acute effects of ovariectomy on pituitary lh, uterine weight, and vaginal cornificatiod 2€¦ ·...

Acute effects of ovariectomy on pituitary LH, uterine weight, and vaginal cornificatiod 2

SCHWARTZ, NEENA B. Acute effects of ouariectomy on pituitary LH, uterine weight, and vaginal corn$kation. Am. J. Physiol.

NEENA B. SCHWARTZ Department of Physiology, Uniuersity of Illinois College of Medicine, Chicago, Illinois

207(6): rqv-1259. Ig64.- In the cyclic rat, uterine weight increases at proestrus, pituitary LH content drops from a maximum at proestrus to a minimum at estrus, and ovulation and vaginal cornification then occur. To investigate the timing of the ovarian secretion presumably responsible for these changes, ovariectomy or sham ovariectomy was performed at increasing intervals before the day of estrus. Results in both 4- and s-day cyclers indicate that the ovary must be in situ between diestrus 4 PM (day before proestrus) and proestrus IO AM in order for estrous vaginal cornification to occur; it must be in situ between diestrus IO AM and 4 PM (day before proestrus) for the uterine weight increase and pituitary LH discharge to occur. In some rats sham ovariectomy, when performed at diestrus I o AM, delayed the proestrous-estrous changes by at least 24 hr. The “extra” day which occurs in normal s-day cycles results at least partly from a delay in the steroid discharge necessary to cause release of the ovulating surge of LH.

pentobarbital gonadotrophin progesterone ovary estrogen estrous cycle hormonal feedback rat

T HAT ESTROGEN (as well as progesterone (2, 24)) secretion occurs during follicular growth in the rat is clear from the following: a) uterine weight and water content increase at proestrus (2); b) vaginal cornification occurs by the morning following ovulation in the 4-day rat and on the preceding morning as well in many 5-day rats (7, 2 I) ; c) behavioral estrus occurs early in the evening between proestrus and estrus (5); d) all of the above are abolished by ovariectomy and can be restored by estrogen (I, I 2) or, in the case of mating behavior, most satis- factorily by estrogen plus progesterone (4). Recent evidence‘ (I 7, 2 I) has demonstrated that an ovulatory

Received for publication 20 March 1964. 1 This work was supported in part by Grant AM 06145 from

the National Institute of Arthritis and Metabolic Diseases, and in part by the University of Illinois Graduate Research Fund.

2 A preliminary report on some of this work was presented at the American Physiological Society Meetings, August I 963 (Physi-

ologist 6 : 270, 1963).

surge of LH is released between 2 PM and 4 PM only on the afternoon of proestrus, in spite of a daily “2 PM-~ PM" facilitatory period for LH release (7), suggesting that a second facilitatory event occurs on the day of proestrus. Single injections of estrogen or progesterone induce ovulation (7), suggesting that the proestrous facilitation of LH release is due to a preceding steroid discharge. The purpose of the present study was to investigate the time of release from the ovary of the steroid(s) responsible for the uterine growth, vaginal cornification, and LH discharge of proestrus and estrus. The technique utilized was to ovariectomize rats at various times before the day of estrus and then determine whether the above-mentioned alterations had taken place in spite of ovary removal.

METHODS

A. Experimental Design

Figure I (groups r-qb) indicates the times when rats were ovariectomized or sham ovariectomized before autopsy at “proestrus” or “estrus.” The degree of pituitary LH discharge was assessed, in the absence of ovaries, by measuring pituitary LH content at estrus, since a significant drop in pituitary LH content occurs between the mornings of proestrus and estrus (2 I), accompanying ovulation. However, the use of pituitary LH content at estrus as a criterion of release in ovariectomized rats depends on the validity of two assumptions. The first is that pituitary LH content will not drop between proestrus and estrus if release is blocked. The second is that if pituitary LH content is found to be high at estrus it is a result of blocking the acute release and not just a reflection of the beginning “pile-up” of LH seen I week after ovariectomy (20).

As a test of the first assumption some rats were injected with pentobarbital at 2 PM on the afternoon of proestrus to determine whether pituitary LH content remains high at estrus, correlating with ovulation blockade (7, 22). To test the possibility of an early accumulation of pituitary LH content because of ovariectomy, pituitary LH was measured in rats ovariectomized and autopsied at cycle stages not overlapping the time of proestrous

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1252

(1)

(2a)

(2b)

(30)

(3b)

(4)

(50)

Wb)

@a)

(6b)

*“24* ovt 18 HR 4A

ovr 42 HR IA

I O”’ 24HR *

OVI IA 48 HR

*“Yz7ic*

*“I 18 HR’*

OVI 42 HR

IA

*“’ 24HR ‘*

*vi IA 40 HR

Sham operafed groups were u/so fun af fhe some fimes

OV - Time of Ovufiecfomy A - Time of Autopsy

FIG. I. Experimental plan: timing of operations and autopsies. Time of cyclic LH release is between 2 PM and 4 PM on the afternoon of proestrus. Hatched area (7 PM-~ AM) indicates lights off. Groups 1-36 are the basic experimental animals, with 4-6b providing “time after ovariectomy” controls. Group ga was the only ovariectomy group not provided with a companion sham-operated group. Unoperated controls were autopsied at each ovariectomy and autopsy time.

