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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 24, 2001 as doi:10.1096/fj.00-0814fje. |
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Departments of
* Biological Chemistry and
Genetics, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel;
Department of Gynecology and
Institute for the Study of Fertility, Surasky Medical Center and Sackler School of Medicine, Tel Aviv, Israel; and
¶ Institute of Animal Science, ARO, Volcani Center, Bet Dagan, Israel
2Correspondence: Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel. E-mail: soreq{at}cc.huji.ac.il
SPECIFIC AIMS
The molecular pathways translating psychological stress into depressed male reproductive potency are not yet known, but likely involve stress hormone-induced alterations in gene expression. We previously reported activation of the gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE) by forced swim stress and pharmacological inhibitors of AChE in brain, muscle, hematopoietic cells, and intestinal epithelium. Therefore, we explored its influence on spermatogenesis and sperm properties.
PRINCIPAL FINDINGS
1. AChE-R is overexpressed in testes from mice exposed to forced
swimming
To examine the effects of acute stress on AChE expression in male
gonads, we subjected adult male FVB/N mice to four successive daily
sessions of confined swim. Stressed mice displayed elevated serum
corticosterone levels that were accompanied by mildly increased AChE
activity in testicular extracts (0.3±0.03 nmol substrate/min/mg
protein after stress vs. 0.1±0.01 in controls). A selective cRNA probe
revealed a circumferential distribution of AChE-R mRNA in testicular
tubules from naive mice. In stressed mice 24 h after the last swim
session, AChE-R mRNA signals were notably intensified and extended into
all cell layers. Immunolabeling of the stress-induced AChE-R variant
produced no detectable staining in testes from control mice. In
contrast, anti-AChE-R antibodies intensively labeled internal cell
layers containing maturing spermatozoa in the tubules of stressed mice.
Stress therefore induced AChE-R mRNA overproduction during early
spermatogenesis and caused accumulation of AChE-R protein at later
stages of sperm formation.
2. Transgenic mice, a model for chronic testicular overexpression
of AChE-R, display complex patterns of AChE-R overexpression in
spermatogenic cells
To establish a model for chronic gonadal overexpression of AChE-R,
we exploited transgenic mice overexpressing human AChE-R with up to
700-fold excess testicular AChE activity. Using In situ hybridization,
we detected high levels of AChE-R mRNA in the peripheral layers of
testicular tubules from transgenic compared with control FVB/N mice
(Fig. 1A
, B
). Elevation of AChE-R mRNA levels in
the circumference of tubules was similar to that observed in
nontransgenic mice after stress. The peripheral layers harbor both
mitotic spermatogonia and postmitotic spermatocytes. Using anti-AChE-R
antibodies, we could not detect the AChE-R protein in sections from
control nonstressed mice (Fig. 1C
). In contrast, most of
stained tubules from transgenic mice displayed pronounced deposition of
AChE-R in a single peripheral cell layer (Fig. 1D
). The
remaining tubules displayed AChE-R deposits (similar to the stress
pattern) or labeling in the tubular cavity into which the spermatozoa
tails project (data not shown). Some tubules stained in the periphery
were also stained in the inner layer or the cavity. Thus, AChE-R
expression in testes of transgenic mice resembled that of mice
subjected to repeated acute stress at the level of the mRNA, but
exhibited a more complex pattern of cellular distribution at the level
of the protein. In contrast, the expression of the ‘synaptic’ AChE-S
splicing variant appeared largely unaffected by either stress or
transgenic overexpression of AChE-R.
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3. Accumulated AChE-R in postmitotic sperm progenitors imposes a
partial block to postmeiotic differentiation
We considered the possibility that overproduction of AChE-R may
affect the proliferation of male germ cell progenitors. To quantify
replicating cells, we stained for proliferating cell nuclear antigen
(PCNA). In both naive FVB/N and AChE-R transgenic mice, PCNA staining
was confined to the most peripheral cell layer comprised exclusively of
spermatogonia, the proliferative spermatogenic progenitors (Fig. 1E
and data not shown). AChE-R was undetectable in this cell
layer (Fig. 1F
), being concentrated in the next inner layer
of postmitotic spermatocytes of transgenic mice. The average number of
PCNA-labeled cells was identical in transgenic and control mice. In
contrast, the number of postmeiotic, differentiating spermatozoa
surrounding the tubular cavity was significantly reduced in transgenic
compared with control mice (82±12 vs. 97±15 cells/normalized tubule
perimeter, respectively; n=3, 8–10 tubules per mouse,
P < 0.0005, Student’s t test).
