BIOLOGY

OF

REPRODUCTION

21,

Maintenance

of the

281-288

(1979)

Corpus

Luteum

I. Luteotropic W.

E.

Properties

ELLINWOOD,’

T.

Department

M.

NETT

of Physiology Colorado State

Fort

of Early

Pregnancy

of Embryonic

Collins,

and

in the

Ewe.

Homogenates G.

D.

NISWENDER

and Biophysics, University,

Colorado

80523

ABSTRACT Experiments were carried out to examine the Iuteotropic properties of embryonic homogenates in ewes. Homogenates prepared from embryos collected 14-15 days postcoitum were infused into the uterine lumen of cycling ewes (n = 4) from Days 12 through 18 postestrus. Four control ewes received isotonic saline only. Embryonic homogenate, infused at a rate of 3 embryonic equivalents/24 h, prolonged the lifespan of corpora lutea through Day 18 postestrus. Mean serum progesterone levels on Days 16, 17 and 18 were 2.2 ± 0.5 ng/ml (±SEM), 2.0 ± 0.4 ng/ml and 1.6 ± 0.4 ng/ml in the treatment group and 0.33 ± 0.29 ng/ml 0.04 ± 0.01 ng/ml and 0.06 ± 0.01 ng/mI in the control group. Luteal weights on Day 18 were 626 ± 22 mg in the treatment group and 194 ± 46 mg in the control group. To determine if the luteotropic property of embryonic homogenate was due to the presence of Ll-I or prolactin-like molecules, we examined embryonic protein in sensitive radioreceptor assays for these hormones. However, 100 pg of embryonic protein had no activity in either the LH-hCG or prolactin radioreceptor assay. In a second study, 100 pg of embryonic protein had no effect on synthesis of progesterone or cAMP in dispersed ovine luteal cells, either alone or in combination with LH and/or prolactin. Synthesis of both progesterone and cAMP could be stimulated in these cells by NIH-LH-S19. The data indicate that homogenates of 14-15-day-old embryos are capable of prolonging luteal function when infused into the uterine lumen, but the effect does not appear to be exerted via an LH or prolactin-like molecule.

INTRODUCTION Maintenance requires

of

for

at

at

duced

by

the a

of

uterine

a local to

1974

ovarian and

Horton

known uterine

to

1966a,b),

the

comes

an

be

(1967)

factor

extended

of

Rowson,

into of of

at

level

a

nonpreg-

like

and

Accepted Received Present Center,

April January address:

Beaverton,

1979. 3, 1979. Oregon Regional

OR

have

sensitive

16,

bility Primate

Research

or

that

281

the

luteo-

ovary

via

a local

vein

to

LH

ruled

re-examined

the

embryonic

or

prolactin-

luteotropic

prop-

homogenates to

test

homogenates molecules.

homogenate

was

possibility

out.

assays

embryonic

ovarian

factor the

an not

The on

the

Mapletoft’s

luteotropic

secretes

by

is exerted

that

molecule,

radioreceptor

or

Although

the

was

ovine

prolactin-like

embryonic

97005.

that

later

blood-borne

its effect

uteroovarian

embryo

of

a

the

at

1976)

embryo

that

reach

gonadotropin We

the

mean

luteolysis

(1975,

that

ovary.

lipid-soluble

erties

by

without to

species-specific

demonstrated

from

the

al.

cycle

also

overcomes

the

suggested

that

estrous

were

et

but

embryo,

homogenates

a

and

must

whole,

estrous

of

secretes

horn of

factor

small

on

evidence

which

artery,

of

the

uterine

pathway

daily

frozen

gravid

tropic

a

interpreted

which

homog25-day-old

sheep

were

is secreted

the

of

effect

Mapletoft direct

studies,

over-

homogenates

length

injections

obtained

for

that

level.

luteotropin

Rowson

embryos the

Intrauterine

it in

embryo

demonstrated

sheep

sheep

labile

Though

no

data

a local

present

or

embryos

These

heat

Ginther,

serum,

transfer

14-15-day-old

see

via

or

embryos

injections

uteroovarian

unknown.

injections

had

embryo

and

the

remains

14-15-day-old nant

which

or

Systemic

effect.

postestrus

(Moor

in

of

in-

cells pig

12-14-day-old

the

be

13

prevented way

intrauterine

cycle.

