0013-7227/79/1042-()839$02.00 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 3 Printed in U.S.A.
Regional Brain Content of Luteinizing HormoneReleasing Hormone in Sheep during the Estrous Cycle, Seasonal Anestrus, and after Ovariectomy* JON E. WHEATON Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108
ABSTRACT. Twenty-three sheep were killed during seasonal anestrus or on different days of the estrous cycle. The stalkmedian eminence (SME), hypothalamic tissue (HP) overlying the SME, and preoptic-suprachiasmatic tissue (PO-SC) were assayed for LHRH using RIA. LHRH contents of the SME during anestrus and days 3-8 of the estrous cycle were 35 ± 3 and 33 ± 5 n.g (x ± SEM), respectively. Higher levels (59 ± 8 ng) were detected in proestrous-estrous ewes (days 1, 16, and 17), and intermediate contents were measured in ewes on days 10-14. LHRH content of the HP (11 ± 1 ng) was not different during the reproductive stages examined, although it was correlated (r = 0.65) with that in the SME. In the PO-SC area, LHRH increased from 1.3 ± 0.2 ng on days 3-8 to 2.6 ± 0.4 ng by days 10-14, but by days 1, 16, and 17 it had fallen to 0.9 ± 0.2 ng. LHRH content of the PO-SC area of anestrous ewes was 2.1 ± 0.4 ng. Anterior pituitary content of LH was greater on days 1, 10-14, 16, and 17 than on days 3-8 of the estrous cycle or during anestrus. In another experiment, 12 ovariectomized (ovx) and 12
L
H-RELEASING activity was first detected in extracts of ventral, medial hypothalamic tissue of the rat brain and to a lesser extent in more rostral tissue overlying the optic chiasm (1). The content of LHRH in the hypothalamic region has been shown to change during the estrous cycle. Chowers and McCann (2) reported that the LH-releasing activity increased on the second day of diestrus, peaked late that evening, then declined to basal levels by noon of proestrus. Other reports have also described a rise in midhypothalamic LHRH content during diestrus, which was followed by a decline on the day of proestrus (3-6). In addition, the content of LHRH in preoptic-suprachiasmatic (PO-SC) tissue has been reported to undergo changes during the estrous cycle which seem to precede those in the midhypothalamic region of the rat brain (6). LHRH has been bioassayed in extracts of ovine hypoReceived November 28, 1977. Address requests for reprints to: Dr. Jonathan Wheaton, Department of Animal Science, University of Minnesota, 495 Animal Science and Veterinary Medicine Building, St. Paul, Minnesota 55108. * Scientific Journal Series Paper number 10,061 of the Minnesota Agricultural Experiment Station, St. Paul.
intact ewes were exposed to the natural photoperiod of the breeding season, while 12 other ovx sheep were exposed to 18 h of light/day for 10 weeks. Six ovx ewes in each group were treated daily with im estradiol benzoate (EB; 138 /xg) or oil. Ovariectomy resulted in a 47% decrease in the LHRH content of the SME, while that in the HP and PO-SC area remained unchanged. Ten-week exposure to 18 h of light/day did not alter the levels of LHRH in the SME, HP, or PO-SC area. Likewise, EB did not affect the LHRH contents of these tissues. LHRH contents of the HP and SME were correlated (r = 0.42). These data demonstrate that the contents of LHRH in the SME and LH in the AP are low during anestrus and the early stages of the estrous cycle and that they both increase near the time of the preovulatory release of LH. LHRH in the PO-SC region undergoes changes in a pattern unlike those of LHRH in the SME. Ovariectomy resulted in a reduced content of LHRH in the SME which was not restored by treatment with EB. (Endocrinology 104: 839, 1979)
thalamic tissue (HP) during the estrous cycle and during the seasonal reproductive quiescent period (anestrus). LH-releasing potency increased during the estrous cycle, reaching highest levels at or near proestrus (7,8). Activity of hypothalamic extracts from anestrous ewes was found to be as high as that detected during the estrous cycle (8, 9). The present investigation used RIA to quantitate LHRH in extracts of the stalk-median eminence (SME), HP overlying the SME, and PO-SC tissue from sheep during the estrous cycle and seasonal anestrus. An experiment was also conducted to determine the effects of ovariectomy, treatment with estradiol benzoate (EB), and photoperiod on the content of LHRH in these brain regions. Materials and Methods Twenty-three mature Suffolk ewes, weighing 48 ± 5 kg (x ± SD), were used in the determination of the regional brain content of LHRH during the estrous cycle and seasonal anestrus. Ewes were checked for behavioral estrus twice daily using vasectomized rams equipped with a marking device. Seasonal polyestrous activity was detected in most ewes beginning in late September. Estrous cycle length averaged 16 ± 1 days (x ± 839
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Kndo • 1979 Vol 104 • No 3
WHEATON
SD), with the first day of estrus being counted as day 1. Results from a previous study with these sheep revealed that the preovulatory release of LH generally peaked 6-12 h after the onset of estrus (10). Ewes studied during the breeding season had exhibited at least two consecutive estrous periods. Five ewes were killed during midanestrus (June 24), and 18 ewes were killed during the breeding season (November 7). Numbers of ewes killed on each day of the estrous cycle are indicated in parentheses: day 3 (2), 4 (2), 7 (1), 8 (1), 10 (2), 12 (2), 14 (1), 16 (1), 17 (4), 1 (2). Data were arranged into groups for statistical analysis depending on the day of the estrous cycle at the time of slaughter: early to midluteal phase (days 3-8), mid to late luteal phase (days 10-14), and proestrus estrus (days 1, 16, and 17). A blood sample was taken from the jugular vein just before slaughter. Twenty-four mature, dark-faced, cross-bred ewes, weighing 49 ± 4 kg, were used in another experiment designed to determine the effects of ovariectomy, treatment with EB, and photoperiod on the regional brain content of LHRH. Sheep were ovariectomized (ovx) in late September and allowed a 2-week recovery period before being assigned randomly to treatment groups. Twelve ovx ewes were exposed to the natural photoperiod of the breeding season, which averaged 10 h of daylight for the experimental period. The other ovx sheep were maintained in an environmental room (4.3 X 6.1 m) with 12 150-watt incandescent bulbs suspended 2.7 m above the floor (light intensity was 366 lux when measured 0.6 m above the floor). Lights were on 18 h/day beginning at 0600 h. Cyclic activity has been shown to cease in ewes when exposed to similar conditions (11). After 5 weeks, all ovx ewes were injected im with EB (138 jug) = 100 jiig-17/3-estradiol or 1 ml corn oil. Injections were administered once a day for 5 weeks, after which the ewes were killed. Venous blood samples were taken at weekly intervals throughout the experiment for serum LH assay. Additional blood samples were taken at 15-min intervals for 1 h on the day before termination of the experiment. Twelve intact ewes also were penned with the 12 ovx sheep that were exposed to the environmental photoperiod. The intact sheep, which were not injected with oil, were bled at 15-min intervals for 1 h and killed the next day on unknown days of the estrous cycle. Similar rations were fed to the ewes maintained in the barn (with adjacent outside pens) or in the environmental room; at the end of the study, body weights were not different (48 ± 4 and 49 ± 3 kg, respectively). At the abattoir, ewes were killed by exsanguination, and the ventral portion of the brain was removed. The SME was separated from the pituitary and adjoining hypothalamus. The HP was defined by cuts made at the caudal edge of the optic chiasm, the rostral limit of the mammillary bodies, 3 mm on either side of the opening to the third ventricle, and to a depth of 4 mm. The PO-SC area included tissue overlying and extending 4 mm anterior to the rostral border of the optic chiasm. Lateral and dorsal limits of the PO-SC region were made by extending the corresponding HP cuts. A sample of cortex (C) about the size of the HP and the pituitary gland were also removed. Approximately 5 min elapsed from the time of death until the tissue samples were removed and frozen in petri dishes on dry ice. Tissues were freeze dried and stored under vacuum until homogenized. The SME, HP, PO-SC area, C, and posterior
