Endocrinology Vol. 139, No. 9 3691-3695
Copyright © 1998 by The Endocrine Society
Neuroendocrine and Reproductive Functions in Male Mice with Targeted Disruption of the Prolactin Gene1
R. W. Steger,
V. Chandrashekar,
W. Zhao,
A. Bartke and
N. D. Horseman
Department of Physiology, Southern Illinois University School of
Medicine (R.W.S., V.C., A.B.), Carbondale, Illinois 62901-6512; and the
Department of Physiology and Biology, University of Cincinnati (N.H.,
W.Z.), Cincinnati, Ohio 45267-0576
Address all correspondence and requests for reprints to: Dr. R. W. Steger, Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-1625. E-mail:
rsteger{at}som.siu.edu
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Abstract
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Mice with a targeted disruption (knock-out) of the PRL gene (PRL-KO)
were used to study the physiological role of PRL in the control of male
neuroendocrine functions related to reproduction. Compared with normal
males, PRL-KO mice had significant reductions in median eminence
dopamine content, plasma LH levels, LH and FSH secretion in
vitro (per mg pituitary), and weights of seminal vesicles and
ventral prostate. PRL was not detectable in incubation medium with
pituitaries from PRL-KO mice. No alterations were detected in PRL-KO
mice in median eminence norepinephrine, plasma testosterone levels, or
testosterone release (per mg testis) in vitro with or
without LH. No differences were detected in PRL-KO vs.
normal male mice in the interval from housing with normal female mice
until conception, rate of pregnancy, or the number of live pups per
litter. Pituitary weight in PRL-KO mice was increased (1.78 ±
0.22 vs. 3.35 ± 0.20 mg; P <
0.001), presumably due to reduced feedback inhibition and hypertrophy
and/or hyperplasia of nonfunctional lactotrophs. These results indicate
that the absence of PRL reduces pituitary LH release, attenuates median
eminence dopaminergic activity, and affects the growth of seminal
vesicles and ventral prostate. Although it was previously shown that
PRL can repair the reproductive defect in male pituitary dwarf mice,
our current results imply that the PRL deficiency alone is not
sufficient to cause male infertility, although there are obvious
alterations in reproductive neuroendocrine function in PRL-KO males.
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Introduction
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THE EFFECTS of pathological elevation of
peripheral PRL levels, hyperprolactinemia, on male reproductive
function in the human and in several species of experimental animals
have been characterized in considerable detail (1, 2, 3, 4). In contrast, the
normal role of physiological levels of PRL in the male is much less
clearly defined because models of PRL deficiency are limited. In the
mouse, congenital PRL deficiency caused by recessive mutations at the
pit-1 (Snell dwarf; dw) or prophet of pit-1 (Ames
dwarf; df) loci is associated with reduced plasma gonadotropin and
testosterone (T) levels, reduced levels of testicular LH and PRL
receptors (PRL-R), and severe suppression of fertility (5, 6, 7, 8). These
deficits can be partially corrected by PRL replacement therapy (5, 6, 8, 9). Although these findings suggest an important role of PRL in male
reproductive development and function, their interpretation is greatly
complicated by the combined deficiency of GH and TSH as well as PRL in
both Snell and Ames dwarf mice (10, 11).
Targeted gene disruption (knock-out) offers new possibilities for
defining the physiological roles of hormones and other chemical
messengers. Disruption of PRL signaling by knocking out the PRL-R gene
resulted in some delay of male fertility without producing any obvious
abnormalities in the male reproductive system (12) (Kelly, P., personal
communication). Animals with a targeted disruption of the PRL gene
(PRL-KO mice) are viable, and initial studies detected no abnormalities
in male reproductive functions of these animals (13, 14). The present
studies were undertaken to characterize neuroendocrine functions
related to reproduction in PRL-KO mice compared with those in their
normal siblings.
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Materials and Methods
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The animals were produced by crossing PRL-KO (-/-) males with
phenotypically normal (N; +/-) females. The progeny was classified as
PRL-KO or N by PCR analysis of DNA extracted from tail clippings (14).
