BIOLOGY OF REPRODUCTION 78, 648–660 (2008)
Published online before print 19 December 2007.
Developmental Programming: Impact of Prenatal Testosterone Excess on Pre- and
Postnatal Gonadotropin Regulation in Sheep1
Mohan Manikkam,4,7Robert C. Thompson,5,7Carol Herkimer,4,7Kathleen B. Welch,8Jonathan Flak,3,4,7
Fred J. Karsch,6,7and Vasantha Padmanabhan2,4,6,7
Departments of Pediatrics,4Psychiatry,5and Molecular and Integrative Physiology,6the Reproductive Sciences
Program,7and the Center for Statistical Consultation and Research,8University of Michigan, Ann Arbor,
The goal of this study was to explore mechanisms that mediate
hypersecretion of LH and progressive loss of cyclicity in female
sheep exposed during fetal life to excess testosterone. Our
working hypothesis was that prenatal testosterone excess, by its
androgenic action, amplifies GnRH-induced LH (but not FSH)
secretion and, thus, hypersecretion of LH in adulthood, and that
this results from altered developmental gene expression of
GnRH and estradiol (E2) receptors, gonadotropin subunits, and
paracrine factors that differentially regulate LH and FSH
synthesis. We observed that, relative to controls, females
exposed during fetal life to excess testosterone, as well as the
nor-aromatizable androgen dihydrotestosterone, exhibited en-
hanced LH but not FSH responses to intermittent delivery of
GnRH boluses under conditions in which endogenous LH
(GnRH) pulses were suppressed. Luteinizing hormone hyperse-
cretion was more evident in adults than in prepubertal females,
and it was associated with development of acyclicity. Measure-
ment of pituitary mRNA concentrations revealed that prenatal
testosterone excess induced developmental changes in gene
expression of pituitary GnRH and E2receptors and paracrine
modulators of LH and FSH synthesis in a manner consistent with
subsequent amplification of LH release. Together, this series of
studies suggests that prenatal testosterone excess, by its
androgenic action, amplifies GnRH-induced LH response,
leading to LH hypersecretion and acyclicity in adulthood, and
that this programming involves developmental changes in
expression of pituitary genes involved in LH and FSH release.
activin, developmental biology, estradiol receptor, follistatin, FSH,
gonadotropin-releasing hormone receptor, inhibin, LH,
neuroendocrinology, pituitary, pituitary responsiveness
Treatment of pregnant sheep with testosterone from Days 30
to 90 of the 150-day gestation period leads to reproductive
dysfunction in adult female offspring [1–8]. Disruptions
include a progressive deterioration of estrous cycles [1–3],
development of multifollicular ovaries [4, 5] and, ultimately,
infertility [1, 3]. Associated with these abnormalities is an
alteration in gonadotropin secretion manifested as hypersecre-
tion of LH but not FSH [7, 8]. Hypersecretion of LH associated
with reproductive abnormalities is also evident following
prenatal exposure to excess testosterone in female rhesus
monkeys , mice , and rats , and it is also seen in
women with polycystic ovary syndrome [12, 13].
Although reproductive consequences of prenatal exposure
of female sheep to excess testosterone are well described, the
mechanisms producing these detrimental effects are not
understood. For example, it is unclear whether the selective
increase in LH secretion results from androgenic action or
aromatization of androgens to estrogens. In addition, prenatal
testosterone treatment leads to reduced responsiveness to E2
negative feedback, characterized by increased frequency and a
tendency toward an increase in amplitude of LH pulses . It is
not known whether the increased LH pulse amplitude is due to
heightened pituitary responsiveness to GnRH, altered amounts
of GnRH or E2receptors, and/or changes in gonadotropin gene
expression. Further, the reduced responsiveness to E2negative
feedback in sheep exposed to excess testosterone during fetal
life is evident only for LH; feedback effects of E2on FSH are
not affected. This suggests involvement of pituitary paracrine
regulators, such as activin or inhibin, and their receptors or
binding proteins (e.g., follistatin).
