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THE EFFECTS OF INSULIN AND INSULIN-LIKE GROWTH FACTOR I
ON BOVINE CORPORA LUTEA.
BY
Alan Dale Ealy

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Science

1990

:QQSrGQ&$

ABSTRACT
THE EFFECT OF INSULIN AND INSULIN-LIKE GROWTH FACTOR I
ON BOVINE CORPORA LUTEA
BY
Alan Dale Ealy

To determine the effect of low insulin on serum
progesterone in heifers maintaining energy balance, sixteen
Holstein heifers were assigned to isocaloric diets of 100% hay
or 90% corn silage:10% soybean meal (CS). Jugular blood was
sampled to quantify insulin and progesterone in serum. Area
of insulin profiles was less in heifers fed hay than in
heifers fed CS. Diet did not affect concentrations of
progesterone. Thus, low postprandial concentrations of
insulin do not affect serum concentrations of progesterone.

To determine effects of IGF-I on basal and
LH-induced secretion of progesterone and number of luteal
cells, corpora lutea were collected from ten Holstein heifers
at d5 or d10 postestrus. Corpora lutea were incubated for
24h with LH (0, .1 or 1 ng) and IGF-1 (0 or 500 ng). At d5
and d10 postestrus, media concentrations of progesterone and
DNA content of cultures increased with 1 ng LH compared with
basal and .1 ng LH. However, 500 ng IGF-I did not affect
progesterone secretion or DNA content from luteal cells at d5
or d10 postestrus. Hence, secretion of progesterone and

luteal cell numbers were not affected by IGF-I.

ii

The encouragement and support of my parents during my Masters
program was immeasurable. To demonstrate my appreciation, I

dedicate this thesis to Harold and Suzanne Ealy.

iii

ACKNOWLEDGEMENTS

I wish to extend my deepest gratitude to my major
professor, Dr. Roy Fogwell. Dr. Fogwell's patience combined
with his knowledge of animal science made him an ideal
graduate student educator. Thanks for everything Roy. Drs.
James Ireland and H. Allen Tucker were also inspirational by
instilling their perspectives of research and education to me.
I thank members of my guidance committee; Drs. Benson, Ireland
and Wells for assistance with my research and education at
Michigan State University.

Support from Larry Chapin and Trudy Hughes made my
Masters seem "almost bearable", rather than "completely
overwhelming." The generous aid of fellow graduate students
in research, seminars and classes were always appreciated.
I thank members of our physiology group: Brent Buchanan, Geoff
Dahl, Jill Evers, Trudy Hughes, Jackie Newbold, Teri Martin,
Debbie Peters and Steve Zinn for their comradary. Finally, I
would like to thank Monica and Lee Houghtaling for always
being there when I needed them. I know there's no place as
great as Pennsylvania but they sure made Michigan more

appealing.

iv

LIST OF FIGURES

LIST OF ABBREVIATIONS

INTRODUCTION

REVIEW OF LITERATURE

DEVELOPMENT AND FUNCTION OF CORPORA
Function of corpora lutea
Development and maintenance of

Bovine luteal cells in_xit:g_

Summary

INSULIN

Structure and general function

TABLE OF CONTENTS

Control of insulin secretion .

Action of insulin

effects of insulin
mechanism of insulin action

Effects of insulin on ovarian cells

corpora

granulosal-thecal cells cells .
luteal cells

Summary

vii

viii

10
10
10

12
12
13

14

INSULIN-LIKE GROWTH FACTOR I (IGF-I)

Structure and general function .

Control of IGF-I secretion .

Actions of IGF-I . . . .
effects of IGF-I .

mechanism of IGF-I action

Effects of IGF-I on ovarian cells

granulosal-thecal cells .

luteal cells . . .
summary 0 0 O O O O O O

GENERALSUMMARY.......

° POSTPRANDIAI.CONCENTRATIONS OF

2092

O O O O O 15
O O O O O 15
O O O O O 17
O O O O O 17
O O O O O 18
O O O O O 19
O O O O O 19
O O O O O 20
O O O O O 21

O O O O O 23

INSULIN DOES NOT

AFFECT SERUM CONCENTRATIONS OF PROGESTERONE IN HEIFERS.

INTRODUCTION . . . . . . . .
“THO DS 0 O O O O I O O O O 0
RESULTS 0 O O O O O O O O O 0

DISCUSSION . . . . . . . . .

EKREBINENT_III. THE EFFECT OF

BASAL AND LUTEINIZING HORMONE

O O O O O 25

O O O O O 26

O O O O O 28

O O O O O 38

INSULIN-LIKE GROWTH FACTOR I ON

INDUCED

SECRETION OF

PROGESTERONE FROM DEVELOPING BOVINE LUTEAL CELLS.

INTRODUCTION . . . . . . . .
MATERIALS AND METHODS . . . .
RESULTS 0 O O O O O O O O O 0

DISCUSSION . . . . . . . . .

FOOTNOTES . . . . . . . . . .
SUMMARY AND CONCLUSIONS . . .

LIST OF REFERENCES . . . . .

vi

Figure

Figure

Figure

Figure

Figure

Figure

LIST OF FIGURES

Body weight of heifers fed hay . . .
or corn silage.

Effect of diet on serum concentrations
of insulin in heifers from 2h before
to 6h after feeding..

Effect of postprandial insulin on . .
serum concentrations of progesterone.

The acute effect of postprandial . .
insulin with progesterone in heifers.

Effect of LH and IGF-1 on DNA in . .
media and cells at 24h.

Effect of LH and IGF-I on secretion of

progesterone from bovine luteal cells
cultured at d5 or d10 postestrus.

vii

31

33

35

37

47

49

CAMP
DNA
FSH

IGF-I

NEB
PEB
PGFZG

RIA

LIST OF ABBREVIATIONS

cyclic Adenosine Monophosphate
Deoxyribonucleic Acid
Follicle-Stimulating Hormone
Insulin-like Growth Factor I
Low Density Lipoprotein
Luteinizing Hormone

Negative Energy Balance
Positive Energy Balance
Prostaglandin F2 alpha
Radioimmunoassay

Ribonucleic Acid

viii

INTRODUCTION

Fertility is the ability of females to produce offspring
(Hensyl, 1987). Fertility is determined in dairy cows by
detection of estrus, conception and survival of embryos.
Interestingly, the previous determinants of fertility in dairy
cows are correlated positively with decreased serum
concentrations of progesterone (Rosenberg et al., 1977; Erb
et al., 1976; Folman et al., 1973; Hill et al., 1970).
Therefore, it is important to investigate luteal development
and function.

Serum concentrations of progesterone are decreased in
negative energy balance (NEB) heifers compared with positive
energy' balance (PEB) heifers (Villa-Godoy' et al., 1990;
Harrison and Randel, 1986). Negative energy balance occurs
when calories needed to support body functions exceed calories
ingested. During NEB, body fat is mobilized, causing
decreased.body weight (Villa-Godoy et al., 1989). During PEB,
calories ingested exceed calories needed to support body
functions. Thus, nutrients are stored and. body' weight
increases (Villa-Godoy et al., 1989).

During NEB in heifers, serum concentrations of

progesterone and lutyeal weight are decreased (Villa-Godoy et

2
al., 1990; Harrison and Randel, 1986). Eighty-one to 92% of
dairy cows experience NEB during early lactation (Villa-Godoy
et al., 1989; Reid et al., 1966). Because of the high
preveance of NEB and its negative effects on luteal
development and function, defining mechanisms of NEB induced
retarted luteal function are merited.

