NOVEL INSIGHTS ON THE ROLE OF SELENOPROTEIN P IN SPERM VIABILITY A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CELL AND MOLECULAR BIOLOGY AUGUST 2012 By Elizabeth Quynh-Mai Dao Nguyen-Wu Thesis Committee: Frederick P. Bellinger, Chairperson Marla J. Berry Martin Rayner Keywords: Selenoprotein, genetic rescue, fertility, sperm, GPx4, Cre-LoxP
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NOVEL INSIGHTS ON THE ROLE OF SELENOPROTEIN P IN SPERM VIABILITY
A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE
testes and epididymides have similar GPx4 protein expression to Sepp1+/+ confirming
genetic restoration of Sepp1. These results show that Sepp1 is necessary for normal
GPx4 protein expression and that Sepp1r/r CMV+ mice have normal GPx4 protein levels
thus complete restoration of a functional Sepp1 gene.
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Figure 9. GPx4 immunoreactivity is significantly decreased in epididymal sperm of
Sepp1-/- mice. Representative images show DAPI (blue) nuclear staining and that GPx4
(red) staining is absent in Sepp1-/- epididymal sperm. Sepp1r/r CMV+ epididymal sperms
have GPx4 immunoreactivity similar to Sepp1+/+ confirming genetic restoration of Sepp1.
These results correlate with western blot analysis that GPx4 levels are decreased to the
point that GPx4 is not detectable compared to sperm from Sepp1 wild type and rescue
mice. Images taken by Zeiss Axioskop Plus 2 40X objective.
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CHAPTER IV
DISCUSSION and CONCLUSION
Sepp1 plays an important role in spermatogenesis and has been shown to be
essential to normal sperm morphology. Surprisingly, the issue of sperm DNA viability
due to the absence of Sepp1 has not been addressed. The results of this study
demonstrate that the infertility of male Sepp1-/- mice is partly due to sperm viability. Our
ICSI data clearly demonstrates for the first time that sperm heads, consisting mostly of
nuclei, are defective and result in impaired fertilization. Further, the developmental
defects exhibited in early stages of the fertilization process ultimately lead to a 72.3%
reduction in progeny number from oocytes injected with KO sperm compared to oocytes
injected with heterozygous control sperm. In addition, we show that GPx4 protein levels
in Sepp1 KO testes and epididymides are dramatically decreased overall and particularly
in sperm nuclei. Western blot shows decreased GPx4 immunoreactivity in Sepp1-/-
epididymal sperm compared to both Sepp1+/+ and Sepp1r/r CMV+ mice, and
immunofluorescence indicates that GPx4 colocalizes with the DAPI stained nucleus. We
also show that our unique strategy of using the existing Cre-LoxP sites in the Sepp1-/-
mice to restore the Sepp1 gene was completely successful as the Sepp1r/r CMV+ mice
had restored neuromotor function, spatial learning and memory, and fertility in males.
Furthermore, GPx4 expression was restored to normal levels similar to those of Sepp1+/+
mice.
Sepp1 is a plasma protein thought to mainly function to transport Se from the liver to
testes and other organs [18]. Studies show that Se is preferentially retained in brain and
testes when dietary Se intake is low. Reduced Se levels and glutathione peroxidase
(GPx) activity in reproductive organs of Se deficient male mice have been reported [81].
During spermatogenesis, Se content is noticeably increased in testes [82]. This requires
increased uptake of Se to testes. As a transporter of Se, Sepp1 may be responsible for
Se distribution during the reproductive process. Se deficiency is associated with
abnormal testicular mass, sperm morphology, and is essential for biosynthesis of
testosterone.
Sepp1 KO males are infertile and several studies have indicated that one of the main
contributing factors to this condition is defective sperm morphology [61]. Sepp1 KO
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males have acute kinks in their sperm flagella rendering them unable to swim to the
oocyte, leading to infertility. Impairments from selenium deficiency can be restored in
wild type animals by dietary supplementation; however this is not the case for Sepp1-/-
mice. With dietary supplementation, neurological function of
Sepp1-/- mice is improved, but other deficits remain. Sepp1-/- mice have abnormal sperm
morphology regardless of supplementation. Sepp1 has direct antioxidant properties as
well as facilitating synthesis of antioxidant selenoproteins such as GPx4.