LH release (Fig. I, groups +-6b). Control data for all variables were obtained from 4-day or 5-day cyclic unoperated rats autopsied at each of the cycle stages which served as an operation or autopsy time for the experimental rats (Fig. I).

B. Adult Donor Groups

Ninety- or sixty-day-old female rats were obtained in seven separate lots from Sprague-Dawley Inc., in Madison, Wis., and were housed in a temperature- controlled, artificially lighted (5 AM-~ PM) room for IO-

20 days before the start of vaginal smearing. Smears were thereafter made each morning before IO AM until autopsy. Each of the 247 rats finally selected (from more than 500) had demonstrated at least two regular cycles before the cycle in which it was used. The previous (2 I)

criteria for determining cycle stage were rigidly adhered to. Rats running both 4- and 5-day cycles were used in almost all groups, with pituitaries being assayed separately for the most part.

I. Untreated controls. Untreated rats were decapitated at estrus (10-1 I AM or 4-5 PM), metestrus (10-r 1 AM), diestrus (day after metestrus) (IO-I I AM), diestrus (day before proestrus) (I O-I I AM or 4-5 PM) and proestrus (I O-I I AM). There is only I day of diestrus for 4-day rats. The uterus was grossly inspected for intraluminal fluid (“ballooning”), trimmed, and weighed before and after

N. B. SCHWARTZ

drying. Oviducts were inspected under a dissecting microscope for swelling and presence of ova, and in some cases were serially sectioned. The ovaries were weighed and discarded in most cases. The neurohypophysis was discarded and the anterior pituitary was weighed and quickly frozen (21). Each pituitary “pool” consisted of two or three pituitaries.

2. Pentobarbital-injected rats. Pentobarbital (3 mg/ I oo

g) was administered intraperitoneally to previously untreated 4-day proestrous rats at 2 PM and complete autopsies were performed between IO and I I AM the following morning. It was necessary to use 4-day rats since pentobarbital administration at this time and dose level does not block ovulation in 5-day rats (N. B. Schwartz, unpublished observations) owing to a pro- longation of the critical period of LH release (7).

3. Ovariectomized and sham-ovariectomized rats (Fig. I). Bilateral ovariectomies were performed using ether anesthesia under nonsterile conditions. The uterus was inspected in situ for ballooning and the oviducts were checked for presence of ova. Ovaries were weighed and discarded. Bilateral sham ovariectomies were performed in a similar fashion with the uterus and oviducts being observed in situ. At the time of autopsy the same pro- cedures were carried out as described for the untreated controls.

In addition to the operated groups described in Fig. I,

some rats ovariectomized or sham ovariectomized on the morning of estrus were autopsied after a 72- or g6-hr interval, and other ovariectomized rats were not autopsied until I month had elapsed.

C. Pituitary Assays

A modification (2 I) of the ovarian ascorbic acid depletion technique (19) was used. Each recipient rat was used once, the pituitary material or standard LH being injected intravenously 16 I hr post HCG, and both ovaries being removed 4 hr later for ascorbic acid measurement. The doses of pituitary administered (to five rats) were ?& and >i pituitary equivalent; NIH- LH-SI or -S2 served as standards at doses of 0.4 pug and I .6 pug per recipient rat. On repeated tests we have found no significant difference in potency between these two LH preparations. Pituitary potencies were calculated (3) on the individual pools (X = .23g & .064, N = 86) and all potencies within a given group were combined statistically using a weighting technique, after first testing for homogeneity. Where significant heterogeneity occurred this is indicated in the tables and the combined potency presented includes the variance among the pools (3) l

RESULTS

I. &operated rats: comparison of p-and s-day cycles. Table I summarizes pituitary, vaginal smear, and uterine data obtained for the unoperated rats. There is no consistent difference in pituitary LH content between the two types, both showing maximum contents at

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OVARIECTOMY ON PITUITARY LH, UTERUS, AND VAGINA 1252 JJ

proestrus and minimum values 24 hr later. The seven estrous AM pools were heterogeneous (as seen previously (21)) but the source of heterogeneity was within the 5- day cyclers. The three diestrous PM pools, however, also showed heterogeneity, and for these it appeared that the 5-day cyclers had a higher pituitary LH content. The striking aspect of the LH data in Table I is that on the morning of the 2nd day of diestrus (day before proestrus), LH content in the 5-day rats is still at diestrous values, not at the high proestrous values seen in the 4-day rats on the same day with respect to the time of last ovulation. Pituitary (and ovarian) weights were not significantly different either with respect to type of rat or stage of cycle (Table I).

In 4-day cyclers, vaginal cornification is present only on the day when ova are found (Table I). Frequently at the time of the proestrous morning smear, nucleated cells are already predominating, although this is not always the case. By contrast, most of the 5-day cyclers show 2

days of cornification, the first on the day of proestrus (Table I).

