Reproductive organ DNA and RNA of male and female rats from birth to 100 days of age.код для вставкиСкачать
Reproductive Organ DNA and RNA of Male and Female Rats from Birth to 100 Days of Age' C. DESJARDINS,z K. L. MACMILLAN? AND H. D. HAFS Animal Reproduction Laboratory, Dairy Department, Michigan State University, East Lansing, Michigan ABSTRACT Male and female rats were adjusted to six pups per litter and weaned at 20 days of age. Groups of ten males were killed at five- to ten-day intervals between one and 100 days of age and groups of 12 to 45 females were killed between ten and 100 days. Accumulation with age of gonadal, seminal vesicular, and uterine weight, DNA, and RNA, were compared with body growth in relative growth analyses to delimit puberty and quantify reproductive growth. Males and females possessed similar body growth to 40 days, after which age females grew more slowly while males continued rapid growth to 60 days. Relative to body growth, testes grew more rapidly from ten days whereas ovarian growth generally paralleled body growth until 50 days of age. A shift i n gonadal RNA/DNA ratio from less than to greater than unity occurred early in puberty in both sexes. Nucleic acid contents of accessory reproductive organs indicated that effective levels of gonadal steroids promote pubertal growth beginning at about 40 days in males and about 33 days of age in females. These marked pubertal growths ended at 70 days i n males and 60 days of age in females. The weight and nucleic acid content of reproductive organs decline following gonadectomy, but can be restored and even elevated above pre-operational levels with injected sex steroids (Velardo, '59; Kochakian and Harrison, '62; Williams-Ashman, '65). The restorational changes in the nucleic acids are rapid and may be detected before significant increases occur in organ weight. Telfer ('53) demonstrated that a single injection of 5 pg estradiol almost doubles RNA content of ovariectomized rat uteri within 48 hours, but responses in DNA and organ weight are neither as rapid nor as dramatic. When testosterone propionate pellets were implanted into castrated male mice, weights of the ventral prostates and seminal vesicles were increased twenty-fold within 30-40 days (Kochakian and Harrison, '62). Levels of RNA and DNA were similar to those in the organs of intact control mice within five days after testosterone implantation. This sensitivity of nucleic acid levels in reproductive organs to the sex steroids was the basis for the following experiment which was designed to measure changes in the nucleic acid contents of gonads and main accessory reproductive organs during reproductive development. These changes, especially those for RNA in accessory organs, may reflect fluctuations ANAT. REC., 161: 17-22. in steroidal titers during puberty more precisely than organ weight or histological criteria previously used (e.g., Moore, '39). An additional object was to provide bases for comparism of future treatments designed to alter reproductive growth. METHODS Male and female Sprague-Dawley rats were adjusted at birth to six pups per litter and weaned at 20 days of age. After weaning, males and females were reared separately with standardized numbers per cage, with feed and water ad libitum, in a room at 24" 1°C and provided with a 14-hour photoperiod daily. Ten male rats were killed by cervical dislocation at 1, 10, 20, 25, 30, 35, 40, 45, 50, 60, 80, 90 and 100 days of age. Forty-five females were killed at ten days, 24 females at 15, 20, and 25 days and 12 females at 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, and 100 days of age. The testes, seminal vesicles, ovaries and uteri were removed, isolated from extraneous tissue, weighed, placed in 0.25M sucrose at 5"C, and stored at 1 Published with the approval of the Director of the Michigan Agricultural Experlment Station as paper 4101. This .~. _ research was suuuorted by U.S.P.H.S. grant HD-01374. 2,Present address: Jackson Laboratory, Bar Harbor, Maine. 