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Effects of luteinizing hormone on steroidogenesis by thecal tissue from human ovarian follicles in vitro
53
2617 EFFECTS OF LUTEINIZING HORMONE ON STEROIDOGENESIS BY THECAL TISSUE FROM HUMAN OVARIAN FOLLICLES IN VITRO Kenneth P. McNatty*, Anastasia Makris, Rapin Osathanondh and Kenneth J. Ryan From the Laboratory of Human Reproduction and Reproductive Biology Harvard Medical School 45 Shattuck Street Boston, Massachusetts 02115 * Present address Wallaceville Animal Research Centre, Private Bag, Upper Hutt, New Zealand Received
2-6-80 ABSTRACT
The steroidogenic responsiveness of human thecal tissue to different doses of LH was investigated in vitro in relation to the health of the follicle and to the responsiveness of stromal tissue, The results show that small incremental increases in LH, over a low range of concentrations (1 to 10 ng/ml), markedly increased the thecal output of androstenedione from healthy and/or atretic follicles. Theta from healthy follicles were also stimulated to increase their output of progesterone and estradiol in response to small increases in LH whereas theta from atretic follicles produced more variable amounts of progesterone and were unable to generate estradiol. In contrast, relatively high concentrations of LH (50 ng/ml) reduced the total steroid output from the theta of both healthy and atretic follicles while 'switching on' a low level of steroidogenesis in stromal tissue. These data suggest that the steroidogenic response of thecal tissue is related to the mass of tissue (i.e., the size of the follicle), the health of the follicle and the amount of LH to which it is exposed. INTRODUCTION Previous studies on human ovaries have shown that thecal tissue produces mainly androstenedione
(androst-4-ene-3,17-dione, A,+) although it
is also capable of producing some testosterone (17S-hydroxy-4-androsten3-one) (T) and estrogen
(estradiol, Eg, and estrone, El) (1,2,3).
From
studies in sheep, it has been deduced that the pattern of secretion of follicular A4 and E2 is regulated by luteinizing hormone (LH):
basal
levels of LH maintain a steady secretion-rate; small pulses stimulate an increased secretion rate; while large pulses, such as the preovulatory LH
Vohme 36, Number 1
S
TDROXDd
July, 1980
surge, inhibit secretion (4,5,6).
Thus it is possible that the
secretory status of thecal tissue is not only dependent on the health of the follicle (2) but also on the amount of I.2 to which it is exposed as well as the nature of the LH stimulus (i.e., pulsatile vs constant level. In the present study, thecal.tissue from different-sizedhealthy
and atretic follicles and stromaf tissues were recovered from human ovaries and incubated in a medium with differing amounts of LH. concentrations of progesterone
The
(4-pregnene-3,20-dione, P), A,+, T,
dihydrotestosterone (17&hydroxy-5o-andtostan-3-one,DKTiT>, El and E2 in the medium were determined at both the start and end of 15 h of incubation.
From the amounts of steroids produced, it was hoped that
information could be obtained on the effects of different constant levels of LH on theta and stromal steroidogenesis. MATERIALS AND METHODS Source of ovarian tissue: Eleven women {aged 26-51 years) who were at varying stages of the menstrual cycle were undergoing hysterectomy for a variety of gynecological disorders. The indications for surgery were: abnormal uterine bleeding (AUB), 5 women, or intractable pelvic pain (IPP), 6 women. The menstrual cycles before surgery varied from 21 to 48 days in length and 5 (45.5%) of the women had ovulated in the cycle under study, as indicated by the presence of a secretory endometrium, and of at least one corpus luteum at the time of operation. The remaining women were assessed to be in the proliferative phase of the menstrual cycle, by their endometrial histology, the absence of a corpus luteum, and also in some cases, by the date of the last menstrual period. Assessment of follicles as healthy or atretic: Follicles from normal ovaries were classified as healthy if they contained more than 50% of the number of granulosa cells present in a fully developed follicle at Follicles with 250% of their complement each follicle diameter reached. of cells per follicle diameter have been suggested to have the potential Follicles were classified as atretic if for further development (9). they contained less than 50% of the granulosa cells present in a fully These follicles developed follicle at each follicle diameter reached. have been suggested to be undergoing irreversible degeneration (9). Collection of follicular and stromal tissues: Follicular and stromal tissues were obtained from the whole ovaries or wedge biopsies of the
Ovarian tissue was callected into eS.evensubjects described above, chilled Medium 199 containing Hank's salts with HEPES (N-2-hydroxyethyl
S
TEIROXDrn
