- Email: [email protected]
The control of puberty in the dietary restricted female rat
Mechanisms of Ageing and Development, 32 (1985) 179-191 Elsevier Scientific Publishers Irelatid Ltd.
179
THE CONTROL OF PUBERTY IN THE DIETARY RESTRICTED FEMALE RAT
ANNE M. HOLEHAN* and BJ. MERRY I¢olfson Institute, University of Hull. Hull, HU6 7RX (U.K.) (Received January 30th, 1985) (Revision receivedJuly 12th, 1985) SUMMARY Food intake, body weight and serum LH, FSH, progesterone and oestradiol-17/3 were monitored from weaning to puberty in fully fed females housed in groups of four or individually and in females individually housed and dietary restricted. Restriction of food intake from weaning delayed the onset of puberty (34-39 days fully fed, 63-189 days dietary restricted) which was achieved at the same body weight as in the fully fed females. Individual housing of fully fed rats resulted in a significant increase in relative and absolute food intake (bet not body weight) and a decrease in serum FsH when compared to group housed fully fed animals. Serum FSH and progesterone were significantly decreased in restricted females and serum oestradiol-1713 significantly increased.
Key words: Dietary restriction; Puberfy; Growth rate; Serum hormones
INTRODUCTION Restriction of food intake from weaning has been used by many workers to modify the rate of ageing and extend the lifespan of rodents [ 1 - 5 ] . Merry and Holehan [4] showed that female rats in which lifespan had been extended by 36% through dietary restriction were capable tff breeding and showed a delay of 400-500 days in the timing of reproductive senescence. This extension in reproductive lifespan was, however, accompanied by a delay of up to 186 days in the onset of puberty. Frisch [6,7] has suggested that a critical body weight or particular body composition triggers menarche in the human female. When the food intake of rats was reduced by 25% from weaning, puberty was delayed and occurred at the same body weight and length as in control animals [8]. In the human female loss of 10-15% of body fat causes amenorrhea, urinary and
*To whom reprint requests and correspondence should be addressed.
0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 ElsevierScientific Publishers Ireland Ltd.
180 plasma gonadotrophins are low and the response to LHRH is diminished [7]. Levels of oestrogen excretion and metabolism in adult women may vary directly with increased body weight and percentage of estimated fat [9]. It has been suggested that late maturing rats may have a diminished pituitary function associated with low levels of food intake and metabolic rate [10]. In an attempt to elucidate the factors associated with delayed puberty we have monitored food intake and body weight and established serum profiles for pituitary gonadotrophins and ovarian steroids in fully fed and dietary restricted f~male rats. MATERIALSAND METHODS
Animals Female Sprague-Dawley (CFY strain) rats were weaned at 21 days and randomly allocated to one of three groups. In group (G) animals were fed at/libitum and housed 4 animals to a cage. Group (I) females were fed ad libitum but housed individually. Group (DR) animals were caged individually and growth was retarded by limiting intake of the pelleted diet (Rat Breeder diet, E.B. Bradshaw & Sons, Ltd, Driffield, England) such that body weight was maintained at 50% that of the ad libitum fed group housed controls [5]. The restricted diet was supplied at 1100 h (B.S.T) each day. All animals had unrestricted access to water, temperature was maintained at 21 + I°C, humidity within the range 50-70% and a light regimen of 12 h light/24 h.
Experimental design Serum concentrations of luteinisin8 hormone (LH), follicle stimulating hormone (FSH), progesterone and oestradiol-17~ were measured from 21 days to puberty and through the first oestrous cycle in 50 (G), 50 (I) and 33 (DR) animals. Blood was removed from the infraorbital sinus of rats lightly tranquillized with diethyl ether and the sera obtained stored at --25°C until assayed. Animals were bled between 1400 h and 1500 h colony time. Groups of 10 (G and I) females were bled at 2 daily intervals from 21 days to puberty, each animal being bled once every I0 days. All animals were checked daily for vaginal opening and 20 individuals were randomly selected from each of the two groups and were bled daily over the first oestrous cycle. Each of the 33 dietary restricted females were bled at intervals of 7 days from weaning to puberty and then daily over the first oestrous cycle. Food intake and body weight were monitored in all three groups.
Radioimmunoassay Luteinising and follicle stimulating hormone. LH and FSH concentrations in 0.l-ml serum samples were measured by the double antibody technique of Niswender et a£ [ 11 ] using materials generously supplied by the NIADDK National Hormone and Pituitary Program, Bethesda, U.S.A. Purified rat LH or FSH was labelled with [12sI] Opec. act. I00 Ci ml -I, Amersham International, U.K.) using 1,3,4,6-tetrachloro-3~, 6~-diphenylglycoluril, (Pierce Chemical Co., Rockford, U.S.A.). All samples were measured in
181 duplicate with an intra-assay variance of 7.7% (LH), 7.9% (FSH) and an inter-assay variance of 10.0% (LH) and 9.1% (FSH). Results are expressed as ng m1-1 of NIADDKrat LH-RP-1 or NIADDK-rat FSH-RP-1. Progesterone. Duplicate aliquots of 0.005 ml serum were extracted into 2 × 0.1 ml absolute ethanol, the precipitated proteins spun down and the supernatants pooled. After evaporation to dryness the sample was reconstituted in 0.25 ml phosphate buffered saline (0.05 M phosphate, 0.15 M sodium chloride, pH 7.6) containing 0.1% sodium azide and O. 1% gelatin (PBS). Recovery of progesterone was 98.5 -+ 1.76% (S.E.M., n = 100) and was not, therefore, monitored in individual samples. Progesterone was assayed with an antisera raised in rabbits against 4-pregnen-llc~-ol-3,20-dione hemisuccinate: BSA according to the method of Vaitukaitis et al. [12] which had a cross reactivity of 12.87% with 1 la-hydroxy-progesterone, 1.53% with 17a-hydroxy-progesterone, 0.24% with 20a-hydroxy-4-pregnen-3-one and 0.23% with corticosterone. Standards (range 0.0125-1.6 ng 0.1 m1-1 were made up to 0.25 ml with assay buffer. To all tubes, excluding the non-specific blanks, in which 0.5 ml of assay buffer was substituted, 0.5 ml of antisera was added. After 30 rain preincubation at room temperature (21°C), 10 000 cpm of [3H]progesterone (spec. act. 81 Ci/mmol) in 0.1 ml assay buffer was added to all tubes, mixed and incubated at 37°C for 1 h, followed by a further incubation at 4°C overnight. Antibody bound and free hormone were separated by the addition of 0.2 ml of 0.5% w/v charcoal and 0.05% w/v dextran-T 70 suspension. After incubation at 4°C for 15 min, the charcoal was precipitated by centrifugation at 1500g for 15 min. The supernatant was decanted into scintillation vials to which 7 ml Fisofluor 2 (Fisons Ltd, U.K.) was added. Samples were counted to an error of less than 2% and the minimum detectable dose per tube was 0.0052 ng with an intra assay variance of 7.0% and an inter assay variance of 7.0%. Oestradiol-17~. Oestradiol-17/~ concentrations were determined in 0.4-ml serum aliquots. To each aliquot t000 cpm [aH]oestradiol-17/3 (spec. act. 89 Ci/mmol) were added and the samples diluted to 1 ml with water. The steroid was extracted into 2 X 3 ml peroxide free diethyl ether, the extracts pooled, evaporated to dryness at 37°C under N2 and reconstituted in 0.85 ml of the assay buffer (PBS). An aliquot of 0.25 ml was taken to determine procedural losses, the calculated recovery for oestradiol-17/~ was 85.21 -+ 0.24% (S.E.M., n = 605). Duplicate aliquots of 0.25 ml were taken for mass determination and oestradiol-17# was assayed with an antisera raised in rabbits against 17fl-estradiol-6-(CMO) BSA (Steranti Research Ltd., St. Albans, U.K.). Standards were in the range 2.5-150.0 pg 0.1 m1-1 and the assay procedure was identical to that for progesterone. The minimum detectable dose of oestradiol-17~ per tube was 2.00 pg with an intra assay variance of 11.0% and an inter assay varaince of 11.5%.