3 Present address: New Zealand Dairy Board, Newstead, Hamilton, N. Z . ~~ ~ ~~~~ _- 17 18 C. DESJARDINS, K. L . MACMILLAN A N D H. D. HAFS -20". Seminal vesicles were too small to 20-day level was less than the comparable measure reliably at one and ten days of increase in organ weight; suggesting tesage. Tissue DNA and RNA were deter- ticular hypertrophy coincidental with hymined by the procedures of Schmidt and perplasia. DNA concentration (mg DNA/ Thannhauser ('45) as modified by Tucker gm testis) declined at a diminishing rate ('64). In some younger age groups, or- after 20 days of age. Seminal vesicular weight, DNA, and gans from several animals were pooled to obtain sufficient tissue for assay purposes. RNA increased most rapidly from 40 to 70 days of age (table 2). Increases in RESULTS RNA with age were greater than comparThe growth rate of male rats (table 1 ) able increases in DNA and, consequently, was not reduced until 60 days of age (256 the RNA/DNA ratio increased from 0.57 gm body weight), but standard errors at 30 days of age to 3.46 at 100 days of associated with mean body weights in- age. Standard errors associated with mean creased considerably from 70 to 100 days organ weights and nucleic acid contents of age. Average testes weight (table 1 ) increased dramatically beyond 60 days of increased most rapidly between 20 (206 age. The body growth rate of females (table mg) and 70 (3,291 mg) days and declined slightly thereafter. Although total testicu- 3 ) was similar to that for males to 40 days lar DNA also increased from 20 (1.77 mg) of age. Thereafter, females grew less rapdays of age, the increase relative to the idly and their body weight apparently staTABLE 1 Body weight and testicular weight, D N A and R N A / D N A ratio Age dags 1 10 20 25 30 35 40 45 50 60 70 80 90 100 Bqdv weight m 720.1 2010.1 43t-1 58% 1 8011 11512 12622 16422 174t-4 25626 27519 265212 274k12 309t-15 Testicular weight m g 72:1 5922 20625 370213 600% 13 945237 1,194*78 1,911260 2,167255 3,009r72 3,291247 2,712279 2,828287 2,9092116 DNA RNA/DNA qng 0.01~0.00 0.32-CO.03 1.77t-0.05 2.4510.08 3.50*0.14 4.55k0.25 5.5220.24 6.72a0.32 9.1520.26 9.40*0.37 9.7020.33 8.2710.44 7.7510.59 7.0520.32 1.78 0.84 0.77 0.80 1.01 1.26 1.27 1.60 1.40 1.27 1.47 1.43 1.70 1.93 TABLE 2 Seminal vesicular weight, D N A , R N A and R N A / D N A ratio Age Weight DNA dags m g nw 20 25 30 35 40 45 50 60 70 80 90 100 8*l 821 6* 1 16t-1 2022 52*3 6517 174210 330230 278528 308*21 449245 0.05*0.01 0.06*0.01 0.07t-0.01 0.10~0.01 0.11*0.01 0.2410.02 0.34W.03 0.63k0.04 0.9Ot-0.12 0.6710.12 0.6320.05 0.6320.05 RNA RNAiDNA m g 0.03t0.01 0.05-CO.01 0.04-CO.01 0.1220.01 0.12r0.01 0.48r0.06 0.54r0.06 1.1420.07 1.9920.19 1.28-CO.24 1.43-CO.10 2.2020.3 1 0.65 0.79 0.57 1.13 1.17 1.94 1.63 I.84 2.40 1.90 2.33 3.46 19 REPRODUCTIVE GROWTH O F RATS bilized after 80 days of age (189 gm body weight). Ovarian weight increased at a constant rate from 15 (8 mg) to 80 (83 mg) days of age (table 3 ) . Although ovarian DNA content showed considerable variation throughout, the generally linear increase in DNA with advancing age was interrupted by a plateau between 30 and 50 days. There was little change in either total DNA or total RNA after 60 days of age. Uterine criteria (table 4 ) continued to increase to 90 days of age. Both testicular and ovarian RNA/DNA ratios generally changed from values of about 0.8 before 30 days to values over 1.0 after 35 days of age. Greatest changes in seminal vesicular and uterine weights and nucleic acid contents also occurred after 35 days of age (tables 2, 4), but the elevation in gonadal RNA/DNA ratios apparently preceded increments in accessory organ criteria. DISCUSSION The decline in growth rate of female rats occurs soon after puberty; vaginal opening occurred at 38 days of age. Similar sexual differences in rate of body growth were TABLE 3 Body weight and ovarian weight, DNA and RNA/DNA ratio Age Body weight days 10 15 20 25 30 31 32 33 34 35 40 50 60 70 80 90 100 Ovarian weight DNA gm mg mg 2020.3 3221 4421 5222 7423 84fl 8723 9023 9522 12523 14725 16725 18023 18926 19925 19124 720.1 820.2 1721 2221 2921 2922 28k2 3021 3021 3322 3922 4822 6423 7023 8326 72*3 6623 4421 5524 16126 142i8 252213 256211 236-22 236-24 182211 186214 194-13 295233 459530 385-27 538-38 437516 419-20 RNA/DNA 0.87 0.83 0.82 0.88 0.75 0.74 0.75 0.93 1.06 1.03 1.06 1.24 1.07 1.25 1.06 1.05 0.95 TABLE 4 