PROSTAGLANDINS

EFFECTS OF SYSTEMIC AND INTRAFOLLICULAR INJECTIONS OF LH, PROSTAGLANDINS, AND INDOMETHACIN

ON THE LUTEINIZATION OF RABBIT GRAAFIAN FOLLICLES

Long T. Phi, Young S. Moon, and David T. Armstrong*

Departments of Physiology and Obstetrics & Gynaecology University of Western Ontario

London, Ontario, Canada

ABSTRACT

The possibility that initiation of luteinization in ovarian follicles by luteinizing hormone (LH) is mediated by prostaglandins (PG's) was investigated in rabbits. Estrous rabbits, given an ovula- tory dose of LH (50 ug) intravenously, were administered indomethacin

(IM), an inhibitor of PG biosynthesis, by various routes. Proges- terone levels in the serum and in the induced corpora lutea (CL) were subsequently measured by radioimmunoassay. Continued daily subcutane- ous injections of IM from 2 days before through 2 days after LH treat- ment reduced the corpus luteal level, measured at 72 hours post-LH, of PGF from 208 f 43 to 98 2 20 pg/CL (P < 0.025) and that of PGE from 272 + 31 to 115 f. 9 pg/CL (P < 0.005). At the same time, progesterone levels were 72 + 12 and 93 i: 10 ng/CL (P > 0.05) in the oil-treated and IM-treated rabbits, respectively. Serum progesterone continued to rise in a linear fashion during the period from 24 to 72 hours following LH treatment, whether IM was injected or not. Intrafollicu- lar treatment with LH (100 ng/follicle) raised the progesterone content in the treated follicles 72 hours later from 1.1 f 0.5 to 50.1 f 13.5 ng. (P < 0.01). This progesterone content reached 21.5 f 15.8 ng (P < 0.05) in follicles similarly treated with PGE2 (5 ng/follicle), but remained meagre at lower doses of PGE2 ( 100 ng/follicle and 2 ng/follicle). Serum progesterone increased from 0.5 + 0.1 to 1.2 + 0.1 ng/ml (P < 0.005) within 72 hours in rabbits treated intrafollicularly with LH, but remained unaltered in those similarly treated with PGE2 (P > 0.1). Intrafollicular injections with PGF2, failed to induce changes in either level of progesterone. It is concluded that prosta- glandins probably do not mediate the luteinizing action of LH in rabbit Graafian follicles, although some degree of luteinization can be induced by high levels of exogenous PGE2.

ACKNOWLEDGEMENTS

We thank Dr. H. R. Behrman, Yale Univ., New Haven, Conn., for generous donations of indomethacin and of antisera to progesterone and to prostaglandins; Dr. John Pike, The Upjohn Co., Kalamazoo, Mich., for supplying purified prostaglandins; and the Hormone Distribution Office, NIAMDD, Bethesda, Md., for providing the LH used in these studies. We also thank Dr. T. G. Kennedy for valuable statistical consultations. This research was supported by World Health Organization Grant i/74228.

* Associate of the Medical Research Council (Canada).

MARCH 1977 VOL. 13 NO. 3 543

PROSTAGLANDINS

INTRODUCTION

The extensive role of prostaglandins in reproductive physiology has been recently reviewed (1). There is good evidence that prostaglandins play a role in the ovulatory action of luteinizing hormone (LH) at the follicular level in rabbits and rats (Z-5). Whether these substances play a role in the luteinizing effect of LH has not been well estab- lished. Various in vitro studies have shown prostaglandin E2 to mimic the action of LH in stimulating the luteinization of incubated follicles from rats (6) or in stimulating in vitro progesterone synthesis by incubated follicles, granulosa cells or corpus luteal slices from mice (7), rhesus monkeys (8), cows (9). Other in vitro studies with rabbits and rats have shown prostaglandin F2u to inhibit (lO,ll), or fail to exhibit (12) the steroidogenic property of LH.

