Ovine Luteinizing Hormone: Isoforms in the Pituitary DGring the Follicular and Luteal Phases of the Estrous Cycle and During Anestrusli2 D. D. Zalesky*J, T. M. Nett+, and H. E. Grotjan*r4
ABSTRACTI Pituitaries were collected from late follicular phase (n = 51, mid-luteal phase (n = 51, and anestrous ewes In = 4) to assess changes in intrapituitary LH heterogeneity a t selected reproductive states. After homogenization, a n aliquot of each pituitary extract was desalted by flow dialysis against water and chromatofocused on a pH 10.5 to 4.0 gradient. Concentrations of LH in pituitary extracts and chromatofocusing fractions were determined by RIA. The LH in pituitary extracts resolved into 13 isoforms during chromatofocusing, which were coded with letters beginning with the most basic isoform. Follicular and mid-luteal phase ewes exhibited similar distributions of intrapituitary LH among its isoforms.
Relative to follicular and luteal phase ewes, anestrous ewes had lower percentages of isoforms D and E as well as higher percentages of isoforms G, H, J and K. Isoform F, the predominant molecular form of LH, constituted a similar percentage in all treatment groups (P > .05).Thus, the distribution of intrapituitary LH among its isoforms did not change significantly between the mid-luteal and follicular phases of the estrous cycle, but higher percentages of the weakly basic and acidic forms of LH were present during anestrus. These observations suggest that intrapituitary LH heterogeneity changes minimally throughout the estrous cycle of ewes during the breeding season.
Key Words: Sheep, LH, Estrous Cycle, Reproduction, Anestrus
J. Anim. Sci. 1992. 70:3851-3856
Introduction The frequency and amplitude of LH pulses change with the reproductive state of the animal (Lincoln, 1976; Schanbacher and Ford, 1978; Karsh et al., 1977; Rahe et al., 1980; Imakawa et al., 1986). During the follicular phase of the estrous cycle,
‘Published as paper no. 9831, Journal Series, Nebraska Agric. Res. Div. This research was supported in part by NIH grant HD 18879 and NSF grant DCB-9104988. 2Appreciationis extended to Laura Rife for her assistance in preparation of this manuscript as well as to D. N. Ward and L. E. Reichert Jr., who provided the purified LH used in the radioimmunoassays. 3hesent address: Anim. Sci. Dept., Knapp Hall, Louisiana State Univ., Baton Rouge 70803-1900. ‘To whom correspondence should be addressed. Received March 23, 1992. Accepted August 3, 1992.
the frequency of LH pulses increases in associa tion with increases in circulating concentrations of estradiol (Baird, 1978; McNeilly et al., 1982; Imakawa et al., 1986). In contrast, the frequency of LH pulses is reduced during the luteal phase of the cycle when progesterone concentrations are elevated and estradiol concentrations are lower (Scaramuzzi et al., 1971; Karsh et al., 1977; Baird, 1978; Rahe et al., 1980). During anestrus, the secretion of LH is markedly reduced, presumably because of the increased sensitivity of the hypothalamic-pituitary axis to the negative feedback effects of estradiol (Legan et al., 1977). Luteinizing hormone, a glycoprotein with variable carbohydrate moieties attached to its subunit peptides (Baenziger and Green, 19881, exists as a series of isoforms. Castration results in a higher percentage of basic isoforms, whereas estrogen replacement of castrates results in a higher
385 1
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*Department of Animal Science, University of Nebraska, Lincoln 68583-0908 and +Department of Physiology and Biophysics, Colorado State University, Fort Collins 80523
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ZALESKY ET AL.
Materials and Methods Animals and Collection of Pituitaries Pituitary glands were collected from ewes in the late follicular (n = 5) or mid-luteal phase of their estrous cycles (n = 5) as well as during anestrus (n = 4). The stage of the reproductive cycle was established by observation of estrus. Pituitaries in the late follicular phase group were collected on the morning of estrus. All ewes were euthanatized with a n overdose of pentobarbital. Blood was collected from luteal and follicular phase ewes at the time of slaughter to measure concentrations of serum progesterone. The anterior lobe was separated from the remaining portion of the pituitary gland by dissection and weighed. Intracellular LH was obtained by homogenizing the anterior pituitary gland in a buffered saline solution containing protease inhibitors (Zalesky and Grotjan, 1991al. Tissue e x tracts were clarified by centrifugation a t 100,000 x g for 1 h, divided into .5-mL aliquots, and stored at -70' C until they were chromatofocused.
Sample Preparation and Chromatofocusing An aliquot of each pituitary extract (.5 mL representing 50 mg of tissue equivalents) was desalted by flow dialysis against water using membranes with a 6 to 8,000 molecular weight cutoff (Spectra/Por 1; Spectrum Medical Industries, Los Angeles, CAI. Exogenous proteins (2 mg each of cytochrome c and myoglobin) and Pharmalyte 8-10.5-HC1(pH 7.0; final concentration of 2% vol/vol) were added to each sample before chro-
matofocusing. Each sample was then chromatofocused on a pH 10.5 to 4.0 gradient using a .6-cm x 18-cm column b e d volume = 5 mL; Spectrum) of PBE-118 resin previously equilibrated with 25 mM triethylamine-HC1 (pH 11.0). After sample application, the pH gradient was sequentially developed with Pharmalyte 8-10.5-HC1(pH 7.01 diluted 1:45 with distilled water and Polybuffer 74-HC1 (pH 4.0) diluted 1:8 with distilled water. Initially, a pH gradient from 10.5 to 7.0 was developed with the Pharmalyte, until a lower limiting pH of approximately 7.0 was attained. Then the elution buffer was changed to the Polybuffer to extend the pH gradient from 7.0 to 4.0. Columns were eluted a t 3 mL/h and .75-mL fractions were collected (n = 150) until a stable, lower limiting pH of approximately 4.0 was attained. Proteins bound to the column at the lower limiting pH were eluted with 1.0 M NaCl and collected as 20 additional .75-mL fractions. All fractions collected during the pH 10.5 to 7.0 gradient (n = 80) were neutralized by the addition of .075 mL of 1.1 M Tris (pH 7.01, whereas fractions collected during the pH 7.0 to 4.0 gradient (n = 90) were neutralized by adding .075 mL of 1.1 M imidazole (pH 7.4). Columns were re-equilibrated with 50 to 60 column volumes of triethylamine between samples. All buffers were degassed before use and contained 1.0% (vol/vol) glycerol. Recovery of immunoreactive LH from the columns averaged 86%. Chromatofocusing reagents were obtained from Pharmacia/LKB Biotechnology (Piscataway, NJI.