Changes in the weight, DNA and RNA contents and RNA/DNA ratio o f the uterus Age Weight DNA Content RNA Content days mg mg mg 10 15 20 25 30 31 32 33 34 35 40 50 60 70 80 90 100 18a0 3921 5021 4721 7324 8223 77c7 7923 127215 141218 234226 289217 348227 384223 413218 493222 435221 10528 190213 25829 236213 39341 399*46 435*41 393228 539254 560260 981299 1,326258 1,918291 1,633296 1,980296 2,036297 1,925*70 60C1 125C8 162*5 16225 304k22 311k24 419k67 282-t21 605290 611295 1,136f182 1,3642123 2,3782247 2,2232178 2,628f172 2,8102193 2,266-C153 RNA/DNA 0.64 0.65 0.64 0.74 0.77 0.89 0.96 0.73 1.07 1.09 1.12 1.01 1.21 1.38 1.32 1.37 1.18 20 C . DESJARDINS, K. L. MACMILLAN AND H. D. HAFS published by Donaldson (’24) and Jack- starting as early as day 20. While size of testes, as measured by weight, DNA son (’13). Because growth of reproductive organs content or RNA content, increased until is selectively stimulated at puberty, ex- 70 days, the nucleic acid changes were pression of their sizes either in absolute not large beyond 50 days. In fact, DNA values or on body weight bases as has decreased appreciably beyond 70 days. been previously done may be misleading. Jackson (’13) also noted a decline in tesConsequently, we have chosen to express ticular weight, but at a later age than we increments in weight, DNA, and RNA of observed and Drori and Folman (’64) reeach reproductive organ relative to its re- ported a marked reduction in rate of inspective weight, DNA, or RNA at a base crease of testicular size beyond 67 days. age. Comparison of these measures of reo n e may compute, from the data in productive “growth with similarly ex- table 1, that the concentration of testicupressed body growth aids interpretation of lar DNA declines nearly continuously but the tabular data because units are elimi- at a declining rate from a high of 8.65 nated with this method and the different mg/gm at 20 days to 2.45 mg/gm at 110 criteria, organs, and sexes may be com- days, similarly to that reported by Fujii pared directly rather than on body weight and Koyama (’62). However, our data rebases. Size at age ten days was arbitrarily veal a dramatic increase from the lowest selected as the base for growth compari- value observed (1.57 mg/gm) at birth to son in all criteria except seminal vesicular that at 20 days of age. The decline in criteria where 20 days was selected be- DNA concentration beyond 20 days is no cause this organ was too small for accu- doubt due to increased diameter of semirate evaluation at earlier ages. niferous tubules. Since the growth of Size of the testes with advancing age these tubules is under the influence of (fig. 1 ) relative to that at ten days of FSH (Nelson, ’52), we interpret the obage increased at rates more than double served decline in DNA concentration to that for body weight, indicating that the reflect increased FSH secretion. testes may be stimulated by gonadotropins The relative growth analysis of seminal vesicles (fig. 2) revealed no marked dif60 0 Body Weight ferences between rate of seminal vesicular 0 Testes Weight growth and rate of body growth between 20 and 30 days. Beyond these ages, OTestes DNA growth of seminal vesicles became pro50 A T e s t e s RNA gressively greater than body growth to 70 ZI IrJ 0 days. RNA content at 35 days is more -0 than double that at 30 days, but greatest 040 increments in each seminal vesicular pac 0 rameter occur between 40 and 70 days of age. The large changes in RNA and weight relative to DNA undoubtedly reflect accumulated seminal vesicular secretion. Thus, although androgens may affect seminal vesicular activity (RNA) as early as 30 days of age, marked pubertal stimulation occurs beginning at 40 days of age, about ten days later than testicular pubertal growth. Both testicular and seminal vesicular parameters peaked at 70 days of age. These responses are reminiscent of responses to exogenous testosterone propionate implants which produce Age (days) dramatic increases in the RNA contents of seminal vesicles, prostates and kidneys Fig. 1 Changes in testes weight, DNA and of castrate mice followed by declines in RNA relative to ten days of age. 21 REPRODUCTIVE GROWTH O F RATS A 'O a - Body Weight Seminal Vesicle weigw 0 Seminal Vesicle DNA 60 - uted to the variation among rats within an age for ovarian and uterine criteria. In conclusion, relative to body growth, testes grow more rapidly from ten days of A Seminal Vesicle RNA i 14 50 0 Body Weight - i" Ovary Weight 0 Ovary DNA u h l 12 A O v a r y RNA Q 0 0 cu 40 10 + 0 0 30 0 0 + 9. 