Uterine wet weight (Table I), water content, and dry weight were highly correlated with each other. Analyses of variance were carried out for wet weight and water content, testing the significance of a) differences among cycle sta’ges, b) differences between 4-day and 5-day cyclers, and G) interaction between cycle stage and animal type. For both wet weight and water content there was no significant difference between 4-day and 5- day rats nor was there a significant interaction between cycle stage and type of rat; however, cycle stages were highly significantly different from each other. On the morning before proestrus for the 5-day rats uterine weight had already started up toward proestrous values in some rats (Table I). Maximum weights, however, were seen on the morning of proestrus for both types of rats, an abrupt fall in weight occurring by estrus (Table I). For 4-day rats uterine ballooning was seen only on the morning of proestrus, but for some 5-day rats it was seen on the previous day as well (Table I).

2. Effects of pentobarbital treatment. As can be seen in Table 2, pentobarbital administration at 2 PM on the day of proestrus blocked ovulation in all animals (7, 22).

Pituitary LH content remained at proestrous values, indicating that with neurogenically induced failure of LH release, estrous values provide a good criterion of blockade, correlating with ovarian findings. Vaginal cornification and the expected drop in uterine weight took place in spite of the blocked ovulation in all of the eight injected rats. However, four of the rats still showed at least some degree of uterine ballooning.

3. Acute effects of ovariectomy (within 48 hr). Data sum- maries for the animals ovariectomized within 48 hr before estrus are seen in Table 2. The data on pituitary LH content and uterine weight are also presented in Fig. 2, against a background of the g5 % confidence limits of unoperated rats.

The rats ovariectomized at IO AM on the morning of proestrus showed (Table 2, Fig. 2) the normal drop in

uterine weight and also a drop in pituitary LH potency from 9.0 to 6.6 pg at estrus. Vaginal cornification was seen in the majority of these rats (Table 2). The only two rats not demonstrating cornification at autopsy were 5- day rats which had been cornified at the time of surgery, at which time they were definitely in proestrus, not in estrus.

A quite different picture was seen following ovariectomy at 4 PM on the afternoon before proestrus (Table 2,

Fig. 2). Uterine weight at the proestrous autopsy time failed to show the pronounced rise seen in unoperated rats (Table I) but all four rats showed some degree of proestrous ballooning. By estrus, uterine weights had dropped to the minimum seen during the cycle (Fig. 2).

Pituitary LH content on the morning of proestrus was significantly heterogeneous between the pools (Table 2) :

4-day pool = 4.3 ,ug (1.9-7.9); 5-day pool = 10.3 pg

(6.2-19.5). This is reminiscent of the heterogeneity seen in diestrous 4 PM controls (Table I) and suggests a delay in LH storage. However, no heterogeneity occurred among the LH potencies as measured at estrus (Table 2); the mean potency at this time was closer to control proestrous than estrous values (Table I). Six of the eight 5-day cyclers ovariectomized at diestrus 4 PM

showed vaginal cornification on the morning of proestrus but only two of the twelve rats autopsied at estrus showed cornification at autopsy (Table 2), these being one 4-day rat and one 5-day rat (cornification blocked at estrus in a/ I5 vs. 1 o/I 2 following surgery at proestrus IO AM and diestrus 4 PM, respectively; P < .o I by chi- square analysis).

Finally, following ovariectomy on the morning of diestrus, uterine weight shows a fall instead of a rise at proestrus and has dropped even lower 24 hr later (Table 2, Fig. 2). Pituitary LH content shows the normal rise at proestrus and does not change at all from this value at estrus (Table 2, Fig. I), resembling the effect of pentobarbital (Table 2). Of the five 5-day cyclers ovariectomized at this time only one showed cornification at proestrus; but not a single animal of either cycle length showed cornification at estrus (Table 2).

Data for the rats ovariectomized after the time of LH release are summarized in Table 3 and Fig. 2. A fall in uterine weight from already low values occurred within the first 18-24 hr after surgery, regardless of the stage of the cycle when ovariectomy was performed, and then did not fall further during the next 24 hr (Table 3, Fig. 2).

Pituitary LH content rose 3.1-3.3 pug during the 24 hr from metestrus to diestrus in all three groups of operated rats (compare 6.4 to 9.5 pg, 6.8 to 9.9 pug, and 4.4 to 7.5 pug; see Tables I and 3). This might be called the base- line rate at which pituitary LH will rise over a 24-hr period following ovariectomy, since over this time span in the unoperated rat it did not change at all (Table I).