Radioimmunoassays Serum progesterone concentrations were determined as previously described (Niswender, 19731. This assay had a lower limit of detection of .12 ng/ mL and a n intraassay CV of 8.4%. The ovine LH (oLH) in pituitary extracts and chromatofocusing fractions was quantified by d o u ble antibody RIA. The standard preparation used was oLH-DNW-HSN-10-124(generously provided by D. N. Ward, Houston, TXI and the primary antibody was anti-hCGaoLHP-EG7N (Zalesky and Grotjan, 199 lb). Relative cross-reactivities of various hormones on a molar basis were as follows: oLH, 100%; oLHP, 113%; oLHa, .14%; ovine FSH, 3.4%; porcine FSHP, e .03%; bovine thyroidstimulating hormone CbTSHI, 2.0%; bTSHP, .4%; bovine growth hormone, .5%; bovine prolactin, c .04%; and porcine ACTH, .01% (Zalesky and Grotjan, 199lb). The purified hormone preparation oLH-LER-1374A (generously provided by Leo Reichert Jr., Albany, NY) was iodinated with immobilized oxidant (Iodobeads, Pierce, Rockford, IL; Markwell, 1982). The immunoassay buffer contained 150 mM NaC1, 50 mM Tris (pH 7.01,s mM Na2 EDTA, 1 mg/mL of gelatin, and ,1% (wt/vol)
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percentage of acidic components (Keel et al., 1987; Stumpf et al., 1992). The biological potencies of specific LH isoforms differ (Keel et al., 1987; Stumpf et al., 1992). These observations demonstrate that gonadal hormones modulate the pattern of LH isohormones in the pituitary and suggest that changes in the LH isohormone profile could play a role in regulating reproductive function. Consistent with this supposition, LH heterogeneity has been reported to change during the estrous cycle of rodents (Wakabayashi, 1977; Uchida and Suginami, 1984; Ozawa and Wakabayashi, 19881. Thus, the present study was performed to characterize more fully LH heterogeneity in ovine females. Our objective was to examine the distribution of LH among its isoforms in the pituitary glands of ewes a t selected reproductive states. Our hypothesis was that changes in concentrations of ovarian steroids would modulate the distribution of LH among its isohormones, yielding distinctive patterns at the selected reproductive states.
3853
OVINE LH ISOFORMS DURING THE ESTROUS CYCLE
sodium azide. Sensitivity of the assays was .025 ng/mL. Intra- and interassay CV were 8.2 and 7.39'0, respectively. Tests of parallelism were conducted using various doses of tissue extracts and LH isoforms. The LH in tissue extracts as well as each LH isoform yielded dose-response curves parallel (P > .OS)to the LH standard (Zalesky and Grotjan, 1991b).
FOLLICULAR
a
12
-
v 0
-10 v CI - I D
-
c 0
The percentage of each oLH isoform present in each extract was calculated and subjected to arc sine transformation (arc sine of the square root of the percentage) before statistical analysis. Oneway analysis of variance (SAS,1985) was used to determine whether differences between the treatment groups existed in the distribution of LH among its isoforms. When differences existed, Duncan's new multiple range test GAS, 1985) was used to determine which means were different. A P-value of < -05 was considered statistically significant.
CHROMATOFOCUSING F R A C T I O N NUMBER
>
0 0
u
10; c,
m
8 1 a
Results
- 600-
Mean pituitary weights (rt SEI for the follicular phase, luteal phase, and anestrous ewes were 1,289 f 39, 1,063 f 93, and 982 f 90 mg, respectively. The mean concentration of immunoreactive oLH (f SEI was greater (P < ,051 in pituitaries of luteal phase ewes ( 3 6 f .10 pg/mgI than in the pituitaries of follicular phase (.38 f .08 pg/mg) or anestrous (.23 f .04 pg/mg) ewes. Serum progesterone concentrations in luteal phase ewes averaged 1.48 f .32 ng/mL, whereas follicular phase ewes had low concentrations @our of five nondetectable at < .12 ng/mL; the fifth had 20 ng/mL). Luteinizing hormone in pituitary extracts resolved into 13 identifiable peaks on pH 10.5 to 4.0 chromatofocusing gradients. Each peak was coded with a letter beginning with the most basic component and was assumed to represent one isoform. Representative chromatofocusing profiles for pituitary extracts of ewes in the follicular and luteal phases of their estrous cycles as well as during anestrus are shown in Figures la, b, and c, respectively. The distributions (mean percentage f SEI of LH eluting as basic isoforms (elution pH > 7.0) are given in Table 1. Similar percentages of the most basic isoforms, A', B, and C, were observed for the three experimental groups (P > .051. Smaller percentages of isoforms D and E as well as higher percentages of isofonns G and H were present in aliquots of pituitary extracts from anestrous ewes (P < .OS)than were present in pituitary extracts
's 0
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6
3
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0
20
40 60 80 100 120 140 160 CHROMATOFOCUSING F R A C T I O N NUMBER
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6
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12
800
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ID
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k 600
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d
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400
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4
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Figure 1. Representative chromatofocusing elution profile of the immunoreactive LH in the pituitary of a ewe in the (a)follicular phase or (b)mid-luteal phase of an estrous cycle or (c)during anestrus. A n aliquot of the pituitary extract was chromatofocused on a pH 10.5 to 4.0 gradient. Each peak was coded with a letter beginning with the most basic form (A' through L). Proteins bound to the column at the lower limiting pH were eluted with 1 M NaCl and designated peak S.
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Statistics
ZALESKY ET AL.
3854
Table 1. Distribution of basic ovine pituitary isoforms of LHa Phase of estrous cycle Isoform
Follicular
A' B C
.08 f
D E F G H
.06 f .07 f .20 f .6y f .9y f 1.2 f .8Y f .1y
.04 f .02 .4 f .07 .9 f .17 4.1 f .5y 16.2 f .8y 50.8 f .7 18.7 f 1.5y 1.5 f .2y
Anestrus .08 f
.4 f .4 f 2.2
f
11.5 f 48.9 f 21.3 f 2.9 f
.08 .12 .05 .1z .5' 1.7 1.6' .2'
Elution pHb 10.7 f .08 10.0 f .03 9.7 f .03 9.6 f .03 9.4 f .02 9.3 f .03 9.1 f .OB < 9.0 to 7.0
&Valuesrepresent mean percentages f SE for each isoform. Follicular (n = 5);luteal (n anestrus (n = 4). bValues represent mean elution pH f SE for each isoform (n = 14). YpzMeans within a row lacking a common superscript letter differ (P < ,051.