Statistics Body weight and food intake data were tested for significant differences with the Wilcoxon test for two samples, ranked observations, not paired, Radioimmunoassay and quality control data were processed by computer programmes supplied by the National
182
+1 +l +1 +] +1
+~ +1 +~ +1 +1
-'FI +1 +1 +1 +1
+1 +1 + 1 + 1 + 1
+1 +1 + l + l
+l+l+l+l+l
+ 1 + 1 + 1 +1+1
+1 +1 +1 +1 +1
+1+1+1+1+1
+1 +1 +1 +1 +1
+1 +1 +1 +1 +t
4-1 +1
+1
+1
p~
E
o
o
c~
[.., o Z
0 p,, 0
o 0
183 Technical Information Services, US Department of Commerce, according to the criteria proposed by Rodbard and Frazier [13] and Rodbard et al. [14]. Correlation and regression analyses were carried out using the programmes of Davies [15]. Onset of puberty data was analysed by Generalised Linear Modelling (GLIM 3.53, 1983, Royal Statistical Society, London, U.K.). Analysis by two-way factorial analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA) was conducted according to the algorithms in the Genstat 4-01 computer programmes published by the Lawes Agricultural Trust [16]. The significance of treatment terms in MANOVA was determined by the Wilk's Lambda and Roy's maximum root test [17]. The Statistical Package for the Social Science (SPSS mark 9) were used for discriminant analysis. RESULTS Vaginal opening and first oestrus Both groups of ad libitum fed females commenced vaginal opening at 34 days and 90% of animals in both groups had open vaginas and first oestrus by 37 days. In contrast the In'st dietary restricted female demonstrated vaginal opening and first oestrus at 63 days and it was not until 147 days that more than 90% were demonstrating these criteria of puberty. In all three groups the increasing percentage of females reaching puberty with age was best described by fitting a logistic curve. Although the ages at onset of puberty in fully fed and restricted rats were significantly (P < 0.01) different, as can be seen from Fig. 1 by plotting the fitted curves on overlapping time scales, the curves were essentially similar. 100-
o =" a. o~ c
~-
II. 80-
O y=-0"2642 X105+XI1981"O)+X2(-49-25)*X3(0-40741
60-
u ca Ir
~.," 40-
]" y = 0.1025 X 106+X (_0.1017X 1051+ X2 (285.7)+X3 (_2.6661
.,,e
OR y = - 330.7 + X 18.6041 + X 21- O.5850 x 10"1 } + X 310.1324 x 10-3)
E
U. 2 0 .
3'4 60
3~ 80
3~ lOO Age
3'7 12o
3~ 14o
3'9 16o
4b Q÷z 18o oR
(days)
Fig. 1. Percentage of females demonstrating the criteria of puberty with increasing age in group housed ad libitum fed (a), individually housed ad libitum fed (e) and dietary' restricted (o) females. The equations for the logistic curves which provide the best fit of the data axe shown. ( ) Group housed; (------) Individuallyhoused; and (. . . . --) Dietary restricted.
184
,.= ~ "o
0
;
I
1
(b)
I
I
|
o
~ ~o ~ . ~ ~
e~e;,Ul poo..-I o
o ..~ .o
" ~ ~.~Q o
~~
m o
~.~=
03
i
!
I
I
I
O~l
O~
,-
,-
,-
I
o
~~o~
o 0,I
=[E< 0 • "'
o
~
¢,q
~qb!eM /~PO8 bOOl./eNel.Ul poo_-I b
o
o !
i
!
0 u3
0 0
0
(b)
; q b ! e ~ ~po8
~o
"~
,.~ ,~:=
o~=~
O~
185
200 ~' ~oo
"1-
¢~
1
, ~°° t 200
C
uJ cL
1 ~ 20
%
t
23 25 27 29 31 33 34 Days
Fig. 3. Serum LH, FSH, progesterone and oestradiol-17# in group housed ad libitum fed rats from 21 days to puberty (mean and S.E.M.).
Body weight and food intake No significant difference in body weight was shown between the two groups of fully fed females from 21 to 40 days (Fig. 2a) and puberty was achieved at a weight range of 101.0-138.0 g (G) and 100.5-125.0 g (I). However, both absolute (P < 0.002) and relative (P < 0.01) food intake were significantly less in group housed ad libitum fed females when compared to individually housed. Dietary restricted rats were allocated 6.5-7.0 g of food from 21 to 48 days and 8.5-9.0 g thereafter, the resulting slow rate of growth producing a significantly lower body weight (P < 0.001) and relative food intake (P < 0.01). Body weight in all three groups on the day of vaginal opening and first oestrus was not singificantly different. There was a significant (P < 0.01) negative correlation between the age at vaginal opening and the average rate of growth from 21 days to vaginal opening in the restricted group only. Serum hormones from weaning to puberty The serum hormone profiles for LH, FSH, progesterone and oestradiol-17~ from weaning to puberty are shown in Figs. 3 (G), 4 (I) and 5 (DR). In contrast to the other two groups serum LH in dietary restricted females remained around the weaning level although individual animals did exhibit higher levels (313.80-1062.60 ng m1-1) at
186
5-~ :
-1- ~
2°t ~o
1
c
- -
50 "3-
::trr/ 15
23 25 27 29 3t 33 35 Doys
Fig. 4. Serum LH, FSH, progesterone and oestradiol-17# in individually housed ad libitum fed rats from 23 days to puberty (mean and S.E.M.).
