P U B E R T Y IN B E E F HEIFERS. 1. T H E I N T E R R E L A T I O N S H I P B E T W E E N P I T U I T A R Y , H Y P O T H A L A M I C AND O V A R I A N H O R M O N E S 1'2 E. Gonzalez-Padilla,4 J.N. Wiltbank s and G.D. Niswender

Colorado State University,6 Fort Collins 80521 Summary Blood serum concentrations of LH, FSH, GnRH, Prolactin, progesterone and estradiol17/3 were measured by radioimmunoassay in samples collected from six half-sib, Angus heifers before and at attainment of puberty. Blood samples were collected daily at 6-hr intervals and once a week at 20-min intervals for 4 hours. Pituitary and hypothalamic hormones were analyzed in each sample; steroid hormones were measured in daily pools. The period of collection started 30 to 64 days before the first preovulatory peak of LH (day 0) and continued until t h e first mid-cycle. There were no marked changes in FSH, prolactin or GnRH concentrations as puberty approached or during the first cycle. Levels of estradiol-17r were high prior to day 40, then decreased gradually for 3 to 4 days to a level that was rather constant through the remainder of the sampling period. There was no elevation of estradiol-17/~ associated with the LH peaks. Prolactin was negatively correlated with estradi-

o1-17/3 (r = - . 2 6 ) and GnRH (r =, -.31) during the prepuberal period, and these negative correlations disappeared after day 0. Prolactin and FSH were positively correlated prior to (r = .15) and after day 0 (r = .63). The prepuberal period was characterized by levels of LH that fluctuated markedly with an average concentration higher than that observed after day 0. Besides pubertal peak of LH (day 0), each heifer showed another peak of LH (priming)of similar duration and magnitude between days -11 to --9. Progesterone levels were very low (300 pg/ml) in the prepuberal period, but there were two distinct elevations in every heifer before day 0. The return of the first elevation to baseline levels was always followed by the priming peak of LH, while the second preceded the pubertal peak of LH. The profile of levels of LH between the two major LH peaks, coincident with the second progesterone elevation, appeared as a transition between prepuberal and postpuberal LH baseline level. This suggested that progesterone plays a key role in the changes leading to the establishment of the phasic LH release characteristic of the cyclic bovine female. Pituitary and hypothalamic hormones were released in bursts. LH bursts appeared of larger magnitude and lower frequency than those of the other hormones. The frequency of LH bursts did not diminish after puberty, but their magnitude was reduced during the mid-cycle.

i Published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 1966. 2Research supported in part by Rockefeller Foundation. s Research supported in part by Western Regional Project WI 12 Improving Reproductive Performance in Beef Cattle. 4present Address: I.N.I.P. Km. 15% de la Carr. Mexieo-Toluca, Pale Alto, 10 D.F. Mexico. Spresent Address: Texas A & M University, AgriIntroduction cultural Research Station, Star Rt. 2 Box 43 C, Beeville, Texas 78102. There is little information regarding the ~Department of Physiology and Biophysics and Department of Animal Sciences. endocrine events associated with puberty in 1091 JOURNAL OF ANIMAL SCIENCE, vol. 40, no. 6, 1975