Day

respectively, length.

is

ovary

1977). must

by

luteolysis Moor

Poyser,

embryo

lumen

luteolysis

and

and

that

In

blood

14-day-old

that

the

from

embryos,

heat-inactivated,

(probably

reaches

reviews

sheep

of

corpus

cycle

factor

(for

the

days

the

estrous

pathway artery

ewe

of

1955). of

the

which

venoarteria!

vein

50

white

enates

by

Martinet,

luteolytic

F2cw)

the

first

regression

end

prostaglandin

the

and

ewes,

luteum

in

progesterone least

(Denamur

nonpregnant

the

of

luteum

gestation

is

pregnancy

secretion

corpus

thawed

synthesis

using the

possi-

contain direct

LII

effect of

proges-

of

ELLINWOOD

282

terone was

and also

cAMP

by

dispersed

ovine

examined.

MATERIALS Intrauterine

In fusion

Embryonic

Homogenates

AND of

METHODS

luteal

cells

ET AL.

thawed and placed into the infusion flasks. All glassware and connecting tubing were sterilized prior to infusion. The infusate contained 1000 IU penicillin/ ml, 500 pg streptomycin/mI and a final protein concentration of 160 pg/mI as determined by the method of Lowry et al. (1951). The infusate was maintained at 6-9#{176}C with continuous stirring and was administered at a rate of 6 mI/h using a Sage model 375 peristaltic pump. Ewes in the control group (n = 4) received isotonic saline (6 mI/h) containing antibiotics; the infusion procedures were identical in all ways to those for embryo infused ewes. Blood samples for analysis of progesterone were taken twice daily by means of an indwelling jugular cannula. On the morning of Day 18 postestrus, ewes were laparotomized and the marked corpora lutea were removed, weighed and frozen for later analysis of progesterone. Concentration of progesterone in serum and luteal tissue was determined by radioimmunoassay (Niswender, 1973).

Western range ewes were visually checked for estrous behavior twice daily using vasectomized rams. The first day that a ewe stood to be mounted was designated Day 0 of the estrous cycle. Ewes from which embryos were subsequently collected were hand-mated to 2 fertile rams on Day 0, then kept penned with the rams until surgery on Day 14 or 15. Fifteen of the 70 donor ewes were superovulated with a s.c. injection of 500 IU PMSG on Day 13 of the estrous cycle; this treatment increased the average ovulation rate from 1.3 to 2.2 ovulations/ewe. For collections of embryos, ewes were anesthetized with Radioreceptor Assay of 20-25 ml sodium pentobarbital (65 mg/mI) and the Embryonic Homogenate uterus exposed via a midventral abdominal incision. Radioreceptor assay for LH-like hormones was The uterine wall was punctured with mosquito hemocarried Out with minor modifications according to the stats at a point just below the intercornual bifurcation method of Haour and Saxena (1974). Ewes were and the hole enlarged by opening and closing the hemostats as necessary. A 15 cm length of Tygon superovulated by the method of Sheridan et al. (1975) tubing with a specially shaped glass tip (o.d. ‘\1.0 cm) to obtain large numbers of corpora lutea. Corpora was inserted into the hole and held in place by an lutea were removed surgically 9-11 days after ovuassistant. Each uterine horn was flushed with 25 ml lation and homogenized in 5 volumes (w/v) 0.025 M sterile isotonic saline introduced near the uterotubal sucrose containing 10 mM Tris, 1 mM MgCI2 and 1 junction and the uterus was “milked” in the direction mM CaCI2 (pH 7.2). The homogenate was filtered of the collecting tube. The entire contents of the through 4 layers of cheesecloth and centrifuged at 480 X g for 10 mm. The supernatant was centrifuged at flush were collected into a sterile culture tube and snap frozen in a dry ice-acetone bath. Embryos 10,000 X g for I h and the resulting pellet resuspended collected in this manner were stored frozen at -20#{176}C in 10 mM Tris, 1 mM MgCI2 and 0.1% bovine serum until used. Following surgery, donor ewes were albumin (BSA) (pH 7.2) for the assay. Highly purified penned with a large herd of normally cycling ewes and hCG (CR 117, 13,000 lU/mg) was radioiodinated by visually checked for estrous behavior twice daily. the lactoperoxidase method (Diekman et al., 1979). Ewes infused with embryonic homogenate or saline The specific activity of this preparation was \60 were laparotomized on the morning of Day 11 postpci/pg and was 50-60% bindable to receptor in the presence of excess membranes. The assay utilized “v20 estrus and a sterile 20 gauge polyvinyl chloride cannula was inserted “‘.‘2 cm into the tip of the uterine lumen pg of membrane protein/tube (Lowry et al., 1951) which bound 20-25% of the I’25I1-hCG added. Tubes ‘\‘l cm from the tubouterine junction of the side ipsilateral to the ovary bearing the corpus luteum. The were incubated at 23#{176}Cfor 24 h. For the separation of membrane-bound and free [‘25Il-hCG, 3 ml ice cold cannula was secured by means of a clove-hitch around Tris buffer were added to each tube and the tubes the cannula and a purse string in the uterine serosa were centrifuged at 2250 X g for 30 mm. Supernatants using 5-0 cardiovascular silk. The cannula was also were decanted and radioactivity associated with the secured to the mesovarium and externalized through a pellets was quantified in an automatic gamma counter stab wound in the flank of the ewe. Corpora lutea were marked with charcoal for future identification. Ewes (Nuclear Chicago, Model 1185). The radioreceptor assay for prolactin-like horwere allowed to recover from anesthesia and were placed in metabolic crates where they received hay or mones was carried out according to the method of alfalfa pellets twice daily and water ad libitum. Shiu et al. (1973). Membranes were prepared from rabbit mammary tissue obtained during late lactation. Embryonic homogenate was prepared from a pool The assay utilized 200 pg of membrane protein/tube of 126 embryos. The contents of each vial were (Lowry et at., 1951). Radioiodination of NIH-PROLthawed in a warm-water bath, homogenized 5 strokes in a ground glass homogenizer and homogenates were SIl (20 pg) was similar to that described for hCG. After purification on Sephadex G-150, [‘2511-prolactin pooled and large particles allowed to settle. The solution was transferred to sterile vials (3 embryonic was 40-60% bindable to rabbit mammary receptors in equivalents/vial) and snap frozen in a dry ice-acetone the presence of excess membranes. All other procebath. The homogenate was composed of the entire dures were as described for the LH-hCG radioreceptor assay. Ovine placental extract was prepared according uterine flushing and thus contained both uterine and embryonic factors. Beginning on the morning of Day to the method of Chan et al. (1976) from ovine 12 postestrus, each ewe (n = 4) was infused continuplacentomes collected on Day 70 of pregnancy. ously with 3 embryonic equivalents/24 h until Day 18 Embryonic homogenate was assayed in both assays in postestrus. Every 24 h a fresh vial of homogenate was 10-fold dilutions from 100 pg-b ng total protein.

