Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 721-728
doi:10.1111/j.1745-7270.2008.00451.x
The spatiotemporal expression changes of 16
epididymis-specific genes induced by testosterone, heat, and combination
treatment in cynomolgus monkey
Xiangqi Li1,2#, Qiang Liu2#, Shigui
Liu1, Xuesen Zhang4, Yixun Liu4, and Yonglian Zhang2,3*
1
College of Life Science,
Sichuan University, Chengdu 610064, China
2
Shanghai Key Laboratory
for Molecular Andrology, State Key Laboratory of Molecular Biology, Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
3
Shanghai Institute of
Planned Parenthood Research, Shanghai 200032, China
4
State Key Laboratory of
Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences,
Beijing 100080, China
Received: April 10, 2008
Accepted: April 25,
2008
This study was
supported by the grants from the National Natural Science Foundation of China
(30230190 and 30570684), the National Basic Science Research and Development
Project (2006CB504002 and 2006CB944002 ) and the Chinese Academy of Sciences
Knowledge Innovation Program (No. KSCX1-YW-R-54)
#
These
authors contributed equally to this work
*Corresponding
author: Tel/Fax, 86-21-54921163; E-mail, [email protected]
The experimental infertility model of
treatments involving testicular warming, testosterone implant, and a
combination of the two was developed to confirm a synergistic action induced by
the combination treatment on germ cell apoptosis in cynomolgus monkey testis.
Using this model, the spatiotemporal expression changes of 16 reported or novel
genes in epididymis were investigated to examine the treatments effect on
epididymal genes. It was demonstrated that these region-specific genes, some of
which were not regionally fixed, changed greatly with these treatments. The
expression levels of these epididymal genes fluctuated, and the expression of
most of the genes returned to nearly normal level at the end of treatments.
Moreover, the expression changes resulting from the combination treatment were
not more significant than those resulting from the single treatment. This
suggests that the combination treatment has an antagonistic action on the
expression of epididymal genes and that its effect is not as adverse on
epididymis as that of the two single treatments.
Keywords epididymis; gene expression;
androgen; temperature; infertility model
Currently, male contraception addresses an important social need, however,
there is no medicine for clinical practice except condoms (reversible but not
reliable) and vasectomies (reliable but not reversible) [1]. One important
reason for the lack of male contraception is that the basic research on male
fertility is far from complete, restricting the development of male
contraception methods. Earlier studies demonstrated that a single exposure (43
?C for 15 min) of the rat testis to heat resulted in selective and reversible
damage to the seminiferous epithelium through germ cell apoptosis. This
predominantly occurred at early (I–IV) and late (XII–XIV) stages [2]. A single
administration of testosterone implant induced loss of germ cells through
apoptosis at mid (VII–VIII) stages in rats [3,4]. The combination of heat exposure and
testosterone implant in rats had a synergistic action, markedly reducing the
number of pachytene spermatocytes and round spermatids through germ cell
apoptosis in all stages (early, mid, and late) [5]. These findings in rats have
made it possible to design an infertility model to determine whether the same
events would happen in non-human primates. Indeed, transient testicular warming
and testosterone implant had similar suppressive effects on spermatogenesis in
the testis of adult cynomolgus monkeys (Macaca fascicularis), and they
also had a synergistic action on germ cell apoptosis in testis [6–8]. However, did these treatments also affect epididymis? The
epididymis, consisting of grossly caput, corpus, and cauda (which was viewed as
a useless organ in the past), has been shown to provide a specific intraluminal
environment for spermatozoa concentration, maturation, transport, and storage
[9,10]. Each region expresses specific proteins, and this regionalized gene
expression is critical for the epididymis to acquire normal functions [11]. The
epididymis is so important to sperm maturation that it may be an ideal target
organ of male contraception [12]. Accordingly, many epididymal-specific
proteins, such as GPX5 [13], CD52 [14], EAPI [15], ESP14.6 [16], ESP13.2 [17],
CRISP1-L [18], and LCN6 [19], have been identified and characterized recently
in monkey. Also, we previously reported that 11 epididymal-specific genes were
isolated and sequenced from a subtracted M. mulatta epididymis-specific
complementary DNA (cDNA) library [20]. These epididymal-specific genes might
serve as potential targets for male contraception if their close relationships
with male fertility were demonstrated completely. However, the physiological
importance of most of these genes still remains unknown, and there is no male
contraceptive medicine based on epididymis for clinical practice at present.To our knowledge, there have been no investigations on the effects
of these infertility treatments on epididymis in non-human primates, although
several similar studies had been made at the physiological level in rats and
mice [21–24]. Therefore, using the same monkey model that our coworkers
previously used to examine infertility [7], we set out to determine, on the
molecular level, whether testicular warming, testosterone administration or the
combination of the two has a special effect on the expressions of 16
epididymal-specific genes. Moreover, this study hopes to provide a basis for
further understanding of these genes as potential targets for male
contraception.
