Institute for General and Experimental Pathology, University of Innsbruck, Medical School, A-6020 Innsbruck, Austria

PROGESTERONE AND 20\g=a\-HYDROXYSTEROID\x=req-\ DEHYDROGENASE REGULATION IN THE CORPUS LUTEUM OF THE PREGNANT RAT

By K. Loewit and N. Zambelis

ABSTRACT Female Wistar rats were mated and passively immunized against bovine luteinizing hormone (bLH) between days 7\p=n-\12 of gestation. In a first series of experiments the effect of simultaneous progesterone treatment on the histochemically detectable activity of 20\g=a\-hydroxysteroiddehydrogenase (20\g=a\-OHSD) in the corpora lutea of pregnancy was evaluated. In other experiments the exact time course was established between the first histologically demonstrable signs of termination of pregnancy and the appearance of 20\g=a\-OHSD activity in corpora lutea of pregnancy. It was found that progesterone substitution not only prevented the deleterious effects of the anti-bLH-serum on gestation but also the re-appearance of 20\g=a\-OHSD activity in the corpora lutea of pregnancy up to day 12 of pregnancy. Moreover in animals injected with anti-bLH-serum from day 7 on and sacrificed on days 8\p=n-\2, signs of foetal destruction clearly preceded the pathological re-appearance of 20\g=a\-OHSD activity by 2\p=n-\3 days. These results support previous findings on a direct or indirect role of progesterone in the complex regulation of 20\g=a\-OHSD activity. Possible mechanisms of action are discussed.

The

functioning of the corpus luteum of pregnancy (CEP), in particular the regulation of progesterone (P) synthesis and degradation is still not completely understood. An original hypothesis according to which luteal function may be

Reprint address: K. Loewit, M. D., Institute for General and Experimental Pathology, Fritz-Pregl-Str. 3, 6020 Innsbruck, Austria. This study was supported by Grant No. M 71.032C from the Population Council, New York and a Dr. Legerlotz-Foundation-Grant.

terminated by conversion of to 20u-hydroxypregn-4-en-3-one (20a-OHP) by the enzyme 20a-hydroxysteroiddehydrogenase (20«-OHSD) (Wiest et al. 1968; Wiest 8c Kidwell 1969) has been abandoned in favour of the view that "luteal regression is a more complex phenomenon than the regulation of a single enzyme" (Lacy et al. 1976). This conclusion is based on the observation of a fall in progesterone secretion on day 18 without any increase either in luteal 20«-OHSD activity or in ovarian 20a-OHP secretion in normal pregnant rats (Uchida et al. 1970) or similar discrepancies between serum and 20«-OHP concentrations and ovarian 20a-OHSD activity during late rat pregnancy, though the stimulus responsible for the increased activity of 20«-OHSD at this time of gestation being unknown (Lacy et al. 1976). In previous studies on the corpus luteum function in the pregnant rat using passive immunization with rabbit anti-bovine LH sera (ALS) (Loewit 8cLaurence 1969) it has been shown that luteinizing hormone (LH) is an essential luteotrophic stimulus between day 7 to 12 of pregnancy and that its neutralization leads to increased 20a-OHSD activity and termination of gestation, day 11 being the most susceptible time for this kind of intervention (Kofler et al. 1977). In the course of these studies preliminary experiments indicated that during the first half of pregnancy concomitant treatment with progesterone could not only prevent the deleterious effects of the ALS, but also the re-appearance of strong 20«-OHSD activity in the CLP which normally are devoid of any histochemically detectable enzyme activity at this time. It has been speculated whether the gonadotrophin dependent progesterone level could regulate the 2()a-OHD activity rather than result from it (Loewit et al. 1969; Loewit 1970). When carrying these experiments further, using the hitherto employed experi¬ mental model of rat pregnancy and passive immunization against LH, the suppressive effect of on 20«-OHSD activity should be re-examined and the tem¬ poral relationship established between first histological signs of foetal reabsorption and histochemical appearance of 20«-OHSD activity in CLP should be studied as a further step towards clarification of the role of in 20«-OHSD

regulation. MATERIAL AND METHODS

a)