pipirazine-N1-ethanesulfonic acid) buffer 20 mM/I1 (Grand Island Biological Company). Nineteen follicles ranging in diameter from 3 to 12 mm, and stromal tissue (from 5 ovaries) were dissected from the ovaries within 3 hours of the operation. The method of recovering the thecal tissue from the isolated follicles is described elsewhere (2). The thecal tissue used in this study is considered to be follicular tissue separated from most of its granulosa cells but containing the entire thecal layer as well as some stromal elements (2). In contrast, the stromal tissue used in this study is considered to be that ovarian tissue which was devoid of any follicles (~0.5 mm diameter), corpora lutea, corpora albicantia and cortex tissue. In Vitro incu3ations: Theta from individual follicles or stroma were minced into fine pieces with scissors and subdivided into six equal Each fraction was transferred into a petri dish fractions (5 mg). (35 x 10 mm) of known weight, then the dish was reweighed so that the One ml of wet weight of each tissue fraction could be determined. incubation medium with or without human luteinizing hormone (LH) (LER 960: at a concentration of either 1, 5, 10 or 50 ng/ml was added to The incubation medium for all studies consisted of each petri dish. 20% fetal calf serum with 80% Medium 199, Hank's salts and HEPES buffer, supplemented with penicillin, fungizone and streptomycin (Antibiotic-Antimycotic mixture, Grand Island Biological Company, New The incubation medium contained an endogenous 'FSH- and LH-like' York). activity since the progesterone response of granulosa cells in the medium was equivalent to that present in cells cultured in a human serum with 2 mIU of LH and FSH activity (9). The concentrations of free P, AI+, T, The DHT, El and E2 in the medium with 20% serum were all ~250 pg/ml. medium was further tested for the presence of other endogenous free steroids or conjugate which might be metabolized by the various cellThe medium containing 20% fetal calf serum was types to estrogen. subjected to enzymatic hydrolysis with glusulase (Endo Laboratories Inc., Garden City, New York) to liberate free steroids from possible sulfate and glucuronide conjugates. After hydrolysis the concentrations of P, AI+, DHT and E2 in 1 ml of the above medium were still undetectable, i.e., co.25 ngfml, but 0.4 ng of T and 1.6 ng of El were found. The total amount of substrate for estrogen production was determined by incubating 1 ml of the culture medium with a human placental microsomal fraction This microsomal preparation was capable of metabolizing 92% of (10). dehydroepiandrosterone (DHEA, 500 ng) and >82% of DHEA sulfate (500 ng) to estrogen (El plus Ep) under the same incubation conditions. After the incubation experiments, 3.75 ng of El and ~0.25 ng of E2 were Thus although the culture media contained undetectable generated. levels of free El and E2, it was possible for about 4 ng of estrogen (El + E2) to be generated during the culture period providing the stromal and/or thecal tissues aromatized the androgen substrate(s) Incubation of LH (identity unknown) and hydrolysed the El conjugates. (at 10 or 50 ng/ml) with the culture medium for 15 h without ovarian Compared tissue did not result in the liberation of any free steroid. to human serum or human follicular fluid, the culture medium with 20% fetal calf serum contained relatively little substrate for estrogen It was not determined if the various ovarian tissues formation (2). actually utilised the endogenous substrates in the culture medium.
S
TDEOXDI
Every thecal wall or stroma was incubated in quadruplicate and two further tissue preparations for each experiment were frozen to -20 OC at the start of the experiment: these latter preparations provided information on steroid levels in the medium and tissues at time zero. The incubations took place at 37 'C, overnight, and were stopped the next day after 15 h. The media were then transferred to test tubes and stored frozen at -20 'C until assayed for steroids. In order to compare results from different experiments, the mean amount of steroid accumulated in the medium was expressed as that amount produced per 5 mg tissue after 15 h minus the mean amount at the start of the experiment. Assays of steroid hormones in culture medium: The P, AL+, T, DHT, EI and E2 in the culture medium were extracted twice with ether (4/l, v/v), separated from one another by celite chromatography (2,8) and quantitated by radioimmunoassay (RIA) techniques (2,8). P was assayed using an antiserum (S-25712) supplied by Dr G. E. Abraham (Harbor General Hospital, Torrance, California). Crossreactions of other steroids in the assay were deoxycorticosterone 35%, 2Oa-dihydroprogesterone (20a-hydroxypregn-4-en-3-one) 7%, T 5%, AI+ 5%, The minimum detectable level of P in DHT 0.5%, El and E2 both ~0.1%. the incubation medium was 100 pgiml. AI+was assayed using an antibody (No.5) raised in our laboratory. Cross-reactions of other steroids were T 3%, DHT 0.05%, P, El and Eg all The minimum detectable level of AI+ in the incubation medium
Although the thecal and
S
%?XBEOIDI
57
10-l
Fig.1. Amount t 1 S.E.M. of P, A4, T, DHT, El and E2 produced in 15 h by stromal tissue (5 mg) (black histograms), thecal tissue (5 mg> from a 5 xan healthy follicle (white histograms) and thecal tissue (5 mg> from an 11 mm follicular cyst (speckled histograms) after exposure to different doses of human LH in vitro. All tissues were recovered from the same ovary. No experiments were performed with thecal tissue from the follicular cyst in the presence of 1 ng LB. Patient was 50 years old and had a proliferative phase endometrium. Reason for surgery was abnormal uterine bleeding.