4. Acute effects of sham ovariectomy (within 48 hr). The data from the sham-ovariectomized animals are shown in Tables 2 and 3 and Fig. 3. Sham ovariectomy on the morning of proestrus or on the preceding afternoon did not block ovulation, the expected vaginal cornification,

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TABLE I. Data summary of untreated control rats

Pituitary

Autopsy Time Cycle No. of Length Rats No. of

pools LH content, pg/pituitary W mg

---

Uterus Corr&kra$3n

Autopsy Dva at Autopsy

Wet wt, mg Ballooned

7P

9/IId

r6/rgd

019

019

o/18

O/3

013

O/II

48

47

o/15

1/5

1/13

013

213

46

Proestrus IO AM

Estrus IO AM

Estrus 4 PM

Metestrus IO AM

Diestrush IO AM

Diestrusj IO AM

Diestrusj

4 PM

o/8

7/1*

7119 o/*9

919

9/9

9/9

9/9

18/18 18/18

3/3

3/3

313

3/3

6/6

46

o/5

O/II

48

47

o/15

015

o/13

o/3

O/3

O/II

o/8

47

o/15

o/5

o/13

o/3

o/3

o/6

9*7a II .6 (7*1-13*3)b zt2.5”

9-3 10. I

(6.4-13-7) rt2.1

7.8 (5.4-11.5)

Combined data on all rats

509 %I 16~

538 k67

528 A85

434 A76 434

A41

434 &Go

413 *41

349 rf42

381 h51

390 h78 354

*58

374 A69

407 *40

352 A81

381 A67

455 *I15

425 *77

348 k47

368 1k82

358 =t61

4 8 3

5 II 3

4+5” I

I9

9

9

9-o (7.3-11 .I)

3.8 (2.7-5.5)

4*7f (2 -3-9-3)

4.9 (2.4-8-g)

Combined data

4*3f (3.0-6.1)

10.7 rt2.3

10.4 %I .g

II .o

&I .g

4

5

4+5”

on all rats

18 7

4 3 I

5 3 1g

4+5” I

4.0 (2.2-6.6)

10.5 &I .3

10.4 ho.4

4.6 (2.3-8.0)


10.5 zt0.g

6 3g

4

5

4+5”

6

5

II 5

4 8 3

5 7 3

6

5

4

5

4.3 (2.8-6.5

5.4 (3.9-7-5)

8.4 (5.7-12.3)

5-9 (3.6-8.6)

Combined data

6.4 (5.1-8.0)

6.5 (4*5-94

6.9 (4.7-10.0)

12.0

h2.6 II .8

33.4

on all rats

II .g

It2 .o

9-I &I .3

9-8 zt2.2

Combined data on all rats’

6-7 9.4 (5.1-8.7) &I .8

4-7 9-o kg-7-7) ZfII .5

Combined data on all rats’

5.8 9.1 (4.3-7-7) *I .3

9-6 ho.8

II .4 3x1 .g

3.2 (2.0-4.8)

10.6 (6.8-18.5)

7-4 (4.2-14.0)

4+5”


6.2f (3.1-12.7)

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OVARIECTOMY ON PITUITARY LH, UTERUS, AND VAGINA 1255

TABLE 2. Data summarv of rats oberated before time of LH release J J I J - -

Treatment Autopsy No. of Rats Pituitary Uterus

LH content, j&pituitary

-

- W mg

8.1” 10.4 (6.5-10. I )b &I .8”

6.6 10.5

(5.1-8.6) &I .8 6.7f 10.8

(2.8-16.5) *I .o

7.8 9-9 (5.8-10.5) &I .4

9.3 IO. I

(7.3-11.8) ho.5

9-o 9.5 (7.0-11 .S) zt0.g

4-9 10.7 (3.7-6.4) rt2.3

8.8 10.5 (6.1-12.8) &I .2

5-o 10.6 (3.7-6.8) h2.5

5.5 10.0

(4.1-7 l 4) zt0.g

4*3 10.8 b-7-6*9) =tI .o

7*5f 10.3 (5*3-10-g) &I .6

Cornifica- tion Smear at Autopsy

Ova at Autopsy

-- f:;1;’ Wet

wt, mg

412 zt82c

451 9=58 399

*37 333

*85 305

+59 247

=t43 426

*74 455

*74 407

A51 376

A82

353 j=53

321 h71

Bal- looned

4

0

4

0

0

0

0

4

0

4

0

2

Process Time Time A Hr Total 4-h - - . .

Pent

ovx

ovx

ovx

ovx

ovx

Sham

Sham

Sham

Sham

Sham

Proestrus 2 PM

Proestrus IO AM

Diestrusj

4 PM Diestrusj

4 PM Diestrusj

IO AM

Diestrusj IO AM

Proestrus IO AM

Diestrusj 4 PM

Diestrusj 4 PM

Diestrusj IO AM

Diestrusj IO AM

Estrus IO AM

Estrus IO AM

Proestrus IO AM

Estrus

20

24

18

IO AM

Proestrus IO AM

Estrus IO AM

Estrus IO AM

Proestrus IO AM

Estrus IO AM

Proestrus IO AM

Estrus

42

24

48

24

18

42

24

48 IO AM

8

15

4

12

4

IO

12

4

4

4

6

IO

- _ -

8/8

13/15

114

2/I2

o/4

O/IO

12/12

214

4/4

o/4

6/6

I/IO

. .

o/8

12/12

o/4

414

o/4

6/6 O/IO

4

6

2

5

2

5

5

2

2

2

3

5

Pent = pentobarbital; Ovx = ovariectomized ; Sham = sham ovariectomized; A Hr = time elapsed between operation and autopsy. Data on 4-day and s-day rats combined. Number of 4-day rats in each experimental group indicated in table. All other symbols and explanations as in Table I.

or the estrous drop in pituitary LH content (Table 2).

