Intrauterine Migration of the Porcine Embryo

AN ABSTRACT OF THE THESIS OF

WILLIAM FREDERICK POPE for the degree of DOCTOR OF PHILOSOPHY in ANIMAL SCIENCE (PHYSIOLOGY) presented on November 6, 1981

Title: INTRAUTERINE MIGRATION OF THE PORCINE EMBYRO

Abstract appoved: Redacted for privacy Fredrick Stormshak

The mechanism(s) involved with intrauterine migration of the porcine embryo was investigated. Twenty-four gilts on Days 6, 9 and 12 of gestation (Day 0 = 1st day of estrus) were utilized to examine the

relationship between myometrial activity and intrauterine migration.

On Day 2, embryos were flushed from one oviduct and transferred to the opposite oviduct. Myometrial contractility increased concomitant with embryo migration through the uterus (day x side interaction, P<.10).

Uterine flushings contained a short-acting component that mimicked, in part, the stimulatory influence(s) of the porcine embryo on myometrial activity. Furthermore, only flushings from the uterine segment containing the Day 12 embryos could overcome the inhibitory effects of

indomethacin (P<.01) on myometrial activity. The porcine embryo coincubated with myometrial strips could not directly stimulate myometrial activity.

The role of estradio1-17B (E2) and histamine in migration of the porcine embryo was examined. The involvement of E2in embryo migra-

tion was examined by observing the distribution of silastic beads

impregnated with cholesterol (five gilts) or E2 (five gilts) on Day 12 of the estrous cycle (after 5 days in utero). Beads impregnated with

E2 migrated further (P<.05) than those impregnated with cholesterol. Twenty additional gilts and sows were assigned to four groups (n = 5) and were used to determine if histamine was involved with intrauterine migration. Cromolyn sodium (an inhibitor of histamine release) treatment when administered on Day 6 restricted (P<.05) Day 10 embryos to the tip of the uterine horn but Day 12 embryos had gained the ability to overcome this restriction. Injection of histamine concomitantly with cromolyn sodium restored migration of Day 10 embryos.

Day 5 and 7 porcine embryos recovered from 24 donors were compared for migration and survival ability in 16 nonpregnant Day 6 recipients. The percentage of Day 5 and 7 embryos surviving the transfer procedures (Day 11) did not differ. However, by mid- gestation (Day 60) more Day 7 (P<.001) than Day 5 embryos had survived. Migration of the embryos may be of importance because the distance between adjacent fetuses was significantly correlated with fetal weight (r = .47, P<.01). Intrauterine Migration of the Porcine Embryo

William Frederick Pope

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Completed November 6, 1981

Commencement June 1982 APPROVED:

Redacted for privacy Professor or Animal science in charge of major

Redacted for privacy

Head of Department of Ani 1 Science

Redacted for privacy

Dean of Gradu e School

Date thesis is presented November 6, 1981

Typed by William Frederick Pope ACKNOWLEDGEMENTS

The author wishes to extend appreciation to the United States

Department of Agriculture for providing the funding for this coopera-

tive agreement between Oregon State University, Corvallis, and Roman L.

Hruska U. S. Meat Animal Research Center, Clay Center, Nebraska. A

special thanks to Drs. Joe Ford, Dan Laster, Jim Oldfield and Bob

Oltjen for their roles in coordinating this agreement. Thanks also to

Ms. Mary Zelinski for her invaluable assistance with the administra-

tive paper shuffling.

I also wish to acknowledge Dr. Fred Stormshak, Professor of

Animal Science, 0.S.U., and Dr. Ralph Maurer, Research Physiologist,

U. S. Meat Animal Research Center, for their counseling and patience

throughout this study. Through their efforts they have impressed upon me the priceless value of well-designed, well-written and unique experimentation.

I feel compelled to dedicate this thesis to the following people

for providing me with the incentive to pursue a doctoral degree in

Animal Science. First, to my high school teacher, Harlan Englerth,

for impressing me with the enjoyment of studying the biological

sciences. Secondly, to my parents for incorporating that inquisitive

interest in biology with the science of animal production.

Finally, I wish to thank my wife, Kristy, for her willingness to accept the opportunity costs of graduate school and her understanding

support throughout this study. TABLE OF CONTENTS

Page REVIEW OF LITERATURE

Importance of Spacing 2 Mechanism of Spacing 5 Innervation of the Uterus 10 Physiology of Myometrial Contraction 18 Modulating Role of Cyclic Nucleotides 23 Hormonal Modification of Myometrial Function 23 Possible Embryonic Stimulants of Myometrial Activity 27

STATEMENT OF THE PROBLEM 32

EXPERIMENT I,II and III: INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO -- INTERACTION OF EMBRYO, UTERINE FLUSHINGS AND INDOMETHACIN ON MYOMETRIAL FUNCTION IN VITRO 33

Introduction 33 Materials and Methods 34 Results 40 Discussion 46

EXPERIMENT IV and V: INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO: INFLUENCE OF ESTRADIOL-17a AND HISTAMINE 51

Introduction 51 Materials and Methods 52 Results and Discussion 55

EXPERIMENT VI and VII: SURVIVAL OF PORCINE EMBRYOS AFTER ASYNCHRONOUS TRANSFER 60

Introduction 60 Materials and Methods 61 Results and Discussion 63

GENERAL DISCUSSION 68

BIBLIOGRAPHY 70 LIST OF FIGURES

EXPERIMENT I,II and III

INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO -- INTERACTION OF EMBRYO, UTERINE FLUSHINGS AND INDOMETHACIN ON MYOMETRIAL FUNCTION IN VITRO

Figure Page

1 Treatment sequence (left to right) and exposure time 36 (min) of P and NP muscle strips of Day 6, 9 and 12 gilts to electrical stimulation (ES), isoproterenol (ISO, 80 ng/m1), wash (4), propranolol (PROP, 200 ng/m1) and norepinephrine (NE, 20 ng/ml, Levophed bitartrate).

2 Treatment sequence (left to right) and exposure time 37 (min) of NP muscle strips of Day 6, 9 and 12 gilts to P or NP flushings (F), wash (+) and indomethacin (INDO, 10 pg/m1).

3 Distribution of embryos within the uterine horns of 40 gilts on Days 6, 9 and 12 (Day 0 = 1st day of estrus) of gestation.

4 Quantity of estradiol -176 (pg) within the P and NP 41 flushings of Days 6, 9 and 12 gilts.

5 Frequency of contractions of myometrial strips excised 42 from Day 6, 9 and 12 pregnant gilts at-1 and 5 g tension.

6 Activity (montevideo units) of myometrial strips excised 42 from Day 6, 9 and 12 pregnant gilts at 1 and 5 g tension. LIST OF FIGURES (Continued)

EXPERIMENT I,II and III

INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO -- INTERACTION OF EMBRYO, UTERINE FLUSHINGS AND INDOMETHACIN ON MYOMETRIAL FUNCTION IN VITRO

Figure Page

7 Changes in contractile frequency of myometrial strips 44 excised from Day 6, 9 and 12 pregnant gilts at 1 g tension. The initial frequency has been subtracted from all the illustrated responses.

EXPERIMENT IV and V

INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO: INFLUENCE OF ESTRADIOL-170 AND HISTAMINE

8 Distribution of silastic beads (10/horn) impregnated 56 with cholesterol (top) or estradio1-17 (bottom) after 5 days in utero (n = 5). Beads were inserted into the uterus drTlYi37 7 of the estrous cycle. Uteri are drawn to scale with the body of the uterus aligned at the center. For illustrative purposes the side originally containing the beads is depicted on the left.

9 Mean distance migrated (cm) of embryos following expo- 58 sure of the gravid uterus to control (vehicle, a-lactose), cromolyn sodium (16 mg), cromolyn sodium plus histamine (16 and 2 mg, respectively) and cromolyn sodium (16 mg) on Day 6. Day in parenthesis denotes the day of hysterectomy. The last group received additional cromolyn sodium (16 mg) on Day 10. LIST OF TABLES

REVIEW OF LITERATURE

Table Page

1 Incidence of embryonic mortality in fertile pigs 3

2 Effect of epinephrine (E) and norepinephrine (NE) 14-17 on uterine motility

EXPERIMENT I,II and III

INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO -- INTERACTION OF EMBRYO, UTERINE FLUSHINGS AND INDOMETHACIN ON MYOMETRIAL FUNCTION IN VITRO

3 Percentage change in frequency of uterine 43 contractions following exposure toe( and 8 adrenergic agonists

4 Activity (montevideo units) of myometrial strips 45 following incubation with or without embryos and flushings

5 Quantity of estradio1-178(pg) in the incubation 46 medium following exposure of myometrial strips to flushings and embryos

EXPERIMENT IV and V

INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO: INFLUENCE OF ESTRADIOL -178 AND HISTAMINE

6 Mean (+SE) percentage of embryos recovered and mean 57 length (cm) of uteri of gilts treated with cromolyn sodium or cromolyn sodium plus histamine on Days 6 and 10. LIST OF TABLES (Continued)

EXPERIMENT VI and VII

SURVIVAL OF PORCINE EMBRYOS AFTER ASYNCHRONOUS TRANSFER

Table Page

7 Modified Krebs-Ringer-Bicarbonate 63

8 Percentage survival of Day 5 and 7 embryos 64 to Day 11 and 60 of gestation INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO

REVIEW OF LITERATURE

In the United States, at present, 13 hogs are marketed per sow per year but this figure is far short of the attainable goal of 20 to

24 (Cunha, 1979). The potential to reach this goal rests with the ability of the swine producer to increase the number of live pigs born, decrease post-natal death loss and decrease the interval between suc- cessive farrowings. Overcoming the harrier imposed by uterine capacity (gazer et al., 1969) is a prerequisite to increasing the number of offspring born at each parturition.

All nolytocous species inherently attempt to reduce this barrier of a finite uterine capacity by spacing their embryos equidistantly before attachment or implantation. Pigs, for example, distribute their embryos throughout the uterus such that the distance between adjacent embryos is essentially the same. Likewise, twin ovulations in cattle and sheep usually result in embryos being distributed to each uterine horn, regardless of whether the ova originate from one or both ovaries (Curson, 1934; Boyd et al., 1944; Wallace, 1948; Robinson,

1951; Hafez, 1964h; Casida et al., 1966). Thus, through this spacing phenomenon the embryos are provided an equivalent intrauterine area in which to develop and grow. Understanding the mechanism(s) responsible for intrauterine migration may contribute to an understanding of the causes of embryonic mortality. 2

Importance of Spacing

Definitive attachment, interlocking microvilli, of the porcine conceptus (embryo plus extraembryonic membranes) to the uterine epithe-

lium, is completed by Day 18 (Perry et al., 1973). During this preat-

tachment stage, the embryo must rely on intrinsic and diffusible maternal nutrients to sustain growthand development. Minimal amounts

of extrinsic nutrients are required during the first six days fol-

lowing conception since hyperplasia is confined to the boundary of the circumscribing zona pellucida (Heuser and Streeter, 1929; Green and

Winters, 1946; Pitkjanen, 1955; Hunter, 1973). After Day 6, the

embryo "hatches" from the zona and rapid growth occurs as character-

ized by increases in protein content and a transition from a spherical

and tubular to a filamentous embryo (Anderson, 1978a). It is during

this latter stage of preattachment that the embryo increases its

requirement for nutrients from the endometrium. In addition, it is

during this preattached stage that the majority of the embryonic mortality occurs (table 1).