S
TDROXDI
stromal tissues were exposed to an identical endocrine environment in
vitro,
the steroid-production profiles for each of the tissues were
different from one another at every dose of LH tested.
Irrespective of
the concentration of LH, the thecal tissues from the healthy and atretic follicles were active in producing steroids, whereas only trace amounts of steroid accumulated in the presence of stromal tissue.
The theta from
the healthy and atretic follicles were steroidogenically most responsive to LH when the LH concentration was 10 mg/ml.
LH concentrations of 50
ng/ml resulted in steroid accumulations that were comparable to the unstimulated controls.
Irrespective of the concentration of LH, the
major steroid produced by the theta of the healthy and atretic follicles was hq.
In the case of theta from the healthy follicle, the second major
steroid produced was estradiol, but for the atretic follicle (i.e., the follicular cyst) it was progesterone. The differential effects of LH,
as well as the effect of different
doses of LH, on thecal and stromal steroidogenesis for all of the tissues tested are all summarized in Tables 1 and 2.
The distribution
of the data was found to be non-normal and skewed to the lower levels. The data in Tables 1 and 2 were therefore summarized showing the median values and the 95% confidence levels around the median. Statistical comparisons were made using the Mann-Whitney Test. Irrespective of the ovaries from which the theta were recovered, the patterns of steroidogenesis for theta from healthy and atretic follicles (see Table 1) were similar to those shown in Fig 1.
Theta
from healthy follicles were stimulated by LH to produce significantly more P and E2 than the controls (~~0.05, pcO.05 respectively) but only when the medium was supplemented with 10 ng of LH per milliliter.
Theta from healthy follicles also produced larger amounts
(1.::;0.9)
(1.23;?S.4)
(,.f::.,,
40.aef (23.0, 79.7)
( 2.:;634.0)
,,*:;;.5,
1)
f
:::A,
El
g
h
3.2gh (0.9,3.2)
,0.:;;.9,
(O.F;:.Z)
,o*~;'r.o,
7)
C
Control
Treatment
(o.:;56.1)
d
(2.::630.1)
cd
(o.92;23.6)
(1.;;l3.2)
+ LH 50 ng/ml
+ LH 10 ng/ml
Control
+ LH 50 ng/ml
+ LH 10 ng/ml
(o.:;93.7)
E2
,o.i?::.
(0.:;;.2)(O.",;:.,)
(0.
(0.:;:. (o.:;L)
(o.:::.,,
,1.2::.9,
( 4.3, 29.0)
11.6e
23.0 (11.5,143.9)
77.4 (33.8,220.3)
DHT
,o.:;L3)(o.~;:.,,
T
In. V’hO
All numbers sharing a common alphabetical letter are significantly different from one another: a,b,c,d,e,f = PcO.05; g,h = PcO.01 (Nann-Whitney U-Test)
Theca from atretic follicles (14)
(1.ij92.2)
b
(l.:;4g.9)
ab
24.9 ( 8.0,120.1)
(0.:;31.8)
Theca from healthy follicles (5)
a
A4
P
Amounts of steroid produced by thecal tissues from healthy and atretic follicles (ng/15 h; median and 95% confidence limits)
Tissue (n)
Table 1.
a
u
n
0
ti
60
S
TDEOXDI
h
5;
Ln
-0
Lno .a
00
.
.
. a-4 .a.
.
om
. r-4 ..a
of A4 and T than either the controls or the tissues which were stimulated with 50 q/ml significant.
LH, however, the differences were not statistically By contrast theca from atretic follicles were stimulated
by LH to produce significantly more A4 and DHT: the most effective dose of LH for A4 production was 10 ng/ml (~~0.05) and for DHT it was 50 ng/ml (pcO.01).
The stromal tissues were stimulated by LH to produce
significantly more P and A4 but only at the highest dose of LH tested, i.e., 50 ng/ml (~~0.05; Table 2). DISCUSSION The data show that the steroidogenic capacity of thecal tissue is dependent on the health of the follicles and also on the amount of LH to which it is exposed.