=
5);
Zalesky and Grotjan, 1991a) and bovine (Trout and Schanbacher, 1988; Hawkins et al., 1989; Zalesky and Grotjan, 1991a,b; Stumpf et al., 1992) LH are similar, each existing as at least nine isoforms. The LH isoforms eluting in the basic pH range (pH 10.5 to 7.0) correspond to those previously reported. When ovine pituitary extracts were chromatofocused on pH 7.0 to 4.0 gradients, a n additional three or four isoforms of LH eluting in the acidic range were recognized (Keel et al., 1990). Chromatofocusing gradients were extended into the acidic range in the present study to characterize more fully LH heterogeneity. With the long gradients, 13 LH isoforms are identifiable in the ovine (present study) and bovine (Stumpf et al., 1992). Earlier studies reported that some of the LH in ovine pituitary extracts flowed through PBE-118 chromatofocusing columns unrestricted (for exam ple, see Keel et al., 1987). Recent studies have established that the PBE-118 resin exhibits a reduced binding capacity at high pH (Grotjan et al., 19911. Hence, the chromatofocusing method was revised by applying the samples in the elution buffer. Under the revised conditions, no LH flows through the columns unrestricted and a basic form
from ewes in the follicular and luteal phases of their estrous cycles. The relative abundance of the most predominant isoform, F, was similar for all treatment groups (P > .05). The distributions of LH eluting as acidic isoforms (elution pH < 7.0) are illustrated in Table 2. Similar percentages of isoforms I, L, and S were observed in all three treatment groups (P > ,051. The principal acidic isoforms J and K were present in greater percentages in pituitaries collected from anestrous ewes than in those obtained from ewes in the follicular and luteal phases (P < .05). Thus, the distribution of LH isoforms was similar in follicular and luteal phase ewes, whereas anestrous ewes had higher percentages of some of the weakly basic and acidic isoforms of LH.
Discussion Pituitary extracts derived from the ewes in each treatment group resolved into 13 identifiable isoforms when chromatofocused on pH 10.5 to 4.0 gradients. Earlier studies utilizing pH 10.5 to 7.0 chromatofocusing gradients revealed that the charge heterogeneity of ovine (Keel et al., 1987;
Table 2. Distribution of acidic ovine pituitary isoforms of LHa Phase of estrous cycle Isoform I
J K L S
Follicular 1.7 f 3.0 f 3.0 f 2.4 f .5 f
.3 .2y .2y .2 .09
Luteal 1.3 3.1 2.0 2.4 .5
f f f f f
.1
.1y
.1y .4 .08
Anestrus
Elution pHb
.1 . 3 ' .32
6.8 f .06 6.4 f .07 6.1 f .07 6.0 to 4.0 c 4.0
2.2 4.1 3.3 2.2
f f f f .5 f
.1
.06
&Valuesrepresent mean percentages f SE for each isoform. Follicular (n = 5);luteal (n anestrus [n = 4). bValues represent mean elution pH f SE for each isoform (n = 141. YsZMeans within a row lacking a common superscript letter differ (P e ,051.
=
5);
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.5 .7 3.9 15.3 51.7 18.1 1.6
Luteal
OVINE LH ISOPORMS DURING THE ESTROUS CYCLE
not clear, but it is interesting to note that anestrous ewes have greater numbers of estrogen receptors in their pituitaries than do ewes in the breeding season (Glass et al., 19841, and that anestrous ewes are particularly sensitive to the feedback effects of estrogens (Legan et al., 1977). All the isoforms of ovine LH except the extremely basic forms are released in vitro, and similar distributions of LH isohormones are released basally and in response to LHRH (Zalesky and Grotjan, 1991a).Although the pattern of LH isoforms released in vitro is related to the pituitary components, the mid-alkaline forms F and G are selectively secreted (Zalesky and Grotjan, 1991a). Thus, it is likely that all but the more basic forms of LH (A', B, and C)are secreted into circulation and that the heterogeneity of circulating LH is related to heterogeneity in the pituitary. However, the precise relationship between intrapituitary and circulating LH remains to be delineated. In summary, anestrous ewes had a n intrapituitary pattern of LH isohormones enriched in acidic forms, whereas the pituitaries of ewes in the follicular and mid-luteal phases of their reproductive cycles had similar distribution of LH among its isohormones. Minimal changes in the percentage of LH a s isoforms F and G, which are thought to be particularly potent (Keel et al., 1987; Stumpf et al., 19921, were observed. Thus, there seem to be minimal changes in the heterogeneity of intrapituitary LH in ewes a t diverse reproductive states.
Implications Several studies have demonstrated that luteinizing hormone is heterogeneous and that the distribution of luteinizing hormone among its isoforms is subject to hormonal regulation. This study tested the hypothesis that luteinizing hormone in the anterior pituitary gland would undergo changes in heterogeneity in conjunction with the hormonal fluctuations in the estrous cycle of the ewe and during anestrus. Although anestrous ewes had a slightly different pattern of luteinizing hormone heterogeneity, similar patterns were observed in the pituitaries of ewes in the late follicular and luteal phases of their estrous cycles. These observations suggest that luteinizing hormone heterogeneity changes minimally during the estrous cycle of ewes.