Embryonic mortality is defined as the proportion of missing or

necrotic embryos relative to the number of corpora lutea. In gilts

and sows, the fertilization rate is rather constant between 95 to

100 percent (Robertson et al., 1951; Squiers et al., 1952; Day et al.,

1959; Spies et al., 1959; Perry and Rowlands, 1962), thus, embryonic mortality, as defined is not comparable to fertilization rate during early gestation. Table 1. Incidence of embryonic mortality in fertile pigs

Day of Embryonic Examination Mortality (%) Reference

1 22.1 Pomeroy (1960) 2 28.1 Pomeroy (1960) 2 2 Spies et al. (1959) 3 33.2 Pomeroy (11-60) 4 37.1 Pomeroy (1960) 5 38.5 Pomeroy (1960) 6 39.3 Pomeroy (1960) 6-9 22 Perry and Rowlands (1962) 7 39.9 Pomeroy (1960) 8-14 42.4 Pomeroy (1960) 9-18 17 Anderson (1978a) 13-18 28.4 Perry and Rowlands (1962) 15-28 45.1 Pomeroy (1960) 17 25 Lerner et al. (1957) 18 28 Spies eIMT: (1959) 25 30-43 Baker TE-0-. (1956) 25 36 Bazer -e-FiT. (1969) 25 33 Day et a1:(1959) 25 37.9 Mortii-sette et al. (1963) 25-40 34.8 Perry and RoV4Tands (1962) 25 45 Robertson et al. (1951) 25 26 Sorenson aTid.r37-)ssett (1956) 25 17 Spies et al. (1959) 25 35 SquierT-eial. (1952) 28 39 King and-Folig (1957) 29-56 47.3 Pomeroy (1960) 30-60 32.6 Hammond (1921) 40 38 Day et al. (1959) 40 34 Lerner et al. (1957) 40 33 Sorenson ana Gossett (1956) 55 31 Rathnasahapathy et al. (1956) 55 23 Reddy et al. (197-) 70 50 Baker et IT. (1956) 105 35 Bazer et -i-T-.(1969) term 44 CasidaTI-M) term 20-30 Corner (1923) term 40 Hammond (1914) term 40.1 Lasley (1957) term 40 Perry (1954) term 31.3 Phillips and Zeller (1941) term 33 Rathnasahapathy et al. (1956) term 46 Squiers et al. (-175-2T 4

Coincident with this period of high embryonic mortality, embryos become spaced equidistantly within the uterine lumen. Patten (1948) proposed that spacing of the embryos was associated with the onset of blastocyst elongation in the pig. Anderson (1978a) observed that initial transformation of spherical to ovoid blastocysts occurred as early as Day 10 and that by Day 14 the embryos were spaced. By Day 9,

Anderson (1978a) observed a significant correlation between the actual and expected distribution of embryos. This latter observation was supported by Dhindsa et al. (1967), Waite and Day (1967) and Polge and

Dziuk (1970) who demonstrated that distribution of porcine embryos occurred between Days 7 and 12 of gestation. In sheep and cattle embryos migrated between Days 12 and 14 (Abenes and Woody, 1971;

Cummins, 1979) and before Day 20 (Hafez, 1964b), respectively.

Equidistant spacing of embryos is a natural phenomenon in all polytocous pregnancies, however, failure of embryos to become spaced has never been demonstrated to be directly associated with early embryonic mortality. Experiments have been conducted to examine the relationship between spacing and embryonic mortality by comparing embryonic death loss to varying numbers of embryos within the uterine lumen. It has been demonstrated that increasing the number of embryos, in polyovular species, by superovulation (Fowler and Edwards, 1957;

Adams, 1960; Hafez, 1964a; Longenecker and Day, 1968) or by superinduction (McLaren and Michie 1959b; Adams, 1960; Bazer et al., 1969) failed to increase the number of offspring horn. In some experiments involving superovulation or superinduction (Adams, 1960; Hafez, 1964a) 5 litter size was reduced. Anderson (1978a) suggested that embryonic survival was unrelated to the space available during the first 18 days of gestation. Further investigations have demonstrated that space does not limit embryonic survival until after Day 30 (Longenecker and

Day, 1968; Pope et al., 1968, 1972; Bazer et al., 1969; Wehel and

Dziuk, 1974). Fenton et al. (1970) observed an apparent exception because superinduction on Day 7 failed to result in an increased litter size on Day 25. Nevertheless, since embryos can not be substantially spaced after attachment, the association can still be made between spacing of the embryos and the ultimate crowding and loss of the fetuses.

The primary importance of intrauterine migration in swine is thus twofold; it maximizes the luminal environment for each embryo to develop in, thereby increasing litter size (Lasley et al., 1963; Bazer et al., 1969; Gentry et al., 1973) and it results in distribution of emhryos sufficiently to maintain pregnancy (Dhindsa and Dziuk, 1968).

Casida et al. (1966), Doney et al. (1973) and Rizzoli and White (1978) observed a higher twinning rate in ewes that shed ova bilaterally versus unilaterally. Likewise, cattle have a higher twinning rate if emhryos are transferred to each horn versus a unilateral transfer

(Gordon et al., 1962; Rowson et al., 1971; Sreenan et al., 1975).

Mechanism of Spacing

The mechanism by which embryos become equally spaced has long been 6

an enigma of biological science. Kussmaul (1859) observed that the final position of the embryo was not always adjacent to the ovary from which the ovum originated. At that time, there existed much confusion as to whether the ovum migrated transabdominally (external migration) or from one horn to the other through the uterine body (internal migration). Leopold (1880) supported the external pathway since exci- sion of the oviduct on one side folloWed by removal of the opposite ovary resulted in occasional pregnancy in rabbits. Some of the earliest evidence that internal migration of the ovum was the prin- ciple route was observed by Kupfer (1920) in sows. Corner (1921) and

Warwick (1926) confirmed this latter observation and refuted external migration as the predominant route of ovum migration.

Another early misconception was that embryos became equidistantly spaced because of predetermined implantation sites. This hypothesis was found to he inconsistent with the fact that embryos are spaced regardless of the number present in the horn. In addition, it failed to explain the midpoint location of the embryo within the uterine horn when a single blastocyst was present in the rabbit (Roving, 1971), cow

(Hafez, 1964h) and ewe (Curson, 1934).

One of the first hypotheses assumed that embryos attach serially down the uterine horn (Mossman, 1937). The more anterior embryo

(closest to the oviduct), by attaching earlier was presumed to be larger, more developed, and thus established a local refractory zone such that the older embryos could only attach further down the horn. 7

McLaren and Michie (1959a) and Dhindsa et al. (1967) demonstrated that there was no correlation between the size of the embryo and its location in the uterus, thus, refutinq Mossman's assumption.

Furthermore, when mouse embryos on flay 3.5 after mating were transferred into the tip of the uterine horn of Day 2.5 pregnant recipients, the transferred embryos tended to implant in the posterior portion of the uterine horn (McLaren and Michie, 1956). The other aspect of Mossman's hypothesis, concerning the refractory zone, may still be valid since it is rare to find adjacent embryos occupying the same space.

Embryo spacing may be due to an electrical charge interaction between adjacent embryos and between the embryo and endometrium.

Clemetson et al. (1971) demonstrated that the Day 5 rat blastocyst, after it loses the zona pellucida, behaves as a negatively charged body in vitro. Likewise, activation of the mouse hlastocyst results in a decreased ability of the trophoderm to hind positively charged iron particles (Nilsson et al., 1975). The susceptibility of these cells to hind colloidal iron indicates that at least some of the negatively charged sites represent sialic acid groups of surface glycoproteins. Anderson (1978a) observed that by Day 12 the cumula- tive length of all elongating porcine embryos exceeded 600 cm within a uterine horn of 100 cm in length, thus, these embryos were highly con- voluted occupying only a fraction of the uterus. Furthermore, he observed no overlapping of extraembryonic membranes. This end-to-end association without any overlapping might be explained by repulsive 8

electrical charges between adjacent embryos. The tendency of rabbit

trophoblasts to adhere near endometrial capillaries rather than over

an intervening space (Boving, 1962) could also be explained by a local

difference in tissue oxidation-reduction potentials due to the state

of oxygenation of the blood in the capillaries.

A more controversial hypothesis contends that the embryos become

uniformly spaced by differential growth of the uterus (Perry and

Rowlands, 1962; Finn, 1968; Defeo and Kleinfeld, 1971). This may be

involved later in gestation because no difference in length of the por-

cine uterus was observed between Days 7 and 12, the period of rapid

intrauterine migration of embryos (Dhindsa et al., 1967).

The hypothesis that formed the basis for the research to be

reported in this thesis was first proposed by Corner (1921), who

stated, "there can be little doubt that the embryos are shifted by the

peristaltic action of the uterine musculature". "Peristalsis" refers

to propulsion of the contents through the long axis of a tube by

circumferential contractions. The major objection to this hypothesis

has been the generally accepted dogma that the uterine myometrium,

under the influence of progesterone, is quiescent during that period when embryo migration is known to occur (Keye, 1923; Wislocki and

Guttamacher, 1924; King and Young, 1957; Zerobin and Sporri, 1972).

Wislocki and Guttamacher (1924) removed uteri from immature, estrous

and pregnant gilts and visually observed peristaltic waves of contrac-

tion of the freshly excised uteri in a large bath. These investiga-

tors concluded that the porcine embryo can maintain the increased 9 activity following mating. Pusey et al. (1980) demonstrated that jugular infusion of relaxin, a potent myometrial inhibitor, caused embryos of the rat to be restricted to the anterior third of the uterus.

Peristaltic contractions of the uteri of estrous ewes and gilts are directed towards the uterotubal junction (Brinsfield and Hawk,

1969; Mann, 1969; Zerobin and Sporri,,1972; Croker and Shelton, 1973;

Hawk and Echternkamp, 1973; Hawk, 1973). This direction of contraction is probably related to the role of the uterus in moving the ejaculate towards the site of fertilization in the oviduct (Einarsson et al., 1980; Miring et al., 1980). Croker and Shelton (1973) and

Hawk (1975) observed contractions shifting towards the cervix during the luteal phase. The reverse direction of contractions during the luteal phase may be related to displacing the embryos down the horn.

Estrogen is involved in initiating uterine contractions towards the oviducts (Croker and Shelton, 1973). Interestingly, continued exposure to exogenous estrogens after estrus maintains contractions that move towards the uterotubal junction in ewes (Hawk, 1975).Greenwald

(1965) also found rabbit embryos crowded into the upper 3 cm of the uterus following estrogen treatment from Day 3 to 8 postcoitum.

By speeding up motion pictures of the exteriorized rabbit uterus

Boving (1971) observed that uterine contractions associated with migration of the embryo could be divided into three phases.The early phase consisted of minimum impaired contractions, consistent with the rapid transport of small hlastocysts randomly along the horn. During 10

the intermediate phase the force of myometrial contractions decreased

proportionately to the distance from its source (blastocyst or end of

the horn). This gradation of propulsive forces could be the key to equidistant spacing of embryos.Finally, the late phase consisted of uncoordinated contractions concomitant with a lack of appreciable

force. It is during this phase that the embryo attaches antimesometrially. Boving (1971) speculated that uterine contractions move blastocysts by increasing the pressure behind the blastocyst relative to the pressure ahead of it, thus, the blastocyst moves in the direction of the propagating wave to equalize this pressure differential. Although fluid volume in utero is minimal he postulated that the endometrium might possess enough fluid-like properties to act as the conveying medium.

Before proceeding with a discussion of possible factors associated with the presence of the embryo that could stimulate myometrial function and initiate migration, a better understanding of myometrial function is required. The following sections will be devoted to discussions of the innervation of the uterus and the physiology of myometrial contraction.

Innervation of the Uterus

The uterus is capable of rhythmic contractions in the absence of innervation. Gruber (1933) observed that any afferent nerve stimulation, if sufficiently painful, will result in contraction of the human uterus. There exists, therefore, little doubt that the nervous system is capable of modifying uterine activity. Although innervation of the uterus has been a subject of investigation for over

300 years its physiological significance is still virtually unknown

(Krantz, 1959). Part of the confusion can be attributed to variation between species, experimental techniques, hormonal modification of neural function and the apparent interaction of acetylcholinomimetic and sympathomimetic drugs (Burn, 1945; Schofield, 1952).

Innervation of the myometrium, as determined with fluorescent histochemistry, has been found to be adrenergic in the rabbit, cat, dog, guinea pig, human and rat (Sjoberg, 1967, 1969; Nakanishi et al.,

1968; Adham and Schenk, 1969). Miller and Marshall (1965), Vogt

(1965), Theobald (1968) and Marshall and Kroeger (1973) contended that the uterus was innervated exclusively bv adrenergic fibers. A controversy has existed as to the presence and extent of cholinergic innervation. Positive staining for acetylcholinesterase has been observed in the myometrium of the rabbit (Sjoberg, 1967), human

(Coupland, 1962), cat (Jacobowitz and Koelle, 1965), guinea pig

(Gerebtzoff, 1959) and rat (Adham and Schenk, 1969). In contrast,

Jacobowitz and Koelle (1965) demonstrated that acetylcholinesterase activity in the cat myometrium was confined to the adrenergic fibers and could not observe any cholinergic innervation. Coupland (1962) noted a rich supply of cholinergic fibers concentrated mainly in the region of the human cervix, but structures other than nerve fibers adsorbed the cholinesterase stain. 12

Any cholinergic supply to the uterus would have to be either hypogastric or parasympathetic in origin. Setekleiv (1964) demonstrated that the excitatory response of the rabbit uterus to hypogastric stimulation was potentiated by anticholinesterases, but unaffected by atropine. Responses of the guinea pig uterus to stimulation of the hypogastric nerve were also unaffected by atropine

(Isaac et al., 1969). Varaqic (1956), however, noted potentiation of uterine responses by anticholinesterases and inhibition by atropine, suggesting that both cholinergic and adrenergic fibers were present in the hypogastric nerve of the rabbit. In the dog, Sherif

(1935) reported the presence of acetylcholine in effluent of the perfused uterus after hypogastric nerve stimulation. However, the possibility of both anticholinesterase and atropine effects being at the ganglionic synapse must he considered (Lahate and Sheehan, 1943;

Bell, 1967; Holman et al., 1971).

