DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 8(2):179-187, 1991
LUTEINIZING HORMONE IN THE BOVINE PARS TUBERALIS: SECRETION IN RESPONSE TO LUTEINIZlNG HORMONE RELEASING HORMONE AND INTRACELLULAR ISOFORMS 1 D.D. Zalesky and H.E. Grotjan 2 Department of Animal Science, University of Nebraska, Lincoln, NE 68583 ReceivedJuly2, 1990 ABSTRACT The objective of this study was to examine the physiological characteristics of gonadotropes in the bovine (b) pars tuberalis as assessed by their ability to release Luteinizing Hormone (LH) in response to LH-Releasing Hormone (LHRH) and the intracellular distribution of LH isoforms. At slaughter, the stalk median eminence and associated pars tuberalis as well as the anterior pituitary gland were collected from each of 7 castrate males. Each stalk median eminence and pituitary gland was mid-sagitally sectioned and weighed. One half of each tissue was immediately frozen and subsequently homogenized to determine the intracellular distribution of bLH isoforms. Tissue extracts were desalted by flow dialysis against water and chromatofocused on pH 10.5-7.0 gradients. The remaining half of the pituitary was sliced with a Staddie-Riggs slicer. The pituitary slices and the remaining half of the stalk median eminence were perifused (0.1 ml/min) for a total of 360 min with effluent samples (1.0 ml) collected every 10 min. At 130 min tissues were stimulated with 5 x 10-s M LHRH. Concentrations of LH in the effluent samples and the fractions collected from chromatofocusing were determined by radioimmunoassay. The release of LH in response to LHRH was 43.9% and 47.0% above basal secretion for the pars tuberalis and pituitary, respectively, suggesting similar degrees of responsiveness. Pars tuberalis and pituitary extracts resolved into nine LH isoforms during chromatofocusing and were coded with letters beginning with the most basic form. No differences (P > .05) were observed in distribution of LH isoforms between the pars tuberalis and the pituitary gland. These data suggest that the gonadotropes of the bovine pars tuberalis are physiologically similar to those of the anterior pituitary as assessed by similar distributions of LH isoforms and responsiveness to LHRH. INTRODUCTION The pars tuberalis is adjacent to and continuous with the adenohypophysis but is an anatomically distinct structure which forms a sleeve around the median eminence and stalk of the pituitary (1,2,3,4). A large percentage of gonadotropes are found within this structure in the sheep (2) and rat (3). Although no specific endocrine function has been ascribed to the pars tuberalis, it is capable of releasing LH in vitro both basally and in response to LHRH (2,5). Furthermore, gonadotropes within the pars tuberalis and anterior pituitary respond in a similar fashion following castration (hypertrophy and hyperplasia) or steroid replacement (inhibition of hypertrophy and hyperplasia) (6). These observations suggest that the gonadotropes within the pars tuberalis are physiologically similar to those in the anterior pituitary. The pituitary gonadotropins, LH and Follicle Stimulating Hormone (FSH) from several species exist as families of isohormones which differ in their isoelectric points, receptor binding and biological activities (7,8,9,10,11). For each hormone, multiple molecular forms appear to result from heterogeneity in their oligosaccharide moieties (12,13). The distribution of the isoforms of the gonadotropins is dependent upon the endocrine status of the animal. Gonadal steroids modulate the pattern of isohormones because changes are effected by stage of the reproductive cycle (14), castration (15), pubertal development (16) or cryptorchidism (15). Because relatively little is known about the physiology of the gonadotropes in the bovine pars tuberalis, the following experiments were performed to compare: 1) the basal release of
Copyright© 1991by Domendo,Inc.
179
0739-7240/91/$3.00
180
ZALESKY AND GROTJAN
LH and the response to LHRH in vitro, and 2) the intracellular distribution of LH isoforms in the pars tuberalis and anterior pituitary. The results provide further evidence that the gonadotropes within the pars tuberalis and anterior pituitary are physiologically similar.
