Biomedical subjects

P Licht

Publications and source records attributed to P Licht.

At least 109 records · Page 6Linked to original sources

PubMed1986

Effects of temperature and mode of delivery on responses to gonadotropin-releasing hormone by superfused frog pituitaries.

An in vitro superfusion system was used to examine the effects of temperature on the responsiveness of frog, Rana pipiens, hemipituitaries to chronic superfusion with gonadotropin-releasing hormone (GnRH). The effects of pulsatile as opposed to continuous delivery of GnRH on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion were also studied at 30 degrees. Secretion of both gonadotropins (FSH and LH) increased significantly in a dose-dependent manner in response to a brief exposure to 0.1-1000 ng/ml (nM) GnRH at both 18 and 32 degrees, but the magnitude of response at each dose was temperature dependent. Moreover, the temporal pattern of the chronic response to a standard dose of GnRH (100 ng/ml) also varied markedly between 10 and 32 degrees: Hemipituitaries superfused at 10 or 18 degrees showed greatly attenuated responsiveness to GnRH after the first hour of GnRH treatment, whereas responsiveness was maintained or even continued to increase for many hours at higher temperatures. These temperature effects may be related to seasonal changes in reproductive activity. Pulsatile administration of GnRH was more effective in inducing elevated gonadotropin (GtH) secretion during the first few hours of superfusion, but continuous superfusion with GnRH was more effective at maintaining elevated GtH levels over long periods. The results support and extend previous studies by indicating that, unlike the situation in many mammals and birds, the frog pituitary is highly resistant to desensitization by even high doses of GnRH, and in fact, self-priming and maintained responsiveness to GnRH is better supported by chronic treatment than by pulsatile delivery of GnRH.

Animals↗

PubMed1986

In vitro ovarian responses to pulsatile and continuous gonadotrophin administration on steroid secretion and oocyte maturation in the frogs, Rana pipiens and Rana catesbeiana.

An in vitro superfusion system was used to study the relative effects of pulsatile and continuous gonadotrophin administration on steroid secretion and oocyte maturation in Rana pipiens ovaries. Pulsatile (10 min pulse/hr) delivery of pituitary extract (PE) resulted in a slightly (insignificantly) lower level of testosterone (T) secretion over a 12-hr period. Administration of a fivefold lower, subliminal amount of PE instead of hormone-free media between pulses did not change the pattern of T secretion. When the interpulse frequency was increased to 2 or 3 hr, there were notable oscillations in T secretion which corresponded to the peaks in luteinizing hormone associated with the PE. In four experiments ovarian fragments underwent oocyte maturation, but this occurred only in fragments that received continuous PE stimulation. Progesterone (P) secretion was measurable only when oocyte maturation was observed in the ovarian fragment. Rana catesbeiana ovarian fragments exposed to continuous superfusion with homologous PE produced more T than those receiving hourly pulses of PE over a 6-hr period. Oocyte maturation accompanied by P secretion was observed in one experiment under continuous, but not pulsatile administration of PE. These results suggest that the frog ovary may be more resistant than the mammalian ovary to "down-regulation" under continuous gonadotrophic stimulation. The implications of these results on the frog pituitary--gonadal axis are discussed.

Animals↗

PubMed1986

Effects of cycloheximide on in vitro testosterone secretion from RANA catesbeiana ovaries.

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.

Animals↗

PubMed1986

The influence of hibernation on testis growth and spermatogenesis in the golden-mantled ground squirrel, Spermophilus lateralis.

Testis size and spermatogenesis were monitored serially in individual golden-mantled ground squirrels before, during, and after the hibernation season. During hibernation, animals spent 81% of days in torpor at body temperatures of 3-4 degrees C. Torpor bouts of 6 days duration were interspersed with brief arousals from torpor during which animals were normothermic. In the 5 mo between December (when animals entered hibernation) and April (when torpor was spontaneously terminated), the estimated mass of testes increased gradually from 500 to 1100 mg, but spermatogenesis did not advance beyond pachytene spermatocytes, which were present before hibernation began. In contrast, during the month after torpor was terminated, testes increased rapidly to 3500 mg and after 31 days, spermatozoa were found in the epididymides. We suggest that the limited testis growth that occurred during the hibernation season was restricted to intervals during which squirrels were aroused from torpor. The major portion of gonadal growth and spermatogenesis in the laboratory, and presumably in the field, occurs after ground squirrels have regained the normothermic state. Since males are reproductively mature when first trapped in spring, these findings suggest that males are normothermic for several weeks before they emerge from their hibernacula in the spring.