In viva studies (13-15) showed that in the LH-treated rabbit follicles, PGF level reached a peak just prior to ovulation which normally occurs about 10 hours following LH, while that of PGE was main- tained at high values until 16 hours after gonadotropin treatment, well past the expected ovulation time. This increased synthesis of prosta- glandins in response to the exogenous gonadotropin was confirmed by in vitro incubation of the rabbit follicle (16,18) as well as by measure- ments in the proestrous rats (17), suggesting that PGE might be present to mediate the luteinizing action of LH.

This study was carried out to investigate further the possibility that prostaglandins may mediate the action of LH in the luteinization of rabbit Graafian follicles, utilizing an intrafollicular injection technique for administration of hormones and inhibitors. Using this approach, it was possible to evaluate luteinization of individually- treated follicles quantitatively by measuring their progesterone content 72 hours after exposure to the luteinizing stimulus.

MATERIALS AND METHODS

Virgin female New Zealand white rabbits, weighing from 3.5 to 5 kg, were studied under four experiments.

In the first experiment, the ability of indomethacin (Merck), admin- istered on different regimens and by different routes, to affect lutein- ization induced by systemic injection of LH (50 pg NIH-LH-B8 intraven- ously (i.v.) in 1 ml of 0.9% NaCl solution (saline)) was investigated in 12 rabbits. Indomethacin regimens tested included: 'i.v. at 20 mg/kg body weight (BW) in phosphate buffer, pH 8, about 1 hour before or 1 hour after LH; intrafollicularly (i.f.) at 5 pg/follicle in phosphate buffer,

PH 8, immediately prior to LH injection; and subcutaneously (s.c.) at 20 mg/kg in sesame oil for 5 consecutive days beginning 48 hours preceding LH treatment. Control rabbits were injected with LH alone.

In Experiment 2, 8 rabbits were given indomethacin at 20 mgfkg BW daily in sesame oil or the oil alone S.C. for 5 consecutive days. On the third day of this treatment period, all rabbits also received an i.v. injection of 50 ug LH in saline, and an i.f. injection of either indomethacin at 5 ugffollicle or its phosphate buffer vehicle.

544 MARCH 1977 VOL. 13 NO. 3

PROSTAGLANDINS

In Experiment 3, 12 rabbits were given i.f. injections, in 1 ~1 of saline, with LH (100 ng/follicle), or PGE2 (Upjohn) (5 ug/follicle), or PGF

1" (5 ug/follicle). Controls received saline injections at 1 ul/fol-

lit e.

In Experiment 4, 6 rabbits were evenly divided into two groups to be injected intrafollicularly with PGE2 at either 100 ng/follicle or 2 ng/follicle in 1 ~1 of saline. These injections were given to follicles of both ovaries in any one animal.

Intrafollicular injections were performed using a flame drawn capillary tube mounted, through a short piece of polyethylene tubing, on a 25 ul Hamilton syringe. Rabbits were anaesthetized with Surital (sodium thiamylal, Parke-Davis), and the ovaries exposed through a mid- ventral incision. The tip of the micropipette, filled with the solution to be injected, was inserted' into the ovarian stroma some distance away from the mature follicle selected for treatment. Next, the follicular wall was penetrated and the micropipette tip directed into the antrum. This procedure helped avoid rupturing of the follicles and minimized leakage of follicular fluid and the injected solution. Graafian follicles protruding on the surface of the ovary were selected for injections, and their locations were mapped for later identification at autopsy.

In all experiments, rabbits were sacrificed with an overdose of sodium pentobarbital, at 72 hours following LH or PG or their vehicle treatment. Treated follicles were identified according to previous mapping and examined under a dissecting microscope for the presence or absence of rupture points. With the aid of fine pointed forceps, they were then isolated from the surrounding ovarian tissue and homogenized using a glass rod in absolute ethanol and kept at -2O'C until hormone levels were measured, using radioimmunoassays for progesterone (20) and prostaglandins (21,22). Prostaglandin assays were performed within 48 hours and progesterone within one week after the follicles had been isolated. Progesterone levels were also determined from peripheral serum samples collected via marginal ear vein prior to, and 24, 48, 72 hours following LH or PG treatment.