MATERIALS AND METHODS Tissue: At slaughter, the stalk median eminence and associated pars tuberalis as well as the anterior pituitary gland were collected from seven castrate males. Each stalk median eminence and anterior pituitary was mid-sagitally sectioned and weighed. One half of each tissue was immediately frozen (-70 C) and subsequently used for the extraction of intracellular LH. The remaining half of the pituitary was sliced into 0.5 mm sections using a Stadie-Riggs tissue slicer. Perifusion: All slices obtained from one hemi-pituitary or the remaining half of the stalk median eminence were placed in an in vitro perifusion chamber (Acusyst-S; Endotronics Inc., Coon Rapids, MN) and perifused at 0.1 ml/min at 37 C for 360 min. Medium 199 (pH 7.4, Sigma Chemical Co., St. Louis, MO) was equilibrated with 95% oxygen and 5% CO~ and used as the perifusion medium. Effluent samples (1.0 ml) were collected every 10 min. After attainment of stable basal secretion (130 min), tissues were stimulated with 5 x 10 8 M LHRH for 30 min. This dose of LHRH was shown in preliminary experiments in this lab to consistently stimulate pituitary slices in our in vitro perifusion system. Effluent samples were frozen (-20 C) until concentrations of LH were quantified. Tissue Extraction: Frozen stalk median eminence or pituitary tissue was homogenized (Polytron at a setting of 6 tbr 30 sec) in 150 mM NaC1 buffered with 50 mM tris (hydroxymethyl) aminomethane (Tris), pH 7.4, containing 0.5% (vol/vol) Triton X-t00, 5 mM Na2ethylenediaminetetraacetate (EDTA), 1 mM phenyl-methylsulphonyl flouride, 0.5 mg/L Leupeptin and 200 U/ml Aprotinin (1.0 ml per 100 mg wet tissue weight). Tissue extracts were clarified by centrifugation at 100,000 x g for 1 hr, aliquoted into 0.5 ml aliquots and stored at -70 C until chromatofocused. Chromatofocusing: Aliquots (0.5 ml representing 50-mg equivalents) of stalk median eminence or anterior pituitary were desalted by flow dialysis against water, utilizing membranes with a 6-8,000 molecular weight cutoff (Spectra/Pot; Spectrum Medical Industries, Inc., Los Angeles, CA). Desalted extracts were supplemented with 2% (v/v) Pharmalyte (pH 7.0) and chromatofocused on pH 10.5-7.0 gradients. Samples were then applied to 0.7 x 26 cm columns (volume = 10 ml; Kontes, Vineland, N J) of PBE-118 (Pharmacia LKB Biotechnology Inc., Pistcataway, N J), equilibrated with 25 mM triethylamine (pH 11.0). The columns were eluted at 5.0 ml/h with Pharmalyte 8-10.5 (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) diluted 1:45 with distilled water and adjusted to pH 7.0 with 6 N HCI. Chromatofocusing fractions ( 1.5 ml) were collected (n=70 or 80) until a stable, lower limiting pH was attained. Proteins bound to the column at the lower limiting pH were eluted with 1.0 M NaCI and collected as an additional twenty 1.5 ml fractions. Fractions were neutralized by addition of 0.15 ml 1.1 M riffs (pH 7.0). Columns were re-equilibrated between samples with 50- to 60-column volumes of triethylamine. All buffers were completely degassed before use and contained 5% glycerol. Recovery of immunoreactive LH from the columns averaged 71.4 + 3.1%. Radioirnmunoassay: lmmunoreactive LH in perifusates, chromatofocusing fractions and tissue extracts was quantified by double antibody radioimmunoassay. Tests of parallelism were conducted for all isoforms and tissue extracts at varying doses in the radioimmunoassay (Figure 1). All isoforms and extracts were parallel (P > .05) to the standard (USDA-bLH-II) utilized in the assay. Bovine LH (USDA-bLH-I 1) was utilized as the standard. The first antibody was anti-hCGcxoLHB-EG7N (H.E. Grotjan, S. Glenn and D.N. Ward, manuscript in preparartion) and the purified hormone preparation, oLH-LER-1374A, was used for iodina-
:K
O. O
"Io r:3 O m
v
0
2
3
4
5
6
7
8
0
.156
.625
.50
I
1.25
1.0
I
2.0
2.5
Mass Added (ng)
.313
.25
u,-..~4.,i fw ~ I I • A x v
5.0
10.0
bmfm'm D bx~rm F Iim4m. G Iim4mn Z
II USDA-bLH-I 1 S
÷ mJ~w ~ v ~ t
Fig. 1. Tests for parallelism of bovine luteinizing hormone (bLH) isoforms and tissue extracts in the bLH radioimmunoassay. Bovine LH in all isoforms and tissue extracts were parallel (P > .05) to USDA- bLH-I1, the standard used. For illustrative purposes, only isoforms D, E G and Z and the pituitary extract are shown.