To test our first hypothesis whether other factors aside from defective sperm
morphology were involved in the infertility of male Sepp1-/- animals, we investigated
whether isolation of the sperm head from the defective flagella followed by ICSI would
produce normal fertilized oocytes and resulting litter size. As evident by our ICSI results,
genetic deletion of Sepp1 reduces viability of the sperm. We observed reduced
fertilization rates and impairments of early cell division stages in oocytes injected with
Sepp1-/- sperm compared to the oocytes injected with Sepp1+/- control sperm. Our data
show that Sepp1-/- male infertility is not entirely due to morphological sperm flagella
defects as suggested by other studies, as absence of flagella in the ICSI procedure did
not result in restored litter size.
It is not surprising that deletion of the Se transporter results in reduction in the levels
of other selenoproteins, such as GPx4. Previous studies have shown that GPx4 levels
are reduced in Sepp1-/- [75]. A 2009 study suggests that of the three GPx4 isoforms
(cytosolic GPx4, nGPx4, mGPx4), the mitochondrially expressed form of the GPx4 gene
is the most relevant one in spermiogenesis [77] while the nuclear form, nGPx4, is not
important for fertility [74,77]. However, a more recent study by Puglisi et. al, shows
convincing evidence that nGPx4 is associated with the sperm nuclear matrix and is
essential for sperm chromatin decondensation [71]. The nuclear isoform of GPx4 is
reportedly expressed in male germ cells. GPX4 contains a selenocysteine as well as
several cysteines with the ability to reduce protein thiols [71]. Our study supports
previous findings that impaired GPx4 biosynthesis, due to selenium deficiency or to
genetic defects in GPx4 itself or in proteins involved in Se distribution and selenoprotein
biosynthesis, results in male infertility [80]. We examined the GPx4 levels in whole
epididymis, epididymal sperm, and testes of the Sepp1 rescue and KO mice compared
to wild type controls to confirm the complete restoration of Sepp1 in our rescue model
and to confirm that the Sepp1-/- mice exhibited alterations in GPx4 levels as reported by
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other groups. Our results confirmed reduced GPx4 protein expression in testes, where
testosterone and spermatozoa are synthesized. Moreover, we also showed that GPx4
protein expression was reduced notably in the epididymis, where sperm undergo a
maturation process and mature active sperm are stored in the cauda epididymis. To
further confirm the correlation of GPx4 levels, we used immunofluorescence microscopy
to assess GPx4 immunoreactivity. In support of our western blot data, we observed
drastically diminished GPx4 signal in Sepp1-/- epididymal sperm and restored levels of
GPx4 in our rescue mice. Our findings suggest that rendering the Sepp1-/- gene inactive
leads to decreased levels of GPx4. Sperm viability is clearly dependent on Sepp1 to
deliver Se and regulate biosynthesis of GPx4.
The latter part of our study was aimed to genetically restore Sepp1 gene expression
in Sepp1 knockout mice. We describe a strategy that takes advantage of the Cre-LoxP
system, using the start codon in the LoxP site, to restore gene expression in the Sepp1
KO mouse model. Previously the Schweizer lab generated a hepatically targeted Sepp1
transgene to rescue Sepp1-/- mice [75]. However, our approach is novel in that we use
the start codon in the existing LoxP site to genetically restore Sepp1 function, and can
therefore rescue Sepp1 in any tissues with the appropriate Cre and without additional
transgenic manipulations. One of our primary interests in generating this Sepp1 rescue
model is to apply this method to restrict gene expression to specific cells or tissues in
order to study the gene’s function within those cells or tissue. This unique application
can allow researchers to study any gene of interest in highly specific cell populations. A
researcher interested in such an approach to elucidate a specific function of a gene can
specifically design the knockout mouse to have LoxP sites in which the LoxP start codon
will remain in frame with the gene of interest following Cre recombination. Subsequent
mating of mice expressing Cre in specific cell types,, such as a subset of
cardiomyocytes or neurons, will excise the KO construct, leaving behind a LoxP site with
an in frame start codon. Following the successful generation of the Sepp1r/r CMV+ mice,
in which we restored global expression of Sepp1, we have used this unique strategy to
generate a brain specific Sepp1 rescue mouse model. Using a Cre expressing mouse
with a promoter that drives expression in forebrain neurons, this mouse model
expresses Sepp1 restricted to neurons in the forebrain, while Sepp1 gene expression
remains inactive in all other tissues. We confirmed the complete restoration of the Sepp1
gene by performing a battery of behavior tests which resulted in Sepp1r/r CMV+ mice
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having restored neuromotor function as observed in the pole and stride tests.