Materials and Methods
Materials
Healthy, fertile adult (7 to 10 years old) male cynomolgus monkeys (M.
fascicularis) were obtained and housed at the Guangxi Hongfeng Primate Research
Center, Institute of Zoology, Chinese Academy of Sciences (Kunming, China).
Animal handling and experimentation were in accordance with the recommendations
of the American Veterinary Medical Association and were approved by the Animal
Care and Use Review Committee of Institute of Zoology, Chinese Academy of
Sciences. The monkeys were housed in a standard animal facility under
controlled temperature (22 ?C) and photoperiod (12 h of light and 12 h of
darkness) with free access to water and monkey chow.
Animal groups
Testicular warming, testosterone administration, semen collection
and hormone assays were conducted as reported previously [7,8]. Eight of 24
adult cynomolgus monkeys (M. fascicularis) were randomly assigned to
each of the following treatments for 84 d: (1) H group was given daily
testicular exposure to heat (43 ?C for 30 min) on 2 consecutive days (1 and 2
d); (2) T group was subjected to two testosterone implants on 1 d; and (3) H+T
group was the combination of H and T groupa. Epididymal tissue was collected
from one epididymidis in five monkeys from each group before treatment and at
3, 8, 28, and 84 d during treatment phase. The materials were snap-frozen in
liquid nitrogen for RNA isolation and Northern blot analysis. The remaining three
monkeys from each group were used for semen collection.
Hybridization probe preparation
Two primers for each one of the 16 epididymis-specific genes (SC-6,
SC-9, SC-13, SC-42, SC-112, SC-342, SC-384,
SC-461, SC-513, SC-615, LCN6, ESP14.6, CD52,
GPX5, ESP13.2, CRISP1-L), and the 18S ribosomal RNA were
designed and used to amplify cDNA fragments with the forward primer (F) and the
reverse primer (R), respectively. The detailed descriptions of these primers
and cDNA fragments are shown in Table 1. These cDNA fragments were
verified by automated sequencing, and then used as probes for Northern blot
analysis. Two primers for each one of the 16 epididymis-specific genes (SC-6,
SC-9, SC-13, SC-42, SC-112, SC-342, SC-384,
SC-461, SC-513, SC-615, LCN6, ESP14.6, CD52,
GPX5, ESP13.2, CRISP1-L), and the 18S ribosomal RNA were
designed and used to amplify cDNA fragments with the forward primer (F) and the
reverse primer (R), respectively. The detailed descriptions of these primers
and cDNA fragments are shown in Table 1. These cDNA fragments were
verified by automated sequencing, and then used as probes for Northern blot
analysis.
RNA isolation and Northern blot analysis
Total RNA was extracted with Trizol (Invitrogen, Carlsbad, USA)
following the manufacturers recommendations. Northern blot analysis was
carried out according to the procedure described previously [25]. Total RNA (12
mg)
from each sample was loaded in each lane. The probe was a 32P-labeled cDNA fragment of each gene. An 18S ribosomal RNA
hybridization signal was used as a loading control. Autoradiographs with
pronounced differences in expression were analyzed by densitometry. The
relative intensity of hybridization was analyzed using Gelwork 3.01 software.
Results
Changes in the regional expression pattern of the 16 selected genes
Regionalization is a feature of epididymal genes expression. All the
examined genes in untreated monkeys exhibited a regionalized pattern of
expression in the epididymis, with different levels in the caput, corpus, and
cauda regions (Fig. 1). With H, T, and H+T treatments, a newly found
regionalized expression for each gene was observed and reported. However, the
expression features of regionalization in tested groups changed significantly
during the whole test period, and some were not even regionally fixed. For
example, the maximum level of SC-615 varied between the cauda region in
the control and the corpus region after the heat stress at 84 d. The same thing
happened to SC-461 in H group, from no expression in the caput region in
the control to marked expression at 84 d.
Changes in the expression levels of the 16 selected genes in their
main expression regions
The expression of the 16 epididymal genes in the three regions of
treated epididymis are very complex. However, analysis of the specific changes
in the main expression region, which has the highest level of expression of all
three epididymal regions, revealed some interesting results (Fig. 1). In
H group and T group, the expression of all tested genes decreased to their
lowest levels at 3 d, except ESP13.2 in T group which reached its lowest
level on 8 d. In H+T group, all the caput-expressed genes, corpus-expressed SC-342,
and cauda-expressed SC-615 fell to their lowest levels at 3 d, but seven
other genes dropped to their lowest levels at 8 d (Table 2).
Intriguingly, the expressions in H+T group were milder than those in the
individual treatment groups. For instance, the expression of SC-9
changed obviously in H group and T group, while in H+T group the variation was
not as remarkable, especially between 30 and 84 d. Similar results were
observed with the expression of SC-342. At 3 d, the expression levels of
ESP14.6 and SC-112 in H+T group increased, while all other genes
in all tested groups decreased significantly (Fig. 2). From 8 to 84 d,
in H group, the expression levels of seven genes (GPX5, CD52, SC-13,
SC-42, SC-112, SC-384, SC-615) were above the
control levels, while three genes (CRISP1-L, SC-342, SC-461)
were lower than the control. In H+T group, the expressions of five genes (LCN6,
GPX5, SC-13, SC-513, SC-615) were above the
control, whereas that was not the case for four genes (CRISP1-L, SC-6,
SC-342, SC-461) that were lower than the control (Fig. 2).
