Stimulation of prostagladin F synthesis by luteinizing hormone in rabbit ovarian follicles grown in organ culture

Stimulation of prostagladin F synthesis by luteinizing hormone in rabbit ovarian follicles grown in organ culture

Pergamon Press Life Sciences Vol . 15, pp . 1731-1738 Printed in the U.S .A . STIMULATION OF PROSTAGLANDIN F SYNTHESIS BY LUTEINIZING HORMONE IN RAB...

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Pergamon Press

Life Sciences Vol . 15, pp . 1731-1738 Printed in the U.S .A .

STIMULATION OF PROSTAGLANDIN F SYNTHESIS BY LUTEINIZING HORMONE IN RABBIT OVARIAN FOLLICLES GROWN IN ORGAN CULTURE Young S . Moon, Jiri Zamecnik and David T . Armstrong l Department of Physiology and Department of Obstetrics and Gynaecology University of Western Ontario, London, Ontario, Canada (Received in final form 15 October 1974)

Summary Pre-ovulatioy follicles of rabbits were cultivated in organ culture dishes for 3 days in media with or without luteinfzing hormone (LH) . Prostaglandin F (PGF) levels were measured in the media harvested after various incubation periods . PGF levels found in media were very low (between 0 .55 t 0 .19 ng and 0 .91 t 0 .15 ng per follicle) throughout incubation periods in the absence of LH . In the LH-treated group, PGF levels were significantly higher than those in corresponding controls, at every time interval . Within the treated group, PGF concentration rose sharply, reaching maximal levels between 12 and 36 hours (11 .25 t 3 .08 ng and 11 .33 t 5 .04 ng per follicle), then fell but still remained above basal level (1 .63 t 0 .55 ng per follicle) by 72 hours . Prostaglandins have recently been implicated as playing a role in ovulation (1,2,3) .

An inhibitor of prostaglandin bio-

synthesis, and antiserum prepared against prostaglandin F2a have been found effective in blocking ovulation when administered to LH-treated rabbits via intrafollicular injection, suggesting that the follicle is the site of involvement of proataglandins (1,2,3) . In addition, elevated levels of prostaglandins F (PGF) in rabbit Associate of the Medical Research Council of Canada .

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Stimulation of PGF Synthesis by LH

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follicles have been observed following coitus or LH injection, and this elevation could be prevented by systemic (4,5) or intrafollicular injection of indomethacin, suggesting that the follicle was the site of increased prostaglandin synthesis in response to LH .

However, the alternative possibility that

prostaglandina of extrafollicular origin were merely concentrated in follicular fluid in response to LH could not be excluded .

The

present studies were undertaken to determine if a direct action of LH in increasing production of PGF by isolated follicles could be demonstrated . Materials and Methods Estrous New Zealand white rabbits weighing about 4 kg were used in this study.

Animals were killed by an overdose of sodium

pentobarbital aministered by i .v . injection .

The ovaries were

immediately removed aseptically and transferred to Hanks' balanced salt solution, where pre-ovulatory follicles were gently teased apart under the dissecting microscope . The organ culture methods used were similar to those for other tissues described by Moon and Hardy (6) .

Each Falcon

plastic organ culture dish containing 1-2 follicle (s) was exposed to 58 C0 2 in air and maintained at 36 °C .

The medium used was

BGJb (Grand Island Biological Co .) or BGJb with 108 horse serum (Grand Island Biological Co .) .

Media were changed every 12 hours

and follicles were grown up to 72 hours in every experiment . Luteinizinq hormone (NIB-LH-B7) was dissolved in 0 .98 NaCl (saline) and serial dilutions made using medium to obtain the desired concentration (1 ug/ml) .

Control follicles were grown

without LH, in media with the same concentration of saline . All media and follicles from cultures were stored at -15°C until analysis .

Samples were extracted and prostaglandina of the

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Stimulation of PGF 5yntha~ia by LH

F series were measured by radioimmunoassay using methods described by Orcayk and Hehrman (7) .

