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382. Regulation of the onset of puberty in the female rat
386
Abstracts
‘iC-testosterone (T) (I Jligl formed primarily androstenedione and 5a-androstanediols (DIOL), and lesser amounts ofdihydrotestosterone (DHT) and androsterone (BBRC 54, 1425. IY73). Our objective was to determine which cell types in the tubule had the capacity to form the 5x-reduced metabolites. Ceil suspensions containing over 90”,, spermatocytes. prepared from tubules of immature testes, formed DHT as the major metabolite of “C-T. Tubule preparations rich in Sertoli cells prepared from testes of regressed hypophysectomized or cryptorchid rats, or from testes of busulphail-tr~ted rats, converted “C-T to both DHT and DIOL. Tubules from adult testes incubated with ‘*C-DHT (1 fig) formed 2OOng DIOL/mg protein/h. spermatocyte preparations formed only 20 n? DIOL:mg protein/h, and Sertoli cell-enriched prcparatlons formed 500-600ng DIOLimg protein/h. We conclude that 5xreductase activity is present in spermatocytes and Sertoli cells. The Sertoli cell appears to be the main site of DIOL production in the tubule.
1OG. Biological rhythms 379. Daily rhythmic secretion of progestin by the ovary in lactating rats YOSHINAGA, K.. Laboratory of Human Reproduction and Reproductive Biology and Department of Anatomy, Harvard Medical School, Boston. Mass. 02115, U.S.A. Daily secretory pattern of progestin by the ovary was examined in rats nursing 8 pups during the first I6 days of lactation. Blood was collected from both the right and left ovaries for IO min at 09.00 h and 18.00 h on different days of lactation. Contents of progesterone (P) and 20a-hydrkxyprego-4-en-3-one @Jr-OH-P) in ovarian venous plasma were measured by gas-liquid chromatography and radioimmunoassay. Secretion rates of both progestins showed a clear daily diurnal fluctuation: low in the morning and high in the evening on each day of lactation. except on days 5 and I2 which were characterized by a sudden increase in P/ZOr-OH-P ratio in the morning of day 5 and a maximal P secretion on day I?. These findings show that ovarian progestin secretion during lactation has a different pattern from that reported for pseudopregnant rats where P secretion is high in the morning and low in the evening. Diurnal fluctuation of ovarian progestin secretion suggests a possible existence of diurnal fluctuation of luteotrophic factors during lactation. (Supported by grants NIH HD06047 and 06467 and the Population Council. N.Y.). Persistence of circadian periodicity in BALB/C mice after adrenalectomy and during inanition and dehydration LAKATUA, D. J., HAUS, E. and HALBERG, F.. Department of Anatomic and Clinical Pathology, St. PaulRamsey Hospital, St. Paul, Minnesota and Chronobiology Laboratories. Dept. of Pathology. University of Minneapolis. Minneapolis, Minnesota. IJ.S.A. Adrenalectomized mice with a subcutaneous implant of a corticosterone pellet, kept on a LD 12: 12 lighting regimen (L 06.00-18.00) maintain a circadian rhythm in liver glycogen and rectal temperature 21 days after surgery. The circadian serum corticosterone rhythm was abolished but non-circadian higher frequencies -in serym corticosterone levels did persist. After a 12h phase shift in lighting
regimen (LD 12: 12 with L lX.W ~~.()I~) accompllshcd h? prolongation of a light period the circadian rhythm in rectal temperature of these animals phase shifted with ;I shift time identical to that in the hham-operated control%. In groups of intact mice kept for up to 3 day\ ci!her t%iihtlttt food or without food and water under a LD 12: 12 lighting regimen a circadian periodicit> in pituitar! ACTH content. serum corticosterone and adrenal corticosteronc content persist with markedly elevated amplitude. 381. Seasonal rhythm in plasma testosterone and LH of male laboratory rats MOCK. E. J. and FRA~KI.L, A. I.. Department of Biological Sciences, State L~niverrity of New York at Binghamton. N.Y.. kJ.S.4. Although seasonal cycles in reproduction are commonly attributed to male field animals. little attention has been paid to the possibility that reproductive hormones in the male fabotatory rat follow a seasonal cycle. A study was therefore made of 8 male rats, more than 80 days of age. during a nine-month period beginning in Janwary. 