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Molecular and Cellular Biology, December 2000, p. 9337-9345, Vol. 20, No. 24
Institute for Molecular Biology and
Biotechnology1 and Department of
Biology,2 McMaster University, Hamilton,
Ontario, Canada L8S 4K1; Department of Cell Biology, University
of Virginia Health Sciences Center, Charlottesville, Virginia
229083; and Department of Pharmacology,
Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia,
Canada B3H 4H74
Received 7 July 2000/Returned for modification 29 August
2000/Accepted 27 September 2000
PEA3, a member of the Ets family of transcriptional regulatory
proteins, is expressed in a unique spatial and temporal pattern during
mouse embryogenesis; its overexpression is positively correlated with
HER2-mediated breast tumorigenesis in both humans and mice. To
determine whether PEA3 plays a part in development and oncogenesis and
to uncover its normal physiological role, we generated mice lacking
functional PEA3 by gene targeting in embryonic stem cells. PEA3 The ets genes, which
currently comprise nearly 30 paralogs in mammals, encode transcription
factors bearing conserved sequence-related DNA binding domains (the ETS
domain) (16). Ets proteins are capable of regulating
transcription by binding to sites in the promoters of their cognate
target genes. DNA binding is achieved by interaction between the ETS
domain and an ~10-bp sequence element termed the Ets binding site
(EBS) comprising a highly conserved core sequence, 5'-GGA(A/T)-3'.
Individual Ets proteins demonstrate specificity for sequences flanking
this core, but it is not uncommon for different Ets proteins to bind to
the same EBS.
PEA3 (36) is the founding member of a subfamily of Ets
proteins, which also includes ER81 (6) and ERM (11,
26). Members of this subfamily possess nearly identical ETS
domains and harbor additional regions of sequence similarity. Analyses
of the transcriptional properties of individual PEA3 subfamily members
reveal that they commonly activate transcription (6, 11, 26,
36). Whereas few bona fide PEA3 target genes have been
identified, transient-transfection studies suggest that PEA3 is capable
of regulating the transcription of genes whose products facilitate cell
motility and invasion (reviewed in reference 13).
PEA3 is expressed in a spatially and temporally restricted pattern
during mouse development in cells derived from each of the three germ
layers and in regions of the embryo undergoing cellular proliferation
and migration (11). PEA3 appears to be preferentially
expressed at sites of epithelium-mesenchyma interactions. In the
developing chick, PEA3 is selectively expressed in specific classes of
motor neurons and corresponding muscle afferent sensory neurons at limb
levels of the spinal cord (24). PEA3 expression by both
motor and sensory neurons is governed by signals derived from the limb
muscle. In adult mice PEA3 RNA is most abundant in the brain and
epididymis (36).
Overexpression of PEA3 is associated with breast cancer in both humans
and mice, suggesting a role for PEA3 in this malignancy (3,
34). Seventy-six percent of all human breast tumors contain elevated levels of PEA3 RNA; 93% of the c-ERB-B2 (also known as HER2)-positive subclass of these tumors overexpress PEA3
(3). PEA3 is also overexpressed in all mouse mammary tumors
arising in transgenic mice engineered to overexpress murine c-Erb-B2
(also known as Her2) in their mammary glands (34). These
findings suggest the possibility that PEA3 plays a role in mammary
oncogenesis or its progression.
To assess the role of the PEA3 gene in embryonic
development, adult physiology, and oncogenesis, we introduced a
loss-of-function mutation in this gene in the mouse germ line.
PEA3 mutant mice were viable, but analyses of males revealed
an unexpected role for the protein in male sexual function.
Construction of a PEA3 targeting vector.
Bacteriophage lambda recombinants bearing mouse genomic PEA3 DNA were
isolated from a 129/sv library using a full-length murine PEA3 cDNA as
a probe (36). The entire mouse PEA3 gene was
sequenced, and this information was used to construct a targeting
construct whose structure is illustrated in Fig. 1A. The targeting
vector comprised 6.0 kb of PEA3 DNA on one side and 10.8 kb on the
other; these sequences were separated by a PGKneoPA expression cassette (25). The targeting vector was linearized with
NotI prior to its introduction into embryonic stem (ES) cells.
Isolation of PEA3 mutant mice.
