdown over a wide dose range of gonadotropin. The effects of supramaximal doses of pituitary homogenate on R. pipiens and the effects of homologous LH on T secretion by fragments of R. catesbeiana ovaries were also unaffected by the presence of GnRH. The lack of a GnRH effect on the dynamics of T secretion in the bullfrog was further confirmed in a superfusion system with homologous pituitary homogenate. This study fails to demonstrate an action of GnRH at the level of the ovary in two ranid species.

A 1 hr exposure to 20 micrograms/ml of the protein synthesis inhibitor, cycloheximide (CHX), essentially abolished secretion of testosterone (T) by bullfrog ovarian fragments during simultaneous administration of homologous pituitary extract and CHX. Removal of CHX from the medium after 4 hr of treatment reversed the inhibition of T secretion, allowing it to attain control levels. Pre-exposure of ovarian fragments to CHX was not required to obtain an inhibition of T secretion. These data supported the hypothesis that protein synthesis is required for acute and chronic gonadotropic stimulation of steroidogenesis by the bullfrog ovary.

Male plasma testosterone (T) and thyroxine (T4) were monitored over several annual cycles in a captive breeding colony of green sea turtles, Chelonia mydas. Daily and annual water temperatures varied by only approximately 1 and 3 degrees, respectively. A pronounced season cycle in plasma T was evident in the population as a whole and in individual animals: plasma T was at a nadir (approximately 3 ng/ml) in September-November and then increased progressively to a peak (27-39 ng/ml) in April; levels began declining immediately thereafter, coincident with the onset of copulatory behavior. By contrast, plasma T4 remained uniform (approximately 9 ng/ml) throughout the year and, thus, could not readily account for the decline in androgen levels. Plasma hormones were relatively stable over a 24-hr period at three times a year, and there was a correlation for individual plasma T levels sampled in April and May. Thus, limited sampling should allow identification of seasonal rhythms and individual variability in plasma T levels. Testis mass and spermatogenic activity were significantly greater in January than in September; i.e., spermatogenesis and androgen secretion were not "uncoupled." Copulatory activity began in April but did not peak until May-June, after plasma T had significantly declined. However, there was a significant (but weak) correlation between individual peak levels of plasma T (i.e., in April) and the quantitative level of mating activity (time spent mounting and number of mates) measured for the entire subsequent season. Thus, green turtles do not exhibit the "postnuptial" type of testis cycle typical of many temperate-zone turtles, and the levels of plasma androgen may be important for initiating and maintaining sex behavior, although they are not tightly linked during the mating season.