During NEB in heifers, decreased serum concentrations of
progesterone and decreased luteal weight are associated with
decreased.postprandial concentrations of insulin (Villa-Godoy
et al., 1990; Harrison and Randel, 1986). Exogenous insulin
increased luteal content of progesterone and luteal weight in
NEB but not PEB heifers (Harrison and Randel, 1986).
Therefore, reduced luteal development and function may be due
to low postprandial secretion of insulin, but these data were
confounded by NEB.

Serum concentrations of IGF-I are less during early
lactation than during late lactation (Range and Blum, 1988).
Since a majority of dairy cattle experience NEB during early
lactation, decreased serum concentrations of IGF-I are
coincident with decreased luteal development and function.
Insulin-like growth factor I increases basal secretion of
progesterone from cultured bovine luteal cells (McArdle and
Holtorf, 1989). But, IGF-I did not affect.basal or LH-induced
secretion of progesterone from bovine luteal cells in other

studies (Leavitt and Condon, 1989; Schams et al., 1988).

3
These disceptancies may be due to differences in dose of IGF-
I.

Knowledge of development and function of corpora lutea
and effects of insulin and IGF-I on bovine corpora lutea are
presented in this review of literature. current knowledge
does not answer several questions. Does low postprandial
concentrations of insulin decrease serum concentrations of
progesterone in heifers maintaining energy balance? Also,
does IGF-I increase basal and LH-induced secretion of
progesterone and number of cells from cultured bovine luteal

cells? These questions will be addressed in this thesis.

B§!I§!_QE_LII§BAIEB§

DEVELOPMENT AND FUNCTION OF CORPORA LUTEA

FUNCTION OF CORPORA LUTEA

Length of the bovine estrous cycle averages 21d. One
corpus luteum develops from cells of each ovulated follicle
and is maintained for an average of 17d (Kaltenbach and Dunn,
1980). Progesterone is secreted from corpora lutea.
Immunoneutralization of progesterone caused.precocious estrus
and ovulation in heifers (Roche, 1976). Therefore, secretion
of progesterone from corpora lutea maintains length of estrous
cycle in cattle.

In cattle, if pregnancy is not recognized by the uterus
from d16 to d18 postestrus, uterine secretion of prostaglandin
F2“ (PGFZG) increases (Kaltenbach and Dunn, 1980) . In
combination 'with estradiol, PGFa, decreases secretion of
progesterone from corpora lutea, causing luteal regression
(Kaltenbach and Dunn, 1980). If pregnancy is recognized by
the uterus during this time, uterine secretion of PGFhlis not
increased and corpora lutea are maintained throughout
pregnancy (Jainuden and Hafez, 1980) . Thus, secretion of
progesterone from corpora lutea also maintains pregnancy in
cattle.

A putative mechanism for maintenance of estrous cycle

length and pregnancy by the secretion of progesterone is by

5
inhibition of ovulation. A peri-ovulatory increase in serum
concentrations of LH is essential for ovulation in cattle
(Kaltenbach and Dunn, 1980). In ovariectomized heifers,
administration of progesterone decreases frequency of LH
pulses (Price and Webb, 1988) . Consequently, spontaneous
ovulation is inhibited by luteal secretion of progesterone

(Price and Webb, 1988).

DEVELOPMENT AND MAINTENANCE OF CORPORA LUTEA

The preovulatory surge of LH causes ovulation and
initiates differentiation of granulosal and thecal cells into
luteal cells (Donaldson and Hansel, 1965). During ovulation,
cytologic changes and vascularization of follicular tissue are
characteristic of luteinization (Donaldson and Hansel, 1965).

After ovulation, luteal weight in cattle (Ireland et al.,

1973) and sheep (Farin et al., 1986; Schwall et al., 1986:
Parry et al., 1980) and steroidogenic cell numbers in sheep
(Farin et al., 1986; Schwall et al., 1986; Parry et al., 1980)
increase until d8 to d12 postestrus.

Luteinizing hormone maintains secretion of progesterone
from corpora lutea. Hypophysectomy in ewes (Kaltenbach et
al., 1968) or immunoneutralization of LH in heifers (Hoffman
et al., 1974) causes luteal regression. Exogenous LH
maintains serum concentrations of progesterone in
hypophysectomized ewes (Kaltenbach et al., 1968).

Additionally, LH maintains epithelial morphology

6
(Gospodarowicz and Gospodarowicz, 1974) and increases
secretion of progesterone (Rodgers et al. , 1988) from cultured
bovine luteal cells.

Luteinizing hormone may mediate decreased secretion of
progesterone during NEB since concentrations of LH in serum
are decreased.in.NEB»cows (Rutter and.Manns, 1987). However,
in other studies, serum concentrations of LH did not differ
between NEB and PEB heifers (Villa-Godoy et al. , 1990: Knutson
and Allrich, 1988). Thus, factors other than LH also mediate

decreased luteal function during NEB.

BOVINE LUTEAL CELLS m

In vigrg, bovine luteal cells collected at st
postestrus respond to LH (Rodgers et.al., 1988). In addition,
LH maintained epithelial morphology of cultured bovine luteal
cells (Gospodarowicz and Gospodarowicz, 1974). At d8 to d12
postestrus, basal and LII-induced secretion of progesterone
from bovine luteal cells decrease with increased duration of
culture (Pate and Condon, 1982). Also, luteal cells collected
at d8 to d12 postestrus are responsive to LH from 0'to 48h and
from 5 to 7d in culture but not between 3 to 4d in culture
(Pate and Condon, 1982) . Conversely, basal secretion of
progesterone from bovine luteal cells collected at d2 to d4
postestrus is maintained for up to 5d in culture (McArdle and
Holtorf, 1989). McArdle and Holtorf (1989) purified luteal

cells to remove red blood cells and cellular debris. It is

7
not known whether sustained secretion of progesterone from
cultured.bovine luteal cells.is dependent.on.corpora lutea.age
or purity of cell preparation. Investigation of basal and
LH-induced secretion of progesterone from.non-purified.bovine
luteal cells is limited to luteal cells collected at 2 d5

postestrus and cultured for 0 to 48h or 5 to 7d.

SUMMARY

Secretion of progesterone from corpora lutea maintains
length of the estrous cycle and pregnancy in cattle
(Kaltenbach and Dunn, 1980; Jainuden and Hafez, 1980).
Decreased concentrations of progesterone and luteal weight
during NEB are not due only to decreased concentrations of LH
(Villa-Godoy et al., 1990; Knutson and Allrich, 1988).
Additional mediators of decreased luteal development and
function during NEB are not defined.

ln_yi;:g, bovine luteal cells are responsive to LH from
0 to 48h and 5 to 7d in culture (Pate and Condon, 1982).
Also, bovine luteal cells from corpora lutea 2 d5 postestrus
respond to LH in culture (Rodger et al., 1988). Thus several

constraints preside when investigating luteal development and

function in_yi§;g.

INSULIN

STRUCTURE AND GENERAL FUNCTION

In the early 20th century, the Islets of Langerhans were
determined to secrete a product that prevented diabetes (Orci
et al., 1988). In 1921, the protein was isolated and named
insulin, from the latin "insulae" (islands) (Orci et al.,
1988).

Insulin consists of two polypeptide chains connected by
disulfide bonds and labelled as A and B chains (Orci et al.,
1988). Insulin is a‘glucoregulatory hormone which increases
glucose, amino acid and lipid uptake into adipose, muscle and
hepatic tissues (Froesch et al., 1986). Insulin also has
stimulatory effects on ovarian tissue (Adashi et al., 1988a:

Maruo et al., 1988).