7 0 - 7 7 and 154-161 days. No significant regression of LH against age was shown in any of the three groups. Serum FSH was higher in the group housed animals than in either of the other two groups. There was a signficant negative regression of serum FSH against age from weaning to puberty in group housed (r = - 0 . 3 9 , P < 0.01) and dietary restricted (r = - 0 . 2 9 , P < 0.01) rats but not in the individually housed group. A significant positive regression of serum progesterone with age from 23 days to puberty was demonstrated in fully fed females (G r = 0.49, P < 0.01 ; I r = 0.54, P < 0.01). Serum levels of progesterone in individually housed rats were lower overall than in the group housed. Restriction of food intake resulted initially in higher levels of serum progesterone but by 35 days serum progesterone had dropped to 2.81 ng m1-1 and remained at very low levels until puberty. In those animals which had not reached puberty by 140 days there was a further fall in serum progesterone to 0.64--1.97 ng m1-1 . Serum progesterone declined significantly with age (r = --0.37, P < 0.01) in restricted animals. Serum oestradiol-17/3 was higher in dietary restricted rats from weanLng to puberty than in the other two groups. Serum ocstradiol-17~ in the ad ltbgtum fed females rose prior to puberty but in the restricted rats there was an overall fall although there was
187
300 -
200
2O4 ~ ~ c
~."
10
17
28 /*2 56 70
84 96
112 126 140 154 168 182
Doys
Fig. 5. Serum LH, FSH, progesterone and oestradiol-17p in dietary restricted' rats from 21 days. All values are for prepubertal animals only ( 2 1 - 5 6 days n = 33, 63 days n = 28, 70 days n = 23, 77 days n = 19, 84 days n = 12, 91 days n = l l , 9 8 - 1 1 2 days n = 8, 1 1 9 - 1 4 0 days n = 6 , 1 4 7 - 1 8 9 days n= 3).
no significant regression of serum oestradiol-17j5 on age from weaning to puberty in any of the three groups.
Discriminant analysis Using discriminant function analysis comparisons between each of the three groups on the basis of all four hormones were significant. The high level of serum FSHin group housed animals provided a basis for efficient discrimination of this group from the other two. When individually housed and dietary restricted females were compared, the higher oestradiol-17B in dietary restricted rats allowed a discrimination of this group from the individually housed.
The first oestrous cycle There was a significant difference (P < 0.001) in serum progesterone between groups on all days of the oestrous cycle (G > I > DR). Only in the group housed rats was there any significant difference (P < 0.005) between days of the cycle. Serum oestradiol-17/3
188 was consistently higher on all days of the oestrous cycle in restricted animals when compared to fully fed. These differences in serum progesterone and oestradiol-17~ were reflected in the MANOVA. In the comparison between groups the highest loading for the discriminant root was on progesterone when group housed females were compared with the other two groups and on oestradiol-17/~ when individually housed animals were compared with dietary restricted. The effect of restricted feeding on the first oestrous cycle was to increase serum oestradiol-17/3 and decrease serum progesterone to an extent which allowed an efficient discrimination between dietary restricted and ad libitum fed animals on the basis of the four variates studied. Individual housing resulted in a decrease in serum progesterone sufficient to produce an efficient discrimination from the group housed females. DISCUSSION AND CONCLUSIONS Several factors have been suggested as the trigger for puberty. Frisch [6] postulates a critical "pubertal" body weight, subsequently modified to a critical fat/lean ratio [7] and the finding that there was no significant difference in body weight at puberty between the three groups would seem to support this theory. This is in agreement with the findings of Wren and Naftolin [18] who reduced food intake of rats by 25% from weaning and found that vaginal opening and first oestrus occurred at a later age than in the controls but at the same body weight and length. Food intake and its correlate metabofic rate have also been cited as triggers for puberty onset. Kennedy and Mitra [10] found that growth retardation without chronic underfeeding delayed the onset of cycling which began when the animals reached the same weight as the ad libitum fed female rats at puberty. Food intake in both groups was same at puberty. This finding is not in agreement with the work of Wren and Naftolin [18] that heavier, fatter, late maturing rats consume less food per unit of body weight and is not confirmed by the results reported in this paper. Food intake in dietary restricted females, both relative and absolute was consistently lower than in both groups of fully fed rats (Fig. 2b,c). There was no difference in growth rate from 21 days to puberty in the fully fed females and puberty occurred over the same age range but both absolute and relative food intake were decreased in the group housed animals. This increased food intake in individually housed ad libitum fed females may be a consequence of the individual housing. In both groups of fully fed rats there would be an initial high metabolic rate associated with a high rate of growth. However, the rats in these experiments w e r e housed at 21 + 1°C which is several degrees below thermoneutrality point (28°C). Group housing leads to huddling behaviours to conserve heat and this option is not open to an animal which is singly housed. Therefore the possibility exists that to maintain body temperature individually housed rats need to raise their metabolic rate and this in turn leads to increased food intake. In the group housed animals, although not at thermoneutrality point, huddling behavior would result in heat conservation and a higher
189 proportion of the food intake could then be directed into growth. Dietary restricted rats would also have a constraint to increase metabolic rate because of single housing but would not be able to do so because of their limited food intake. The significantly lower serum FSH in individually housed and dietary restricted females may reflect this thermal regulatory problem. Glass, Harrison and Swerdloff [19] found that the delay in puberty seen in underfed animals was inversely related to growth rate. The body weight at puberty (vaginal opening and first oestrus) was not constant among the undernourished groups but varied quadratically with the growth rate. They suggested that the timing of puberty is more related to growth rate than to the attainment of any fixed weight. However, the results reported here would not support this view. Dietary restricted females reach puberty over a wide age range but at the same body weight as fully fed rats. Therefore, there was a siginficant negative correlation between growth rate and the age at onset of puberty. It seems improbable that the body can determine a specific growth rate at any time and it is more likely that a set body weight reflects a set body composition and is a trigger for puberty onset. When considering the prepubertal hormonal profiles of the two fully fed groups (Figs. 3 and 4) there is general agreement with the results of other workers. Progesterone values rise as puberty approaches and there is a fall in the levels of serum FSH [20,21]. In all groups, mean values of serum LH showed no change with age but there were 2-3 animals in each group which produced high levels of serum LH, some compatible with those of the adult preovulatory surge. This was also shown by other workers [21] who stated that 13% of their females exhibited this phenomenon. In the fully fed rats there is a rise in oestradiol-17fl prior to pube~r3y and it is this rise acting by a positive feedback response which is thought to trigger the first preovulatory surge of gonadotrophins [22,23]. Mackinnon, Puig-Duran and Laynes [24] found very high levels of serum oestradiol-17fl before 21 days which then fell from approximately 150 pg ml -t at 21 days to approximately 50 pg m1-1 by 30 days. However, due to the presence of an oestrogen binding protein (o~-feto protein) not all this oestradiol-17fl is likely to be biologically active. Before 18 days less than 1% is unbound, this percentage increases until at 28 days greater than 4% is unbound. Therefore the biological activity of the oestradiol-17fl increases prior to puberty and probably accounts for the decrease in serum FSH [25[. It has been suggested [26] that the inability of the ovary of dietary restricted females to secrete oestrogen in quantities large enough to trigger a gonadotrophin surge was the mechanism by which puberty was delayed in these animals. Obviously the results reported here would not appear to support this view. However, as the oestro~n binding has not been measured in any of the groups, it is not known how much of the secreted oestradiol1713 is biologically active. It is unclear at present whether the decreased circulating levels of FSH result from a negative feedback induced by the elevated oestradiol.171~ or a direct result of underfeeding on gonadotrophin release.