Animals

Adult female Wistar rats (Bäumler, Wolfratshausen, FRG) weighing between 200 g and 300 g each were kept under a 12:12 h light-dark schedule at 20°C with free access to rat pellets and tap water. Vaginal smears were examined daily and animals show¬ ing regular cycles were placed overnight with males of proven fertility on the evening of pro-oestrus. If sperm and/or a vaginal plug were found the next morning, this day was designated day 1 of pregnancy. An exploratory laparotomy was performed on all animals under ether anaesthesia on day 7 to verify the pregnancy and to determine the umber of implantations. This procedure does not interfere with gestation (Loewit 1970: Euker Se Riegle 1973). Animals were assigned to either of two groups:

Table I. Effects of exogenous progesterone

pregnancy and 20a-OHSD treated with rabbit anti-bovine LH-serum. on

VII NF

ALS

Untreated Controls

I) 2)

4/2 9/4

Antiserum Controls

3) 4)

8/4

6) 7) S)

6/5 7/6 10/4 5/3 4/5

9)

6/6

5)

Antiserum

10)

Progeste- 11)

0.5 0.5 0.5 0.5 0.5 0.5 0.5 5 mg P 0.5 4-5 mg P

8/7

0.5 5 mg P 0.5 4-5 mg P

5/0

+

13)

5/7

8/6

0.5 5 mg P 0.5 + 5 mg +

14) VII/XII NF ALS DP NI SP EA

=

=

=

=

=

=

=

SP

NF 4 _'

rone

12)

NI

11

4/8

7-10 7-10 7-10 7-10 7-10 7-10

0/0

res

0

res

•H-

0/0 0 u 0 0 0

res

++

7-10

7/8

7-10

8/7

7-10

5 II

7-10

6/8

7-10

6/6

7-10

2/7

+:

some, ++:

+

res

4-4-

res

4-4-

sc

sc

sc

se

sc

p.res

sc

=

none,

L

res

day 7/12 of pregnancy number of foetuses in the left/right horn of the uterus amount of antiserum against LH days of pregnancy with treatment number of injections state of pregnancy; normal, res résorption, p.res partial 20ot-OHSD activity in corpora lutea of pregnancy; —:

EA

9/4

+

+

in rats,

XII

DP

(ml ip)

activity

=

=

re-absorption

strong

Fig.

1

a.

lutea of pregnancy of an animal injected 4 times with 0.5 ml anti-bovine-LH on days 7 to 10 of pregnancy, showing strong 20a-OHSD activity (dark areas) on day 12. All the original 12 foetuses have been resorbed, 48.

Corpora serum

Fig. 1 b. Corpora lutea of pregnancy of an animal injected 4 times with 0.5 ml anti-bovine-LH serum plus 5 mg progesterone on days 7-10 of pregnancy, showing no 20u-OHSD activity like on day 12 in normal controls. Fifteen foetuses appeared undisturbed. Some 20a-OHSD activity in an old, degenerating corpus luteum (top), 48.

Fig.

1

Fig.

1 b.

a.

Group 1. days 7

Passive immunization against LH and concomitant treatment with 10, sacrifice after the second laparotomy on day 12. Normal pregnant rats and pregnant animals treated with antiserum only served as controls. from

-

to

Group 2. Passive immunization against LH for 1 to 5 days between days 7 to 12 of pregnancy. Experimental animals and normal rabbit-serum controls were sacrificed on each of these days for the study of foetal alterations and luteal 20a-OHSD activity. As indicated below, experimental animals were injected with 0.5 ml of undiluted antiserum ip once or twice daily, controls with 0.5 ml of normal rabbit serum. Pro¬ gesterone was administered sc in sesamic oil at a dose of 5 mg/day. Before sacrifice, a second laparotomy was performed to evaluate the state of the foetuses, and uteri and ovaries were removed for histological or histochemical examination. —

b)