ES cells of the
J1 line were cultured and electroporated as described previously
(5). Homologous recombination events at the PEA3
locus were identified by simultaneous cleavage of ES cell DNA with
ClaI and EcoRI, followed by Southern blotting
(31) and hybridization with a radiolabeled probe
corresponding to an 840-bp ClaI/KpnI DNA fragment
(Fig. 1A). The structure of the targeted locus was confirmed by
EagI digestion of ES cell DNA and Southern analysis using
the same DNA probe. Chimeras were generated by blastocyst injection as
described previously (5). A male chimera was mated with
BALB/c females to generate an outbred mouse line and with 129/sv
females to generate an inbred 129/sv line.
RNase protection analysis of tissue RNA.
RNA was isolated
from tissues and organs using the guanidinium thiocyanate method
(10). RNase protection experiments were performed as
described previously (36).
Analysis of PEA3 protein in mouse embryonic fibroblast cell
lines.
Fibroblast cell lines were established from individual
mouse embryos harvested at 13.5 days postcoitus using a 3T3 protocol (33). Nuclear extracts were prepared from these cells as
described previously (23). Protein samples (25 µg) were
electrophoresed through a sodium dodecyl sulfate-10% polyacrylamide
gel and transferred to a membrane. Membranes were incubated with a
mixture of two PEA3-specific monoclonal antibodies (MP-13-MP-16)
followed by a goat anti-mouse secondary antibody. The Western blots
were developed by chemiluminescence.
Histological analyses.
Mice were euthanized and perfused
with 4% paraformaldehyde prior to dissection. Organs were fixed
overnight in 4% paraformaldehyde, dehydrated through a graded series
of ethanol baths, and embedded in paraffin wax. The paraffin blocks
were cut into 8.0-µm-thick sections, adhered to glass slides,
dewaxed, counterstained with hematoxylin and eosin, and mounted with
Permount. Slides were photographed using a Zeiss Axioskop microscope.
Analysis of male fertility.
In vitro fertilization of mouse
oocytes was performed as described previously (4). Eggs that
had cleaved were scored as fertilized; one-celled oocytes were recorded
as unfertilized. Observations were made using an Olympus S-30
dissecting microscope. In vivo fertilization was performed as described
previously (18). BALB/c mice were superovulated and paired
with a male overnight. The following morning, females were examined for
the presence of a copulatory plug and for the presence of spermatozoa
in their uterine horns and vagina. Pronucleation was scored as
described above.
Determination of hormone concentrations by RIA.
Blood was
collected from the inferior vena cava using a 21-gauge needle attached
to a 3-ml syringe containing 0.1 ml of heparin. Blood samples were
transferred to 1.5-ml tubes and centrifuged for 15 min at 4°C. The
plasma was collected and stored at Induced erections.
The procedure used to evoke erections in
male mice has been described previously by Sachs (30).
Briefly, male mice were allowed to run partway into a plastic cylinder
(56 mm long by 26 mm wide) and then placed in a supine position with
their hind legs held by the tester. The penile sheath was retracted and
held in this position using a cotton-tipped applicator held at the base
of the penis. Gentle pressure was applied to the mouse's abdomen with
the tester's finger for 10 to 15 s, and the occurrence of penile
erection was noted. Erections were scored as either strong
(engorgement, color change, and change in length and circumference) or
weak (poor engorgement and little change in dimensions).
In vitro relaxation of corpora cavernosa.
Mice were
sacrificed by cervical dislocation, and the corpora cavernosa was
dissected in situ. One corpus cavernosum from each mouse was suspended
in a jacketed organ bath (1 ml, 37°C) containing a modified
Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM
MgSO4, 1.2 mM KH3PO4, 25 mM NaHCO,
2.5 mM CaCl2, 5.6 mM glucose). A mixture of 95% oxygen and
5% carbon dioxide was bubbled through the solution. The tissues were
subjected to 0.2 g of tension and allowed to equilibrate for 30 min. Isometric relaxation responses to single concentrations of
acetylcholine were measured after contracting the corpus with
phenylephrine (10 µM). The tissues were then washed by overflow for
10 min. The tissue was equilibrated for 5 min and then contracted again with phenylephrine before another concentration of acetylcholine was
tested. No more than five different concentrations of acetylcholine were used per experiment. Drugs were prepared freshly on the day of the
experiment. Phenylephrine (Sigma) was dissolved in Krebs-Henseleit solution; acetylcholine (Sigma) stock solution was prepared in 0.1 mN
HCl and diluted in Krebs-Henseleit solution.
Isolation of mice with targeted loss-of-function PEA3
alleles.