pituitary gland (PP) were homogenized in 2 ml 0.2 N acetic acid-absolute ethanol (1:1). Duplicate aliquots of two dilutions of the supernatants (centrifugation, 15,000 x g for 30 min) were dried under vacuum, and the residues were dissolved in 200 jul LHRH RIA buffer. Recovery of synthetic LHRH was 85% when it was added to C and subjected to the extraction procedure. Anterior pituitary glands (AP) were homogenized in 15 ml phosphate-buffered saline and centrifuged (15,000 X g for 15 min). Supernatants were diluted 1:2000 with phosphate buffered solution. Ovarian and uterine weights and the diameter of the largest ovarian follicle were recorded. RIA of LHRH was conducted according to the procedure of Nett et al. (12). Synthetic LHRH (Beckman) was used for reference and iodination. Binding inhibition curves for preparations of the SME, HP, and PO-SC area were parallel to that of synthetic LHRH. In the first experiment, two RIAs were conducted; one contained the tissue extracts from the anestrous sheep, and the other included the samples from the ewes killed during the estrous cycle. Average intraassay and interassay coefficients of variation were 12% and 10%, respectively. Assay sensitivity was approximately 1 pg/tube. In the second experiment, tissue samples were measured in a single RIA. Ovine LH was assayed using the RIA procedure described by Niswender et al. (13). NIH-LH-S19 was used for reference material, and results are expressed in terms of this preparation. Serum (100and 200-JUJ duplicate aliquots) and AP samples (10-, 50-, and 100-jiil duplicate aliquots) from each experiment were measured in a single assay. Data from the seasonally anestrous and reproductively cycling sheep were statistically analyzed in a completely randomized design using analysis of variance (df = 3, 19). Data from the 24 ovx sheep were initially analyzed in a 2 X 2 factorial design to determine if there was a significant interaction between photoperiod and treatment with EB. Since there was none, these data and the data from the 12 intact sheep were analyzed together in a completely randomized design (df = 4, 31). Differences between treatment means within F test were examined using Duncun's new multiple range test.
Results Reproductive tracts from the anestrous sheep were typical of ewes in the middle of this seasonal reproductive state: ovaries without corpora lutea or large follicles (>6 mm in diameter) and a pale, flaccid uterus (Table 1). Ewes killed during the estrous cycle had ovaries with corpora lutea and/or follicles ranging up to 8 mm in diameter and a more vascular, firm uterus. Corpora lutea were present on the ovaries from all ewes in the early and midstages of the estrous cycle, the mass of which is reflected in the ovarian weight. Five ewes killed on days 16 or 17 of the estrous cycle also had corpora lutea, but these were smaller and less vascular, indicative of regression. Ovaries from two animals detected in estrus on the day of slaughter were devoid of corpora lutea. Extracts of the SME from the anestrous ewes and ewes during days 3-8 of the estrous cycle contained similar amounts of LHRH (Fig. 1). Higher (P < 0.01) LHRH
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REGIONAL BRAIN CONTENT OF LHRH IN SHEEP TABLE 1. Uterine and ovarian characteristics of anestrous and cyclic ewes Days of the estrous cycle Anestrus (n = 5)
3-8 (n = 6)
10-14 (n=5)
1, 16, and 17 (n = 7)
Uterine wt (g)
18 ± 3"
32 ± 2
26 ± 2
29 ± 2
Ovarian wt (g)
1.1 ±0.1
1.6 ± 0.3
2.0 ± 0.2
1.3 ± 0.2
Diameter of largest ovarian follicle (mm)
3.9 ± 0.8
4.3 + 0.6
5.3 + 0.8
6.2 + 0.5
100
100
Ewes with a corpus luteum
71
" Values given are \ ± SEM.
levels were measured in proestrous-estrous ewes (days 1, 16, and 17), whereas contents intermediate between these two were detected in ewes during days 10-14. LHRH was not detected in extracts of the C, nor of the PP. The mean LHRH content of the HP was 11 ± 1 ng (x ± SEM) and did not vary significantly during the reproductive stages examined (Fig. 1). Nonetheless, the contents of LHRH in the HP and SME were correlated (r = 0.65; P < 0.01 ). In PO-SC tissue, the LHRH content changed during the estrous cycle (Fig. 1). It increased (P < 0.01) from days 3-8 to days 10-14, but by days 1, 16, and 17, it had decreased to levels similar to those on days 3-8. Anestrous ewes had an LHRH content in the PO-SC area intermediate between those detected during the early and midstages of the estrous cycle. The concentration of LH in the AP was greater (P < 0.05) on days 10-14 and 1, 16, and 17, than on days 3-8 of the estrous cycle or anestrus (Table 2). Serum LH was consistently less than 1 ng/ml, with the exception of one of the two ewes in estrus at the time of slaughter. This ewe had a serum LH concentration of 55 ng/ml and was excluded in the calculation of the mean for ewes killed on days 1, 16, and 17. LHRH contents of the SME, HP, and PO-SC area of the ewe with the elevated levels of LH were similar to those of other ewes during days 1,16, and 17. Regional brain content of LHRH in the ovx sheep treated with EB or oil and exposed to either the natural photoperiod of the breeding season or to an artificially extended photoperiod are presented in Table 3. The content of LHRH in the SME of ovx sheep (n = 24) was 23 ± 2 ng, 53% of that detected in the SME of the 12 intact ewes (P < 0.01). In contrast to the reduction of LHRH in the SME, ovariectomy did not result in a decreased content of LHRH in the HP or PO-SC area. LHRH contents of the SME and HP were correlated (r
841
= 0.42; P < 0.05). Exposure to 18 h of light/day for 10 weeks did not alter the contents of LHRH in the SME, HP, or PO-SC area when compared to those of ovx ewes kept under environmental photoperiod. Daily administration of EB to ovx sheep for 5 weeks was not effective in restoring the reduced content of LHRH in the SME or altering the levels of LHRH in the HP or PO-SC area. During the course of the experiment, serum LH levels were similar in the ewes exposed to either 18 or approximately 10 h of light/day; EB was effective in reducing concentrations of LH in both groups of ewes. Plasma LH levels in sheep on the day before slaughter and the AP content of LH and wet weight at the termination of the experiment are shown in Fig. 2. Plasma LH levels in the ovx EB-treated sheep that were exposed to the natural photoperiod were less than those of intact ewes. Plasma LH and the content of LH in the AP reflected the same pattern of changes and were correlated (r = 0.60; P < 0.01). Treatment with EB decreased the LH content of 70 SME
60
CH PO-SC a,b
40
O
30
3 (/)
6 20
a.