However, the rise in uterine weight at proestrus was somewhat reduced by surgery at diestrus 4 PM.

By contrast, sham ovariectomy on the morning of diestrus had quite drastic sequelae. The four 4-day rats autopsied at proestrus did not show the expected rise in uterine weight, two showed only partial uterine ballooning, and both pools of pituitaries revealed low LH contents (Table 2). Of the 16 rats which were autopsied at estrus, only 6 showed vaginal cornification, visible ova, and low pituitary LH content (Table 2). The other IO

had no tubal ova by gross inspection, 2 of them (4-day rats) had ballooned uteri, and only I of the latter showed vaginal cornification at autopsy (Table 2). Pituitary LH contents were significantly heterogeneous in these rats; the mean potencies were 3.8, 6.7, 7.7, 8. I, and I I .8 pg. Uterine weights in the nonovulating rats were somewhat lower than in the ovulating rats at estrus (Table 2), but all were reduced from control estrous levels.

ovariectomy, six had shown the expected cornification at proestrus. For these six rats sham ovariectomy could conceivably have induced ovulation 24 hr early (as progesterone does (7))) so that it took place on the night of surgery, with the ova no longer grossly visible at autopsy (Table I). On the other hand, the surgery may have delayed ovulation, as in the $-day rats, perhaps by reducing estrogen secretion soon enough to block the estrous cornification, but not soon enough to block the proestrous cornification (Table 2). I f the former were true, then ova should still be detectable in serial sections of the oviducts (16; unpublished observations) ; if the latter were true, the ova would no longer be present in the oviduct. Serial sections of one oviduct from such a 5- day rat revealed no intact ova, suggesting that this rat had not ovulated recently. Thus it is probable that subsequent events are delayed, not speeded up, by sham ovariectomy at diestrus IO AM in both the 4-day and 5- day rat.

From the data in the 4-day rats (autopsied at either Sham ovariectomy after the time of LH release did not proestrus or estrus) it is clear that sham ovariectomy at alter pituitary LH contents from normal values (Table diestrus I-O AM can delay ovulation and vaginal cornifica- 3, Fig. 3). However, uterine weights showed a reduction tion. However, of the seven 5-day rats which failed to from unoperated levels; in fact, this reduction was fully show ova or vaginal cornification 48 hr after sham as severe for the rats sham ovariectomized on the morn-

8 Combined potency where more than one pool is represented (Bliss, 4). b 95% confidence limits. ’ SD. d Observation not made on one 4-day and two s-day rats. 8 Pool contained one 4-day and one s-day pituitary- data other than LH content for these rats included with other 4-day or s-day rats of same cycle stage. f Heterogeneity in potencies among pools. g As- say on one pool showed divergent slopes, so potency not calculated. h Day after metestrus. i Data from 4-day rats included in both diestrous combinations. j Day before proestrus.

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N. B. SCHWARTZ

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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. : . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

600-7

E” 500-

g 450-

3 400- u, g 350- s s 300-

g 250-

.I... . . . . . . In :ji.::ii ‘::.YA 95 % CQn f j&n

Diestrus Proestrus Estrus Metes tr us Diestrus loam loom loam loam loam

FIG. 2. Changes in pituitary LH content (upper curve) and uterine weight (lower curve) following ovariectomy. The 95% confidence limits for these variables throughout the cycle for unoperated control rats (d-day and s-day combined data from Table I) are shown in background. Starting value for operated rats at time of surgery ( l ) is simply the mean potency or uterine weight for unoperated rats autopsied at that time. Autopsy values for ovariectomized rats (0) are taken from Tables 2 and 3.

ing of metestrus or on the afternoon of estrus as for the ovariectomized rats (Table 3, Figs. 2 and 3).

5. More chronic efects of ouariectomy and sham ouariectomy. Pituitary LH content and uterus weight 3 and 4 days after ovariectomy (at IO AM on the morning of estrus) are shown at the bottom of Table 3. Uterine weight continued to drop from the value at 48 hr, but pituitary LH content did not change. One month after ovariectomy, uterine weight had dropped to a minimal value, and pituitary LH content showed the expected, very large postovariectomy rise.

By contrast, 3 days following sham ovariectomy, uterine weight had not dropped further and by the 4th day showed recovery (Table, 3), some animals (5-day cyclers) being in proestrus. Pituitary LH contents also remained at diestrous values 3 and 4 days following the sham operation.

DISCUSSION

It seems clear that the critical time during which the ovary must be secreting in order to produce the proestrous-estrous changes in uterine weight, vaginal smear,

and pituitary LH content is the 24-hr period between IO AM of diestrus and IO AM of proestrus, since ovariectomy at the beginning, but not at the end, of this period blocks virtually completely all of the expected changes (Table 2). Since the pituitary LH content rose about 3 pg between metestrus and diestrus following ovariectomy (Table 3), the LH contents at estrus of animals ovariectomized on the morning of proestrus, or the afternoon before, might conceivably represent a normal LH discharge with a 3-pg increment superimposed. However, the rats ovariectomized at diestrus IO AM show almost a 5-pg increment over control estrous LH contents suggesting at least some inhibition of the ovulatory discharge and substantiating the hypothesis (21) that the timing of the cyclic LH discharge in the rat is due to a daily (ccclock”) facilitation which occurs between 2 PM and 4 PM and an ovarian steroid feedback which reaches thres- hold levels at proestrus.