Data were analyzed by means of analyses of variance and Duncan's New Multiple-Range Test, for significant effects of treatments on serum and corpus luteal levels of progesterone as well as luteal content of prostaglandins. Logarithmic transformation of data was carried out where a heterogeneity of variance was indicated by Bartlett's test.

RESULTS AND DISCUSSION

Peripheral serum levels of progesterone in the four separate experiments are summarized in Tables I and II. As indicated in these tables by analyses of variance, intravenous or intrafollicular adminis- tration of LH significantly increased serum progesterone concentrations; levels increased linearly with time from 0 to 72 hours after LH admin- istration. The levels reached 72 hours after i.f. administration of LH (Table II) were not as high as those attained following i.v. injection of this hormone (Table I). Intrafollicular injection of PGE2 or PGF2u

MARCH 1977 VOL. 13 NO. 3 545

TABLE I

Effect of Indomethacin (IM) Administration on Peripheral Serum Levels of

Progesterone (ng/ml ?r SE) in Rabbits Administered Luteinizing Hormone (LH) Systemically

N

Treatment

-

Experiment 1:

3

LH i.:. (50 pg)

3

LH i.v. + IM i.v.

(20 m

g/kg

SW

>

4 LH i.v. + IM i.f.

(5 ug/follicle)

2

LH i.v. + IM S.C.

(20 mg/kg)

Experiment 2:

2

LH i.v. -t IM S.C.

+ IM i.f.

0 24

48

0.69 * 0.03

0.89 + 0.48

0.60 f 0.19

0.67 + 0.41

0.95 k 0.30

2.04 f 0.26

0.96 + 0.41

1.87 + 0.15

0.73 ?r.

0.26

1.23 + 0.42

0.85 f 0.43

2.05 + 0.86

1.26 f 0.11

0.55 f 0.26

0.78 f 0.16

0.73 + 0.12

1.71 f. 0.43

2.19 f. 0.64

%

2

LH i.v. -t IM S.C.

+ buffer i.f.

0.72 + 0.30

0.95 + 0.35

3

2

LH i.v. + oil S.C.

+ IM i.f.

;;

=:

2

LH i.v. + oil S.C.

+ buffer i.f.

1.23 + 0.21

1.15 f 0.23

0.77 + 0.21

1.54 + 0.11

N = number of rabbits

PI = probability of a linear increment with time within treatment.

Hours following LH Treatment

-.n

IL

2.84 + 0.43

3.84 + 0.61

2.31 ?r 0.84

3.18 f 1.53

3.00 + 0.29

1.75 ?r 0.04

1.81 f 0.09

2.05 f. 0.25

0.005

0.005

0.05

0.05

0.05

0.05

0.025

0.005

NS

NS

0.05

NS

NS

C

il Pi = probability of a significant difference from controls (C) at 72 hours.

P

NS = not significant

LH = NIH-LH-B8

W

TABLE II

Effects of Intrafollicular Injections of LH, Prostaglandin E2 (PGE2) and

Prostaglandin F2c (PGF2u) on Peripheral Serum Levels of Progesterone (ng/ml + SE) in Rabbits

Hours following Treatment

N

Treatment

0

24

48

-JQ

IL

-

d

Experiment 3:

! 3

saline

L

g

3

LH (1 pi/follicle)

0.69 f 0.06

0.49 + 0.01

0.50 + 0.08

W

(100 ng/follicle)

0.55 + 0.06

0.61 f 0.09

0.69 f 0.16

3

PGE2

(5 ng/follicle)

0.36 * 0.12

0.30 f 0.16

0.58 + 0.14

3

PGF2u

(5 ug/follicle)

0.44 ?r 0.06

0.43 f 0.09

0.34 f 0.15

Experiment 4:

3

PGE

(160 ng/follicle)

1.06 ?r 0.44

0.72 f 0.20

1.16 + 0.32

3

PGE2

(2 ng/follicle)

1.55 f. 0.43

0.60 f 0.14

0.98 + 0.20

N = number of rabbits

Pl = probability of a linear increment with time within treatment.