We constructed a gene targeting vector by deleting
PEA3 gene sequences spanning exons 6 through 11 and
replacing these sequences with an expression cassette bearing the
neo gene (Fig. 1A). We selected this region for disruption because it includes exon 11, which
encodes an essential region of the ETS DNA binding domain (36). The linearized targeting vector was electroporated
into J1 ES cells, and G418-resistant clones were screened by Southern analysis for homologous recombination events. The wild-type (WT) and
targeted PEA3 alleles were distinguished by cleavage of ES cell DNA with ClaI and EcoRI and hybridization
with an appropriate DNA probe (Fig. 1A). An 8.2-kb DNA fragment or a
7.6-kb fragment is expected to result from cleavage of the cellular DNA
of WT and PEA3 mutant mice, respectively. Three ES cell
clones (CF11, DD1, and DE3) of the 210 that were screened yielded both
8.2- and 7.6-kb fragments, suggesting that they contained the targeted allele (Fig. 1B, lanes 3, 5, and 7). Cleavage of the DNA of a representative G418-resistant ES cell clone (BE8) that had not undergone homologous recombination is also shown (Fig. 1B, lane 1).
Homologous recombination was confirmed by cleavage of genomic DNA from
these same ES cell lines with EagI, which cleaves once within exon 2 in the WT PEA3 gene (Fig. 1A). A second
EagI site (present in the PGKneoPA cassette) should be
present in the appropriately targeted mutant PEA3 allele.
Southern analyses revealed the presence of the predicted 10-kb fragment
in EagI-cleaved ES cell DNA from the three clones bearing
the targeted PEA3 allele (Fig. 1B, lanes 4, 6, and 8); this
fragment was not detected in the EagI-cleaved DNA from the
B8 ES cell line (lane 2).
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Male Sexual Dysfunction in Mice Bearing Targeted
Mutant Alleles of the PEA3 ets Gene
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice arose from heterozygous crosses
with the expected Mendelian frequency, revealing that PEA3 is
dispensable for embryogenesis. PEA3 mutant mice displayed
no overt phenotype and lived a normal life span. However,
PEA3-deficient males failed to reproduce. PEA3 is expressed in several
male sexual organs, but gross and histological analyses of the organs
from PEA3/ mice revealed no abnormalities.
Spermatogenesis and spermiogenesis also appeared normal in mice
homozygous for the PEA3 mutation, and their sperm were
capable of fertilizing eggs in vitro. PEA3/
males engaged in normal mating behavior, but they did not set copulatory plugs and sperm could not be detected in the uteri of
females that had mated with PEA3/ males.
Erections could be evoked by abdominal pressure in PEA3-deficient male
mice, and the results of in vitro experiments revealed that the corpus
cavernosum isolated from PEA3 mutant males relaxed in
response to acetylcholine. Therefore, the infertility of
PEA3 mutant males involves either mechanisms proximal to
the cavernosal smooth muscle or an ejaculatory dysfunction. However,
PEA3 mutant mice are phenotypically distinguishable from
other knockout mice with such deficits and thus provide a unique model
for further investigation of male sexual dysfunction.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
20°C. The levels of luteinizing
hormone (LH) and follicle-stimulating hormone (FSH) in plasma were
measured by radioimmunoassay (RIA) with reagents provided by the
National Hormone and Pituitary Program of the National Institute of
Health and Human Development. Anti-rat LH (S-11), anti-rat FSH (S-11),
and reference preparations RP-3 (LH) and RP-2 (FSH) were used in these
assays. The intra-assay coefficient of variation for the quality
controls ranged from 6.65 to 13.99% for LH and 6.2 to 14.3% for FSH.
Testosterone levels were measured by RIA using antibody-coated tubes
manufactured by ICN Biomedicals. The assay had a sensitivity of 0.22 ng/ml and less than 7.8% reactivity with other relevant hormones
including dihydrotestosterone. The intra-assay coefficient of variation for the quality controls ranged from 2.5 to 8.2%.
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Generation of a mouse line bearing a targeted deletion
at the PEA3 locus. (A) Schematic diagram of the
PEA3 gene and the targeting construct (pBSneo9331) used to
generate the mutant allele. The location of the probe (840-bp
ClaI/KpnI fragment) used in Southern blot
analyses is marked on the schematic. A naturally occurring
NcoI site in exon 6 was converted to an EcoRI
site to facilitate molecular cloning of the targeting construct. All
other restriction enzyme cleavage sites shown on the schematic occur
naturally in the PEA3 gene. The length of the expected
EcoRI digestion products (RI) representative of the WT
PEA3 gene (8.2 kb) and targeted PEA3 allele (7.6 kb) that would be detected in Southern analyses using the appropriate
hybridization probe are indicated by horizontal lines. mut, mutant. (B)
Southern analysis of the genomic DNA of four G418-resistant ES cell
clones recovered after transfection of the targeting construct. The BE8
ES cell line did not undergo a homologous recombination event and is
included here for comparative purposes. The odd-numbered lanes contain
the products homologous to the probe in
EcoRI/ClaI double digestions of cellular DNA,
whereas the even-numbered lanes contain products of
EagI-cleaved cellular DNA that were homologous to the probe.