Furthermore, Seppr/r CMV+ mice had normal spatial learning and memory and did not
exhibit any of the deficits seen in Sepp1-/- mice when administered the Morris Water
Maze test. Ultimately, the ability of Sepp1 rescue mice to sire pups that were used in the
behavior tests was indication that infertility seen in male KO mice was restored.
Furthermore, the sperm morphology studies confirmed that the rescue mice had normal
sperm morphology and motility.
Further research is essential to understanding the mechanisms involved in Se
function and the roles of various selenoproteins, such as Sepp1 and GPx4, in male
reproductive health. Some studies have suggested that Se deficient Balb/c mice show
decreased mRNA and protein expression patterns for both cJun and cFos (components
of transcription factor AP-1, activator protein-1) [82]. These factors regulate cellular
growth and differentiation and also have regulatory roles in spermatogenesis and
steroidogenesis [82]. Jun has been detected during specific stages of testes
differentiation [1,82]. cFos activity has also been observed in premeiotic germ cells of
mammalian testes during mouse spermatogenesis [46,82]. Studies show that Jun D
knockout mice have impaired spermatogenesis as well as reduced reproductive aptitude
[82,90]. cJun expression has been observed in Leydig cells, potentially enhancing
testosterone secretion, whereas in Se deficient mice, these cells as well as the
seminiferous tubules showed abnormal morphology [82]. Alteration in Se supply may
lead to these altered cJun and cFos patterns in the testicular germ cells, which might be
responsible for decreased germ cell number, differentiation and reduced fertility [82].
The study suggests that this may be an explanation for the mechanism of Se action in
regulating spermatogenesis [82].
As previously mentioned, Sepp1 and especially GPx4 have been reported to have
antioxidant properties protecting cells from oxidative stress and similar insults.
Investigation in sperm damage suggests a link between DNA fragmentation and
oxidative base damage. Lipid peroxidation also induces sperm damage [101]. There is
evidence showing that a significant proportion of the free-radical induced DNA damage
observed in human spermatozoa is due to oxidative processes [102]. Antioxidants such
as Sepp1 and GPx4 may have beneficial effects in alleviating sperm damage and may
be instrumental as a treatment. A study by Shalini et. al showed that sperm from dietary
Se deficient mice exhibited incomplete chromatin decondensation and increased
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incidence of DNA breaks [81]. Sperm chromatin condensation in spermatogenesis is a
complex process that involves sequential replacement of the majority of histones by
transition proteins and protamines in testis [13,14,24]. During spermatozoa transit
through the epididymis from caput (head) to cauda (tail), protamine thiol oxidation is
completed and intra- and intermolecular cross-links are formed. Subsequently, a
transcriptionally inactive and tightly packed haploid genome results and renders sperm
nuclei more resistant to mechanical and chemical insults and mature [24]. There are
many potential molecular mechanisms underlying this sperm damage, but certainly
taking all these findings into consideration is an important factor.
The exciting results of this study increase our knowledge of the role of Sepp1 in
relation to GPx4 and male fertility. Significantly, our ICSI results highlight that Sepp1 KO
male mice are infertile due to sperm viability issues other than abnormal flagella
morphology. Our findings contribute to the further understanding of the function of Sepp1
in mammalian sperm infertility issues.
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Concluding Remarks and Future Studies
Selenoprotein synthesis is dependent primarily on dietary intake of the trace element
Se. Selenoproteins have a unique energetically costly expenditure for biosynthesis. The
25 human (24 in mice) selenoproteins have highly diverse tissue and subcellular
localization with each exerting specific functions in the subcompartments in which they
reside [9,74]. The unique aspect of Sepp1 having 10 Sec residues suggests its
importance for synthesis of other selenoproteins and its role in preventing loss of Se
levels in brain and testes during low dietary Se intake [6,32,50]. Future experiments that
may help further elucidate the mechanism of Sepp1 in sperm and male infertility include
examining sperm chromatin decondensation and sperm fragmentation in the Sepp1-/-
mouse model. Elucidating the functional roles and significance of individual
selenoproteins, such as Sepp1 and GPx4, will eventually provide crucial insight into how
dietary Se and selenoproteins affect human health.
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