The adrenergic innervation of the uterus arises from the lumbar region (1st to 6th segments) of the spinal cord and exits in the white rami communicantes to the 4th and 6th lumbar ganglia of the paraver- tebral chain. These nerve bundles extend from the lumbar vertebral ganglia to the inferior mesenteric ganglia. In the cat, rabbit, dog, rat and guinea pig, two discrete hypogastric nerves arise from the inferior mesenteric ganglia (Marshall, 1970). Each hypogastric nerve divides into a dorsal and a ventral branch. The dorsal branch extends to the pelvic plexus, while the ventral branch joins ganglion forma- tions in the dorso-lateral wall of the vagina. The pelvic plexus, 13 sometimes called Frankenhauser's plexus, is located on either side of the uterus at the base of the broad ligament near the cervix. Most of the adrenergic nerves supplying the uterus extend from the pelvic plexuses to the uterus in juxtaposition to the uterine vasculature.

Within the uterus large bundles of myelinated and unmyelinated nerve fibers penetrate both muscular layers, branch repeatedly and extend parallel to the muscle bundles. These axons lose their myelin sheaths before terminating adjacent to a group of muscle cells (Paine et al., 1954; Krantz, 1959). Adrenergic nerves within the uterus are characterized by elongated, bead-like varicosities situated along the entire length of the terminal axons. Neurotransmitter is stored and released from these varicosities following threshold activation of the neuron (Falck, 1962; Norherg and Hamberger, 1964; Malmfors, 1965). In the rat, rabbit, cat, guinea pig and human uterus this neurotransmitter is norepinephrine (Sjoberg, 1967).

Released neurotransmitter appears to bind to two populations of

adrenergic membrane receptors designated a and B . It has been postulated that a- and 8- adrenergic receptors are structurally related and may in fact represent alternate configurations of the same active site (Kunos _et al., 1973). However, quantification of receptor numbers re- futes this postulate (Roberts et al., 1977) and suggests that the receptors are not structurally interrelated. The order of activity of various sympathomimetic agents for a-receptors in the uterus is epinephrine>norepinephrine>isoproterenol, while for 8-receptors isoproterenol>epinephrine>norepinephrine (Miller, 1967). In the uterus, Table 2. Effect of epinephrine (E) and norepinephrine (NE)on uterine motility

Si5eETes -Tondition ---"E-1 7A-a-renergic receptor bloEker Reference agent effect

Rabbit in vitromature Ergot extract inhibits Dale (1906), Gaddum (1926) nonpregnant in vitro immature t DHE inhibits Greeff and Holtz (1951) immature and fi t DHE inhibits Greeff and Holtz (1951) estrogen in vitro pregnant (term) t DHE inhibits Greeff and Holtz (1951) Ti vivo immature and Phenoxyhenzamine reverses Willems and DeSchaep- estrogen dryver (1966) in vivo immature and Propranolol reverses Willems and DeSchaep- estrogen and dryver (1966) progestational agent in vitromature, Dibenamine reverses NiCkerson and Goodman (1947) nonpregnant

Guinea pig in vitro immature Balassa (1940), Greeff and Holtz (1951), Miller (1967), Davidson and Ikoku (1966) in vitro immature Hermansen (1961) fresh DCI reverses 6-8 h at 39 C t 18 h at 4 C in vitro immature Balassa (1940) 2 h at 37 C in vitro immature and DHE inhibits Greeff and Holtz (1951) gonadotropin in vitrovirgin Balassa (1940) Table 2. (Continued)

Species Condit-T-6n Mt -,kdrenergic receptor blocker Reference agent effect

in vitro immature f Balassa (1940) estradiol in vitro virgin and 4- 4, Hermansen (1961) estradiol and progesterone in vitro pregnant DHE inhibits Greeff and Holtz (1951) parturient in vitro virgin or f Bulbring et al. (1968) pregnant in vitro mature f DHE inhibits Clegg (1962) di estrus DCI inhibits

Rat

--in vitro mature 4 4- Pronethalol inhibits Le'vy and Tozzi (1963) DCI inhibits Phentolamine no effect

in vitro mature and 4- DCI reverses Jensen and Vennerod (1961) estrogen

in vitro mature and .4 4. Phentolamine inhibits Rudzik and Miller (1962) estrogen DHE inhibits

in vitro mature 4, 4- MJ-1999 reverses Brooks et al. (1965) in- Tift76 mature and 4, 4, Propranolol inhibits DiamondingBrody (1966) spayed

in vitro spayed and 4, 4 Propranolol stimulates Diamond and Brody (1966) estrogen reverses Phentolamine stimulates reverses in vitro spayed and Propranolol reverses Diamond and Brody (1966) estrogen Table 2. (Continued)

Species Condition E Eft Adrenergic receptor bloCker ---Te-ference agent effect

in vitro mature and + Propranolol reverses Paton (1968) estrogen in vitromature and 4 Phentolamine inhibits Tothill (1967) estrogen in vitro pregnant, + Greeff and Holtz (1951) parturient in vitro pregnant, near + Phentolamine reverses Marshall (1967) term Propranolol reverses

Cat in vivo mature, Ergot extract inhibits Dale (1906), Cushny (1906), nonpregnant Rulbring et al. (1968)

early pregnant 4. late pregnant 4 in vitromature, virgin 4 DCI reverses Tsai and Fleming (1964), Phentolamine no effect Vogt (1965) Phenoxyhenzamine no effect

in vitro early pregnant I- Phenoxyhenzamine reverses Tsai and Fleming (1964) Tri. mature Tsai and Fleming (1964) estrogen 4. 4, DCI reverses Graham and Gurd (1960) progesterone DCI no effect Phenoxyhenzamine inhibits extract of f DCI no effect pregnant or P Phenoxyhenzamine inhibits treated uterus Table 2. (Continued)

Species Condition E NE AdrenergiTreceptor blocker Reference agent effect

Human in -vivo mid to early + Kaiser and Harris (1950), Stroup pregnant (1962), Pose et al. (1962), Barden and SfidFF (1968) in vivo in labor Eskes et al. (1965) 77iTio pregnant (term) 4, Garrett (T54),Cibils et al. (1962), Barden and StanA-Er (1968) in vivo nonpregnant Garrett (1955a), Wansbrough et proliferative al. (1967) secretory + Propranolol reverses in vitro pregnant Garrett (19555), Adair and mid + + .Haugen(1939), Lehrer (1965) term + in vitro nonpregnant Milleretal. (1937), Wanshrough proliferative + + Phentolamine inhibits etar.-TT967) secretory + Phentolamine inhibits

aArrow pointing upward: increase, downward: decrease Allyl-estrenol DHE = dihydroergotamine methanesulfate DCI = 1-(3',4'-Dichorlpheny1)-2-isopropylaminoethanol hydrochoride MJ-1999 = 4-(2-isopropylamino-1-hydroxyethyl) methanesulfonanilide 18 a-receptors are stimulatory and s- receptors are inhibitory (table 2).

Though Miller (1967) suggests that all mammalian uteripossess both

types of adrenergic receptors this has not been confirmed in swine.

Myometrial response to nerve stimulation can be modified by alter-

ing the number and ratio ofa- and s- receptors. The primary modifier

of this ratio is the endocrine status of the female (table 2). A classical example of this is the response of the uterus to epinephrine

referred to as "pregnancy reversal". Exogenous epinephrine causes

beta-mediated inhibition in the uterus of prepuberal, estrous and estrogen-treated cats, but alpha mediated excitation in the pregnant

and progesterone-treated cat (Cushny, 1906; Dale, 1906; Robson and

Schild, 1938; Balassa and Gurd, 1941; Tsai and Fleming, 1964). In contrast, estradiol can reverse the inhibitory effects of epinephrine and norepinephrine on uteri of rats and immature guinea pigs to that of stimulation (Mann, 1949; Van Der Pol, 1956; Hermansen, 1961).

Physiology of Myometrial Contractions

The resting potential of the myometrium is defined as the maximum level of membrane depolarization between contractions. Myometrium of ovariectomized rats primed with estrogen has a resting potential of

-50 mV (Marshall, 1959; Kao and Nishiyama, 1964). Smooth muscle cells maintain this resting electronegative charge across the membrane by controlling ion movement. Specifically, the plasma membrane can selec- tively alter the permeability of ions, thus, creatingan ionic con- 19

centration gradient indicative of the electrical gradientas measured

with electrolyte filled micropipettes placed insidethe cell. Dif- + fusion of K ions in and out of the highly permeable membrane results

in a localized negative potential at the internal membrane surfacedue

to the entrapment of large impermeable anions attempting to efflux

with the cation. The permeability of the resting myometrium to Na

ions is about one-tenth that of K ions (Kao and Nishiyama, 1964;

Casteels and Kuriryama, 1965). The resulting intracellular concentra- + tion of K ions is thus greater than in the extracellular fluids. In contrast, the concentration of Na and Cl ions is greater on the out- side of a myometrial cell relative to the inside. These cells can augment this concentration gradient by altering the coupling of Na + + and K ions to the sodium pump such that more Na is exchanged for K ions, thus resulting in a net transfer of cations out of the cell. If this coupling of Na /K ions is greater than unity the condition is considered electrogenic and the cell will become hyperpolarized

(membrane potential<-50mV). The Nernst equation allows a separate

+ -I- expression of the contribution of K , Na and Cl to the equilibrium potential of the myometrial cell at rest; -80, 25 and -20 mV, respectively. These three ions, therefore, establish a complex chemical electrical gradient across the resting membrane indicative ofa net -50 mV potential.

Denervated myometrium has an intrinsic canacity to control both its in vitro and in vivo contraction activity. These spontaneous contractions have three distinctive characteristics; 1) slow rhythmic 20

fluctuations in the magnitude of the electrical potentialacross the

cell membrane, referred to as slow wave or pacemaker potentials, 2)

subthreshold spike depolarizations occurring at the crest of each slow

wave potential, followed by 3) activation of an action potential

(Anderson, 1978b).

Much of the intrinsic oscillatory nature of the myometrium is

related to the activity of the pacemaker potential. Although little

is actually understood regarding the control of pacemaker function,

three models have been proposed. The first model involves cyclic

activity of the electrogenic sodium pump (Connor et al., 1974).

According to this model, initiation of the pacemaker potential results

from a decrease in electrogenic sodium pumping. Ouahain, a specific

inhibitor of the sodium pump, abolishes spontaneous activity of the

intestinal smooth muscle (Connor et al., 1974). Mills and Taylor

(1971) suggest a second mechanism whereby the ionic permeability

changes such that increased Na+ and C1 conductance could initiate

a pacemaker potential. These two models fail to explain the close

cellular association between pacemaker and metabolic activity (Tomita

and Watanabe, 1973). Capp and Berridge (1977) suggest that

oscillating levels of intracellular adenosine 3',5'-monophosphate ++ (cAMP) and Ca ions, feedback in some, as yet undefined, manner to control pacemaker potentials.

The spike depolarizations and subsequent action potentials are mediated by both voltage and time dependent changes in ionic permeability. Specifically, at the threshold potential (" -35 mV) a 21

++ rapid and transient influx of Na and Ca ions causes depolarization

of the cell raising the membrane potential abovezero millivolts. A

fraction of a millisecond later K ions diffuse out of the smooth

muscle cell bringing the membrane potential back to -50 millivolts.

The next process, repolarization, includes changing membraneperme-

ability and activation of the sodium pump to reestablish the ionic

environment indicative of the resting. potential.The repolarization

process is delayed (.3 s) in uterine smooth muscle and differs from

the rapid (3 ms) repolarization observed with other excitable bio-

membranes (Guyton, 1976).

Contraction of smooth muscle is caused by biochemical processes

which appear similiar to those initiating contraction of striated ++ muscle cells. The rapid influx of Ca plus the release of internal ++ Ca , pooled by the sarcoplasmic reticulum and plasma membrane

(Bianchi, 1969; Devine et al., 1972), increases the intracellular concentration of calcium. In striated muscle, calcium binds to troponin stereohindrically allowing the myosin ATPase head to bind to the actin filament to initiate a contraction (Ehashi et al., 1967,

1968; Tonomura, 1972).

Troponin is composed of three subunits, two of which may be phosphorylated by the protein kinase phosphorylase kinase (Stull et al., 1972; England et al., 1972; Perry and Cole, 1973). The activity of this phosphorylase kinase is regulated by the cAMP dependent protein kinase. The phosphorylated form of troponin can thus be altered ++ by cAMP to become more or less sensitive to Ca binding (England et 22

al., 1974). Though troponin has not yet been identified in smooth

muscle, Bremel (1974) suggests the Ca++ sensitivity of actomyosin

ATPase in smooth muscle is associated with the myosin molecule.

