PROSTAGLANDINS

THE POSS IBlE MODE OF ACTION OF PROSTAGLANDINS: X. ANTAGONISM BETWEEN PROSTAGIANDIN F2a AND PROLACTIN OR HUMAN CHORIONIC GONADCIROPIN , A

COMPARATIVE STUDY *

Amar Chatterjee

Department of Physiology, Rajla Peary Mohan College Uttarpara, Hooghly, W.Bengal 712258, India.

ABSTRACT

A comparative assessment of the efficacy of prolactin or HCG in reversing the luteolytic property of a single dose (2.0 mg/kg) of PGF2a was conducted in pregnant (day 10) rats. It was found that prolactin maintained pregnancy in 90% of the test animals. HCG though found to maintain pregnancy in -la% of the PGF2a-treated pregnants, the fetal survival rate was, however, recorded to be 62% against the control value of 98%. Conwrsely, prolactin replacement maintained the fetal survival rate as high as 93.4%. Moreover, the growth of the fetuses, placentae and corpus luteum in the pregnants having prolactin in conjunction with PGF2a was also found to be greater compared to the animals which had a combined regimen of PGF2a and HCG, but identical to controls. On the other hand, similar combined regimen when applied to hysterectomized pregnant rats, it was observed that though the vaginal diestrus was main- tained by the prolactin or HC3 in the presence of PGF2a, the prolactin regimen was found to be superb compared to HOG in the maintenance of luteal weight and functional activity. It was concluded that the antifertility effect of PGF2a in the rat is primarily the consequence of luteolysis and prolactin seems to be a much more appropriate hormonal replacement compared to HC3, a long-acting LH, in antagonizing the luteolytic property of ffiF2a.

* The expenses of this investigation were defrayed by Research Grants from the Pooulation Council, New York (M.74.51/M.76.18), Indian Council of Medical Research, World Health Orqanisation (Human Reproduction Unit, Small Supplies Programme), and Ford Foundation (ICMR).

OCTOBER 1976 VOL. 12 NO. 4 525

PROSTAGLANDINS

INTRODUCT ION

Since the first report by Pharris et al. (1) that prostaglandin modifies corpus luteum function , a large number of publications have appeared demonstrating the luteolytic efficacy of prostaglandin F2a ( PGF2a ) . This action of PGF2a has been virtually shown in all laboratory (2,3,4,5) and large (6,7) animals including nonhuman primates (8), but in human, FGF2a-induced luteolysis has not yet been unanimously accepted (9,lO). The mechanism of luteolytic efficacy of PGF2a followed by loss of progesterone secretion remains unresolved, however, several hypotheses have been forwarded. Those include I constriction of ovarian veih (ll), depression of ovarian blood flow (2), stimulation of pituitary luteolysin (12) and antagonism of gonadotropin action (13,14). Through a series of experiments, it is documented that constriction of ovarian velh or depre- ssion of ovarian blood flow by KiF2a is not the mechanism responsible in inducing functional luteolysis (15,16,17). However, secretion of IH at luteolytic dose level (18), antagonism of prolactin action at the luteal level (14) by PGF2a, antagonism of luteolytic efficacy of FGF2a by exce- ssive prolactin (19) or in the presence of pituitary heterotransplant-under the kidney capsule (20) are very much paradoxical events. Present study reveals the effectiveness of either prblactin or human chorionic gonadotro- pin (Ha), a long-acting LH-like hormone, in the reversal of the luteolytic efficacy of FGF2a.

MATERIALS AND METHODS

Animal preparation I About three-month old laboratory bred albino rats were housed in a controlled environment and the lighting schedule maintained at 12 hr of light per day. The animals had free access to pelleted food and water. Pregnancy was induced by caging females on evening of proestrus with males of proven fertility. The day of sperm positive smear was designated as day 1 of pregnancy.

Laparotomy for confirming pregnancy and counting of embryonic swellings or complete hysterectomy was done under light ether anesthesia and semi- aseptic State. At autopsy (Tables 1,3), the fetuses, placentae and ovaries were dissected out and weighed on a torsion balance, the 4 largest corpora lutea (CL) from each group microdissected and combined weight determined. The CL were taken from the same ovary and the establishment of 4 CL as the number weighed was a purely arbitrary choice.

For assessing the functional aspect of experimental CL, histochemical estimation of enzymes concerned with steroidogenesis such as A5-3B-hydro- xysteroid dehydrogenase (AS-3B-OHSD) and 20a-hydroxysteroid dehydrogenase (20a-OHSD) was made.

526 OCTOBER 1976 VOL. 12 NO. 4

PROSTAGLANDINS

Chemicals used I Prostaglandin F2a (tromethamine Salt) was made avail- able throuqh the courtesy of Dr. John E. Pike of Upjohn Company, Michigan. Prolactin (NIH-Ovine Prolactin - SlO) was generously supplied by the National Institute of Arthritis and Metabolic Diseases, Bethesda, Md, Human Chorionh Gonadotropin (HCG - “Pregnyl”) was kindly donated by Organon Laboratories Ltd London. Progesterone (hydroxypmgesterone caproate - “Roluton Depot”) was obtained through German Remedies Private Limited, India.

