Physiology & Behavior 69 (2000) 391397

Hypothyroidism increases prolactin secretion and decreases the

intromission threshold for induction of pseudopregnancy
in adult female rats
A. Toheia,b,*, K. Tayaa, G. Watanabea, J. L. Voogtb
a
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 1838509, Japan;
b
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160-7401, USA

Abstract
In order to understand the mechanism by which thyroid hormones alter prolactin (PRL) secretion, we investigated the role of tubero-
infundibular dopamine (TIDA) neurons and pituitary and hypothalamus vasoactive intestinal peptide (VIP) in thiouracil- (0.03% in
drinking water for 16 days) inducedhypothyroid adult female rats. The intromission threshold for induction of pseudopregnancy also
was examined to evaluate the PRL response to coital stimulation in hypothyroid rats. Hypothyroidism in adult female rats did not affect
TIDA neuronal activity as measured by tyrosine hydroxylase activity (DOPA accumulation 30 min after administration of m-hydroxy-
benzylhydrazine dihydrochloride, 100 mg/kg, i.p.) in the stalk-median eminence compared with that in euthyroid rats, whereas pituitary
concentration of VIP was dramatically increased. Plasma concentration of PRL was higher at 1100 h of proestrus and estrus in hypothy-
roid rats as compared with that of euthyroid rats. The proportion of female rats exhibiting pseudopregnancy was higher in hypothyroid
animals (100%) receiving seven intromissions than in euthyroid animals (43%). Administration of L-thyroxine in hypothyroid rats de-
creased the proportion of pseudopregnancy (40%) to the level of euthyroid animals. These results indicate that the increased level of pi-
tuitary VIP probably affects PRL secretion in a paracrine or autocrine manner and account for the hyperprolactinemia induced in hy-
pothyroid female rats. No role for TIDA neurons in PRL elevation can be ascribed. A decrease in the intromission threshold for
induction of pseudopregnancy might be due to increased levels of PRL in hypothyroid female rats. 2000 Elsevier Science Inc. All
rights reserved.
Keywords: Hypothyroidism; Hyperprolactinemia; VIP; Dopamine

1. Introduction concentration of PRL increased on proestrus and estrus in

thiouracil-induced, hypothyroid adult female rats [41]. In
The lack of thyroid hormone has been known to produce
this study, we showed that hypothyroidism induced hyper-
reproductive abnormalities, including irregular menstrual
prolactinemia in intact adult female rats without additional
cycle, amenorrhea, and galactorrhea accompanied by hyper-
estrogen treatment.
prolactinemia in women [18,31,39]. Studies using male rats
It is well established that the secretion of PRL is under
[19,35,40] or ovariectomized [32] or immature female rats
tonic inhibitory hypothalamic control exerted by dopamine
[22], on the other hand, reported that hypothyroidism re-
[7,8,25]. In addition to the inhibitory dopaminergic tone, it
sulted in decreased levels of plasma and pituitary prolactin
is generally accepted that PRL secretion is influenced by
(PRL). Pan et al. have reported that ovariectomized, thy-
thyrotrophin-releasing hormone (TRH), vasoactive intesti-
roidectomized rats treated with estrogen show increased
nal peptide (VIP), and other neuropeptides acting as PRL-
levels of plasma PRL, suggesting that estrogen plays a sig-
releasing factors [8,25]. Recent studies demonstrated that
nificant role in the induction of hyperprolactinemia in hy-
both TRH and VIP are also synthesized within the rat ante-
pothyroidism [32,44]. We also demonstrated that plasma
rior pituitary gland [5,10]. VIP has an autocrine or paracrine
role in basal PRL release, as shown in vivo and in both pri-
mary culture of anterior pituitary and GH3 cells [6,17,29].
Present address: Department of Pharmacology, Tokyo University of
Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-
Hypothyroidism has been shown to increase VIP content
0392, Japan. [22] and release [23], and VIP-mRNA content [24,38] in
* Corresponding author. Tel.: 81-426-76-4530; Fax: 81-426-76-4529. anterior pituitary as well as immunoreactivity and gene ex-
E-mail address: tohei@ps.toyaku.ac.jp pression in the paraventricular nucleus (PVN) [42].
0031-9384/00/$ see front matter 2000 Elsevier Science Inc. All rights reserved.