a,b
2 £ 10
Anestrus
I
1
Si 3-8
10-14
1
X DC I
16,17,1
Days of the estrous cycle FIG. 1. LHRH contents (X ± SEM) of the SME, HP, and the PO-SC region of the ovine brain during seasonal anestrus (n = 5) and days 3-8 (n = 6), 10-14 (n = 5), and 1, 16, and 17 (n = 7) of the estrous cycle, a, b, and c, Mean differences at the P < 0.01 level. LHRH content of the HP was not significantly different. TABLE 2. AP and serum concentrations (x ± SEM) of LH in anestrous and cyclic ewes Days of the estrous cycle Anestrus 3-8
10-14
1, 16, and 17
AP concentration of LH (jug/mg)
1.0 ± 0.2"
1.2 ± 0.2" 4.4 ± 1.5* 4.1 ± 1.3*
Serum LH (ng/ml)c
0.4 ± 0.1
0.7 ± 0.2
0.3 ± 0.1
"• * Different from one another at the P < 0.01 level. c Levels are not different (P > 0.05).
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0.7 ± 0.3
842
WHEATON
Endo • 1979 Vol 104 • No 3
TABLE 3. LHRH contents (nanograms) of the SME, HP, and PO-SC area of ewes maintained under the natural photoperiod of the breeding season (n = 12) or exposed to an artificially extended photoperiod for 10 weeks (n = 12) Photoperiod Treatment
Artificial (18 h of light/day)
Natural (-10 h of light/day) SME
HP
PO-SC
SME
HP
PO-SC
EB
19.7 ± 4.2 21.9 ± 3.4
6.6 ± 2.0 5.3 ± 1.1
3.0 ± 1.3 3.7 ± 1.1
28.6 ± 4.5 23.4 ± 5.5
9.3 ± 2.8 8.2 ± 2.3
3.4 ± 0.6 4.3 ± 1.0
Intact
43.8 ± 1.8
7.0 ± 1.7
4.0 ± 0.6
Ovariectomized Oil
Ovx ewes were injected im daily with EB (138 fig; n = 12) or oil (n = 12) for 5 weeks before slaughter. Intact ewes (n = 12) were kept under natural photoperiod and killed on unknown days of the estrous cycle. " Values given are x ± SEM.
the AP and increased its weight. Uteri from ovx oiltreated, ovx EB-treated, and intact ewes were 4.3 ± 0.4, 11.7 ± 0.7, and 18.4 ± 1.7 g, respectively.
evidence to support a relationship between a decrease in hypothalamic content of LHRH and an increase in the
Discussion The medial basal region of the ovine brain contained a total of 57 ng LHRH when averaged across anestrus and the estrous cycle. Of this amount, 77% was concentrated in the SME, 19% in adjacent tissue overlying the SME, and 3% in more rostral PO-SC tissue. White et al. (14) reported a similar content of LHRH in extracts of ovine SME. Wheaton et al. (15) measured approximately 3 ng LHRH in the rat brain, of which 52% was located in the SME, 41% in tissue immediately above the SME, and 7% in PO-SC tissue. Palkovits et al (16) reported that the amount of LHRH in the rat median eminence is about twice that found in the arcuate nucleus. In a subsequent publication from the same laboratory, it was reported that the content of LHRH in a fragment of suprachiasmatic tissue was 13% of that concentrated in the SME (17). The increased content of LHRH detected in the SME during proestrus-estrus could have been brought about by adjustments in the net rate of LHRH synthesis and/or release. Simultaneous determination of one or the other of these processes would be necessary to reveal the basis for the change in content. Use of circulating LH concentrations as an indirect estimate of LHRH release rate is complicated by the changing response of the AP to LHRH during the estrous cycle (18). LHRH in the SME and LH in the AP were both elevated during the latter half of the estrous cycle and, conversely, were both reduced by days 3-8. Roche et al. (19) previously reported that the AP concentration of LH increased during the ovine estrous cycle. The increases in contents may be important in providing sufficient LHRH and LH for the preovulatory release of LH. Further research is needed to determine if the drop in LHRH content in the SME that occurs by days 3-8 of the estrous cycle is related to the surge release of LH. In the rat, there is
LHRH in the HP did not change significantly during anestrus, during the estrous cycle, or after ovariectomy. There was a positive correlation between the content of LHRH in the HP and that in the SME. This indicates that similar, but relatively minor, changes may also occur in the LHRH content of the HP. An arcuate-median eminence LHRH neuronal system has been identified in the rat brain (22). If an analogous tract exists in the ovine brain, it may account for the positive relationship. In the PO-SC area, LHRH was highest during days 10-14 of the estrous cycle and then fell to low levels by days 1,16, and 17, the time when LHRH in the SME was greatest. LHRH in the PO-SC region of the rat brain has been shown to increase during the estrous cycle until proestrus, at which time the content had nearly vanished (23). It has been suggested that LHRH of PO-SC origin is involved with eliciting the cyclic discharge of LH in the rat (24). The drop in content of the PO-SC area, coincident with the rise in content of the SME during the latter part of the estrous cycle, is consistent with such a function for LHRH located in the PO-SC area of the ovine brain. Participation of the PO-SC area in bringing about the preovulatory release of LH in sheep, however, is less certain than it is in rats (25). LHRH content of the PO-SC area was not correlated significantly with that in the HP or SME, nor was it altered after ovariectomy and treatment with EB. LHRH in homologous rat tissue also remains constant after ovariectomy (6) and has been reported to have a different ontogeny from that in the median eminence (26). Experimentally, the function of this more rostral LHRH will be difficult to establish. Ovariectomy was followed by a 47% reduction in the LHRH content of the SME. Daily treatment for 5 weeks with EB, beginning 7 weeks postovariectomy, did not restore the content. EB treatment did reduce the elevated levels of serum LH and partially restored uterine
release of LH (20, 21).
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REGIONAL BRAIN CONTENT OF LHRH IN SHEEP
O)
a,b
Q.
a
a 1.2 •^5
b.c
1 O
of ovariectomy (31, 32). Several studies indicate that both the synthesis (33, 34) and release (35) rates of LHRH are accelerated in castrate rats. It is possible that chronic exposure to EB diminishes both the synthesis and release rates of LHRH such that the content remains unchanged. Midanestrous ewes were found to have as much LHRH as ewes during the early stages of the estrous cycle. Hypothalamic LH-releasing activity of anestrous ewes has been reported to be as high as that of cyclic ewes (8, 9). Exposure to 18 h of light/day for 10 weeks did not affect the content of LHRH in ovx sheep. Marked depletion of the LHRH content of the brain clearly does not underlie seasonal anestrus in sheep.