A previous study in the rat also demonstrated that ovariectomy 48 but not 24 hr before the time of vaginal cornification prevents its occurrence (I 2). Observations that estrogen induces vaginal cornification after 24-40 hr (I 2), maximum uterine weight increase in 20 hr (I),

and ovulation by 48 hr which can be blocked at 20 hr by atropine (7) support the results of the ovariectomy experiments with regard to the timing of steroid secretion.

If estrogen is secreted on the day before proestrus, gonadotrophin secretion must, of course, be responsible. Indirect evidence for this gonadotrophin release is as follows: a) between late diestrus and early proestrus, ovarian ascorbic acid and lipid concentrations drop (I 7),

luteolysis begins, and the corpora lutea are no longer capable of supporting pseudopregnancy (7) ; b) LH can induce luteolysis and is necessary for estrogen secretion by the FSH-stimulated follicle (7) ; c) antibodies to gonadotrophins block ovulation but not cornification when administered at proestrus (I 5), resembling the effects of pentobarbital (Table 2), but when administered on the day before proestrus they block the expected cornification (6), as did ovariectomy (Table 2).

A significant reduction in uterine weight occurred following ovariectomy at estrus or metestrus (Table 3, Fig. 2), suggesting that normally within 48 hr after ovulation some estrogen begins to be released. However, the effect of ovariectomy on uterine weight, as well as on other variables, is complicated by the effects of sham ovariectomy (Fig. 3). Since laparotomy or splenectomy does not reduce the uterotrophic effects of exogenous estrogen (23), the reduction in uterine weight seen following sham ovariectomy was probably due to a relative reduction in estrogen secretion secondary to a. reduction in gonadotrophin secretion. “Nonspecific’” laparotomy stressors might reduce gonadotrophin secretion by stimulating a competitive ACTH release (I 8, 22). Presumably the initial input of sham ovariectomy (as well as of ovariectomy), whatever its nature, is the same in all rats, but the final manifestations would differ depending on how much estrogen would normally be

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OVARIECTOMY ON PITUITARY LH, UTERUS, AND VAGINA

TABLE 3. Data summary of rats operated after time of LH release

I”57

Treatment

Process Time Time A Hr Total

-

_ - h-day To. of pool: Wt, mg LH content,

pg/pi tui tary Wet wt, mg

ovx Diestrush 24 9 4 4 g.yp f 9-7 IO AM (6.2-14.7)b *I .2O

ovx Metestrus 18 4 2 2 6.8 9*8 IO AM (4.8-W) %I .4

ovx Diestrush 42 7 2 3 9.9 10.7 IO AM (6.6-14.7) &I .g

ovx Metestrus 24 4 2 2 4.4 8.3 IO AM (2.8-7.1) zt2.1

ovx Diestrush 48 8 5 4 7.5 9.3 IO AM W-9-5) 3x1 .g

Sham Diestrush 24 4 2 2g 7.2 10.4 IO AM (3 -8-q 4) ItI .4

Sham Metestrus 18 IO AM

Sham Diestrush 42 4 2 2 5.4 10.4 IO AM (3.5-8.5) &I .6

Sham Metestrus 24 4 4 2 5.8 12.0

IO AM (3.8-g-o) 3x1 .g

Sham Diestrush 48 8 2 4g 8.6 10.2

IO AM (6.1-12.2) zt0.g

ovx 72 4 I 2g 5.8 II .6 IO AM (2.2-13.6) *I .3

ovx 96 4 0 2 6.2 10.3 IO AM (3.9-10.0) *I .g

Sham 72 4 I 2 5.1 II .5 IO AM (3.2-8.0) &I .8

Sham 96 4 0 2g 6.1 13.1 IO AM (3. I-I I .8) &I .6

ovx 30 6 2 34.8 II .6 (22 l I-54*9) ZtI .3

II AM days

All symbols and explanations as in Tables I and 2. No animal autopsied at 48 hr or less after surgery showed cornification, ova, or uterine ballooning. One s-day sham-operated rat autopsied after 72 hr and two s-day sham-operated rats autopsied after g6 hr had ballooned uteri, one of the latter rats also demonstrating vaginal cornification. None had ova at autopsy.