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1092

GONZALEZ-PADILLA, WILTBANK AND NISWENDER

reproductive organs was performed weekly throughout the sampling period. At approximately 1489 months of age, one jugular vein of each heifer was cannulated under local anesthesia, with a 14 gauge indwelling catheter equipped with an obturator. This catheter was secured to the skin of the neck by two sutures. A sample of 35 ml of blood was collected from each heifer at 0600, 1200, 1800 and 2400 hr each day and at 20-min intervals for 4 hr every Saturday morning. Collection was continued for at least 10 days after the heifers showed the first estrus (in one heifer, collections were stopped 3 days after estrus). Blood samples were allowed to clot and were maintained at 4C for 18 to 26 hr until serum was collected by centrifugation at 5C. Serum samples were stored frozen in plastic vials. Concentrations of LH, FSH, Prolactin, and GnRH were measured in every sample; while progesterone and estradiol-17/3 were determined in pools of the four samples collected in one day. Serum LH, FSH and GnRH were analyzed by the double antibody radioimmunoassays (RIA) described by Niswender et al. (1969), Akbar et al. (1973) and Nett et al. (1973), respectively. Progesterone was measured with the progesterone 11 oc RIA system described by Niswender (1973). Materials and Methods Prolactin determinations were Performed usThe experimental animals weie six paternal ing the RIA system described for sheep by half-sib Angus heifers born in a 45-day calving 9 Davis et al. (1971). Validation of this system season. From 6 months of age until 14 to 15 for bovine prolactin was done by demonstratmonths of age, the heifers were fed to gain ing: 1 ) t h a t bovine FSH (LER-1640-3), LH approximately 150 g daily. This low level of (NIH-LH-B8), TSH (NIH-TSH-B6) and GH feed was continued until 6 days after the first (NIH-GH-B17), did not influence the RIA for blood sample was collected. At this time, the prolactin; 2) that serum samples produced inamount of feed was gradually increased over a hibition curves which were parallel to those 8-day period until the heifers were eating alfalfa obtained with the prolactin standard (NIH-Phay ad libitum and 2 to 3 kg of shelled corn per B3); and, 3) quantitative recovery of known head per day. A mixture of salt and minerals amounts of bovine prolactin added to bovine was provided free choice throughout the experi- sera. Estradiol-17/~ was measured by double extraction of 6 ml of serum in five volumes of ment. In the interval between weaning and the first benzene-hexane (! :2) and assaying the extract bleeding, the heifers were haltered and trained in duplicate in a system without chromatogto the restraining procedures to be used for the raphy as described by England et al. (1974). All sampling of blood, so that the heifers were bled values were corrected for procedural losses with a minimum of stress. Observations for calculated by using aH estradiol-17/3 (3,000 estrus were made twice daily starting 40 days cpm per 6 ml of serum) as an internal standard. before the first blood sample was collected. To demonstrate that the assay was suitable for After sampling started, observations were made quantification of estradiol-17/3 in bovine samevery 6 hours. Rectal examination of the pies, several samples were assayed with or

heifers. Desjardins and Hafs (1968) studied the pituitary content of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in heifers from 0 to 12 months of age. FSH was highest at 1 month, declined at 2 months and remained relatively constant from 2 to 12 months of age. LH increased from 1 to 4 months and fluctuated considerably, thereafter. Serum LH and prolactin were measured in heifers bled monthly from 7 months of age until first estrus (Swanson et al., 1972). LH levels increased as the first estrus approached, then declined, being lowest during mid cycle. Prolactin levels before first estrus were higher than during subsequent cycles. Plasma progesterone concentrations comparable to cyclic levels have been reported in 15-to 18-monthold Brahma x Shorthorn heifers prior to their first estrus and palpable corpora lutea (CL) (Donaldson et al., 1970). We found no reports on serum estradiol or gonadotropin releasing hormone (GnRH) associated with the onset of puberty in heifers. The purpose of this experiment was to study the interrelationship in levels of major reproductive hormones in the blood occurring before and at the onset of puberty.

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PUBERTY OF BEEF HEIFERS without chromatographic purification of the extract on 6 X 137 mm columns of Sephadex LH-20. Parallelism between the inhibition curves of an estradiol-17/3 standard and extracts of sera assayed without chromatography at three dilutions was also determined, as well as recovery of known amounts of estradiol-17/3 added to different bovine sera prior to extraction. Statistical Analysis. Once the hormone levels in individual heifers were obtained and plotted, the time periods in which data were going to be divided were selected, and the data from the six heifers were combined (unless otherwise stated in the text) to study the differences between periods. The data on hormonal levels were statistically analyzed by testing the homogeneity of variances between the different periods for each hormone studied and by comparing the means by tests for means with unequal number of observations and equal or unequal variances, depending on the means being compared. Simple correlations between the different hormones were run within each period in the data from samples collected at 6-hr intervals in all heifers. Besides, simple correlations were obtained by grouping all the data collected before or after the preovulatory LH peak, from samples obtained at either 6 hr or 20-min intervals. In the case of the correlations between hormones studied at 6-hr intervals, with those of hormones analyzed on a daily basis, the covariances were obtained by the cross products of each of the 6-hr observations of 1 day with the single observation of the hormone being measured on a daily basis.