P = probability of a significant difference from controls (C) at 72 hours.

NS = not significant

NT = not tested

LH = NIH-LH-B8

0.81 ?r 0.15

1.21 + 0.11

0.58 f 0.21

0.56 ?r 0.11

1.69 f 0.01

1.34 + 0.14

NS

C

0.005

0.05

NS

NS

NS

NS

NS

NT

NS

NT

PROSTAGLANDINS

failed to elevate peripheral serum levels of progesterone significantly (Table II). Indomethacin, whether given i.v., i.f., S.C. or both S.C. and i.f., failed to prevent the LH-induced rise in serum progesterone (Table I). At the same time, these i.v. and S.C. injections of indo- methacin significantly reduced the levels of prostaglandins E and F in the induced corpora lutea, as evidenced in Table III. This inhibitory effect on PG syntheses of indomethacin at the given dose had also been demonstrated at 5, 9 (14), and 9 l/2 (5) hours following LH treatment.

Although elevating peripheral serum levels of progesterone is undoubtedly a valid index of luteinization, it was felt that this might not be a sufficiently sensitive parameter to detect possible partial luteinization, or luteinization of individual follicles which may have occurred, especially following i.f. injections of either LH or prosta- glandins. Therefore, progesterone contents of individual injected follicles (or corpora lutea which developed from them) were measured. The results of these measurements are presented in Table IV. Although exhibiting considerable variability, the follicular/luteal tissue levels of progesterone observed 72 hours after i.v. administration of LH were not significantly altered by administration of indomethacin, irrespect- ive of the route and dosage of administration of this inhibitor of prostaglandin synthesis. In fact, there was a tendency, although not statistically significant, for indomethacin actually to increase the levels of progesterone observed in these structures.

Intrafollicular injection of LH increased follicular/luteal tissue levels of progesterone to'about the same level as that observed follow- ing i.v. injection of LH. Statistically significant (P < 0.05), although smaller increases were observed in follicles injected with 5 pg of PGE2, as compared to those injected with saline or PGF . injections of PGE2 at lower doses (100 ng or 2 npper

However, similar follicle) failed

to induce any increase in the progesterone contents therein.

It has been previously observed that indomethacin, at the doses used in this study, did not alter the LH-induced pattern of either peripheral progesterone level or the morphology of luteinized granulosa cells in rabbits (2,4), but inhibited both PGE and PGF syntheses in the follicles of these animals (5,14). It has also been demonstrated that i.f. injections of LH could induce progesterone production comparable to that by the pseudopregnant rabbits, as measured in the plasma collected from ovarian veins (24). In vitro incubation of rat follicles with PGE2 has been shown to result in subsequent luteinization (appraised histo- logically) when these follicles were transplanted beneath the kidney capsule of hypophysectomized recipient rats (6). The present study was the first to evaluate the functional corpus luteum, newly formed in response to LH or to directly injected PG's, by determining the content of progesterone at the follicular level, in addition to peripheral serum measurements. The results suggest that measurement of luteal tissue levels of progesterone may provide a more convenient, sensitive and quantitative index of luteinization of individual follicles than is provided by histological examination or by measurement of peripheral or ovarian vein blood levels of progesterone.

548 MARCH 1977 VOL. 13 NO. 3

Effect of Indomethacin (IM)

Expt.

w

3

4

2

2

2

LH i.v. + IM S.C. + IM i.f.