Small incremental increases in LH over a low range
of concentrations progressively enhanced the thecal output of A4 in atretic follicles and A4 and Eg in healthy follicles without influencing stromal steroidogenesis.
In contrast, relatively high concentrations of
LH appear to 'turn-off' thecal steroidogenesis except for DHT and/or El in atretic follicles (Table 1, Fig 1) while 'switching on' stromal steroidogenesis although at a relatively low level (Table 2).
The latter
finding is consistent with the observation that stromal tissues recovered from post-menopausal women or women during the late follicular phase were steroidogenically
more active than similar tissues recovered from other
phases of the menstrual cycle (2). The estrogen accumulation in culture medium containing healthy thecal tissue was enhanced with LH.
This finding suggests that a certain level
of aromatase activity exists in the theta from healthy follicles and moreover, that it is enhanced specifically by LH since FSH has previously been shown to be ineffectual (11).
It seems unlikely that the thecal
output of estrogen was due to granulosa-cell contamination:
it can be
calculated, from the amounts of oestrogen in culture (Tables 1 and 2 and Fig.1) and from the known steroidogenic capacity of granulosa cells (Z), that about 10 000 granulosa cells would need to be present on every milligram of thecal tissue to generate the amount of estrogen found, assuming the theta was devoid of aromatase activity.
Nevertheless
conclusive proof for thecal aromatase activity must await the isolation of a 'pure' line of thecal cells. Every vascularized follicle within an ovary is probably exposed to similar concentrations of gonadotropins since they are delivered to the thecal capillaries of every follicle via the arterial circulation (13). However, as no two antral follicles contain an identical hormonal milieu (14,15), it seems probable that the follicles respond to the gonadoRecent results (2) together
tropins individually and not identically.
with those of the present study suggest that the differential responsiveness of follicles is due in part to the differing functional status of the theca in the individual follicles.
In conclusion, the data show
that the steroidogenic capacity of thecal tissue is related to the mass of tissue present (i.e., the size of the follicle) (Z), the health of the follicle and the amount of LH to which the theta is exposed. Moreover, it is also likely to be influenced by the pufsatile nature of the LH stimulus (4,5,6,7>. ACKNOWLEDGEMENTS This research was supported by a U.S. Public Health Service Grant HDo7923. We gratefully acknowledge the gift of human LH (LER 960) from the National Pituitary Agency (NIAMD). Dr K. P. McNatty was a recipient of a Harkness Fellowship from the Commonwealth Fund of New York. REFERENCES 1. 2.
Channing, C.P.: J. Endocrinol. 45, 297 (1969). McNatty, K.P., Makris, A., DE Grazia, C., Osathanondh, R., and Ryan, K.J.: J. Clin. Endocrinol. Metab. 5, 687 (1979).
S 3.
4. 5. 6. 7. 8. 9. 10.
11. 12. 13.
14. 15.
TmROXDI
63
McNatty, K.P., Moore Smith, D., Makris, A., De Grazia, C., Tulchinsky, D., Osathanondh, R., Schiff, I. and Ryan, K.J.: J. Clin. Endocrinol. Metab. (in press). Moor, R.M.: J. Endocrinol. 61, 455 (1979). Seamark, R.F., Moor, R.M. and McIntosh, J.E.A.: J. Reprod. Fert. 5, 143 (1974). Baird, D.T., Swanston, I., and Scaramuzzi, R.J.: Endocrinology 98, 1490 (1976). Scaramuzzi, R.J., and Baird, D.T.: Endocrinology 101, 1801 (1977). McNatty, K.P., Moore Smith, D., Makris, A., Osathanondh, R. and Ryan, K.J.: J. Clin. Endocrinol. Metab.: 49, 851 (1979). McNatty, K.P. and Sawers, R.S.: J. Endocrinol. 66, 291 (1975). Ryan, K.J.: J. Biol. Chem. 234, 268 (1959). McNatty, K.P., Makris, A., Reinhold, V.N., De Grazia, C., Osathanondh, R., and Ryan, K.J.: Steroids 34, 429 (1979). Challis, J.R.G., Davies, I.J., Benirschke, K., Hendrickx, A.G. and Ryan, K.J.: Endocrinology 96, 333, 1975. Reynolds, S.R.M.: In: Handbook of Physiology, Ed. R. 0. Greep, American Physiological Society, Section 7, Vol. 2, Part 2, p.261 (1973). Edwards, R.G., Steptoe, P.C., Abraham, G.E., Walters, E., Purdy, J.M. and Fotherby, K.: Lancet ii, 611 (1972). McNatty, K.P.: In: ClinicTin Endocrinology and Metabolism Ed. G. T. Ross and M. B. Lipsett, Vo.7, Number 3, p.577 (1978).