.078
• 125
I
N
r-
--I c w rn
m
0
-4 "1" m
"n
0
O0 0
"I"
0
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z
O
"100 m -n i"11 --4
r-"
182
ZALESKY AND GROTJAN
tion. The relative cross reactivities of various hormones on a molar basis were: ovine LH (oLH) 100%, oLHI3 113%, oLHa 0.14%, ovine FSH 3.4%, porcine FSH8 < 0.03%, bovine thyroid-stimulating hormone (b TSH) 2.0%, bTSHf5 0.4%, bovine growth hormone 0.5%, bovine prolactin < 0.04% and porcine ACTH < 0.01%. lodinated hormone was prepared with immobilized oxidant (Iodobeads; Pierce, Rockford, IL) (17). The immunoassay buffer contained 150 mM NaC1, 50 mM Tris (pH 7.0), 5 mM Na2EDTA, 1 mg/ml gelatin and 0.1% (wt/vol) sodium azide. Sensitivity of the assays was 0.08 ng/ml with intra- and interassay coefficients of variation of 7.9% and 8.8%, respectively. Statistics: Areas under the LH release curve (trapezoid rule) were determined for basal (5 samples prior to LHRH treatment) and response to LHRH (5 samples during and after LHRH treatment). Statistical differences between areas under the curve for basal and LHRHinduced secretion were determined by paired t-test (18). One-way analysis of variance (18) was used to determine if statistical significance existed between distribution of LH isoforms in the pars tuberalis and anterior pituitary. Probabilities of less than 0.05 were considered significant. Percentage values were subjected to arc sine transformations (arc sine of the square root of the percentage) prior to analysis.
RESULTS Average tissue weights were 101 + 7 mg for the stalk median eminence and 1,788 + 134 mg for the anterior pituitary gland. Mean LH content was 6.6 + 1.5 and 1,275 + 260 lag for the stalk median eminence and anterior pituitary, respectively. The concentration of LH in the pars tuberalis (0.06 _+0.01 lag/mg) was less (P < .05) than that in the anterior pituitary (0.70 + 0.11 lag/mg). Mean (_+ s.e.) secretion of LH from the pars tuberalis and the anterior pituitary gland during perifusion is illustrated by the profiles in Figure 2. The pars tuberalis was capable of secreting LH basally and in response to LHRH stimulation. The general pattern of LH secretion by the pars tuberalis was similar to that observed for the anterior pituitary. Initially, an increased release of LH was observed from both the pars tuberalis and pituitary, followed by a relatively stable basal rate of secretion. Areas under the curves for LH release representing basal and LHRH-induced secretion for tissues are shown in Table 1. Both the pars tuberalis and anterior pituitary responded (P < .05) to LHRH with increases of 43.9% and 47.0% above basal secretion, respectively. Extracts of both the pars tuberalis and anterior pituitary resolved into nine LH isoforms (Figure 3) following chromatofocusing over pH 10.5-7.0 gradients. The isoforms were coded with letters beginning with the most basic form. The mean percentage of intracellular LH in each isoform is shown in Table 2. No differences (P > .05) were observed in the distribution of LH among the various isoforms between the pars tuberalis and the anterior pituitary gland. DISCUSSION The study reported herein indicates that the bovine pars tuberalis contains gonadotropes capable of synthesizing and secreting LH. The pattern of secretion during in vitro perifusion was similar to that of the anterior pituitary gland. Both basal secretion and the response to LHRH stimulation were similar between the two tissues. These cells also had a similar distribution of LH isoforms. In some mammals, the pars tuberalis has been estimated to make-up 1-3% of the total adenohypophysial volume, while the ovine pars tuberalis has been estimated to comprise 1012% of the total tissue (2). Because we did not attempt to dissect the pars tuberalis from the remaining portion of the stalk median eminence, an estimate of the size or volume of this structure was not attained. Nonetheless, the content of LH (6.6 +_ 1.5 lag) is believed to be a reasonable estimate of the amount of LH within the bovine pars tuberalis. While the concen-
LH SECRETION AND LH ISOFORMS OF THE BOVINE PARS TUBERALIS
183
tration of LH in the bovine pars tuberalis was less than that for the anterior pituitary, it is possible that a greater concentration of LH would have been found in the pars tuberalis if it had been dissected away from the stalk median eminence. The ovine pars tuberalis was previ-
901
. Pots Tuberahs
] I
Q.