Literature Cited Baenziger, J. U.,and E. D. Green. 1088. Pituitary glycoprotein hormone oligosaccharides: Structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin,
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of LH that elutes before peak B but is retarded slightly becomes apparent (Grotjan et al., 1991). This peak was coded as A' LA prime) herein to provide a unique designation (see Grotjan et al., 1991 for additional details). Pituitaries were collected from ewes a t three critical phases of their reproductive cycles: during the late follicular phase when circulating estrogens were highest, during the mid-luteal phase when progesterone was the predominant ovarian steroid, and during anestrus when the reproductive system was quiescent. These times were chosen because the secretory pattern of LH is markedly different a t different stages of the reproductive cycle (Scaramuzzi et al., 1971; Lincoln, 1976; Schanbacher and Ford, 1976; Karsh et al., 1977, Baird, 1978; Rahe et al., 1980; McNeilly et al., 1982; Imakawa et al., 1986). Our hypotheses were that the intrapituitary profile of LH isohormones would change with reproductive state, as has been reported for rodents (Wakabayashi, 1977; Uchida and Suginami, 1984; Ozawa and Wakabayashi, 19881, and that the pattern of intrapituitary LH heterogeneity would reflect circulating concentrations of gonadal steroids. In particular, we hypothesized that the relatively high circulating concentrations of estrogens present during the follicular phase of the cycle would result in a higher percentage of acidic forms of LH, analogous to the changes that occur in gonadectomized animals treated with estrogens (Keel et al., 1987; Stumpf et al., 1992). However, no differences were observed when the distribution of intrapituitary LH among its isoforms was compared between follicular and mid-luteal phase ewes. One interpretation is that the intracellular pattern of LH isohormones is subject to minimum endocrine regulation. However, changes in castrates and estrogen-replaced castrates demonstrate that intrapituitary LH heterogeneity is regulated by steroids and perhaps by other gonadal products. Thus, an alternative explanation is that there were sufficient estrogens in luteal phase ewes (Pant et al., 1977; Rieger and Rawlings, 19851 to maintain a pattern of LH isohormones similar to that found in follicular phase ewes. Irrespective of the mechanisms involved, the results of this study and of a comparable study in the bovine (Stumpf et al., 1992) demonstrate that the pattern of intrapituitary LH isohormones changes minimally in intact females with functional gonads during periods when the pulsatile pattern of LH secretion exhibits marked diversity. It was also hypothesized that anestrous ewes would have an intracellular pattern of LH isohormones similar to that of castrates (Le., a shift toward basic forms). Exactly the opposite was observed: anestrous ewes had a significant shift in the distribution of LH toward acidic isoforms. Again, the endocrine mechanism(s1 involved are
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Karsh, F. J., S. J. Legan, R. L. Hauger, and D. L. Foster. 1977. Negative feedback action of progesterone on tonic luteinizing hormone secretion in the ewe: Dependence on the ovaries. Endocrinology 101:800. Keel, B. A., R.. L. Harms, and H. E. Grotjan, Jr. 1990. Characterization of the acidic forms of ovine pituitary luteinizing hormone. Acta Endocrinol. (Copenhl. 123563. Keel, B. A,, B. D. Schanbacher, and H. E. Grotjan, Jr. 1987. Ovine luteinizing hormone. I. Effects of castration and steroid administration on the charge heterogeneity of pituitary luteinizing hormone. Biol. Reprod. 36:1102. Legan, S.J., F. J. Karsh, and D. L. Foster. 1977. The endocrine control of seasonal reproductive function in the ewe: A marked change in response to the negative feedback action of estradiol on luteinizing hormone secretion. Endocrinology 101:818. Lincoln, G. A. 1976. Seasonal variation in the episodic secretion of luteinizing hormone and testosterone in the ram. J. Endocrinol. 69:213. Markwell, M.A.K. 1982. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labelling of antiserum. Anal. Biochem. 125:427. McNeilly, A. S., M. O’Connell, and D. T. Baird. 1982. Induction of ovulation and normal luteal function by pulsed injections of luteinizing hormone in anestrous ewes. Endocrinology 110:1292.
Niswender, G. D. 1973. Influence of the site of conjugation on the specificity of the antibodies to progesterone. Steroids 22:4 13.
Ozawa, K., and Wakabayashi, K. 1988. Dynamic change in charge heterogeneity of pituitary FSH throughout the estrous cycle in female rats. Endocrinol. Jpn. 35421. Pant, H. C., C.R.N. Hopkinson, and R. J. Fitzpatrick. 1977. Con centration of oestradiol, progesterone, luteinizing hormone and follicle-stimulating hormone in the jugular venous plasma of ewes during the oestrous cycle. J. Endocrinol. 73: 247.
Rahe C. H., R. E. Owens, J. L. Fleeger, H. J. Newton, and P. G. Harms. 1980. Pattern of plasma luteinizing hormone in the cyclic cow: Dependence upon the period of the cycle. Endocrinology 107:498. Rieger, D., and N. C. Rawlings. 1985. The influence of estradiol17j3 and progesterone on serum levels of LH and FSH in the ewe before and after GnRH treatment. Domest. Anim. Endocrinol. 2:17. SAS.1985. SAS User’s Guide: Statistics (5th Ed.). SAS Inst. Inc., Cary NC. Scaramuzzi, R. J., S. A. Tillson, I. H. Thorneycroft, and B. V. Caldwell. 1971. Action of exogenous progesterone and estrogen on behavioral estrus and luteinizing hormone levels in the ovariectomized ewe. Endocrinology 88:1184. Schanbacher, B. D., and J. J. Ford. 1976. Seasonal profiles of plasma luteinizing hormone, testosterone and estradiol in the ram. Endocrinology 99:752. Stumpf, T. T., M. S. Roberson, M. W. Wolfe, D. D. Zalesky, A. S. Cupp, L. A. Werth, N. Kojima, K. Hejl, R. J. Kittok, H. E. Grotjan, and J. E. Kinder. 1992. A similar distribution of gonadotropin isohormones is maintained in the pituitary throughout sexual maturation in the heifer. Biol. Reprod. 46:442.
Trout, W. E., and B. D. Schanbacher. 1988. Characterization of bovine luteinizing hormone charge microheterogeneity by chromatofocusing. J. Anim. Sci. 66 (Suppl 11:399 CAbstr.1. Uchida, H., and H. Suginami. 1984. Subpopulations of luteinizing hormone (LH)possessing various ratios of bioactivity to immunoreactivity in the female rat pituitary glands and their changes during the estrous cycle. Endocrinol. Jpn. 31: 605.
Wakabayashi, K. 1977. Heterogeneity of rat luteinizing hormone revealed by radioimmunoassay and electrofocusing studies. Endocrinol. Jpn. 24:473. Zalesky, D. D., and H. E. Grotjan. 1991a. Comparison of intracellular and secreted isoforms of bovine and ovine luteinizing hormone. Biol. Reprod. 44:1018. Zalesky, D. D., and H. E. Grotjan. 1991b. Luteinizing hormone in the bovine pars tuberalis: Secretion in response to luteinizing hormone releasing hormone and intracellular isoforms. Domest. h i m . Endocrinol. 8:179.
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follitropin and thyrotropin. Biochim. Biophys. Acta 947:287. Baird, D. T. 1978. Pulsatile secretion of LH and ovarian estradiol during the follicular phase of the sheep estrous cycle. Biol. Reprod. 18359. Glass J. D., R. P. Amann, and T. M. Nett. 1984. Effects of season and sex on the distribution of cytosolic estrogen receptors within the brain and the anterior pituitary gland of sheep. Biol. Reprod. 30394. Grotjan, H. E., B. D. Schanbacher, and B. A. Keel. 1991. Ovine luteinizing hormone V: Significance of flow through peaks observed during chromatofocusing as revealed by various methods of sample preparation and application. J. Chromatogr. 549:141. Hawkins, D. E., P. G. Harms, and D. W. Forrest. 1989. Characterization of bovine luteinizing hormone IbLH) isoforms by chromatofocusing. J. Anim. Sci. 67 (Suppl 11:393 [Abstr.). Imakawa, K., M. L. Day, D. D. Zalesky, M. Garciawinder, R. J. Kittok, and J. E. Kinder. 1988. Regulation of pulsatile LH secretion by ovarian steroids in the heifer. J. Anim. Sci. 63:
ABSTRACTI Pituitaries were collected from late follicular phase (n = 51, mid-luteal phase (n = 51, and anestrous ewes In = 4) to assess changes in intrapituitary LH heterogeneity a t selected reproductive states. After homogenization, a n aliquot of each pituitary extract was desalted by flow dialysis against water and chromatofocused on a pH 10.5 to 4.0 gradient. Concentrations of LH in pituitary extracts and chromatofocusing fractions were determined by RIA. The LH in pituitary extracts resolved into 13 isoforms during chromatofocusing, which were coded with letters beginning with the most basic isoform. Follicular and mid-luteal phase ewes exhibited similar distributions of intrapituitary LH among its isoforms.