2617 EFFECTS OF LUTEINIZING HORMONE ON STEROIDOGENESIS BY THECAL TISSUE FROM HUMAN OVARIAN FOLLICLES IN VITRO Kenneth P. McNatty*, Anastasia Makris, Rapin Osathanondh and Kenneth J. Ryan From the Laboratory of Human Reproduction and Reproductive Biology Harvard Medical School 45 Shattuck Street Boston, Massachusetts 02115 * Present address Wallaceville Animal Research Centre, Private Bag, Upper Hutt, New Zealand Received
2-6-80 ABSTRACT
The steroidogenic responsiveness of human thecal tissue to different doses of LH was investigated in vitro in relation to the health of the follicle and to the responsiveness of stromal tissue, The results show that small incremental increases in LH, over a low range of concentrations (1 to 10 ng/ml), markedly increased the thecal output of androstenedione from healthy and/or atretic follicles. Theta from healthy follicles were also stimulated to increase their output of progesterone and estradiol in response to small increases in LH whereas theta from atretic follicles produced more variable amounts of progesterone and were unable to generate estradiol. In contrast, relatively high concentrations of LH (50 ng/ml) reduced the total steroid output from the theta of both healthy and atretic follicles while 'switching on' a low level of steroidogenesis in stromal tissue. These data suggest that the steroidogenic response of thecal tissue is related to the mass of tissue (i.e., the size of the follicle), the health of the follicle and the amount of LH to which it is exposed. INTRODUCTION Previous studies on human ovaries have shown that thecal tissue produces mainly androstenedione
(androst-4-ene-3,17-dione, A,+) although it
is also capable of producing some testosterone (17S-hydroxy-4-androsten3-one) (T) and estrogen
(estradiol, Eg, and estrone, El) (1,2,3).
From
studies in sheep, it has been deduced that the pattern of secretion of follicular A4 and E2 is regulated by luteinizing hormone (LH):
basal
levels of LH maintain a steady secretion-rate; small pulses stimulate an increased secretion rate; while large pulses, such as the preovulatory LH
Vohme 36, Number 1
S
TDROXDd
July, 1980
surge, inhibit secretion (4,5,6).
Thus it is possible that the
secretory status of thecal tissue is not only dependent on the health of the follicle (2) but also on the amount of I.2 to which it is exposed as well as the nature of the LH stimulus (i.e., pulsatile vs constant level. In the present study, thecal.tissue from different-sizedhealthy
and atretic follicles and stromaf tissues were recovered from human ovaries and incubated in a medium with differing amounts of LH. concentrations of progesterone
The
(4-pregnene-3,20-dione, P), A,+, T,
dihydrotestosterone (17&hydroxy-5o-andtostan-3-one,DKTiT>, El and E2 in the medium were determined at both the start and end of 15 h of incubation.
From the amounts of steroids produced, it was hoped that
information could be obtained on the effects of different constant levels of LH on theta and stromal steroidogenesis. MATERIALS AND METHODS Source of ovarian tissue: Eleven women {aged 26-51 years) who were at varying stages of the menstrual cycle were undergoing hysterectomy for a variety of gynecological disorders. The indications for surgery were: abnormal uterine bleeding (AUB), 5 women, or intractable pelvic pain (IPP), 6 women. The menstrual cycles before surgery varied from 21 to 48 days in length and 5 (45.5%) of the women had ovulated in the cycle under study, as indicated by the presence of a secretory endometrium, and of at least one corpus luteum at the time of operation. The remaining women were assessed to be in the proliferative phase of the menstrual cycle, by their endometrial histology, the absence of a corpus luteum, and also in some cases, by the date of the last menstrual period. Assessment of follicles as healthy or atretic: Follicles from normal ovaries were classified as healthy if they contained more than 50% of the number of granulosa cells present in a fully developed follicle at Follicles with 250% of their complement each follicle diameter reached. of cells per follicle diameter have been suggested to have the potential Follicles were classified as atretic if for further development (9). they contained less than 50% of the granulosa cells present in a fully These follicles developed follicle at each follicle diameter reached. have been suggested to be undergoing irreversible degeneration (9). Collection of follicular and stromal tissues: Follicular and stromal tissues were obtained from the whole ovaries or wedge biopsies of the
Ovarian tissue was callected into eS.evensubjects described above, chilled Medium 199 containing Hank's salts with HEPES (N-2-hydroxyethyl
S
TEIROXDrn