The contractile proteins are not organized in smooth muscle to

the same extent as in striated muscle cells, however, contraction is

considered to occur in accordance with the sliding filament theory.

This lack of such an intensive organization in smooth muscle contrac-

tile proteins allows these cells to contract 75% their stretched

length verses 25 to 35% for striated muscle cells (Guyton, 1976).A

smooth muscle cell will also remain contracted longer than striated

muscle cells due to the slower ability of the calciumpump to remove

the cation.

Coordination of myometrial function is accomplished by synchro-

nizing a group or bundle of adjacent cells (Tomita, 1975) through

intercellular electrical coupling. These nexuses or gap junctions

serve as low resistance pathways for propagation of the action poten-

tial (Barr et al., 1968). Taylor et al. (1977) suggest that an

additional intercellular bridge exists between the longitudinal and circular muscle layers. This latter observation might explain how

intact (both layers) tissue can propagate pacemaker potentials, whereas separately only slow waves of low amplitude (Bortoff, 1965;

Kobayashi et al., 1966; Connor et al., 1974) and spontaneous spikes are observed with longitudinal and circular muscle layers, respectively. 23

Modulating Role of Cyclic Nucleotides

Based on the previous discussion of pacemaker and troponin activity it is not surprising that cyclic nucleotides are involved in modulating myometrial function. Sutherland (1971) received the Nobel prize for his discovery of the role of cyclic AMP as a second messenger for activated membrane receptors. Adenosine 3',5'-monophosphate (cAMP) is usually associated with relaxation and guanosine

3',5'-monophosphate (cGMP) with contraction of smooth muscle (Anderson and Nilsson, 1977; Diamond, 1977; Hardmann, 1977).

Korenman and Krall (1977) proposed a model whereby increasing amounts of cytosolic cAMP can initiate relaxation. In this model the ++ activated kinase migrates, in a Ca dependent step, to become associated with the mvometrial cell membrane through hydrophobic interactions (Korenman et al., 1974). In this position the kinase phosphorylates specific membrane proteins which in turn sequester ++ Ca , thereby, decreasing the free to bound ratio of intracellular calcium. Lowered amounts of available calcium will cause inactivation ++ of actomyosin Ca -sensitive ATPase hence resulting in relaxation

(Korenman and Krall, 1977).

Hormonal Modification of Myometrial Function

Exposure to estrogen usually transforms the electrically and 24

mechanically quiescent uterus (resting potential -30 mV) intoa

rhythmically contracting tissue (resting potential -50 mV; Marshall,

1959). Estradio1-176 administration to ovariectomized rats decreases the cAMP to cGMP ratio in the whole uterus (Chew and Rinard, 1974;

Goldberg et al., 1975; Kuehl et al., 1976; Flandroy and Galand, 1978), and causes a decline in cAMP-phosphodiesterase activity (Gardner et al., 1976). Estrogens also might activate uterine contractions in the rat by increasing nexus formation (Dahl and Berger, 1978; Garfield et al., 1980; Merk et al., 1980) as indicated by the increased propagation of action potentials (Rortoff and Gilloteaux, 1980). Estrogen increases a-adrenergic receptors in rat myometrium (Krall et al.,

1978) and increases a-adrenergic receptors in rabbit myometrium

(Roberts et al., 1977; Williams and Leftowitz, 1977). Estrogen may also block neuronal reuptake of neurotransmitter (Sjoberg, 1968;

Hamlet et al., 1980).

Oxytocin, a potent stimulant of the uterine smooth muscle, increases intracellular cGMP (Goldberg and Haddox Hartel, 1973; Dousa,

1977) and calcium (Anderson and Nilsson, 1977). Oxytocin also increases pacemaker potentials (Anderson and Ramon, 1976), lowers the threshold potential and increases both amplitude and frequency of action potentials (Kleinhaus and Kao, 1969). Oxytocin may prevent a-adrenergic induction of cAMP generation (Bhalla et al., 1972).

Estrogen can augment the action of oxytocin presumably by increasing the number of myometrial receptors for this posterior pituitary hormone (Soloff, 1975). 25

The ability of prostaglandin to stimulate uterine contractions was first discovered by Von Euler (1937). Prostaglandin has subsequently been demonstrated to bind to specific membrane receptors in the hamster, rat and human myometrium (Soloff et al., 1973; Wakeling and Wyngarden, 1974; Kimball and Wyngarden, 1975). Prostaglandin has been found to increase intracellular cGMP (Kuehl, 1974), initiate and increase frequency of action potentials (Reiner and Marshall, 1976), release intracellular ionized calcium and inhibit ATP-dependent calcium binding (Carsten, 1974). Prostaglandin E1 (PGE1) and E2

(PGE2) but not prostaglandin Fla (PGF2a) initially elevated myometrial cAMP (Harbon and Clauser, 1971). Isoproterenol-induced elevation of cAMP in rat myometrium was inhibited by PGE2 and PGF2a (Shalla et al.,

1972); PGF2a being a more potent inhibitor (Kroeger and Marshall,

1974). Garfield et al. (1980) observed the ability of prostaglandin

H2, but not PGE1,E2, F2a or thromhoxane 02, to overcome the inhibitory effects of indomethacin on nexuses formation. Estrogen can stimulate uterine PGF2a synthesis in ewes (Ford et al., 1975) and gilts (Frank et al., 1977).

Progesterone antagonizes the action of estrogens, oxytocin and

PGF2a on uterine contractions (Lye and Porter, 1978). The threshold potential, for example, is increased by progesterone and decreased by estrogen (Csapo, 1961), oxytocin and prostaglandin (Kleinhaus and Kao,

1969). Csapo (1956) coined the phrase "progesterone block" to describe the inhibitory effects of progesterone on uterine contractility. Progesterone is primarily thought to maintain quiescence of 26

myometrial activity. Under the influence of progesterone the uterine

myometrium is characterized by an increase in junctional resistance

concomitant with a decrease in the degree of electrical coupling

between cells (Ichikawa and Bortoff, 1970). This might he explained

by the ability of progesterone, in the presence of estrogen, to reduce

the number of gap junctions as compared to the effects of estrogen

alone (Garfield et al., 1980). Progesterone is also considered to

stabilize membrane-bound pools of calcium (Torok and Csapo, 1976). In

comparison to the estrogen-dominated uterus, the progesterone-

dominated myometrium is hyperpolarized (Kuriyama and Csapo, 1961;

Bulbring et al., 1968) and has slower developing pacemaker potentials

(Marshall, 1959).

Another hormone closely associated with altering myometrial

activity, especially during the last trimester of gestation, is

relaxin. The primary source of relaxin is the corpus luteum (Belt et

al., 1971; Anderson et al., 1973) but a recent investigation also

indicates a follicular source (Matsumoto and Chamley, 1980). Relaxin

is a potent inhibitor of smooth muscle contraction both in vitro

(Sawyer et al., 1952; Griss et al., 1967; Chamley et al., 1977) and in

vivo (Porter et al., 1979). The activity of this polypeptide is not mediated via the e-adrenergic nervous system (Wigvist, 1959; Paterson,

1965; Sanborn et al., 1980) nor through an inhibition of oxytocin's action (Miller et al., 1957; Porter, 1971; 1972). Relaxin does, however, inhibit PGF2a activation of uterine contraction (Porter et al., 1979). These latter observations support the supposition that 27 oxytocin and PGF2a act viadifferent mechanisms (Roberts and

McCracken, 1976; Laudanski et al., 1977). Relaxin does increase myometrial concentration of cAMP (Braddon, 1978; Sanborn et al., 1980) without changing the cGMP concentration (Sanborn et al., 1980).

Estrogen potentiates the inhibitory effect of relaxin by increasing the myometrial sensitivity to the hormone (Miller et al., 1957;

Wiqvist, 1959).

Possible Embryonic Stimulants of Mynmetrial Activity

Of the possible embryonic metabolites that could influence myometrial activity, estrogens seem the most plausible.The porcine embryo is capable of estrogen synthesis as early as Day 12 (Perry et al., 1976; Heap et al., 1977); early enough to be involved in the process of embryo migration and spacing. The local effect of estrogen could counteract the dominant suppressive effects of progesterone in such a manner as to stimulate uterine contractions at a site adjacent to the preattached embryo. Such a supposition could explain the observations of Roving (1971), that uterine contractions originated from wherever an embryo was located. Additionally, the existence of an estrogen concentration gradient could explain the observed reduction in myometrial contractions with increasing distance from the embryo.

The influence of estrogens may not be direct on myometrial function but rather indirectly through a local increase in uterine blood flow. Previous investigations have demonstrated a positive rela- 28

tionship between blood flow to the porcine uterus and myometrial

activity (Keye, 1923; Wislocki and Guttamacher, 1924; King and Young,

1957; Dickson et al., 1969a; Zerobin and Sporri, 1972; Ford and

Christenson, 1979). Senger et al. (1967) demonstrated that reduced

blood flow to a portion of the mouse uterus prevented embryo migration

beyond the ischemic area. Though estrogen has been shown to be

vasodilatory in swine (Dickson et al., 1969b), the steroid may be

acting through the release of histamine (Holden, 1939; Spaziani and

Szego, 1958; Shelesnyak, 1959; Szego, 1966; McKercher et al., 1973).

Castro-Vazquez et al. (1976) and Brandon and Raval (1979) suggested

the action of estrogen on the uterus was mediated, in part, through histamine. Because prostaglandins have been found to be involved in

increasing vascular permeability (Kennedy, 1980), the effect of estrogen might be mediated through increased prostaglandin synthesis.

Alternatively, the porcine embryo may synthesize histamine and prostaglandin has been detected in the embryos of the mouse (Dey and

Johnson, 1980) and rat (Dey et al., 1980), respectively.

The rabbit embryo, during the period of migration (between Days 4 and 8), increases in volume 10,000-fold (Boving, 1971). Likewise, the porcine embryo grows exponentially coincident with intrauterine migration. These observations led Boving (1971) to postulate that distension of the endometrium by the expanding embryo signals a wave of contractions. He supported this assumption by demonstrating that spherical beads, the size of unattached blastocysts, could stimulate contractions like those of the gravid uterus. The mechanism whereby 29 distension of the uterus by the blastocyst initiates myometrial contractions has not been determined. However, it has been established that irritation of the uterus will initiatea contraction at the site of the irritation. Prostaglandins (Poyser et al., 1971) and histamine (Szego and Sloan, 1961) have been shown to be released following distension and irritation of the uterus.

The porcine embryo enters the uterus on Day 3 (Hancock, 1961;

Oxenreider and Day, 1965) but fails to migrate toany appreciable extent until Day 7. Coincident with this initial migration the blastocyst hatches from the zona pellucida. Vasodilation, as measured by uterine uptake of stain (Pontamine Sky Blue), is also closely associated with loss of the zona pellucida in rats (Ljungkvist and

Nilsson, 1974), hamsters (Orsini, 1963) and mice (Orsini and McLaren,

1967). Searle et al. (1976) observed that H-2 antigens exist on the surface of mouse trophectoderm and that these proteins are unmasked by the hatching process. It is plausible, therefore, that concomitant uterine vasodilation may be a result of histamine release following an immunological recognition of antigenic proteins on the blastocyst.

Sea urchin ova injected into the anterior uterine horn of the rabbit are distributed throughout the horn within a few hours versus the normal three to four day period required for migration of rabbit embryos (Markee, 1944).

In contrast, it is possible that the porcine embryo synthesizes and liberates a specific, and as yet undiscovered, compound(s) that stimulates myometrial activity. Uteroferrin, for example, is a 30

pregnancy specific uterine protein observed 12 to 16 days post-mating

in gilts (Bazer, 1975) that mightserve such a function. Finally, it

is possible that some by-product of embryonic metabolism,such as CO2,

adenosine or inorganic phosphate, could by itself be vasodilatory and

thus promote migration of embryos.

By identifying Day 90 fetuses according to skin pigmentation,

Dziuk et al. (1964) observed that synchronous transfer of porcine

embryos resulted in fetuses being mixed throughout both uterine horns.

The mixing of white and colored fetuseswas not random and they

concluded that the site of transfer (left or right uterine horn)

influenced the side to which the embryos attached. Webel et al.

(1970) observed that transferring porcine embryos +one day out of

synchrony with the recipients had no detrimental effectson fetal

survival. No investigations, however, have been conducted to examine

the competitive nature of embryo migration and survival utilizing

asynchronous transfer procedures. This approach would allow examina-

tion of the interaction between embryoage and uterine environment.