The goal of this study was to explore mechanisms that
mediate hypersecretion of LH in prenatal testosterone-treated
female sheep. Our working hypothesis was that prenatal
testosterone excess, by its androgenic action, amplifies
GnRH-induced LH (but not FSH) secretion and, thus,
hypersecretion of LH in adulthood. This increased pituitary
responsiveness to GnRH results from developmental changes
in GnRH and E2receptors, gonadotropin subunits, and
paracrine factors that differentially regulate LH and FSH
MATERIALS AND METHODS
Breeding, Maintenance, and Prenatal Treatment
All procedures were approved by the Institutional Animal Care and Use
Committee of the University of Michigan and were in conformity with the
National Institutes of Health Guide for Use and Care of Animals. Suffolk sheep
ages 2–3 yr were purchased from local farmers and were maintained in a farm
inspected by the US Department of Agriculture and approved by the University
of Michigan Department of Laboratory Animal Medicine. Prior to breeding,
sheep were group fed with 0.5 kg shelled corn and 1 kg alfalfa hay per female
sheep per day. During October to November of three successive years (2001–
2003), ewes were mated by raddled rams. Pregnant sheep were maintained on
pasture under natural photoperiods with a supplement feed of 1.25 kg alfalfa/
brome mix hay per female sheep. After birth, each mother with its lambs was
individually housed for the first 3 days and then group housed in a barn under
natural photoperiods except for a 60-watt bulb in the lamb feed area at night.
All lambs were weaned at 8 wk and maintained in Sheep Research Facility
1Supported by USPHS Grants P01-HD44232 and R01 HD41098 to V.P.
2Correspondence: Vasantha Padmanabhan, Department of Pediatrics
and Reproductive Sciences Program, University of Michigan, 300 N.
Ingalls Bldg., Rm. 1109 SW, Ann Arbor, MI 48109-0404.
FAX: 734 936 8620; e-mail: email@example.com
3Current address: Department of Psychiatry, University of Cincinnati,
Cincinnati, OH 45267.
Received: 7 June 2007.
First decision: 6 July 2007.
Accepted: 9 December 2007.
? 2008 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
(Ann Arbor, MI), where they were maintained outdoors and fed commercial
feed pellets ad libitum.
Prenatal treatments consisted of intramuscular injections twice a week of
testosterone (T1875; Sigma-Aldrich Corp., St. Louis, MO) or dihydrotestosterone
(DHT) propionate (A2595–000; Steraloids Inc., Newport, RI), 100 mg in 2 ml
corn oil, to pregnant sheep from Day 30 to Day 90 of gestation. Control pregnant
sheep received no injections because we have found no difference in cycle
dynamics of offspring born to vehicle-injected and noninjected mothers .
Study 1: Pituitary Responsiveness to GnRH
This study tested the hypothesis that prenatal testosterone, via its
androgenic action, increases pituitary responsiveness to GnRH and that this
action is specific for LH (i.e., the FSH response is not affected). The approach
was to compare pituitary responsiveness to GnRH in control females and
females exposed prenatally to exogenous testosterone or DHT as well as
aromatizable and nonaromatizable androgens, respectively (Fig. 1). Only one
offspring from each mother was used when twin female births were involved.
Responsiveness to GnRH was tested twice, first at 5.5 wk (prepubertal), when
the lambs were ovary intact, and second at 23 mo of age, after they had been
ovariectomized at the end of breeding season and studied 2 mo later during the
Prepubertal GnRH testing involved seven control, six prenatal testosterone-
treated, and five prenatal DHT-treated females. Adult testing involved five
control, four prenatal testosterone-treated, and three prenatal DHT-treated
females. To block pulsatile LH/GnRH release, sheep were inserted subcuta-
neously with one 30-mm Silastic E2implant . Prepubertal and anestrous
control females are extremely sensitive to E2negative feedback [16, 17], and
this E2treatment is known to abolish endogenous LH/GnRH pulsatility [16,
18]. Jugular blood samples (3 ml) were taken at 24-min intervals for 6 h at 1–2
wk after insertion of E2implants to confirm the absence of LH pulses (pre-
GnRH bleed). GnRH (2 ng/kg; Luteinizing hormone-releasing hormone
human, acetate salt L7134; Sigma, St. Louis, MO) was then administered
intravenously every 90 min for 48 h via a jugular catheter. This dose of GnRH
produces LH responses of magnitude similar to those seen in prepubertal
females . Blood samples (3 ml) were collected into heparinized tubes at 10-
min intervals for 9 h (spanning six GnRH injections) beginning 42 h after initial
injection for characterization of LH and FSH responses.