pipirazine-N1-ethanesulfonic acid) buffer 20 mM/I1 (Grand Island Biological Company). Nineteen follicles ranging in diameter from 3 to 12 mm, and stromal tissue (from 5 ovaries) were dissected from the ovaries within 3 hours of the operation. The method of recovering the thecal tissue from the isolated follicles is described elsewhere (2). The thecal tissue used in this study is considered to be follicular tissue separated from most of its granulosa cells but containing the entire thecal layer as well as some stromal elements (2). In contrast, the stromal tissue used in this study is considered to be that ovarian tissue which was devoid of any follicles (~0.5 mm diameter), corpora lutea, corpora albicantia and cortex tissue. In Vitro incu3ations: Theta from individual follicles or stroma were minced into fine pieces with scissors and subdivided into six equal Each fraction was transferred into a petri dish fractions (5 mg). (35 x 10 mm) of known weight, then the dish was reweighed so that the One ml of wet weight of each tissue fraction could be determined. incubation medium with or without human luteinizing hormone (LH) (LER 960: at a concentration of either 1, 5, 10 or 50 ng/ml was added to The incubation medium for all studies consisted of each petri dish. 20% fetal calf serum with 80% Medium 199, Hank's salts and HEPES buffer, supplemented with penicillin, fungizone and streptomycin (Antibiotic-Antimycotic mixture, Grand Island Biological Company, New The incubation medium contained an endogenous 'FSH- and LH-like' York). activity since the progesterone response of granulosa cells in the medium was equivalent to that present in cells cultured in a human serum with 2 mIU of LH and FSH activity (9). The concentrations of free P, AI+, T, The DHT, El and E2 in the medium with 20% serum were all ~250 pg/ml. medium was further tested for the presence of other endogenous free steroids or conjugate which might be metabolized by the various cellThe medium containing 20% fetal calf serum was types to estrogen. subjected to enzymatic hydrolysis with glusulase (Endo Laboratories Inc., Garden City, New York) to liberate free steroids from possible sulfate and glucuronide conjugates. After hydrolysis the concentrations of P, AI+, DHT and E2 in 1 ml of the above medium were still undetectable, i.e., co.25 ngfml, but 0.4 ng of T and 1.6 ng of El were found. The total amount of substrate for estrogen production was determined by incubating 1 ml of the culture medium with a human placental microsomal fraction This microsomal preparation was capable of metabolizing 92% of (10). dehydroepiandrosterone (DHEA, 500 ng) and >82% of DHEA sulfate (500 ng) to estrogen (El plus Ep) under the same incubation conditions. After the incubation experiments, 3.75 ng of El and ~0.25 ng of E2 were Thus although the culture media contained undetectable generated. levels of free El and E2, it was possible for about 4 ng of estrogen (El + E2) to be generated during the culture period providing the stromal and/or thecal tissues aromatized the androgen substrate(s) Incubation of LH (identity unknown) and hydrolysed the El conjugates. (at 10 or 50 ng/ml) with the culture medium for 15 h without ovarian Compared tissue did not result in the liberation of any free steroid. to human serum or human follicular fluid, the culture medium with 20% fetal calf serum contained relatively little substrate for estrogen It was not determined if the various ovarian tissues formation (2). actually utilised the endogenous substrates in the culture medium.
S
TDEOXDI
Every thecal wall or stroma was incubated in quadruplicate and two further tissue preparations for each experiment were frozen to -20 OC at the start of the experiment: these latter preparations provided information on steroid levels in the medium and tissues at time zero. The incubations took place at 37 'C, overnight, and were stopped the next day after 15 h. The media were then transferred to test tubes and stored frozen at -20 'C until assayed for steroids. In order to compare results from different experiments, the mean amount of steroid accumulated in the medium was expressed as that amount produced per 5 mg tissue after 15 h minus the mean amount at the start of the experiment. Assays of steroid hormones in culture medium: The P, AL+, T, DHT, EI and E2 in the culture medium were extracted twice with ether (4/l, v/v), separated from one another by celite chromatography (2,8) and quantitated by radioimmunoassay (RIA) techniques (2,8). P was assayed using an antiserum (S-25712) supplied by Dr G. E. Abraham (Harbor General Hospital, Torrance, California). Crossreactions of other steroids in the assay were deoxycorticosterone 35%, 2Oa-dihydroprogesterone (20a-hydroxypregn-4-en-3-one) 7%, T 5%, AI+ 5%, The minimum detectable level of P in DHT 0.5%, El and E2 both ~0.1%. the incubation medium was 100 pgiml. AI+was assayed using an antibody (No.5) raised in our laboratory. Cross-reactions of other steroids were T 3%, DHT 0.05%, P, El and Eg all The minimum detectable level of AI+ in the incubation medium
Although the thecal and
S
%?XBEOIDI
57
10-l
Fig.1. Amount t 1 S.E.M. of P, A4, T, DHT, El and E2 produced in 15 h by stromal tissue (5 mg) (black histograms), thecal tissue (5 mg> from a 5 xan healthy follicle (white histograms) and thecal tissue (5 mg> from an 11 mm follicular cyst (speckled histograms) after exposure to different doses of human LH in vitro. All tissues were recovered from the same ovary. No experiments were performed with thecal tissue from the follicular cyst in the presence of 1 ng LB. Patient was 50 years old and had a proliferative phase endometrium. Reason for surgery was abnormal uterine bleeding.