PII: S0031-9384(00)00 2 2 4 - 9
392 A. Tohei et al. / Physiology & Behavior 69 (2000) 391397
Given the conflicting data on the effect of hypothyroid-
ism on PRL and the positive effect on VIP, the present set of
experiments were designed. The present study was designed
to explore PRL regulation during hypothyroidism. Specifi-
cally, we examined the activity of the rate-limiting enzyme
for dopamine synthesis, tyrosine hydroxylase (TH) in the
stalk-median eminence (SME), and VIP concentrations in
the anterior pituitary, SME, and PVN in hypothyroid adult
female rats. The intromission threshold for induction of
pseudopregnancy also was examined to evaluate the effect
of elevated PRL on the response to coital stimulation in hy-
pothyroid rats.
2. Materials and methods
2.1. Animals and treatments
Adult female (230280 g) and male (300350 g) Wistar
strain rats were obtained from Harlan (Indianapolis, IN),
Charles River Breeding Labs, Inc. (Kingston, NY), and the
Imamichi Institute for Animal Reproduction (Ibaraki, Ja-
pan). Rats were housed under controlled temperature and
lighting conditions and supplied with food and water ad libi-
tum. The estrous cycle of female rats was followed by daily
vaginal lavage. Hypothyroidism was induced by administra-
tion of 0.03% 4-methyl-2-thiouracil (Wako Pure Chemical
Industries, LTD; Osaka, Japan) in the drinking water.
2.2. Effects of thiouracil on the plasma
concentration of PRL
To examine the effects of thiouracil on plasma concen-
tration of PRL and spontaneous ovulation, five animals of
each group were killed by decapitation at 1100 h on the day
of estrus. Hypothyroidism was induced by administration of
thiouracil for two (8 days) or four estrous cycles (16 days).
Trunk blood was collected and centrifuged for the determina-
tion of plasma levels of PRL, triiodo-thyronine (T3), L-thyrox-
ine (T4), and thyroid-stimulating hormone (TSH). After de-
capitation, the oviducts were examined for oocytes.
2.3. Effects of thiouracil on the intromission threshold for
induction of pseudopregnancy and lordosis behavior in
adult female rats
Three groups of animals (seven control, seven thiouracil-
treated and five thiouracil-treated + T4-treated rats) were
used in this experiment. Thyroid hormone replacement was
performed daily by intraperitoneal (i.p.) injection of T4 (5
g per injection, Sigma Chemical Co., St. Louis, MO) for 8
days before the experiment. Tests of copulatory behavior
were conducted from 20002200 h (14 h light, 10 h dark;
light on, 0500 h) under dim illumination from a 25-W red
bulb, by direct observation of male paired with proestrous
female rats in semicircular aquariums with sawdust bedding
on the floor. A single vasectomized male was placed into
the test chamber 5 min prior to introduction of the female.
Four estrous cycles (16 days) after administration of thiou-
racil, or two estrous cycles (8 days) after replacement of T4
to thiouracil-treated rats, each female was placed into the
test chamber with a cage-adapted stimulus male. During
each test, the occurrence of each mount and intromission
was recorded, and the occurrence of the lordotic response to
each coital contact from the male also was recorded. The
behavioral measures of sexual receptivity were the percent
occurrence of lordosis in response to copulatory mounts or
mounts with intromission from males (Lordosis Quotient,
LQ). Each female was mated until it received seven in-
tromission from males, which would be expected to induce
pseudopregnancy in 50% of normal rats [12,15,16]. Follow-
ing behavioral tests, the animals were returned to their home
cages, and their vaginal estrous cycles were monitored daily
for 14 days after the testing day.
2.4. Effects of thiouracil on the TH activity in vivo and
pituitary concentrations of VIP
Hypothyroidism was induced by administration of thiou-
racil for four estrous cycles (16 days). Eight rats of each
group were injected with m-hydroxybenzylhydrazine dihy-
drochloride (NSD 1015; 100 mg/kg, i.p.), an L-aromatic
amino acid decarboxylase inhibitor, at 1030, 1630, 1930,
and 2230 h on the day of proestrus and at 0430 h on the day
of estrus. Rats were killed by decapitation after a 30-min in-
terval. The SME was dissected with a pair of fine scissors,
homogenized in 120 L 0.1 N perchloric acid, and centri-
fuged at 10,000 g for 3 min [14]. The content of DOPA in
the supernatant was determined by high-pressure liquid
chromatography with electrochemical detection, as de-
scribed previously [1,2,3]. The pellet was solubilized in 0.5
N sodium hydroxide and analyzed for protein content by the
method of Bradford [9]. Anterior pituitary glands were sep-
arated from neural lobe, weighed, and homogenized in 250
L 0.1 mol acetic acid at 4C. The homogenates were then
heated at 85C for 5 min, freeze thawed in an acetonedry
CO2 bath, and centrifuged at 10,000 g for 30 min at 4C.