1093 Results

Validation of Assays. The ability of different bovine sera and hormonal pt~,parations to inhibit the binding of l zSI-labeled ovine prolactin (LER-860-2) to the antibody is depicted in figure 1. The binding of labeled ovine prolactin in the absence of competing hormones was considered 100% binding. All sera produced curves parallel to that obtained with NIH-P-B3. Neither LH, FSH, or TSH, appeared to interfere with the binding of labeled bovine prolactin at levels of 50 g per tube. The NIH-GH-B17 did appear to cross react in this assay system with" an inhibition curve parallel to that observed in the prolactin standard. However, this apparent cross reaction was ascribed to the contamination of this GH preparation with prolactin, since the radioimmunoassay and bioassay estimates of the prolactin content of NIH-GH-B17 were in good agreement (.015 ng/ng in RIA vs .020 ng/ng in bioassay). The recoveries of added prolactin to bovine sera are shown in table 1. The slope for the regression line was 1.06 Z- .14 ng/rrd, and the correlation coefficient was .94, suggesting that prolactin could be quantitatively recovered from serum. The analysis of 16 bovine serum samples containing from 20 to 240 pg of estradiol-17/3 per milliliter, with or without the chromatography step showed the equivalence of the two methods. The slope of the regression line of values obtained without chromotography on values obtained with chromatography was 1.13

INHIBITION CURVES IN PROLACTIN RADIOIMMUNOASSAY

,oof 8o 7O

6o I--

50 3o 20 I0

~176 I ,o,o

,o pQ

,'.g

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AMOUNT OF HORMONE PER ASSAY TUBE

Figure 1. Cross reactivity of different bovine hormones with the prolactin assay and parallelism of the inhibition ciarves of bovine prolactin (NIH-P-B 3) with bovine sera.

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1094

GONZALEZ-PADILLA, WlLTBANK AND NISWENDER

TABLE 1. RECOVERY OF PROLACTIN (NH-P-B3) ADDED TO BOVINE SERA

TABLE 2. RECOVERY OF ESTRADIOL-17/~ ADDED TO BOVINE SERA

Added (nglml)

(n)

Recovered (ng/ml) x + SE

Added (pg/ml)

(n)

20 40 80

3 3 3

20.6 + .76 5 2 . 5 +- 5 . 0 6 86.3 + 2.18

10 50 250

3 3 3

b -- 1 . 0 6 -+ . 1 4 r = .94

Recovered (pg/ml) X + SE 13 +- 4.6 46 + 4.5 224 + 9.8

b = .88 + .04 r = .99

-+ .17 pg/pg, and the correlation coefficient was .98. The typical inhibition curve of the estradiol-17/3 standard and the parallelism of bovine serum extracts assayed without chromatographic separation are shown in figure 2. The recovery of estradiol-17/~ added to bovine serum samples prior extraction is shown in table 2. The slope of the regression line of amount recovered on amount added was .88 + .04 pg/pg, and the correlation coefficient was .99. Estrus Presentation. Three of the six heifers failed to show estrus before the first CL was detected by rectal palpation or by serum levels of progesterone. Therefore, the data were normalized using day 0 as the day of the LH peak preceding the formation of the first detectable CL. All heifers showed behavioral estrus 18 to 21 days after day 0. When the level of feed was increased, all heifers reached puberty'within a relatively short time. Day 0 occurred between 50 and 55 days