Relative to follicular and luteal phase ewes, anestrous ewes had lower percentages of isoforms D and E as well as higher percentages of isoforms G, H, J and K. Isoform F, the predominant molecular form of LH, constituted a similar percentage in all treatment groups (P > .05).Thus, the distribution of intrapituitary LH among its isoforms did not change significantly between the mid-luteal and follicular phases of the estrous cycle, but higher percentages of the weakly basic and acidic forms of LH were present during anestrus. These observations suggest that intrapituitary LH heterogeneity changes minimally throughout the estrous cycle of ewes during the breeding season.
Key Words: Sheep, LH, Estrous Cycle, Reproduction, Anestrus
J. Anim. Sci. 1992. 70:3851-3856
Introduction The frequency and amplitude of LH pulses change with the reproductive state of the animal (Lincoln, 1976; Schanbacher and Ford, 1978; Karsh et al., 1977; Rahe et al., 1980; Imakawa et al., 1986). During the follicular phase of the estrous cycle,
‘Published as paper no. 9831, Journal Series, Nebraska Agric. Res. Div. This research was supported in part by NIH grant HD 18879 and NSF grant DCB-9104988. 2Appreciationis extended to Laura Rife for her assistance in preparation of this manuscript as well as to D. N. Ward and L. E. Reichert Jr., who provided the purified LH used in the radioimmunoassays. 3hesent address: Anim. Sci. Dept., Knapp Hall, Louisiana State Univ., Baton Rouge 70803-1900. ‘To whom correspondence should be addressed. Received March 23, 1992. Accepted August 3, 1992.
the frequency of LH pulses increases in associa tion with increases in circulating concentrations of estradiol (Baird, 1978; McNeilly et al., 1982; Imakawa et al., 1986). In contrast, the frequency of LH pulses is reduced during the luteal phase of the cycle when progesterone concentrations are elevated and estradiol concentrations are lower (Scaramuzzi et al., 1971; Karsh et al., 1977; Baird, 1978; Rahe et al., 1980). During anestrus, the secretion of LH is markedly reduced, presumably because of the increased sensitivity of the hypothalamic-pituitary axis to the negative feedback effects of estradiol (Legan et al., 1977). Luteinizing hormone, a glycoprotein with variable carbohydrate moieties attached to its subunit peptides (Baenziger and Green, 19881, exists as a series of isoforms. Castration results in a higher percentage of basic isoforms, whereas estrogen replacement of castrates results in a higher
385 1
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*Department of Animal Science, University of Nebraska, Lincoln 68583-0908 and +Department of Physiology and Biophysics, Colorado State University, Fort Collins 80523
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ZALESKY ET AL.
Materials and Methods Animals and Collection of Pituitaries Pituitary glands were collected from ewes in the late follicular (n = 5) or mid-luteal phase of their estrous cycles (n = 5) as well as during anestrus (n = 4). The stage of the reproductive cycle was established by observation of estrus. Pituitaries in the late follicular phase group were collected on the morning of estrus. All ewes were euthanatized with a n overdose of pentobarbital. Blood was collected from luteal and follicular phase ewes at the time of slaughter to measure concentrations of serum progesterone. The anterior lobe was separated from the remaining portion of the pituitary gland by dissection and weighed. Intracellular LH was obtained by homogenizing the anterior pituitary gland in a buffered saline solution containing protease inhibitors (Zalesky and Grotjan, 1991al. Tissue e x tracts were clarified by centrifugation a t 100,000 x g for 1 h, divided into .5-mL aliquots, and stored at -70' C until they were chromatofocused.
Sample Preparation and Chromatofocusing An aliquot of each pituitary extract (.5 mL representing 50 mg of tissue equivalents) was desalted by flow dialysis against water using membranes with a 6 to 8,000 molecular weight cutoff (Spectra/Por 1; Spectrum Medical Industries, Los Angeles, CAI. Exogenous proteins (2 mg each of cytochrome c and myoglobin) and Pharmalyte 8-10.5-HC1(pH 7.0; final concentration of 2% vol/vol) were added to each sample before chro-
matofocusing. Each sample was then chromatofocused on a pH 10.5 to 4.0 gradient using a .6-cm x 18-cm column b e d volume = 5 mL; Spectrum) of PBE-118 resin previously equilibrated with 25 mM triethylamine-HC1 (pH 11.0). After sample application, the pH gradient was sequentially developed with Pharmalyte 8-10.5-HC1(pH 7.01 diluted 1:45 with distilled water and Polybuffer 74-HC1 (pH 4.0) diluted 1:8 with distilled water. Initially, a pH gradient from 10.5 to 7.0 was developed with the Pharmalyte, until a lower limiting pH of approximately 7.0 was attained. Then the elution buffer was changed to the Polybuffer to extend the pH gradient from 7.0 to 4.0. Columns were eluted a t 3 mL/h and .75-mL fractions were collected (n = 150) until a stable, lower limiting pH of approximately 4.0 was attained. Proteins bound to the column at the lower limiting pH were eluted with 1.0 M NaCl and collected as 20 additional .75-mL fractions. All fractions collected during the pH 10.5 to 7.0 gradient (n = 80) were neutralized by the addition of .075 mL of 1.1 M Tris (pH 7.01, whereas fractions collected during the pH 7.0 to 4.0 gradient (n = 90) were neutralized by adding .075 mL of 1.1 M imidazole (pH 7.4). Columns were re-equilibrated with 50 to 60 column volumes of triethylamine between samples. All buffers were degassed before use and contained 1.0% (vol/vol) glycerol. Recovery of immunoreactive LH from the columns averaged 86%. Chromatofocusing reagents were obtained from Pharmacia/LKB Biotechnology (Piscataway, NJI.