190 REFERENCES 1 C.M. McCay, Chemical aspects of ageing and the effect of diet upon ageing. In A.F. Lansing (ed.), Cowdry's Problems of Ageing, 3rd ed., Williams and Wilkins Co., Baltimore, 1952, 139-202. 2 M.H. Ross and G. Bras, Tumor incidence patterns and nutrition in the rat. J. Nutr., 87 (1965) 245-260. 3 B.N. Berg and H.S. Simms, Nutrition and longevity in the rat. III. Food restriction beyond 800 days. J. Nutr., 74 (1961) 23-32. 4 B.J. Merry and A.M. Holehan, Onset of puberty and duration of fertility in rats fed a restricted diet. J. Reprod. Fertil., 57 (1979) 253-259. 5 B.]. Merry and A.M. Holehan, Serum profiles of LH, FSH, testosterone and 5a-DHT from 21 to 1000 days ofagein ad ltbitum fed and dietary restricted rats. Exp. Gerontol., 16 (1981) 431-444. 6 R.E. Frisch, Weight at menarche: Similarity for well nourished and undernourished girls at differing ages, and evidence for historical constancy. Pediatrics, Springfield, 50 (1972) 445-450. 7 R.E. Frisch, Pubertal adipose tissue: is it necessary for normal sexual maturation? Evidence from the rat and human female. Fed. Prec., 39 (1980) 2395-2400. 8 R. Wilen and F. Naftolin, Pubertal food intake and body length, weight and composition in the feed-restricted female rat: Comparison with well fed animals. Pediatr. Res., 12 (1978) 263-267. 9 R.E. Frisch and J.W. McArthur, Menstrual cycles: fatness as a determinant of minimum weight for height necessary for their maintenance and onset. Science, 185 (1974) 949-951. 10 G.C. Kennedy and J. Mitra, Body weight and food intake as initiating factors for puberty in the rat. J. Physiol. /Lend.J, 166 (1963) 408-418. 11 G.D. Niswender, A.R. Midgley, S.E. Monroe and L.E. Reichert, Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-131 I. Prec. Soc. Exp. Biol. Med., 128 (1968) 807. 12 J. Valtukaitis, J.B. Robbins, E. Nieschlag and G.T. Ross, A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. Metab., 33 (1971) 988-991. 13 D. Rodbard and C.R. Frazier, Radioimmunoassay Data Processing: Listings, 2nd edn. National Technical Information Service, U.S. Department of Commerce, 1972. 14 D. Rodbard, V.B. Fader, D.M. Hutt and S. Knisley, Radioimmunoassay Data Processing: Listings and Documentation, 3rd edn., Vol. 2, National Technical Information Service, U.S. Department of Commerce, 1975. 15 R.G. Davies, Computer Programming in Quantitative Biology, Academic Press, London, 1971. 16 Lawes Agricultural Trust (Rothamsted Experimental Station), Genstat 4.01. 1977. 17 L.V. ]ones, Analysis of variance in its multivariate developments. In R.B. Cattell (ed.), Handbook o f Multivariate Experimental Psychology, Rand McNally & Company, Academic Press, London, 1966, pp. 244-266. 18 R. Wilen and F. Naftolin, Pubertal food intake, body length, weight and composition in the well fed female rat. Pediatr. Res., 11 (1977) 701-703. 19 A.R. Glass, R. Harrison and R.S. Swerdloff, Effect of undernutrition and amino acid deficiency on the timing of puberty in rats. Pediatr. Res., 10 (1976) 951-955. 20 K.D. Dohler and W. Wuttke, Changes with age in levels of serum gonadotrophins, proiactin and gonadal steroids in prepubertal male and female rats. Endocrinology, 97 (1975) 899-907. 21 P.C.B. Mackinnon, J.M. Mattock and M.B. ter Haar, Serum gonadotropin levels during development in male, female and androgenized female rats and the effect of general disturbance on high luteinizing hormone levels. J. Endocrinol., 70 (1976) 361-371. 22 H.M.A. Meijs-Roelofs, J.ThJ. Uilenbroek, W.]. de Greef, F.H. de Tongs and P. Kramer, Gonadotrophin and steroid levels around the time of first ovulation in the rat. Z Endocrinol., 67 (1975) 275 -282. 23 S.R. Ojeda, I.E. Wheaten, H.E. Jameson and S.M. McCann, The onset of puberty in the female rat: changes in plasma prolactin, gonadotropins, luteinizing hormone-releasing hormone (LHRI-I) and hypothalamic LHRH content. Endocrinology, 98 (1976) 630-638. 24 P.C.B. Mackinnon, E. Puig-Duran and R. Laynes, Reflections on the attainment of puberty in the
191 rat: have circadian rhythms a role to play in its onset? Symposium Report No. 11, Puberty. J.
Reprod. Fertil., 52 (1978) 401-412. 25 W.W. Andrews and S.R. Ojeda, On the feedback actions of estrogen on gonadotrophin and prolactin release in infantile female rats. Endocrinology, 101 (1977) 1517-1523. 26 O.K. Ronnektiev, S.R. Ojeda and S.M. McCann, Undernutrition, puberty and development of estrogen positive feedback in the female rat. Biol. Reprod., 19 (1978) 414-424.