of bleeding in four heifers and at 64 and 30 days in the other two. Hormonal Levels. Serum concentrations of the different hormones are shown in figures 3 to 7. Figures 3 and 4 depict the averages -+ 1 standard deviation for all the samples collected at 6-hr intervals (progesterone and estradiol-17/3 were analyzed in a pool of the samples collected each day). The values observed in a representative heifer are shown in figures 5 and 6 for samples collected at 6-hr intervals, and in figure 7 for samples collected at 20-min intervals for 4 hr once a week. The means for prolactin concentration ranged from 40 to 63 ng/ml in the different phases of the experimental period (table 3), and there was large variation between heifers. In one heifer most values fluctuated between 20 and 40 ng/ml, while in another they fluctuated between 80 and 140 ng/ml; each heifer appeared to have an inherent level. Prolactin levels were lowest ( P < .01)before day - 4 0 , the period when GnRH and estradiol-17/3 were highest. In fact, there was an inverse relationship ( P < .01) of prolactin levels with GnRH INHIBITIONCURVESIN ESTRADIOL 17/3 RADIOIMM~JNOASSAY IOO (' and estradiol-17/3 levels during the prepuberal period. This negative correlation was not observed during the first 10 days of the first ~ RUM2 estrous cycle (tables 4 and 5). r r B ~ \,a, The levels of GnRH tended to decrease prior ~ 60 50 o ~ SERUMI to day - 1 2 (figure 3). When analyzed by periods, the GnRH levels prior to day - 4 0 were higher (P < .01) than those between day - I 1 and 0, and during the luteal phase of the first estrous cycle (table 3). The variation of GnRH I0 levels between heifers was also high; one heifer I,Q I~,Q Iob.~ I.g had values consistently 4 to 5 times higher than ESTRADIOL IrE PER the other five. Every heifer appeared to have its Figure 2. Parallelism between the inhibition curves individual baseline levels. There was no correlation between GnRH and LH or FSH of estradiol 17~ and bovine sera extracts without chromatography. levels (P > .05) when considering all values

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PUBERTY OF BEEF HEIFERS

1095

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Figure 3. Average concentrations of prolactin, GnRH and FSH in samples collected at 6-hr intervals in six prepuberal heifers. Each dot represents the standard deviation at a given period.

during the prept~beral period or the first half of the first estrous cycle (table 5); however, when correlations were studied in smaller intervals of time, GnRH and FSH were negatively correlated (P < .01) between days - 5 and 0 (table 4). No elevation of GnRH associated with the conspicuous LH peaks was observed. FSH levels were fairly constant, except for a decrease (P < .01) between days - 3 8 to - 2 0 . There was a tendency for FSH levels to be higher between days - 5 to 0; however, this was not consistent among heifers, as shown by the larger variability in this period. There was a small positive correlation (P < .01) between FSH and prolactin during the prepuberal period (.15), that became higher during the first cycle (.63, P < .01).

The change s in LH as puberty approached were similar in all heifers. The mean concentration of LH and the variation between samples collected at either 6-hr or 20-min intervals before day - 1 1 were higher (P < .01) than in those collected after day - 6 . Two distinct LH peaks were observed in every heifer: one, from 11 to 52 ng/ml, occurred on the designated day 0 (arbitrary referred to as "pubertal LH peak"), and the other, of similar magnitude, occurred 8 to 10 days earlier in all heifers, and will be referred to as "priming LH peak". The LH values shown in table 3 for the period - 1 1 to - 6 days include the priming LH peak and thus have a large mean and standard error. The value of the pubertal LH peak was not considered in the period including day 0. The LH values

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1096 32.;

GONZALEZ-PADILLA, WILTBANK AND NISWENDER LH

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between the two LH peaks (day - 5 to 0), whether in samples collected at 6-hr or 20-min intervals, had a mean and variance different (P < .01) to those observed before day - 1 2 , or after day 0 (table 3). Progesterone levels were low prior to day - 2 0 , with some variation between heifers. Fo{Jr had levels usually below 100 pg/ml, while the other two fluctuated between 200 and 300 pg/ml. Between days - 2 0 and 0, every heifer showed two periods when progesterone was elevated (P < .01) for 2 to 5 days; the first period occurred between days - 1 8 to -11 and always preceded the priming LH peak, and the second occurred between the priming and prepuberal LH peaks. This is illustrated in

figure 6. The first progesterone elevation was usually briefer and lower in magnitude than the second. The progesterone levels during the luteal phase of the first cycle were always higher than any values previously observed within the same animal (P < .01). Progesterone showed a small negative correlation (r -- .11, P < .01) with prolactin in the prepuberal period but they were positively correlated (r = .40, P < .01) after the pubertal LH peak. FSH and progesterone were always positively correlated (r = .16, r = .21, P < .01) (table 5). There was no correlation (P > .05) between LH and progesterone. Estra~iiol-17/3 concentrations showed the same trend in all animals; they were high prior