1973. All rats were housed in windowless animal rooms with lights on from 06.00 to 18.00. Temperature was maintained at 71 _t 3 C; food and water were provided trll /i~j~~~~~f.Blood was collected monthly from 1000 to 1100h r)y, cardiac puncturrr and the plasma was analyzed by radrolmmunoassay for testosterone and LH concentrations. Plasma testosterone concentration during Fehruarq-March (4.3 & 0.5 ng,‘ml) was significantly elevated (/J < 0.0001) when compared to other sampling periods (2.3 t O.Zng:ml). Data from X7 additional male rats sampled infrequently during the Ymonth period confirm this result. Plasma testosterone concentrations in male rats during the maturational surge at 60-65 days of age were similar. whether during February March (3-6 & 0.4ng!ml. N = 36) or during other months ol the year (3.9 f 0.3 ng!ml. N = 5X) and were comparable to those of older rata during the seasonal curge. Scasonat variation of plasma concentration of LH was synchront& to testosterone concentration. aithouyh LH Icvels declined more slowly than did testosterone in the months followinrr February and March. (Supported in part by L!SPHS HDO4081 and NIMHT MHl305S.)
382. Regulation of the onset of puberty in the female rat HOCHMAK J. and ESHEL, S.. Department of Zoology. Hebrew University, Jerusalem. Israel The induction of precocious puberty with non-estrogenic steroids was studied in the immature female rat. 5%. androstane-3~~-17~-diol enhanced specifically the onset of puberty, the 3/I and 17/j hydroxyls both being essential for the induction process, as judged by the ineffectiveness of similar compounds differing onlv in one or both (?/I and 17h) positions. The induction is ovarian dependent as shown by the inability of the /i compound to enhance vaginal opening in unilateral and bilateral ovariectomized immature female rats. Introduction of a double bond at C-5 into the compound increased its effectiveness fourfold. Lower and upper time limits for the induction process were found to be when steroid administration started between days425 postnatatly. Ineffectiveness of the compound after day 25 suggests some sort of maturation in the ovary itself (change in receptor sites or metabolizing enzyme’.‘). In addition we found that the compound was unable either to induce androgeni~dtioll in neonates or to inhibit the andrcl-
Abstracts
387
genizing capacity of testosterone propionate thus suggesting that a center in the brain, other than the one responsive to TP, takes part in the regulation of the onset of puberty.
384. Long-lasting effects of prepuberal administration of
383. Sites of steroid ~~~t~~s and ageing changes in the human p~meno~u~i ovary GURAYA, S. S., Department of Zoology, Punjab Agricultural University, Ludhiana, India
of Anatomy and Brain Research Institute, UCLA Schooi of Medicine, Los Angeles, California, U.S.A.
During early postmenopausal years of women varying in age from 45 to 54 years, theta interna cells of normal follicles, theta lutein and granulosa lutein cells of atretic lollicles (or corpora atretica), and stromal theta cells constitute active sites of steroid biosynthesis as revealed by their histochemical reactions for lipids, cholesterol and steroid dehydrogenases. They secrete steroid hormones apparently as a result of menopausal gonadotrophin (HMG) stimulation. Their involution is closely accompanied by the storage of ab~dant lipid droplets composed mainly of cholesterol and/or its esters, trigIy~~d&s, pigments and very little phospholipids, and by the disappearance of their enzyme activities indicative of steroid biosynthesis. Evolution and involution of these steroid gland cells continue in early postmenopausal years. With ageing, a stromal lipid band is developed by the fatty degeneration of cortical stromal cells, which is not involved in steroid biosynthesis. Corresponding with its development, the medullary blood vessels and primordial oocytes also start showing ageing changes accompanied by the accumulation of mostly neutral lipids.