.8
Acknowledgments
a,b
.6
Appreciation is expressed to Dr. T. M. Nett (Colorado State University) for providing LHRH antiserum (no. 42), to Dr. G. D. Niswender (Colorado State University) for ovine LH antiserum (no. 15), and to Dr. L. E. Reichert (Emory University) for highly purified ovine LH used for iodination. NIH-LH-S19 was kindly supplied through the NIAMDD Pituitary Hormone Program. The author wishes to thank Sheila E. Harsdorf and David K. Combs for their technical assistance.
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References 1. McCann, S. M., S. Taleisnik, and H. M. Friedman, LH-releasing activity in hypothalamic extracts, Proc Soc Exp Biol Med 104: 432, 1960. 2. Chowers, I., and S. M. McCann, Content of luteinizing hormonereleasing factor and luteinizing hormone during the estrous cycle and after changes in gonadal steroid titers, Endocrinology 76: 700,
b,c
.4
1965.
.2 18 hr
10hr
Ovx-oil
18hr
10hr
Ovx-EB
10 hr
Intact
FIG. 2. Plasma LH levels in ewes sampled at 15-min intervals for 1 h the day before slaughter and the AP LH contents and wet weights in these ewes. The multiple serum samples from each ewe were averaged, and the mean was used in the calculation of the treatment (X ± SEM). a, b, and c, Differences between means at the P < 0.05 level. Ovx sheep were exposed to either an artificially extended photoperiod of 18 h of light/day for 10 weeks or to the natural photoperiod of the breeding season (~10 h of light/day). After 5 weeks, six ewes in each treatment group were injected im daily with EB (138 jug). Intact ewes (n = 12) were exposed to natural photoperiod and killed on unknown days of the estrous cycle.
weight. Decreases in hypothalamic LHRH stores have been reported to occur after castration in rats (27). Hypothalamic LHRH content of long term ovx rats chronically treated with EB has not been published. Conflicting reports have appeared in the literature regarding the effects of a single injection of EB (21, 28-30) or its ability to maintain LHRH stores if administered from the time
3. Asai, T., and K. Wakabayashi, Changes of hypothalamic LH-RH content during the rat estrous cycle, Endocrinol Jap 22: 319,1975. 4. Kalra, P. S., and S. P. Kalra, Temporal changes in the hypothalamic and serum luteinizing hormone-releasing hormone (LH-RH) levels and the circulating ovarian steroids during the rat oestrous cycle, Ada Endocrinol (Kbh) 85: 449, 1977. 5. Ramirez, V. D., and C. H. Sawyer, Fluctuation in hypothalamic LH-RH (luteinizing hormone-releasing hormone) during the rat estrous cycle, Endocrinology 76: 282, 1965. 6. Araki, S., M. Ferin, E. A. Zimmerman, and R. L. Vande Wiele, Ovarian modulation of immunoreactive gonadotropins-releasing hormone (Gn-RH) in the rat brain: evidence for a differential effect on the anterior and mid-hypothalamus, Endocrinology 96: 644, 1975. 7. Crighton, D. B., B. M. Hartley, and G. E. Lamming, Changes in luteinizing hormone releasing activity of the hypothalamus, and in pituitary gland and plasma luteinizing hormone during the oestrous cycle of the sheep, J Endocrinol 58: 377, 1973. 8. Jackson, G. L., J. F. Roche, D. L. Foster, and P. J. Dziuk, Luteinizing hormone releasing activity in the hypothalamus of anestrous and cyclic ewes, Biol Reprod 5: 5,1971. 9. Pelletier, J., and R. Ortavant, Influence de la duree quotidienne d'eclairement sur l'activite hypothalamique LRF du Belier, C R Acad Sci [D] (Paris) 266: 1604, 1968. 10. Wheaton, J. E., T. I. Raabe, and M. J. Burrill, Characteristics of the ovulatory release of LH in Finn, Finn x Suffolk and Suffolk ewes, J Endocrinol 75: 449, 1977. 11. Palmer, W. M., G. D. Phillips, B. E. Howland, and E. A. Ibrahim, Effect of daylength on reproduction of ewes, In Proceedings of the Western Section of the American Society of Animal Sciences, vol. 23, 1972, p. 318.
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WHEATON
12. Nett, T. M., A. M. Akbar, G. D. Niswender, M. T. Hedlund, and W. F. White, A radioimmunoassay for gonadotropin-releasing hormone (Gn-RH) in serum, J Clin Endocrinol Metab 36: 880, 1973. 13. Niswender, G. D., L. E. Reichert, Jr., A. R. Midgley, Jr., and A. V. Nalbandov, Radioimmunoassay for bovine and ovine luteinizing hormone, Endocrinology 84: 1166, 1969. 14. White, W. F., M. T. Hedlund, G. F. Weber, R. H. Rippel, E. S. Johnson, and J. F. Wilber, The pineal gland: a supplemental source of hypothalamic-releasing hormones, Endocrinology 94: 1422, 1974. 15. Wheaton, J. E., L. Krulich, and S. M. McCann, Localization of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay, Endocrinology 97: 30, 1975. 16. Palkovits, M., A. Arimura, M. Brownstein, A. V. Schally, and J. M. Saavedra, Luteinizing hormone-releasing hormone (LH-RH) content of the hypothalamic nuclei in rat, Endocrinology 95: 554,1974. 17. Kizer, J. S., M. Palkovits, and M. J. Brownstein, Releasing factors in the circumventricular organs of the rat brain, Endocrinology 98: 311, 1976. 18. Reeves, J. J., A. Arimura, and A. V. Schally, Pituitary responsiveness to purified luteinizing hormone-releasing hormone (LH-RH) at various stages of the estrous cycle in sheep, J Anim Sci 32: 123, 1971. 19. Roche, J. F., D. L. Foster, F. J. Karsch, B. Cook, and P. J. Dzuik, Levels of luteinizing hormone in sera and pituitaries of ewes during the estrous cycle and anestrus, Endocrinology 86: 568, 1970. 20. Smith, E. R., and J. M. Davidson, Luteinizing hormone releasing factor in rats exposed to constant light: effects of mating, Neuroendocrinology 14: 129, 1974. 21. Baram, T., and Y. Koch, Evidence for the dependence of serum luteinizing hormone surge on a transient, enhanced secretion of gonadotropin-releasing hormone from the hypothalamus, Neuroendocrinology 23: 151, 1977. 22. Setalo, G., S. Vigh, A. V. Schally, A. Arimura, and B. Flerko, LHRH-containing neural elements in the rat hypothalamus, Endocrinology 96: 135, 1975. 23. Naik, D. V., Immuno-histochemical localization of LH-RH during different phases of estrus cycle of rat, with reference to the preoptic and arcuate neurons, and the ependymal cells, Cell Tissure Res 173: 143, 1976. 24. Schneider, H. P. G., D. B. Crighton, and S. M. McCann, Supra-