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PUBERTY OF BEEF HEIFERS PROL

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Figure 5. Serum levels of prolactin, GnRH and FSH in samples collected at 6-hr intervals in heifer 588. to day - 3 9 , then declined gradually in a period of 2 to 3 days and were maintained fairly constant at a lower level for the rest of the experimental period. No distinct estradiol-17/3 elevations were found associated with the major LH peaks in this study. This may have been the result of frequency of sampling. Estradiol-17/3 was slightly correlated with GnRH during the prepuberal period (r = .16, P < .01). The levels of prolactin, GnRH, FSH and LH in samples collected at 20-min intervals showed the same general trend indicated by the samples collected at 6-hr intervals (figure 7). The levels of the hypothalamic and pituitary hormones fluctuated markedly in samples collected at 20-rain intervals, indicating that these hormones are secreted in episodic bursts. The magnitude of the LH bursts was always greater

than that of the other hormones; however, the frequency of the bursts appeared higher in FSH, prolactin and GnRH than in LH. There were no detectable changes in the frequency of the bursts in the different periods studied; thus, with the sampling schedule used, it would appear that elevated levels were associated with bursts of higher magnitude, rather than with a change in their frequency. Discussion

The levels of prolactin in prepuberal heifers were similar to those during the estrous cycle, which disagrees with the report of Swanson et al. (1972). The prolactin levels in our study were lower than those they reported during the prepuberal period but agree with their values

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1098

GONZALEZ-PADILLA, WILTBANK AND NISWENDER LH -- HEIFER 588

20. 16. 12, Z

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DAYS FROM START OF FIRST CYCLE Figure 6. Se•m LH levels measured every 6 hr and progesterone and estradJo]-t7~ measured daily

heifer 588. during the first cycle. The discrepancy during the prepuberal period may reflect differences in the sampling procedure and age and breed of the heifers. The higher prolactin levels they observed may have been associated with the phase of rapid mammary development reported to occur at about 9 months of age in Holstein heifers (Sinha and Tucker, 1969). The lowest levels of prolactin occurred during the time when estradiol levels were highest (before day - 4 0 ) . Furthermore, they were negatively correlated during this period (r = .24, P < .01). The facilitating effect of estradiol, on prolactin secretion in the rat (Nicoll and Meites, 1962; Ratner and Meites, 1964; Chen and Meites, 1970) and rabbit (Kane-matsu and Sawyer, 1963) may not exist

in the prepuberal heifer. Similarly, the periods of increased progesterone levels were not associated with decreased prolactin, which would be expected if progesterone stimulated the release of prolactin inhibiting factor (PIF), as suggested for the rat (Chen and Meites, 1970). The change in the relationship between prolactin and the hypothalamic and ovarian hormones after the pubertal LH peak suggests that certain modifications in the physiology of the hypothalamo-pituitary axis are associated with the onset of puberty. The periods of lower concentration of GnRH were coincident with the periods when progesterone was elevated, while the highest values of GnRH concentration occurred when estradiol levels were higher. However, GnRH

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1099

PUBERTY OF BEEF HEIFERS LH

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Figure 7. Levels of pituitary and hypothalamic hormones in samples collected at 20-thin intervals for 4 hr once a week in heifer 588.

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1100

GONZALEZ-PADILLA, WILTBANK AND NISWENDER

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1101

PUBERTY OF BEEF HEIFERS TABLE 4. CORRELATION COEFFICIENTS IN DIFFERENT PERIODS BETWEEN HORMONES IN SAMPLES COLLECTED AT 6-HR INTERVALS IN SIX HEIFERS BEFORE AND AFTER PUBERTY D a y s f r o m first p r e o v u l a t o r y lh p e a k Hormones

Prol. Prol. Prol. Prol. Prol. GnRH GnRH GnRH GnRH FSH FSH FSH LH LH Prog.

--GnRH --FSH --LH --Prog. --E2 --173 --FSH --LH --Prog.