morphinein female rats ZIM~~MA~N, E.. YOUNG. J., BRANCH,B.. TAYLOR, A. N., PANG, C. N. and SAWYER,C. H., Department
To study effects of prepuberal administration of morphine on body growth and pituitary-adrenal function. newborn Sprague-Dawley female rats were given subcutaneous saline (S) or morphine (M) (to 8 mg/kg, b.i.d.) from l-21 days of age. Compared with controls, body weights of M-treated animals were reduced (p < 0.01) by day 7 and remained significantly depressed for approximately 120 days. Puberty (vaginal opening) occurred simultaneously in M- and Streated rats on days 36 f 2. On day 56. both groups showed comparable diurnal patterns of non-stress levels of plasma corticosterone (B). Compared with controls on day 72, M-treated rats showed intact B responses to ether stress, but reduced (p < 0.05) B responses to M (20 or 40 mgjkg). Similarly, on day 80, M-treated rats showed reduced 0, c 0.05) analgetic response to M (IO mg/kg) using the hot-plate test. Finally, on day 92. M-treated rats, but not controls, showed increased (p < 0.05) plasma B levels 30min after injection of naloxone (5 mg/kg). Persistence of growth deficit, tolerance to M. and sensitivity to naloxone suggest that exposure of the immature female rat to morphine results in prolonged M-specific alteration of neuroendocrine and nociceptive brain mechanisms. (Supported in part by the Ford Foundation.)
11. Steroids in neur~ndo~rin~ fun~tion$ 11A. Steroid modulation of the pituitary response to hypothalamic hormones 385. Effect of sex steroidson pituitary responsesto luteiuiziug hormone releasing hormone (LH-RH) in amenorrheic women TUOMPSON, I. E., PATTON,W. C., CHONG,A. P. and TAYMOR,M. L., Department of Surgery (Gyn), Peter Bent Brigham Hospital, Boston, Ma., U.S.A. Synthetic LH-RH in a dose of ISOpg was administered intravenously on two successive days to seventeen women with secondary amenorrhea of hypothalamic origin. Three patients received LH-RH injections only, and in addition to LH-RH five were treated with estradiol (E,), four with aqueous progesterone (P), three with E,+P, and two with testosterone (T). Each steroid was administered at various dose levels by intramuscular injection 4h after the first injection of LH-RH. The patients receiving successive LH-RH tests without steroids showed a similar LH response in both tests, each response being greater than that seen in the normal menstrual cycle. The patients receiving sex steroids in addition showed a significant suppression of LH response in the second LH-RH test, which was performed 20 h after the administration of the steroid. FSH responses to LH-RH were much more variable, but in those cases where a pronounced initial FSH response was obtained,
the second test after administration of steroid also revealed suppression of the FSH response. The results indicate a direct inhibitory effect of Ez, P. E,+P, and T on the response of the pituitary gland to synthetic LH-RH in amenorrheic women. 386. Response of LH and FSH to LH-RH in exogenous and endogenous hypercortisolism MACIA, A. L., CHARRO,A. L., DURANGO.R. F., PUENTE,M., AVILA, C. and CRUZ. A. F.. 1 Catedrd de Patologia Gral. Hospital Clinico, Madrid A control group of normal women in the follicular phase, another group of short-term steroid-treated women, also in the first half of the cycle, two females with adrenal hyperplasia, one with Sheehan’s syndrome, and one with acromeaalv were given 100 11~of LH-RH i.v. : blood samples were taken at 0’, 15’, 3O’,’45’, 60’. 90’ and 120’ intervals from LH-RH administration. and RIA of LH and FSH was performed by a double antibody technique. All the normal women tested had a clear response of LH and FSH with a mean increase of A LH = 18.0 i_ 7.9 S.D. and d FSH = 15,l + 3,4S.D. The steroid treated women had values of LH and FSH and a response to LH-RH that were normal and not statistically different from controls (d LH = 18.2 + IO,6 S.D..d FSH = 216 r~:15,8). In endogenous hypercortisolism as well as in Sheehan’s syndrome and acromegaly, the values of LH and FSH were low and the response I_
-
Abstracts
‘iC-testosterone (T) (I Jligl formed primarily androstenedione and 5a-androstanediols (DIOL), and lesser amounts ofdihydrotestosterone (DHT) and androsterone (BBRC 54, 1425. IY73). Our objective was to determine which cell types in the tubule had the capacity to form the 5x-reduced metabolites. Ceil suspensions containing over 90”,, spermatocytes. prepared from tubules of immature testes, formed DHT as the major metabolite of “C-T. Tubule preparations rich in Sertoli cells prepared from testes of regressed hypophysectomized or cryptorchid rats, or from testes of busulphail-tr~ted rats, converted “C-T to both DHT and DIOL. Tubules from adult testes incubated with ‘*C-DHT (1 fig) formed 2OOng DIOL/mg protein/h. spermatocyte preparations formed only 20 n? DIOL:mg protein/h, and Sertoli cell-enriched prcparatlons formed 500-600ng DIOLimg protein/h. We conclude that 5xreductase activity is present in spermatocytes and Sertoli cells. The Sertoli cell appears to be the main site of DIOL production in the tubule.