E lOOO. Anterior Pituitary
0
O~ V
-I- 500 -J .t3
LHRH 0
5
10 15 20 25 ~ Per|fusion Sample Number
35
Fig. 2. Perifusion profiles for bovine LH secretion (means + s.e.) by the pars tuberalis (upper panel) and anterior pituitary gland (lower panel). Tissues were perifused at 0.1 ml/min and 1.0 ml (10 min) fractions were collected. At 130 rain, tissues were challenged with 5 x 10-8 M LHRH for 30 min. Immunoactive LH was quantified by radioimmunoassay.
ously reported (2) to contain similar concentrations of LH on a per mg of tissue basis to that of the anterior pituitary when pars tuberalis tissue was separated from the median eminence. The hypothalamus of the rat (5,19) has been reported to possibly contain LH. However, these studies (5,19) included the pars tuberalis along with the hypothalamus. The pattern of LH secretion from the bovine pars tuberalis provides direct evidence that this structure is capable of basal and LHRH-stimulated release of LH. Similar patterns of LH
184
ZALESKY AND GROTJAN
TABLE l. AREAS UNDERTHE CURVEFOR BASALANDLHRH-INDUCEDLH RELEASE FROMTHE BOVINEPARSTUBERALISANDANTERIORPITUITARY'. Areas Under the Curve (ng• ml ' • 50 min ~)b Pars Tuberalis
Anterior Pituitary
Basal Release
1,158 + 267
10,727 + 2,038
LHRH-induced Release
1,753 _+301
21,676 _+4,201
43.9
47.0
%increase
"Values represent means _+s.e. (n=7). areas for basal secrtion were determined from the 5 samples prior to LHRH treatment and areas for LHRH-induced secretion from the 5 samples during and after LHRH treatment. bValues of areas under the curve for LHRH-induced LH secretion are significantly greater than corresponding basal secretion (P < .05).
18o~ A~.'B~ClDI r IF I 0 I H 15oi ~ P=r= Tuber=li=
I Z
r 12 -11
.o
..lo
,o i
\
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,.
,oo 0
~"
J._,
o
.
.z
12
o
A'"r° "u'°oA t,o
I
t 10
--
20
30
40
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Chromofofocusing
t
50
60
70
m~Rlll~, 7 80
90
Froction Number
Fig. 3. Representative profiles for the distribution of bovine LH isoforms in the pars tuberalis and anterior pituitary gland of a castrate male. Tissue extracts were desalted and chromatofocused on p H l 0.5-7.0 gradients. Chromatofocusing fractions (1.5 ml) were collected (n=70 or 80) until a stable, lower limiting pH was attained. Proteins bound to the column at the lower limiting pH were eluted with 1.0 M NaC1 and collected as an additional twenty 1.5 ml fractions. Immunoreactive LH in each fraction was quantified by radioimmunoassay. Isoforms are coded with letters (A, B, C, D, E, E G, H, Z) beginning with the most basic isoform.