(C) Southern analysis of ClaI/EcoRI-cleaved
cellular DNA from the tails of mice of the F2 generation,
illustrating germ-line transmission of the targeted PEA3
allele. Results for three genotypes (PEA3+/+
[+/+], PEA3+/ [+/], and
PEA3/ [/]) (D) RNase protection
products from brain RNA of PEA3+/+ (+/+),
PEA3+/ (+/), and
PEA3/ (/) mice. The sizes of the
observed RNase-protected products as well as that of the
phosphoglycerate kinase (PGK-1) internal control are shown on the right
of the autoradiogram. Marker DNAs of the indicated size (in base pairs)
were electrophoresed in lane 1. The products of digestion of the
riboprobes following hybridization to tRNA are shown in lane 2. Lanes 3 to 5 contain the products of digesting hybrids between the probes and
total cellular RNA from the brains of mice of the three genotypes. The
asterisk denotes the expected RNase digestion product from the mutant
PEA3 gene. (E) Immunoblot analysis of PEA3 protein in
nuclear lysates of mouse embryo fibroblast (MEF) cell lines derived
from WT (+/+) and PEA3/ (/) mice. PEA3
protein was detected with a mixture of two different monoclonal
antibodies. Lane 1, MEF-4; lane 2, MEF-D; lane 3, MEF-1; lane 4, MEF-H.
The various PEA3 protein species migrate at apparent molecular masses
of 69, 64, and 63 kDa.
and
PEA3/ mice. A protected fragment of this
size was observed following analysis of the brain RNA samples from
PEA3+/ and PEA3/
mice, suggesting that the targeted locus is transcribed and that the
resulting transcript is stable (Fig. 1D, lanes 4 and 5). As expected,
the 323-bp species, representative of the WT PEA3 gene transcript, was not present in the RNA samples from the
PEA3/ mice (lane 5). It is noteworthy that
none of the other protected species, presumably representative of
alternatively spliced mRNAs, were observed in RNA samples from the
PEA3/ mice. The failure to detect these
species is likely due to the absence of the appropriate donor and
acceptor splice sites in the targeted allele.
To determine whether expression of the PEA3 protein occurred in the
organs of mice representative of each genotype, we carried out
immunoblot analyses of extracts prepared from their brains and
epididymides using PEA3-specific monoclonal antibodies. We were
invariably unsuccessful in detecting the PEA3 protein in these samples
even after immunoprecipitation of lysates followed by immunoblot
analysis of the resulting immunocomplexes (M. A. Laing and J. A. Hassell, unpublished data). Our inability to detect the protein
likely resulted from the fact that PEA3 is expressed in only a small
fraction of the cells within most tissues and organs.
In an attempt to circumvent this problem, we established mouse embryo
fibroblasts from each genotype and assayed nuclear extracts prepared
from these cells for the PEA3 protein, because previous analyses of 3T3
mouse fibroblasts revealed the occurrence of relatively abundant PEA3
transcripts in these cells (36). We used two monoclonal antibodies that recognize different antigenic determinants encoded by
exon 8; all of our monoclonal antibodies bind to epitopes that are
encoded by exons that are missing from the targeted PEA3
gene. Two independent PEA3+/+ mouse embryo cell
lines expressed the three species of PEA3 protein commonly detected in
other mouse cell lines (Fig. 1E, lanes 1 and 2). These PEA3 proteins
were not detected in two independent PEA3/
cell lines (lanes 3 and 4). Because we do not have antibodies capable
of detecting amino-terminal epitopes in the PEA3 protein, we have been
unable to determine whether the targeted locus encodes a truncated
amino-terminal fragment of the PEA3 protein. However, as noted below,
the phenotype conferred by mutation of the PEA3 gene is
discernible only in PEA3/ mice and not in
PEA3+/ mice. Hence, it is unlikely that the
targeted locus encodes a dominant-acting, truncated PEA3 protein.
PEA3 mutant mice are viable, but males are
sterile.