1OG. Biological rhythms 379. Daily rhythmic secretion of progestin by the ovary in lactating rats YOSHINAGA, K.. Laboratory of Human Reproduction and Reproductive Biology and Department of Anatomy, Harvard Medical School, Boston. Mass. 02115, U.S.A. Daily secretory pattern of progestin by the ovary was examined in rats nursing 8 pups during the first I6 days of lactation. Blood was collected from both the right and left ovaries for IO min at 09.00 h and 18.00 h on different days of lactation. Contents of progesterone (P) and 20a-hydrkxyprego-4-en-3-one @Jr-OH-P) in ovarian venous plasma were measured by gas-liquid chromatography and radioimmunoassay. Secretion rates of both progestins showed a clear daily diurnal fluctuation: low in the morning and high in the evening on each day of lactation. except on days 5 and I2 which were characterized by a sudden increase in P/ZOr-OH-P ratio in the morning of day 5 and a maximal P secretion on day I?. These findings show that ovarian progestin secretion during lactation has a different pattern from that reported for pseudopregnant rats where P secretion is high in the morning and low in the evening. Diurnal fluctuation of ovarian progestin secretion suggests a possible existence of diurnal fluctuation of luteotrophic factors during lactation. (Supported by grants NIH HD06047 and 06467 and the Population Council. N.Y.). Persistence of circadian periodicity in BALB/C mice after adrenalectomy and during inanition and dehydration LAKATUA, D. J., HAUS, E. and HALBERG, F.. Department of Anatomic and Clinical Pathology, St. PaulRamsey Hospital, St. Paul, Minnesota and Chronobiology Laboratories. Dept. of Pathology. University of Minneapolis. Minneapolis, Minnesota. IJ.S.A. Adrenalectomized mice with a subcutaneous implant of a corticosterone pellet, kept on a LD 12: 12 lighting regimen (L 06.00-18.00) maintain a circadian rhythm in liver glycogen and rectal temperature 21 days after surgery. The circadian serum corticosterone rhythm was abolished but non-circadian higher frequencies -in serym corticosterone levels did persist. After a 12h phase shift in lighting
regimen (LD 12: 12 with L lX.W ~~.()I~) accompllshcd h? prolongation of a light period the circadian rhythm in rectal temperature of these animals phase shifted with ;I shift time identical to that in the hham-operated control%. In groups of intact mice kept for up to 3 day\ ci!her t%iihtlttt food or without food and water under a LD 12: 12 lighting regimen a circadian periodicit> in pituitar! ACTH content. serum corticosterone and adrenal corticosteronc content persist with markedly elevated amplitude. 381. Seasonal rhythm in plasma testosterone and LH of male laboratory rats MOCK. E. J. and FRA~KI.L, A. I.. Department of Biological Sciences, State L~niverrity of New York at Binghamton. N.Y.. kJ.S.4. Although seasonal cycles in reproduction are commonly attributed to male field animals. little attention has been paid to the possibility that reproductive hormones in the male fabotatory rat follow a seasonal cycle. A study was therefore made of 8 male rats, more than 80 days of age. during a nine-month period beginning in Janwary. 