CONTROL OF INSULIN SECRETION

Glucose stimulates secretion of insulin from beta cells
of the Islets of Langerhans in nonruminants (Orci et al.,
1988) and ruminants (Mannsrand Boda, 1967). But, most.glucose
is metabolized during ruminal digestion so butyrate,
propionate and acetate are main ruminal products used for
calories. In sheep, acetate does not affect secretion of
insulin (Manns and Boda, 1967). But, propionate and butyrate

increases beta cell secretion of insulin (Brockman and

9
Laarveld, 1986) and increases serum concentrations of insulin
(Trenkle, 1971; Manns and Boda, 1967).

Serum concentrations of insulin can be altered by
qualatative nature of diet. Diet affects the molar proportion
of volatile fatty acids from rumen digestion. For example,
grain diets increase rumen production of propionate and
butyrate, but hay diets decrease propionate and butyrate
production from the rumen (Trenkle, 1971) . Production of
acetate is not affected by diet (Trenkle, 1971). iIn cattle
(McAtee and Trenkle, 1971) and sheep (Trenkle, 1971)
postprandial concentrations of insulin increase with feeding
grain and hay but does not increase with feeding only hay.
Consequently, diets with high starch increase postprandial
concentrations of insulin. Whereas, diets with high fiber and
low starch do not increase postprandial insulin.

In NEB heifers, postprandial concentrations of insulin
are decreased compared with PEB heifers (Villa-Godoy et al.,
1990; Harrison and Randel, 1986). Likewise, serum
concentrations of insulin are lower during early lactation
than during late lactation (Smith et al., 1976; Koprowski and
Tucker, 1973). Consequently, insulin secretion is altered by

NEB.

ACTION OF INSULIN
W

Insulin increases uptake of glucose, lipids and/or amino
acids in every tissue investigated to date. However, actions
of insulin have been studied mostly in adipose, skeletal
muscle and hepatic tissues.

Insulin increases number of intracellular and membrane
bound transporters for glucose in adipose and muscle tissue
(Simpson and Cushman, 1986) . In adipocytes, synthesis of
fatty acids from glucose (Jungas, 1975; Simpson and Cushman,
1986) and acetate (Brockman and Laarveld, 1986) are increased
by insulin. In skeletal muscle, insulin increases uptake of
amino acids and reduces protein breakdown (Fukagawa et al.,
1985). Insulin also increased glycogen synthesis in
hepatocytes (Brockman and Laarveld, 1986). Consequently,
insulin facilitates transfer and storage of nutrients and
precursors from blood into tissues in adipose, muscle and
hepatic tissues.

MW

Insulin acts by binding to receptors on target cells
(Carpentier, 1989; Czech, 1985). Receptors for insulin are
protein complexes with two extracellular (a) and two
transmembrane (3) subunits. Membrane receptors are arranged
in a fi-a-a-fi configuration by disulfide bonds (Carpentier,
1989; Czech, 1985). Insulin binds to alpha subunits of

receptors, causing phosphorylation of tyrosine and.methionine

10

11
residues on beta subunits (Czech et al., 1988; Czech, 1985).
After binding, insulin and receptor are internalized, insulin
is degraded and receptors are either recycled or degraded
(Carpentier, 1989).

Tyrosine kinase action and hormone-receptor
internalization provide post-receptor mechanisms for actions
of insulin (Carpentier, 1989; Caro et al., 1988). During
insulin binding, various enzymes, including glycogen synthase
(Sheorain et al. , 1982) , pyruvate dehydrogenase (Hughes et
al., 1980) and acetyl CoA carboxylase (Witters, 1981) are
phosphorylated-dephosphorylated to active forms. Tyrosine
kinase action may be one of the kinase promotors involved with
enzyme activation (Carpentier, 1989; Czech et al., 1988;
Czech, 1985) , but only coincidental evidence supports this
hypothesis. Internalization of insulin-receptor complex is
not essential for acute intracellular responses (Carpentier,
1989) . However, with large doses of insulin, receptor
internalization increases and causes decreased number of
membrane insulin receptors (Carpentier, 1989) .
Internalization of receptors may reduce sensitivity of tissues
to insulin, but it is not certain whether internalization of
receptors occurs in yiyg in response to endogenous
concentrations of insulin.

Several intracellular compounds are associated with
actions of insulin (e.g. decreased cAMP, calcium influx),

suggesting involvement of second messengers (Saltiel and

12
Cuatrecass, 1988; Cheng and Larner, 1985). But, there is no
evidence that these compounds are endogenous post-receptor

mediators for actions of insulin.

EFFECTS OF INSULIN ON OVARIAN CELLS

Diabetes in humans is associated with decreased
fertility. For example, exogenous insulin, given to
diabetics, increases fertility (Poretsky and Kalin, 1987).
In addition, hyperinsulinemia is associated with polycystic
ovarian disease in humans (Poretsky and Kalin, 1987). Thus
both, low and high concentrations of insulin decrease
fertility.
WW

Receptors for insulin are in human ovarian tissue
(Poretsky et al. , 1988) and porcine granulosal cells (Rein and
Schomberg, 1982). Insulin increases basal and LH- or
follicle-stimulating hormone- (FSH) induced secretion of
progesterone from.cultured.porcine granulosal cells (Veldhuis
and Kolp, 1985; May and Schomberg, 1981'; Channing et al.,
1976). Insulin also increases LH-induced secretion of
androgens from rat thecal cells (Cara and Rosenfield, 1988;
Magoffin and Erickson, 1988). Insulin, therefore, increases
steroidogenesis in granulosal and thecal cells.

Positive steroidogenic effects of insulin on granulosal
cells are caused by multiple actions of insulin. Insulin

increases numbers of receptors for LH from porcine granulosal

13

cells (Maruo et al., 1988) and rat leydig cells (Charreau et
al. , 1978) . Insulin also increases number of cultured bovine
and ovine granulosal cells (Peluso and Hirshel, 1987) and
sustained morphology of porcine granulosal cells (May and
Schomberg, 1981; Channing et al., 1976). Therefore, insulin
is both steroidogenic and mitotic in cultured granulosal
cells.
W

Receptors for insulin are in rat luteal cells (Ladenheim
et al., 1984). Insulin increases basal (0'Shaughnessy and
Wathes, 1985) and LH-induced (Leavitt and Condon, 1989; Poff
et al., 1988; O'Shaughnessy and Wathes, 1985) secretion of
progesterone from cultured bovine luteal cells. Mechanisms
for luteotropic actions of insulin have not been determined.

In yivg, decreased postprandial secretion of insulin is
coincident with decreased serum concentrations of progesterone
in NEB heifers compared with PEB heifers (Villa-Godoy et al. ,
1990; Harrison and Randel, 1986). In addition, exogenous
insulin increases luteal weight and luteal content of
progesterone in NEB, but not PEB, heifers (Harrison and
Randel, 1986) . Therefore, postprandial concentrations of
insulin may stimulate serum concentrations of progesterone in
m. However, it is not certain why postprandial insulin
affects luteal development and function during NEB, but not
PEB. Perhaps exogenous insulin is therapeutic only during

NEB.

SUMMARY

Insulin is a glucoregulatory hormone and increases uptake
of nutrients and precursors into adipose, muscle and liver.
Serum concentrations of insulin are controlled positively by
propionate and butyrate absorbed from the rumen (Trenkle,
1971; Manns and Boda, 1967). Serum concentrations of insulin
are low during NEB (Villa-Godoy et al., 1990; Harrison and
Randel, 1986) and.early lactation7(Koprowski and'Tucker, 1973)
compared with PEB or later lactation. Consequently,
concentrations of insulin are dependent on diet and
metabolic/physiologic state.