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1979 No 3
chiasmatic LH-releasing factor, Neuroendocrinology 5: 271, 1969. 25. Przekop, F., and E. Domanski, Hypothalamic centers involved in the control of gonadotropin secretion and ovulation in sheep, Acta Physiol Pol 21: 34, 1970. 26. Araki, C, D. Toran-Allerand, M. Ferin, and R. L. Vande Wiele, Immunoreactive gonadotropin-releasing hormone (Gn-RH) during maturation in the rat: ontogeny of regional hypothalamic differences, Endocrinology 97: 693, 1975. 27. Wheaton, J. E., and S. M. McCann, Luteinizing hormone-releasing hormone in peripheral plasma and hypothalamus of normal and ovariectomized rats, Neuroendocrinology 20: 296, 1976. 28. Kalra, S. P., Tissue levels of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus, and serum concentrations of gonadotropins following anterior hypothalamic deafferentation and estrogen treatment of the female rat, Endocrinology 99: 101, 1976. 29. Libertun, C, K. J. Cooper, C. P. Fawcett, and S. M. McCann, Effects of ovariectomy and steroid treatment on hypophyseal sensitivity to purified LH-releasing factor (LRF), Endocrinology 94: 518, 1974. 30. Aijika, K., L. Krulich, C. P. Fawcett, and S. M. McCann, Effects of estrogen on plasma and pituitary gonadotropins and prolactin, and on hypothalamic releasing and inhibiting factors, Neuroendocrinology 9: 304, 1972. 31. Piacsek, B. E., and J. Meites, Effects of castration and gonadal hormones on hypothalamic content of luteinizing hormone releasing factor (LRF), Endocrinology 79: 432, 1966. 32. Kobayashi, R. M., K. H. Lu, R. Y. Moore, and S. S. C. Yen, Regional distribution of hypothalamic luteinizing hormone-releasing hormone in proestrous rats: effects of ovariectomy and estrogen replacement, Endocrinology 102: 98, 1978. 33. McKelvy, J. F., and Y. Grimm-Jorgensen, Biosynthesis and degradation of hypothalamic hypophysiotropic peptides, In James, V. H. T. (ed.), Proceedings of the Fifth International Congress of Endocrinology, vol. 1, Excerpta Medica, Amsterdam, 1976, p. 175. 34. Reichlin, S., Biosynthesis and degradation of hypothalamic hypophysiotrophic factors, In Naftolin, F., K. J. Ryan, and I. J. Davies (eds.), Subcellular Mechanisms in Reproductive Neuroendocrinology, Elsevier Scientific Purblishing Co., New York, 1976, p. 109. 35. Ben-Jonathan, N., R. S. Mical, and J. C. Porter, Superfusion of hemipituitaries with portal blood. I. LRF secretion in castrated and diestrous rats, Endocrinology 93: 497, 1973.
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Vol. 104, No. 3 Printed in U.S.A.
Regional Brain Content of Luteinizing HormoneReleasing Hormone in Sheep during the Estrous Cycle, Seasonal Anestrus, and after Ovariectomy* JON E. WHEATON Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108
ABSTRACT. Twenty-three sheep were killed during seasonal anestrus or on different days of the estrous cycle. The stalkmedian eminence (SME), hypothalamic tissue (HP) overlying the SME, and preoptic-suprachiasmatic tissue (PO-SC) were assayed for LHRH using RIA. LHRH contents of the SME during anestrus and days 3-8 of the estrous cycle were 35 ± 3 and 33 ± 5 n.g (x ± SEM), respectively. Higher levels (59 ± 8 ng) were detected in proestrous-estrous ewes (days 1, 16, and 17), and intermediate contents were measured in ewes on days 10-14. LHRH content of the HP (11 ± 1 ng) was not different during the reproductive stages examined, although it was correlated (r = 0.65) with that in the SME. In the PO-SC area, LHRH increased from 1.3 ± 0.2 ng on days 3-8 to 2.6 ± 0.4 ng by days 10-14, but by days 1, 16, and 17 it had fallen to 0.9 ± 0.2 ng. LHRH content of the PO-SC area of anestrous ewes was 2.1 ± 0.4 ng. Anterior pituitary content of LH was greater on days 1, 10-14, 16, and 17 than on days 3-8 of the estrous cycle or during anestrus. In another experiment, 12 ovariectomized (ovx) and 12
L
H-RELEASING activity was first detected in extracts of ventral, medial hypothalamic tissue of the rat brain and to a lesser extent in more rostral tissue overlying the optic chiasm (1). The content of LHRH in the hypothalamic region has been shown to change during the estrous cycle. Chowers and McCann (2) reported that the LH-releasing activity increased on the second day of diestrus, peaked late that evening, then declined to basal levels by noon of proestrus. Other reports have also described a rise in midhypothalamic LHRH content during diestrus, which was followed by a decline on the day of proestrus (3-6). In addition, the content of LHRH in preoptic-suprachiasmatic (PO-SC) tissue has been reported to undergo changes during the estrous cycle which seem to precede those in the midhypothalamic region of the rat brain (6). LHRH has been bioassayed in extracts of ovine hypoReceived November 28, 1977. Address requests for reprints to: Dr. Jonathan Wheaton, Department of Animal Science, University of Minnesota, 495 Animal Science and Veterinary Medicine Building, St. Paul, Minnesota 55108. * Scientific Journal Series Paper number 10,061 of the Minnesota Agricultural Experiment Station, St. Paul.