Sixteen PRL-KO and nine N animals were studied. All animal protocols
were approved by the local committee on animal care. After reaching
adulthood (2–3 months of age), each male was placed in a cage with two
normal young adult virgin females. The females were checked daily for
birth of litters, the numbers of live and dead pups were recorded, and
females with litters were removed from the male’s cage. Six weeks
after placing the males with the females (i.e. 2.5–3 weeks
after most of the females had been removed), the males were moved to a
laboratory at 0645 h and, starting 1.5 h later, were
anesthetized with ether, bled by cardiac puncture, and killed by
decapitation. The blood plasma was saved for measurements of LH and T
by RIAs previously validated for the use in mouse plasma (7). At the
time of death, the brain was rapidly removed, and the median eminence
was dissected free and frozen, as was the remaining brain for
subsequent determinations of DA and norepinephrine (NE) content by HPLC
with electrochemical detection (15). Before assay, the brains were
allowed to thaw partially to allow dissection of the medial basal
hypothalamic area. This area consisted of a tissue block 2.0 mm deep
extending from the caudal border of the optic chiasm to the rostral
margin of the mammillary bodies and laterally to the hypothalamic
sulci. The pituitaries were weighed, cut in half, and used for
determinations of LH, FSH, and PRL contents or were incubated for
1 h in medium 199 after a 1-h preincubation (16). The levels of
LH, FSH, and PRL in the media were measured by RIAs. The testes were
weighed, decapsulated, preincubated for 30 min, and incubated for
4 h in 2 ml Krebs-Ringer-bicarbonate buffer containing glucose in
an atmosphere of 95% oxygen-5% carbon dioxide in the presence of 0,
2.5, or 12.5 ng ovine LH (oLH NIH-26)/ml, and the levels of T in the
media were measured by RIA. The results of RIAs were expressed in terms
of NIH standards: LH RP-3 for LH, FSH RP-2 for FSH, and AFP-6476C for
PRL. In each study, all samples were processed in the same assay, and
the intraassay coefficients of variation were 4.99% for T, 2.84% for
plasma LH, and 3.3% for PRL.
The male accessory reproductive glands, seminal vesicles (SV),
coagulating glands, and ventral prostate (VP), were removed and weighed
(SV and coagulating glands were weighed both with and without their
secretions). The epididymides as well as the liver, spleen, and
adrenals were also removed and weighed.
The significance of the differences between the values measured in
PRL-KO and N mice was calculated using Student’s t
test.
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Results
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Fertility
All of the PRL-KO and N males were fertile. Seventeen of 18
females mated to N males delivered litters, with 83% of the litters
arriving within 24 days after placing the females with the male.
Thirty-one of 32 females mated to PRL-KO males delivered litters, with
88% of the litters arriving within 24 days after mating. The number of
live pups per litter did not differ between the 2 groups (9.68 ±
0.39 and 10.06 ± 0.63 for PRL-KO and N males, respectively).
Organ weights
There were no significant differences between PRL-KO and N animals
in body weight or in the weights of the testes, epididymides, or
coagulating glands. The weights of the SV and VP were significantly
reduced in PRL-KO compared with those in N mice. In contrast, the
weight of the pituitary was increased by approximately 100% in PRL-KO
males. The weight of the liver was significantly reduced in PRL-KO
animals, and there were no significant differences in the weights of
the spleen, or adrenals (Table 1
). The
results of histological analysis of multiple organs from PRL-KO animals
from this same line of mice have been described previously (14).
Hypothalamic catecholamines
Dopamine (DA) content in the median eminence was significantly
reduced in the PRL-KO mice compared with that in controls (Fig. 1). DA levels in the medial basal
hypothalamus (Fig. 1) and anterior hypothalamus (data not shown) were
similar. There were no significant differences in NE content in any of
these areas.
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Figure 1. Median eminence (top panel) and
medial basal hypothalamic (bottom panel) NE and DA
contents determined in PRL-KO mice and their normal littermates. Values
are expressed as the mean ± SEM.