Metestrus IO AM

Estrus 4 PM

Estrus 4 PM

Estrus IO AM

Estrus IO AM

Metestrus IO AM

Estrus 4 PM

Estrus 4 PM

Estrus IO AM

Estrus IO AM

Estrus IO AM

Estrus IO AM

Estrus IO AM

Estrus IO AM

Proestrus IO AM

290 rt31”

279 *40 284

*38 290

A42 276

j=36 292

It56

289 *40

337 &I5

336 *35

259 A32

209 *56 352

*20

433 -9

131 &II

Autopsy No. of Rats Pituitary Uterus

secreted at the particular cycle stage. When ovariectomy (at proestrus IO AM) was ineffective with respect to uterine weight the sham operation was also (Table 2);

when ovariectomy reduced uterine weight at the other cycle stages (Fig. 2), sham ovariectomy did also, but to a lesser extent (Fig. 3). However, on the highly critical day of diestrus sham ovariectomy prevents, in many rats, much of the expected uterine, vaginal, and pituitary change of proestrus and estrus (Table 2), thus delaying the completion of the cycle. Similarly, in hamsters, which are usually highly regular 4-day cyclers, unilateral ovariectomy during the day before proestrus can induce a delay (24 hr) in ovulation time ( I I ) .

The picture which emerges for the 4-day rat with respect to pituitary and ovarian events is as follows. Soon after ovulation, follicular growth begins, and proceeds at a linear rate until the preovulatory swelling (5). The follicles begin secreting estrogen as early as the day of estrus (Table 3). Pituitary LH content rises from the time of estrus to the morning of proestrus (2 I, Table I)

in spite of the fact that release must be occurring in order for estrogen secretion to occur (7). On the day before proestrus a final larger release of estrogen occurs (Table

2, Fig. 2), which is responsible for the obvious estrogen- induced changes (uterine, pituitary, and vaginal) of proestrus and estrus. Finally, when the ovulating surge of LH begins acting on the follicle, estrogen production probably decreases to minimal values, since a) ovariectomy at proestrus had no effect on uterine weight, which drops rapidly anyway at this time in normal rats (Tables I and 2, Fig. 2); and b) vaginal cornification disappears by metestrus (Table I). Not all of the drop in uterine weight in unoperated animals between proestrus and estrus (Table I) can, however, be explained on the basis of a decrease in estrogen secretion, since Astwood showed (2) that the uterine weight response to exogenous estro gen is also reduced at this time. He suggested that this is due to a preovulatory secretion of progesterone. That progesterone is, in fact, secreted before ovulation has recently been demonstrated by measurements of progesterone in rat plasma (24).

In the 5-day rat, follicular growth follows the same course as in the 4-day rat except that preovulatory swelling is delayed by 24 hr (5). Some estrogen secretion starts as early as the day of estrus (Table 3), but the accumulation of LH in the pituitary is delayed in the 5-day rat

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1258 N. B. SCHWARTZ

q g~%Confide/lce L hits

for mopefated Rufs 600

g 500

g 450 .- s 400 m g 350. 5 G 300 z )- 250

Diestrus loam

Proestrus loam

Estrus loam

Metes t rus Diestrus loam loam

FIG. 3. Changes in pituitary LH content (upper curve) and uterine weight (lower curve) following sham ovariectomy (see legend for Fig. 2). “Estrous” autopsy data for rats sham ovariectomized at rats which had

AM On the day of diestrus are shown separately for ova at the time of autopsy (uninterrupted line) and

for those which did not (interrupted line).

(Table I). However, it is unlikely that this is the primary cause of the 24-hr delay in ovulation in all 5-day cyclers, since the lag m LH storage was not uniformly observed in a previous study (21) and, moreover, electrical stimulation of the preoptic area at 2 PM on this day can cause ovulation in some 5-day rats (8), indicating that adequate pituitary LH release is possible. Since most 5-day rats are cornified at proestrus (7, 2 I, Table I) and a few show an

REFERENCES

I. ASTWOOD, E. B. A six-hour assay for the quantitative deter- mination of estrogen. Endocrinology 23 : 25-3 I, 1938.

2. ASTWOOD, E. B. Changes in the weight and water content of the uterus of the normal adult rat. Am. J. Pliysiol. I 26: 162- 1707 19390

3. BLISS, C. I. Analysis of the biological assays in U.S.P. XV. Drug Std. 24: 33-68, I 956.

4. BOLING, J. L., AND R. J. BLANDAU. The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25 : 359-364, I 939.

5. BOLING, J. L., R.J. BLANDAU, A. L. SODERWALL, AND W. C. YOUNG. Growth of the Graafian follicle and the time of ovulation in the albino rat. Anat. Record 79: 313-331, 1941.

6. BOURDEL, G., AND C. H. LI. Effect of rabbit antiserum to sheep pituitary interstitial-cell stimulating hormone in adult female rats. Acta Endocrinol. 42 : 473-479, 1963.

7. EVERETT, J. W. The mammalian female reproductive cycle and its controlling mechanisms. In: Sex and Internal Secretions,

increase in uterine weight before proestrus (Table I),

some increase in estrogen secretion must have occurred by diestrus day I (as in the 4-day rat). However, since ovariectomy on the day before proestrus had the same consequences for 4- and 5-day rats (Table 2) most of the critical steroid secretion must have been delayed in the 5-day rats. Progesterone administration to 5-day rats on the day before proestrus causes ovulation 24 hr early (T), suggesting a relative progesterone deficiency. The progesterone might be acting by inducing LH storage (IO, I 3) or by lowering neural thresholds (I 4). Taking all of the data into account it seems likely that the 5-day rat secretes estrogen or progesterone, or both, earlier in the cycle than the 4-day rat with respect to the day which will be proestrus, but later with respect to the time of last ovulation, thus delaying uterine weight increase and vaginal cornification by less than 24 hr, but delaying the beginning of LH release (7) by exactly 24 hr, the latter function depending not only on steroid levels but on a clock facilitation as well.