Care must be taken when conducting such an experiment because

various hormones being synthesized by the transferred embryosmay

advance or retard uterine secretions. This alteration of uterine

secretions could influence the degree of asynchrony between the

transferred embryos and favor continued development ofone age of embryos more than another as Beier et al. (1972) and Adams (1973) observed in the rabbit. Wilmut and Sales (1981) complemented this

research by demonstrating that younger ovine embryoswere able to 31

develop better than older embryos in a second recipient, following

an intermediate transfer to an advanced recipient. Presumably the

"older" uterus and (or) embryos, by being synthetically more active,

advanced embryonic development and the degree of asynchrony more than

the younger counterparts.

Elucidation of the embryonic factor(s) which affects the uterine musculature and distributes the embryos is imperative to achieving the ultimate goal of increasing the number of pigs born at each parturition.

The logical sequence of events for such an elucidation dictates verification of an embryonic effect on the myometrium and examination of the mechanism of such an effect. 32

STATEMENT OF THE PROBLEM

Computer simulation studies, which are programmed to incorporate the economic variables of importance to swine production, have demonstrated that increasing litter size will increasea producer's profit margin more than any other variable.The average number of piglets born/farrowing in the United States has unfortunately remained ata constant eight to nine. The average number of embryos present initially in the uterus is, however, 13 to 15. A large portion of embryos are, thus, lost during gestation. Review of the literature demonstrated that one of the primary causes of this embryonic mortalitywas a lack of sufficient uterine space to accomodate all the embryos.

Cattle and certain breeds of sheep also appear to experiencea limitation in their capacity to supportan abnormal number of embryos.

As attempts are made to increase calving and lambing ratesover one calf and lamb/parturition, respectively, embryonic mortality gains importance. Because only one embryo is necessary to maintain pregnancy the ability of cows and ewes to produce more offspring is not always evident.

Polytocous species such as pigs attempt.to circumvent this restriction of uterine space by distributing their embryos such that each embryo occupies essentially the same intrauterine volume.

Consequently, investigation of the mechanism whereby embryos are distributed may provide more insight into our understanding of embryo mortality. 33

EXPERIMENT I,II and III: INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO -- INTERACTION OF EMBRYO, UTERINE FLUSHINGS AND INDOMETHACIN ON MYOMETRIAL FUNCTION IN VITRO

Introduction

Overcoming the barrier imposed by uterine capacity (Bazer et al.,

1969) is a prerequisite to increasing the number of offspring born at

each parturition. All litter bearing species attempt to reduce this

barrier by spacing their embryos before attachment or implantation.

Patten (1948), Dhindsaet al. (1967), Waite and Day (1967), Polge and

Dziuk (1970) and Anderson (1978a) determined that intrauterine migra-

tion of porcine embryos occurs between Days 7 and 12 (Day 0 = 1st day

of estrus). Intrauterine migration of porcine embryos not only maxi-

mizes the luminal environment within which each embryo can develop,

thereby increasing litter size (Lasley et al., 1963; Bazer et al.,

1969; Gentry et al., 1973), but also results in distribution of

embryos sufficiently and early enough to maintain pregnancy (Dhindsa

and Dziuk, 1968).

A number of hypotheses have been proposed to explain intrauterine migration of embryos. These postulations range from the existence of

regional refractory zones (Mossman, 1937), differential growth (Defeo

and Kleinfeld, 1971), and repulsive electrical charge (Clemetson et al.,

1971) to the more popular hypothesis of peristaltic contractions of

the myometrium (Corner, 1921; Keye, 1923; Wislocki and Guttamacher,

1924; Kho-Seng Lim and Chao, 1927; Boving, 1971; Pusey et al., 1980). 34

This latter hypothesis is confounded by the quiescent nature ofmyo-

metrium during the luteal phase of the estrous cycle (Keye, 1923;

Wislocki and Guttamacher, 1924; King and Young, 1957; Zerobin and

Sporri, 1972). If the peristaltic contraction of uterine smooth

muscle is involved then it would seem that the porcine embryos stimu-

late myometrial function.

The present experiments were conducted to determine whether the

presence of the porcine embryo increased myometrial activity in vitro

and the mechanisms by which the embryo affects the function of the

myometrium.

Materials and Methods

Twenty-four landrace and Yorkshire gilts, checked twice daily for

estrus were used in this experiment. Gilts were assigned to one of

three groups (n = 8) as they were detected in estrus (Day 0) and were

either mated to fertile boars or artificially inseminated 12 and 24 h

later. On Day 2 of gestation gilts were anesthetized with sodium pen-

tobarbital and halothane and subjected to linea alba laparotomy.

Embryos were flushed from one oviduct chosen at random, and transfer-

red to the opposite oviduct. Single ligatures using umbilical tape were subsequently placed firmly around the uterine horn at the fol-

lowing locations; at the uterotubal junction of the flushed oviduct

and 40 and 50 cm posterior to the uterotubal junction of each horn.

This procedure created a "pouch" in the tip of each uterine horn such 35 that the effects of the embryo on uterine physiology were initially concentrated on one side.

Four, 7 or 10 days later (Day 6, 9 or 12 of gestation) gilts were again laparotomized and the uterine horn, to which the embryos had been transferred, was flushed from the external bifurcation to the most posterior ligation (50 cm from the uterotubal junction). This procedure permitted detection of any embryos that had migrated through the ligations. Subsequently, the anterior half of both horns was excised and transported to the laboratory in oxygenated

Krebs-Ringer-Bicarbonate solution (39 C; DeLuca and Cohen, 1964). At the laboratory, the middle of each pouch was clamped with a hemostat and the resulting anterior and posterior halves of each pouch flushed

(35 ml) to recover the embryos.The area between the 40 and 50 cm ligations was also flushed. Tissue samples were immediately removed for utilization in one of three experiments.

Medium used for transferring embryos, flushing the uterus and incubation (Exp. 3) was Modified Ham's F-10 (Kane and Foote, 1970).

Exp. 1. Circumferential strips of myometrium (.5 x 4.0 cm) were excised from the uterine segment containing the most embryos (pregnant horn) and the corresponding segment of the nonpregnant horn (P and MP muscle strips, respectively). The sequence of treatments to which the myometrium was subjected is schematically presented in figure 1.

Muscle strips were suspended in baths containing 60 ml oxygenated

Krebs-Ringer-Bicarbonate solution (39 C) and allowed 40 min to equilibrate to a base tension of 1g. Changes in tension due to isometric 36

INITIAL Es EQUILIBRATE ACTIVITY ISO RECOVER PROP NE RECOVER TENSIONSYENSIuN /\ 30 10 0.5 43 10 15 10 15 15

Figure 1. Treatment sequence (left to right) and exposure time (min) of P and NP muscle strips of Day 6, 9 and 12 gilts to electrical stimulation (ES), isoproterenol (ISO, 80 nq/m1), wash (+), propranolol (PROP, 200 ng/ml) and norepinephrine (NE, 20 nq/ml, Levophed bitartrate).

contractions were monitored for frequency and amplitude with Grass

Force-Displacement Transducers (FT10C) and recorded on a Gould Brush

2400 two channel chart recorder. A contraction was defined as an

increase in tension following a relaxation. The initial activity of

the muscle strips was monitored for the last 10 min of the 40 min equilibration period. Intramural nerves were subsequently subjected

to electrical field stimulation throunh two bipolar platinum elec- trodes situated parallel to the muscle strips (30 s, 70 V, 10 Hz and

1 ms duration). After 5 min the muscle was exposed to a bolus injection of isoproterenol(a 0-adrenergic agonist, concentration in the bath 80 ng/ml) and monitored for 10 min. The baths were then flushed twice and refilled with Krebs-Ringer-Bicarbonate solution and the muscle strips allowed 15 min to recover to the initial activity.

Subsequently, the muscle strips were exposed to propranolol (a

-adreneroic antagonist, 200 nq/m1) and Levophed hitartrate (norepinephrine, an of and 3-adrenergic agonist, 20 nq /ml) and activity monitored for another 10 min. The concentrations of drugs used in this experiment were predetermined to be within the linear portion of 37

the dose-response curve for each respective drug. Finally, the tissue

was washed and the base tension increased from 1 to 5 g. Increasing

the base tension from 1 to 5 g provided an indication of the work

performance of the muscle (King, 1927).

Data were expressed as either frequency (the number of contrac-

tions during a 10 min period) or montevideo units (frequency times

amplitude; Caldeyro- Rarcia and Poseiro, 1960). Due to heterogeneity

of variance, data were subjected to a logarithmic transformation and

analyzed by use of a split-split-plot analysis of variance. Trans-

formed means were compared by a protected least Significant Difference

test.

Exp. 2. Two additional muscle strips of the nonpregnant horn were excised from the same location as in Exp. 1 and placed into

Krebs-Ringer-Bicarbonate solution (4 C) until they could be placed into the muscle baths. The schematic representation of the sequence of treatments to which the muscle strips were subjected is presented in figure 2. As the myometrium warmed to 39 C, contractile activity

INITIAL EQUILIBRATE ACTIVITY F RECOVER INDO RECOVER F INDO

30 10 5 15 10 15 5 10

Figure 2. Treatment sequence (left to right) and exposure time (min) of HP muscle strips of Day 6, 9 and 12 gilts to P or HP flushings (F), wash (+) and indomethacin (Ivo, 10 vq/m1). was restored after which the muscle strips were allowed 40 min to equilibrate to 1 g of tension. The initial activity of the muscle 38

strips was monitored during the last 10 min of the 40 min equilibra-

tion period. Subsequently, 5 ml of Krebs-Ringer-Bicarbonate solution

in the muscle bath was replaced with 5 ml of flushings (39 C) from

either the uterine segment containing the most embryos (P flushings)

or the corresponding segment of the nonpregnant horn (NP flushings).

The flushings were centrifuged at 800x g for 20 min to remove cellu-

lar dehris and kept at 39 C until placement into the muscle baths.

The remaining flushings were refrigerated (4 C) until utilization in

Exp. 3. The flushings were allowed 5 min to affect myometrial activ-

ity after which they were removed and the muscle stripswere washed.

Fifteen minutes after washing, indomethacin in 100 Pl ethanolwas

placed into the muscle baths for 10 min (final concentration, 10

Pg/ml). Indornethacin, an inhibitor of prostaglandin synthesis, was

included because the porcine embryo, at the time of migrationcan

increase the concentration of intrauterine prostaglandins (Zavy et

al., 1980). The tissue was again washed and allowed 15 min to recover

before flushinqs were added. In the presence of the flushings,

indomethacin was again added to the muscle baths and theresponse of the muscle monitored for another 10 min.

Due to the consistency of amplitudes, data were expressed only as frequency of contractions. The initial frequency was subtracted from all the subsequent responses and the resulting data analyzed as in

Exp. 1.

Exp. 3. Uterine tissue from the nonpregnant horn was removed circumferentially at a width of .5 cm and trimmed of endometrium and 39 incubated with or without embryos and either P or NP flushings resulting in a 2 x 2 factorial design. The incubation conditions were optimized in a series of preliminary trials. These conditions consisted of maintaining a neutral pH under an atmosphere of 95% 02-5%

CO2 (3 mm pressure). Tissue was incubated (39 C) for 20 h in Modi- fied Ham's F-10 (20% flushings by volume) at a volume of 5 and 15 ml for the first 6 and the remaining 12 h, respectively. Incubations were terminated by opening the uterine tissue mesometrially and transferring these muscle strips to baths as described above. Contractile activity was then observed for 20 min at 1 g of tension.

Data were expressed as montevideo units and analyzed by use of a split-split-plot analysis of variance with the sub-sub-plots parti- tioned into a 2 x 2 arrangement (embryo x flushings).

Estradio1-17$ Assay. The remaining uterine flushings and the media following the 20 h incubation were subsequently analyzed for estradio1-1745 by radioimmunoassay procedures previously described by

Redmer and Day (1981). The procedure was modified by extracting 5 to

10 ml of flushings or media three times with 10 ml of diethyl ether

(extraction efficiency, > 95%) and the final concentration of antisera used was 1:60,000. Intra-assay coefficient of variation (n = 7) was

11.4%. Final amounts of estradiol in NP flushings, P flushings and incubation media were subjected to a logarithmic transformation and compared by use of an analysis of variance. Differences between transformed means were tested by a protected Least Significant

Difference test.

The quantity of estradiol -178 in uterine flushings from the P and 40

Results

Examination of the uterine flushings revealed limited intra-

uterine migration of Day 6 embryos (figure 3). However, Day 9 embryos

DOM

ow *0 t

t DAY 9 M f WWI t MOP ON r meOM r

00 000

DAY 12

LIGAT1ONS BODY OF UTERUS UGAT1ONS Figure 3. Distribution of embryos within the uterine horns of gilts on Days 6, 9 and 12 (Day 0 1st day of estrus) of gestation. For illustrative purposes the left horn contains the embryos. had begun to migrate through the ligations and by Day 12 some of the embryos had migrated sufficiently past the ligatures to be recovered in the posterior portion of the uterine horn.

