CHEMICAL ESTIMATION OF URINARY

ESTROGENS OF GILTS

by

Alan Marquis Bowerman

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment

The Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Major Subject: Animal Reproduction

Approved:

Signature was redacted for privacy.

Signature was redacted for privacy. Hëàd of TfêTjor Department

Signature was redacted for privacy.

Dean/5f Gradua

Iowa State University Of Science and Technology Ames, Iowa

1963 il

TABLE OF CONTENTS

Page

INTRODUCTION 1

REVIEW OF LITERATURE . . 2

EXPERIMENTAL PROCEDURE 13

RESULTS AND DISCUSSION 22

.SUMMARY 32 LITERATURE CITED 34

ACKNOWLEDGMENTS 41 1

INTRODUCTION

The physiological mechanisms controlling the endocrine activities of the mammalian ovary are complex and relatively little is known about them. In the non-pregnant female corpora lutea remain functional for a relatively short interval in marked contrast to the pregnant animal in which these glands usually persist for the duration of pregnancy. Further know­ ledge of the factor or factors responsible for the failure or maintenance of corpora lutea would be valuable to animal scien­ tists for improving reproductive efficiency and to clinicians for treatment of reproductive disorders. It has been known for many years that corpora lutea persist similar to pregnancy in the absence of the uterus. Recent evidence has suggested that uterine metabolism of some substance or substances is involved in the control of the life span of corpora lutea. Since estrogen administration has been demonstrated in many cases to be bene­ ficial to luteal function it was the object of this investiga­ tion to examine the role of the uterus in the metabolism of estrogen. Chemical estimation of estrogen metabolites occurring in urine was utilized to evaluate estrogen metabolism in the gilt. 2

REVIEW OF LITERATURE

Natural Estrogens

Numerous reviews have been presented on the occurrence,

chemistry and biology of mammalian estrogens. Among the most

recent and comprehensive are those of Boscott (1962), Eckstein

(1962), Emmens (1959) and Velardo (1958). All estrogens iso­

lated from mammalian tissues and fluids are characterized by

the cyclopentanoperhydrophenanthrene nucleus with a phenolic A ring with a hydroxyl group on C-3 and the absence of a 19 car­

bon atom.

HO'

Figure 1. Basic structure of estrogens

These estrogens have been isolated from ovaries, testes, adre­ nals, placentae, blood and principally urine. The urinary estrogens are predominently conjugated with glucuronic or sul­ phuric acid. The most commonly occurring ones are estradiol-l? beta, estrone and estriol. In addition estradiol-l^ alpha, equilin, equilenin, l6-epiestriol and the three most common estrogens with various substitutions including hydroxyl and 3 methoxyl groups at the 2 position, a hydroxyl group at the 6 position and various combinations of hydroxyl and ketonic groups at the 16 and 17 positions have been isolated and characterized for the most part from human pregnancy urine.

Estrogenic activity of sow ovaries was first demonstrated by Allen and Doisy (1923) in the follicular fluid. The active principles in this preparation were subsequently identified as estradiol-l^ beta (MacCorquodale et al., 1936) and estrone

(Westerfeld et al., 1938). Estrogenic, activity was observed in sow urine by Struck (1935)• In an attempt to characterize the estrogens in swine urine, Bredeck and Mayer (1958) reported estrone and estriol in ether extract of acid hydrolyzed urine.

Lunaas (1962), Raeside (1961a) and Velle (1959) have been un­ able to confirm the presence of estriol in sow urine but agree that estrone is the main excretory estrogen with possibly a small amount of estradiol-l^ beta.

Bioassay

Most techniques for biological assay of estrogens involve the administration of the test subtance to ovariectomized or immature female rodents. One widely used method is the Allen-

Doisy test which depends on the appearance of a cornified vaginal smear in ovariectomized mice or rats; this responce is typical of estrogen stimulation (Allen and Doisy, 1923). In the original method the test subtance was administered subcutaneously 4

in oil. Many modifications of the Allen-Doisy test have been

devised (Emmens, 1962). The method of administration may be

subcutaneous, peracutaneous, oral, intraperitoneal, intravenous

or intravaginal with the latter being of considerable impor­

tance due to its greater sensitivity. The number and spacing

of administrations varies from a single one to 6 or 8 spaced

over a 2 to- 3 day period. Different methods are used for taking

and evaluating vaginal smears.

Another assay is based on the increase in uterine weight

following subcutaneous administration of the test subtance in

oil to ovariectomized or immature female rats. Other methods used to a lesser extent involve observations on vaginal open­ ing, mitotic and metabolic changes of the vagina in rats and

mice, oral dosage to chicks with the end point based on oviduct

weight and an in vitro method based upon the reduction of DPN

with the reduced DPN measured by spectrophotometry (Emmens,

1962). The disadvantages of these bioassays are numerous. Fore­

most are cost and time consumed due to maintenance of an animal colony and the large number of animals which must be injected and/or smeared. Different estrogens give different responses due to varying potencies. Likewise, potencies vary with the

type of bioassay used. It has also been demonstrated the non­ es trogenic substances added to estrogen extracts can inhibit or potentiate the response. 5

Bioassay procedures have been indispensable in the past due to the lack of chemical methods with sufficient sensitivity to measure estrogens and estrogen metabolites normally occur­ ring in urine and blood. These techniques still remain invalu­ able in the determination of estrogenic activity of chemically unidentified compounds.

Chemical Assay

Until the past several years chemical methods for estrogen assay had sufficient sensitivity only for urine of late preg­ nancy in which several mg. of estrogen are excreted daily.

Since then, at least three methods have been developed which have sufficient sensitivity and specificity to measure estrone, estradiol-17 beta and estriol. All of these methods utilize

15 vol. % HC1 for hydrolysis of the estrogen conjugates.

The first and most widely used of these methods is that of Brown (1955)• Additional steps have been added to this method (Brown et al., 1957; Salokangas and Bulbrook, 1961). It involves a double extraction sequence, formation of the 3-methyl ethers of the 3 estrogens, followed by absorption chromatog­ raphy of these ^ethers on alumina columns and detection of the ethers by the Kober reaction. The Kober chromogens are extract­ ed and analyzed spectrophotometrically. The sensitivity of this method is 3 meg. of each of the estrogens per 24 hr. sam­ ple of human urine. 6

The method of Bauld (1956) although developed Independent­

ly Is basically the same as that of Brown. It differs in that

the estrogens are not methylated and that they are separated by

partition chromatography on Celite columns. This method also

has a sensitivity of 3 meg. of each estrogen per 24 hr. human urine sample.

A single extraction sequence, partition column chromatog­

raphy and detection of the estrogens directly by sulphuric

acid fluorescence are used by Preedy and Aitken (1961). A

Celite column used in this method is developed with three suc­

cessive mobile phases and is collected in one ml. fractions.

This method can measure down to 1.0 meg. estrone, 1.8 meg.

estradiol-17 beta and 3-° meg. estriol per 24 hr. sample. The

chromatography in this method requires 36 hr. and each urine

sample analyzed requires 75 to 80 fluorescent determinations.

• • Ichii et al. (1963) have recently described a method for

blood plasma based on fluorescent determination which has a

sensitivity of 0.002 meg. estrogen/lOOml. plasma. This method,

if proven practical, has the sensitivity required to measure circulating estrogens during the menstrual cycle. Recently gas chromatographic methods have been developed for estrogen assay (Fishman and Brown, 19*52; Lukkainen et al.,

1962; Wotiz and Martin, 1962). However, these methods have sensitivity down only to 150 meg. estrogen per 24 hr. sample. The chromatographic and other purification techniques which 7

have been developed during the past several years have brought

the routine use of chemical assay of menstrual cycle urine in

clinical laboratories. Continuous improvement of these tech­ niques makes it highly probable that blood estrogens during the

menstrual and estrual cycles will be studied in the near future, further elucidating the processes of reproductive phenomema.

~ ifluence of Estrogen and the Uterus on Luteal Function

The effects of exogenous estrogen and progesterone on luteal function have been studied in several species in various physiologic and experimental conditions. The results of pro­ gesterone administration have varied from no effect in some species to complete luteal failure in others (Amoroso and Finn,

1962). However, exogenous estrogen has generally been bene­ ficial to luteal function.

Estrogen treatment has been shown to induce formation of corpora lutea in prepubertal rats (Hohlweg, 193^) and to in­ crease the size and cause persistance of these glands in cycling rats (Maekawa, 1956; Merckel and Nelson, 19^0; Selye et al.,

1935; Wolfe, 1935). Chu and You (19^5) reported inhibition of ovarian activity by estrogen administration to rabbits which were neither pregnant nor pseudopregnant but Sawyer (1959) observed spontaneous ovulation following estrogen treatment during winter and spring but not in summer and early autumn 8 indicating that the response may be dependent on season in this species. Gardner et al. (1963) and Kidder et al. (1955) observed a lengthening of the estrous cycle in gilts when daily administration of estrogen was began on day 11 of the cycle but not when it was began on day 6. Corpora lutea persisted for prolonged periods in dairy cattle implanted with stilbestrol for 34 to 190 days (Hammond and Day, 1944). This observation was not confirmed by Greenstein et al. (1958) and Loy et al.

(i960) in dairy heifers injected with estrogen from days 2 to 12 and 1 to 13, respectively, of the estrous cycle. These experiments suggest that the time of initiation and the dura­ tion of estrogen treatment may be important in prolonging the life of corpora lutea in swine and cattle.

Exogenous estrogen has been shown to prolong the life span of corpora lutea in pseudopregnant (Hammond, 1956; Heckel and Allen, 1936), hysterectomized-pseudopregnant (Chu et al., 1946;

Greep, 1941) and pregnant rabbits (Heckel and Allen, 1939)• Estrogen administration hastened implantation in pregnant, lactating rats (Weichert, 1942) and induced ovulation in preg­ nant rats (Everett, 1947). Recently Barnes and Meyer (i960) have shown that estrogen is essential for implantation in rats. There is evidence that the luteal stimulating influence of exogenous estrogen acts in two modes, indirect stimulation by causing release of gonadotropic hormones from the pituitary and by direct stimulation of the ovary or luteal tissue. Everett 9

(1947) could induce ovulation with injections of either estrogen

or L H (luteinizing hormone) in intact pregnant rats but could

induce ovulation only with L H in hypophysectomized pregnant rats with corpora lutea maintained with L H - free lactogen,

suggesting that estrogen caused ovulation by effecting the re­ lease of L H from the pituitary. Estrogen administered to immature rats evoked an increase in pituitary weight followed by a decrease in gonadotropic potency and an increase in ovar­ ian weight. However, when animals were spayed prior to estro­ gen treatment the increase in pituitary weight followed but its gonadotropic content did not decrease suggesting that the ovary potentiates the release of hypophyseal gonadotropins by estro­ gen secretion (Bradbury, 1947). Estradiol added to a culture of rat anterior pituitary cells stimulated production of pro­ lactin which is luteotrophic in the rat (Nicoll and Meites, 1962). When estradiol was implanted in the arcuate nucleus of the rat hypothalamus (Lisk and Newlon, 1963) and in the poste­ rior median eminence of rabbits (Davidson and Sawyer, 1961) ovarian atrophy similar to that of hypophysectomy followed, indicating that pituitary gonadotropin secretion was suppressed.

Several workers have presented evidence that the bene­ ficial effect of estrogen on corpora lutea is a direct one.

Estrogen administration retarded ovarian atrophy following hypophysectomy in rats (Williams 1944; 1945) and maintained corpora lutea following pituitary ablation in pregnant rats 10

(Lyons et al., 1943), pseudopregnant rabbits (Westman and

Jacobsohn, 1937)> hysterectomized-pseudopregnant rabbits (Greep,

19^1) and pregnant rabbits (Robson, 1939)• The ovarian re­ sponse to exogenous gonadotropins was enhanced in intact and hypophysectomized rats by estrogen treatment (Meyer and Brad­ bury, I960; Pencharz, 1940; Williams, 1940). HBhn and Robson (1949) observed equal maintenance or degeneration between con­ trol corpora lutea and those implanted with crystalline estro­ gen in pseudopregnant rabbits while Hammond and Robson (1951) subsequently found that direct application of estrogen to rab­ bit corpora lutea maintained adjacent luteal cells while luteal cells in other corpora failed following hysterectomy, parturi­ tion or the end of pseudopregnancy. Bradbury (1961) applied crystalline estrogen to one ovary of immature rats leaving the other ovary untreated and observed a greater increase in weight, formation of corpora lutea and an increased response to endo­ genous and exogenous gonadotropins in the treated ovary.

Results of Heckel (1942) suggested estrogen metabolism by the uterus was related to the life span of corpora lutea. Re­ moval of part of the uterus in rabbits prolonged pseudopreg­ nancy in proportion to the amount of uterus extirpated. An amount of exogenous estrogen insufficient to increase the life span of corpora lutea in intact animals prolonged pseudopreg­ nancy in partially hysterectomized animals passed that of simi­ larly hysterectomized controls. The presence of the uterus 11 under progesterone stimulation was reported to facilitate the conversion of estrone to estriol in rabbits (Pincus, 1937) and in humans (Smith et al., 1938). The absence of the uterus or progesterone stimulation of the uterus resulted in less estriol and less total estrogen recovered in the urine after adminis­ tration of exogenous estrone indicating that the progesterone stimulated uterus influenced estrogen metabolism. However, using advanced chemical techniques, Heard et al. (1941) could not confirm these observations in the rabbit.

Loeb (1923) observed an increase in the life span of corpora lutea in the guinea pig following hysterectomy during the luteal phase of the estrous cycle. Uterine removal at this time also increases the life span of corpora lutea in the cow, ewe and sow. Autotransplants of the uterus in pigs (Anderson et al., 1963b) and guinea pigs (Butcher et al., 1962a) resulted in a normal or only slightly prolonged estrous cycle in these species. Butcher et al. (1962b) observed a relationship between the life span of corpora lutea and the amount of uterine and luteal tissue in guinea pigs. Unilateral ovariectomy at the time of partial hysterectomy shortened the estrual interval to 18 days as compared to 22 days following hysterectomy alone.

Unilateral ovariectomy with the uterus intact had no effect on the life span of the remaining corpora lutea. These results suggest that some ovarian substance which acts independently of uterine innervation may be involved in the uterine influence on 12 the life span of corpora lutea. This possibility is augmented by the results of Du Mesnil Du Buisson (1961) and Bathmacher.

Severing one of the uterine horns from the body of the uterus in pigs prevented conception in these animals except when functional corpora lutea were absent from the ovary on the side of the sterile uterine horn. Conception was not prevented in control operations in which one horn of the uterus was com­ pletely removed. Corpora lutea were always present on both ovaries in these control animals.

1Rathmacher, R. P., Ames, Iowa. Sterile uterine horn and embryonic survival in pigs. Private communication. 19&3. 13

EXPERIMENTAL PROCEDURE

Urine Collection

Urine was analyzed from crossbred gilts weighing 240 to

350 lb. when urine collection was initiated. Four intact, two

subtotally hysterectomized (tubo-uterine junction to mid-cer­ vix), two ovariectomized and two ovariectomized-subtotally

hysterectomized animals were used. Corpora lutea in ovaries

of hysterectomized animals were marked with sterile animal

charcoal at surgery, 5 or 6 days following the onset of estrus.

The intact and hysterectomized animals were checked daily for

the occurrence of estrus with vasectomized boars.

Urine was collected for 11 days from two of the intact

animals commencing at day 15 of the estrous cycle. The other

two intact gilts were bred and urine was collected days 15 to

25, 30, 31, 40, 41, 50, 51, 75, 76, 100, 101 and 110 to farrow­ ing. A similar collection schedule was followed in the hyster­

ectomized animals except that the collections had to be inter­ rupted between days 15 and 25 due to cystitis and that the animals were collected 110, 111, 120, and 121 days following

the estrus prior to surgery. Urine was collected from the

ovariectomized and ovariectomized-hysterectomized•animals for

two-six day periods, one in which they were injected with 400

meg. estradiol benzoate per 100 lb. body wt. and the other in 14

which they were injected with estradiol benzoate at the same

rate in combination with 100 mg. progesterone per 100 lb. body

wt. The hormones were administered in a single injection 24

hr. after initiation of urine collection which was continued

for 4 or 5 days thereafter. One animal in each group received

estradiol benzoate first while the other two received the com­

bination first. The hormones were administered intramuscularly in sesame oil containing 100 meg. estradiol benzoate per ml. and in addition, 25 mg. progesterone per ml. when this steroid was given concurrently. A minimum of 10 days was allowed to elapse between injections.

Samples were collected and analyzed on a 24 hr. basis from days 15 to 25 with the cycling, pregnant and hysterectomized animals, days 110 to farrowing with the pregnant animals and all days with the ovariectomized and ovariectomized-hyster- ectomized animals. The remainder of the samples were collected and analyzed on a 48 hr. basis.

Urine was collected by means of a self-retaining catheter with a 30 ml. bulb filled with sterile water placed in the bladder. A size 20 or 28 (French scale) catheter was used depending on the condition of the animal. The catheter was connected to an 8 or 20 liter polyethylene bottle with a length of 1/4 in. I. D. polyvinyl tubing. The animals were restrained in farrowing crates during collection periods. Bladder inflam­ mation and irritation were reduced by irrigating the bladder 15

twice daily with 50 to 100 ml. of a one to seven dilution of a

solution of 5 lb. citric acid, 8 oz. magnesium oxide, heavy,

and 11 oz. sodium carbonate monohydrate in 5 gal. water^.

Chemical Assay

Aliquots of the urine taken for analysis generally amounted to approximately 10$ of the 24 hr. urine output. The principal exception to this was late pregnancy where only 5 to 25 ml. aliquots were taken. With the exception of samples from late pregnancy which were diluted to 100 or 200 ml. with water, aliquots were diluted to 500 ml. for acid hydrolysis. Dupli­ cate analyses were run on each sample.

All chemicals used in the chemical analysis were reagent grade with further purification as described by Bauld (1956).

The estrogens in the urine were hydrolyzed, extracted, purified and separated as described by Baeside (196la). The method in­ cluded hydrolysis by refluxing for 1 hr. after the addition of

15% concentrated hydrochloric acid. Hydrolysates were ex­ tracted once with 300 ml. and twice with 125 ml. diethyl ether.

The pooled ether extracts were washed with 100 ml. concentrated

"'"Solution G., Urinary tract irrigant, Sunnybrook Hospital, Toronto.

p J. Baeside, J. I. Ontario Veterinary College, Guelph, Ontario. A chemical estimation of urinary estrogens in the pig. Private communication. 1962. 16

carbonate buffer (130 ml. 5N. sodium hydroxide made up to 1 liter

with 1 M. sodium bicarbonate). The wash was discarded and the

ether extract was partitioned with 25 ml. 2 N. sodium hydroxide and without discarding the sodium hydroxide, 100 ml. of 1 M.

sodium bicarbonate was added and the extract was partitioned

again. The aqueous solution was discarded and the ether ex­

tract was washed successively with 25 ml. of 1 M. sodium bicar­ bonate and 25 ml. water. Ether extracts were then evaporated

to dryness under reduced pressure. Columns for adsorption chromatography were prepared by pouring 5 g. deactivated alumina (Merck 71707 containing 9.5% water) into a glass chromatography tube (10 mm. I. D. with a sintered glass disk) containing water saturated benzene. The top of the column was protected by the addition of 1 to 2 mm. of acid-washed sand added after the alumina had settled under gentle tapping. The dried ether extracts were added to the columns with 3 times 2 ml. 2% ethanol in benzene and the columns were eluted with an additional 20 ml. of the solution. Eluates were collected in 125 ml. separatory funnels containing 25 ml. n-hexane and 25 ml. water. After partitioning the organic phase against the water the latter was discarded and the estrogens were extracted from the hexane-benzene mixture with 2 times 25 ml. 1.6% sodium hydrox­ ide. The alkaline solutions were collected in 250 ml. flat bottom flasks and saponified by refluxing for 30 min. following 17

the addition of 1.2 g. sodium hydroxide pellets. The solutions

were cooled, acidified with 5 ml* 12 N. sulphuric acid and ex­

tracted once with 50 ml. benzene. The benzene was washed once

with 10 ml. 1 M. sodium bicarbonate and twice with 20 ml. water and then evaporated in vacuo at 60° C.

Partition chromatography was accomplished on columns pre­

pared with diatomaceous silica (Celite 535» Johns-Manville). The solvent system was composed of 80$ aqueous methanol as the

stationary phase, 3 parts'n-hexane plus 2 parts benzene as

mobile phase I and 2 parts n-hexane with 3 parts benzene as

mobile phase II. The phases were equilibrated by shaking in one liter separatory funnels and allowing them to separate overnight. Columns were prepared by adding 25 ml. of the

stationary phase to 25 g. Celite and stirring for 3 min. Mobile

phase I was then added to form a slurry. The slurry was added in small quantities to chromatography tubes 300 mm. long and

10 mm. I. D. The tops of the tubes had 14/35 standard taper

joints for attachment of reservoirs and the bottoms were sealed,

flattened and perforated with about 8 pinholes. The added

slurry was homogenized by rapid strokes of the packing plunger (a disk with 0.71 mm. perforations which was only slightly

smaller in diameter than the tube and was attached to a rod

with a stirrup) and packed in 1-2 mm. segments by slower strokes. The columns were packed to a height of 15 cm. and had flow

rates of approximately 15 ml. per hr. Columns required 18 approximately 5 g. Celite with 5 ml. stationary phase.

The purified urine extracts were added to the columns with 3 times 1 ml. mobile phase I. Reservoirs containing 30 ml. this solution were then attached to the columns. After discarding the first 10 ml. of eluate and collecting the next

2 ml. as a "pre-cut" fraction the remainder was collected as the estrone fraction. The reservoir was then filled with 40 ml. mobile phase II and the eluate collected in a flask as the estradiol fraction. The solvents were evaporated from the estrogen fractions in vacuo at 60° C.

The estrone fraction was further purified with Girard1s reagent T. Two ml. absolute ethanol, 0.5 g. Girard's reagent

T. and 0.5 ml. glacial acetic acid were added to the residue and the mixture was heated for 15 min. at 80° C. in a water bath. The contents of the flask were cooled, 50 ml. water was added and the whole was extracted with 75 ml. diethyl ether. The ether was discarded and the aqueous portion was returned to the flask, acidified with 7.5 ml. concentrated HC1 and heated on a water bath at 100° C. for 15 min. After hydrolysis of the complex the free estrone was extracted with 50 ml. benzene and the benzene was washed twice with 10 ml. water and evaporated in vacuo at 60° C.

The estradiol and estrone fractions were transferred to glass stoppered centrifuge tubes containing 50+5 mg. hydro- quinone with 3 times 2 ml. benzene and taken to dryness under 19 nitrogen on a water bath at 55° C.

The color reagents for developing the Kober chromogens of estrone and estradiol consisted of 1 liter of 66 and 60% sulphuric acid (v/v), respectively, containing 10 mg. sodium nitrate, 20 mg. resublimed quinone and 20 g. hydroquinone.

Duplicate standards for each color reaction were prepared by evaporating 0.1 ml. ethanol containing 5 meg. recrystallized estrone and estradiol-l? beta"'" in the presence of 50 + 5 mg. hydroquinone. Blank tubes containing hydroquinone alone were also prepared.

The color reagents were added to the appropriate tubes with 10 ml. burettes (3.0 ml. per tube). Color tubes were then heated in a boiling water bath for 20 min. with shaking during the third and tenth min. of heating. The tubes were then cool­ ed in an ice bath and 50 + 5 additional mg. hydroquinone was added. Water (0.3 ml.) was then added to each of the estrone tubes and 0.3 ml. of estradiol color reagent was added to each of the estradiol tubes. All tubes were shaken and replaced in the water bath for 15 min. with shaking again during the third and tenth min. of heating. After heating the tubes were

/ again removed to the ice bath.

After cooling, 3*3 ml. ice water was added to each color tube and the Kober chromogens were extracted from the aqueous

"'"Kindly supplied by Dr. J. K. McGowan; G. D. Sear le and Co., Chicago. 20

solution with 4 ml. tetrachloroethane containing 2% p-nitro- phenol. Optical densities of the solutions at 508, 539 and

570 millimicrons were then determined with a Beckman DU spectro­ photometer. The concentration of estrone or estradiol in each sample was calculated using Allen's correction formula where

the mean of the optical densities at the short and long wave lenths, 508 and 570 millimicrons, was subtracted from the optical density at the median wavelength, 539 millimicrons, which is the wavelength of maximum absorbance of the Kober chromogens in tetràchloroethane containing 2% p-nitrophenol.

In recovery experiments 4 aliquots of urine were hydro- lyzed and 5 or 10 meg. of estrone and estradiol-17 beta were added to 2 of the aliquots following hydrolysis.

Placentae were collected from one of the animals studied through pregnancy at the time of farrowing. Two 75 g. aliquots of this tissue were extracted by the method of Diczfalusy and

Magnusson (1958). Their method included 25$ saturation of the tissue with ammonium sulphate and disintegration with a Waring blender, extraction twice with 100 ml. 80$ (v/v) ethanol, centrifugation, further disentegration of the tissue by grind­ ing in a mortar with quartz sand until a fine paste was obtain­ ed and extraction of the paste 3 times with 25 ml. portions of absolute ethanol. The ethanol extracts were combined, stored at -17° C. for 24 hr. and centrifuged at -12° C. The clear supernatent was evaporated under reduced pressure, brought to a 21 volume of 5° ml. with water and extracted 4 times with 25 ml. ether and once with 50 ml. toluene. This toluene-ether extract contained the free estrogens. The aqueous phase containing the conjugated estrogens was brought to a volume of 100 ml. with water and the conjugated estrogens were hydrolyzed by reflux­ ing for 1 hr. after the addition of 15 volume percent hydro­ chloric acid. The hydrolysate was extracted 4 times with 50 ml. ether and once with 50 ml. toluene. Both ether-toluene extracts were evaporated to dryness, and handled in the same manner as the ether extracts of hydrolyzed urine.

Biological Assay

Estrone was isolated from the urine of animals in three physiological states. These samples were from untreated ovariectomized and ovariectomized-hysterectomized animals, an animal at the onset of estrus and an animal just prior to farrowing. The chemical isolation described previously for urine was used and the estrone fractions were collected from several Celite columns, pooled and dissolved in sesame oil for bioassay. Bioassay was accomplished in ovariectomized rats (Allen and Doisy, 1923) by subcutaneous administration in two injections spaced approximately 8 hr. apart and vaginal smears obtained approximately 24, 40, 48 and 64 hr. following the last injection1.

"'"Assay performed by The Endocrine Laboratories, Madison 1, Wisconsin. 22

RESULTS AND DISCUSSION

In recovery experiments 88.8 +6.3 (S. D.)% of the estrone and 72.5 + 7.3$ of the estradiol-17 beta added to urine hydro- lysates were recovered. Evidence has been presented which suggests that free estrogens are less labile to destruction during acid hydrolysis than the conjugated forms. (Katzman et al., 1954). Therefore the estrogens were added following hydrolysis and no adjustment for recovery was made to the chemical estimates obtained.

Estrone samples subjected to bioassay had biological potencies equal to 7^.4 +5-9 (S. D.), 77.1 + 11.1 and 57.2 + 9.8$ of the chemically estimated quantities of estrone in ex­ tracts of urine collected at the onset of estrus, during the last week of pregnancy and prior to hormone administration in ovariectomized and ovariectomized-hysterectomized animals, respectively. These results suggest that urine containing large quantities of estrogen possessed approximately 75$ of the chemically estimated quantities of estrone while urine containing lower concentrations possessed in excess of 5°$ of the chemical estimates of estrone. The results of chemical determination of estrone in urine of cycling, pregnant and hysterectomized gilts are presented in Table 1. Estrone excretion in one of the cycling animals, C-l, increased from 0.016 mg. per day at day 15 to 0.113 mg. per day on the day preceeding estrus, day 20. During estrus Table 1. Milligrams of estrone in 24 hour urine of cycling, pregnant and hysterectomized gilts

Day 15 16 17 18 19 20 21 22 23 24 25 30 40 50

Cycling C-l 0.016 0.018 0.025 0.036 0.056 0.113 0.034* 0.009* 0.006 0.010 0.011 C-2 0.018 0.027 0.037 0.034 0.069 0.104 0.152* 0.063* 0.019* 0.012 0.009 Mean 0.017 0.022 0.031 0.035 0.062 0.108 0.093 0.036 0.012 0.011 0.010

Pregnant P-l 0.015 0.019 0.032 0.051 b 0.115 0.171 0.255 0.346 0.590 0.935 1.196 1.39 0.027 0.044 P-2 0.013 0.015 0.022 0.038 0.070 0.128 0.201 0.275 0.477 0.453 0.762 1.10 0.025 0.031 Mean 0.014 0.017 0.027 0.044 0.092 0.150 0.228 0.310 0.534 0.694 0.979 1.20 0.026 0.038

Hysterectomy H-l 0.015 0.012 0.014 0.013 0.010 0.009 0.010b 0.014 0.014 0.017 H-2 0.016 0.011 0.012 0.016 0.016 0.040 0.019 Mean 0.016 0.012 0.010 0.015 0.027 0.018

Day 75 100 110 111 112 113 114 115 116 117 120 140 170 200

Pregnant P-l 0.227 6.70 9.62 8.64 11.36 11.29 12.99 14.70 16.52 13.71 P-2 0.769 9.16 20.40 20.47 26.77 19.21 19.10 Mean 0.498 7.93 15.01

Hysterectomy H-l 0.020 0.045 0.022 0.020 H-2 0.016 0.020 0.021 0.020 0.023 0.077 0.037 Mean 0.018 0.032 0.022 0.020

*Estrus as determined by male acceptance.

^Estimated 24 hour excretion based on hourly excretion rate for interval collected during 24 hour period. 24 the level dropped rapidly to a low of 0.006 mg. estrone per day on the day following estrus, day 23. In the other cycling gilt, C-2, estrone excretion increased from 0.018 mg. on day 15 to a high of 0.152 mg. on the first day of estrus, day 21.

Daily urinary estrone then decreased until urine collection was terminated on the 25th day following the pre-collection estrus when 0.009 mg. estrone was observed. These data suggest that estrogen production decreased prior to ovulation in C-l and probably in C-2 for ovulation is normally believed to occur

24 to 36 hr. after the onset of psychic estrus. Raeside (1961b) has presented evidence from analysis of 8 hr. collection periods that estrogen production drops prior to ovulation.

Similar results were observed in pregnant animals from day

15 through day 20 with values of 0.015 and 0.013 mg. at day 15 and 0.171 and 0.128 mg. at day 20 in urine from gilts P-l and P-2 respectively. However from days 21 to 25 the amount of estrone in the 24 hr. urine continued to increase to 1.195 mg. from P-l and 0.762 mg. from P-2 at day 25. By day ]0 the amount of estrone was 1.39 and 1.10 mg. respectively from these animals. Between days 30 and 40 a precipitous drop occurred and on the latter day only 0.027 and 0.025 mg. estrone were observed in 24 hr. samples from animals P-l and P-2 respectively. Urinary estrone again increased from this stage of pregnancy to highs of

16.5 mg. from P-l two days before farrowing and 26.8 mg. from P-2 three days before parturition. Gilt P-l delivered 11 25

piglets on the llôth day of pregnancy and P-2 farrowed 7 on the

115th day. Delivered placentae from P-2 contained 102 meg.

free estrone, 18 meg. conjugated estrone, 5»° meg. free estra­ diol, presumably the beta form and less than 1 meg. conjugated

estradiol per 100 g. These values indicate that swine placentae

probably produce estrogens. Results of Faiermark (1935) with bioassay of swine placentae also suggest that they produce estrogen during late pregnancy and in addition during the third

and fourth weeks of pregnancy. These data from 24 hr. urine

samples from cycling and pregnant animals were in agreement with those of Lunaas (1962) and Raeside (1961a).