LH SECRETION AND LH ISOFORMS OF THE BOVINE PARS TUBERALIS
185
secretion were observed for the anterior pituitary and pars tuberalis. Following an initial increased rate of LH release, both tissues reached a steady state release and then responded in an analagous manner to LHRH. Secretion of LH above baseline was not different between the two tissues (43.9% vs 47.0%). Our results for the in vitro secretion of LH from the bovine
TABLE2. DISTRIBUTIONOF BOVtNELH ISOFORMSIN PARSTUBERALISANDANTERIORPITUITARYTISSUE ANDELUTIONPH OF EACHISOFORM. Tissuea Iso~
A B C D E F G H Z
b
(elufionpH)
Pars~bo~is
An~fior~mitary
(>10.0±0.2) (9.8±0.1) (9.6±0.1) (9.4±0.~) (9.2±0.02) (9.1±0.02) (8.9±0.02) (7.0) (
Vol. 8(2):179-187, 1991
LUTEINIZING HORMONE IN THE BOVINE PARS TUBERALIS: SECRETION IN RESPONSE TO LUTEINIZlNG HORMONE RELEASING HORMONE AND INTRACELLULAR ISOFORMS 1 D.D. Zalesky and H.E. Grotjan 2 Department of Animal Science, University of Nebraska, Lincoln, NE 68583 ReceivedJuly2, 1990 ABSTRACT The objective of this study was to examine the physiological characteristics of gonadotropes in the bovine (b) pars tuberalis as assessed by their ability to release Luteinizing Hormone (LH) in response to LH-Releasing Hormone (LHRH) and the intracellular distribution of LH isoforms. At slaughter, the stalk median eminence and associated pars tuberalis as well as the anterior pituitary gland were collected from each of 7 castrate males. Each stalk median eminence and pituitary gland was mid-sagitally sectioned and weighed. One half of each tissue was immediately frozen and subsequently homogenized to determine the intracellular distribution of bLH isoforms. Tissue extracts were desalted by flow dialysis against water and chromatofocused on pH 10.5-7.0 gradients. The remaining half of the pituitary was sliced with a Staddie-Riggs slicer. The pituitary slices and the remaining half of the stalk median eminence were perifused (0.1 ml/min) for a total of 360 min with effluent samples (1.0 ml) collected every 10 min. At 130 min tissues were stimulated with 5 x 10-s M LHRH. Concentrations of LH in the effluent samples and the fractions collected from chromatofocusing were determined by radioimmunoassay. The release of LH in response to LHRH was 43.9% and 47.0% above basal secretion for the pars tuberalis and pituitary, respectively, suggesting similar degrees of responsiveness. Pars tuberalis and pituitary extracts resolved into nine LH isoforms during chromatofocusing and were coded with letters beginning with the most basic form. No differences (P > .05) were observed in distribution of LH isoforms between the pars tuberalis and the pituitary gland. These data suggest that the gonadotropes of the bovine pars tuberalis are physiologically similar to those of the anterior pituitary as assessed by similar distributions of LH isoforms and responsiveness to LHRH. INTRODUCTION The pars tuberalis is adjacent to and continuous with the adenohypophysis but is an anatomically distinct structure which forms a sleeve around the median eminence and stalk of the pituitary (1,2,3,4). A large percentage of gonadotropes are found within this structure in the sheep (2) and rat (3). Although no specific endocrine function has been ascribed to the pars tuberalis, it is capable of releasing LH in vitro both basally and in response to LHRH (2,5). Furthermore, gonadotropes within the pars tuberalis and anterior pituitary respond in a similar fashion following castration (hypertrophy and hyperplasia) or steroid replacement (inhibition of hypertrophy and hyperplasia) (6). These observations suggest that the gonadotropes within the pars tuberalis are physiologically similar to those in the anterior pituitary. The pituitary gonadotropins, LH and Follicle Stimulating Hormone (FSH) from several species exist as families of isohormones which differ in their isoelectric points, receptor binding and biological activities (7,8,9,10,11). For each hormone, multiple molecular forms appear to result from heterogeneity in their oligosaccharide moieties (12,13). The distribution of the isoforms of the gonadotropins is dependent upon the endocrine status of the animal. Gonadal steroids modulate the pattern of isohormones because changes are effected by stage of the reproductive cycle (14), castration (15), pubertal development (16) or cryptorchidism (15). Because relatively little is known about the physiology of the gonadotropes in the bovine pars tuberalis, the following experiments were performed to compare: 1) the basal release of
Copyright© 1991by Domendo,Inc.
179
0739-7240/91/$3.00
180
ZALESKY AND GROTJAN
LH and the response to LHRH in vitro, and 2) the intracellular distribution of LH isoforms in the pars tuberalis and anterior pituitary. The results provide further evidence that the gonadotropes within the pars tuberalis and anterior pituitary are physiologically similar.