Radioimmunoassays Serum progesterone concentrations were determined as previously described (Niswender, 19731. This assay had a lower limit of detection of .12 ng/ mL and a n intraassay CV of 8.4%. The ovine LH (oLH) in pituitary extracts and chromatofocusing fractions was quantified by d o u ble antibody RIA. The standard preparation used was oLH-DNW-HSN-10-124(generously provided by D. N. Ward, Houston, TXI and the primary antibody was anti-hCGaoLHP-EG7N (Zalesky and Grotjan, 199 lb). Relative cross-reactivities of various hormones on a molar basis were as follows: oLH, 100%; oLHP, 113%; oLHa, .14%; ovine FSH, 3.4%; porcine FSHP, e .03%; bovine thyroidstimulating hormone CbTSHI, 2.0%; bTSHP, .4%; bovine growth hormone, .5%; bovine prolactin, c .04%; and porcine ACTH, .01% (Zalesky and Grotjan, 199lb). The purified hormone preparation oLH-LER-1374A (generously provided by Leo Reichert Jr., Albany, NY) was iodinated with immobilized oxidant (Iodobeads, Pierce, Rockford, IL; Markwell, 1982). The immunoassay buffer contained 150 mM NaC1, 50 mM Tris (pH 7.01,s mM Na2 EDTA, 1 mg/mL of gelatin, and ,1% (wt/vol)
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percentage of acidic components (Keel et al., 1987; Stumpf et al., 1992). The biological potencies of specific LH isoforms differ (Keel et al., 1987; Stumpf et al., 1992). These observations demonstrate that gonadal hormones modulate the pattern of LH isohormones in the pituitary and suggest that changes in the LH isohormone profile could play a role in regulating reproductive function. Consistent with this supposition, LH heterogeneity has been reported to change during the estrous cycle of rodents (Wakabayashi, 1977; Uchida and Suginami, 1984; Ozawa and Wakabayashi, 19881. Thus, the present study was performed to characterize more fully LH heterogeneity in ovine females. Our objective was to examine the distribution of LH among its isoforms in the pituitary glands of ewes a t selected reproductive states. Our hypothesis was that changes in concentrations of ovarian steroids would modulate the distribution of LH among its isohormones, yielding distinctive patterns at the selected reproductive states.
3853
OVINE LH ISOFORMS DURING THE ESTROUS CYCLE
sodium azide. Sensitivity of the assays was .025 ng/mL. Intra- and interassay CV were 8.2 and 7.39'0, respectively. Tests of parallelism were conducted using various doses of tissue extracts and LH isoforms. The LH in tissue extracts as well as each LH isoform yielded dose-response curves parallel (P > .OS)to the LH standard (Zalesky and Grotjan, 1991b).
FOLLICULAR
a
12
-
v 0
-10 v CI - I D
-
c 0
The percentage of each oLH isoform present in each extract was calculated and subjected to arc sine transformation (arc sine of the square root of the percentage) before statistical analysis. Oneway analysis of variance (SAS,1985) was used to determine whether differences between the treatment groups existed in the distribution of LH among its isoforms. When differences existed, Duncan's new multiple range test GAS, 1985) was used to determine which means were different. A P-value of < -05 was considered statistically significant.
CHROMATOFOCUSING F R A C T I O N NUMBER
>
0 0
u
10; c,
m
8 1 a
Results
- 600-
Mean pituitary weights (rt SEI for the follicular phase, luteal phase, and anestrous ewes were 1,289 f 39, 1,063 f 93, and 982 f 90 mg, respectively. The mean concentration of immunoreactive oLH (f SEI was greater (P < ,051 in pituitaries of luteal phase ewes ( 3 6 f .10 pg/mgI than in the pituitaries of follicular phase (.38 f .08 pg/mg) or anestrous (.23 f .04 pg/mg) ewes. Serum progesterone concentrations in luteal phase ewes averaged 1.48 f .32 ng/mL, whereas follicular phase ewes had low concentrations @our of five nondetectable at < .12 ng/mL; the fifth had 20 ng/mL). Luteinizing hormone in pituitary extracts resolved into 13 identifiable peaks on pH 10.5 to 4.0 chromatofocusing gradients. Each peak was coded with a letter beginning with the most basic component and was assumed to represent one isoform. Representative chromatofocusing profiles for pituitary extracts of ewes in the follicular and luteal phases of their estrous cycles as well as during anestrus are shown in Figures la, b, and c, respectively. The distributions (mean percentage f SEI of LH eluting as basic isoforms (elution pH > 7.0) are given in Table 1. Similar percentages of the most basic isoforms, A', B, and C, were observed for the three experimental groups (P > .051. Smaller percentages of isoforms D and E as well as higher percentages of isofonns G and H were present in aliquots of pituitary extracts from anestrous ewes (P < .OS)than were present in pituitary extracts
's 0
LI:
6
3
fi
W
4 +
0
20
40 60 80 100 120 140 160 CHROMATOFOCUSING F R A C T I O N NUMBER
c
-I+
>
0 0
6
-A
12
800
c,
u
V
ID
0
k 600
10
CI ID
d
E
400
8 % c
-c 200
.?I
0 \
0
0,
6 3 r(
I
W
J
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c >
-4
0
X
0
20 40 60 80 100 120 140 160 CHROMATOFOCUSING F R A C T I O N NUMBER
4
0
Figure 1. Representative chromatofocusing elution profile of the immunoreactive LH in the pituitary of a ewe in the (a)follicular phase or (b)mid-luteal phase of an estrous cycle or (c)during anestrus. A n aliquot of the pituitary extract was chromatofocused on a pH 10.5 to 4.0 gradient. Each peak was coded with a letter beginning with the most basic form (A' through L). Proteins bound to the column at the lower limiting pH were eluted with 1 M NaCl and designated peak S.
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Statistics
ZALESKY ET AL.
3854
Table 1. Distribution of basic ovine pituitary isoforms of LHa Phase of estrous cycle Isoform
Follicular
A' B C
.08 f
D E F G H
.06 f .07 f .20 f .6y f .9y f 1.2 f .8Y f .1y
.04 f .02 .4 f .07 .9 f .17 4.1 f .5y 16.2 f .8y 50.8 f .7 18.7 f 1.5y 1.5 f .2y
Anestrus .08 f
.4 f .4 f 2.2
f
11.5 f 48.9 f 21.3 f 2.9 f
.08 .12 .05 .1z .5' 1.7 1.6' .2'
Elution pHb 10.7 f .08 10.0 f .03 9.7 f .03 9.6 f .03 9.4 f .02 9.3 f .03 9.1 f .OB < 9.0 to 7.0
&Valuesrepresent mean percentages f SE for each isoform. Follicular (n = 5);luteal (n anestrus (n = 4). bValues represent mean elution pH f SE for each isoform (n = 14). YpzMeans within a row lacking a common superscript letter differ (P < ,051.