intact ewes were exposed to the natural photoperiod of the breeding season, while 12 other ovx sheep were exposed to 18 h of light/day for 10 weeks. Six ovx ewes in each group were treated daily with im estradiol benzoate (EB; 138 /xg) or oil. Ovariectomy resulted in a 47% decrease in the LHRH content of the SME, while that in the HP and PO-SC area remained unchanged. Ten-week exposure to 18 h of light/day did not alter the levels of LHRH in the SME, HP, or PO-SC area. Likewise, EB did not affect the LHRH contents of these tissues. LHRH contents of the HP and SME were correlated (r = 0.42). These data demonstrate that the contents of LHRH in the SME and LH in the AP are low during anestrus and the early stages of the estrous cycle and that they both increase near the time of the preovulatory release of LH. LHRH in the PO-SC region undergoes changes in a pattern unlike those of LHRH in the SME. Ovariectomy resulted in a reduced content of LHRH in the SME which was not restored by treatment with EB. (Endocrinology 104: 839, 1979)
thalamic tissue (HP) during the estrous cycle and during the seasonal reproductive quiescent period (anestrus). LH-releasing potency increased during the estrous cycle, reaching highest levels at or near proestrus (7,8). Activity of hypothalamic extracts from anestrous ewes was found to be as high as that detected during the estrous cycle (8, 9). The present investigation used RIA to quantitate LHRH in extracts of the stalk-median eminence (SME), HP overlying the SME, and PO-SC tissue from sheep during the estrous cycle and seasonal anestrus. An experiment was also conducted to determine the effects of ovariectomy, treatment with estradiol benzoate (EB), and photoperiod on the content of LHRH in these brain regions. Materials and Methods Twenty-three mature Suffolk ewes, weighing 48 ± 5 kg (x ± SD), were used in the determination of the regional brain content of LHRH during the estrous cycle and seasonal anestrus. Ewes were checked for behavioral estrus twice daily using vasectomized rams equipped with a marking device. Seasonal polyestrous activity was detected in most ewes beginning in late September. Estrous cycle length averaged 16 ± 1 days (x ± 839
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840
Kndo • 1979 Vol 104 • No 3
WHEATON
SD), with the first day of estrus being counted as day 1. Results from a previous study with these sheep revealed that the preovulatory release of LH generally peaked 6-12 h after the onset of estrus (10). Ewes studied during the breeding season had exhibited at least two consecutive estrous periods. Five ewes were killed during midanestrus (June 24), and 18 ewes were killed during the breeding season (November 7). Numbers of ewes killed on each day of the estrous cycle are indicated in parentheses: day 3 (2), 4 (2), 7 (1), 8 (1), 10 (2), 12 (2), 14 (1), 16 (1), 17 (4), 1 (2). Data were arranged into groups for statistical analysis depending on the day of the estrous cycle at the time of slaughter: early to midluteal phase (days 3-8), mid to late luteal phase (days 10-14), and proestrus estrus (days 1, 16, and 17). A blood sample was taken from the jugular vein just before slaughter. Twenty-four mature, dark-faced, cross-bred ewes, weighing 49 ± 4 kg, were used in another experiment designed to determine the effects of ovariectomy, treatment with EB, and photoperiod on the regional brain content of LHRH. Sheep were ovariectomized (ovx) in late September and allowed a 2-week recovery period before being assigned randomly to treatment groups. Twelve ovx ewes were exposed to the natural photoperiod of the breeding season, which averaged 10 h of daylight for the experimental period. The other ovx sheep were maintained in an environmental room (4.3 X 6.1 m) with 12 150-watt incandescent bulbs suspended 2.7 m above the floor (light intensity was 366 lux when measured 0.6 m above the floor). Lights were on 18 h/day beginning at 0600 h. Cyclic activity has been shown to cease in ewes when exposed to similar conditions (11). After 5 weeks, all ovx ewes were injected im with EB (138 jug) = 100 jiig-17/3-estradiol or 1 ml corn oil. Injections were administered once a day for 5 weeks, after which the ewes were killed. Venous blood samples were taken at weekly intervals throughout the experiment for serum LH assay. Additional blood samples were taken at 15-min intervals for 1 h on the day before termination of the experiment. Twelve intact ewes also were penned with the 12 ovx sheep that were exposed to the environmental photoperiod. The intact sheep, which were not injected with oil, were bled at 15-min intervals for 1 h and killed the next day on unknown days of the estrous cycle. Similar rations were fed to the ewes maintained in the barn (with adjacent outside pens) or in the environmental room; at the end of the study, body weights were not different (48 ± 4 and 49 ± 3 kg, respectively). At the abattoir, ewes were killed by exsanguination, and the ventral portion of the brain was removed. The SME was separated from the pituitary and adjoining hypothalamus. The HP was defined by cuts made at the caudal edge of the optic chiasm, the rostral limit of the mammillary bodies, 3 mm on either side of the opening to the third ventricle, and to a depth of 4 mm. The PO-SC area included tissue overlying and extending 4 mm anterior to the rostral border of the optic chiasm. Lateral and dorsal limits of the PO-SC region were made by extending the corresponding HP cuts. A sample of cortex (C) about the size of the HP and the pituitary gland were also removed. Approximately 5 min elapsed from the time of death until the tissue samples were removed and frozen in petri dishes on dry ice. Tissues were freeze dried and stored under vacuum until homogenized. The SME, HP, PO-SC area, C, and posterior
pituitary gland (PP) were homogenized in 2 ml 0.2 N acetic acid-absolute ethanol (1:1). Duplicate aliquots of two dilutions of the supernatants (centrifugation, 15,000 x g for 30 min) were dried under vacuum, and the residues were dissolved in 200 jul LHRH RIA buffer. Recovery of synthetic LHRH was 85% when it was added to C and subjected to the extraction procedure. Anterior pituitary glands (AP) were homogenized in 15 ml phosphate-buffered saline and centrifuged (15,000 X g for 15 min). Supernatants were diluted 1:2000 with phosphate buffered solution. Ovarian and uterine weights and the diameter of the largest ovarian follicle were recorded. RIA of LHRH was conducted according to the procedure of Nett et al. (12). Synthetic LHRH (Beckman) was used for reference and iodination. Binding inhibition curves for preparations of the SME, HP, and PO-SC area were parallel to that of synthetic LHRH. In the first experiment, two RIAs were conducted; one contained the tissue extracts from the anestrous sheep, and the other included the samples from the ewes killed during the estrous cycle. Average intraassay and interassay coefficients of variation were 12% and 10%, respectively. Assay sensitivity was approximately 1 pg/tube. In the second experiment, tissue samples were measured in a single RIA. Ovine LH was assayed using the RIA procedure described by Niswender et al. (13). NIH-LH-S19 was used for reference material, and results are expressed in terms of this preparation. Serum (100and 200-JUJ duplicate aliquots) and AP samples (10-, 50-, and 100-jiil duplicate aliquots) from each experiment were measured in a single assay. Data from the seasonally anestrous and reproductively cycling sheep were statistically analyzed in a completely randomized design using analysis of variance (df = 3, 19). Data from the 24 ovx sheep were initially analyzed in a 2 X 2 factorial design to determine if there was a significant interaction between photoperiod and treatment with EB. Since there was none, these data and the data from the 12 intact sheep were analyzed together in a completely randomized design (df = 4, 31). Differences between treatment means within F test were examined using Duncun's new multiple range test.
Results Reproductive tracts from the anestrous sheep were typical of ewes in the middle of this seasonal reproductive state: ovaries without corpora lutea or large follicles (>6 mm in diameter) and a pale, flaccid uterus (Table 1). Ewes killed during the estrous cycle had ovaries with corpora lutea and/or follicles ranging up to 8 mm in diameter and a more vascular, firm uterus. Corpora lutea were present on the ovaries from all ewes in the early and midstages of the estrous cycle, the mass of which is reflected in the ovarian weight. Five ewes killed on days 16 or 17 of the estrous cycle also had corpora lutea, but these were smaller and less vascular, indicative of regression. Ovaries from two animals detected in estrus on the day of slaughter were devoid of corpora lutea. Extracts of the SME from the anestrous ewes and ewes during days 3-8 of the estrous cycle contained similar amounts of LHRH (Fig. 1). Higher (P < 0.01) LHRH
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REGIONAL BRAIN CONTENT OF LHRH IN SHEEP TABLE 1. Uterine and ovarian characteristics of anestrous and cyclic ewes Days of the estrous cycle Anestrus (n = 5)
3-8 (n = 6)
10-14 (n=5)
1, 16, and 17 (n = 7)
Uterine wt (g)
18 ± 3"
32 ± 2
26 ± 2
29 ± 2
Ovarian wt (g)
1.1 ±0.1
1.6 ± 0.3
2.0 ± 0.2
1.3 ± 0.2
Diameter of largest ovarian follicle (mm)
3.9 ± 0.8
4.3 + 0.6
5.3 + 0.8
6.2 + 0.5
100
100
Ewes with a corpus luteum
71
" Values given are \ ± SEM.