S
TDROXDI
stromal tissues were exposed to an identical endocrine environment in
vitro,
the steroid-production profiles for each of the tissues were
different from one another at every dose of LH tested.
Irrespective of
the concentration of LH, the thecal tissues from the healthy and atretic follicles were active in producing steroids, whereas only trace amounts of steroid accumulated in the presence of stromal tissue.
The theta from
the healthy and atretic follicles were steroidogenically most responsive to LH when the LH concentration was 10 mg/ml.
LH concentrations of 50
ng/ml resulted in steroid accumulations that were comparable to the unstimulated controls.
Irrespective of the concentration of LH, the
major steroid produced by the theta of the healthy and atretic follicles was hq.
In the case of theta from the healthy follicle, the second major
steroid produced was estradiol, but for the atretic follicle (i.e., the follicular cyst) it was progesterone. The differential effects of LH,
as well as the effect of different
doses of LH, on thecal and stromal steroidogenesis for all of the tissues tested are all summarized in Tables 1 and 2.
The distribution
of the data was found to be non-normal and skewed to the lower levels. The data in Tables 1 and 2 were therefore summarized showing the median values and the 95% confidence levels around the median. Statistical comparisons were made using the Mann-Whitney Test. Irrespective of the ovaries from which the theta were recovered, the patterns of steroidogenesis for theta from healthy and atretic follicles (see Table 1) were similar to those shown in Fig 1.
Theta
from healthy follicles were stimulated by LH to produce significantly more P and E2 than the controls (~~0.05, pcO.05 respectively) but only when the medium was supplemented with 10 ng of LH per milliliter.
Theta from healthy follicles also produced larger amounts
(1.::;0.9)
(1.23;?S.4)
(,.f::.,,
40.aef (23.0, 79.7)
( 2.:;634.0)
,,*:;;.5,
1)
f
:::A,
El
g
h
3.2gh (0.9,3.2)
,0.:;;.9,
(O.F;:.Z)
,o*~;'r.o,
7)
C
Control
Treatment
(o.:;56.1)
d
(2.::630.1)
cd
(o.92;23.6)
(1.;;l3.2)
+ LH 50 ng/ml
+ LH 10 ng/ml
Control
+ LH 50 ng/ml
+ LH 10 ng/ml
(o.:;93.7)
E2
,o.i?::.
(0.:;;.2)(O.",;:.,)
(0.
(0.:;:. (o.:;L)
(o.:::.,,
,1.2::.9,
( 4.3, 29.0)
11.6e
23.0 (11.5,143.9)
77.4 (33.8,220.3)
DHT
,o.:;L3)(o.~;:.,,
T
In. V’hO
All numbers sharing a common alphabetical letter are significantly different from one another: a,b,c,d,e,f = PcO.05; g,h = PcO.01 (Nann-Whitney U-Test)
Theca from atretic follicles (14)
(1.ij92.2)
b
(l.:;4g.9)
ab
24.9 ( 8.0,120.1)
(0.:;31.8)
Theca from healthy follicles (5)
a
A4
P
Amounts of steroid produced by thecal tissues from healthy and atretic follicles (ng/15 h; median and 95% confidence limits)
Tissue (n)
Table 1.
a
u
n
0
ti
60
S
TDEOXDI
h
5;
Ln
-0
Lno .a
00
.
.
. a-4 .a.
.
om
. r-4 ..a
of A4 and T than either the controls or the tissues which were stimulated with 50 q/ml significant.
LH, however, the differences were not statistically By contrast theca from atretic follicles were stimulated
by LH to produce significantly more A4 and DHT: the most effective dose of LH for A4 production was 10 ng/ml (~~0.05) and for DHT it was 50 ng/ml (pcO.01).
The stromal tissues were stimulated by LH to produce
significantly more P and A4 but only at the highest dose of LH tested, i.e., 50 ng/ml (~~0.05; Table 2). DISCUSSION The data show that the steroidogenic capacity of thecal tissue is dependent on the health of the follicles and also on the amount of LH to which it is exposed.