Asterisks denote a statistical difference from the
respective control value (P < 0.05).
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Hormone levels
Plasma LH levels were significantly lower in PRL-KO males than in
the normal controls (1.17 ± 0.19 vs. 1.75 ± 0.24
ng/ml; P < 0.05), whereas plasma T levels in these two
groups were not significantly different (Fig. 2).
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Figure 2. Plasma LH and T concentrations in PRL-KO mice and
their normal littermates. Values are expressed as the mean ±
SEM. Asterisks denote a statistical
difference from the respective control value (P <
0.05).
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The contents of LH and FSH per mg pituitary were markedly reduced in
PRL-KO animals compared with those in normal mice (Table 2). However, when the LH and FSH contents
were expressed on a per pituitary basis, the differences between PRL-KO
and normal mice became numerically small and not statistically
significant (Table 2). PRL was not detectable in pituitary homogenates
of PRL-KO mice.
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Table 2. Pituitary LH, FSH, and PRL contents of
hemipituitaries collected from normal and PRL-KO mice at the time of
death
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Hormone release in vitro
The in vitro secretion of LH and FSH were not different
between groups when calculated on a per pituitary basis (Table 3). However, the pituitaries from the
PRL-KO mice released significantly less LH and FSH when calculated on a
weight basis. PRL levels in the incubation medium from the PRL-KO
pituitaries were not detectable.
The amounts of T released into the media by testicular parenchyma from
PRL-KO mice did not differ from the amounts of T released by testicular
parenchyma from normal animals incubated under identical conditions
(Table 4). This was true of both basal
and LH-stimulated release. Although T release was numerically less
after LH stimulation of PRL-KO testes, the differences were not
significant at either dose of LH.
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Discussion
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Some of the characteristics of the reproductive and neuroendocrine
phenotypes of adult PRL-KO male mice are consistent with the previously
reported effects of PRL in this species. However, other characteristics
of these animals were not anticipated.
The reduced DA content in the median eminence of the hypothalamus is
compatible with the well documented negative feedback relationship of
PRL and tuberoinfundibular DA (TIDA) neurons in which PRL provides a
stimulatory input to the function of this neuronal group (17, 18). The
median eminence contains terminal fields of TIDA neurons.
Suppression of plasma LH levels in PRL-KO animals is consistent with
the reduced LH levels in PRL-deficient dwarf mice (5, 7) and with the
ability of PRL treatment or PRL-secreting ectopic pituitary transplants
to stimulate LH release in mice (4). This is in contrast to rat and
man, where hyperprolactinemia leads to decreases in LH secretion (2).
Curiously, treatment of dwarf mice with PRL or pituitary transplants
failed to increase plasma LH levels (5, 19).
The reduction in plasma LH levels could be secondary to changes in
either pituitary or hypothalamic function. Although in vitro
LH secretion was not different between the PRL-KO and control mice,
responses to LHRH stimulation were not evaluated. However, previous
studies suggest that the effects of hyper- or hypoprolactinemia on
gonadotropin release are most likely due to PRL effects on the
hypothalamus rather than to effects on the pituitary (2).
Hyperprolactinemia may reduce LH secretion in the rat by reducing
noradrenergic stimulation of LHRH release while in the mouse,
hyperprolactinemia increases NE turnover and LH secretion (20). The
hypothalamic NE content was unchanged in the present experiment, but
content does not necessarily reflect neuronal activity, and additional
studies measuring NE turnover need to be completed to address this
question. Alternatively, the LHRH neuron or numerous other neuronal
products affecting LHRH release could be affected by PRL.