1973. All rats were housed in windowless animal rooms with lights on from 06.00 to 18.00. Temperature was maintained at 71 _t 3 C; food and water were provided trll /i~j~~~~~f.Blood was collected monthly from 1000 to 1100h r)y, cardiac puncturrr and the plasma was analyzed by radrolmmunoassay for testosterone and LH concentrations. Plasma testosterone concentration during Fehruarq-March (4.3 & 0.5 ng,‘ml) was significantly elevated (/J < 0.0001) when compared to other sampling periods (2.3 t O.Zng:ml). Data from X7 additional male rats sampled infrequently during the Ymonth period confirm this result. Plasma testosterone concentrations in male rats during the maturational surge at 60-65 days of age were similar. whether during February March (3-6 & 0.4ng!ml. N = 36) or during other months ol the year (3.9 f 0.3 ng!ml. N = 5X) and were comparable to those of older rata during the seasonal curge. Scasonat variation of plasma concentration of LH was synchront& to testosterone concentration. aithouyh LH Icvels declined more slowly than did testosterone in the months followinrr February and March. (Supported in part by L!SPHS HDO4081 and NIMHT MHl305S.)
382. Regulation of the onset of puberty in the female rat HOCHMAK J. and ESHEL, S.. Department of Zoology. Hebrew University, Jerusalem. Israel The induction of precocious puberty with non-estrogenic steroids was studied in the immature female rat. 5%. androstane-3~~-17~-diol enhanced specifically the onset of puberty, the 3/I and 17/j hydroxyls both being essential for the induction process, as judged by the ineffectiveness of similar compounds differing onlv in one or both (?/I and 17h) positions. The induction is ovarian dependent as shown by the inability of the /i compound to enhance vaginal opening in unilateral and bilateral ovariectomized immature female rats. Introduction of a double bond at C-5 into the compound increased its effectiveness fourfold. Lower and upper time limits for the induction process were found to be when steroid administration started between days425 postnatatly. Ineffectiveness of the compound after day 25 suggests some sort of maturation in the ovary itself (change in receptor sites or metabolizing enzyme’.‘). In addition we found that the compound was unable either to induce androgeni~dtioll in neonates or to inhibit the andrcl-
Abstracts
387
genizing capacity of testosterone propionate thus suggesting that a center in the brain, other than the one responsive to TP, takes part in the regulation of the onset of puberty.
384. Long-lasting effects of prepuberal administration of
383. Sites of steroid ~~~t~~s and ageing changes in the human p~meno~u~i ovary GURAYA, S. S., Department of Zoology, Punjab Agricultural University, Ludhiana, India
of Anatomy and Brain Research Institute, UCLA Schooi of Medicine, Los Angeles, California, U.S.A.
During early postmenopausal years of women varying in age from 45 to 54 years, theta interna cells of normal follicles, theta lutein and granulosa lutein cells of atretic lollicles (or corpora atretica), and stromal theta cells constitute active sites of steroid biosynthesis as revealed by their histochemical reactions for lipids, cholesterol and steroid dehydrogenases. They secrete steroid hormones apparently as a result of menopausal gonadotrophin (HMG) stimulation. Their involution is closely accompanied by the storage of ab~dant lipid droplets composed mainly of cholesterol and/or its esters, trigIy~~d&s, pigments and very little phospholipids, and by the disappearance of their enzyme activities indicative of steroid biosynthesis. Evolution and involution of these steroid gland cells continue in early postmenopausal years. With ageing, a stromal lipid band is developed by the fatty degeneration of cortical stromal cells, which is not involved in steroid biosynthesis. Corresponding with its development, the medullary blood vessels and primordial oocytes also start showing ageing changes accompanied by the accumulation of mostly neutral lipids.