Aging↗

PubMed1986

Suitability of the mammalian model in comparative reproductive endocrinology.

Perhaps one should be impressed by the basic generality of many of the features of the mammalian HHG system. The applicability of this model to other groups argues for the generally conservative nature of the basic organization of the reproductive endocrine system. Even in cases where exceptions are apparent, we must be cognizant of the relatively narrow base of mammalian species on which the classical mammalian model is built. While reference to the mammalian system provides a useful first approach to studies on new groups, in view of the many exceptions in details of this system, it is important that we avoid undue constraints that might be imposed by the the mammalian model. In this connection it would seem advisable to test each principle carefully before assuming that it exists or is as important as in mammals. A particularly significant issue in this regard is the question of temporal organization of endocrine secretions: Both the existence and relative importance of discontinuous, pulsatile, stimulation must be explored in more groups. It must be emphasized that past experience has already shown us that a simple phylogenetic approach is not likely to provide a clear pattern for the similarities and differences in the reproductive endocrine system. Thus, we should not be too hasty in our extrapolations of data, even among closely related species.

Amphibians↗

PubMed1985

Effects of gonadectomy and steroid treatment on plasma gonadotropins and the response of superfused pituitaries to gonadotropin-releasing hormone in the turtle Sternotherus odoratus.

Gonadectomized (gonadex) turtles, Sternotherus odoratus, had significantly elevated plasma FSH, but LH was less consistently affected. Estradiol (E2)-implants suppressed plasma FSH in gonadex females but not in males: testosterone (T) partially suppressed FSH in males. In contrast, E2-treatment markedly suppressed pituitary LH content and in vitro LH secretion in gonadex and intact turtles (inhibitory effects of E2 were less in intact than ovariectomized females). These steroid effects were relatively specific for gonadotropin; pituitary TSH content was not altered. In vitro, pituitary LH secretion responded to doses of GnRH greater than or equal to 1 ng/ml and LH output remained elevated for at least 3 hr of continuous superfusion with gonadotropin-releasing hormone (GnRH). In general, gonadectomy elevated pituitary responsiveness to GnRH while E2 and T suppressed this responsiveness; the effects of E2 are greater in gonadectomized than intact turtles. Thus, negative gonadal feedback appears to be involved in the secretion of gonadotropins in turtles, and steroidal actions may be partly due to suppression of pituitary hormone content and responsiveness to GnRH.

Animals↗

PubMed1985

Reevaluation of the relative activities of the pituitary glycoprotein hormones (follicle-stimulating hormone, luteinizing hormone, and thyrotrophin) from the green sea turtle, Chelonia mydas.

The discovery that the follicle-stimulating hormone (FSH) previously prepared from the green sea turtle, Chelonia mydas, contained a major neurohypophysial contaminant prompted a repurification and characterization of the glycoprotein hormones in this turtle. Results reaffirmed the physicochemical distinctiveness of the three hormones. Minimal cross-contamination between hormones (less than 2%) was achieved by ion-exchange chromatography, subunit dissociation (of contaminating luteinizing hormone (LH], gel filtration, and immuno-affinity chromatography. New preparations of FSH and thyrotrophin (TSH) derived from adult pituitaries proved to be more potent than those described previously (the degree depending on the nature of the assay); FSH showed the expected increase in activity based on estimated contamination of previous preparations. LH was similar to original preparations except for enhanced activity in FSH radioreceptor assays. Binding assays (in heterologous and homologous systems) again demonstrated the general absence of an FSH-specific receptor in the reptilian (chelonian and squamate) testes. In an in vivo bioassay in the lizard Anolis, the turtle FSH was orders of magnitude more potent than LH in stimulating both testis growth and androgen secretion, but in vitro LH was considerably more potent than FSH in stimulating androgen secretion in squamate and chelonian testes. Thus, the possibility exists that androgen secretion in some chelonian systems may exhibit a high degree of LH specificity like that of mammals and birds.

Androgens↗

PubMed1985

Annual and diurnal cycles in plasma testosterone and thyroxine in the male green sea turtle Chelonia mydas.