SHEEP

This

quantity

of

equivalent

embryonic

to 1.2%

protein

of 1 embryo

EMBRYONIC

(100

on a dry

pg)

LUTEOTROPIN

was

weight

basis.

283

Luteal

weight

terone

were

and

embryonic Effect

of

on

Synthesis

and

cAMP

Ovine

Embryonic of by

Luteal

Homogenate

Dispersed ovine luteal cells were prepared according to the method of Simmons et at. (1976). Incubations were carried out in tissue culture Medium 199 (Grand Island Biological Co.) with 25 mM HEPES and room air as the gaseous phase. Separate experiments were run in parallel for determinations of progesterone and CAMP. All treatment groups were tested in triplicate. For determination of progesterone, each tube contained 15,000 viable large luteal cells and test substances as required in a final volume of 800 p1. The large lutcal cells (average diameter 28 pm) comprised 4-5% of the total cell population (Simmons et at., 1976). Viabilities were determined by staining with 0.2% trypan blue (Tennant, 1974). For determination of cAMP, each tube contained 30,000 viable large luteal cells in Medium 199 containing 0.5 mM isobutyl methyl xanthine to inhibit phosphodiesterase activity. Incubations were carried out for 2 h at 37#{176}Cwith continuous shaking (60 cycles/mm) in a Dubnoff metabolic incubator. After incubation, tubes were immersed in a boiling water bath for 20 mm to disrupt the cells and destroy enzyme activity. Progesterone and cAMP were quantified by radioimrnunoassay (Jordan et at., 1978). One-way analysis of variance and Tukey’s hsd procedure (Steel and Torrie, 1960) were used to test for statistical significance of differences

of

ovulation.

In

ipsilateral

to

fresh

there ewe.

terone

in 14

Day

did and

not

(mean

15

SEM)

Nine

included was

within in

not

which

TABLE. I. Luteal onic homogenate

no

the

extended

were

mated

embryos

weights or isotonic

in

61

additional

in

analysis.

of

the

4

onic

± 0.4

and

flushed,

were

in

receptor

assay,

with

different

In

due

which

to

In

human

placental

ovine gen)

but

not

the

placental as well

had

[‘25ll-prolactin NIH

on

Day

be

4

ovine

times

greater This

LII

in ewes

assay,

(P