levels were measured in proestrous-estrous ewes (days 1, 16, and 17), whereas contents intermediate between these two were detected in ewes during days 10-14. LHRH was not detected in extracts of the C, nor of the PP. The mean LHRH content of the HP was 11 ± 1 ng (x ± SEM) and did not vary significantly during the reproductive stages examined (Fig. 1). Nonetheless, the contents of LHRH in the HP and SME were correlated (r = 0.65; P < 0.01 ). In PO-SC tissue, the LHRH content changed during the estrous cycle (Fig. 1). It increased (P < 0.01) from days 3-8 to days 10-14, but by days 1, 16, and 17, it had decreased to levels similar to those on days 3-8. Anestrous ewes had an LHRH content in the PO-SC area intermediate between those detected during the early and midstages of the estrous cycle. The concentration of LH in the AP was greater (P < 0.05) on days 10-14 and 1, 16, and 17, than on days 3-8 of the estrous cycle or anestrus (Table 2). Serum LH was consistently less than 1 ng/ml, with the exception of one of the two ewes in estrus at the time of slaughter. This ewe had a serum LH concentration of 55 ng/ml and was excluded in the calculation of the mean for ewes killed on days 1, 16, and 17. LHRH contents of the SME, HP, and PO-SC area of the ewe with the elevated levels of LH were similar to those of other ewes during days 1,16, and 17. Regional brain content of LHRH in the ovx sheep treated with EB or oil and exposed to either the natural photoperiod of the breeding season or to an artificially extended photoperiod are presented in Table 3. The content of LHRH in the SME of ovx sheep (n = 24) was 23 ± 2 ng, 53% of that detected in the SME of the 12 intact ewes (P < 0.01). In contrast to the reduction of LHRH in the SME, ovariectomy did not result in a decreased content of LHRH in the HP or PO-SC area. LHRH contents of the SME and HP were correlated (r
841
= 0.42; P < 0.05). Exposure to 18 h of light/day for 10 weeks did not alter the contents of LHRH in the SME, HP, or PO-SC area when compared to those of ovx ewes kept under environmental photoperiod. Daily administration of EB to ovx sheep for 5 weeks was not effective in restoring the reduced content of LHRH in the SME or altering the levels of LHRH in the HP or PO-SC area. During the course of the experiment, serum LH levels were similar in the ewes exposed to either 18 or approximately 10 h of light/day; EB was effective in reducing concentrations of LH in both groups of ewes. Plasma LH levels in sheep on the day before slaughter and the AP content of LH and wet weight at the termination of the experiment are shown in Fig. 2. Plasma LH levels in the ovx EB-treated sheep that were exposed to the natural photoperiod were less than those of intact ewes. Plasma LH and the content of LH in the AP reflected the same pattern of changes and were correlated (r = 0.60; P < 0.01). Treatment with EB decreased the LH content of 70 SME
60
CH PO-SC a,b
40
O
30
3 (/)
6 20
a.
a,b
2 £ 10
Anestrus
I
1
Si 3-8
10-14
1
X DC I
16,17,1
Days of the estrous cycle FIG. 1. LHRH contents (X ± SEM) of the SME, HP, and the PO-SC region of the ovine brain during seasonal anestrus (n = 5) and days 3-8 (n = 6), 10-14 (n = 5), and 1, 16, and 17 (n = 7) of the estrous cycle, a, b, and c, Mean differences at the P < 0.01 level. LHRH content of the HP was not significantly different. TABLE 2. AP and serum concentrations (x ± SEM) of LH in anestrous and cyclic ewes Days of the estrous cycle Anestrus 3-8
10-14
1, 16, and 17
AP concentration of LH (jug/mg)
1.0 ± 0.2"
1.2 ± 0.2" 4.4 ± 1.5* 4.1 ± 1.3*
Serum LH (ng/ml)c
0.4 ± 0.1
0.7 ± 0.2
0.3 ± 0.1
"• * Different from one another at the P < 0.01 level. c Levels are not different (P > 0.05).
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0.7 ± 0.3
842
WHEATON
Endo • 1979 Vol 104 • No 3
TABLE 3. LHRH contents (nanograms) of the SME, HP, and PO-SC area of ewes maintained under the natural photoperiod of the breeding season (n = 12) or exposed to an artificially extended photoperiod for 10 weeks (n = 12) Photoperiod Treatment
Artificial (18 h of light/day)
Natural (-10 h of light/day) SME
HP
PO-SC
SME
HP
PO-SC
EB
19.7 ± 4.2 21.9 ± 3.4
6.6 ± 2.0 5.3 ± 1.1
3.0 ± 1.3 3.7 ± 1.1
28.6 ± 4.5 23.4 ± 5.5
9.3 ± 2.8 8.2 ± 2.3
3.4 ± 0.6 4.3 ± 1.0
Intact
43.8 ± 1.8
7.0 ± 1.7
4.0 ± 0.6
Ovariectomized Oil
Ovx ewes were injected im daily with EB (138 fig; n = 12) or oil (n = 12) for 5 weeks before slaughter. Intact ewes (n = 12) were kept under natural photoperiod and killed on unknown days of the estrous cycle. " Values given are x ± SEM.
the AP and increased its weight. Uteri from ovx oiltreated, ovx EB-treated, and intact ewes were 4.3 ± 0.4, 11.7 ± 0.7, and 18.4 ± 1.7 g, respectively.