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.

Animals↗

PubMed1985

In vivo and in vitro responses to gonadotropin releasing hormone in the turtle, Chrysemys picta, in relation to sex and reproductive stage.

In vivo and in vitro responsiveness to gonadotropin releasing hormone (GnRH) was studied in the turtle, Chrysemys picta, after manipulation of reproductive condition by temperature: Warm temperatures (28 degrees) induced testicular growth and ovarian regression compared to cold (17 degrees) treatment. Only males (and primarily from cold treatment) responded to GnRH injection (40 micrograms/100 g body wt intracardiac); correlated increases occurred in plasma LH and testosterone. Effects of GnRH (10 and 100 ng/ml) on LH and FSH secretion by hemipituitaries were studied in a superfusion system; tissues responded to between 0.1 and 1 ng/ml GnRH. Sex differences were evident in both acute and chronic effects of GnRH. Although both groups of females had significantly (sixfold) higher pituitary LH content, basal secretion rates of gonadotropins were similar, and LH and FSH secretion in males was more responsive to GnRH. Gonadotropin secretion rates by male glands showed high initial increments (approx four- to sevenfold) followed by an attenuation (especially LH) during 5 hr of GnRH superfusion. In contrast, tissues from warm-treated females showed a smaller initial response (approx twofold) followed by a progressive increase in output over time, and glands from cold-treated females did not respond to GnRH. Total LH secretion by superfused male hemiglands represented almost half the total LH recovered (secreted + stored); whereas, females secreted only 5% ("cold-treated") or 10% ("warm-treated") of total LH. Thus, the capacity of the pituitary to respond to GnRH is influenced by both sex and reproductive condition in the turtle. Secretion of both FSH and LH were similarly stimulated by GnRH, but thyrotropin (TSH) secretion was independent of GnRH.

Animals↗

PubMed1985

In vitro study of the direct ovarian effects of gonadotropin-releasing hormone (GnRH) in the frogs, Rana pipiens and Rana catesbeiana.

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.

Animals↗

PubMed1985

Changes in hormone levels in relation to breeding behavior in male bullfrogs (Rana catesbeiana) at the individual and population levels.

Fluctuations in plasma androgen (testosterone and 5 alpha-dihydrotestosterone), corticosterone (B), and luteinizing hormone (LH) of male bullfrogs in central California were measured during the spring mating season. Androgen and LH levels generally increased in the population prior to the initiation of chorusing and establishment of territories, whereas plasma B peaked in a 2-week period at the start of heavy chorusing; this coincided with a transitory, 1 week decline in androgen. Individual males showed fluctuations in plasma androgen and LH levels throughout the breeding season, often within 1 day, but there was no clear correlation between changes in the two hormones. No time of day effect was apparent on any of the hormones. B and androgen levels were significantly but weakly correlated, (r = 0.35) but LH and androgen were not. Although the seasonal trend of increasing androgen corresponded with the start of intense chorusing (and presumably sexual activity) by the population, behavior of individuals and their circulating androgen levels did not correlate. After acquiring territories, males showed no overall trend of increased plasma androgen. Moreover, males that showed no vocal or territorial behavior had significantly higher androgen and lower B levels than calling males. Increased B levels suggest that territorial behavior and especially direct agonistic encounters represent stresses that could have an inhibitory effect on androgen secretion.

Androgens↗

PubMed1985

Pituitary responsiveness to superfused GnRH in two species of ranid frogs.

Dynamics of pituitary responsiveness to gonadotropin-releasing hormone (GnRH) as measured by LH and FSH secretion rates were examined for two species of ranid frogs in an in vitro superfusion system. The influence of sex was studied with juvenile bullfrogs, Rana catesbeiana, and responsiveness to long-term continuous superfusion with GnRH was examined in R. catesbeiana and R. pipiens. Female juvenile bullfrogs showed virtually no response to doses of GnRH up to 1000 ng/ml, whereas males responded to doses of GnRH as low as 10 ng/ml, and very large increments in LH and FSH were observed with 100 and 1000 ng/ml GnRH (responsiveness in the males was dependent on body size). Adult R. pipiens were more sensitive than bullfrogs to GnRH; 0.2 ng/ml GnRH was an effective dose. These data indicate that, relative to mammals, sensitivity to GnRH in frogs is higher than suggested by previous in vivo studies. As in mammals, "self-priming" in response to GnRH was evident; but unlike mammals, the "self-priming" occurred only after 7-12 hr of continuous superfusion with GnRH, and the R. pipiens glands were more resistant to desensitization during 48 hr of GnRH treatment. This study confirms that sex- and age-dependent differences in responsiveness to GnRH as well as "self-priming" found in previous in vivo studies are evidently dependent on properties of the anterior pituitary per se.