To learn whether the loss of PEA3 affected
embryogenesis, we measured the occurrence of the PEA3
mutation among the progeny of heterozygous crosses. These crosses
yielded offspring that segregated PEA3 alleles in the
expected Mendelian ratio (33+/+:57+/:26/), as determined by
Southern analyses, suggesting that loss of PEA3 is not associated with
embryonic lethality. Analyses of the sex of these mice revealed
that PEA3/ mice of both sexes were
viable. Moreover, there were no overt differences in the appearance,
growth rate, behavior, state of health, or life span of
PEA3/ mice compared to their
PEA3+/ or WT littermates (Laing and Hassell, unpublished).
/ male failed to produce litters, even
after pairing more than 30 different PEA3/
males with multiple females over a 12-month period (Laing and Hassell,
unpublished). Heterozygous males fathered pups at the same frequency as
WT males did, and females of all three genotypes reproduced
normally. Moreover, the average litter size of
PEA3/ females was similar to those of their
PEA3+/+ and
PEA3+/ counterparts.
PEA3/ females nursed their pups, and they
developed at the same rate as the offspring of
PEA3+/ and PEA3+/+
females did (Laing and Hassell, unpublished).
PEA3 is expressed in male reproductive organs.
To aid
elucidation of the cellular and molecular bases for the reproductive
failure of PEA3/ males, we determined the
expression profile of PEA3 by examining a larger repertoire of
organs than we had previously analyzed (36). We measured
PEA3 RNA levels by RNase protection analysis from the
organs of male mice at weekly intervals from 3 to 8 weeks of age, a
period during which males achieve sexual maturity. PEA3 RNA was
expressed in the brain, spinal cord, kidney, small intestine, skeletal
muscle, testis, and epididymis of 3- to 8-week-old males (a
representative example from a 4-week-old male mouse is shown in Fig.
2A). The highest levels of expression
occurred in the brain (lanes 2 and 3), spinal cord (lane 4), thymus
(lane 6), small intestine (lane 12), skeletal muscle (lane 13), testis
(lane 14), and epididymis (lane 15). Much lower levels of PEA3 RNA were also detected in the lung (lane 7), kidney (lane 11), and salivary gland (16) after prolonged exposure of the autoradiogram. We were unable to detect PEA3 RNA in the liver, spleen, and pancreas after
this or later exposure times (Laing and Hassell, unpublished). The only
change in the PEA3 expression profile between 3 and 8 weeks of age was
in the heart. PEA3 RNA was expressed in the heart at low but detectable
levels in the 3-week sample, but not in samples prepared thereafter
(Laing and Hassell, unpublished).
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Histological analyses of the testes and epididymides.
The
expression of PEA3 in these male reproductive organs raised the
possibility that male infertility in PEA3/
mice might be associated with a deficit in one or both of these tissues. To learn whether the development and cellularity of the testis
and epididymis were affected by disruption of the PEA3 gene,
we performed histological analyses of these organs from WT and
PEA3/ littermates. The gross structure of
the testes appeared to be the same for mice of both genotypes (Fig.
3A and B). The architecture of the
testicular seminiferous epithelium also appeared to be the same in mice
of both genotypes; Sertoli cells, spermatogonia, primary spermatocytes,
spermatids, and spermatozoa were all present in approximately
equivalent numbers in sections of testes from each genotype (Laing and
Hassell, unpublished). The sections also revealed the presence of
Leydig cells and stromal cells outside of the basal laminae surrounding
the seminiferous tubules. Hence, there appeared to be no discernible
morphological or cellular differences between the testes of
PEA3+/+ and PEA3/
mice.
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/ males.
Sperm from PEA3/ males is capable of
fertilizing eggs in vitro.
In vitro fertilization analyses were
performed to address whether the spermatozoa from
PEA3/ males are capable of fertilizing
oocytes. A series of six experiments were performed comparing sperm
samples from an outbred WT mouse strain (ICR) (as a positive control)
with those of PEA3+/+ and
PEA3/ mice. This analysis revealed that
spermatozoa from the PEA3/ mice fertilized
oocytes as efficiently in vitro as did the sperm samples from the
PEA3+/+ and ICR control mice (Table
1). Statistical analyses showed that the
mean fertilization rates were not significantly different between the
sperm samples of the two genotypes. Hence, under conditions of these in
vitro fertilization experiments, sperm from
PEA3/ males did not appear to harbor any
intrinsic defects.
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In vivo fertilization analyses.