Although luteal tissue similarly persists in a functional

state during pregnancy and following hysterectomy (Anderson et

al., 1961; Duncan et al., 1961) the quantities of urinary

estrone were very dissimilar. The quantity of estrone in 24 hr. urine samples remained at a consistently low level at the days

studied with the exceptions to be noted. It was impossible to

collect continuously between days 15 and 25 following the pre­ operative estrus as both animals were very susceptible to cystitis. At day 15, 24 hr. estrone excretion was similar to

cycling and pregnant animals with 0.015 mg. from H-1 and 0.016

mg. from H-2. However at day 21 the values were 0.009 and

0.012 mg. respectively and at day 25 there were 0.016 mg. in the 24 hr. sample from H-2. At day 30 there were only 0.014 mg. estrone in urine from H-1 and 0.016 in urine from H-2 in marked 26

contrast to the quantities of over 1 mg. in pregnant animals.

Through the remainder of the study the amounts excreted remain­

ed relatively constant with a tendency to increase slightly

with the exception of the samples from H-1 at day 100 and H-2

at days 40, 170, and 200. The increases at days 40 and 100 could be an indication that their may be some sub-threshold

cyclic activity of the ovaries in these animals. The increase

to 0.077 mg. per day at day 170 was possibly associated with

the failing of the corpora lutea in this animal for her vulva

was pink and swollen and boars showed interest in her for

several days thereafter although boar acceptance was never

noted. When slaughtered 214 days following her pre-operative estrus the ovaries were greatly enlarged with luteinized cysts

up to 75 mm. diameter and carbon marks in the ovarian stroma.

These cysts probably account for the elevated estrone excre­

tion at day 200. Gilt H-1 exhibited estrus on the 134th and 135th days following the preoperative estrus and when slaugh­

tered 9 days later her ovaries contained carbon marked corpora

albicantia and unmarked functional-appearing corpora lutea.

Estrone was also the principal estrogen found in the urine

of ovarlectomized and ovarlectomized-hysterectomized gilts in­

jected with estradiol benzoate alone or in combination with

progesterone. The percentage of injected estrogen, computed on a molecular basis, that was recovered as estrone from the urine

of these animals for one day prior to and 4 or 5 days following 27

administration is set forth in Table 2. The amount of the

steroids administered to these animals at the beginning of the

second 24 hr. collection period is also presented. Severe cystitis developed in gilt 0-2 during both collection periods

and the catheter had to be removed at the end of the fourth post-injection day.

Small amounts of estrone were detected in the urine of all

4 animals on the day prior to injection (day 0). This value is

expressed as a percentage of the amount of estrogen injected

and it was not used to adjust the values for the post-injection days. Bioassay results of pooled extracts of hydrolyzed pre-

injection urine from these animals indicated that over half of

the chemically estimated potency was estrone. Bulbrook and

Greenwood (1957) observed low levels of estrogen in urine from

ovarlectomized women.

In the ovarlectomized gilts 46.9$ of the injected estrogen was recovered over a 4 day period when estradiol benzoate was given alone compared to 41.4$ when these animals were injected with both progesterone and estradiol benzoate. The results were more variable in the ovarlectomized-hysterectomized animals with OH-1 having a higher recovery rate of 55*0$ when given estradiol benzoate alone compared to 47.7$ when both hormones were administered. The opposite occurred in OH-2 with 39*0$ recovered when only estradiol was injected compared to 60.3$ when progesterone was administered with the estradiol benzoate. Table 2. Percent of injected estrogen in urine as estrone

Injected with : Estradiol Day Benzoate Progesterone Total percent mg. mg. 0 1 2 3 4 5 4 days 5 days Ovariectomy Percent/day 0-1 1.24 0 0.4 24.0 14.0 7.1 3.7 2.3 48.8 51.3 0-2 1.24 0 0.4 19.8 14.4 7.8 3.0 45.0 Mean * 0.4 21.9 14.2 7.4 3.4 2.5 46.9 49.4 0-1 1.16 290 1.1 16.3 15.l b 7.2 2.7 1.6 41.3 42.9 0-2 1.26 315 1.0 23.9 10.0 5.3 2.4 41.6 Mean 1.0 .20.1 12.6 6.2 2.6 1.6 41.4 43.0

Ovariectomy- Hysterectomy 0H-1 1.08 0 1.5 36.9 12.2 3.8° 2.1 1.6 55.0 56.6 OH-2 1.04 0 1.3 13.3 15.5 6.3 3.9 2.6 39.0 41.6 Mean 1.4 25.1 13.8 5.0 3.0 2.1 46.9 49.0 0H-1 1.04 260 1.2 23.9 14.4 6.0 3.4 2.4 47.7 50.1 OH-2 0.96 240 2.2 29.1 19.2 7.4 4.6 3.6 60.3 63.9 Mean 1.7 26.5 16.8 6.7 4.0 3.0 54.0 57.0

^Administered at a rate of 0.40 mg. estradiol benzoate per 100 lb. and 100 mg. progesterone per 100 lb.