Aging↗

PubMed1985

LH secretion in response to gonadotropin releasing hormone (GnRH) by superfused pituitaries from two species of turtles.

A superfusion system was employed to study the dynamics of the responses of LH secretion to gonadotropin releasing hormone (GnRH) by anterior pituitary glands (quartered) from adult turtles, Pseudemys scripta and Chrysemys picta. Responsiveness was highly variable in both species, but in many cases, the tissues showed marked responses to relatively low doses (2-20 ng/ml) of GnRH. GnRH had no effect on LH secretion in five female P. scripta tested in the month of August, whereas it stimulated most glands tested in June and September; the few males tested were not as responsive as females in the same season. Female P. scripta invariably showed no or only very transient responses (less than 1 hr) to continuous GnRH superfusion, although glands recovered rapidly from this state of "desensitization" as evidenced by their ability to respond to subsequent stimuli. In some cases, pulsatile GnRH was effective in preventing this rapid desensitization. In contrast, glands from female C. picta were highly responsive in August. In March, they were similar to June P. scripta in showing a rapid desensitization to GnRH, whereas in August, the tissues of C. picta maintained full responsiveness to continuous GnRH for many hours. There was little evidence of GnRH "self-priming" in the turtles. The high individual variability in the magnitude of the response of LH to GnRH, the variable but often rapid desensitization to GnRH, as well as possible sexual, seasonal, and species differences in GnRH responsiveness may underlie the discrepancies previously observed among in vivo and in vitro studies in reptiles. However, the turtle is clearly not as unresponsive to mammalian GnRH as suggested by some previous studies.

Animals↗

PubMed1985

Seasonal cycles in testicular activity, gonadotropin, and thyroxine in the painted turtle, Chrysemys picta, under natural conditions.

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.

Analysis of Variance↗

PubMed1985

Steroid biosynthesis in turtle testes.

Testicular metabolism of radiolabelled substrates (pregnenolone, progesterone and androstenedione) was examined in two species of turtles, Pseudemys scripta and Sternotherus odoratus. Chemical methods (chromatography, derivative formation and crystallization) identified testosterone (in both species) and 5 alpha-androstane-3 beta,17 beta-diol (in S. odoratus) as products. Tentative identifications were also made of 20 beta-hydroxypregn-4-en-3-one in both turtles. Chromatographic profiles indicated the presence of several other C19- and C21-steroids. In S. odoratus, changes in proportions of the two major C19-compounds (testosterone and the 5 alpha-androstanediol) suggest a "switch" in the enzymatic pathways which resembles that associated with maturation in the rat. This enzymatic switch may be associated with the annual testicular cycle of the adult turtle.

Androstenedione↗

PubMed1984

Studies on the specificity of thyroid response to pituitary glycoprotein hormones.

Thyrotropins (TSH) and gonadotropins (luteinizing hormone, LH, and follicle-stimulating hormone, FSH) purified from ovine, bovine, ostrich, sea turtle, and bullfrog pituitary glands were tested for their ability to simulate the thyroid glands of four species of amphibians (three anurans and a urodele), a reptile (the slider turtle), and a bird (the cockerel). All animals tested responded specifically to mammalian, sea turtle, and ostrich thyrotropins; any thyroid-stimulating activity of the gonadotropins from these species could be accounted for by their contamination with thyrotropin. In addition, the three anuran species showed a high degree of specificity for bullfrog TSH; bullfrog LH exhibited low thyrotropic activity in amphibians. In contrast, bullfrog LH was more potent than bullfrog TSH in stimulating the thyroid gland of the cockerel. This bird therefore resembles reptiles in showing a "heterothyrotropic" response to bullfrog LH. The hormonal specificity of thyroid response to these pituitary glycoprotein hormones therefore varies distinctly among representatives of different vertebrate classes. Although the tetrapods tested were generally specific for thyrotropin in their thyroid responses to homologous and heterologous pituitary hormones, occasionally a lack of specificity for heterologous hormones existed which could be shared by species from closely-related classes. The varied patterns seen indicate that independent evolution of the functional characteristics of the thryotropin receptor has occurred to different degrees within each vertebrate class.

Animals↗

PubMed1984

The role of androgen in the development of sexual differences in pituitary responsiveness to gonadotropin releasing hormone (GnRH) agonist in the bullfrog, Rana catesbeiana.

Sexual differences in pituitary responsiveness to acute injection of gonadotropin-releasing hormone (GnRH) agonist, as measured by increments in plasma FSH and LH, were examined throughout development in the bullfrog, Rana catesbeiana. Untreated tadpoles, in various stages of metamorphosis, were unresponsive to GnRH agonist. Postmetamorphic males showed a progressive increase in the magnitude of pituitary response with age, whereas females remained relatively insensitive until after sexual maturation; males were always more responsive than females. Chronic (1-2.5 week) Silastic implants containing 5 alpha-dihydrotestosterone (DHT) significantly augmented the pituitary response (for both gonadotropins) in intact postmetamorphic females at all ages; a similar, though less pronounced, action of testosterone in subadult females may have been due to its conversion to DHT. (Silastic implants of comparable size always produced higher circulating levels of DHT in females than in males as was observed in previous studies with gonadectomized frogs.) DHT enhanced the responsiveness of intact tadpoles (sexes undetermined); only the treated tadpoles responded to the GnRH agonist. Supplemental DHT did not enhance pituitary response in intact males; in fact, it attenuated the response in FSH. GnRH responsiveness paralleled changes in pituitary gonadotropin content; pituitary content of FSH and LH was higher in males than females; it showed a marked increase with age from tadpole to adult; and it was increased by DHT treatment. The potentiating effect of DHT on GnRH responsiveness and the significantly higher levels of DHT observed in males of all ages suggest that the nonaromatizable androgen DHT may be responsible for the early establishment and maintenance of sexual dimorphism in pituitary GnRH responsiveness in the bullfrog.

Animals↗

PubMed1984

Effects of gonadectomy and sex steroids on pituitary gonadotrophin release and response to gonadotrophin-releasing hormone (GnRH) agonist in the bullfrog, Rana catesbeiana.

Gonadectomy of adult bullfrogs, Rana catesbeiana, elevated plasma levels of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Profiles of plasma gonadotrophins after gonadectomy were similar in the two sexes; however, FSH increased faster in females than males. Both gonadotrophins continued to rise for several months and remained elevated after 1 year, but there was some dissociation between the two hormones; FSH increased sooner (1 week vs 3 weeks), reached higher levels (FSH/LH much greater than 1), and did not show the secondary decline exhibited in LH. Similarly, in subadult males and females, gonadectomy increased plasma FSH by 1 week but LH levels were less affected. Postgonadectomy increases in both gonadotrophins were prevented by chronic (6-week) implantation of capsules containing estradiol-17 beta (E2) or 5 alpha-dihydrotestosterone (DHT), and treatment with DHT and E2 within physiological ranges starting 1 year after gonadectomy also suppressed chronically elevated levels of plasma gonadotrophins. Compared to untreated gonadectomized frogs, the rate of increase in both plasma gonadotrophins. Compared to untreated gonadectomized frogs, the rate of increase in both plasma gonadotrophins was accelerated after removal of DHT at 6 weeks. Acute pituitary responsiveness to a gonadotrophin-releasing hormone (GnRH) agonist was markedly reduced in short-term (3.5-8 weeks) gonadectomized subadult males (but not females) and in long-term (1 year) gonadectomized males and females. Treatment with E2 had no effect on GnRH responsiveness in these frogs, but DHT implants significantly enhanced the response to agonist in both sexes. Thus, the nonaromatizable androgen--DHT--may have both negative feedback effects (at the hypothalamic level) and positive effects at the level of the pituitary, whereas, estrogen exhibited only the former, negative feedback activity. Sex differences in circulating DHT, which are detectable even in juveniles, may account for the sexual dimorphism in pituitary responsiveness to GnRH in the bullfrog.

Aging↗