The quantity of estradio1-176 in uterine flushings from the P and 41

NP flushings increased by Day 12, but the quantity in P flushingsof Day

12 gilts was greater than that of NP flushings (dayx side interaction,

P<.01, figure 4).

1200-

1101

1030

900

1300 no_

6cc

soe

400.-

300

2CO

100.-

Figure 4. Quantity of estradio1-17E (pg) within the P and NP flushings of Days 6, 9 and 12 gilts. Each point represents the mean + SE of eight gilts.

Exo. 1. Results of this experiment indicated that concomitant with migration of the embryos there was an increase in the frequency of muscle contractions at 1 g of tension (figures 3, 5 and 6). The contractile activity of P muscle strips steadily increased from Day 6 through 12 but that of NP muscle strips failed to increase until Day

12 of gestation (day x side interaction, P<.10). At 5 g base tension the frequency of contractions of P or NP muscle strips did not increase until Day 12 (figure 5). Likewise, when the muscle activity was 42

/REGNANT NONPREGNANT

so SO Sg Tension

40 40

,..30 30

sc 20 20

10 10

0 Day 6 Day 9 Day 12 Day 6 Day 9 Day 12 Figure 5. Frequency of contractions of myometrial strips excised from Day 6, 9 and 12 pregnant gilts at 1 and 5 g tension. Each point represents the mean + SE of eight gilts.

/REGNANT NON/REGNANT

19 TO MI iC01 5g Tension

0 Day 6 Day 9 Day 12 Day 6 Day 9 Day 12

Figure 6. Activity (montevideo units) of myometrial strips excised from Day 6, 9 and 12 pregnant gilts at 1 and 5 g tension. Each point represents the mean + SE of eight gilts. 43

expressed as montevideo units (figure 6)a day x side interaction

(P<.10) was evident at both base tensions. As the embryos migrated

down the uterine horn the muscle activity (montevideo units)of the P

muscle strips increased almost linearly while that of the NPmuscle

strips did not increase until Day 12.

Electrical field stimulation produced no alterations in contractile activity of the myometrium. The addition of isoproterenol reduced

(P<.05) the frequency of contractions of both P and NP musclestrips

(table 3). Reduction in the number of contractions was indicative of

TABLE 3. PERCENTAGE CHANGE IN FREQUENCY OF UTERINE CONTRACTIONS FOLLOWING EXPOSURE TO a AND 0 ADRENERGIC AGONISTSa,b

Day 6 Day 9 Day 12

P NP NP P NP

ISO(%4)c 45.3e 40.7e 82.7f 64.9ef 73.5ef 77.8f

NE (%f)d 33.3 23.5 22.1 38.9 28.4 29.9

a Numbers represent the mean of eight myometrial strips. b Standard error basedon the common estimate of variance = 10.6. Isoproterenol (80 ng/m1). d Propranolol (200nq /ml) plus levophed bitartrate (norepinephrine, 20 ng/ml). e,f Means with differentsuoerscript letters are different (P<.05).

e-adrenergic stimulation because the base tension was below 1g.

Furthermore, the response of myometrium of both horns to isoproterenol increased (P<.05) by Days 9 and 12 of gestation. Although this increase in --adrenergic responsiveness did not correspond with the initial activity at 1 and 5 g tension, itmay be consistent with the quiescent nature of the uterine nervous system at this time. Exposure 44

to propranolol (at a dosage sufficient to antagonize the 0-receptors)

and norepinephrine increased (P<.05) the base tension and the fre-

quency of contractions relative to the initial activity.

Exp. 2. Results of this experiment demonstrated that the addi-

tion of P and NP flushings recovered on Days 6, 9 and 12 increased

(P<.01) the frequency of contractions of NP muscle strips of Day 6, 9

and 12 gilts, respectively (figure 7). It is possible that this

PREGNANT 1E3NONPREGNANT

DAY 6 a co 04 'sax Elbe wii CC z-2 0

0-8 DAY 9 zC.) O UJ cc z

W Z

C.)

W CC

Fiushings Indomethacin Fiushing FU shinQs Indomethocin Figure 7. Changes in contractile frequency of myometrial strips excised from Day 6, 9 and 12 pregnant gilts at 1 g tension. The initial frequency has been subtracted from all the illustrated responses. Each bar represents the mean + SE of eight gilts. The open bars represent responses of muscle strips exposed to P flushings and the cross-hatched bars NP flushings. 45

increase was due to ionic differences after the replacement of

Krebs-Ringer-Bicarbonate solution with Modified Ham's F-10. The

greatest increase (P<.05) in number of contractions, however,was

observed with P flushings of Day 12 gilts. Indomethacin reduced

muscle activity in all cases to the same frequency of contractions,

thus, the reduction was greater with Day 12 myometrium exposed to Day

12 flushings than Day 6 or 9 myometrium and flushings. Reintroducing

flushings increased the frequency of contractions above theresponses

to indomethacin in all cases by the same magnitude. The ultimate test

was to determine if indomethacin could reduce the muscle activity in

the presence of flushings. Under these conditions, only the P flush-

ings of Day 12 gilts overcame the effects of indomethacin (P<.01).

Exp. 3. Results of this experiment are presented in table 4.

TABLE 4. ACTIVITY (FONTEUCEC UNITS) OF MYOMETRIAL STRIPS.b FOLLOWING IN.SIBATITI WITH CR WITHOUT EMBRYOS AND FLUSWINGS"'

P tissue P tissue NP tissue NP tissue P flushings rP flushings P flushings NP flushings

With Without With Without Uith Without With Without embryo embryo embryo embryo embryo embryo erbnm embryo Day 6 6.4 7.4 5.1 5.2 5.53 4.8 6.03 5.99 Day 9 4.9 9.5 11.1 9.3 11.9 10.2 11.8 10.1

Day 12 6.3 3.7 3.4 3.5 5.3 5.4 9.8 9.4

a bNumbers represent the mean of eight myometrial strips. Standard error based on the commo,1 estimate of variance 5.5.

Activity of P and NP muscle strips, though depressed following the 20 h incubation in the presence of corresponding flushings and in the presence and absence of the embryos, was greater (P<.05) for Day 9 gilts than either Day 6 or 12 gilts. There were, however, no side differences (P vs NP) in muscle activity. Likewise, neither the 46

addition of flushings nor the embryo altered muscle activity.

The embryos remained viable under the experimental conditions

because Day 6 embryos hatched from the zona pellucida. Additionally,

the embryos synthesized estradiol during the 20 h incubation (table 5)

TABLE 5. QUANTITY OF ESTRAOIOL-176 (P9) IN THE INCUBATION MEDIUM FOLLOWING EXPOSURE OF PYOMETRIAL STRIPS TO FLUSHINGS AND EMBRYOS4'

P tissue P tissue NP tissue MP tissue. P flushirgs MP flustinos P flushings NP flushings

With Without with Without With Without With Without embryo embryo er.tryo e-bryo embryo embryo embryo embryo

Day 6 36.8 33.4 35.4 38.3 28.8 28.7 37.4 28.6

Day 9 180.7 27.9 49.2 32.0 39.9 24.5 45.5 33.1

Day 12 3512.3 2677.4 1119.5 56.7 2352.3 2279.9 2000.1 76.1

tNatters represent the rean of eight otservations. Standard error based on the comqon estimate of variance 63.8.

in a manner similar to that synthesized by the embryos in vivo (figure

4).

Discussion

Results of the first experiment demonstrated that intrauterine migration of the porcine enhyro through uterine ligations coincided with increased myometrial activity. By Day 9 of gestation embryos migrated through the first ligation (40 cm from the uterotubal junction), three days later, Day 12, embryos had migrated through the second and most posterior ligation. Dhindsa and Dziuk (1968) observed that porcine embryos could migrate through double ligations placed one-third but not through similar ligations place two-thirds the dis- 47 tance from the uterotuhal junction. The activity, frequency of contractions or montevideo units, of P muscle strips of Days 9 and 12 gilts increased relative to P muscle strips of Day 6 gilts. Muscle strips removed from an area of the uterus devoid of embryos (NP muscle strips) failed to increase in frequency of contractionsor montevideo units until Day 12. Although it is not possible to conclude that embryo migration was due to increased myometrial function, these data do support the supposition of myometrial involvement in embryo migration set forth earlier (Corner, 1921; Keye, 1923; Wislocki and

Guttamacher, 1924; Roving, 1971; Pusey et al., 1980).

The increase in frequency of contractions of NP muscle strips of

Day 12 gilts was not expected and is not consistent with the quiescent nature of the uterus as observed in gilts on Day 12 of the estrous cycle (Keye, 1923; Wislocki and Guttamacher, 1924; King and Young,

1957; Zerobin and Sporri, 1972). Perhaps by Day 12, the stimulatory influence of the embryo becomes systemic. Alternatively, embryos may have migrated through the uterine body to a position posterior to the ligations on the nonpregnant horn, thereby influencing myometrial function.

It does not appear from these data that the presence of the embryo stimulated muscle activity through the autonomic nervous system because the porcine uterine nerves were quiescent at this time. This apparent quiescence of intramural nerves was confirmed by two independent procedures (W. F. Pope, data not shown). First, electrical field stimulation, similar to that used in the present experiment, 48

increased the contractile activity of striated muscle from ratsas

well as bovine myometrium. Second, the addition of physiological (100

ng/ml) and then pharmacological doses (>10 ;KIM) of both imipramine

and tyramine failed to significantly alter responses of the porcine

myometrium. These drugs, in combination, concentrate the neuro-

transmitter present in the varicosities within the neuromuscular

junction. Spies et al. (1960) demonstrated that denervation of the

porcine uterus failed to interfere with normal cyclic behavior.

Additionally, sows can parturate normally following transection of the

spinal cord (Theobald, 1968).

Alpha- and beta-adrenergic stimulation was sufficient to conclude

that the porcine myometrium contains receptors for both these agonists. Response of P and HP muscle strips to the 0-adrenergic agonist increased concomitant with the descent of the blastocysts.

However, this response might be due to an increase in myometrial

$-receptors caused by the increased levels of estrogen being synthesized by the embryo or endometrium. Krall et al.(1978) demonstrated that estrogen increased 6-adrenergic receptors in the myometrium of the rat.

Because the stimulatory influence of the embryo was not neural it was concluded that the factor(s) responsible for increasing muscle activity was hormonal and affected the smooth muscle cells of the uterus.

Results of Exp. 2 demonstrated that some short-acting component of the uterine flushings increased the frequency of muscle contractions.

This factor(s) found in the flushings mimicked, in nart, the changes 49

in intrinsic muscle activity observed in Exp. 1. The Day 12 embryo

was able to alter uterine flushings sufficiently to overcome the inhib-

tory effects of indomethacin. Vane and Williams (1973) observed the

ability of indomethacin to reduce spontaneous activity of rat myometrium. Indomethacin has been shown to inhibit: 1) prostaglandin

synthesis (Vane, 1971), 2) protein kinase (Catalan et al., 1980;

Goueli and Ahmed, 1980), 3) localized increases in endometrial vascular permeability (Kennedy, 1980), 4) and prostaglandin binding to

serum proteins, thus increasing the rate of oxidation of prostaglandin

(Attallah and Lee, 1980). Results of the present experiment suggest the nature of the active component in Day 12 flushings as prostaglandin or some compound with comparable activity. Zavy et al. (1980) observed an increase in the concentration of luminal PGF2a in uteri of pregnant gilts beginning 12 days post-mating.

Uterine flushings coincuhated with myometrium for 20 h (Exp. 3) failed to alter muscle activity. However, flushings used immediately after recovery from the uterus were able to affect myometrial function within a 5 min exposure period (Exp. 2). Perhaps the reduced activity of the muscle strips after a 20 h incubation precluded any change in activity produced by the flushings. Alternatively, these observations suggest either some sort of destabilization of the factor(s) found in flushings or destabilization of an endometrial intermediate. The increased activity following incubation of myometrium of Day 9 gilts may be due to chance alone or some component of uterine physiology not investigated in the present experiment. 50

Estradiol synthesis by the blastocystwas also coincident with migration of the blastocyst through the ligations. The Day 12 embryos coincubated with myometrium synthesized estradiol in culture butwere unable to increase muscle activity as determined after coincubation.

In a separate trial, a 20 h incubation of muscle strips of Day 12 nonpregnant gilts either in the presence or absence of estradiol (1 ng/ml media) failed to increase muscle activity, 12.8+6.4 vs 19.6+10.5 montevideo units (n = 5), respectively (W. F. Pope, data not shown).