Media and tissues were

extracted with the mixture of ethyl acetate :isopropanol :l N HC1 (3 :3 :1, v :v :v) .

After addition of saline and another volume of

ethyl acetate, the organic layer was evaporated to dryness in a stream of N2 and the mixture was applied on a silicic acid column .

Columns were eluted using benzene :ethyl acetate :methanol .

[5,6~H]Prostaglandin F2a (PGF 2a ) was used to allow correction for losses during extractions and chromatography . taining PGF

The fraction con-

was assayed by radioimmunoassay, using prosta-

glandin F antiserum obtained from H . R . Behrman .

Prosta-

(3000 dpm/tube) glandin F2a was used as a standard and (9, eH]PGF2 was used as tracer .

All values were corrected for losses and

expressed as nq of PGF per 1 ml of media or per follicle . Some of the follicles were fixed in Bouin's fixative for histological studies, embedded in paraffin, sectioned at 6

u

and

stained with Mayer's hematoxylin and eosin . Analysis of variance was performed on logarithmicallytranaformed data, because of significant heterogeneity of variance of the untransformed data . Results Pre-ovulatory follicles grown in organ culture dishes with a chemically defined medium (BGJb) or a chemically defined medium with 108 horse serum appeared histologically healthy at the end of 72 hours of cultivation .

The follicles cultured in the

presence of LH contained more granulosa cells which were well luteinized, than those cultured in its absence (Figs . 1 and 2) . Prostaglandin F levels measured in the media harvested after various incubation periods are shown in Table 1 .

The presence of

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FIG . 1 Control follicle after 72 hours of culture . luteinization of follicular wall . (x 200)

Note slight

FIG . 2 LH-treated follicle after 72 hours of culture . Note the heavily-luteinized follicular wall compared with that shown in Fig . 1 . (x 200)

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Stimulation of PGF Syntheois by LH

horse serum in media had no significant effect on PGF production ; therefore, the results of incubation in both types of media were combined for statistical analysis .

Analysis of variance indicated

highly significant differences in PGF production due to LH (P < 0 .001) and to time of incubation (P < 0 .001) .

Prosta-

glandin F levels found in media were very low (between 0 .55 t 0 .19 nq and 0 .92 t 0 .15 ng per follicle) throughout incubation periods when follicles had been cultured in the absence of LH . In the LH-treated group, PGF levels were significantly higher than those in corresponding controls, at every time interval . Within the treated group, PGF concentration rose sharply, reaching maximal levels between 12 and 36 hours (11 .25 t 3 .08 ng and 11 .33 t 5 .04 ng per follicle), then fell but still remained significantly (P < 0 .02) above basal level (1 .63 t 0 .55 ng per follicle) by 72 hours . Since PGF levels in unstimulated follicles of estrous rabbits, after dissection but without incubation, are less than 0 .02 ng per follicle (Armstrong et al ., 1974), it is clear that the total PGF present at the end of incubation (in tissue plus media) represents almost entirely PGF which was synthesized during the incubations .

Because of the possibility that a portion

of the PGF may have been metabolized to other compounds during incubation, the sum of the PGF in the six changes of media plus the increment in tissue content during incubation must be considered as a conservative estimate of total PGF synthesis . Discussion The results of the present studies provide conclusive evidence that rabbit ovarian follicles respond to LH with increased net synthesis of proataglandins of the F series .