--64 to --39

--38 to --20

--19 to --20

--11 to --6

--5 to 0

1 to 4

5 to 10

--.28** .27** n.s. n.s. --.24**

--.32** .17"* n.s. n.s. --.25**

--25** .19"* n.s. --.16 ~ --.29**

--.32** n.s. n.s. n.s. n.s.

--.35** n.s. n.s. n.s. --.20**

n.s. .37** .54** .50** .30**

n.s. .78** n.s. .36** n.s.

n.s.

n.s.

n.s.

n.s.

--.26**

nss.

n.s.

n~. n.s.

n-~. n.s.

n.s. .31"*

n~. .42**

n~. .17"*

n~. n.s.

nJs. n.s.

--E 2-17#

--LH --Prog. - E 2 --17# --Prog. --E 2 --17# --E 2 --17#

*

(P < . o 5 ) .

** n.s.

(P < .Ol). (P > .05).

.20**

n.s. n.s. n.s. n.s. n.s. n.s.

n.s.

.45**

.24** n.s. .21"* n.s. n.s. n.s.

n.s. --.50** n.s. n.s. n.s. .21"*

showed a slight positive correlation (r = .17 and r = .16, P < .01) with progesterone and estradiol-173 during the prepuberal period, which disappeared after day 0. This suggests that the ratio o f the two steroids may be involved in the regulation o f G n R H secretion. There were no G n R H elevations associated with the major LH peaks, even in one heifer that had an LH peak when samples were being collected

n.s.

n.s. .65** n.s. n.s. n.s. n.s.

.18"*

n.s. .49** n.s. n.s. n.s. .38**

n.s.n.s.

n.s. .51"* .23** n.~. n.s. .42**

n.s. .50** n.s. n.s. .39** n.s.

at 20-rain intervals. This contrasts with the spikes o f G n R H in jugular b l o o d observed in two ewes on the day o f estrus by Crighton et al. (1973) but agrees with the lack o f jugular serum G n R H peaks associated with LH peaks observed by Nett et at (1974) in cycling and estradiol-injected, anestrous ewes. The serum c o n c e n t r a t i o n s o f F S H reported here are slightly lower than those observed in

TABLE 5. OVERALL CORRELATION COEFFICIENTS BETWEEN HORMONES IN SAMPLES COLLECTED AT 6-HR AND 20-MIN INTERVALS IN SIX HEIFERS BEFORE AND AFTER PUBERTY

Samples collected e v e r y 20 min for 4 hr o n c e a w e e k '

6 hr

Hormones Prol. Prol. Prol.

Prol. Prol. GnRH FSH FSH

FS H

------

Prog.

--.11"*

E2--173

--.26**

-- Prog. -- LH

.17"* .09**

- - Prog.

.16"* .12"* .15"*

-

Prog.

GnRH FSH LH

Before day 0 --.31"* .15"* n.s.

-

E2--17~

-- E2--17# *

(P < . 0 5 )

** rLs.

(P < .01) (P > .05)

A f t e r day 0

Before day 0

After day0

n.s. .63** n.s. .40**

-- .30** n.s. .17"*

n.s. .43** n.s.

.17" n.s.

.27** .31"* n.$. n.s.

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.22**

n.s.

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GONZALEZ-PADILLA, WILTBANK AND NISWENDER