0 /\ 1 8 - + 0 .c 4- o e! t 0) 6- al > .t 0 5 I 30 50 70 4 - N Q) I .- 90 v) 2- Age (days) Fig. 2 Changes in seminal vesicular weight, DNA and RNA relative to 20 days of age. 0 I , , , , 50 90 10 each after the maximum response was produced (Kochakian and Harrison, '62). In contrast to testes, relative growth of ovaries is similar to that for the body, at least until age 50 days (fig. 3 ) . In fact, even beyond 50 days, ovarian weight changes with advancing age do not differ greatly from those for the body. Each measured ovarian criterion attains maximal value at 80 days of age. The relative growth analysis of uteri (fig. 4 ) revealed that until 30 days of age, its rate of growth paralIeled body growth. Beyond that age, the uterus grew much more rapidly than the body and increases in uterine weight and especially RNA were more rapid than increases in uterine DNA. In fact, DNA changed little after 60 days while weight and RNA continued to accumulate at least until 90 days. Because this experiment was designed to measure the rats at given ages, it was not possible to standardize the stage of estrous cycle at slaughter of the post-pubertal females. This undoubtedly contrib- 70 30 Age (days) Fig. 3 Changes in ovarian weight, DNA and RNA relative to ten days of age. 50 - Body Weight 0 Uterine Weight 0 Uterine DNA u) g40 U AUUterine RNA 0 c 4- 30 - 0 f 220al > _c -0 E l0- 0) N .cn r OW I0 30 50 70 90 Fig. 4 Changes in uterine weight, DNA and RNA relative to ten days of age. 22 C . DESJARDINS, K. L. MACMILLAN AND H. D. HAFS age whereas ovarian growth generally parallels body growth until 50 days of age. A marked shift in gonadal RNA/DNA ratio of both males and females from less than unity to greater than unity, occurred early during puberty. This gonadal hypertrophy apparently preceded sex steroid secretion. Based upon nucleic acid content of rat accessory reproductive organs, gonadal sex steroids begin to promote marked pubertal development at about 40 days of age in males and about 33 days of age in females. Terminal ages for these marked pubertal changes are about 70 days in males and 60 days in femalesages a t which rats may be considered to be postpubertal. LITERATURE CITED Donaldson, H. H. 1924 The Rat. Memoirs Wistar Inst. Anat. Biol., Philadelphia, p. 301. Drori, D., and Y. Folman 1964 Effects of cohabitation on the reproductive system, kidneys, and body composition of male rats. J. Reprod. Fertil., 8: 351-359. Fujii, T., and R. Koyama 1962 Nucleic acid contents of various organs i n rats during postnatal growth. Endocrinol. Japon., 9: 66-73. Jackson, C. M. 1913 Postnatal growth and variability of the body and of the various organs in the albino rat. Am. J. Anat., 15: 1-68. Kochakian, C. D., and D. G. Harrison 1962 Regulation of nucleic acid synthesis by androgens. Endocrinology, 70: 99-108. Moore, C. R. 1939 Biology of the testes. In: Sex and Internal Secretions. 2nd Ed., edited by A. D. Doisy. Williams and Wilkins Co., Baltimore. chapt. 7. Nelson, W. 0. 1952 Interrelations of gonadotrophic and gonadal hormones in the regulation of testicular functions. Ciba Found. Colloquia Endocrinol., 4: 271-281. Schmidt, G., and S. J. Thannhauser 1945 A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem., 161: 83-89. Telfer, M. A. 1953 Influence of estradiol on nucleic acids, respiratory enzymes and the distribution of nitrogen in the rat uterus. Arch. Biochem., 44: 111-119. Tucker, H. A. 1964 Influence of number of suckling young on nucleic acid content of lactating rat mammary gland. Proc. SOC.Exptl. Biol. Med., 116 : 218-220. Velardo, J. T. 1959 Steroid hormones and uterine growth. Ann. New York Acad. Sc., 75: 441462. Williams-Ashman, H. G. 1965 Ribonucleic acid and protein synthesis in male accessory reproductive glands and its control by testosterone. In: Mechanisms of Hormone Action. Edited by P. Karlson. Academic Press, New York. p. 214221.