4. Transgenic testicular AChE-R overproduction is associated with
sperm impairments
As predicted by the reduced number of spermatozoa in testicular
tubules, epididymal sperm counts were also significantly lower in
transgenic than control mice. Motility of surviving sperm appeared
compromised in transgenic mice compared with controls, although not
statistically significant. Transgenic mice also displayed reduced
seminal gland weight compared with controls.
5. AChE-R may serve as a marker of stress-related male infertility
Testicular tubules from stressed mice and a large portion of those
from transgenic mice displayed accumulation of AChE-R in differentiated
spermatozoa residing at the innermost cell layer. Anti-AChE-R
antibodies failed to label testicular spermatozoa from control mice
(Fig. 2A
). In contrast, repeated acute stress facilitated strong,
punctate intracellular labeling that was limited to spermatozoa heads
(Fig. 2B
), with a few cells also stained at the neck.
Spermatozoa from AChE-R transgenic mice were either unlabeled or
stained at the neck or head (Fig. 2C
). We then examined
human sperm in air-dried smears of ejaculates from fertile donors or
from the male partner of couples with unexplained infertility who did
not display any abnormal sperm parameter to which the infertility could
be attributed. Sperm from fertile donors were stained in both the head
and neck regions. In contrast, samples from male partners of infertile
couples presented a significantly decreased proportion of head labeling
(Fig. 2D
).
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CONCLUSIONS AND SIGNIFICANCE
Our current findings associate stress with testicular
overproduction of AChE-R and suggest that stress insults of varying
duration or severity may initiate graded increases in AChE-R in
spermatogenic cells (Fig. 3
). The increase in AChE expression after stress is consistent with the
presence of a recently discovered consensus sequence for a putative
glucocorticoid response element in the upstream promoter region of the
human ACHE gene locus. AChE overexpression after stress was
highly selective as it was observed only for the AChE-R isoform, which
suggests active diversion of 3' alternative splicing of AChE mRNA. This
pattern of stress-mediated ACHE gene expression parallels that observed
in other tissues and strengthens the concept of shifted alternative
splicing and the resultant AChE-R protein as universal stress response
elements in multiple mammalian organs, including the gonads.
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Compared with psychologically stressed FVB/N mice, transgenic mice
exhibiting massive AChE-R overproduction displayed heterogeneity in the
cellular and subcellular localization of AChE-R (Fig. 3)
. It is unclear
whether this difference between stressed and transgenic mice reflects
the high levels of overexpression achieved in the transgenic model or
results from chronic congenital overexpression of the transgenic
protein. The greatly elevated expression of AChE-R in transgenic mice
was accompanied by decreased sperm counts, sperm motility, and seminal
gland weight. The decline in postmitotic spermatozoa numbers in AChE-R
transgenic mice suggests that AChE-R excess may impose yet undefined
restrictions on spermatogenesis after mitotic cell division (Fig. 3)
.
The high accumulation of AChE-R in sperm cell progenitors suggests that
the primary effect of its transgenic overexpression on spermatogenesis
and/or sperm properties results from direct effects of the protein on
cellular processes. In this light, noncatalytic cell-signaling
capacities now well established for nervous system AChE may be
relevant. Our findings therefore emphasize the need to identify the so
far unknown protein partner(s) of AChE-R and its putative signal
transduction pathways.
The transgenic mouse model demonstrates the potentially detrimental effects of high levels of dispersed testicular AChE-R on mammalian sperm maturation and/or properties. The absence of AChE-R from heads of testicular spermatozoa from transgenic mice was characteristic of sperm from the male partners of couples with unexplained infertility, but not of sperm from fertile controls. The differential AChE-R staining patterns observed in the two human groups suggest AChE-R labeling as a possible useful marker for stress-related male infertility and strengthen the notion that stress-associated overexpression of AChE-R may be a risk factor in fertility disturbances.
In conclusion, our data suggest a previously unperceived mechanism for the negative effects of psychological stress on male fertility. This points to AChE-R as a previously unrecognized target for studying, analyzing, and treating stress and anticholinesterase-induced human male infertility.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0814fje ; to cite this
article, use FASEB J. (July 24, 2001)
10.1096/fj.00-0814fje 
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40 cells from each of 3 healthy
human donors (C) and 3 male partners from infertile couples (INF).
*P<0.05, Mann Whitney. The outside columns show
representative compound confocal images of a sperm from a donor (right)
or infertility patient (left). Note labeling of the postacrosomal
region of sperm heads and neck in control cells (black arrowheads) vs.
limited sperm head labeling in the patient (white
arrowhead).