=
5);
Zalesky and Grotjan, 1991a) and bovine (Trout and Schanbacher, 1988; Hawkins et al., 1989; Zalesky and Grotjan, 1991a,b; Stumpf et al., 1992) LH are similar, each existing as at least nine isoforms. The LH isoforms eluting in the basic pH range (pH 10.5 to 7.0) correspond to those previously reported. When ovine pituitary extracts were chromatofocused on pH 7.0 to 4.0 gradients, a n additional three or four isoforms of LH eluting in the acidic range were recognized (Keel et al., 1990). Chromatofocusing gradients were extended into the acidic range in the present study to characterize more fully LH heterogeneity. With the long gradients, 13 LH isoforms are identifiable in the ovine (present study) and bovine (Stumpf et al., 1992). Earlier studies reported that some of the LH in ovine pituitary extracts flowed through PBE-118 chromatofocusing columns unrestricted (for exam ple, see Keel et al., 1987). Recent studies have established that the PBE-118 resin exhibits a reduced binding capacity at high pH (Grotjan et al., 19911. Hence, the chromatofocusing method was revised by applying the samples in the elution buffer. Under the revised conditions, no LH flows through the columns unrestricted and a basic form
from ewes in the follicular and luteal phases of their estrous cycles. The relative abundance of the most predominant isoform, F, was similar for all treatment groups (P > .05). The distributions of LH eluting as acidic isoforms (elution pH < 7.0) are illustrated in Table 2. Similar percentages of isoforms I, L, and S were observed in all three treatment groups (P > ,051. The principal acidic isoforms J and K were present in greater percentages in pituitaries collected from anestrous ewes than in those obtained from ewes in the follicular and luteal phases (P < .05). Thus, the distribution of LH isoforms was similar in follicular and luteal phase ewes, whereas anestrous ewes had higher percentages of some of the weakly basic and acidic isoforms of LH.
Discussion Pituitary extracts derived from the ewes in each treatment group resolved into 13 identifiable isoforms when chromatofocused on pH 10.5 to 4.0 gradients. Earlier studies utilizing pH 10.5 to 7.0 chromatofocusing gradients revealed that the charge heterogeneity of ovine (Keel et al., 1987;
Table 2. Distribution of acidic ovine pituitary isoforms of LHa Phase of estrous cycle Isoform I
J K L S
Follicular 1.7 f 3.0 f 3.0 f 2.4 f .5 f
.3 .2y .2y .2 .09
Luteal 1.3 3.1 2.0 2.4 .5
f f f f f
.1
.1y
.1y .4 .08
Anestrus
Elution pHb
.1 . 3 ' .32
6.8 f .06 6.4 f .07 6.1 f .07 6.0 to 4.0 c 4.0
2.2 4.1 3.3 2.2
f f f f .5 f
.1
.06
&Valuesrepresent mean percentages f SE for each isoform. Follicular (n = 5);luteal (n anestrus [n = 4). bValues represent mean elution pH f SE for each isoform (n = 141. YsZMeans within a row lacking a common superscript letter differ (P e ,051.
=
5);
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.5 .7 3.9 15.3 51.7 18.1 1.6
Luteal
OVINE LH ISOPORMS DURING THE ESTROUS CYCLE
not clear, but it is interesting to note that anestrous ewes have greater numbers of estrogen receptors in their pituitaries than do ewes in the breeding season (Glass et al., 19841, and that anestrous ewes are particularly sensitive to the feedback effects of estrogens (Legan et al., 1977). All the isoforms of ovine LH except the extremely basic forms are released in vitro, and similar distributions of LH isohormones are released basally and in response to LHRH (Zalesky and Grotjan, 1991a).Although the pattern of LH isoforms released in vitro is related to the pituitary components, the mid-alkaline forms F and G are selectively secreted (Zalesky and Grotjan, 1991a). Thus, it is likely that all but the more basic forms of LH (A', B, and C)are secreted into circulation and that the heterogeneity of circulating LH is related to heterogeneity in the pituitary. However, the precise relationship between intrapituitary and circulating LH remains to be delineated. In summary, anestrous ewes had a n intrapituitary pattern of LH isohormones enriched in acidic forms, whereas the pituitaries of ewes in the follicular and mid-luteal phases of their reproductive cycles had similar distribution of LH among its isohormones. Minimal changes in the percentage of LH a s isoforms F and G, which are thought to be particularly potent (Keel et al., 1987; Stumpf et al., 19921, were observed. Thus, there seem to be minimal changes in the heterogeneity of intrapituitary LH in ewes a t diverse reproductive states.
Implications Several studies have demonstrated that luteinizing hormone is heterogeneous and that the distribution of luteinizing hormone among its isoforms is subject to hormonal regulation. This study tested the hypothesis that luteinizing hormone in the anterior pituitary gland would undergo changes in heterogeneity in conjunction with the hormonal fluctuations in the estrous cycle of the ewe and during anestrus. Although anestrous ewes had a slightly different pattern of luteinizing hormone heterogeneity, similar patterns were observed in the pituitaries of ewes in the late follicular and luteal phases of their estrous cycles. These observations suggest that luteinizing hormone heterogeneity changes minimally during the estrous cycle of ewes.