179
THE CONTROL OF PUBERTY IN THE DIETARY RESTRICTED FEMALE RAT
ANNE M. HOLEHAN* and BJ. MERRY I¢olfson Institute, University of Hull. Hull, HU6 7RX (U.K.) (Received January 30th, 1985) (Revision receivedJuly 12th, 1985) SUMMARY Food intake, body weight and serum LH, FSH, progesterone and oestradiol-17/3 were monitored from weaning to puberty in fully fed females housed in groups of four or individually and in females individually housed and dietary restricted. Restriction of food intake from weaning delayed the onset of puberty (34-39 days fully fed, 63-189 days dietary restricted) which was achieved at the same body weight as in the fully fed females. Individual housing of fully fed rats resulted in a significant increase in relative and absolute food intake (bet not body weight) and a decrease in serum FsH when compared to group housed fully fed animals. Serum FSH and progesterone were significantly decreased in restricted females and serum oestradiol-1713 significantly increased.
Key words: Dietary restriction; Puberfy; Growth rate; Serum hormones
INTRODUCTION Restriction of food intake from weaning has been used by many workers to modify the rate of ageing and extend the lifespan of rodents [ 1 - 5 ] . Merry and Holehan [4] showed that female rats in which lifespan had been extended by 36% through dietary restriction were capable tff breeding and showed a delay of 400-500 days in the timing of reproductive senescence. This extension in reproductive lifespan was, however, accompanied by a delay of up to 186 days in the onset of puberty. Frisch [6,7] has suggested that a critical body weight or particular body composition triggers menarche in the human female. When the food intake of rats was reduced by 25% from weaning, puberty was delayed and occurred at the same body weight and length as in control animals [8]. In the human female loss of 10-15% of body fat causes amenorrhea, urinary and
*To whom reprint requests and correspondence should be addressed.
0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 ElsevierScientific Publishers Ireland Ltd.
180 plasma gonadotrophins are low and the response to LHRH is diminished [7]. Levels of oestrogen excretion and metabolism in adult women may vary directly with increased body weight and percentage of estimated fat [9]. It has been suggested that late maturing rats may have a diminished pituitary function associated with low levels of food intake and metabolic rate [10]. In an attempt to elucidate the factors associated with delayed puberty we have monitored food intake and body weight and established serum profiles for pituitary gonadotrophins and ovarian steroids in fully fed and dietary restricted f~male rats. MATERIALSAND METHODS
Animals Female Sprague-Dawley (CFY strain) rats were weaned at 21 days and randomly allocated to one of three groups. In group (G) animals were fed at/libitum and housed 4 animals to a cage. Group (I) females were fed ad libitum but housed individually. Group (DR) animals were caged individually and growth was retarded by limiting intake of the pelleted diet (Rat Breeder diet, E.B. Bradshaw & Sons, Ltd, Driffield, England) such that body weight was maintained at 50% that of the ad libitum fed group housed controls [5]. The restricted diet was supplied at 1100 h (B.S.T) each day. All animals had unrestricted access to water, temperature was maintained at 21 + I°C, humidity within the range 50-70% and a light regimen of 12 h light/24 h.
Experimental design Serum concentrations of luteinisin8 hormone (LH), follicle stimulating hormone (FSH), progesterone and oestradiol-17~ were measured from 21 days to puberty and through the first oestrous cycle in 50 (G), 50 (I) and 33 (DR) animals. Blood was removed from the infraorbital sinus of rats lightly tranquillized with diethyl ether and the sera obtained stored at --25°C until assayed. Animals were bled between 1400 h and 1500 h colony time. Groups of 10 (G and I) females were bled at 2 daily intervals from 21 days to puberty, each animal being bled once every I0 days. All animals were checked daily for vaginal opening and 20 individuals were randomly selected from each of the two groups and were bled daily over the first oestrous cycle. Each of the 33 dietary restricted females were bled at intervals of 7 days from weaning to puberty and then daily over the first oestrous cycle. Food intake and body weight were monitored in all three groups.
Radioimmunoassay Luteinising and follicle stimulating hormone. LH and FSH concentrations in 0.l-ml serum samples were measured by the double antibody technique of Niswender et a£ [ 11 ] using materials generously supplied by the NIADDK National Hormone and Pituitary Program, Bethesda, U.S.A. Purified rat LH or FSH was labelled with [12sI] Opec. act. I00 Ci ml -I, Amersham International, U.K.) using 1,3,4,6-tetrachloro-3~, 6~-diphenylglycoluril, (Pierce Chemical Co., Rockford, U.S.A.). All samples were measured in
181 duplicate with an intra-assay variance of 7.7% (LH), 7.9% (FSH) and an inter-assay variance of 10.0% (LH) and 9.1% (FSH). Results are expressed as ng m1-1 of NIADDKrat LH-RP-1 or NIADDK-rat FSH-RP-1. Progesterone. Duplicate aliquots of 0.005 ml serum were extracted into 2 × 0.1 ml absolute ethanol, the precipitated proteins spun down and the supernatants pooled. After evaporation to dryness the sample was reconstituted in 0.25 ml phosphate buffered saline (0.05 M phosphate, 0.15 M sodium chloride, pH 7.6) containing 0.1% sodium azide and O. 1% gelatin (PBS). Recovery of progesterone was 98.5 -+ 1.76% (S.E.M., n = 100) and was not, therefore, monitored in individual samples. Progesterone was assayed with an antisera raised in rabbits against 4-pregnen-llc~-ol-3,20-dione hemisuccinate: BSA according to the method of Vaitukaitis et al. [12] which had a cross reactivity of 12.87% with 1 la-hydroxy-progesterone, 1.53% with 17a-hydroxy-progesterone, 0.24% with 20a-hydroxy-4-pregnen-3-one and 0.23% with corticosterone. Standards (range 0.0125-1.6 ng 0.1 m1-1 were made up to 0.25 ml with assay buffer. To all tubes, excluding the non-specific blanks, in which 0.5 ml of assay buffer was substituted, 0.5 ml of antisera was added. After 30 rain preincubation at room temperature (21°C), 10 000 cpm of [3H]progesterone (spec. act. 81 Ci/mmol) in 0.1 ml assay buffer was added to all tubes, mixed and incubated at 37°C for 1 h, followed by a further incubation at 4°C overnight. Antibody bound and free hormone were separated by the addition of 0.2 ml of 0.5% w/v charcoal and 0.05% w/v dextran-T 70 suspension. After incubation at 4°C for 15 min, the charcoal was precipitated by centrifugation at 1500g for 15 min. The supernatant was decanted into scintillation vials to which 7 ml Fisofluor 2 (Fisons Ltd, U.K.) was added. Samples were counted to an error of less than 2% and the minimum detectable dose per tube was 0.0052 ng with an intra assay variance of 7.0% and an inter assay variance of 7.0%. Oestradiol-17~. Oestradiol-17/~ concentrations were determined in 0.4-ml serum aliquots. To each aliquot t000 cpm [aH]oestradiol-17/3 (spec. act. 89 Ci/mmol) were added and the samples diluted to 1 ml with water. The steroid was extracted into 2 X 3 ml peroxide free diethyl ether, the extracts pooled, evaporated to dryness at 37°C under N2 and reconstituted in 0.85 ml of the assay buffer (PBS). An aliquot of 0.25 ml was taken to determine procedural losses, the calculated recovery for oestradiol-17/~ was 85.21 -+ 0.24% (S.E.M., n = 605). Duplicate aliquots of 0.25 ml were taken for mass determination and oestradiol-17# was assayed with an antisera raised in rabbits against 17fl-estradiol-6-(CMO) BSA (Steranti Research Ltd., St. Albans, U.K.). Standards were in the range 2.5-150.0 pg 0.1 m1-1 and the assay procedure was identical to that for progesterone. The minimum detectable dose of oestradiol-17~ per tube was 2.00 pg with an intra assay variance of 11.0% and an inter assay varaince of 11.5%.