Study 2: Prenatal Testosterone Effects on Adult
In view of the progressive deterioration of the reproductive axis seen in
prenatal testosterone-treated females [1–3], study 2 was performed during the
second breeding season to determine whether the selective increase in LH
release seen in prepubertal females in the first year of life [7, 8] persists into
adulthood (second year of life). Adult control (n¼6) and prenatal testosterone-
treated (n¼8) females (22 mo old) were given two intramuscular injections of
prostaglandin F2a(PGF2a, 20 mg; Lutalyse; Pfizer Inc., Kalamazoo, MI) 11
days apart to induce regression of the corpora lutea, and thus synchronize the
follicular phase of the cycle within a group of females (Fig. 1). Jugular blood
samples were collected at 10-min intervals from 23 to 28 h after the second
PGF2ainjection for measurement of LH (all samples) and FSH (hourly
samples). Twice-weekly progesterone measures were taken from the start of the
second breeding season to determine whether the prenatal testosterone-treated
females were cycling at the time of the experiment.
Study 3: Prenatal Testosterone Effects on Gonadotropin
Subunits, GnRH Receptor, and Estrogen Receptors
Study 3 was undertaken to examine whether prenatal testosterone excess
caused developmental changes in pituitary gene expression that might account
for the selective increase in LH secretion and pituitary responsiveness to
GnRH. For this purpose, real-time PCR was used to quantify changes in mRNA
expression patterns of gonadotropin subunits (glycoprotein hormones, alpha
peptide [CGA], LHB, FSHB), GnRH receptor (GNRHR), and E2receptors
(ESR1, ESR2) at four developmental stages. Table 1 summarizes the treatments,
developmental stages, and number of animals studied. Where possible,
umbilical arterial levels of LH and FSH were measured to relate changes in
fetal gonadotropin subunit mRNA expression with fetal LH and FSH secretion.
When twin female fetuses were involved, both fetuses were studied. Values of
these two female fetuses were averaged to ensure independence of individual
measures in all analyses.
Pregnant ewes were anesthetized with 1000–1500 mg pentobarbital
intravenously (Nembutal Na; Abbott Laboratories, Chicago, IL); anesthesia
was maintained by inhalation of 1%–2% halothane (Halocarbon Laboratories,
Riveredge, NJ) in oxygen-nitric oxide mixture (2:1). A midventral laparatomy
was performed to expose the uterus with fetuses. Blood samples (;5 to 10 ml)
were collected from umbilical arteries using a Butterfly-21 infusion set (no.
4492; Abbott Laboratories, North Chicago, IL). The ewes then were killed by a
barbiturate overdose (Fatal Plus; Vortech Pharmaceuticals, Dearborn, MI), and
fetuses were removed. The entire procedure took less than 30 min. The adult
female sheep used in this study were the ones used in study 2; they were also
killed by a barbiturate overdose. Pituitaries collected from female fetuses and
adult females were frozen in liquid nitrogen and stored at?808C until real-time
Study 4: Prenatal Testosterone Effects on Pituitary Paracrine
Regulators of Gonadotropins
This study was undertaken to determine whether prenatal testosterone
excess causes developmental changes in the mRNA expression pattern of
pituitary paracrine modulators of gonadotropins that might contribute to
differential changes in LH and FSH secretion and pituitary responsiveness to
GnRH. Pituitary mRNA expression patterns of the following modulators were
determined by real-time PCR on RNA obtained from pituitaries collected in
Study 3: inhibin alpha (INHA), inhibin beta A (INHBA), inhibin beta B
(INHBB), their binding proteins—follistatin (FST), immunoglobulin superfam-
ily member I, also known as inhibin binding protein (IGSF1), transforming
responsiveness to GnRH; and study 2, adult hypergonadotropism. In study
1, one 30-mm E2implant was inserted subcutaneously in each sheep and
retained throughout the experiment. Shaded boxes indicate periods of blood
sampling. Pre-GnRH samples were taken 5 days after implant insertion in
prepubertal sheep and 11 days after implant insertion in adult sheep.
Schematic showing experimental design of study 1, pituitary
at various ages used in the pituitary gene expression study.