Small incremental increases in LH over a low range
of concentrations progressively enhanced the thecal output of A4 in atretic follicles and A4 and Eg in healthy follicles without influencing stromal steroidogenesis.
In contrast, relatively high concentrations of
LH appear to 'turn-off' thecal steroidogenesis except for DHT and/or El in atretic follicles (Table 1, Fig 1) while 'switching on' stromal steroidogenesis although at a relatively low level (Table 2).
The latter
finding is consistent with the observation that stromal tissues recovered from post-menopausal women or women during the late follicular phase were steroidogenically
more active than similar tissues recovered from other
phases of the menstrual cycle (2). The estrogen accumulation in culture medium containing healthy thecal tissue was enhanced with LH.
This finding suggests that a certain level
of aromatase activity exists in the theta from healthy follicles and moreover, that it is enhanced specifically by LH since FSH has previously been shown to be ineffectual (11).
It seems unlikely that the thecal
output of estrogen was due to granulosa-cell contamination:
it can be
calculated, from the amounts of oestrogen in culture (Tables 1 and 2 and Fig.1) and from the known steroidogenic capacity of granulosa cells (Z), that about 10 000 granulosa cells would need to be present on every milligram of thecal tissue to generate the amount of estrogen found, assuming the theta was devoid of aromatase activity.
Nevertheless
conclusive proof for thecal aromatase activity must await the isolation of a 'pure' line of thecal cells. Every vascularized follicle within an ovary is probably exposed to similar concentrations of gonadotropins since they are delivered to the thecal capillaries of every follicle via the arterial circulation (13). However, as no two antral follicles contain an identical hormonal milieu (14,15), it seems probable that the follicles respond to the gonadoRecent results (2) together
tropins individually and not identically.
with those of the present study suggest that the differential responsiveness of follicles is due in part to the differing functional status of the theca in the individual follicles.
In conclusion, the data show
that the steroidogenic capacity of thecal tissue is related to the mass of tissue present (i.e., the size of the follicle) (Z), the health of the follicle and the amount of LH to which the theta is exposed. Moreover, it is also likely to be influenced by the pufsatile nature of the LH stimulus (4,5,6,7>. ACKNOWLEDGEMENTS This research was supported by a U.S. Public Health Service Grant HDo7923. We gratefully acknowledge the gift of human LH (LER 960) from the National Pituitary Agency (NIAMD). Dr K. P. McNatty was a recipient of a Harkness Fellowship from the Commonwealth Fund of New York. REFERENCES 1. 2.
Channing, C.P.: J. Endocrinol. 45, 297 (1969). McNatty, K.P., Makris, A., DE Grazia, C., Osathanondh, R., and Ryan, K.J.: J. Clin. Endocrinol. Metab. 5, 687 (1979).
S 3.
4. 5. 6. 7. 8. 9. 10.
11. 12. 13.
14. 15.
TmROXDI
63
McNatty, K.P., Moore Smith, D., Makris, A., De Grazia, C., Tulchinsky, D., Osathanondh, R., Schiff, I. and Ryan, K.J.: J. Clin. Endocrinol. Metab. (in press). Moor, R.M.: J. Endocrinol. 61, 455 (1979). Seamark, R.F., Moor, R.M. and McIntosh, J.E.A.: J. Reprod. Fert. 5, 143 (1974). Baird, D.T., Swanston, I., and Scaramuzzi, R.J.: Endocrinology 98, 1490 (1976). Scaramuzzi, R.J., and Baird, D.T.: Endocrinology 101, 1801 (1977). McNatty, K.P., Moore Smith, D., Makris, A., Osathanondh, R. and Ryan, K.J.: J. Clin. Endocrinol. Metab.: 49, 851 (1979). McNatty, K.P. and Sawers, R.S.: J. Endocrinol. 66, 291 (1975). Ryan, K.J.: J. Biol. Chem. 234, 268 (1959). McNatty, K.P., Makris, A., Reinhold, V.N., De Grazia, C., Osathanondh, R., and Ryan, K.J.: Steroids 34, 429 (1979). Challis, J.R.G., Davies, I.J., Benirschke, K., Hendrickx, A.G. and Ryan, K.J.: Endocrinology 96, 333, 1975. Reynolds, S.R.M.: In: Handbook of Physiology, Ed. R. 0. Greep, American Physiological Society, Section 7, Vol. 2, Part 2, p.261 (1973). Edwards, R.G., Steptoe, P.C., Abraham, G.E., Walters, E., Purdy, J.M. and Fotherby, K.: Lancet ii, 611 (1972). McNatty, K.P.: In: ClinicTin Endocrinology and Metabolism Ed. G. T. Ross and M. B. Lipsett, Vo.7, Number 3, p.577 (1978).