MATERIALS AND METHODS Tissue: At slaughter, the stalk median eminence and associated pars tuberalis as well as the anterior pituitary gland were collected from seven castrate males. Each stalk median eminence and anterior pituitary was mid-sagitally sectioned and weighed. One half of each tissue was immediately frozen (-70 C) and subsequently used for the extraction of intracellular LH. The remaining half of the pituitary was sliced into 0.5 mm sections using a Stadie-Riggs tissue slicer. Perifusion: All slices obtained from one hemi-pituitary or the remaining half of the stalk median eminence were placed in an in vitro perifusion chamber (Acusyst-S; Endotronics Inc., Coon Rapids, MN) and perifused at 0.1 ml/min at 37 C for 360 min. Medium 199 (pH 7.4, Sigma Chemical Co., St. Louis, MO) was equilibrated with 95% oxygen and 5% CO~ and used as the perifusion medium. Effluent samples (1.0 ml) were collected every 10 min. After attainment of stable basal secretion (130 min), tissues were stimulated with 5 x 10 8 M LHRH for 30 min. This dose of LHRH was shown in preliminary experiments in this lab to consistently stimulate pituitary slices in our in vitro perifusion system. Effluent samples were frozen (-20 C) until concentrations of LH were quantified. Tissue Extraction: Frozen stalk median eminence or pituitary tissue was homogenized (Polytron at a setting of 6 tbr 30 sec) in 150 mM NaC1 buffered with 50 mM tris (hydroxymethyl) aminomethane (Tris), pH 7.4, containing 0.5% (vol/vol) Triton X-t00, 5 mM Na2ethylenediaminetetraacetate (EDTA), 1 mM phenyl-methylsulphonyl flouride, 0.5 mg/L Leupeptin and 200 U/ml Aprotinin (1.0 ml per 100 mg wet tissue weight). Tissue extracts were clarified by centrifugation at 100,000 x g for 1 hr, aliquoted into 0.5 ml aliquots and stored at -70 C until chromatofocused. Chromatofocusing: Aliquots (0.5 ml representing 50-mg equivalents) of stalk median eminence or anterior pituitary were desalted by flow dialysis against water, utilizing membranes with a 6-8,000 molecular weight cutoff (Spectra/Pot; Spectrum Medical Industries, Inc., Los Angeles, CA). Desalted extracts were supplemented with 2% (v/v) Pharmalyte (pH 7.0) and chromatofocused on pH 10.5-7.0 gradients. Samples were then applied to 0.7 x 26 cm columns (volume = 10 ml; Kontes, Vineland, N J) of PBE-118 (Pharmacia LKB Biotechnology Inc., Pistcataway, N J), equilibrated with 25 mM triethylamine (pH 11.0). The columns were eluted at 5.0 ml/h with Pharmalyte 8-10.5 (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) diluted 1:45 with distilled water and adjusted to pH 7.0 with 6 N HCI. Chromatofocusing fractions ( 1.5 ml) were collected (n=70 or 80) until a stable, lower limiting pH was attained. Proteins bound to the column at the lower limiting pH were eluted with 1.0 M NaCI and collected as an additional twenty 1.5 ml fractions. Fractions were neutralized by addition of 0.15 ml 1.1 M riffs (pH 7.0). Columns were re-equilibrated between samples with 50- to 60-column volumes of triethylamine. All buffers were completely degassed before use and contained 5% glycerol. Recovery of immunoreactive LH from the columns averaged 71.4 + 3.1%. Radioirnmunoassay: lmmunoreactive LH in perifusates, chromatofocusing fractions and tissue extracts was quantified by double antibody radioimmunoassay. Tests of parallelism were conducted for all isoforms and tissue extracts at varying doses in the radioimmunoassay (Figure 1). All isoforms and extracts were parallel (P > .05) to the standard (USDA-bLH-II) utilized in the assay. Bovine LH (USDA-bLH-I 1) was utilized as the standard. The first antibody was anti-hCGcxoLHB-EG7N (H.E. Grotjan, S. Glenn and D.N. Ward, manuscript in preparartion) and the purified hormone preparation, oLH-LER-1374A, was used for iodina-
:K
O. O
"Io r:3 O m
v
0
2
3
4
5
6
7
8
0
.156
.625
.50
I
1.25
1.0
I
2.0
2.5
Mass Added (ng)
.313
.25
u,-..~4.,i fw ~ I I • A x v
5.0
10.0
bmfm'm D bx~rm F Iim4m. G Iim4mn Z
II USDA-bLH-I 1 S
÷ mJ~w ~ v ~ t
Fig. 1. Tests for parallelism of bovine luteinizing hormone (bLH) isoforms and tissue extracts in the bLH radioimmunoassay. Bovine LH in all isoforms and tissue extracts were parallel (P > .05) to USDA- bLH-I1, the standard used. For illustrative purposes, only isoforms D, E G and Z and the pituitary extract are shown.