To further investigate the
fertility deficiency in male PEA3 mutant mice, we determined
whether these males successfully copulated with females. To this end,
PEA3+/+, PEA3+/, and
PEA3/ males were independently paired
overnight with superovulated female BALB/c mice. The following morning,
females were examined for the presence of a copulatory plug. Copulatory
plugs were found in approximately 70% of the females paired with
either PEA3+/+ or
PEA3+/ males (Table
2). However, not a single copulatory plug
was detected in 113 pairings of females with 36 PEA3/ males. The uteri of females were
routinely flushed after mating and examined for the presence of
spermatozoa. Spermatozoa were detected in the uteri of females
mated with either PEA3+/+ or
PEA3+/ males, but they were not found in the
uteri of females mated with PEA3/ males
(Laing and Hassell, unpublished). The pronucleation rate, assessed by
cleavage of isolated oocytes cultured in vitro, was ~80% in one-cell
embryos recovered from matings of females with PEA3+/+ and PEA3+/
males, whereas it was less than 2% in embryos isolated from females following mating with PEA3/ males (Table 2).
Two eggs from females that had mated with
PEA3/ males did undergo one cleavage.
However, the first cleavage was unequal and both eggs failed to develop
further in culture. This suggests that these cleavages were not
likely the result of a true fertilization event but were parthenogenic
in nature (4).
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Circulating levels of male sex hormones and male sexual behavior
are normal in PEA3 mutant mice.
The levels of various
circulating hormones affect male sexual function (37). To
learn whether the concentrations of sex hormones in the blood of the
PEA3/ male mice were affected by loss of
functional PEA3, we measured the levels of FSH, LH, and testosterone by
RIA. The concentrations of all of these hormones in the
PEA3/ male mice were within normal limits
and did not differ significantly from those of the
PEA3+/+ and ICR control mice (Table
3). This observation suggests that male
infertility is not a consequence of a neuroendocrine disorder.
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/ males and compared it to the behavior
of age-matched PEA3+/+ littermates that
had been paired with superovulated females. PEA3/ males, like their WT counterparts,
displayed normal grooming behavior and mounted females, suggesting that
their sexual behavior was not affected by mutation of the
PEA3 gene (Laing and Hassell, unpublished).
Erections could be induced in male PEA3 mutant mice. The transfer of spermatozoa into the female reproductive tract requires penile erection and subsequent ejaculation. Penile erection is a complex neurovascular event. Within the penis it involves relaxation of the corporal smooth muscle and subsequent engorgement with blood of the paired corpora cavernosa due to compression of the emissary veins against the connective tissue sheath surrounding the corpora. This process is regulated by the central and peripheral nervous systems.
Erections can be induced artificially in mice by a combination of retraction of the penile sheath and gentle abdominal pressure (30). The mechanism of the erection is uncertain but may be reflexic (30) or due to obstruction of the venous outflow from the penis. To determine whether the penile tissue of PEA3 mutant mice was capable of supporting an erection, the abdominal pressure test was applied to male mice of each genotype. Almost all of the male mice tested (n = 34) by this method achieved strong erections; only four males achieved weak erections, which were characterized by poor engorgement and little increase in the length of the penis (Table 4). Importantly, 11 of 13 homozygous PEA3 mutant male mice achieved strong erections by this method. These findings suggest that the PEA3 mutant male mice do not harbor any physical impediments to achieving penile erections. This conclusion was sustained by histological analyses of penis sections from mice of the various genotypes. These analyses failed to reveal any differences in the tissue architecture among males of all three genotypes (Laing and Hassell, unpublished). Taken together, these findings suggest that the penile structures required for erection were normal in PEA3 mutant males.
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Erectile tissue isolated from PEA3 mutant males was functional in vitro. Parasympathetic nerves, via cholinergic and noncholinergic neurotransmission, are responsible for the corporal smooth muscle relaxation underlying erection, but the major part of their effect is due to the liberation of nitric oxide (NO) and subsequent activation of biochemical events in the muscle (2, 8, 19, 21). Deficits in erection could occur at the level of the corpus cavernosum smooth muscle (17) or in the neural organization of relaxation. We directed our attention initially at the level of the corpus, because other attempts to produce genetic models of impotence targeted this level (9, 17).