evidence to support a relationship between a decrease in hypothalamic content of LHRH and an increase in the
Discussion The medial basal region of the ovine brain contained a total of 57 ng LHRH when averaged across anestrus and the estrous cycle. Of this amount, 77% was concentrated in the SME, 19% in adjacent tissue overlying the SME, and 3% in more rostral PO-SC tissue. White et al. (14) reported a similar content of LHRH in extracts of ovine SME. Wheaton et al. (15) measured approximately 3 ng LHRH in the rat brain, of which 52% was located in the SME, 41% in tissue immediately above the SME, and 7% in PO-SC tissue. Palkovits et al (16) reported that the amount of LHRH in the rat median eminence is about twice that found in the arcuate nucleus. In a subsequent publication from the same laboratory, it was reported that the content of LHRH in a fragment of suprachiasmatic tissue was 13% of that concentrated in the SME (17). The increased content of LHRH detected in the SME during proestrus-estrus could have been brought about by adjustments in the net rate of LHRH synthesis and/or release. Simultaneous determination of one or the other of these processes would be necessary to reveal the basis for the change in content. Use of circulating LH concentrations as an indirect estimate of LHRH release rate is complicated by the changing response of the AP to LHRH during the estrous cycle (18). LHRH in the SME and LH in the AP were both elevated during the latter half of the estrous cycle and, conversely, were both reduced by days 3-8. Roche et al. (19) previously reported that the AP concentration of LH increased during the ovine estrous cycle. The increases in contents may be important in providing sufficient LHRH and LH for the preovulatory release of LH. Further research is needed to determine if the drop in LHRH content in the SME that occurs by days 3-8 of the estrous cycle is related to the surge release of LH. In the rat, there is
LHRH in the HP did not change significantly during anestrus, during the estrous cycle, or after ovariectomy. There was a positive correlation between the content of LHRH in the HP and that in the SME. This indicates that similar, but relatively minor, changes may also occur in the LHRH content of the HP. An arcuate-median eminence LHRH neuronal system has been identified in the rat brain (22). If an analogous tract exists in the ovine brain, it may account for the positive relationship. In the PO-SC area, LHRH was highest during days 10-14 of the estrous cycle and then fell to low levels by days 1,16, and 17, the time when LHRH in the SME was greatest. LHRH in the PO-SC region of the rat brain has been shown to increase during the estrous cycle until proestrus, at which time the content had nearly vanished (23). It has been suggested that LHRH of PO-SC origin is involved with eliciting the cyclic discharge of LH in the rat (24). The drop in content of the PO-SC area, coincident with the rise in content of the SME during the latter part of the estrous cycle, is consistent with such a function for LHRH located in the PO-SC area of the ovine brain. Participation of the PO-SC area in bringing about the preovulatory release of LH in sheep, however, is less certain than it is in rats (25). LHRH content of the PO-SC area was not correlated significantly with that in the HP or SME, nor was it altered after ovariectomy and treatment with EB. LHRH in homologous rat tissue also remains constant after ovariectomy (6) and has been reported to have a different ontogeny from that in the median eminence (26). Experimentally, the function of this more rostral LHRH will be difficult to establish. Ovariectomy was followed by a 47% reduction in the LHRH content of the SME. Daily treatment for 5 weeks with EB, beginning 7 weeks postovariectomy, did not restore the content. EB treatment did reduce the elevated levels of serum LH and partially restored uterine
release of LH (20, 21).
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REGIONAL BRAIN CONTENT OF LHRH IN SHEEP
O)
a,b
Q.
a
a 1.2 •^5
b.c
1 O
of ovariectomy (31, 32). Several studies indicate that both the synthesis (33, 34) and release (35) rates of LHRH are accelerated in castrate rats. It is possible that chronic exposure to EB diminishes both the synthesis and release rates of LHRH such that the content remains unchanged. Midanestrous ewes were found to have as much LHRH as ewes during the early stages of the estrous cycle. Hypothalamic LH-releasing activity of anestrous ewes has been reported to be as high as that of cyclic ewes (8, 9). Exposure to 18 h of light/day for 10 weeks did not affect the content of LHRH in ovx sheep. Marked depletion of the LHRH content of the brain clearly does not underlie seasonal anestrus in sheep.
.8
Acknowledgments
a,b
.6
Appreciation is expressed to Dr. T. M. Nett (Colorado State University) for providing LHRH antiserum (no. 42), to Dr. G. D. Niswender (Colorado State University) for ovine LH antiserum (no. 15), and to Dr. L. E. Reichert (Emory University) for highly purified ovine LH used for iodination. NIH-LH-S19 was kindly supplied through the NIAMDD Pituitary Hormone Program. The author wishes to thank Sheila E. Harsdorf and David K. Combs for their technical assistance.
.4 .2
^
1 2
5
i.o
843
"
References 1. McCann, S. M., S. Taleisnik, and H. M. Friedman, LH-releasing activity in hypothalamic extracts, Proc Soc Exp Biol Med 104: 432, 1960. 2. Chowers, I., and S. M. McCann, Content of luteinizing hormonereleasing factor and luteinizing hormone during the estrous cycle and after changes in gonadal steroid titers, Endocrinology 76: 700,
b,c
.4
1965.
.2 18 hr
10hr
Ovx-oil
18hr
10hr
Ovx-EB
10 hr
Intact
FIG. 2. Plasma LH levels in ewes sampled at 15-min intervals for 1 h the day before slaughter and the AP LH contents and wet weights in these ewes. The multiple serum samples from each ewe were averaged, and the mean was used in the calculation of the treatment (X ± SEM). a, b, and c, Differences between means at the P < 0.05 level. Ovx sheep were exposed to either an artificially extended photoperiod of 18 h of light/day for 10 weeks or to the natural photoperiod of the breeding season (~10 h of light/day). After 5 weeks, six ewes in each treatment group were injected im daily with EB (138 jug). Intact ewes (n = 12) were exposed to natural photoperiod and killed on unknown days of the estrous cycle.
weight. Decreases in hypothalamic LHRH stores have been reported to occur after castration in rats (27). Hypothalamic LHRH content of long term ovx rats chronically treated with EB has not been published. Conflicting reports have appeared in the literature regarding the effects of a single injection of EB (21, 28-30) or its ability to maintain LHRH stores if administered from the time
3. Asai, T., and K. Wakabayashi, Changes of hypothalamic LH-RH content during the rat estrous cycle, Endocrinol Jap 22: 319,1975. 4. Kalra, P. S., and S. P. Kalra, Temporal changes in the hypothalamic and serum luteinizing hormone-releasing hormone (LH-RH) levels and the circulating ovarian steroids during the rat oestrous cycle, Ada Endocrinol (Kbh) 85: 449, 1977. 5. Ramirez, V. D., and C. H. Sawyer, Fluctuation in hypothalamic LH-RH (luteinizing hormone-releasing hormone) during the rat estrous cycle, Endocrinology 76: 282, 1965. 6. Araki, S., M. Ferin, E. A. Zimmerman, and R. L. Vande Wiele, Ovarian modulation of immunoreactive gonadotropins-releasing hormone (Gn-RH) in the rat brain: evidence for a differential effect on the anterior and mid-hypothalamus, Endocrinology 96: 644, 1975. 7. Crighton, D. B., B. M. Hartley, and G. E. Lamming, Changes in luteinizing hormone releasing activity of the hypothalamus, and in pituitary gland and plasma luteinizing hormone during the oestrous cycle of the sheep, J Endocrinol 58: 377, 1973. 8. Jackson, G. L., J. F. Roche, D. L. Foster, and P. J. Dziuk, Luteinizing hormone releasing activity in the hypothalamus of anestrous and cyclic ewes, Biol Reprod 5: 5,1971. 9. Pelletier, J., and R. Ortavant, Influence de la duree quotidienne d'eclairement sur l'activite hypothalamique LRF du Belier, C R Acad Sci [D] (Paris) 266: 1604, 1968. 10. Wheaton, J. E., T. I. Raabe, and M. J. Burrill, Characteristics of the ovulatory release of LH in Finn, Finn x Suffolk and Suffolk ewes, J Endocrinol 75: 449, 1977. 11. Palmer, W. M., G. D. Phillips, B. E. Howland, and E. A. Ibrahim, Effect of daylength on reproduction of ewes, In Proceedings of the Western Section of the American Society of Animal Sciences, vol. 23, 1972, p. 318.
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