.078
• 125
I
N
r-
--I c w rn
m
0
-4 "1" m
"n
0
O0 0
"I"
0
Z
z
O
"100 m -n i"11 --4
r-"
182
ZALESKY AND GROTJAN
tion. The relative cross reactivities of various hormones on a molar basis were: ovine LH (oLH) 100%, oLHI3 113%, oLHa 0.14%, ovine FSH 3.4%, porcine FSH8 < 0.03%, bovine thyroid-stimulating hormone (b TSH) 2.0%, bTSHf5 0.4%, bovine growth hormone 0.5%, bovine prolactin < 0.04% and porcine ACTH < 0.01%. lodinated hormone was prepared with immobilized oxidant (Iodobeads; Pierce, Rockford, IL) (17). The immunoassay buffer contained 150 mM NaC1, 50 mM Tris (pH 7.0), 5 mM Na2EDTA, 1 mg/ml gelatin and 0.1% (wt/vol) sodium azide. Sensitivity of the assays was 0.08 ng/ml with intra- and interassay coefficients of variation of 7.9% and 8.8%, respectively. Statistics: Areas under the LH release curve (trapezoid rule) were determined for basal (5 samples prior to LHRH treatment) and response to LHRH (5 samples during and after LHRH treatment). Statistical differences between areas under the curve for basal and LHRHinduced secretion were determined by paired t-test (18). One-way analysis of variance (18) was used to determine if statistical significance existed between distribution of LH isoforms in the pars tuberalis and anterior pituitary. Probabilities of less than 0.05 were considered significant. Percentage values were subjected to arc sine transformations (arc sine of the square root of the percentage) prior to analysis.
RESULTS Average tissue weights were 101 + 7 mg for the stalk median eminence and 1,788 + 134 mg for the anterior pituitary gland. Mean LH content was 6.6 + 1.5 and 1,275 + 260 lag for the stalk median eminence and anterior pituitary, respectively. The concentration of LH in the pars tuberalis (0.06 _+0.01 lag/mg) was less (P < .05) than that in the anterior pituitary (0.70 + 0.11 lag/mg). Mean (_+ s.e.) secretion of LH from the pars tuberalis and the anterior pituitary gland during perifusion is illustrated by the profiles in Figure 2. The pars tuberalis was capable of secreting LH basally and in response to LHRH stimulation. The general pattern of LH secretion by the pars tuberalis was similar to that observed for the anterior pituitary. Initially, an increased release of LH was observed from both the pars tuberalis and pituitary, followed by a relatively stable basal rate of secretion. Areas under the curves for LH release representing basal and LHRH-induced secretion for tissues are shown in Table 1. Both the pars tuberalis and anterior pituitary responded (P < .05) to LHRH with increases of 43.9% and 47.0% above basal secretion, respectively. Extracts of both the pars tuberalis and anterior pituitary resolved into nine LH isoforms (Figure 3) following chromatofocusing over pH 10.5-7.0 gradients. The isoforms were coded with letters beginning with the most basic form. The mean percentage of intracellular LH in each isoform is shown in Table 2. No differences (P > .05) were observed in the distribution of LH among the various isoforms between the pars tuberalis and the anterior pituitary gland. DISCUSSION The study reported herein indicates that the bovine pars tuberalis contains gonadotropes capable of synthesizing and secreting LH. The pattern of secretion during in vitro perifusion was similar to that of the anterior pituitary gland. Both basal secretion and the response to LHRH stimulation were similar between the two tissues. These cells also had a similar distribution of LH isoforms. In some mammals, the pars tuberalis has been estimated to make-up 1-3% of the total adenohypophysial volume, while the ovine pars tuberalis has been estimated to comprise 1012% of the total tissue (2). Because we did not attempt to dissect the pars tuberalis from the remaining portion of the stalk median eminence, an estimate of the size or volume of this structure was not attained. Nonetheless, the content of LH (6.6 +_ 1.5 lag) is believed to be a reasonable estimate of the amount of LH within the bovine pars tuberalis. While the concen-
LH SECRETION AND LH ISOFORMS OF THE BOVINE PARS TUBERALIS
183
tration of LH in the bovine pars tuberalis was less than that for the anterior pituitary, it is possible that a greater concentration of LH would have been found in the pars tuberalis if it had been dissected away from the stalk median eminence. The ovine pars tuberalis was previ-
901
. Pots Tuberahs
] I
Q.