mature cows by Akbar et al. (1973), who reported mean values between 64 and 78 ng/ml of serum. The serum levels of FSH in this study do not indicate an association of changes in blood FSH and the onset of puberty. If blood levels of FSH in the prepuberal period reflect the secretion rate of this hormone, as indicated by Akbar et al. (1974) in cycling ewes, FSH apparently plays a permissive or synergistic role in the onset of puberty. It was observed, however, that low levels of FSH occurred shortly after the decrease in estradiol concentration (P < .01). Meijs-Roelofs et al. (1973) observed that the estradiol levels in rats fall gradually from a high of 55 to 60 pg/ml at 15 days of age, to practically undetectable levels between 25 and 35 days of age, and these changes are associated with parallel changes in levels of FSH. The blood levels of LH did change strikingly as puberty approached. The serum concentration of this hormone was higher and more variable during the prepuberal period than in the luteal phase of the first cycle or those reported as normal for cycling cows and heifers (Christensen et al. 1971; Henricks et al. 1970; Hansel and Echternkamp, 1972). The higher levels of LH during the prepuberal period were suggested in cattle by the results of Odell e t al. (1970) and Swanson et al. (1972); in rats by Kragt and Masken (1972) in sheep by Liefer et al. (1972); and in swine by Chakraborty et al. (1973). However, contrary to the observation of Swanson et al. (1972), our data show that levels of LH do not increase as puberty approaches but rather decrease and fluctuate within a smaller range, especially in the 6 days prior to the pubertal LH peak. This reduction in the concentration and fluctuation of LH levels was apparently due to a decrease magnitude of the bursts of LH secretion, as indicated by the observations made in samples collected at 20-min intervals. The bursts of release of pituitary and hypothalamic hormones are consistent with the observations of Akbar et al. (1974) and Nett et al. (1974) in ewes. Despite the lack of a significant correlation (P > .05) between LH and progesterone, apparently due to the inherent variability in the pattern of secretion of LH, there appeared to be a large degree of association between the two hormones. The priming LH peak always

occurred after the first significant elevation of progesterone had returned to the baseline levels, and the interrelation between progesterone, the mean values and variability of LH concentrations were not drastically affected. These changes were obvious during the second progesterone elevation, and still more pronounced after the pubertal LH peak and the formation of the first apparently normal CL. Thus, it appears that the establishment of the mature pattern of LH secretion in heifers is not a sudden event but gradual. Among other things, this gradual change may be the result of a stepwise increase in the levels of progesterone, with each elevation serving as primer for further "maturation" of the hypothalamo-pituitaryovarian axis. The trigger for the first progesterone elevation is not obvious from the blood levels of the hormones studied. The second elevation of progesterone prior to the pubertal LH peak may be assumed of ovarian origin, since the priming LH peak may induce luteinization of follicles or formation of a small CL which was not detected by rectal examination. It is possible that the first elevation of progesterone was at least partially produced by the adrenals. The adrenal venous blood in cattle contains at least 10 times more progesterone than arterial blood (Balfour and Comline, 1957). Furthermore, in ovariectomized rats the adrenal produces detectable levels of progesterone, and these can be enhanced by ACTH (Resko, 1969), or ACTH, prolactin, GH and some preparations of LH (Piva et al., 1973). In both experiments, the administration of dexamethasone drastically depressed progesterone production by the adrenals. Direct evidence of adrenal involvement in the onset of puberty in female rats has been reported by Gorse and Lawton (1973). Levels of estradiol-17/3 were within the range of values reported in cycling cows and heifers by Henricks et al. (1971), and Hansel and Echternkamp (1972) for total estrogens and estradiol-17/3, respectively. The decrease in levels of estradiol-17/3 about 40 days prior to the pubertal LH peak, may indicate changes in the metabolism of this steroid or actual changes in its production. Increased utilization of estradiol-17/3 in this period may be related to the rapid uterine growth occurring in dairy

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PUBERTY OF BEEF HEIFERS heifers near the first estrus (Sorensen et al. 1959; Desjardins and Hafs, 1969). Changes in the steroidogenic pattern of the prepuberal ovary have been observed in rats as puberty approaches. An epimerization system inducible by FSH and responsible for the formation of a steroid capable of inducing puberty, appears functional about 10 days prior to the onset of puberty (Eckstein etal., 1970; Springer and Eckstein, 1971 ; Eckstein and Springer, 1971; Eckstein and Ravid, 1974). These changes may be related to the observed ability ofprogogesteroneto facilitate induced ovulation in prepuberal rats only when given during the 10 days prior to vaginal opening (McCormack and Meyer, 1964; Caligaris e t al.,1972). The results reported here indicate that the 2 months preceding the onset of puberty in heifers are not characterized by any deficiency in circulating levels of pituitary or hypothalamic hormones. It is, however, characterized by a lack of the cyclic pattern of release of LH, that appears gradually and is apparently mediated by the action of progesterone.

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