Insulin also increases basal and LH- or FSH-induced
secretion progesterone from cultured granulosal (Veldhuis and
Kolp, 1985; May and.Schomberg, 1981; Channing et al.,1976) and
luteal cells (Leavitt and Condon, 1989; Poff et al., 1988;
O'Shaunessy and Whathes, 1985). In_yiyg, exogenous insulin
increases luteal function in NEB, but not PEB, heifers
(Harrison and Randel, 1986). It is not known whether insulin
affects luteal development or function during PEB or if

insulin is therapeutic only during NEB.

14

INSULIN-LIKE GROWTH FACTOR I

STRUCTURE AND GENERAL FUNCTION

Insulin-like growth factor I is named for its
hypoglycemic effects (Froesch et al., 1986). Because IGF-I
"mediates" effects of growth hormone (OH) on bone growth
(Salmon and Daughaday, 1957) , IGF-I is also been termed
somatomedin-C. Insulin-like growth factor I is a 7.5
kilodalton (kd) protein (Rechler, 1989) that consists of four
domains (A,B,C,D) arranged B-C-A-D from the amino terminus
(Baxter, 1988) . Amino acid sequence of the A and B domains of
IGF-I are 40% homologous to the A and B chains of insulin,
respectively (Baxter, 1988) , which may explain some of the

insulin-like actions of IGF-I.

CONTROL OF INSULIN-LIKE GROWTH FACTOR I SECRETION

In this review, IGF-I will be called somatomedin activity
when quantified by bioassay and will be called IGF-I when
quantified by RIA.

Growth hormone regulates hepatic secretion of IGF-I.
Exogenous GH increases hepatic content and serum
concentrations of IGF-I in rats and sheep (Clemmons, 1989;
Davis, 1989). Pituitary secretion of GH is regulated by
negative feedback of IGF-I on somatotropes (Clemmons, 1989;

Mathews et al., 1988; Phillips, 1986; Tannenbaum et al.,

15

16
1983) . Therefore IGF-I, in addition to GH, controls secretion
of IGF-I from liver.

During certain metabolic/physiologic states, GH appears
to have less influence on concentrations of IGF-I in serum.
Serum concentrations of GH increases during early lactation
compared with late lactation (Koprowski and Tucker, 1973).
But serum concentrations of IGF-I (Ronge and Blum, 1988) and
somatomedin activity (Falconer et al., 1980) decrease during
early versus late lactation. Malnutrition or diabetes in
humans also increases concentrations of GH but somatomedin
activity decreases (Winter et al., 1979; Grant et al., 1973).
Number of GH receptors are decreased in hepatocytes from
diabetic rats (Maes et al., 1983), suggesting a mechanism for
decreased GH-induced secretion of IGF-I in hepatocytes during
diabetes and other metabolic/physiologic states.

Ninety percent of serum IGF-I is derived from liver
(Baxter, 1988; Scott et al., 1985). All serum IGF-I is bound
to binding proteins (Baxter, 1988). Most IGF-I in serum is
bound to 150 kd binding proteins (Baxter and Martin, 1987:
Furnanetto, 1980). However some IGF-I is complexed to 34 kd
binding proteins (Baxter, 1988). Both binding proteins are
produced by hepatocytes (Baxter, 1988; Scott et al., 1985:
Walton and Etherton, 19890. Secretion of the 150 kd, but not
the 34kd, species is stimulated by GH in hepatocytes (Baxter,
1988). Production and release of both binding proteins is

independent of IGF-I production and release and does not

17
affect secretion of IGF-I (Baxter, 1988; Schwander et al.,
1983) . Binding proteins for IGF-I decrease rate of IGF-I
clearance from blood (Cascieri et al., 1988; Hodgkinson et
al., 1987). Consequently, binding proteins facilitate

retention of IGF-I in blood.

ACTIONS OF INSULIN-LIKE GROWTH FACTOR I

Effects of IGE-I

Most actions of IGF-I mimic actions of insulin or promote
growth. In adipose tissue, IGF-I increases numbers of glucose
transporters (Zapf et al. , 1978) , increases glucose conversion
to fatty' acids, increases lipid. synthesis and. decreases
lipolysis (Froesch et al., 1986). However, 50- to loo-fold
more IGF-I (moles) than. insulin. is needed to stimulate
insulin-like responses in adipocytes. Insulin-like growth
factor I also causes differentiation of preadipocytes to
adipocytes (Smith et al. , 1988) . Increased serum
concentrations of GH do not accelerate body weight gain in
rats until serum concentrations of IGF-I increases after two
weeks of age (Mathews et al., 1988). In addition, IGF-I
increases cell replication in cultured smooth.muscle (Froesch
et al;, 1986), satellite cells (Dodson et al., 1985) and.ohick
fibroblasts (Zapf et al. , 1978) . Therefore, IGF-I causes
insulin-like effects in adipose tissue and mitosis in muscle
and conceptus.

Both the 150 and 34 kd binding proteins inhibit IGF-I

18

action in adipocytes, chondrocytes and uterine endothelium
(Walton et al., 1989; Ritvos et al., 1988; Rutanen et al.,
1988). But, IGF-I-induced fibroblastic growth increases with
addition of 34 Rd binding proteins (Blum et al., 1989: Elgin
et al., 1987). Although not defined, inhibitory/stimulatory
effects of binding proteins for IGF-I on actions of IGF-I may
be tissue dependent.
Won

Both serum and tissue IGF:Iwbinthowtype I andwziwIGF

receptors and insulin receptors (Czech et al., 1983; Rechler

.M_.-u>-—~~r-.h. «'1‘ NM..-.—~,—v.~

 

and Nissley, 1985). TypehI IGF {FEEEEEE§“PEBQMIQEIImEéPh
highfast affinity 391.111.91891311‘4”1.93711.--an§._-.insulin-with_50. to
109919.95: $985.8.-affiniwtx Phi-m 29%.; (Rechler. 198.9 Rechiér and >2
thissle , 1985; Czech et al., 1983). _Structure of type I IGF
receptors, like receptors for insulin, have four subunits,
arranged in a fi-o-o-B configuration and linked by disulfide
bonds (Rechler and Nissley, 1985; Czech et al., 1983). Type
I IGF receptors also have tyrosine kinase activity on their
beta subunits like receptors for insulin (Rechler, 1989:
Jacobs et al., 1983; Zick et al., 1984). Consequently,
although not defined, post-receptor mechanisms of type I IGF
receptors may be similar to mechanisms of insulin receptors.

Type II IGF receptors bind IGF-I and IGF-II with high
affinity (Rechler, 1989; Rechler and Nissley, 1985; Czech et
al., 1983) but have no detectable affinity for insulin

(Rechler and.Nissley, 1985). Type II IGF receptors are single

19

polypeptide. chains (Rechler, 1989; Czech. et al., 1983).
Post-receptor mechanisms of type II receptors are not defined.

Receptors for insulin also bind IGF-I and IGF-II with 50
to 100 times less affinity than insulin (Rechler and Nissley,
1985). Therefore, observed insulin-like effects of IGF-I may
be due in part to binding of IGF-I to insulin receptors
(Froesh et al., 1986; Zapf et al., 1978). Likewise, growth
promoting effects of insulin may be due in part to binding of

insulin to type I IGF receptors (Fukagawa et al., 1985).

EFFECTS OF INSULIN-LIKE GROWTH FACTOR I ON OVARIAN TISSUE
WWII;

Insulin-like growth factor I increases basal (Schams et
al. , 1988) and FSH-induced secretion of progesterone (Maruo et
al., 1988; Adashi et al., 1988a) from cultured rat and ovine
granulosal cells. Luteinizing hormone-induced secretion of
androgens from rat thecal cells 1311111129 are also increased by
IGF-I (Cara and.Rosenfield, 1988). Thus, IGF-I, like insulin,
stimulates steroidogenesis from cultured granulosal and thecal
cells.