Literature Cited Baenziger, J. U.,and E. D. Green. 1088. Pituitary glycoprotein hormone oligosaccharides: Structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin,
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of LH that elutes before peak B but is retarded slightly becomes apparent (Grotjan et al., 1991). This peak was coded as A' LA prime) herein to provide a unique designation (see Grotjan et al., 1991 for additional details). Pituitaries were collected from ewes a t three critical phases of their reproductive cycles: during the late follicular phase when circulating estrogens were highest, during the mid-luteal phase when progesterone was the predominant ovarian steroid, and during anestrus when the reproductive system was quiescent. These times were chosen because the secretory pattern of LH is markedly different a t different stages of the reproductive cycle (Scaramuzzi et al., 1971; Lincoln, 1976; Schanbacher and Ford, 1976; Karsh et al., 1977, Baird, 1978; Rahe et al., 1980; McNeilly et al., 1982; Imakawa et al., 1986). Our hypotheses were that the intrapituitary profile of LH isohormones would change with reproductive state, as has been reported for rodents (Wakabayashi, 1977; Uchida and Suginami, 1984; Ozawa and Wakabayashi, 19881, and that the pattern of intrapituitary LH heterogeneity would reflect circulating concentrations of gonadal steroids. In particular, we hypothesized that the relatively high circulating concentrations of estrogens present during the follicular phase of the cycle would result in a higher percentage of acidic forms of LH, analogous to the changes that occur in gonadectomized animals treated with estrogens (Keel et al., 1987; Stumpf et al., 1992). However, no differences were observed when the distribution of intrapituitary LH among its isoforms was compared between follicular and mid-luteal phase ewes. One interpretation is that the intracellular pattern of LH isohormones is subject to minimum endocrine regulation. However, changes in castrates and estrogen-replaced castrates demonstrate that intrapituitary LH heterogeneity is regulated by steroids and perhaps by other gonadal products. Thus, an alternative explanation is that there were sufficient estrogens in luteal phase ewes (Pant et al., 1977; Rieger and Rawlings, 19851 to maintain a pattern of LH isohormones similar to that found in follicular phase ewes. Irrespective of the mechanisms involved, the results of this study and of a comparable study in the bovine (Stumpf et al., 1992) demonstrate that the pattern of intrapituitary LH isohormones changes minimally in intact females with functional gonads during periods when the pulsatile pattern of LH secretion exhibits marked diversity. It was also hypothesized that anestrous ewes would have an intracellular pattern of LH isohormones similar to that of castrates (Le., a shift toward basic forms). Exactly the opposite was observed: anestrous ewes had a significant shift in the distribution of LH toward acidic isoforms. Again, the endocrine mechanism(s1 involved are
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Karsh, F. J., S. J. Legan, R. L. Hauger, and D. L. Foster. 1977. Negative feedback action of progesterone on tonic luteinizing hormone secretion in the ewe: Dependence on the ovaries. Endocrinology 101:800. Keel, B. A., R.. L. Harms, and H. E. Grotjan, Jr. 1990. Characterization of the acidic forms of ovine pituitary luteinizing hormone. Acta Endocrinol. (Copenhl. 123563. Keel, B. A,, B. D. Schanbacher, and H. E. Grotjan, Jr. 1987. Ovine luteinizing hormone. I. Effects of castration and steroid administration on the charge heterogeneity of pituitary luteinizing hormone. Biol. Reprod. 36:1102. Legan, S.J., F. J. Karsh, and D. L. Foster. 1977. The endocrine control of seasonal reproductive function in the ewe: A marked change in response to the negative feedback action of estradiol on luteinizing hormone secretion. Endocrinology 101:818. Lincoln, G. A. 1976. Seasonal variation in the episodic secretion of luteinizing hormone and testosterone in the ram. J. Endocrinol. 69:213. Markwell, M.A.K. 1982. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labelling of antiserum. Anal. Biochem. 125:427. McNeilly, A. S., M. O’Connell, and D. T. Baird. 1982. Induction of ovulation and normal luteal function by pulsed injections of luteinizing hormone in anestrous ewes. Endocrinology 110:1292.
Niswender, G. D. 1973. Influence of the site of conjugation on the specificity of the antibodies to progesterone. Steroids 22:4 13.
Ozawa, K., and Wakabayashi, K. 1988. Dynamic change in charge heterogeneity of pituitary FSH throughout the estrous cycle in female rats. Endocrinol. Jpn. 35421. Pant, H. C., C.R.N. Hopkinson, and R. J. Fitzpatrick. 1977. Con centration of oestradiol, progesterone, luteinizing hormone and follicle-stimulating hormone in the jugular venous plasma of ewes during the oestrous cycle. J. Endocrinol. 73: 247.
Rahe C. H., R. E. Owens, J. L. Fleeger, H. J. Newton, and P. G. Harms. 1980. Pattern of plasma luteinizing hormone in the cyclic cow: Dependence upon the period of the cycle. Endocrinology 107:498. Rieger, D., and N. C. Rawlings. 1985. The influence of estradiol17j3 and progesterone on serum levels of LH and FSH in the ewe before and after GnRH treatment. Domest. Anim. Endocrinol. 2:17. SAS.1985. SAS User’s Guide: Statistics (5th Ed.). SAS Inst. Inc., Cary NC. Scaramuzzi, R. J., S. A. Tillson, I. H. Thorneycroft, and B. V. Caldwell. 1971. Action of exogenous progesterone and estrogen on behavioral estrus and luteinizing hormone levels in the ovariectomized ewe. Endocrinology 88:1184. Schanbacher, B. D., and J. J. Ford. 1976. Seasonal profiles of plasma luteinizing hormone, testosterone and estradiol in the ram. Endocrinology 99:752. Stumpf, T. T., M. S. Roberson, M. W. Wolfe, D. D. Zalesky, A. S. Cupp, L. A. Werth, N. Kojima, K. Hejl, R. J. Kittok, H. E. Grotjan, and J. E. Kinder. 1992. A similar distribution of gonadotropin isohormones is maintained in the pituitary throughout sexual maturation in the heifer. Biol. Reprod. 46:442.
Trout, W. E., and B. D. Schanbacher. 1988. Characterization of bovine luteinizing hormone charge microheterogeneity by chromatofocusing. J. Anim. Sci. 66 (Suppl 11:399 CAbstr.1. Uchida, H., and H. Suginami. 1984. Subpopulations of luteinizing hormone (LH)possessing various ratios of bioactivity to immunoreactivity in the female rat pituitary glands and their changes during the estrous cycle. Endocrinol. Jpn. 31: 605.
Wakabayashi, K. 1977. Heterogeneity of rat luteinizing hormone revealed by radioimmunoassay and electrofocusing studies. Endocrinol. Jpn. 24:473. Zalesky, D. D., and H. E. Grotjan. 1991a. Comparison of intracellular and secreted isoforms of bovine and ovine luteinizing hormone. Biol. Reprod. 44:1018. Zalesky, D. D., and H. E. Grotjan. 1991b. Luteinizing hormone in the bovine pars tuberalis: Secretion in response to luteinizing hormone releasing hormone and intracellular isoforms. Domest. h i m . Endocrinol. 8:179.
Downloaded from https://academic.oup.com/jas/article-abstract/70/12/3851/4705775 by Iowa State University user on 19 January 2019
follitropin and thyrotropin. Biochim. Biophys. Acta 947:287. Baird, D. T. 1978. Pulsatile secretion of LH and ovarian estradiol during the follicular phase of the sheep estrous cycle. Biol. Reprod. 18359. Glass J. D., R. P. Amann, and T. M. Nett. 1984. Effects of season and sex on the distribution of cytosolic estrogen receptors within the brain and the anterior pituitary gland of sheep. Biol. Reprod. 30394. Grotjan, H. E., B. D. Schanbacher, and B. A. Keel. 1991. Ovine luteinizing hormone V: Significance of flow through peaks observed during chromatofocusing as revealed by various methods of sample preparation and application. J. Chromatogr. 549:141. Hawkins, D. E., P. G. Harms, and D. W. Forrest. 1989. Characterization of bovine luteinizing hormone IbLH) isoforms by chromatofocusing. J. Anim. Sci. 67 (Suppl 11:393 [Abstr.). Imakawa, K., M. L. Day, D. D. Zalesky, M. Garciawinder, R. J. Kittok, and J. E. Kinder. 1988. Regulation of pulsatile LH secretion by ovarian steroids in the heifer. J. Anim. Sci. 63:
