Pituitary gonadotropin release by graded electrical stimulation of the preoptic area in the male bullfrog, Rana catesbeiana.

Acute (5 min) graded electrical stimulation (biphasic pulses: amplitude, 50, 200, and 500 microA; frequency, 10 Hz; duration, 0.5 msec) of the medial preoptic area (including the medial and lateral septal nuclei and amygdala, pars medialis) induced significant elevations in circulating gonadotropins (more luteinizing hormone, LH, than follicle-stimulating hormone, FSH) in male bullfrogs, Rana catesbeiana. Pituitary response to all currents was rapid and of brief duration; levels of plasma gonadotropins (LH and FSH) peaked at 5 min (the end of stimulation) and decreased rapidly thereafter (LH by at least 50% after 30 min). A stimulus-response relationship was apparent; the magnitude of LH maxima was directly dependent on current intensity between 50 and 500 microA. In general, pituitary responses characteristic of acute electrical stimulation were similar to those induced by injection of mammalian gonadotropin-releasing hormone (GnRH). This is the first direct physiological demonstration of GnRH-like activity in the brain of the bullfrog.

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.

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.

Effects of gonadectomy on polymorphism in stored and circulating gonadotropins in the bullfrog, Rana catesbeiana. I. Clearance profiles.

Marked differences were observed between the clearance profiles of immunoreactive plasma gonadotropins in gonadectomized and intact male bullfrogs (Rana catesbeiana). The disappearance patterns of endogenously secreted follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from plasma of intact animals following chronic (1-4 days) infusion with gonadotropin releasing hormone (GnRH) showed multiple components, but the initial few half-lives were relatively short (less than 1 h) and about 90% of both gonadotropins were cleared from the plasma within 6 h. Hypophysectomy had no effect on gonadotropin clearance rates following the termination of GnRH infusion. Clearance profiles of exogenous gonadotropins after chronic (6 h) infusion of bullfrog pituitary extract were similar to those observed after GnRH infusion. Gonadectomized frogs also cleared these infused pituitary gonadotropins at the same rate as intact animals, confirming that gonadectomy did not impair peripheral clearance mechanisms. Relatively rapid clearance rates were also observed for endogenous FSH and LH in normal untreated frogs. By comparison, the disappearance rates of FSH and LH from plasma of six long-term gonadectomized males following hypophysectomy were extremely slow: first half-lives for FSH and LH were 25.6 h and 17.2 h, respectively, and subsequent half-lives were even longer. Several weeks were required to clear fully the FSH and LH from the circulation in these males. Thus, a significant change in the physicochemical form of the circulating gonadotropins after gonadectomy in the male bullfrog is postulated; the corresponding changes in clearance rates were considerably greater than have been observed in any other species.

Induced ovulation and changes in pituitary responsiveness to continuous infusion of gonadotropin-releasing hormone during the ovarian cycle in the bullfrog, Rana catesbeiana.

Chronic (2-4 days) constant-rate infusions of mammalian gonadotropin releasing hormone (GnRH) were performed in female bullfrogs, Rana catesbeiana. The magnitude and temporal relationship of profiles of plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH) and sex steroids [testosterone (T), estradiol-17 beta (E2) and progesterone (P)] during GnRH infusion were dependent on ovarian stage. However, in all females, the same biphasic increase in plasma gonadotropins was apparent and initial elevations in gonadotropins were accompanied by correlated increments in plasma T and E2. Complete pituitary "desensitization" to chronic GnRH infusion was not observed. Females in early follicular stages were relatively unresponsive to infusions of 1.0-10.0 micrograms/h GnRH; elevations in plasma LH were marginal and FSH was unchanged. Females with fully developed (preovulatory) ovaries were more responsive: infusion of 1.0 micrograms/h GnRH produced significant elevations in plasma LH by 2 h followed by even larger increases ("surges") after 12 h. This LH "surge" was preceded by a decline in plasma T and E2 and was accompanied by abrupt elevations in plasma P and by ovulation. Postovulatory females showed a more gradual and smaller increase in plasma LH. Infusion of GnRH in the female bullfrog establishes a clear relationship between pituitary responsiveness and the ovarian cycle not evident from acute GnRH injection; GnRH was most effective immediately before ovulation. These data are also the first to detail periovulatory changes in plasma gonadotropins and ovarian steroids in an amphibian.

An analysis of the results of mammographically guided biopsies of the breast.

Three hundred and fifty-three women underwent 358 biopsies of the breast for nonpalpable mammographic lesions during a five year period. Cancer was identified in 95 (27 per cent). Mammographic findings in the patients with cancer were calcifications only (54 per cent), a mass (27 per cent), a mass with calcifications (15 per cent), an asymmetric distortion (1 per cent) and an asymmetric distortion with calcifications (3 per cent). Cancer was identified in 29 per cent of the biopsies done for calcifications, 20 per cent of those done for a mass with calcifications, 6 per cent of those done for an asymmetric distortion and 38 per cent of those done for an asymmetric distortion with calcifications. Forty-nine per cent of the mammographically suspicious calcifications were cancer, while 100 per cent of the indeterminate calcifications were benign. Forty-five per cent of the spiculated masses were cancerous, while only 3 per cent of the circumscribed masses were malignant. For those patients with carcinoma undergoing axillary lymph node dissection, 13 per cent had one or more positive nodes. A strategy for increasing the effectiveness of mammographically guided biopsies of the breast is presented.

Relation between acute pituitary responsiveness to gonadotropin releasing hormone (GnRH) and the ovarian cycle in the bullfrog, Rana catesbeiana.

Freshly captured female bullfrogs (Rana catesbeiana) were challenged with a standard dose (2 micrograms) of gonadotropin-releasing hormone (GnRH) shortly before and at the onset of the spawning season (in early April and mid-May) to examine the relationship between pituitary responsiveness and ovarian stage; males were also tested in April. As shown previously, increments in plasma gonadotropins induced by GnRH were consistently lower in females than in males. There was little difference in average responsiveness between the two collections of females and results for these were similar to those obtained at other times of the year. A wide range of ovarian stages was represented at both collection times as evidence by plasma steroid profiles, but there was no correlation between the GnRH-induced increments in plasma FSH and LH and plasma levels of androgens (testosterone and 5 alpha-dihydrotestosterone), estradiol-17 beta, or progesterone in the 71 females tested. Thus, there is no evidence of a feedback effect of ovarian steroids on the pituitary responsiveness to acute GnRH stimulation in the frog as has been demonstrated in mammals.

Seasonal and stress related changes in plasma gonadotropins, sex steroids, and corticosterone in the bullfrog, Rana catesbeiana.

Studies of seasonal gonadal cycles combined with direct measurements of plasma levels of the two gonadotropins (FSH and LH), several gonadal steroids (estrogen, E; progesterone, P; testosterone, T; and 5 alpha-dihydrotestosterone, DHT), and the interrenal steroid corticosterone (B) were made in male and female bullfrogs in central California over a 5-year period (between 1976 and 1981). During the course of these studies, we discovered that levels of plasma gonadotropins and steroids are highly labile and particularly sensitive to the effects of captivity, especially in males. In animals captured and sampled repeatedly in the field over a 3-day period, hormone levels remained constant, but if held in collecting sacks, gonadotropin and gonadal steroids began to drop within 2--4 hr and usually reached "baseline" levels within 20 hr. This effect was apparent in all seasons, except occasionally in early spring when hormone levels dropped by only about half. Hormone levels were also generally depressed by the time commercially collected frogs reached local supply houses. Plasma B increased within 30 min of capture and remained high for days in captive animals. Blood samples taken from several hundred animals at the time of capture reveal pronounced seasonal cycles in all hormones measured. These changes are discussed in connection with other gross changes in gonadal condition and with regard to possible interactions among gonadotropins and steroids. Both sexes show a general elevation of hormone levels in spring and early summer, but the sexes differ somewhat both in timing and in magnitude of the changes, as well as in the nature of the dominant steroids. Pronounced "surges" in gonadotropins are evident around the time of gamate release in bot sexes, but the temporal pattern of these surges is not the same for ovulation and spermiation; an elevation in plasma P is associated with the periovulatory surge in gonadotropins. Results were not entirely consistent with expectation of pituitary--gonadal relationships. Levels of plasma gonadotropins and steroids did not show the reciprocal relationship expected from a simple negative feed-back between gonadal and pituitary secretion, nor did changes in gonadotropins and gonadal activities show the consistent positive correlation expected from a direct dependence of gonadal function on circulating gonadotropins. In females, plasma T, but not E, correlated with ovarian growth. Plasma T in females reached much higher levels than in males, but DHT was higher in males. Androgens were generally elevated during the period of sexual activity in males, but absolute levels did not correlate well with individual differences in sexual behavior. Thus, seasonal changes in testicular and ovarian activities cannot be accounted for solely by seasonal cycles in circulating gonadotropin levels.

Effects of gonadectomy on polymorphism in stored and circulating gonadotropins in the bullfrog, Rana catesbeiana. II. Gel filtration chromatography.

Marked polymorphism was revealed in both stored and circulating forms of immunoreactive follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the bullfrog, Rana catesbeiana, by exclusion chromatography on columns of Sephracyrl-S200. FSH behaved as a more homogeneous and larger molecule than LH from the same pituitary or plasma, but the properties of both hormones in the plasma were markedly affected by gonadectomy. Chromatographic profiles of FSH stored in the pituitaries were similar in intact and gonadectomized frogs, but pituitary LH in the latter was comprised of a larger proportion of early eluting activity. Previously purified preparations of bullfrog FSH and LH were more homogeneous than these extracts. Differences between pituitary hormones in intact and gonadectomized frogs were small compared with those between circulating hormones. Plasma FSH and lH from gonadectomized frogs behaved as more homogeneous and larger molecules than those from intact frogs in which plasma gonadotropins were elevated normally or by injections with gonadotropin releasing hormone (GnRH). Some differences in circulating hormones were also observed between a normal male and female and both differed from gonadectomized an GnRH-treated intact frogs. Chromatographs of plasma gonadotropins in GnRH-treated animals generally resembled those of the hormones stored in the pituitary, whereas plasma FSH and LH in gonadectomized frogs appeared more homogeneous and larger than the pituitary-stored forms. Those pronounced differences in chromatographic properties of gonadotropins in intact and gonadectomized frogs correlate with previously observed effects of gonadectomy on clearance profiles of circulating FSH and LH.

Effects of chicken and mammalian gonadotropin-releasing hormones (GnRH) on in vivo pituitary gonadotropin release in amphibians and reptiles.

The ability of mammalian and chicken gonadotropin-releasing hormones (GnRH) and their agonistic analogs to stimulate in vivo gonadotropin release were tested in a frog (Rana catesbeiana), snake (Naja naja), and turtle (Sternotherus odoratus). In the frog, chicken and mammalian GnRH were equipotent in stimulating the release of FSH and LH. Attendant increases in plasma androgen and the occurrence of spermiation confirmed the release of biologically active gonadotropin. Neither of the GnRH preparations or their agonists produced significant changes in plasma hormones in either of the reptiles. In light of comparable data for the actions of these GnRH preparations in mammals and birds, it appears that species specificity in the response to different GnRHs does not correlate well with the nature of the homologous hypothalamic GnRH molecule.