Although evidence is clear that ovariectomy of I week’s duration or more causes a large increase in pituitary LH content (20, Table 3), as well as an increase in blood levels (20), an increase in pituitary LH above proestrous levels does not occur before at least one cycle length has elapsed (Table 3), suggesting that withdrawal of steroids must be more “chronic” than one cycle length before appreciably increased gonadotrophin storage takes place. When given acutely, estrogen or progesterone can enhance LH release (7) but when given chronically, estrogen suppresses LH storage and release (g), while progesterone suppresses ovulation and increases pituitary LH content (I 3). Any theory of regulation of reproductive cyclicity must ultimately account for these differences in the effects of acute vs. chronic changes in ovarian steroid levels on pituitary gonadotrophin storage and release.

The author thanks the Endocrinology Study Section, National Institutes of Health, for supplies of NIH-LH-SI and NIH-LH-S2; William Talley and Jack Thiel for technical assistance; and Dr. J. P. Marbarger and the staff of the Aeromedical Laboratory for providing environmentally controlled animal housing.

edited by W. C. Young. Baltimore : Williams & Wilkins, I 961, vol- 17 PP. 497-555.

8. EVERETT, J. W. The preoptic region of the brain and the re- lation to ovulation. In: Control of Ovulation, edited by C. A. Villee. New York : Pergamon, I g6 I, pp. I OI -I 2 I.

g. GANS, E., AND G. P. VAN REES. Effect of small doses of oestra- diol benzoate on pituitary production and release of I.C.S.H. in gonadectomized male and female rats. Acta Endocrinol. 39 :

245-252, 1962. IO. GORSKI, R. A., AND C. A. BARRACLOUGH. Adenohypophyseal

LH content in normal, androgen-sterilized and progesterone- primed sterile female rats. Acta Endocrinol. 39 : I 3-2 I, I 962.

I I. GREENWALD, G. S. Temporal relationship between unilateral ovariectomy and the ovulatory response of the remaining ovary. Endocrinology 7 I : 664-666, I 962.

I 2. HEMMINGSEN, A. M. Studies on the oestrus-producing hormone (oestrin). Skand. Arch. Physiol. 65 : 97-250, 1933.

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OVARIECTOMY ON PITUITARY LH, UTERUS, AND VAGINA I259

13, HOFFMANN, J. C., AND N. B. SCHWARTZ. “Progesterone-withdrawal ovulation” in the rat. Federation Proc. 23 : 109, I 964.

I 4. KAWAKAMI, M., AND C. H. SAWYER. Neuroendocrine cor- relates of changes in brain activity thresholds by sex steroids and pituitary hormones. Endocrinology 65 : 652-668, I 959.

15. KELLY, W. A., H. A. ROBERTSON, AND D.A. STANSFIELD. The suppression of ovulation in the rat by rabbit anti-ovine-LH serum. J. Endocrinol. 27 : I 27-128, I 963.

16. LONG, J. A., AND H. M. EVANS. The oestrous cycle in the rat and its associated phenomena. Mem. Univ. CaZif. 6 : I -I 48, 1922.

17. MILLS, J. M., AND N. B. SCHWARTZ. Ovarian ascorbic acid as an endogenous and exogenous assay for cyclic proestrous LH release. Endocrinology 6g : 844-850, I 96 I.

18. MOORE, W. W. Failure of adrenergic and cholinergic blocking agents to block ovulation in the rat. Am. J. Physiol. 200: I 2g3- 1295, 1961.

19. PARLOW, A. F. A rapid bioassay method for LH and factors stimulating LH secretion. Federation Proc. I 7 : 402, 1958.

20. PARLOW, A. F. Plasma and pituitary LH: detection and quantitative bioassay in the rat following gonadectomy. Endocrinol. Sot. Progr. 4rst Meeting pp. 46-47, 1959.

21. SCHWARTZ, N. B., AND D. BARTOSIK. Changes in pituitary LH content during the rat estrous cycle. Endocrinology 71 : 756- 762, 1962.

22. SCHWARTZ, N. B., AND L. S. BOSWELL. Independence of LH and ACTH release from the rat pituitary. Endocrinology 63 :

yg-32% I@* 23. SZEGO, C. M., AND S. ROBERTS. Pituitary-adrenal cortical

antagonism to estrogenic stimulation of the uterus of the ovariectomized rat. Am. J. Fhysiol. 152 : I 3 I -I 40, 1948.

24. TELEGDY, G., AND E. ENDROCZI. The ovarian secretion of progesterone and 20 a hydroxypregn-4-en-3-one in rats during the estrous cycle. Steroids 2 : I 19-I 23, r 963.

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acute effects of ovariectomy on pituitary lh, uterine weight, and vaginal cornificatiod 2€¦ ·...

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