Type I IGF receptors are in rat and ovine granulosal

-.-.— ~w‘ r.

cells( (Monget et al., 1989:- Adashi 'et  alc3 1988b5\‘

__..—-'

 

\hvV-Ah

receptors in swine (Maruo gt ales 1988) and increases number

WWW

 

 

of LH but not FSH receptors in rat.granulosal cells (Adashi et

al., 1988b). Adenylate cyclase activity is also increased

20
with IGF-I in rat granulosal cells (Adashi et al. , 1988a) . in

swine granulofls'aluflcells, IGF-I

J w'nnblai‘r

insgsasssrhlpw. ....<.1ensit¥

 

lipoprotein (LDL) ”receptors, LDL internalization and

———-‘—-— 1‘.”

 

 

—.-HMW‘ ha . 1‘

subs quent “111113;69933153923991esterQl, ,(Yslslhuis .et...a1. .

“v“ ax. if
M. tun- flan“. a- ‘1

1987) . Aromatase activity, inducgdmbyWFSH,is, als.o,,...increas.e.d

”rm‘“--*im p-WL J. "*m- . ‘fi'..“:

 

by IGF-Iqin‘ human granulosalflcells (Erickson et "al. ,fl 1989) .
Consequently, positive steroidogenic effects of IGF-I in
granulosal cells are well defined W.

Ovarian production of IGF-I has also been observed.
Messenger RNA for IGF-I is present in rat and porcine
granulosal cells (Hernandez et al., 1989; Mondschein and
Hammond, 1988). Granulosal cell secretion of IGF-I is
increased by GH (Hsu and Hammond, 1987a; Davoren and Hsueh,
1986) estradiol, FSH and LH (Hsu and Hammond, 1987b).
Immunoneutralization of IGF-I from porcine granulosal cells
decrease FSH-induced secretion of progesterone in the presence
and absence of exogenous IGF-I (Mondschein et al. , 1989) .
Consequently, IGF-I also has autocrine/paracrine modes of
action on secretion of progesterone from cultured granulosal
cells.

11321141915

In 48h cultures of bovine luteal cells collected at d2 to
d4 postestrus, IGF-I increases basal secretion of progesterone
(McArdle and Holtorf, 1989). However IGF-I does not affect

basal or LH-induced secretion of progesterone from bovine

luteal cells cultured for 4h (Schams et al., 1988) or 8d

21
(Leavitt and Condon, 1989). These bovine luteal cells are
derived from corpora lutea at d8 to d12 postestrus. Therefore

age of corpora lutea may affect IGF-I action.

SUMMARY

Hepatic secretion and subsequent serum concentrations of
IGF-I are regulated mainly by GH (Clemmons, 1989: Davis,
1989). Serum concentrations of IGF-I decrease with diabetes
although serum concentrations of GH are increased (Winter et
al., 1979; Grant et al., 1973); most likely because of
decreased number of hepatic GH receptors (Maes et al., 1983).
Serum concentrations of IGF-I (Ronge and Blum, 1988) and
somatomedin activity (Falconer et al., 1980) are lower during
early compared with late lactation. Therefore decreased
concentrations of IGF-I are coincident with NEB in cattle.

Structure of IGF-I is similar to insulin (Baxter, 1988)
and IGF-I cross-reacts with receptors for insulin (Rechler,
1989: Rechler and Nissey, 1985). Basal and FSH-induced
secretion of progesterone from granulosal cells is increased
with IGF-I (Adashi et al., 1988a; Maruo et al., 1988; Schams
et al., 1988). Purified bovine luteal cells collected at d2
to d4 postestrus increase basal secretion of progesterone in
response to IGF-I (McArdle and Holtorf, 1989) . However,
IGF-I does not affect. basal or’ LH-induced secretion. of
progesterone from bovine luteal cells collected at d8 to d12

postestrus (Leavitt and Condon, 1989; Schams, et al., 1988).

22
Hence, steroidogenic effects of IGF-I in vitzg may be
dependent on age of corpora lutea and/or'purity of luteal cell

preparations.

GENERAL SUMMARY

After parturition, 81 to 92% of dairy cows experience NEB
(Villa-Godoy et al. , 1989; Reid et al. , 1966) . Serum
concentrations of progesterone and luteal weight are decreased
in NEB heifers compared with PEB heifers (Villa-Godoy et al.,
1990; Harrison and Randel, 1986) . Therefore, the
coincidiental observations of decreased luteal development and
function with infertility of dairy cows during NEB raises the
question of whether decreased luteal function causes
infertility of dairy cows.

After ovulation in sheep and cattle, serum concentrations
of progesterone (Villa-Godoy et al. , 1990) , luteal weight
(Ireland et al. , 1973) and numbers of steroidogenic cells
(Farin.et.al., 1986) increasen Corpora lutea are comprised of
two steroidogenic cell types (Alila and Hansel, 1984), which
differ in morphology (0'Shea.et.al., 1979), number (Schwall et
al., 1986) and basal and LH-induced secretion of progesterone
(Fitz et al., 1982).

Decreased serum concentrations of progesterone and
decreased luteal weight are coincident with decreased
postprandial concentrations of insulin in NEB heifers
(Villa-Godoy et al., 1990; Harrison and Randel, 1986). In
NEB, but not PEB heifers, exogenous insulin increases luteal
content. of' progesterone .and luteal. weight (Harrison. and

Randel, 1986). However, it is not known whether postprandial

23

24
insulin affects luteal development and function during PEB or
if exogenous insulin affects corpora lutea only during NEB.
The first objective of this thesis was to determine effects of
postprandial concentrations of insulin on serum concentrations
of progesterone in heifers maintaining energy balance.

In early lactation, serum concentrations of IGF-I were
decreased compared with later lactation (Ronge and Blum,
1988) . Therefore, decreased serum concentrations of IGF-I are
coincident with decreased luteal development and function
during NEB. Basal secretion of progesterone is increased
with IGF-I from bovine luteal cells cultured at d2 to d4
postestrus (McArdle and. Holtorf, 1989). But, basal or
LH-induced secretion of progesterone from bovine luteal cells
cultured at d8 to d12 postestrus, is not affected by IGF-I
(Leavitt and Condon, 1989; Schams et al., 1985). Therefore,
corpora lutea age may affect IGF-I actions on cultured bovine
luteal cells. Also, increases in number of bovine luteal
cells are associated positively with duration of culture
(0'Shaughnessy and Wathes, 1985) . Since IGF-I stimulates
mitotic activity in other tissues, IGF-I may also increase
number of luteal cells during culture. The second set of
objectives were to determine the effects of IGF-I on 1) basal
and LH-induced secretion of progesterone, 2) number of cells
after culture and 3) steroidogenic and mitotic response of
cultured bovine corpora lutea at different stages of

development.

EXPERIMENT I:
POSTPRANDIAL INSULIN DOES NOT AFFECT SERUM

CONCENTRATIONS OF PROGESTERONE IN HEIFERS .

INTRODUCTION

Decreased concentrations of progesterone in cattle are
associated with decreased rate of conception (Erb et al. ,
1976; Folman et al. , 1973) and decreased embryo survival (Hill
et al., 1970) . In NEB heifers, decreased postprandial
concentrations of insulin are coincident with decreased serum
concentrations of progesterone (Villa-Godoy et al., 1990).