Antisera

Five female white New Zealand rabbits (Bäumler, Wolfratshausen, FRG) weighing 2-3 kg were used for the production of antibodies to LH. They were immunized with bovine LH, NIH-LH-B6 obtained from the Endocrinology Study Section, National Institutes of Health. Immunization was accomplished by three intradermal injections of 5 mg LH dissolved in saline and emulsified with an equal volume of complete Freund's adjuvant (Difco Laboratories, Detroit, Mich.) in multiple sites in the nuchal region at weekly intervals and one iv booster of 4 mg two weeks later, as previously described in detail (Loewit 8c Laurence 1969; Loewit et al. 1969). Antisera (AS) were absorbed with normal bovine serum (NBS) and were then characterized in double diffusion in gel and in passive haemagglutination tests using sheep red blood cells coated with bLH, oFSH and NBS by means of the chromium chloride method (Gold 8c Fudenberg 1967). The absorbed AS showed titres of 1:5120 and above against bLH in passive haemagglutination and lacked any cross-reactivity with oFSH, bLTH, NBS and rat serum proteins. Thus, these AS showed principally the same serological charac¬ teristics as those of the earlier studies raised with the same immunization protocol.

c) Histology

and

histochemistry

removed at the second laparotomy and implantation sites or foetuses respectively excised from both horns. They were fixed in Bouin's solution and alcohol, embedded in paraffin, cut in serial sections of 10 ftm thickness and stained with haematoxylin and eosin for microscopical examination. Ovaries were removed simul¬ taneously for histochemical evaluation of 20a-OHSD activity. They were immediately frozen on dry ice and kept at -20°C until processing for histochemical analysis which was performed following the slightly modified method of Balogh (1964) as described previously (Loewit et al. 1969). Uteri

were

RESULTS

Group

1

The results are summarized in Table 1, which shows the macroscopically assessed state of pregnancy and the histochemically demonstrable activity of 20a-OHSD in CLP of passively immunized, P-treated animals. Progesterone not only protected the foetuses but also prevented the occurrence of enzyme activity on day 12 of pregnancy, as shown in Fig. 1.

Table 2. foetal and appearance of 20a-OHSD activity between destruction Temporal relationship in the corpora lutea of pregnancy (CLP) of rats passively immunized against bovine LH. State of pregnancy

Treatment

I.

NA

Lap.

Lap.

II.

ALS

1)1'

(ml ip)

NI

NRS-Co

Foetal

histology

0.5, b. i. d. 0.5, b. i. d.

6- 7 7

7 7 7 p.

NRS-Co 0.5, b. i. d.

7- 8

8 p. 8 p.

EA

normal

not

done

m.

not done

m.

normal

m.

beginning destruction

NRS-Co 0.5, b. i.d.

7- 9

9 p.m. 9 p. m.

normal destruction

7- 9 7-10

10 10 10 p.m.

normal destruction destruction

I I

normal

7-10 7-10

11 II p.

NRS-Co

0.5, b. i.d. 0.5, b. i. d. NRS-Co 0.5

0.5, b. i. d. NRS-Co 0.5

7-11

0.5, b. i. d.

7-11

reabsorption m.

reabsorption

12

normal

12

complete reabs.

in

12 p.

m.

complete reabs.

NA I. Lap. 1 . Lap. ALS DP NI EA

=

=

=

=

=

=

=

number of animals day of first laparotomy day of second laparotomy amount of antiserum against LH days of pregnancy with treatment number of injections 20a-OHSD activity in corpora lutea of pregnancy; -:

none,

+:

some, ++:

NRS-Co normal rabbit *: 6 of 7 animals =

=

normal,

res

=

serum

résorption,

strong controls

p.res

=

partial re-absorption

(6/7)« (1/7) (2/3) (1/3) (1/4) -(3/4)

Group

2

The results of passive immunization between days 7 to 12 of pregnancy and the correlation of histological and histochemical findings are summarized in Table 2. Signs of foetal destruction, consisting of haemorrhages at the implanta¬ tion sites, cell degeneration within the decidual and foetal tissue and immigra¬ tion of polynuclear leucocytes, clearly preceded the re-appearance of histochemically detectable 20«-OHSD activity in CLP. While gestation was already severely affected, 6 of 7 animals showed no enzyme activity on day 9, 4 of 5 even on day 10 of pregnancy. Only from day 11 on foetal résorption is cor¬ related with marked enzyme activity.