The supernatants were collected and stored frozen at 20C
until VIP assay. The samples were neutralized by NaOH
prior to radioimmunoassay (RIA).
2.5. Effects of thiouracil on the VIP concentration
in hypothalamus
Hypothyroidism was induced by administration of thiou-
racil for four estrous cycles (16 days). Eight animals of each
group were killed by decapitation at 1100 h on the day of
proestrus. This model was chosen because estradiol level is
at the highest concentration at this time [43]. Blood was col-
lected and centrifuged for the determination of plasma lev-
els of PRL. After decapitation, the SME was dissected with
a pair of fine scissors, homogenized in 0.1 mol acetic acid,
centrifuged, and stored in freezer for VIP assay. The re-
mainder of the brain was frozen on dry ice, and the PVN on
both sides (800-m-diameter punches) were microdissected
(thickness: 1.2 mm). Tissue punches were also extracted in
 A. Tohei et al. / Physiology & Behavior 69 (2000) 391397
0.1 mol acetic acid for VIP assay. The pellets of SME and
PVN were solubilized in 0.5 N sodium hydroxide and ana-
lyzed for protein content by the method of Bradford [9]. An-
terior pituitary glands were separated from neural lobe,
weighed, and homogenized in 0.05 mol phosphate-buffered
saline (PBS) at 4C. The homogenates were centrifuged at
10,000 g for 30 min at 4C, and the supernatants were col-
lected and stored frozen at 20C until PRL assay.
2.6. Radioimmunoassay
Concentrations of TSH and PRL were measured using
NIDDK rat RIA kits for rat TSH and PRL. The intra- and
interassay coefficients of variation were 6.6 and 7.9% for
TSH and 5.5 and 8.3% for PRL, respectively.
T3 and T4 [40] were measured by double-antibody RIAs
using 125I-labeled radioligands as described previously. An-
tisera to T3 and T4 were kindly provided by Dr. M. Suzuki
(Gunma University, Gunma, Japan). The intra- and interas-
say coefficients of variations were 7.2 and 13.4 for T3 and
9.4 and 10.9 for T4.
VIP was measured by double-antibody RIA using 125I-la-
beled radioligands (ICN). Antiserum to VIP was kindly pro-
vided by Dr. Walro (University of Akron, OH). The RIA
tube contained 250 L buffer (0.05 mol PBS, pH 7.4; 3%
bovine serum albumin; 0.1% Triton X-100; and 0.1%
NaN3) plus VIP standard or sample and 100 L VIP antise-
rum diluted 1:16,000 with 1:200 dilution of normal rabbit
serum in EDTA-PBS. After a 48-h incubation at 4C, 50 L
125
I-VIP (8,00010,000 cpm) were added to all tubes, and
the mixture was incubated for an additional 48 h. Antirabbit
-globulin was used to separate bound from unbound iodi-
nated VIP. The intra- and interassay coefficients of varia-
tion were 5.2 and 9.4% for VIP.
2.7. Statistical analyses
The percentage of females showing prolonged periods of
diestrus were compared by chi-square test and Fishers ex-
act probability test. To compare the data among three
groups, chi-square test was used, and Fishers exact proba-
bility test was used to compare between any two groups
(control thiouracil, thiouracil thiouracil T4, and
control thiouracil T4). All results except for frequency
of pseudopregnancy are expressed as the mean SEM.
Hormone and DOPA data were analyzed by one-way analy-
sis of variance (ANOVA), followed by Fishers protected
least significant difference (PLSD) test; a value of p 0.05
was considered significant.
3. Results
3.1. Effects of thiouracil on the plasma concentration of T3,
T4, TSH, and PRL
Plasma concentrations of T3 (control, 680.4 47.8 pg/
mL; thiouracil, 183.8 22.5 pg/mL) and T4 (control; 53.9
12.6 ng/mL; thiouracil, 6.7 1.6 ng/mL) were markedly
393
suppressed in female rats as a result of administration of
thiouracil for 16 days. On the other hand, plasma concentra-
tion of TSH (control, 0.98 0.25 ng/mL; thiouracil, 10.1
2.3 ng/mL) in hypothyroid rats was significantly increased
after administration of thiouracil.
Plasma concentration of PRL increased at 1100 h on the
day of estrus in hypothyroid rats treated with thiouracil for
16 days, although there was no difference in plasma PRL
levels between control rats and rats treated with thiouracil
for 8 days (Fig. 1).
The number of oocytes in oviducts at 1100 h on the day of
estrus was not statistically different in hypothyroid animals
(10.8 0.86) as compared with euthyroid rats (12.4 0.51).
3.2. Effects of thiouracil on the intromission threshold for
induction of pseudopregnancy and lordosis behavior in
adult female rat
All animals received seven intromissions from vasecto-
mized males and did not receive any ejaculations. In addition
to seven intromissions, female rats received 510 mounts
without intromission during the tests. The number of animals
exhibiting prolonged diestrous vaginal smears are shown in
Figure 1. The chi-square p-value was p 0.039, suggesting
a relationship between each treatment and frequency of
pseudopregnancy. The proportion of female rats exhibiting
pseudopregnancy was significantly higher in hypothyroid
animals (7/7; 100%) receiving seven intromissions than in
euthyroid animals (3/7; 43%). Administration of T4 in hy-
pothyroid rats decreased the proportion of pseudopregnancy
(2/5; 40%) to the level of control animals. The length of pro-
longed diestrus was not different between euthyroid, hy-
pothyroid, and T4-replacement groups. Nearly maximal LQs
were displayed by all three groups (control, 0.95 0.03;
thiouracil, 0.96 0.02; T4, 0.98 0.02), and there was no
significant effect of thiouracil treatment on LQ.
Fig. 1. (a) Plasma concentrations of PRL at 1100 h on estrus in control
(open bar) and hypothyroid (solid bar) adult female rats two (8 days) or
four estrous cycles (16 days) after treatment of thiouracil. Each bar repre-
sents the mean SEM of five animals. Asterisks indicate p 0.05 com-
pared with the value for the respective control. (b) The percentage of rats
mated at 2000 h on proestrous that showed pseudopregnancy. Control
(open bar), hypothyroid (solid bar), and hypothyroid T4 (hatched bar)
female rats received seven intromissive stimulations from vasectomized
male rats. *indicate p 0.05 compared with the value for the respective
control, and # indicate p 0.05 compared with the value for the hypothy-
roid T4.
394 A. Tohei et al. / Physiology & Behavior 69 (2000) 391397
3.3. Effects of thiouracil on the TH activity in the SME and
pituitary concentrations of VIP
TH activity in the SME, measured by accumulation of
DOPA 30 min after injection of NSD 1015 (100 mg/kg,
i.p.) was similar at all times examined on proestrus and es-
trus in both control and thiouracil-treated rats except for a
statistically significant decrease in TH activity at 1700 h on
proestrus (compared to 1100, 2300, and 0500 h on estrus)
in control rats (Fig. 2). There were no statistically signifi-
cant effects of thiouracil treatment on TH activity at any of
these times.
Fig. 2. (a) In vivo dopa accumulation 30 min after injections of NSD 1015
(100 mg/kg, i.p.) at 1100, 1700, 2000, and 2300 h on proestrus and at 0500
h on estrus in control (open bar) and hypothyroid (solid bar) adult female
rats. (b) Pituitary concentrations of VIP at 1100, 1700, 2000, and 2300 h on
proestrus and at 0500 on estrus in control (open bar) and hypothyroid (solid
bar) adult female rats. Each bar represents the mean SEM of eight ani-
mals. Asterisks indicate p 0.05 compared with the value for the respec-
tive control (one-way ANOVA followed by Fishers PLSD test).
Pituitary concentrations of VIP were dramatically in-
creased at all times examined on proestrus and estrus in hy-
pothyroid rats as compared with euthyroid rats (Figure 2).