Positive steroidogenic effects of insulin have been
demonstrated in granulosal (Lino et al. , 1985: Veldhuis and
Kolp, 1985; May and Schomberg, 1981) and luteal cells (Leavitt
and Condon, 1989; O'Shaunessy and Wathes, 1985) ill—VISIO-
Administration of insulin also alleviates secondary amenorrhea
in diabetic women (Poretsky and Kalin, 1987) . Decreased serum
concentrations of progesterone and decreased luteal weight are
coincident with decreased postprandial concentrations of
insulin in NEB heifers (Villa-Godoy et al. , 1990; Harrison and
Randel, 1986) . In NEB but not PEB heifers, exogenous insulin
increased luteal weight and luteal content of progesterone
(Harrison and Randel, 1986) . It is not known whether
postprandial secretion of insulin is luteotropic in cattle

during PEB. My objective was to determine the effect of low

25

26
postprandial concentrations of insulin on serum concentrations

of progesterone in heifers maintaining energy balance.

METHODS

Sixteen postpubertal Holstein heifers were blocked
according to body weight and assigned to a diet of 100% hay or
90% corn silage: 10% soybean meal (CS). Both diets were
formulated to support 0.45 kg body weight gain/d (NRC, 1978)
and were presented to heifers at 0700 and 1900h daily.

Each.heifer'was fed.the same diet for the duration of the
experiment (44 days) which included a 21d adjustment period
and a 23d experimental period (do to d22) . Heifers were
weighed on two consecutive days every two weeks throughout the
experiment. The adjustment period was included to allow
ruminal acclimation to diets. Prostaglandin F2 alpha1 (25
mg) was injected intramuscularly 13 and 3d before do to
synchronize estrus for do in all heifers. After PGanI estrus
of heifers was judged to be synchronized if serum
concentrations of progesterone were below 1.0 ng/ml for at
least three days. From 21 to 8 days before d0 heifers were
housed and fed in groups (n=8) and from 7 days before and
throughout the experimental period heifers were housed and fed
in individual stalls.

During the experimental period (do to d22), feed offered
and orts were measured to determine daily intake of diet.

0rts were discarded immediately before the subsequent meal.

27
Daily consumption and nutritive estimates of diets (NRC, 1978)
were used to estimate caloric intake. Heifers that lost body
weight or failed to consume calories required for maintenance
were excluded from all analyses.

A.cannula.was inserted into a jugular vein.of each heifer
on the last day of the adjustment period. On d0, 7, 14 and
21 blood was sampled every 20 min from 0500h to 1300h to
determine basal (2h preprandial) and postprandial (6h)
concentrations of insulin in serum. Jugular blood was also
sampled daily at 0630h and 1830h from do to d22 and hourly
from 0800 to 1200 h on d7 and d14 to quantify progesterone in
serum. Blood was allowed to clot at room temperature (12 to
20 C) and then was stored at 4 C overnight. Serum was
harvested by centrifugation (2000 rpm for 25 min) and stored
at -20 C until analyzed.

A previously validated double antibody radioimmunoassay
(RIA) was used to quantify insulin in serum (Villa-Godoy et
al., 1990). The intra-assay coefficient of variation from
several dilutions of standard sera for the insulin RIA was
5.7%. Progesterone was also quantified by RIA (Spicer et
al., 1981). Mean extraction efficiency was 93.0 i- 1.3%.
Intra and interassay coefficients of variation were 9.3 and
12.3% for standard sera, respectively.

Data were examined by split plot analysis of variance
with repeated measurements (Gill, 1986) and specific contrasts

were by Bonferroni's T test.

RESULTS

One heifer fed CS lost weight during the experimental
period and was excluded from analysis. Mean body weight
(Figure 1) of heifers fed.hay (n=8) or CS (n=7) did not change
throughout the adjustment or experimental periods. Based on
estimates of caloric requirements for heifers (NRC, 1978),
feed consumed by heifers on both diets satisfied or exceeded
maintenance requirements for energy (data not shown).

Basal concentrations of insulin (2h preprandial) at do
and d14 in heifers fed hay were less (P<.05) than in heifers
fed CS (Figure 2). In heifers fed hay, area of periprandial
serum insulin profiles (2h before to 6h after feeding) were
decreased (P<.05) at d0, 7, 14 and 21 compared with heifers
fed CS (Figure 2) . Concentrations of insulin from feeding to
6h after feeding increased (P<.05) in heifers fed CS but not
in heifers fed hay on d7, 14 and 21 (Figure 2).
Consequently, we.examined.the association.of low (hay diet) or
increased (CS diet) postprandial concentrations of insulin on
secretion of progesterone in heifers maintaining energy
balance. Heifers will now be classified as heifers with low
insulin (hay diet) or normal insulin (CS diet).

Based on patterns of progesterone in serum (Figure 3),
estrus was synchronized in all heifers within a period of 3d
(do to d2). Total area of progesterone from do to d22, mean
concentrations of progesterone from do to d22, peak

concentrations of progesterone and interval from nadir to peak

28

29
progesterone did not differ in heifers with low or normal
insulin (Figure 3). Within 5h after feeding, when differences
in concentrations of insulin were greatest, mean
concentrations of progesterone on d7 or d14 were not different

between heifers with low or normal insulin (Figure 4).

30

Figure 1. Body weight of heifers fed hay (0—0) or corn
silage (0——-0). Heifers were weighed on two consecutive days
every other week during the experiment. Pooled standard error

was 2.8 kg.

BODY WEIGHT (kg)

500w

 

31

 

 

 

 

 

475-
450? 3: =9
425-4
A
400 T T I I l l
“'2' "I4 '7 O 7 l4 2|
ADJUSTMENT EXPERIMENTAL
PERIOD ((1) PERIOD (d)

32

Figure 2. Effect of diet on serum concentrations of insulin
in heifers from 2h before to 6h after feeding. Jugular blood
was sampled every 20 min in heifers fed hay (O-——O) or corn
silage (0—0) . Concentrations of insulin were measured on
d0, 7, 14 and 21. Arrow indicates presentation of feed.
Pooled standard error of insulin area was 25.97 ng-ml'1-min"

of serum.

INSULIN (ng/ml)

 

33

 

 

 

 

 

 

 

2.0-1 do
"04 M
T T T T T T T T T
2.0"q 1 d7
|.O-
T T T T T T T T T
2-01 l dl4
|.0-‘
T T r T T T T Ti T
2"“ d2I
..o. 1
T T T T T T T T 7
-2 -| O l 2 3 4 5 6

TIME RELATIVE TO FEEDING (h)

34

Figure 3. Effect of postprandial insulin on serum
concentrations of progesterone. Blood was sampled twice daily
from do to d22 in heifers with low (0—0) or normal (0—0)

insulin. Pooled standard error was .16 ng/ml of serum.

3S

7'79“!"9‘
OUIOUIO
11114

 

 

0.5

 

PROGESTERONE (ng/ml)

’TNW

j
IS 20

OTT T T TT T

1
O 4 8 l2
EXPERIMENTAL PERIOD (d)

34

Figure 3. Effect of postprandial insulin on serum
concentrations of progesterone. Blood was sampled twice daily
from do to d22 in heifers with low (0————0) or normal (0———0)

insulin. Pooled standard error was .16 ng/ml of serum.

35

 

E 3.0-

\

gas-

g 2.0-4

8 I.5‘

E

a, I.o~

3 05

O .

E o—r. . . . . .

 

T I! r T 1
O 4 8 I2 I6 20
EXPERIMENTAL PERIOD (0)

36

Figure 4. The acute effect of postprandial insulin with
progesterone in heifers. Jugular blood was sampled hourly for
the first 5h after feeding. Serum concentrations of
progesterone in heifers with low (0———0) or normal (0———O)
insulin were compared with serum concentrations of insulin in
heifers with low (0---0) or normal (0---0) insulin. Pooled
standard error was .36 ng/ml for progesterone and .35 ng/ml

for insulin.