Treatment schedule : Prostaglandin F2a (PGF2a) was dissolved in cold saline and administered (2.0 mg/kg) SC as per the schedule (Table 1).

HCX and prolactin were also prepared in cold saline and scheduled SC at a daily dose of 10 IU and 4.0 mg (in two equally divided doses) per rat, respectively.

Progesterone was diluted with corn oil and administered SC (5 mg/rat) as a single daily injection in a volume of 0.2 ml of the vehicle.

Histochemistry t Histochemical detection of A'-3E-OHSD and 20a-OHSD was made as per the methods of Deane et al.(21) and Balogh (22). respectively.

Analysis of data : Student’s 2 test was used for analysing the data.

BESULTS AND DISCUSSION

It is very much consistent that PGF2a at our selective dose of 2.0 mg/kg bw on day 10 of pregnancy developed luteolysis and induced complete resorption of fetuses and placentae by day 18. Progesterone replacement concurrently with PGF2a absolutely neutralised the antifertility efficacy of PGF2a and maintained pregnancy in 100% of the test animals (Table 1). This result agrees with the findings of others that PGF2a interrupts pregnancy by lowering blood progesterone levels, the consequence of functional luteolysis (11,15).

Prolactin, one of the primary components of the “luteotropic complex” when scheduled along with PGF2a was found to reverse the detrimental effect of PGF2a in 9% of the test animals and the fetal survival rate was recorded to be 93.4% against the control value of 98% (Table 1). The intrauterine growth of the fetuses and placentae was also found normal (Table 2). Conversely, in- jections of HCG though apparently prevented the luteolytic or antifertility property of PGF2a in 7d of the experimental animals, the percentage of fetal survival was significantly lowered compared to controls or prolactin-treated group. The survival rate as determined in the Ha-treated group was 62%. Furthermore, the growth of the fetuses and placentae was tending to be subnormal and the CL weight was also significantly lowered compared to controls (P< 0.05). These results could be explained on the basis of experimental facts that the luteal progesterone stimulating property of LH (23,241 is being lowered by PGF2a due to a marked fall in the binding capacity of the luteal tissue to HCG (25) and it is the prolactin which, however, could maintain the binding capacity of HC3 with luteal tissue in the presence of PGF2a (26).

OCTOBER 1976 VOL. 12 NO. 4 527

PROSTAGLANDINS

The apparent parallel effect of progesterone, prolactin or to some extent the HG3, in overcoming the luteolytic or antifertility efficacy of PGF2a does not agree with the statement of Lsbhsetwar (18) that PGF2a-induced luteolysis is the consequence of an excessive release of endogenous LH, rather partly support Sehrman et al. (13) who are in opinion that LH not only has no luteolytic effect on ovarian progesterone in intact rat, but to some extent counteracts PGF2a-induced luteolysis. Recent observation of Fuchs et a1.(27) also reveals that the U-l is consistently effective to reverse the ffiF2a- induced functional luteolysis in the rat. Regarding the pituitary involvement in the process of luteolysis, contradictory opinions are on records (12,28). The higher percentage of reversal as obtained following prolactin therapy in the PGF2a-primed pregnants again supports our earlier observations of develo- ping a refractoriness of the animals having established placentae (19) or pituitary heterotransplant under the kidney capsule (20) to the antifertility effect of PGF2a and suggests that PGF2a fails to antagonize the prolactin action at the luteal level as proposed by Behrman et a1.(14) if the prolactin titer in the blood remains high enough.

It is evident that PGF2a stimulates estrogen secretion (21,30) presu- mably by the follicular element of the ovary (31). This estrogen is consis- tently luteolytic in a variety of species (32,33,34,35) and also known to stimulate endometrial ffiF2a (36). Extensive studies in our laboratory on the mechanism of luteolytic efficacy of estrogen show that both of the pro- lactin and indomethacin are consistent in reversing the luteolytic effect of estrogen, however, HO3 fails (37). Therefore, the antagonism of PGF2a by prolactin in the present experiment could be explained as proposed by Hixon and Armstrong (38) that the prolactin may act by interfering the biosynthesis of estrogen or prostaglandin, since the use of a specific antiestrogen is also consistent in reversing the antifertility efficacy of PGF2a (39).

To confirm our assumption that the luteolysis caused by PGF2a is not the consequence of intrauterine fetal death, pregnant rats were subjected to complete hysterectomy on day 10 and given the treatment schedules such as PGF2a, PGF2a with prolactin or FGF2a with HU3. Daily vaginal smearing was programmed as an index of ovarian status. It was observed that by day 14 of pregnancy both the hysterectomized controls and the animals which had PGF2a alone showed proestrus to estrus smears, but none of the experimental animals showed such a vaginal smear which had prolactin or HCG concurrently with PGF2a, conversely, their vaginal smears were found to be identical to that obtained in the intact pregnants.