Statistics Body weight and food intake data were tested for significant differences with the Wilcoxon test for two samples, ranked observations, not paired, Radioimmunoassay and quality control data were processed by computer programmes supplied by the National
182
+1 +l +1 +] +1
+~ +1 +~ +1 +1
-'FI +1 +1 +1 +1
+1 +1 + 1 + 1 + 1
+1 +1 + l + l
+l+l+l+l+l
+ 1 + 1 + 1 +1+1
+1 +1 +1 +1 +1
+1+1+1+1+1
+1 +1 +1 +1 +1
+1 +1 +1 +1 +t
4-1 +1
+1
+1
p~
E
o
o
c~
[.., o Z
0 p,, 0
o 0
183 Technical Information Services, US Department of Commerce, according to the criteria proposed by Rodbard and Frazier [13] and Rodbard et al. [14]. Correlation and regression analyses were carried out using the programmes of Davies [15]. Onset of puberty data was analysed by Generalised Linear Modelling (GLIM 3.53, 1983, Royal Statistical Society, London, U.K.). Analysis by two-way factorial analysis of variance (ANOVA) and multivariate analysis of variance (MANOVA) was conducted according to the algorithms in the Genstat 4-01 computer programmes published by the Lawes Agricultural Trust [16]. The significance of treatment terms in MANOVA was determined by the Wilk's Lambda and Roy's maximum root test [17]. The Statistical Package for the Social Science (SPSS mark 9) were used for discriminant analysis. RESULTS Vaginal opening and first oestrus Both groups of ad libitum fed females commenced vaginal opening at 34 days and 90% of animals in both groups had open vaginas and first oestrus by 37 days. In contrast the In'st dietary restricted female demonstrated vaginal opening and first oestrus at 63 days and it was not until 147 days that more than 90% were demonstrating these criteria of puberty. In all three groups the increasing percentage of females reaching puberty with age was best described by fitting a logistic curve. Although the ages at onset of puberty in fully fed and restricted rats were significantly (P < 0.01) different, as can be seen from Fig. 1 by plotting the fitted curves on overlapping time scales, the curves were essentially similar. 100-
o =" a. o~ c
~-
II. 80-
O y=-0"2642 X105+XI1981"O)+X2(-49-25)*X3(0-40741
60-
u ca Ir
~.," 40-
]" y = 0.1025 X 106+X (_0.1017X 1051+ X2 (285.7)+X3 (_2.6661
.,,e
OR y = - 330.7 + X 18.6041 + X 21- O.5850 x 10"1 } + X 310.1324 x 10-3)
E
U. 2 0 .
3'4 60
3~ 80
3~ lOO Age
3'7 12o
3~ 14o
3'9 16o
4b Q÷z 18o oR
(days)
Fig. 1. Percentage of females demonstrating the criteria of puberty with increasing age in group housed ad libitum fed (a), individually housed ad libitum fed (e) and dietary' restricted (o) females. The equations for the logistic curves which provide the best fit of the data axe shown. ( ) Group housed; (------) Individuallyhoused; and (. . . . --) Dietary restricted.
184
,.= ~ "o
0
;
I
1
(b)
I
I
|
o
~ ~o ~ . ~ ~
e~e;,Ul poo..-I o
o ..~ .o
" ~ ~.~Q o
~~
m o
~.~=
03
i
!
I
I
I
O~l
O~
,-
,-
,-
I
o
~~o~
o 0,I
=[E< 0 • "'
o
~
¢,q
~qb!eM /~PO8 bOOl./eNel.Ul poo_-I b
o
o !
i
!
0 u3
0 0
0
(b)
; q b ! e ~ ~po8
~o
"~
,.~ ,~:=
o~=~
O~
185
200 ~' ~oo
"1-
¢~
1
, ~°° t 200
C
uJ cL
1 ~ 20
%
t
23 25 27 29 31 33 34 Days
Fig. 3. Serum LH, FSH, progesterone and oestradiol-17# in group housed ad libitum fed rats from 21 days to puberty (mean and S.E.M.).
Body weight and food intake No significant difference in body weight was shown between the two groups of fully fed females from 21 to 40 days (Fig. 2a) and puberty was achieved at a weight range of 101.0-138.0 g (G) and 100.5-125.0 g (I). However, both absolute (P < 0.002) and relative (P < 0.01) food intake were significantly less in group housed ad libitum fed females when compared to individually housed. Dietary restricted rats were allocated 6.5-7.0 g of food from 21 to 48 days and 8.5-9.0 g thereafter, the resulting slow rate of growth producing a significantly lower body weight (P < 0.001) and relative food intake (P < 0.01). Body weight in all three groups on the day of vaginal opening and first oestrus was not singificantly different. There was a significant (P < 0.01) negative correlation between the age at vaginal opening and the average rate of growth from 21 days to vaginal opening in the restricted group only. Serum hormones from weaning to puberty The serum hormone profiles for LH, FSH, progesterone and oestradiol-17~ from weaning to puberty are shown in Figs. 3 (G), 4 (I) and 5 (DR). In contrast to the other two groups serum LH in dietary restricted females remained around the weaning level although individual animals did exhibit higher levels (313.80-1062.60 ng m1-1) at
186
5-~ :
-1- ~
2°t ~o
1
c
- -
50 "3-
::trr/ 15
23 25 27 29 3t 33 35 Doys
Fig. 4. Serum LH, FSH, progesterone and oestradiol-17# in individually housed ad libitum fed rats from 23 days to puberty (mean and S.E.M.).