3.4. Effects of thiouracil on plasma and pituitary
concentrations of PRL and hypothalamic
concentration of VIP
The concentrations of PRL in plasma and pituitary at
1100 h on the day of proestrus were significantly increased
in hypothyroid rats 16 days after administration of thiou-
racil as compared with control rats (Fig. 3a,b). The concen-
tration of VIP in SME at 1100 h on the day of proestrus was
similar in both the euthyroid and hypothyroid groups (Fig-
ure 3c). Although the concentration of VIP in PVN was
60% higher in hypothyroid rats, this difference from control
values was not statistically significant (Fig. 3d).
4. Discussion
Hypothyroidism induced by administration of thiouracil
for 16 days in intact female rats resulted in an increase in
plasma concentration of PRL at 1100 h on the day of
proestrus. Studies using male rats [19,35,40], ovariecto-
mized [32], or immature female rats [22], however, reported
that hypothyroidism resulted in decreased levels of plasma
and pituitary PRL. Previous reports indicated that ovariecto-
mized, thyroidectomized rats showed increased levels of
plasma PRL only if estrogen was present [32,44]. A finding
from our laboratory demonstrated that the plasma concen-
tration of PRL was higher at 2300 h on proestrus and at
0500 and 1100 h on estrus in thiouracil-induced hypothy-
roid adult female rats [41]. In this study, we showed that hy-
Fig. 3. PRL concentrations in plasma (a) and pituitary (b) and VIP concentra-
tions in SME (c) and PVN (d) at 1100 h on proestrus in control (open bar) and
hypothyroid (solid bar) adult female rats. Each bar represents the mean
SEM of five animals. Asterisks indicate p 0.05 compared with the value for
the respective control (one-way ANOVA followed by Fishers PLSD test).
 A. Tohei et al. / Physiology & Behavior 69 (2000) 391397
pothyroidism induced hyperprolactinemia in intact adult fe-
male rats without additional estrogen treatment.
The number of oocytes in the oviducts at 1100 h on the
day of estrus was slightly less in hypothyroid as compared
with euthyroid rats, although the difference was not statisti-
cally significant. It has been reported that hypothyroidism
can induce reproductive abnormalities including amenor-
rhea in women [18,31,39]. We also observed that the num-
ber of oocytes after human chorionic gonadotropin chal-
lenge was significantly lower on the day of diestrus in
hypothyroid rats as compared with euthyroid rats, although
there is no difference on the day of proestrus between hy-
pothyroid and control rats [41]. The duration of thiouracil
administration may have been too short to decrease the
number of oocytes which ovulated spontaneously in the
present study.
Pseudopregnancy occurred in 43% of control rats that re-
ceived seven intromissions. This result is similar to those in
reports elsewhere [12,15,16]. The proportion of female rats
exhibiting pseudopregnancy was significantly higher in hy-
pothyroid animals receiving seven intromissions than in eu-
thyroid animals. Administration of T4 in hypothyroid rats
decreased the occurrence of pseudopregnancy to the level of
control animals. Fewer than six intromissions was nearly ef-
fective for induction of pseudopregnancy, whereas pseudo-
pregnancy occurred in 67% of rats that received 69 in-
tromissions in normal adult female rats [12]. We demonstrated
that hypothyroidism decreases the intromission threshold
for induction of pseudopregnancy and that T4 replacement
to hypothyroid rats restored the threshold to that observed in
euthyroid adult female rats. The increased levels of PRL in
hypothyroid rats suggests that the PRL response to coital
stimulation may be activated in hypothyroid female rats to
account for the higher incidence of pseudopregnancy.
TH activity, as measured by DOPA accumulation 30 min
after injections of NSD 1015, was similar at all times exam-
ined on proestrus and estrus in control rats except for a sta-
tistically significant decrease (40% as compared with that at
1100 h) in TH activity at 1700 h in control rats. The de-
crease at 1700 h confirms a report of a reduction in TH ac-
tivity in the SME on proestrus [4]. There were no significant
effects of thiouracil treatment on TH activity. Several previ-
ous reports demonstrated that hypothyroidism increased the
activity of dopaminergic neurons and decreased prolactin
secretion in male rats [19,21,30,35]. There are no reports re-
garding TH activity in intact adult hypothyroid female rats.
In the present study, we demonstrated that hypothyroidism
has no effect on TH activity in the SME throughout
proestrus in adult female rats, suggesting that the increase in
PRL secretion is not due to a change in dopamine secretion
from TIDA neurons.