Details of the number of female sheep from treatment groups
No. of females No. of mothers
Fetal Day 65
Fetal Day 90
Fetal Day 140
aPrenatal T, prenatal testosterone-treated.
PRENATAL PROGRAMMING OF GONADOTROPIN REGULATION
with ACVR2A and the type I receptor [60, 61], the increase in
ACVR2A mRNA at Fetal Day 90 and ACVR1B mRNA at Fetal
Day 140 should provide a stimulatory input to FSH and an
inhibitory input to LH. However, the effects of increase in
activin and activin receptor expression on LH and FSH in the
Day 140 female fetuses appear to be counterbalanced by
increased expression of FST, a neutralizer of activin action.
Our findings provide evidence that prenatal testosterone
excess induces changes in the developmental progression of
various regulatory signals, the sum effect of which directs
differential expression of LH and FSH. Figure 8 summarizes
changes in key integrators of hypothalamic and ovarian inputs
(GNRHR and ESR1) and pituitary paracrine mediators and the
predicted stimulatory or inhibitory input to LH and FSH at
Fetal Days 65, 90, and 140. While the reduction in ESR1 on
Fetal Day 65 is likely to reduce E2negative feedback input to
LH, increase in pituitary activin action stemming from a
reduction in INHA and TGFBR3 may override the positive
effects of reduced ESR1, resulting in reduced expression of
LHB mRNA and increased expression of FSHB mRNA.
Similarly, an increase in the ACVR2A mRNA expression and
consequent increase in activin action A may override the
effects of reduced ESR1 mRNA at Fetal Day 90, resulting in
reduced LHB mRNA expression. The increase in pituitary
GNRHR mRNA expression at Fetal Day 140 may contribute
toward the postnatal amplification of LH release [7, 8]. An
increase in FST, neutralizer of activin action, stemming from
an increase in FST mRNA expression may negate the potential
for increased activin action stemming from increased INHBB
and ACVR1B mRNA expression. Changes in LHB and FSHB
mRNA expression seen in this study parallel reported changes
in circulating LH/FSH of male fetuses (Fig. 8D, open triangles)
[44, 45, 53] and the prenatal testosterone-treated female fetuses
studied at one time point . They are also consistent with the
observed changes in expression of the various modulators,
suggesting that the developmental integration of these
regulatory signals likely sets the stage for the subsequent
increase in LH release seen during postnatal life .
Prenatal Testosterone Programming of Genes Involved in
Gonadotropin Biosynthesis in Adult Females
Since the regulation of gonadotropin secretion in adult
females can be influenced by the cycle stage, the relationships
between gonadotropin changes and relevant subunit mRNAs
are discussed here separately from developmental changes in
fetuses discussed earlier. Clearly, the cycling prenatal testos-
terone-treated females during the presumptive follicular phase
showed increased LH and not FSH secretion. No changes in
mRNA expression patterns of GNRHR or ESR1 were evident,
consistent with lack of changes in LHB and FSHB mRNA
expression. The only increase seen—namely, that of ACVR1
mRNA—is inconsistent with observed changes in LHB and
FSHB mRNA expression. As such, the increased LH release
seen in the adult animals must involve posttranscriptional
events either at the translational (change in mRNA translational
efficiency) or posttranslational levels (i.e., GNRHR and ESR1,
receptor recycling, or degradation).
In summary, prenatal testosterone treatment in sheep, by its
androgenic action, programs increased pituitary responsiveness
and produces pronounced increase in LH secretion in adults.
Furthermore, prenatal testosterone treatment alters developmen-
tal trajectory of pituitary mediators of gonadotropins in a manner
consistent with the role they play in differential regulation of LH
and FSH, the observed changes in LHB and FSHB mRNA
expression, and/or the expected pattern of gonadotropin release.
We are grateful to Mr. Douglas Doop for his role in breeding/lambing,
animal care, and facility management; Drs. Teresa Steckler, Leslie Jackson,
Puliyur S. Mohankumar, and Hiren Sarma, Mr. James Lee, Mr. James
Dell’Orco, Mr. David Han, Ms. Olga Astapova, and Ms. Danielle Djombi
for their assistance with the prenatal testosterone treatment, collection of
blood samples, tissue procurement, and/or hormonal assays; and Mrs.
Rebecca Demo for assistance with RNA analysis.
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