In order to determine whether the erectile tissue of the PEA3 mutant mouse has the biochemical machinery required to support relaxation, we measured the capacity of this tissue to relax in response to acetylcholine in vitro (2). Corpora cavernosa isolated from male mice of each PEA3 genotype did not display spontaneous contractile activity in vitro. The
-adrenoreceptor agonist phenylephrine contracted isolated corpora
cavernosa (average increase in tension, 39.6 ± 5.3 mg)
(n = 20). There was no difference between the magnitude of contraction elicited in tissue from PEA3/
males (41.4 ± 9.0 mg) (n = 11) and that in tissue
from WT and PEA3+/ males combined (37.4 ± 4.6) (n = 9). Acetylcholine added in the presence of
phenylephrine-induced contracted corpora cavernosa, produced a
concentration-dependent relaxation of this tissue (Fig. 4A). Dose-response curves constructed for
acetylcholine-induced relaxation were similar for corporal tissue
isolated from mice of all three genotypes (Fig. 4B). Hence, the
erectile tissue from the PEA3 mutant mice demonstrated a
normal relaxation response to acetylcholine in vitro.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PEA3 appears to be required for normal male sexual function. All
PEA3 mutant males that we have characterized are unable to impregnate females, suggesting that the penetrance of this phenotype is
absolute. Moreover, introgression of the targeted PEA3
allele to other mouse genetic backgrounds (129/sv and FVB) did not
alter the nature or penetrance of the described phenotype of male
PEA3/ mice (Laing and Hassell, unpublished).
Gross and histological analyses of male reproductive organs including
the epididymis, which expresses relatively high levels of PEA3 RNA in
its initial segment, revealed no discernible differences between WT and
PEA3/ males. Hence, the inability of
PEA3/ males to impregnate females does not
appear to result from morphological aberrations or cellular deficits in
these organs.
The sperm of PEA3/ males proved capable of
fertilizing eggs in vitro, suggesting that spermatogenesis,
spermiogenesis, and sperm maturation occur normally in PEA3
mutant males. However, we have not assessed
PEA3/ sperm motility or the rate with which
these sperm bind to and penetrate eggs. Moreover, it is noteworthy that
the sperm/egg ratios used in these in vitro fertilization studies
greatly exceed the ratios common in vivo. Hence, it is formally
possible that some aspect of sperm function is compromised by loss of
functional PEA3.
PEA3 mutant males displayed normal grooming and mating
behavior. These males, like their age-matched WT counterparts,
repeatedly mounted superovulated females shortly after being
paired with them. However, PEA3/ males did
not produce vaginal plugs in females. This finding is consistent
with our inability to detect sperm in the reproductive tracts of
females shortly after mating with PEA3/
males; by contrast, sperm was readily detected in the reproductive tracts of females that mated with PEA3+/+
or PEA3+/ males. These findings are
compatible with an erectile and/or ejaculatory dysfunction in male
PEA3 mutant mice.
The circulating levels of various sex hormones can affect erectile
function (37). However, the concentrations of LH, FSH, and
testosterone were the same in WT and PEA3 mutant males.
Hence, if PEA3/ males have an erectile
dysfunction, it is unlikely to be of neuroendocrine origin. Commonly,
sterility resulting from neuroendocrine malfunction is manifested in
the form of defects in the maturation of secondary sexual
characteristics, such as hypogonadism, a phenotype that was not
apparent in PEA3/ male mice (37).
Indeed, all of the male sexual organs, including the genitalia, of
PEA3-deficient males were identical to those of their WT littermates.
There was no obvious relationship between loss of PEA3 function and lack of fecundity in male mice. Therefore, we initially chose to test two very basic requirements for erectile function, namely, that the penile tissue had the required structure to engorge and become erect when venous outflow was blocked and that a fundamental biochemical step in the mechanism underlying erection was present in corporal tissue.
Abdominal compression produced erection in the majority of male mice of
all three genotypes. Although the mechanistic basis of abdominal
pressure-induced erections is unclear, it probably impairs venous
outflow from the penis. It is an artificial procedure and bypasses the
muscular pumping that underlies erections in rodents (15,
30). Whatever its shortcomings, this test demonstrated that the
penile structures required for erection were present in
PEA3/ males. This was confirmed by
histological analyses of the penises of
PEA3/ males, which also did not reveal any
obvious structural deficits of this organ.
A critical biochemical step for producing the corporal relaxation
underlying erection is the release of NO and subsequent intracellular
generation of cyclic GMP (cGMP) (2, 8, 19). Cholinergic
agonists cause relaxation through release of NO in vascular tissue
including corpus cavernosum (17, 21). In our experiments,
corporal relaxation induced by acetylcholine in tissue from
PEA3/ mice was indistinguishable from that
observed in tissue from PEA3+/+ or
PEA3+/ mice. This result implies that the
molecular targets and biochemical steps through which acetylcholine
causes relaxation in corporal tissue are active and intact in
PEA3/ mice.