37

.2mCCZ A:O\3: IIII

0 O O. O
2 In 2 I
_ _ _ _ _ _ p P

 

 

 

 

 

 

 

 

 

 

7 T 4. T5
.0 2 m n.
. . _
:1 1. _ 1 -4
_ _ _ _
_ _ . .
I - E 1 g
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x n _. n
a Pr .. a e .i
w. d n—U. - W1 anod
2 I

2 T
I 2.59: mzommkmmooma

TIME AFTER FEEDING (h)

DISCUSSION

Calories ingested were adequate for maintenance (NRC,
1978). Furthermore, body weight of heifers fed.diets of hay or
CS did not decrease during the trial. Therefore heifers were
at energy balance, which allowed us to determine effects of
insulin on serum. concentrations of progesterone without
confounding by NEB.

Diets clearly affected postprandial concentrations of
insulin in heifers. In ruminants, secretion of insulin is
controlled primarily by propionate and butyrate, not by
glucose or acetate (Trenkle, 1971; Manns and Boda, 1967) .
Ruminal digestion of diets high in starch (CS diet) produce
more propionate and butyrate. Conversely, diets with high
fiber and low starch (hay diet) produce less propionate and
butyrate (Trenkle, 1971; Manns and Boda, 1967). In addition,
it took longer for heifers to consume a hay diet than a CS
diet, which may also affect patterns of serum concentrations
of insulin.

Insulin increases secretion of progesterone from
granulosal (Lino et al., 1985; Veldhuis and Kolp, 1985; May
and Schomberg, 1981) and luteal cells (Leavitt and Condon,
1989: O'Shaunessy and Wathes, 1985) in_yi§rg and in corpora
lutea in yivg (Harrison and Randel, 1986) . Receptors for
insulin are present in rat luteal tissue (Ladenheim et al.,
1984). Additionally, diabetes in humans decreases fertility

and exogenous insulin is therapeutic (Poretsky and Kalin,

38

39
1987). Thus, there is evidence that insulin is gonadotropic.
But, in the present study, normal postprandial concentrations
of insulin had no effect on serum concentrations of
progesterone.

Decreased postprandial concentrations of insulin are
coincident with decreased serum concentrations of
progesterone in NEB heifers (Villa-Godoy et al., 1990;
Harrison and Randel, 1986). In NEB but notPEB heifers,
exogenous insulin increases luteal weight and luteal content
gimpggg9§gggpneflLHarrison and Randel,1986). From my data,
FEEBEEQIPQEEBEEDQisl c9n¢sntreEi9ns 9t insulin did notalter
luteal development or function in heifers in energy
balance. However, in previous studies of heifers in NEB,
serum concentrations of progesterone were not decreased until
tho estrous cycles after inducing NEB (Villa-Godoy et al.,
1990: Harrison and Randel, 1986). Consequently, if low
postprandial insulin is necessary for reduced luteal
development and/or function, absence of increased postprandial
insulin for one estrous cycle may not be of sufficient
duration to detect effects.

In summary, all heifers fed hay or CS maintained energy
balance during the experiment. Heifers fed CS, but not
heifers fed hay, had increased postprandial concentrations of
insulin. But absence of postprandial insulin did not affect
secretion of progesterone during one estrous cycle or within

5h after feeding. I conclude that low postprandial

40
concentrations of insulin during energy balance are adequate
for sustained secretion of progesterone in heifers maintaining
energy balance. An implication of these data is that low
postprandial insulin may not mediate adverse effects of NEB on

secretion of progesterone.

P :
THE EFFECT OF INSULIN-LIKE GROWTH FACTOR I 0N SECRETION 0F

PROGESTERONE FROM BOVINE LUTEAL CELLS

INTRODUCTION

Serum concentrations of IGF-I are lower during early
lactation compared with later lactation in dairy cows (Ronge
and Blum, 1988; Falconer et al., 1980). During early
lactation, 81 to 92% of dairy cows are in negative energy
balance (Villa-Godoy et al., 1989; Reid et al., 1966), which
is associated with decreased serum concentrations of
progesterone and luteal weight (Villa-Godoy et al. , 1990;
Harrison and Randel, 1986) . Therefore, decreased serum
concentrations of IGF-I coincide with decreased luteal
development and function during NEB.

Perhaps IGF-I affects luteal development and function.
Insulin-like growth factor I increases basal secretion of
progesterone from cultured bovine luteal cells (McArdle and
Holtorf, 1989) in one study but not others (Leavitt and
Condon, 1989 :Schams et al., 1988). Detection of positive
steroidogenic effects of IGF-I may be dependent on dose of
IGF-I administered. Also, since IGF-I effects were not
observed until after 48h of culture (McArdle and Holtorf,
1989), duration of culture may also affect detection of IGF-I
effects from bovine luteal cells.

Increases in bovine luteal cell numbers are associated

41

42
positively with duration of culture (0'Shaughnessy and Wathes,
1985) . Insulin-like growth factor I increases replication of
cells from smooth muscle cells (Froesch et al., 1986),
satellite cells (Dodson et al., 1985) and embryonic chick
fibroblasts (Zapf et al. , 1978) . Mitotic activity of cultured
bovine luteal cells may also be stimulated by IGF-I. Our
objectives were to determine effects of IGF-I on basal and
LH-induced secretion of progesterone and number of bovine

luteal cells during culture.

MATERIALS AND METHODS
Ten postpubertal Holstein heifers were fed corn silage
and hay to gain .6 kg body weight daily (NRC, 1978). Body
weights were measured weekly. Heifers were observed for
estrus three times daily. Estrus was determined by two
observations of a heifer standing to be mounted by another

heifer within a 30 min detection peroid.
0n d5 (n=5) or d10 (n85) postestrus (estrus=d0), Luteal
tissue was removed and dissociated enzymatically as described
previously (Villa-Godoy et al., 1990). Following
dissociation, cells were washed and centrifuged four times
with Hams F12 media2 containing 25 mM Hepesz, 14 mM sodium
bicarbonatez, .1 g/L streptomycin sulfate2 and .07 g/L
potassium penicillin2 (pH 7.35). Viability of cells was
estimated by exclusion of trypan blue. Media (1 ml Hams F12)

containing 5 x 105 cells were placed in 24 well-plates3 and

43

incubated at 37 C in 95% oxygen: 5% carbon dioxide for 24h.
Before culture, some plated cells and media were harvested,
centrifuged (5 min at 1000 rpm), media were aspirated and
cells were resuspended with .05 M phosphate buffered saline
containing 2.0 M sodium chloride2 and .003 M EDTA2 (PBS-EDTA;
pH 7.35) and stored at -20 C until DNA was quantified. These
cells were used to estimate content of DNA before culture.

Immediately before culturing, cells and media were
treated with Iflf (0, .1 or 1 ng) and IGF-I5 (0 or 500 ng).
Luteinizing hormone was solubilized in Hanks buffered saline
solution (pH 7.0), frozen on.dry ice and stored at -70 C until
use. One ng LH increased secretion of progesterone from
bovine luteal cells in preliminary experiments and.was used.in
this experiment to determine effects of IGF-I on LH-induced
secretion of progesterone. Treatment of cells and media with
.1 ng LH was also included to determine if any synergistic or
additive effects between LH and IGF-I occur when the dose of
LH does not stimulate secretion of progesterone.

Insulin-like growth factor I was reconstituted with 10 mM
sodium acetate2 (pH 5.5) and frozen at -70 C until use.
Treatment with 500 ng IGF-I was chosen because it is between
the range of doses used in previous experiments (Leavitt and
Condon, 1989; McArdle and Holtorf, 1989; Schams et al., 1988).
Also, 500 ng IGF-I increased FSH-induced secretion of
progesterone from cultured granulosal cells (Maruo et al.,

1988).