7 0 - 7 7 and 154-161 days. No significant regression of LH against age was shown in any of the three groups. Serum FSH was higher in the group housed animals than in either of the other two groups. There was a signficant negative regression of serum FSH against age from weaning to puberty in group housed (r = - 0 . 3 9 , P < 0.01) and dietary restricted (r = - 0 . 2 9 , P < 0.01) rats but not in the individually housed group. A significant positive regression of serum progesterone with age from 23 days to puberty was demonstrated in fully fed females (G r = 0.49, P < 0.01 ; I r = 0.54, P < 0.01). Serum levels of progesterone in individually housed rats were lower overall than in the group housed. Restriction of food intake resulted initially in higher levels of serum progesterone but by 35 days serum progesterone had dropped to 2.81 ng m1-1 and remained at very low levels until puberty. In those animals which had not reached puberty by 140 days there was a further fall in serum progesterone to 0.64--1.97 ng m1-1 . Serum progesterone declined significantly with age (r = --0.37, P < 0.01) in restricted animals. Serum oestradiol-17/3 was higher in dietary restricted rats from weanLng to puberty than in the other two groups. Serum ocstradiol-17~ in the ad ltbgtum fed females rose prior to puberty but in the restricted rats there was an overall fall although there was
187
300 -
200
2O4 ~ ~ c
~."
10
17
28 /*2 56 70
84 96
112 126 140 154 168 182
Doys
Fig. 5. Serum LH, FSH, progesterone and oestradiol-17p in dietary restricted' rats from 21 days. All values are for prepubertal animals only ( 2 1 - 5 6 days n = 33, 63 days n = 28, 70 days n = 23, 77 days n = 19, 84 days n = 12, 91 days n = l l , 9 8 - 1 1 2 days n = 8, 1 1 9 - 1 4 0 days n = 6 , 1 4 7 - 1 8 9 days n= 3).
no significant regression of serum oestradiol-17j5 on age from weaning to puberty in any of the three groups.
Discriminant analysis Using discriminant function analysis comparisons between each of the three groups on the basis of all four hormones were significant. The high level of serum FSHin group housed animals provided a basis for efficient discrimination of this group from the other two. When individually housed and dietary restricted females were compared, the higher oestradiol-17B in dietary restricted rats allowed a discrimination of this group from the individually housed.
The first oestrous cycle There was a significant difference (P < 0.001) in serum progesterone between groups on all days of the oestrous cycle (G > I > DR). Only in the group housed rats was there any significant difference (P < 0.005) between days of the cycle. Serum oestradiol-17/3
188 was consistently higher on all days of the oestrous cycle in restricted animals when compared to fully fed. These differences in serum progesterone and oestradiol-17~ were reflected in the MANOVA. In the comparison between groups the highest loading for the discriminant root was on progesterone when group housed females were compared with the other two groups and on oestradiol-17/~ when individually housed animals were compared with dietary restricted. The effect of restricted feeding on the first oestrous cycle was to increase serum oestradiol-17/3 and decrease serum progesterone to an extent which allowed an efficient discrimination between dietary restricted and ad libitum fed animals on the basis of the four variates studied. Individual housing resulted in a decrease in serum progesterone sufficient to produce an efficient discrimination from the group housed females. DISCUSSION AND CONCLUSIONS Several factors have been suggested as the trigger for puberty. Frisch [6] postulates a critical "pubertal" body weight, subsequently modified to a critical fat/lean ratio [7] and the finding that there was no significant difference in body weight at puberty between the three groups would seem to support this theory. This is in agreement with the findings of Wren and Naftolin [18] who reduced food intake of rats by 25% from weaning and found that vaginal opening and first oestrus occurred at a later age than in the controls but at the same body weight and length. Food intake and its correlate metabofic rate have also been cited as triggers for puberty onset. Kennedy and Mitra [10] found that growth retardation without chronic underfeeding delayed the onset of cycling which began when the animals reached the same weight as the ad libitum fed female rats at puberty. Food intake in both groups was same at puberty. This finding is not in agreement with the work of Wren and Naftolin [18] that heavier, fatter, late maturing rats consume less food per unit of body weight and is not confirmed by the results reported in this paper. Food intake in dietary restricted females, both relative and absolute was consistently lower than in both groups of fully fed rats (Fig. 2b,c). There was no difference in growth rate from 21 days to puberty in the fully fed females and puberty occurred over the same age range but both absolute and relative food intake were decreased in the group housed animals. This increased food intake in individually housed ad libitum fed females may be a consequence of the individual housing. In both groups of fully fed rats there would be an initial high metabolic rate associated with a high rate of growth. However, the rats in these experiments w e r e housed at 21 + 1°C which is several degrees below thermoneutrality point (28°C). Group housing leads to huddling behaviours to conserve heat and this option is not open to an animal which is singly housed. Therefore the possibility exists that to maintain body temperature individually housed rats need to raise their metabolic rate and this in turn leads to increased food intake. In the group housed animals, although not at thermoneutrality point, huddling behavior would result in heat conservation and a higher
189 proportion of the food intake could then be directed into growth. Dietary restricted rats would also have a constraint to increase metabolic rate because of single housing but would not be able to do so because of their limited food intake. The significantly lower serum FSH in individually housed and dietary restricted females may reflect this thermal regulatory problem. Glass, Harrison and Swerdloff [19] found that the delay in puberty seen in underfed animals was inversely related to growth rate. The body weight at puberty (vaginal opening and first oestrus) was not constant among the undernourished groups but varied quadratically with the growth rate. They suggested that the timing of puberty is more related to growth rate than to the attainment of any fixed weight. However, the results reported here would not support this view. Dietary restricted females reach puberty over a wide age range but at the same body weight as fully fed rats. Therefore, there was a siginficant negative correlation between growth rate and the age at onset of puberty. It seems improbable that the body can determine a specific growth rate at any time and it is more likely that a set body weight reflects a set body composition and is a trigger for puberty onset. When considering the prepubertal hormonal profiles of the two fully fed groups (Figs. 3 and 4) there is general agreement with the results of other workers. Progesterone values rise as puberty approaches and there is a fall in the levels of serum FSH [20,21]. In all groups, mean values of serum LH showed no change with age but there were 2-3 animals in each group which produced high levels of serum LH, some compatible with those of the adult preovulatory surge. This was also shown by other workers [21] who stated that 13% of their females exhibited this phenomenon. In the fully fed rats there is a rise in oestradiol-17fl prior to pube~r3y and it is this rise acting by a positive feedback response which is thought to trigger the first preovulatory surge of gonadotrophins [22,23]. Mackinnon, Puig-Duran and Laynes [24] found very high levels of serum oestradiol-17fl before 21 days which then fell from approximately 150 pg ml -t at 21 days to approximately 50 pg m1-1 by 30 days. However, due to the presence of an oestrogen binding protein (o~-feto protein) not all this oestradiol-17fl is likely to be biologically active. Before 18 days less than 1% is unbound, this percentage increases until at 28 days greater than 4% is unbound. Therefore the biological activity of the oestradiol-17fl increases prior to puberty and probably accounts for the decrease in serum FSH [25[. It has been suggested [26] that the inability of the ovary of dietary restricted females to secrete oestrogen in quantities large enough to trigger a gonadotrophin surge was the mechanism by which puberty was delayed in these animals. Obviously the results reported here would not appear to support this view. However, as the oestro~n binding has not been measured in any of the groups, it is not known how much of the secreted oestradiol1713 is biologically active. It is unclear at present whether the decreased circulating levels of FSH result from a negative feedback induced by the elevated oestradiol.171~ or a direct result of underfeeding on gonadotrophin release.