This conclusion is supported by the phenotype of mice bearing targeted
deletions in the downstream effectors of acetylcholine action in the
corpus cavernosum. Targeted disruption of the gene for cGMP-dependent
kinase I (cGK-I), the likely molecular target for the cGMP
generated by NO action, prevents cholinergically mediated corporal
relaxation (17). Unlike PEA3/
male mice, cGK-I/ male mice have a
substantially reduced but finite reproductive capacity. Similarly,
specific disruption of the neuronal isoform of the NO-synthesizing
enzyme, nitric oxide synthase (nNOS), does not eliminate male potency
(9, 20, 28). Whereas alternative splicing of nNOS
transcripts (8) as well as other mediators and genetic
sources of NOS activity may compensate for nNOS gene disruption in
these mutant mice (14), the sexual phenotype of PEA3/ males is clearly distinguishable from
that of nNOS mutant males.
An alternative interpretation of the observation that PEA3 mutant males are unable to set copulatory plugs in timed matings is that they harbor an ejaculatory dysfunction. However, the phenotype of PEA3 mutant male mice is also distinguishable from that of other knockout mice, which display clear ejaculatory defects. The neural messenger, carbon monoxide (CO), like NO, is implicated in neurotransmission (7). CO is synthesized by heme oxygenase 2 (HO2), whose expression is localized to neurons that mediate ejaculation. HO2 knockout male mice are fertile, despite the fact that they mount females less frequently and display reduced intromission activity compared to their WT counterparts (7). Moreover, the reflex activity of the bulbospongiosus muscle, a muscle that plays a significant role in promoting fertility in the male mouse (15), is substantially reduced in HO2 mutant male mice (7).
Similarly, male mice with a targeted disruption of the P2X1 ATP receptor also display ejaculatory abnormalities that result in a 90% reduction in fertility (27). ATP is released with noradrenaline from sympathetic neurons and acts through P2X1 receptors on smooth muscle to effect contraction. Disruption of the P2X1 gene reduces the contractile response within the smooth muscle of the vas deferens, leading to a reduced sperm count in the ejaculate and resulting in reduced male fertility (27). Importantly, P2X1 null male mice copulate normally and consistently set plugs in timed matings, characteristics that distinguish them from PEA3 mutant male mice (27). Hence, whatever the underlying cellular and molecular bases for the sexual dysfunction of PEA3 mutant males, these mice are clearly phenotypically distinguishable from other mouse mutants with erectile and ejaculatory deficits described so far.
We suspect that the infertility of PEA3/
males has an underlying neuronal basis. PEA3 is expressed in specific
bundles of motor neurons that innervate limb muscles and in afferent
sensory neurons of these same muscles (24). Hence, it is
conceivable that PEA3 is also expressed in neurons that innervate the
penis. To address this, we are now deriving new PEA3 mutant
mice by homologous recombination in ES cells that carry bacterial
beta-galactosidase in place of PEA3 coding sequences.
Analysis of the pattern of beta-galactosidase activity in such male
mice may help to uncover the cellular basis and ultimately the
molecular basis of male infertility in PEA3/
mice. Nerve stimulation experiments to determine whether there is a
peripheral neurotransmission defect in these mice are also under way.
The possibility that there is an ejaculatory defect is also being
considered. Whatever the deficit, these mutant mice afford a unique
model for studies of male sexual dysfunction and its treatment.
Moreover, contingent on the nature of the lesion in
PEA3/ mice, our findings suggest the
potential that PEA3 antagonists may act as effective male contraceptives.
| |
ACKNOWLEDGMENTS |
|---|
We thank Jaquelyn Labus for technical assistance in performing
the in vitro fertilization analysis, Lisa Tabek and William Hardy for
performing the blastocyst injections, Laura Hastings for deriving mouse
embryonic fibroblast cell lines from WT and PEA3/ mice, and Leslie Ingraham and Phil
Barnsley for help with the corpus cavernosum experiments.
This study was supported in part by grants to J.A.H. from the Medical Research Council of Canada and the Canadian Breast Cancer Research Initiative and to B.T.H. from the National Institute of Child Health and Human Development (HD-32979).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institute for Molecular Biology and Biotechnology, McMaster University, 1280 Main St. W., Hamilton, Ontario, Canada L8S 4K1. Phone: (905) 525-9140, ext. 27217. Fax: (905) 521-2955. E-mail: hassell{at}mcmaster.ca.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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