44
After 24h, cells and media were collected and centrifuged
(5 min at 1000 rpm). Media were aspirated and stored at -20
C until progesterone and DNA were quantified. Cells were
resuspended with PBS-EDTA and stored at -20 C until DNA was
quantified.

Quantification of progesterone and DNA: Progesterone in
media was quantified as described previously (Spicer et al.,
1981). Efficiency of progesterone extraction averaged 94.1%.
Intra and interassay coefficients of variation were 5.9 and
14.6% from.pooled media, respectively. Quantification of DNA
in cells and media were described previously (Labarca and
Paigen, 1980) . Intra and interassay coefficients of variation
were 5.4 and 10.9% from pooled cells and 7.7 and 5.9% from
pooled media, respectively.

Statistical analysis: Data were examined by split-plot
analysis of variance with repeat measurements (Gill, 1986).

Specific contrasts were by Bonferroni's T test.

RESULTS
All heifers gained body weight before lutectomy.
Viability of cells was >92.0% following dissociation and
>86.0% after 24h.of’cultureu Cell viability after 24h was not
affected by age of corpora lutea or by presence of LH or
IGF-I.
Percent DNA recovered after 24h did not differ between

luteal cells cultured at d5 (82.1 i 12.8%) and d10 (95.8 i

45

6.77%) postestrus. In luteal cells collected at d5 and d10
postestrus, 1 ng LH increased (P<.05) percent DNA after 24h
compared with basal or .1 ng LH (Figure 5). Percent DNA.after
24h did not differ between basal and .1 ng LH at either day
postestrus. Percent DNA at 24h was not affected by IGF-I and
there were no interactions of IGF-I with LB or day postestrus
(Figure 5).

Concentrations of progesterone in media from luteal cells
collected on d5 postestrus did not differ from that of luteal
cells collected on d10 postestrus. In luteal cells collected
on d5 and d10 postestrus, 1 ng LH increased (P<.01)
concentrations of progesterone in media compared with basal or
.1 ng LH (Figure 6). Basal concentrations of progesterone in
media did not differ from .1 ng LH at either day postestrus.

Insulin-like growth factor I did not affect basal ormLH-

”me-v—mm ‘4 “no I" Jm'mwuwm.d MuT—éW—Pi'dhmurn" ‘M’WAw-uw- “mu-wk

 

induced concentrationsmggwprogesterone in media from luteal

'ManwW\MI-w NF‘HP" m pfr

cellécuguredwat .95....91“....51191rzessssgzys. .I.§iqure 6) .

46

Figure 5. Effect of LH and IGF-I on DNA in media and cells at
24h. DNA was expressed as percent of DNA in cells before

culture. Pooled standard error was 1.78%.

47

I00-

DNA RECOVERED AFTER 24H (°/o)
(D
O
I

 

 

 

 

 

 

 

 

 

60
LHIng) o 0 mm I 110 o O.IO.I I I

 

. l
IGF-I (no) 0500 0500 05001 0500 0500 0500

 

5d POSTESTRUS I l0d POSTESTRUS

 

48

Figure 6. iEffect of LH and IGF-I on secretion.of progesterone
from bovine luteal cells cultured at d5 or d10 postestrus.

Pooled standard error was .26 ng/ug DNA.

49

 

 

 

 

 

   
 

k. . .I
EAT...» Fm“.

\ . Jr... 3 ,... .
.. 1: L. .3... i... i. .T. ..1 .

 

  

    

 

 

 

 

A420 o:\ocv mzomwhwmooma

 

T
0 500 0 500 0 500I 0 500 0 500 0 500

I o 0 0.101 I

L

0 0 0.! OJ

LH(ng)

IGF-I (ng)

 

I IOd POSTESTRUS

5d POSTESTRUS

 

 

DISCUSSION

In this experiment, heifers gained body weight before
lutectomy, so data were not confounded by NEB. After 48h of
culture, positive steroidogenic effects of IGF-I from bovine
luteal cells have been observed (McArdle and Holtorf, 1989).
In our experiment, basal and LH-induced secretion of
progesterone from 24h cultures of bovine luteal cells were not
affected by IGF-I. Consequently, longer durations of culture
may be needed to observe effects of IGF-I on basal or
LH-induced secretion of progesterone. However, a known
luteotropin, LH, increased secretion of progesterone from
bovine luteal cells cultured for 24h. Therefore, positive
steroidogenic effects of IGF-I should have been observed, if
present, in our experiment. McArdle and Holtorf (1989)
administered 20 fold more moles of IGF-I to cultured bovine
luteal cells than in our experiment. Thus dose of IGF-I may
effect detection of IGF-I effects on basal and LH-induced
secretion of progesterone from bovine luteal cells.

' Number of cells in culture did were not affected by IGF-I
in our experiment. Duration of culture may not have been
adequate for detection of IGF-I effects on mitotic activity
since 48 to 96h of culture are needed to observe a 2 fold
increase in number of luteal cells (O'Shaugnessy and Wathes,
1985). However, LH, but not IGF-I, increased maintenance of

luteal cell numbers after 24h of’cultureu Thus IGF-I does not

50

51
affect. maintenance of luteal cell numbers after 24h. of
culture.

In summary, LH increased secretion of progesterone from
luteal cells collected on d5 and d10 postestrus. Also, LH
increased maintenance of luteal cell numbers. But, IGF-I did
not affect basal or LH-induced secretion of progesterone or
maintenance of luteal cell numbers at either day postestrus.
Perhaps greater'doses of IGF-I are needed to detect effects of
IGF-I on steroidogenic and mitotic activity in bovine luteal
cells. In conclusion, 500 ng of IGF-I does not increase basal
or LH-induced secretion of progesterone or number of cells

from cultured bovine luteal cells.

FOOTNOTES

Lutylase, (25 mg); Upjohn Co., Kalamazoo, Michigan
Sigma Chemical Company; St. Louis, Missuori.
Costar; Cambridge, Massachusetts

NIH-bLH4; NIH, Beltsville, Maryland

IMCERA; Terre Haute, Indiana

52

SUMMARY AND CONCLUSIONS

In the first experiment, low’postprandial concentrations
of insulin had no effect of serum concentrations of
progesterone in heifers maintaining energy balance.
Conversely, during NEB, low postprandial insulin was
coincident with decreased serum concentrations of progesterone
and luteal weight (Villa-Godoy et al., 1990: Harrison and
Randel, 1986) . Additionally, exogenous insulin increased
luteal content of progesterone and luteal weight in NEB but
not PEB heifers (Harrison and Randel, 1986). However, low
postprandial insulin was not associated with decreased serum
concentrations of progesterone until after the second estrous
cycle that heifers experienced NEB (Villa-Godoy et al.,
1990). Therefore , it is not known whether low postprandial
insulin decreased luteal function only during NEB or if low
postprandial insulin must exist for 2 two estrous cycles to
decrease luteal function.

In the second experiment IGF-I did not affect basal or
LH-induced secretion of progesterone or number of cells from
24h cultures of bovine luteal cells collected on d5 or d10
postestrus. waever, 20 fold more (moles) IGF-I increased

basal secretion of progesterone from bovine luteal cells

53

54

(McArdle and Holtorf, 1989). Thus, IGF-I effects on

mmwwt-VJflRfllrw-P’! '5 I“ "

AJ—l‘aokdk‘vru'v [M‘n‘ W

steroidogenesismgf bovine lutealmcellsmmgymbefldependentmpn

dose of IGF-I. Bovine luteal cells double in number from 48

 

to 96h of culture (O'Shaunessy and Wathes, 1985). Therfore,
my culture duration may have been inadequate to detect mitotic

activity of IGF-I on bovine luteal cells.

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