^Estimated 24 hr. excretion based on hourly excretion rate for interval collected during 24 hr. period. ^ 29

With one exception the maximum hormone excretion was observed in the first 24 hr. following injection. After the administra­ tion of estradiol benzoate alone to OH-2, the amount of estrone in the urine was unusually low the first day following injec­ tion and the maximum amount of urinary estrone was observed during the second day. However the percentage excreted the second day was very similar to that of the other animals qn the second post-injection day. Lunaas (1963) administered 10 mg. estradiol-17 beta to an ovarlectomized sow and recovered 40$ in the urine as estrone over a 4 1/8 day period. When estrone propionate and estradiol dipropionate were administered to ovarlectomized-hysterectomized rhesus monkeys only 3*3 and

4.4$ respectively of these compounds were recovered in the urine as estrone (Dorfman et al., 1945).

During pregnancy the estrogen production of gilts appears to be inversely related to progesterone content of corpora lutea. The amount of estrone observed in a day's urine at days 40 and 50 of pregnancy was the lowest throughout preg­ nancy exclusive of the first three weeks while Duncan ejb al. (i960) observed the highest endogenous levels of luteal pro­ gesterone at day 48 of pregnancy. Corpora lutea were observed to have the lowest progesterone content at day 102 of preg­ nancy which was the latest day studied while urine collected after the 100th day of pregnancy contained very large quan­ tities of estrone. However, this inverse relationship does not 30 follow for in vitro synthesis by these glands and it does not hold true in hysterectomized animals. In animals subjected to this operation progesterone content of corpora lutea at 25, 50 and 110 days post-hysterectomy was approximately the same as at equivalent stages of pregnancy. The amount of estrone in the daily urine of hysterectomized animals remained at a consis­ tently low level through a period equal in length to pregnancy.

The results with ovarlectomized and ovariectomized-hyster- ectomized animals injected with estradiol benzoate alone and in combination with progesterone gave no indication that the uterus, the progesterone stimulated uterus, or progesterone itself influenced the metabolism of estrogen. Therefore the difference in estrogen excretion between pregnant and hyster­ ectomized animals must represent differences in estrogen pro­ duction rather than estrogen metabolism. These differences can probably be attributed to maintained corpora lutea pre­ venting pre-ovulatory swelling of follicles and estrogen pro­ duction from days 18 to 20 and to the lack of placental pro­ duction of estrogen at later stages. Although evidence has previously been presented (Anderson et al., 1963a; 1963b; Butcher et al., 1962b) that the relation of the uterus to the life span of corpora lutea may be meta­ bolic, the results of this investigation gave no indication that it involves the metabolism of estrogen. Although urine samples were routinely analyzed for 31

estradiol-17 beta the amounts in the aliquots were generally below the minimum sensitivity of the method employed. The

estradiol fraction material gave rise to low corrected optical densities when subjected to spectrophotometry analysis.

Little variation occurred in the corrected optical density of

the estradiol fraction and no patterns were apparent in any

of the animals studied including the ovarlectomized animals following the administration of estradiol benzoate. 32

SUMMARY

Urine was collected from gilts in various physiologic

states on a 24 or 48 hr. basis. The estrone and estradiol-17

beta content of this urine was chemically estimated by spectro­

photometry analysis of their respective Kober chromogens. The preparation of these estrogens for quantitation involved

acid hydrolysis, solvent partition, saponification and chro­

matography on adsorption and partition columns.

The estimates for estradiol were consistently low and without any marked variation in any of the states studied. Animals which were collected for 11 days beginning at day 15

of the estrous cycle exhibited a high estrone content of

urine at the approximate onset of estrus with a low near the

end or shortly after estrus. Estrone content of urine from

pregnant animals was similar to that of cycling animals from

days 15 to 20. After day 20 the estrone content of urine continued to increase in contrast to the cycling animals. At

day 30 a high for early pregnancy was observed but by day 40

estrone quantities had dropped precipitously to levels similar

to late in the estrous cycle. From day 40 to two or three days

prior to parturition the estrone content of urine continued

to increase to the highest levels observed in this study. Delivered placentae from one of these animals contained rela­

tively large amounts of estrone and some estradiol, presumably 33 estradiol-17 beta. Urine from hysterectomized animals was very different in estrone content from that of pregnant animals even though corpora lutea similarly persist in animals in these two states. The quantities of estrone in the urine of hyster­ ectomized animals remained at consistently low levels similar to those observed in the latter part of the estrous cycle. No marked differences were noted in the renal elimination of estrone from ovariectomized versus ovarlectomized-hyster­ ectomized animals when they were injected with estradiol ben­ zoate alone and in combination with progesterone. These re­ sults suggest that estrogen metabolism as evaluated by urine content of metabolites is not affected by the uterus, with or without progesterone stimulation or by the absence of this . structure. 34

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i 41

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation

to Dr. R. M. Melampy for his guidance in the preparation of

this thesis and for his continuous aid and encouragement in

the development of the author's valuation of scientific

investigation and personnel. The author would also like to thank Dr. L. L. Anderson for performing the surgery and for

his timely assistance, Dr. G. W. Duncan for his helpful

training in chemical analysis of steroids, Mr. R. P. Rath-

macher for his part in the surgery and aid in making urine

collections and in caring for animals during collection

periods and Messrs. David Grimm and Mahlon Shell for aid in caring for and handling the gilts. The assistance of Miss

Barbara Murdock in typing and proof-reading the first draft of this thesis is also appreciated.