2

LH i.v. + IM S.C. + buffer i.f.

2

LH i.v. + oil S.C. + IM i.f.

2

LH i.v.

+ oil S.C. + buffer i.f.

at 72

TABLE III

Administration on FollicularfLuteal Content of Prostaglandins (pg * SE)

Hours following Luteinizing Hormone (LH) Treatment

Treatment

PGE

P

PGF

LH i.v.

LH i.v. + IM i.v.

LH i.v. + IM i.f.

LH i.v. + IM s.c.

ND

106.96 ? 24.74

C

ND

51.78 + 23.19

0.01

ND

131.88 + 36.98

NS

ND

62.86 + 13.66

0.01

73.2 +

6.06

0.005

99.5 + 31.36

0.025

115.31 f

9.59

0.005

97.81 f 20.37

0.025

240.75 + 41.74

NS

236.77 + 64.38

NS

272.38 + 31.43

C

207.71 + 42.62

C

N = number of rabbits

P = probability of a significant difference from controls (C) in each experiment.

NS = not significant

ND = not determined

Treatments as indicated on Table I.

P

TABLE IV

Follicular/Luteal Tissue Levels of Progesterone (ng k SE)

at 72 Hours following Treatment with Luteinizing Hormone (LH) or Prostaglandins

Expt.

Progesterone

No.

N

Treatment

-

(ng)

P

1

3

LH i.v.

58.39 * 8.34

C

3

LH i.v. + IM i.v.

02.57 +

2.43

NS

4

LH i.v. + IM i.f.

73.38 f 4.12

NS

2

LH i.v. + I&l S.C.

102.62 f 22.72

NS

2

2

LH i.v. + IM S.C. + IM i.f.

72.44 f 12.7

NS

2

LH i.v. + IM S.C. + buffer i.f.

92.99 t

9.8

NS

2

LH i.v. + oil S.C. + IM i.f.

80.28 f 10.8

NS

2

LH i.v. + oil S.C. + buffer i.f.

72.45 _+ 12.5

C

3

3

saline i.f.

1.07 f 0.49

C

3

LH i.f.

50.14 k 13.47

0.01

3

PGE2 i.f.

21.48 + 15.84

0.05

3

PGF2,

2.42 _+ 0.49

NS

4

3

PCE2 i.f. (100 ng/follicle)

0.96 f

0.30

NT

3

PGE2 i.f. (2 nglfollicle)

0.81 +

0.08

NT

P = probability of a significant difference from controls (C) within each experiment.

N = number of rabbits

NS = not significant

NT = not tested

Treatments are the same as explained in Tables I and II.

PROSTAGLANDINS

The results presented here provide further support for the previous findings that blockade of prostaglandin synthesis did not eliminate the luteinizing and steroidogenic effects of exogenous LH, injected systemically or locally at the follicle. Five micrograms (5 ug) of PGE2, when directly injected into the follicle, apparently induced some degree of luteinization, as quantified by follicular progesterone measurements. However, this dosage was considerably in excess of levels which have been observed to occur in rabbit follicles following systemic injection of an ovulatory dose of gonadotropin (14,15). Furthermore, intra- follicular injection of 100 ng or 2 ng of PGE2, which more closely approximated the physiological range, did not induce any luteinization, as evidenced by its failure to raise the progesterone levels in the treated follicles. From these results, and those in which inhibition of prostaglandin synthesis failed to prevent luteinization, we conclude that prostaglandins do not act as obligatory mediators of luteinizing hormone in the luteinization of rabbit Graafian follicles.

REFERENCES

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23. LeMaire, W.J., T. Mills, Y. Ito, and J.M. Marsh. Inhibition by 3'-5'-cyclic AMP of luteinization induced by intrafollicular in- jection of luteinizing hormone. Biol. Reprod. 6: 109-116, 1972.

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Received November 9th, 1976 - Accepted January 7th, 1977

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