morphinein female rats ZIM~~MA~N, E.. YOUNG. J., BRANCH,B.. TAYLOR, A. N., PANG, C. N. and SAWYER,C. H., Department
To study effects of prepuberal administration of morphine on body growth and pituitary-adrenal function. newborn Sprague-Dawley female rats were given subcutaneous saline (S) or morphine (M) (to 8 mg/kg, b.i.d.) from l-21 days of age. Compared with controls, body weights of M-treated animals were reduced (p < 0.01) by day 7 and remained significantly depressed for approximately 120 days. Puberty (vaginal opening) occurred simultaneously in M- and Streated rats on days 36 f 2. On day 56. both groups showed comparable diurnal patterns of non-stress levels of plasma corticosterone (B). Compared with controls on day 72, M-treated rats showed intact B responses to ether stress, but reduced (p < 0.05) B responses to M (20 or 40 mgjkg). Similarly, on day 80, M-treated rats showed reduced 0, c 0.05) analgetic response to M (IO mg/kg) using the hot-plate test. Finally, on day 92. M-treated rats, but not controls, showed increased (p < 0.05) plasma B levels 30min after injection of naloxone (5 mg/kg). Persistence of growth deficit, tolerance to M. and sensitivity to naloxone suggest that exposure of the immature female rat to morphine results in prolonged M-specific alteration of neuroendocrine and nociceptive brain mechanisms. (Supported in part by the Ford Foundation.)
11. Steroids in neur~ndo~rin~ fun~tion$ 11A. Steroid modulation of the pituitary response to hypothalamic hormones 385. Effect of sex steroidson pituitary responsesto luteiuiziug hormone releasing hormone (LH-RH) in amenorrheic women TUOMPSON, I. E., PATTON,W. C., CHONG,A. P. and TAYMOR,M. L., Department of Surgery (Gyn), Peter Bent Brigham Hospital, Boston, Ma., U.S.A. Synthetic LH-RH in a dose of ISOpg was administered intravenously on two successive days to seventeen women with secondary amenorrhea of hypothalamic origin. Three patients received LH-RH injections only, and in addition to LH-RH five were treated with estradiol (E,), four with aqueous progesterone (P), three with E,+P, and two with testosterone (T). Each steroid was administered at various dose levels by intramuscular injection 4h after the first injection of LH-RH. The patients receiving successive LH-RH tests without steroids showed a similar LH response in both tests, each response being greater than that seen in the normal menstrual cycle. The patients receiving sex steroids in addition showed a significant suppression of LH response in the second LH-RH test, which was performed 20 h after the administration of the steroid. FSH responses to LH-RH were much more variable, but in those cases where a pronounced initial FSH response was obtained,
the second test after administration of steroid also revealed suppression of the FSH response. The results indicate a direct inhibitory effect of Ez, P. E,+P, and T on the response of the pituitary gland to synthetic LH-RH in amenorrheic women. 386. Response of LH and FSH to LH-RH in exogenous and endogenous hypercortisolism MACIA, A. L., CHARRO,A. L., DURANGO.R. F., PUENTE,M., AVILA, C. and CRUZ. A. F.. 1 Catedrd de Patologia Gral. Hospital Clinico, Madrid A control group of normal women in the follicular phase, another group of short-term steroid-treated women, also in the first half of the cycle, two females with adrenal hyperplasia, one with Sheehan’s syndrome, and one with acromeaalv were given 100 11~of LH-RH i.v. : blood samples were taken at 0’, 15’, 3O’,’45’, 60’. 90’ and 120’ intervals from LH-RH administration. and RIA of LH and FSH was performed by a double antibody technique. All the normal women tested had a clear response of LH and FSH with a mean increase of A LH = 18.0 i_ 7.9 S.D. and d FSH = 15,l + 3,4S.D. The steroid treated women had values of LH and FSH and a response to LH-RH that were normal and not statistically different from controls (d LH = 18.2 + IO,6 S.D..d FSH = 216 r~:15,8). In endogenous hypercortisolism as well as in Sheehan’s syndrome and acromegaly, the values of LH and FSH were low and the response I_
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