These data suggest that either estradiol is not involved in increasing muscle activity, or under these conditions, could not directly stimulate smooth muscle cells. The latter supposition might involve estrogen stimulation of an intermediate(s) such as prostaglandin or histamine which in turn activates the myometrium.

Understanding the mechanism(s) involved in the descent and even- tual distribution of embryos in polyovular species might prove valuable to increasing litter size. Results of these experiments suggest an involvement of myometrial function in displacing the porcine embryos through the uterine lumen. This stimulatory influence(s) of the embryo appears to be hormonal rather than neural. The effect of the embryo does not appear to be exerted directly on the smooth muscle cell but rather may involve an intermediate of short half-life. 51

EXPERIMENT IV and V: INTRAUTERINE MIGRATION OF THE PORCINE EMBRYO: INFLUENCE OF ESTRADIOL-17$ AND HISTAMINE

Introduction

Migration and the equidistant spacing of embryos is an inherent characteristic of polyovular species to minimize embryo mortality. In the sow, intrauterine migration of embryos occurs between Days 7 and

12 post-mating (Dhindsa et al., 1967). The more popular explanation for intrauterine migration includes an involvement of the uterine musculature (Corner, 1921; Keye, 1923; Wislocki and Guttamacher, 1924;

Kho-Senq Lim and Chao, 1927; Boving, 1971; Pusey et al., 1980).

Increased synthesis of estradiol by the porcine embryo was found to occur concomitantly with the migration of the embyros and increased myometrial activity in vitro (Exp.1 and 3).

The association between estradiol synthesis and migration of the embryos may be more than coincidentally related. It was observed

(Exp. 1 and 3) that neither the embryo nor estradiol directly stimulated the myometrium in vitro as was observed in vivo. However, uterine flushings of Day 6, 9 and 12 pregnant gilts increased the frequency of myometrial contractions in vitro during a 5 min exposure period. Only flushings of Day 12 gilts overcame the inhibitory effects of indomethacin, suggesting an indirect action of estradiol on myometrial function through prostaglandins (Exp. 2).

Alternatively, the action of estradiol on the uterus could have been 52

mediated, in part, through histamine release (Spaziani and Szego,

1958; Shelesnyak, 1959; Szego, 1966; McKercher et al., 1973).

Histamine, by increasing uterine blood flow (Holden, 1939; Harvey and

Owen, 1979), could increase myometrial activity (Keye, 1923; Wislocki

and Guttamacher, 1924; King and Young, 1957; Dickson et al., 1969b;

Zerobin and Sporri, 1972; Ford and Christenson, 1979) sufficiently to

initiate migration of the embryos. We previously observed that im-

proper sterilization of silastic beads and the resulting inflammatory

response of the uterus, indicative of histamine release, resulted in

migration of some of the beads throughout the entire length of the

uterus.

The present experiments were conducted to examine the role of

estradiol and histamine in migration of the porcine embryo.

Materials and Methods

Exp.4. Ten gilts, having estrous cycles of normal duration, were

assigned randomly to receive ten silastic (Dow Corning, 891) beads

impregnated with either cholesterol or estradio1-17B. These beads were introduced surgically (linea alba incision) via a 1 ml injection

of physiological saline containing 100 units Penicillin, 0.25lig

Fungizone and 25 Pg Strentomycin (Gibco Laboratories) into the tip of

one uterine horn on Day 7 of the estrous cycle (Day 0 = 1st day of estrus). Five days later, Day 12, the migration of the beads was examined by flushing segments (10 to 15 cm) of the excised uterine horns 53

with saline. The total distance the beads migratedper gilt was calcu-

lated by the formula: total distance (cm)= (length of segments tra-

versed by each bead)- 10 x length of the anterior segment (10 to 15

cm) to which the beadswere introduced.

Beads impregnated with cholesterol and estradiolwere manufac

tured containing 1 and 2 g of steroidper ml of silastic glue,

respectively. Cholesterol was impregnated into the beads to control

for the physical properties of beads impregnated with estradiol.

Steroid was mixed with glue to forma slurry and the resulting mixture

partially dried before being formed into spherical beads (1 to 2mm

dia.). A black ink (No. 414) was also incorporated into the slurry to

facilitate the recovery of beads from the uteri.

Beads were preincubated (39 C, 24 h) inserum from an ovariec-

tomized gilt to preclude the initial spike release of steroid after

placement in utero. Subsequently, the beads were washed with saline,

blotted dry, autoclaved (121 C, 35 min) and suspended inthe injection

solution. Preliminary trial results indicated that estradiol

impregnated beads treated in thismanner and incubated (39 C) in a balanced salt media for eight days releaseda constant 3.28+0.27 ug

1 estradiol 10 beads* - ldaye" , well within the physiological range of estradiol leaving the gravid uterus at this time (Ford et al., 1981a).

Thin layer chromatography (developed with benzene:ethanol, 9:1, vol:vol) revealed that only estradiolwas released into the medium during these trials.

Exp. 5. Twenty gilts and sows were assigned randomly to one of 54 four groups (n = 5). On Day 6 of gestation the uteriwere surgically exteriorized (linea alba incision) and the anterior portion (0 to 30 cm posterior to the uterotubal junction) of each horn subjected to multiple subserosal injections (total volume, 3 ml) of sterilized 1% methyl-cellulose in saline containing either: 1) 16 mg a-lactose

(vehicle), 2) 16 mg cromolyn sodium and a-lactose (1:1, w:w), 3) 16 mg cromolyn sodium and a-lactose and 2 mg histamine or 4) 16 mg cromolyn sodium and a-lactose. The quantities of cromolyn sodium and histamine used in this study were similar to those used by Dey et al.

(1978) to investigate the role of histamine in implantation in rabbits. Cromolyn sodium is an inhibitor of histamine release

(Brogden et al., 1974; Dykes, 1974) and does not inhibit myometrial function (Cox and Beach, 1973). Four days later, Day 10, animals within the first three groups were hysterectomized and segments (15 to

20 cm) of the excised uterus flushed with saline to recover the embryos. Females within group 4 were reinjected on Day 10 with 16 mg cromolyn sodium and a-lactose and hysterectomized on Day 12 of gestation and the embryos recovered as above.

Transuterine migration (migration through the uterine body) can occur as early as Day 10 (Dhindsa et al., 1967), however, this experiment was not specifically designed to examine this phenomenon.

Transuterine migration was observed in a limited number of females, in which case the ovary having the fewest corpora lutea (CL) was considered the origin of an equivalent number of the adjacent embryos.

For the remaining gilts and sows the origin of the embryos (left or 55

right ovary) was considered conservatively to be from the adjacent

ovary.

The total distance the embryos migratedper uterus was calculated

by the following formula: total distance (cm)={ (Elength of segments

traversed by each of the embryos from the left ovary)- [ number of

embryos x length of the anterior segment from the left horn ]}

Orlength of segment traversed by each of the embryos fromthe right

ovary) - [ number of embryos x length of the anterior segment from

the right horn ]} . It was necessary to subtract the length of the

anterior segments since all embryos were located in these segments

before their migration.

Due to heterogeneity of variance, datawere subjected to a square

root transformation and compared by using an analysis of variance.

Differences between transformed means were compared by usinga protected Least Significant Difference test.

Results and Discussion

Results of Exp. 4 (figure 8) demonstrated that the estradiol -17h impregnated beads migrated further (P<.05) than cholesterol impregnated beads, 384.2+96.1 vs 68.8+28.6cm, respectively. In a preliminary trial it was determined that cholesterol did not inhibit migration as silastic beads devoid of any steroid failed tomove down the horn of gilts at this stage of the estrous cycle. Migrating porcine embryos synthesized estradiol (Perry et al., 1976; Heap et al., 56

BEADS IMPREGNATED WITH CHOLESTEROL

if It

BEADS IMPREGNATED WITH ESTRADIOL- 178

U so

111.

rSIP

SCALE4ii At 'At A1lA A-4 C }C 2C 3040 50607C SO 900010421330.40 CM. Figure 8. Distribution of silastic beads (10/horn) imnregnated with cholesterol (top) or estradiol -17h (bottom) after 5 days in utero (n = 5). Beads were inserted into the uterus on Day 7 of the estrous cycle. Uteri are drawn to scale with the body of the uterus aligned at the center. For illustrative purposes the side originally containing the beads is depicted on the left.

1977; EXP. 3) and release of this steroid displaced silastic beads the size of these embryos. Therefore, it seems probable to conclude an involvement of estradiol in intrauterine migration of embryos.

In the fifth experiment no deleterious effects of treatment with cromolyn sodium or histamine were observed regarding the morphology and percentage of embryos recovered and length of the uterus (table 6). 57

Table 6. Mean (+ SE) percentage of embryos recovered and mean length (cm) of uteri of gilts treated with cromolyn sodium or cromolyn sodium plus histamine on Day 6 and 10.

Cromolyn sodium Cromolyn plus Cromolyn Control sodiuma histaminp sodium (Day 10)b (Day 10)b (Day 10)° (Day 12)c

Embryo recovery (%)d 69.1+6.4 68.6+4.7 82.7+ 5.3 79.7+6.2

Length of the uterus (cm)e 108.4+5.6 105.8+8.3 130.4+12.3 118.2+7.6

a Cromolyn sodium (Fisons, Intal). b Treated on Day 6 and embryos examinedon Day 10. Treated on Day 6 and 10 and embryos examined on Day 12. d Percentage embryos recovered= (embryos recovered/CL number) x 100. e Length of the uterus = mean of both uterine horns.

Therefore, data as expressed (mean distance the embryos migrated/female) were unbiased. Cromolyn sodium restricted the Day 10 embryos to the tip of the uterine horns but not (P<.05) the Day 12 embryos

(figure 9). Histamine overcame (P<.05) the inhibitory effects of cromolyn sodium alone such that normal intrauterine migration was observed.

Although cromolyn sodium has a number of possible actions, exogenous histamine was able to overcome a well documented effect of cromolyn sodium, specifically, the inhibition of endogenous histamine release (grogden et al., 1974; Dykes, 1974). This experiment was not designed to examine how histamine could be involved with embryo migration. Neither was this experiment designed to determine what factors associated with gestation cause the release of histamine.

However, it is suggested that histamine by increasing uterine blood flow (Holden, 1939; Harvey and Owens, 1979) increased myometrial 58

4110-

430 -

420 -

3410r-

I NSOb- 300- 270- 240- 2l0-

"3°' I50 no- 90-

CO-

0 CONTROL CROMOON CROMOLYN CROMOLYN (DAY 10) SODIUM SODIUM SODIUM (DAY 10) (DAY 12) MtSTAMINE+ (DAY 10) Figure 9. Mean distance migrated (cm) of embryos following exposure of the gravid uterus to control (vehicle, a-lactose), cromolyn sodium (16 mg), cromolyn sodium plus histamine (16 and 2 mg, respectively) and cromolyn sodium (16 mg) on Day 6. Day in parenthesis denotes the day of hysterectomy. The last group received additional cromolyn sodium (16 mg) on Day 10. Each bar represents the mean+SE of 5 females.

activity sufficiently to displace the embryos through the uterine

lumen. Ford et al. (1981b) observed an increase in uterine blood flow

associated with migration of the porcine embryos.

The concentration of intrauterine estradiol increased between Days

10 and 12 of gestation (Zavy et al., 1980). Therefore, if estradiol induced histamine release in the uterus of pigs, as in other species

(Spaziani and Szego, 1958; Shelesnyak, 1959; Szego, 1966; McKercher et _al., 1973), then perhaps insufficient cromolyn sodium was present be- 59 tween Days 10 and 12 to inhibit release of this hdogenic amine. The

Day 12 porcine embryo might also have synthesized histamineas has been demonstrated with rabbit (Dey et al., 1979) and mouse (Dey and Johnson,

1980) embryos. If so, then histamine of embryonic origin, could overcome the cromolyn sodium between Days 10 and 12 as exogenous histamine did between Days 6 and 10 in this experiment. A final possibility might be that the action of estradiol and histamine were independent of each other.

Knowledge of the mechanism(s) whereby embryos migrate throughout the uterine lumen may lead to a better understanding of how polyovular species attempt to reduce mortality associated with crowding of embryos.

Results presented in this manuscript suggest an involvement of estradiol-17s and histamine in intrauterine migration of porcine embryos. Further investigations are necessary to delineate the exact mechanism(s) by which each compound acts. 60

EXPERIMENT VI and VII: SURVIVAL OF PORCINE EMBRYOS AFTER ASYNCHRONOUS TRANSFER

Introduction

Considerable variation exists in embryonic development within all

polytocous females. This is not surprising because in pigs ovulation

extends for a 6 h period (Lewis, 1911). Accordingly, the first

cleavage division of the fertilized ova occurs between 60 and 108 h

after the onset of estrus in sows (Oxenreider and Day, 1965).