Discussion
Previously, the experimental infertility model of treatments with
testicular warming, testosterone implant, and a combination of two had been
developed to confirm a synergistic action induced by the combination treatment
on germ cell apoptosis in cynomolgus monkey testis. Using this model, the
expression changes of 16 reported or novel genes in epididymis were
investigated to examine the effects of these treatments on epididymal genes.
The epididymis ensures sperm concentration, maturation, transport, and storage,
which are regulated by testis via circling testosterone and luminal testicular
factors as well as by ambient temperature [26–31]. It has been confirmed
that the effect of abdominal temperature on the physiology of the cauda region
in the epididymis reduces luminal capacity and sperm numbers, enhances sperm
transport [22], suppresses transepithelial ion and water transport [24], causes
spontaneous lipid peroxidation [32], induces apoptotic cell death in its
proximal segment [33], destroys sperm motility, and induces spermatozoa death
[34]. On the molecular level, temperature has been demonstrated to regulate
CD52 in epididymal cell culture [35]. Here, temperature warming significantly
changed the expression of all genes in all regions, besides the cauda region.
Androgen did not seem to be responsible for the change as no differences in
serum testosterone levels in the H group were noted when compared with the
control group [7]. Additionally, the expression levels of epididymal genes
decreased quickly by hot shock, and they also recovered quickly, which may be
the result of stress reaction. Androgen withdrawal has been physiologically demonstrated to reduce
sperm numbers and sperm motility in cauda epididymidis in some non-primate
animals [22,36]; it has also been shown to induce apoptosis of principal cells
throughout the rat epididymis [37]. Androgen has been shown to regulate CD52
[38], ESP14.6 [39], CRISP1-L [18], and some unknown rat and tammar wallaby
proteins [27,40]. Additionally, androgen together with testicular factor(s) can
regulate SC-13 [41], GPX5 [31], and SC-42 [20]. Here, androgen treatment alone
induced the decrease of gene expressions for most epididymal genes at 3 or 8 d.
After that, gene expressions began to recover quickly, while the androgen level
was maintained in the upper normal range throughout the 12 weeks of treatment
with testosterone implant [7]. It has been shown that exogenous testosterone
implant might have double hits on epididymis, and exert special effects on
epididymis by low intratesticular testosterone and low follicle-stimulating
hormone (FSH) and luteinizing hormone in serum [42–46]. Since the testosterone
implant provided a constant supraphysiological testosterone concentration in
serum, the testosterone implant-induced expression responses of the 16
epididymis-specific genes might be underscored by the complicated regulation
mechanism: the combination of supraphysiological serum testosterone, low
intratesticular testosterone, low serum luteinizing hormone, FSH and/or other
factors.Our model also demonstrated androgen and temperature could
individually regulate all 16 genes, but they could also do so in combination.
Moreover, the combination reduced the expression of epididymal genes quickly,
though expression recovered immediately thereafter. Temperature and androgen
have been shown to have a synergistic effect on sperm motility and sperm counts
in cauda epididymidis in rat [22], and our coworker reported that this
combination treatment also had an additive action on sperm quantity of treated
monkey [7]. However, we found that the expression changes resulting from the
combination treatment in epididymis were not more significant than the changes
resulting from the individual treatments, which suggests that the combination
treatment has a less adverse effect on the epididymis than the individual
treatments. In the combination treatment model, serum testosterone
concentration was the same as that in T group. H group had increased serum FSH
levels, while T group had decreased FSH levels. H+T group had higher serum FSH
levels than T group, but lower than H group. However, the serum FSH levels in
H+T group were still much lower than those in the control [5], which partially
explains the change of epididymal gene expression affected by H+T combination.Intriguingly, several genes, such as CRISP1-L, SC-342,
and SC-461, always had expression levels lower than H group and H+T
group controls, which may indicate that they are potential targets for male
contraception. Also, CRISP1-L has already been confirmed to be directly
involved in sperm maturation in the epididymis by sperm-egg fusion and
capacitation.On the molecular level, the expression changes of epididymal
specific genes under H, T, and H+T treatments demonstrated that these
epididymal genes all had regionalized expression patterns and were able to
recover quickly, with some fluctuation, after the treatment. The expression
changes of the H+T group were not more significant than those of the single
treatment groups, suggesting that the combination treatment has a less adverse
effect on epididymis than the individual treatments.
Acknowledgements
The authors are grateful to the researchers at the State Key Laboratory
of Reproductive Biology, the Institute of Zoology, the Chinese Academy of
Sciences for their assistance in preparing this report. Also, the authors would
like to thank the Guangxi Hongfeng Primate Research Center and the Institute of
Biological Products of Beijing for their assistance with animal healthcare.
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