E lOOO. Anterior Pituitary
0
O~ V
-I- 500 -J .t3
LHRH 0
5
10 15 20 25 ~ Per|fusion Sample Number
35
Fig. 2. Perifusion profiles for bovine LH secretion (means + s.e.) by the pars tuberalis (upper panel) and anterior pituitary gland (lower panel). Tissues were perifused at 0.1 ml/min and 1.0 ml (10 min) fractions were collected. At 130 rain, tissues were challenged with 5 x 10-8 M LHRH for 30 min. Immunoactive LH was quantified by radioimmunoassay.
ously reported (2) to contain similar concentrations of LH on a per mg of tissue basis to that of the anterior pituitary when pars tuberalis tissue was separated from the median eminence. The hypothalamus of the rat (5,19) has been reported to possibly contain LH. However, these studies (5,19) included the pars tuberalis along with the hypothalamus. The pattern of LH secretion from the bovine pars tuberalis provides direct evidence that this structure is capable of basal and LHRH-stimulated release of LH. Similar patterns of LH
184
ZALESKY AND GROTJAN
TABLE l. AREAS UNDERTHE CURVEFOR BASALANDLHRH-INDUCEDLH RELEASE FROMTHE BOVINEPARSTUBERALISANDANTERIORPITUITARY'. Areas Under the Curve (ng• ml ' • 50 min ~)b Pars Tuberalis
Anterior Pituitary
Basal Release
1,158 + 267
10,727 + 2,038
LHRH-induced Release
1,753 _+301
21,676 _+4,201
43.9
47.0
%increase
"Values represent means _+s.e. (n=7). areas for basal secrtion were determined from the 5 samples prior to LHRH treatment and areas for LHRH-induced secretion from the 5 samples during and after LHRH treatment. bValues of areas under the curve for LHRH-induced LH secretion are significantly greater than corresponding basal secretion (P < .05).
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t 10
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t
50
60
70
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Froction Number
Fig. 3. Representative profiles for the distribution of bovine LH isoforms in the pars tuberalis and anterior pituitary gland of a castrate male. Tissue extracts were desalted and chromatofocused on p H l 0.5-7.0 gradients. Chromatofocusing fractions (1.5 ml) were collected (n=70 or 80) until a stable, lower limiting pH was attained. Proteins bound to the column at the lower limiting pH were eluted with 1.0 M NaC1 and collected as an additional twenty 1.5 ml fractions. Immunoreactive LH in each fraction was quantified by radioimmunoassay. Isoforms are coded with letters (A, B, C, D, E, E G, H, Z) beginning with the most basic isoform.
LH SECRETION AND LH ISOFORMS OF THE BOVINE PARS TUBERALIS
185
secretion were observed for the anterior pituitary and pars tuberalis. Following an initial increased rate of LH release, both tissues reached a steady state release and then responded in an analagous manner to LHRH. Secretion of LH above baseline was not different between the two tissues (43.9% vs 47.0%). Our results for the in vitro secretion of LH from the bovine
TABLE2. DISTRIBUTIONOF BOVtNELH ISOFORMSIN PARSTUBERALISANDANTERIORPITUITARYTISSUE ANDELUTIONPH OF EACHISOFORM. Tissuea Iso~
A B C D E F G H Z
b
(elufionpH)
Pars~bo~is
An~fior~mitary
(>10.0±0.2) (9.8±0.1) (9.6±0.1) (9.4±0.~) (9.2±0.02) (9.1±0.02) (8.9±0.02) (7.0) (