Anderson (1978a) noted marked variation in the morphology of porcine

embryos between Days 11 and 13 of gestation. Within a uterine horn

embryos ranged in development from spherical and tubular to filamen-

tous. However, embryo survival was not altered when embryos were

transferred 1 day out of synchrony with the recipient (Wehel et al.,

1970), suggesting that this within-animal variation is normal.

A number of physiological events, important for survival of the porcine embryo, are closely associated with developmental changes of the embryo. These events include estrogen synthesis (Ford et al.,

1981a), maintenance ofpregnancy (Frank et al., 1977) and embryo migration (Patten, 1948). Because the majority of embryonic mortality occurs during early gestation it is possible that embryos more advanced in development (older) have a survival advantage over those less well developed (younger).

The present experiment was conducted to determine if embryos further developed embryologically migrated further and had a greater 61

chance for survival duringpregnancy.

Materials and Methods

Exp. 6. Sixteen gilts and sows, checked daily for estrous

behavior, were utilized in this experiment. Recipients, 6 days after

the onset of estrus, received four to six embryos each from Day 5 and

7 donors (Day 0 = 1st day of estrus). Such a procedure allowed estab-

lishment of pregnancy with embryos 2 days apart inage but only 1 day

out of synchrony with the recipient. The side to which the embryos,

within an age, were introducedwas randomized. Gilts assigned on the

appropriate days as donors were mated 4 and 24 h after the onset of

estrus. To maximize utilization of females, two recipientswere used

for each Day 5 and 7 donor. On Day 11, 5 days post-transfer, the

recipients were slaughtered and the embryos recovered by flushingseg-

ments (10 to 20 cm) of the excised uterus with physiological saline.

Age (Day 10 or 12) and location of the embryos and the length of

uterine segments were recorded. The age of the recovered embryos was

determined by size. Only those recipients that received viable Day 5

and 7 embryos, as determined following examination of the paired

recipients on Day 11 of gestation, were included in the statistics.

The distance the embryos migrated (cm) was determinedas follows: total distance = {(Elength of uterine segments traversed by each embryo)- (the total number of Day 5 or 7 embryos transferred) x [the length of the initial uterine segment (10 to 20 cm) containing the 62

respective embryos]). Distance the embryos migrated was compared by

using a least-squares analysis of variance.

Exp. 7. Twenty-four [eight purebred colored (Duroc, Spotted or

Hampshire), eight purebred white (Yorkshire, Landraceor Large White)

and eight crossbred] gilts and sows were utilized in this experiment.

Day 6 recipients received embryos in accordance with procedures

discribed in Exp. 6 except donors were mated to appropriately pheno-

typed boars. To have sufficient embryos to maintain pregnancy onlyone

recipient was used for each Day 5 and 7 donor. Attempts were made to

balance the number of Day 5 and 7 embryos introduced into each

recipient (10.9+.9 Day-5 and 10.4+1.0 Day-7 embryos, 7(7+ SE).Breed

of donor (colored vs white) was randomized such that four Day-5 and

four Day-7 colored donors were utilized. Fetuses were recovered be-

tween Day 60 to 70 of gestation, identified by skin pigmentation, weighed and the distance between adjacent fetuses noted. One gilt

aborted on Day 60 of gestation, in which case, all fetuseswere recovered immediately and skin pigmentation recorded. Fetal weight was

subsequently correlated with the distance between adjacent fetuses.

The percentage of Day 5 and 7 embryos surviving to Day 11 (Exp. 6) and

60 of gestation were compared bya nonparametric Mann-Whitney U-test.

Embryo Manipulation. Embryos were collected surgically from donors by flushing the anterior half of each uterine horn towards the cannulated (medical grade teflon tubing, 1.50 mm I.D., 2.11mm 0.D.) tip of the uterus. The flushing medium (table 7) was similar to that utilized by Davis and Day (1978) except lactate and pyruvatewere 63

yam 7.MODIFIED KREBS -RINGER -81E48800TE.

logftdient 9/500 .1 mll

84C1 3.500 119.78

Itl 0.178 4.78

CFAC12 2820 0.125 1.71

abe04 0.081 1.19

85504 7820 0.147 1.19

108E03 1.053 25.00

Gimes. 0.500 5.56

Bovine seven albuslob 2.000

Antibiotic-antinycoticc 50,000 onits/5 .1

Davis and 00. 1978.

b ontes bovine elbwein crystallized. Miles Labovotovies.

c Antibiotic-ontisbcotic, lyophilised. Gibco Laboratories.

deleted, antibiotic-antimycotic substituted for penicillin G and streptomycin and sodium chloride increased to maintain physiological osmolarity. Recovered embryos were incubated (39 C, 95%02-5% CO2) for not more than 30 min before being transferred. This transfer procedure consisted of aspirating the embryos and medium into medical grade teflon tubing (.69 mm I.D., .99 mm 0.0.) and effluxing the contents into the tip of the uterus through the posterior 3 to 4 cm of the oviduct. Only morphologically normal appearing embryos were transferred.

Results and Discussion

Results of Exp. 6 (table 8) indicated no difference in the ability of Day 5 and 7 embryos to survive for 5 days after transfer.

However, by mid-gestation (nay 60) more fetuses which developed from 64

TABLE 8.PERCENTAGE SURVIVAL OF DAY 5 AND 7 EMBRYOS TO DAY 11 AND 60 OF GESTATION

Day 5 embryos Day 7 embryos

No No. No. recip-embryos % embryos % cents trans- No. survival/ trans- No. survival/ utilized ferred survived recipient ferred survived recipient

Day 11 8a 45 19 42.3+10.4 42 18 43.1+12.4 d 8b Day 60 87 6 8.2+ 6.9 83 53 62.6+ 7.7

a Two recipients were not includedas five of the 10 transferred Day-7 embryos were recovered and none of the 11 Day-5 embryos. b Eight recipientswere not included after failing to maintain pregnancy following the introduction ofa total of 78 Day-5 and 63 Day-7 embryos. c,d,e Means withdifferent superscripts within rows are different (P<.001).

Day 7 embryos survived than fetuses which developed fromDay 5 embryos

(P<.001). Only two recipients contained fetuses which developedfrom

Day 5 embryos at Day 60 of gestation. Only females that remained pregnant to Day 60 were included in the data. This elevated the survival of Day 7 embryos at Day 60 comparedto Day 11 of gestation, because, the percentage of all transferred Day 5vs Day 7 embryos surviving to Day 11 and 60was 33.9 vs 44.2 and 3.6 vs 36.3, respectively.

Migration of porcine embryos occurs between Days 7 and 12

(Dhindsa et al., 1967). Transferred Day 5 and 7 embryos were mixed within both uterine horns aswas previously observed with synchronous transfer (Dziuk et al., 1964). The older embryos (Day 12) failed to migrate further than theyounger embryos (Day 10) when examined on Day

11, 160.1+29.6 vs 113.5+16.1cm, respectively. However, the distance 65

the embryos migrated may have been different if examined atan earlier

time. Because only about 40% of the transferred embryos were viable

on Day 11, differentiating the healthy from the dying embryos would

have been difficult before this time.

After the variation between recipients was reduced by standard-

izing fetal weight (Day 60), the distance between adjacent fetuses was

highly correlated with fetal weight (r = .47, P<.01). Rathnasabapathy

et al. (1956) ohserved a similar relationship with fetuses examined on

Day 55 of gestation. Knight et al. (1977) demonstrated a significant correlation of fetal weight to placental length suggesting a relationship between migration of the porcine embryo, outgrowth of the pla- centa and the subsequent development of the fetus.

Identification of embryos by size in Exp. 6 was more subjective than skin pigmentation of the fetuses in Exp. 7. Wright and Grammer

(1980) observed a 25-fold increase in protein content of the porcine embryos between Days 8 and 9 of gestation. This exponential growth of embryos continued between Days 9 and 16. Although considerable variation existed, spherical shaped Day 12 embryos were larger and contained fourfold more protein, than spherical Day 10 embryos (Anderson,

1978a). Little difficulty was experienced in the present study in differentiating between Day 10 and 12 embryos. To confirm that Day 5 embryos could survive in Day 6 recipients, three additional recipients received only Day 5 embryos. Two recipients remained pregnant to Day

60 with 36.4 and 12.5% of the transferred embryos surviving. Thus, 2 more (P<.05, X, 1 df) Day 5 embryos survived to Day 60 in the 66

absence of Day 7 embryos than with them (24.5 vs 8.2%, respectively).

Though the majority of porcine embryos destined to die doso

around Day 30 (Longenecker and Day, 1968; Pepe et al., 1968, 1972;

Bazer et al., 1969; Webel and Dziuk, 1974) it is unknown whenor why

the younger embryos (Day 5) died between Days 11 and 60 of gestation.

Since the porcine embryo can elongate rapidly, 3 cm/h (Geisert et al.,

1981), the possibility exists the older embryos (Day 7) elongated

sooner and occupied more of the uterus than the younger (Day 5)

embryos. Anderson (1978a) observed the inability of embryos to over-

lap each other regardless of the uterine space available. Knight et

al. (1977) observed an increase in mortality of crowded fetuses be-

tween Days 40 and 100 due to placental insufficiency. Perhaps in this

experiment the younger embryos (Day 5) died because of placental

insufficiency induced by the older embryos (Day 7).

Another explanation for the loss of the younger embryos

(transferred at Day 5) might include some sort of physiological

advancement in the biochemical development of the recipient's uterus

such that the younger embryos could no longer continue to develop.

Beier et al. (1972) and Adams (1973) demonstrated the fragile rela-

tionship between synchronizing the pattern of uterine secretions and

the age of successfully transferred rabbit embryos. Exogenous estro-

gen extends the length of the estrous cycle of nonpregnant pigs

(Gardner et al., 1963) possibly by altering secretion of uterine proteins (Geisert et al.. 1979), intrauterine accumulation of prostaglandin (Frank et al 1978) and uterine blood flow (Ford and 67

Magness, 1980). It is possible the older embryos (Day 7), by synthe- sizing estrogen earlier, advanced the secretory pattern(s) ofthe uterus resulting in the demise of the younger embryos (Day 5).

The precise mechanism by whichsome embryos survive and others die in polytocous species isan enigma. Understanding this phenomenon would prove valuable to reducing embryonic mortality. These experiments indicated embryos more embryologically advanced havea greater chance to survive and actuallymay have caused the demise of embryos less embryologically developed. 68

General Discussion

Analysis of the results from these experiments indicated an association between myometrial function and migration of the porcine embryo. This seems reasonable since embryos could migrate past ligations previously placed around the uterus. Certainly this was not a random or passive event but rather an active, if not a forceful, process. The mechanism(s) whereby the presence of the embryo stimulates muscle contraction appears to be hormonal since the uterine nervous system was quiescent at this stage of gestation.

Estrogen, a hormone synthesized by the migrating embryo, may be such a stimulant. If I may speculate, it seems possible that embryonic estrogens indirectly stimulate uterine contractions by sequestering prostaglandin within the uterine lumen.The effects of prostaglandin would then be to stimulate contractions of the uterine smooth muscle cells. Alternatively, estrogen, alone or by inducing histamine release, could increase blood flow to an area of the uterus associated with the embryo. This localized increase in uterine blood flow could increase the supply of nutrients to the myometrium which could in turn increase contractile activity. The possibility still exists that the embryo synthesizes prostaglandin or histamine. An alternate hypothesis would involve an immunological recognition of antigenic sites on the embryo and the subsequent release of histamine or prostaglandin by the uterus.

It seems important that the model for stimulation of myometrial 69

function be associated with normal embryonic development, ie., estro-

gen synthesis or hatching from the zona pellucida, such that the grav-

id uterus conservatively expends the energy necessary for embryo

migration. Following eons of natural selection, one must respect the

efficiency of reproductive functions. In addition to understanding of

mechanisms whereby the embryo stimulates myometrial function, further

investigations are required to establish how adjacent embryos are

equidistantly spaced.

Embryos of litter bearing species, such as the pig, are in

constant competition for survival with their litter mates. I

observed through experimentation that the presence of embryos which

were slightly advanced in their development, had a survival advantage

over, and resulted in the destruction of, the less developed embryos.

As we attempt to increase litter size it seems important to continue

investigation of the nature of this embryonic competition. The logi-

cal sequence of events is to examine when and how the interaction

between embryos of different developmental stages occurs.

Once these criteria are established then we can proceed with

overcoming the barriers associated with uterine capacity and begin to

increase litter size. The importance of litter size in swine produc-

tion dictates that we investigate these phenomena. Of course as we

increase litter size further investigation of neonatal survival, management, selection and lactation are required to sustain the

increased number of piglets born. 70

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