Animals were sacrificed on day 16 of pregnancy, ovaries and CL were dissected out and weighed. One ovary from each of the animal was immediately processed for histochemical examination ofh5-3B-OHSD and 20a-OHSD enzyme activity. It was observed that compared to the hysterectomized controls, weight of the ovaries or CL did not differ much following ffiF2a, however, the CL weight of the animals given a combined regimen of prolactin or HCG with ffiF2a was significantly higher (Table 3). Similarly, it was noted that E.5-BEWJHSD activity in the CL of the animals having FGF2a or its combina- tion with prolactin or HOG did not differ much, but the 20a-OHSD activity

528 OCTOBER 1976 VOL. 12 NO. 4

PROSTAGLANDINS

which serves as a marker of luteolysis (40,41) was found to be very high in the PGF2a-treated hysterectomized animals and faintly detectable in the ani- mals having a combined regimen of PGF2a and HCX. However, the enzyme acti- vity of 20a-OHSD was completely absent in the CL of the experimental animals which received prolactin along with PGF2a (Table 3). These data are again proposing that PGF2a at our dose level does not significantly affect ovarian steroidogenesis, but modify the biosynthetic pathway in favouring the ovarian metabolism of progesterone to its less active metabolite, 20a-dihydroproges- terone. Prolactin was found to be much better than HCG in antagonizing 20a- OHSD activity in the luteal tissue which is logical (41,42,43), but our observa- tions do not agree with the statement made by Fuchs and Mok (44) who obtained better results by using a very high dose of LH along with PGF2a. Prolactin, in their experiment, was found to be exclusively ineffective. It is evident that the excessive LH-stimulated follicles subsequently luteinize and start secreting progesterone (45 ). Possibly this new source of progesterone may again help in the maintenance of pregnancy and high levels of progesterone on continuous LH stimulation as recorded by Fuchs and Mok (44) and Fuchs et a1.(27). It has been recently shown that ffiF2a is accompanied by a loss of gonadotropin receptor and prolactin plays a direct role in the regulation of corpus luteum LH receptor (26) and also the functional CL even in the hypophy- sectomized rat (46).

ACKNOWLEDGMENTS

The stimulating cooperation of Dr. S. Tejuja, Dr. S. J. Segal, Dr. R. T. Mahoney, Dr. J. M. Spieler and Dr. A. Bartke in favouring my research activity is gratefully acknowledged.

Vitamins supplementation to the experimental animals has been kindly made by Glaxo Laboratories (India) Ltd., through the courtesy of Dr. K. V. Joshi.

The sincere cooperation of Mr. Bimal Shankar Paul in analyzing the data is appreciated.

OCTOBER 1976 VOL. 12 NO. 4 529

PROSTAGLANDINS

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OCTOBER 1976 VOL. 12 NO. 4 531

TA

BL

E

1

PC

&a

as

an e

ffec

tive

an

tife

rtil

ity

agen

t an

d

its

reve

mal

by

pr

oges

tero

ne,

pr

olac

tin

or

H

O3

Tre

atm

snt

(Day

of

pr

egn

ant

y)

Con

trol

ffi&

a(D

ay

10)

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10)

f P

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ster

one

(Day

s 9-

15

)

No.

of

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cces

sfu

l N

o.of

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able

fe

- S

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bryo

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ngs

at

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ses

at

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i-

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) pa

rato

my(

Day

8)

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ce

(Day

18

) W

J

10

100

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99

98.0

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0 10

6 0

0

10

100

96

96

100

PW

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ay

X0)

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n

(Day

s 9-

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90

92

86

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75

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t 1.

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4 +

0.

1

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82

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7.5

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6.5

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t

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4.1

+

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(Day

s 9-

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) +

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s 9-

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8 +

0.

1

(P<

0.

001)

(P

<

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)

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ay

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2

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G =

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uman

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orio

nic

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.=

Cor

pus

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um.

TABL

E 3

??

Antagonism between PGFoa and prolactin or HCG

at the ovarian level in

th

e hvsterectomized

Pregnant rat

Treatment (Day

No. of

Mean ovarian wt.

Mean C.L.wt.

Enzyme activity

of pregnancy)

animals

(m9 + SE)

(mg t SE)

A53b-CHsD

1

used

20a-CHSD

Control (hys-

terectomy on

day 10)

PGF2a (Day 10)

Hffi(Days 10-15)

control

PGF2a(Day 10) +

HCG (Days 10-15)

Prolactin

(Days 10-15)

control

PGF2a (Day 10)

t Prolactin

(Days 10-15)

6

68.0 + 1.3

2.1 + 0.1

f

+t+

6

70.0 2 2.7

5

6

118.6 ;t 24.7

2.94 + 0

t-t-+

t

114.0 + 16.0

(PC 0.001)

2.9 + 0.1

+i-t

t

(PC 0.005)

5

61.8 2 2.1

(P(O.01)

3.5 F 0.3

-I++

(P< 0.005)

6

64.0 fr 3.9

2.2 + 0.1

+t

++I

3.4 t 0.2

+t+

(PC 0.001)

Animals were sacrificed on Day 16.

C.L. = Corpus luteum.

Hcxj = Human chorionic gonadotropin.