DISCUSSION

al. (1972) have shown that a single injection of LH-AS in day 8has already led to an 80 °/o reduction in the P-secretion rate and pregnant clear of vaginal bleeding, intrauterine haemorrhage and résorption evidence to of foetuses within 24 h. Moreover the process of foetal destruction has been meticulously followed in light and electron microscopic studies in rats biovariectomized at different times after implantation between days 7 to 12 (Clabaut 1972, 1973; Clabaut 8c Métayer 1973). The first signs of résorption occurred at the foetomaternal junction. Vascular disorders were predominant and appeared as early as 8 h after castration. Similarly, it has been reported, that the immediate and most conspicuous effect of ovariectomy in 6 to 9 daypregnant rats is the degeneration of the decidua leading to haemorrhagic col¬ lapse of the whole conceptus. This process could be modified and even stopped by P-substitution (Deansley 1973). These studies confirm that indeed ALS re¬ duces P-secretion and thus leads to termination of pregnancy, recognizable

Moudgal

et

rats

histologically

at a very early stage. Hence, the demonstration of a considerable time lag of

2-3 days between the histological detection of first signs of foetal destruction and the appearance of 20a-OHSD activity in the CLP clearly shows that the decline in P-concentration precedes the increase in 20«-OHSD activity. In addition, the suppressive effect of on 20a-OHSD activity under the described experimental conditions supports the concept that in some way, either directly or indirectly, is in¬ volved in the regulation of this enzyme. However, the observed inhibition of 20a-OHSD activity could also be brought about by some other substance, originating from the viable foetus or pregnant uterus, or by acting via the placenta. This possibility could be excluded by hysterectomizing normal pregnant rats on day 9 of pregnancy and determining luteal 20«-OHSD activity on day 11 and in the morning and evening of day 12

respectively, using the experimental procedures described in this paper. No enzyme activity was detectable in CLP before the evening of day 12 (Loewit 8c Zambelis, unpublished results). The 20«-OHSD thus showed a pattern cor¬ responding to normal pregnancy even 3 days after removal of uterus and foe¬ tuses. Only after the CLP switched from pituitary to placental gonadotrophin support on the evening of day 12 (Pencharz 8c Long 1933; Selye et al. 1933) did the enzyme activity appear. This again speaks in favour of as the effective of 20«-OHSD mechanism which well role in a more a play complex agent, may levels also LH and e. prolactin (Prl) activity regulation, involving g. (Bast 8c this has been An in postulated in Melampy 1972). regard interesting hypothesis a recent paper on the role of Prl in the prostaglandin (PG) induced luteolysis in the super-luteinized rat ovary (Aakvaag 8c Torjesen 1977). The investigators conclude from their study that PG induced luteolysis leads to an immediate decline in serum which probably triggers an increase in pituitary Prl secretion which in turn stimulates the 20«-OHP production. The increase of 20«-OHP after PG could be prevented by 2-Br-«-ergocryptine-treatment and restored by concomitant Prl administration. If this conclusion holds true also for the CLP and if the rise in 20a-OHP reflects an increase in 20«-OHSD activity, then it would explain the suppressive action of on 20a-OHSD activity by suppression of the enzyme-stimulating Prl production and vice versa. A similar sequence of physiological events does indeed occur at the end of pregnancy, when a sharp decline in concentration precedes a "precipitous increase" of Prl (Morishige et al. 1973). Recent evidence suggests even an intracellular site of action of Prl in the rat corpus luteum (Nolin 1978). However, this hypothesis conflicts with accepted views of an inhibitory effect of Prl on 20«-OHSD activity in vivo (Wiest et al. 1968; Lamprecht et al. 1969; Strauss 8c Stambaugh 1974) and in vitro (Lahav et al. 1977), and is difficult to reconcile with results showing that ovariectomy in late pregnancy prevents the normal pre-term rise of Prl in the rat (Bridges 8c Goldman 1975). may, however, not trigger Prl production directly, but via increased ovarian oestrogen secretion. Studies on the effect of anti-prolactin sera or 2-Br-«-ergocryptine on the histochemically detectable 20«-OHSD activity in CLP of passively immunized pregnant rats as well as on a possible role of oestrogen are presently under way in our laboratory.

ACKNOWLEDGMENTS our gratitude to the Population Council, New York and the Dr. Legerlotz-Foundation for financial support and to the Endocrine Study Section Pitui¬ tary Hormone Distribution Program (NIAMD) for generous gifts of pituitary hor¬ mones. We thank Dr. R. Kofler for his help in characterizing the antisera. We also

We want to express

extend our thanks to Mrs. I. Fill for valuable technical and to Miss M. secretarial assistance.

Rappold

for

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on

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Century