190 REFERENCES 1 C.M. McCay, Chemical aspects of ageing and the effect of diet upon ageing. In A.F. Lansing (ed.), Cowdry's Problems of Ageing, 3rd ed., Williams and Wilkins Co., Baltimore, 1952, 139-202. 2 M.H. Ross and G. Bras, Tumor incidence patterns and nutrition in the rat. J. Nutr., 87 (1965) 245-260. 3 B.N. Berg and H.S. Simms, Nutrition and longevity in the rat. III. Food restriction beyond 800 days. J. Nutr., 74 (1961) 23-32. 4 B.J. Merry and A.M. Holehan, Onset of puberty and duration of fertility in rats fed a restricted diet. J. Reprod. Fertil., 57 (1979) 253-259. 5 B.]. Merry and A.M. Holehan, Serum profiles of LH, FSH, testosterone and 5a-DHT from 21 to 1000 days ofagein ad ltbitum fed and dietary restricted rats. Exp. Gerontol., 16 (1981) 431-444. 6 R.E. Frisch, Weight at menarche: Similarity for well nourished and undernourished girls at differing ages, and evidence for historical constancy. Pediatrics, Springfield, 50 (1972) 445-450. 7 R.E. Frisch, Pubertal adipose tissue: is it necessary for normal sexual maturation? Evidence from the rat and human female. Fed. Prec., 39 (1980) 2395-2400. 8 R. Wilen and F. Naftolin, Pubertal food intake and body length, weight and composition in the feed-restricted female rat: Comparison with well fed animals. Pediatr. Res., 12 (1978) 263-267. 9 R.E. Frisch and J.W. McArthur, Menstrual cycles: fatness as a determinant of minimum weight for height necessary for their maintenance and onset. Science, 185 (1974) 949-951. 10 G.C. Kennedy and J. Mitra, Body weight and food intake as initiating factors for puberty in the rat. J. Physiol. /Lend.J, 166 (1963) 408-418. 11 G.D. Niswender, A.R. Midgley, S.E. Monroe and L.E. Reichert, Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-131 I. Prec. Soc. Exp. Biol. Med., 128 (1968) 807. 12 J. Valtukaitis, J.B. Robbins, E. Nieschlag and G.T. Ross, A method for producing specific antisera with small doses of immunogen. J. Clin. Endocrinol. Metab., 33 (1971) 988-991. 13 D. Rodbard and C.R. Frazier, Radioimmunoassay Data Processing: Listings, 2nd edn. National Technical Information Service, U.S. Department of Commerce, 1972. 14 D. Rodbard, V.B. Fader, D.M. Hutt and S. Knisley, Radioimmunoassay Data Processing: Listings and Documentation, 3rd edn., Vol. 2, National Technical Information Service, U.S. Department of Commerce, 1975. 15 R.G. Davies, Computer Programming in Quantitative Biology, Academic Press, London, 1971. 16 Lawes Agricultural Trust (Rothamsted Experimental Station), Genstat 4.01. 1977. 17 L.V. ]ones, Analysis of variance in its multivariate developments. In R.B. Cattell (ed.), Handbook o f Multivariate Experimental Psychology, Rand McNally & Company, Academic Press, London, 1966, pp. 244-266. 18 R. Wilen and F. Naftolin, Pubertal food intake, body length, weight and composition in the well fed female rat. Pediatr. Res., 11 (1977) 701-703. 19 A.R. Glass, R. Harrison and R.S. Swerdloff, Effect of undernutrition and amino acid deficiency on the timing of puberty in rats. Pediatr. Res., 10 (1976) 951-955. 20 K.D. Dohler and W. Wuttke, Changes with age in levels of serum gonadotrophins, proiactin and gonadal steroids in prepubertal male and female rats. Endocrinology, 97 (1975) 899-907. 21 P.C.B. Mackinnon, J.M. Mattock and M.B. ter Haar, Serum gonadotropin levels during development in male, female and androgenized female rats and the effect of general disturbance on high luteinizing hormone levels. J. Endocrinol., 70 (1976) 361-371. 22 H.M.A. Meijs-Roelofs, J.ThJ. Uilenbroek, W.]. de Greef, F.H. de Tongs and P. Kramer, Gonadotrophin and steroid levels around the time of first ovulation in the rat. Z Endocrinol., 67 (1975) 275 -282. 23 S.R. Ojeda, I.E. Wheaten, H.E. Jameson and S.M. McCann, The onset of puberty in the female rat: changes in plasma prolactin, gonadotropins, luteinizing hormone-releasing hormone (LHRI-I) and hypothalamic LHRH content. Endocrinology, 98 (1976) 630-638. 24 P.C.B. Mackinnon, E. Puig-Duran and R. Laynes, Reflections on the attainment of puberty in the
191 rat: have circadian rhythms a role to play in its onset? Symposium Report No. 11, Puberty. J.
Reprod. Fertil., 52 (1978) 401-412. 25 W.W. Andrews and S.R. Ojeda, On the feedback actions of estrogen on gonadotrophin and prolactin release in infantile female rats. Endocrinology, 101 (1977) 1517-1523. 26 O.K. Ronnektiev, S.R. Ojeda and S.M. McCann, Undernutrition, puberty and development of estrogen positive feedback in the female rat. Biol. Reprod., 19 (1978) 414-424.