The lack of changes in either plasma T levels or responsiveness of
testicular T production to LH in vitro was unexpected. PRL
was reported to increase the number of testicular LH receptors and
testicular responsiveness to LH in several rodent species, including
mice (1, 2, 6, 21, 22). Suppression of testicular LH binding in DBA/2
male mice with experimentally induced hyperprolactinemia was believed
to be due to down-regulation by chronically elevated LH levels in these
animals (4). Perhaps the suppression of LH levels in PRL-KO mice leads
to an increase in testicular LH receptors. This could explain the
normal plasma T levels despite reduced LH. However, the testicular T
response to LH in vitro was not augmented in PRL-KO animals.
The maintenance of normal plasma T levels in PRL-KO males in which LH
is reduced could also reflect redundancy of stimulatory inputs to the
Leydig cells. Conceivably, one of the factors involved, such as
insulin-like growth factor I, GH, FSH, or testicular nerves, can
substitute for the functions normally served by PRL (7, 23, 24, 25).
Coexistence of normal plasma T and suppressed LH levels could also be
due to an increased sensitivity of the hypothalamus or pituitary to
negative T feedback. Although not tested in the present study, this
possibility is consistent with the results obtained in Syrian hamsters
(26, 27). In this species, similarly to the mouse, PRL enhances
gonadotropin release (26, 27, 28), and there is evidence that this effect
of PRL may involve reducing the sensitivity to T feedback (26, 27).
The normal testicular weight and breeding performance of the PRL-KO
males suggest that these animals have no major deficits in
spermatogenesis or in copulatory behavior. This interpretation is
consistent with normal T levels in PRL-KO mice. Previous studies of
PRL-KO animals from the same line revealed normal testicular histology
(14).
The striking increase in pituitary weight in PRL-KO mice probably
results from hypertrophy and/or hyperplasia of nonfunctional
lactotrophs in the absence of normal inhibitory input from TIDA
neurons. Although further studies will be required to test the validity
of this hypothesis and to characterize the cellular composition of the
enlarged pituitaries of PRL-KO mice, the data on gonadotropin release
in vitro suggest that the number and/or size of the
gonadotrophs were probably not affected. The release of LH and FSH per
mg pituitary tissue was greatly reduced, but this was an artifact of
increased pituitary weight, as the release of these hormones per
pituitary was not altered.
A reduction in the weights of the SV and VP is consistent with the
ability of PRL to potentiate the effects of androgens on the male
accessory reproductive glands in various species, including mice
(29, 30, 31, 32). Treatment with PRL or ectopic pituitary transplants increases
SV weight in hypophysectomized mice treated with T (30), in dwarf mice
(31), and in normal, intact mice (32). Hypertrophy of different lobes
of the prostate, including VP, was recently described in transgenic
mice overexpressing rat PRL (33).
Thus, the present results suggest that in the male mouse PRL has a
physiological role in the control of LH release and in the regulation
of growth of the accessory reproductive glands, but is not required for
the maintenance of normal plasma T levels or fertility.
Some differences appear to exist between the reproductive consequences
of disrupting the PRL gene and the PRL-R gene (12). Male PRL-KO mice
are fully fertile (Refs. 13, 14 and the present study). Male
PRL-R-KO mice were initially reported to have a high incidence of
infertility (12), but were subsequently shown to exhibit a delay,
rather than an inhibition, of fertility (Kelly, P., personal
communication). PRL-R messenger RNA and PRL binding can be detected in
many organs of fetal mice (34, 35), whereas pituitary PRL secretion
starts postnatally (36, 37). This raises an intriguing possibility that
signaling through PRL-R may be necessary for some early developmental
events that are required for normal male sexual maturation. Perhaps
PRL-R-mediated actions of placental lactogens and/or maternal PRL
delivered via the fetal circulation or the milk are involved in the
development of the male reproductive system, its hormonal regulation,
or hypothalamic centers that control sexual behavior.
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Acknowledgments
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The authors thank Clare Fadden for the excellent technical
assistance. We also thank the Hormone Distribution Branch, NIDDK, NIH,
and Dr. A. F. Parlow for supplying the materials used in the
RIAs.
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Footnotes
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1 This work was supported by NIH Grants HD-20001 and DK-49895. 
Received February 20, 1998.
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Top
Abstract
Introduction