Pituitary concentration of VIP was dramatically in-
creased at all times examined on proestrus and estrus in hy-
pothyroid rats as compared with euthyroid rats. VIP concen-
tration in PVN also increased at 1100 h on the day of
proestrus in hypothyroid rats, though the difference was not
395
statistically significant. Both plasma and pituitary concen-
trations of PRL also increased in hypothyroid female rats as
compared with control rats at this time. Hypothyroidism has
been shown to increase VIP content and/or mRNA in pitu-
itary and PVN in male or immature female rats [2224,38].
No studies have been reported showing a concomitant in-
crease of pituitary VIP and plasma PRL in hypothyroid
adult female rats. In this study, we have shown a concomi-
tant increase of pituitary VIP and PRL secretion in hypothy-
roid female rats as compared with control rats, whereas TH
activity in SME was not different between both groups.
These results suggest that hypothyroidism-induced increase
in PRL may be due to an increase in pituitary VIP, which
can affect PRL secretion by acting as a paracrine or auto-
crine regulator. VIPpituitary adenylate cyclaseactivating
polypeptide (PACAP) receptors have been shown to exist in
anterior pituitary [33] and the somatolactotroph cell lines
GH3/GH4C1, which express mRNA for VIP-PACAP type 3
receptor [33,34]. It also has been reported that anti-VIP se-
rum decreases basal PRL secretion from cultured hypothy-
roid pituitary cells [23]. VIP in the PVN, acting as a hypo-
physiotropic factor, may also increase PRL secretion in
hypothyroid female rats. Lactation, a state in which circulat-
ing PRL concentrations are elevated, has been known to in-
crease VIP immunoreactivity in the parvocellular portion of
the PVN and increase the number of VIP-positive fibers in
the external zone of the median eminence [27].
It is generally accepted that PRL secretion is mediated by
TRH, also acting as a PRL-releasing factor [8,25]. The se-
cretion of TRH would be expected to increase in hypothy-
roid rats if a classical negative-feedback mechanism exists.
The effect of hypothyroidism on TRH secretion from hypo-
thalamus, however, has not shown this to be the case. Some
studies showed that hypothyroidism can increase TRH im-
munoreactivity and gene expression in PVN [26,37]. Other
studies reported that TRH levels in hypophysial-stalk blood
and the hypothalamus, as determined by RIA, did not
change in hypothyroid rats [13,20,28,36]. Main target sites
of negative feedback in thyroid hormone seem to be pitu-
itary because plasma concentrations of TSH dramatically
increase in hypothyroid animals. TRH also may be involved
in the induction of hyperprolactinemia in hypothyroid adult
female rats by an increase in the anterior pituitary respon-
siveness to TRH for PRL release, which has been reported
in ovariectomized, thyroidectomized rats treated with estro-
gen [32].
An isolated report mentioned that hypothyroidism
caused hyperprolactonemia by decreasing the clearance of
PRL in plasma [11]. In the present study, we demonstrated
that hypothyroidism decreased the intromission threshold
for induction of pseudopregnancy and increased pituitary
contents of VIP as a PRL-releasing factor. These results
suggest that hyperprolactinemia observed in hypothyroid
female rats is mainly due to the changes in neuroendocrine
function.
In summary, hypothyroidism did not affect TH activity
396 A. Tohei et al. / Physiology & Behavior 69 (2000) 391397
in SME, whereas it dramatically increased pituitary concen-
tration of VIP in adult female rats. This increased level of
VIP likely affected PRL secretion in a paracrine or auto-
crine manner, inducing hyperprolactinemia in hypothyroid
female rats. A decrease in the intromission threshold for in-
duction of pseudopregnancy is likely due to this increased
level of PRL in hypothyroid female rats.
Acknowledgments
We are grateful to the Rat Pituitary Hormone Distribution
Program, NIDDK, NIH, Bethesda, MD, for providing RIA
materials; Dr. Walro, University of Akron, Akron, Ohio for
providing antisera to VIP; Dr. M. Suzuki, Gunma Univer-
sity, Maebashi, Japan for antisera to T3 and T4; and Dr. R.
Hokao, The Imamichi Institute for Animal Reproduction,
Ibaraki, Japan for Wistar rats. The present study was sup-
ported by NIH Research Grant HD 24190 (to J.L.V.) and a
grant-in-aid for Scientific Research from the Ministry of Ed-
ucation of Japan: 066375 (to K.T.). A.T. received a fellow-
ship from the Japan Society for the Promotion of Science.
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