Extrapolation

of these in vitro findings to conditions in vivo lead to the

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TABLE 1 Effects of LH on in Vitro Production of Prostaglandins F by Isolated Rabbit Ovarian Follicles Hours of Incubation

PGF in Medium (ng t S .E . per foll icle Control (10 follicles)

LH (18 follicles)

0 - 12

0 .55 t 0 .19

1 .34 t 0 .40

12 - 24

0 .75 t 0 .08

11 .25 t 3 .08

24 - 36

0 .92 t 0 .15

11 .33 t 5 .04

36 - 48

0 .83 t 0 .15

4 .55 t 1 .89

48 - 60

0 .71 t 0 .12

2 .47 t 0 .93

60 - 72

0 .70 t 0 .10

1 .63 t 0 .55

Tabular values are means of 5 culture dishes per treatment . Analysis of variance of logarithmically-transformed data indicated significant differences due to treatment (P < 0 .001), to time of incubation (P < 0 .001) and to interaction between treatment and time (P < 0 .05) . conclusion that the pre-ovulatioy elevation of PGF in follicular

fluid is likely the result of increased synthesis by the follicle rather than of increased accumulation of proataglandina of extrafollicular origin . Despite the marked increase in PGF production in response to LH stimulation in culture, none of the cultured follicles was observed to ovulate . failures

Many reasons may be suggested fôr this

e .g ., lack of vasçular supply, with inherent fluctu-

ations in blood pressure which may contribute to pre-ovulatioy swelling and rupture (8) : lack of innervation, which may be necessary for maintenance of contractility of follicular smooth muscle fibres (9) ; lack of surrounding stroma, whose contractile activity may contribute to ovulation (10) . Other follicular functions in which prostaglandina have been suggested as participating, on the basis of in vitro studies, are oöcyte maturation (10,11) and luteinization of granulosa

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cells (12,13) .

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The present in vitro culture technique, employing

rabbit follicles, may prove useful in experiments designed to confirm these actions, and elucidate their mechanisms . Acknowledgeme nts We are grateful to Dr . H . R. Behrman of Merck Institute for Therapeutic Research, Rahway, N . J . for donating the prostaglandin antiserum, to Dr . John Pike of the IIpjohn Co ., Kalamazoo, Mich . for donating the prostaglandin F 2a , and to the Hormone Distribution Office, N .I .A .M .D .D ., Betherda, Md ., for donating the luteinizing hormone used in this study .

This research has

been supported by grants from the Medical Research Council (Canada) and the Ford Foundation . References 1.

D.T . Armstrong, Y .S . Moon and D .L . Grinwich . 709-715 (1972) .

Adv . Biosci . 8 :

2.

D.T . Armstrong, D .L . Grinwich, Y .S . Moon and J . Zamecnik . Life Sai . 14 : 129-140 (1974) .

3.

Y.S . Moon and D.T . Armstrong.

4.

W .J . LeMaire, N .S .T . Yang, H .R . Behrman and J .M . Marsh . ProstagZandine 3 : 367-376 (1973) .

5.

J. Zamecnik, D .L . Grinwich and D.T . Armstrong. Fed. Biol . So a . IB : 29 (1973) .

6.

Y.S . Moon and M.H . Hardy .

7.

G .P . Orczyk and H .R . Behrman .

8.

J .H . Burr, Jr . and J .I . Davier . (1951) .

9.

H.W . Burden .

Ped . Pro a . 33 : 435 (1974) .

Proo . Can .

Am . J . Anat . 13B: 253-267 (1973) . Prostaglandina 1 : 3-20 (1972) . Anat . Reo . 111 : 273-297

Am . J. Anat . 133 : 125-142 (1972) .

10 .

D .T . Armstrong, Y .S . Moon and J . Zamecnik . Conference on Chemistry, Biology and Immunology of Gonadotropins, Bangalore, India. Academic Preae (1974) (in press) .

11 .

H .R . Lindner, A. Tsafriri, M .E . Liberman, U . Zor, Y . Roch, S . Bauminger and A. Barnes . Re a. Prog . Borin. Res . 30 : 79-138 (1974) .

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Stimulation of PGF Synthesis by LH

Reprod . 5 : 87 (1971) .

12 .

C .P . Charming .

13 .

L.R . Ellsworth and D .T . Armstrong .

(1974) .

BioZ .

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Prostaglandine 7 : 165-174