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- Original Article -
Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells
Li Lu, Min Lin, Min Xu, Zuo-Min Zhou, Jia-Hao Sha
Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing
210029, China
Abstract
Aim: To study polyethylenimine (PEI)-mediated
in vivo gene transfection into testis cells and preliminary functional
research of spermatogenic cell-specific gene
NYD-SP12 using this method. Methods:
PEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and
speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The
long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells at
different stages were analyzed with fluorescent microscopy and propidium iodide staining.
Results: With the mediation of PEI, the
GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells (especially in
primary spermatocytes). Transfection into Sertoli cells was not observed. The subcellular localization of the
GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during
spermatogenesis. Conclusion: PEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional
research of spermatogenic-cell specific genes such as the
NYD-SP12 gene. In our study, the NYD-SP12 protein was
visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the
detailed function of NYD-SP12 in spermatocytes.
(Asian J Androl 2006 Jan; 8: 53-59)
Keywords: gene transfer techniques; polyethylenimine; NYD-SP12 gene; spermatogenic cells; spermatogenesis
Correspondence to: Dr Jia-Hao Sha, Laboratory of Reproductive
Medicine, Nanjing Medical University, 140 Hanzhong Road,
Nanjing 210029, China.
Tel/Fax: +86-25-8686-2908
E-mail: shajh@njmu.edu.cn
Received 2005-05-08 Accepted 2005-06-23
DOI: 10.1111/j.1745-7262.2006.00089.x
1 Introduction
Spermatogenesis, the fundamental function of testis,
occurs in successive mitotic, meiotic and postmeiotic
phases of germ cells. The germ cells move from the
periphery to the lumen of the seminiferous tubule during
this process. Spermatogenesis is basically controlled by
the programmed expression of a number of stage-specific genes, some of which have so far been identified as
spermatogenic cell-specific genes that perform intrinsic
regulation on spermatogenesis [1, 2]. Functional research
of these genes would shed light on the understanding of
the biological mechanisms of spermatogenesis and
facilitate the treatment of male infertility. Our laboratory
has identified a number of spermatogenic cell-specific
genes by microchip screening [1, 2]. One of these,
NYD-SP12, was thought to be a Golgi apparatus-associated
protein [1]. In order to know the potential role of these
genes in spermatogenesis, we are seeking a convenient
and efficient method that could circumvent gene
targeting and spermatogenic cell culture.
Although transgenic mice offer ideal models for
research into gene function, the technique of producing
transgenic mice is laborious and time-consuming. Moreover,
lethality before adolescence in transgenic mice precludes
detecting the role of a candidate gene in spermatogenesis.
Even when survival to later stages occurs, extrinsic defects might
confound the interpretation of spermatogenic phenotypes.
Under such circumstan-ces, several in vivo gene transfer
methods were developed during the past decade, such as
microparticle bombardment [3], electroporation [3, 4],
adenoviral mediation [5] and lentiviral mediation [6]. However,
these techniques were found to preferentially transfect
Sertoli cells or both Sertoli and germ cells, but not
spermatogenic cells alone, which disappointed us in
conducting functional research of spermatogenic cell-specific
genes. The recently developed technique of germ cell
transplantation into the seminiferous tubules [7-9] offered an
excellent in vivo model for intrinsic regulation research
of spermatogenesis. Nevertheless, this technique was
greatly limited by the insufficient availability of
spermatogenic stem cells in the testis. Thus, a simple and
effective germ cell-oriented in vivo gene transfer method was
urgently needed for the functional research of
spermatogenic cell-specific genes.
Recently, the linear 22 kDa form of polyethylenimine
(PEI) has shown to function as an effective nonviral vec
tor for both in vitro and in vivo gene transfer
[10, 11]. Because its receptor proteoglycan is a common
component of cell membranes [12], PEI was supposed to be
used to mediate gene transfection into all kinds of cells.
However, it has not been tested in testis. In this study,
we tested whether PEI could mediate gene transfer into
spermatogenic cells using intratesticular injection. Then,
using the same technique, we further investigated the
functional role of the NYD-SP12 gene by its subcellular
localization in spermatogenic cells at different stages. The
NYD-SP12 gene is a spermatogenic cell-specific gene
and its protein has been proved to be associated with
Golgi apparatus [1]. Golgi is an important organelle in
sperm acrosome formation, therefore the functional role
of the NYD-SP12 gene was predicted to be involved in
this process. In this study, long-term observational
results revealed that NYD-SP12 was involved in the
formation of acrosome, which implicated its potential role
in spermatogenesis and sperm-egg fusion.
2 Materials and methods
2.1 Mice
Adult (8-week-old) ICR male mice (weight 30-40
g) were obtained from the Animal Center of Nanjing
Medical University (Nanjing, China). All experiments were
reviewed and approved by the Institutional Animal Care
and Use Committee at the University.
2.2 Plasmids
Plasmid pEGFP, encoding green fluorescent protein
(GFP) driven by the cytomegalovirus (CMV) promoter,
was purchased from Clontech (Mountain View, CA, USA). Plasmid
pEGFP-NYD-SP12 was constructed by inserting the
NYD-SP12 open reading frame (nucleotides 131-1840) into plasmid
pEGFP, as described in detail by Xu et
al. [1]. The plasmids were amplified in DH5a host
bacteria, then extracted and purified with the Qiagen
Plasmid Mini Kit (Valencia, CA, USA) according to the
manufacturer¡¯s instructions. The extracted plasmids were
dissolved in 5 % sterilized glucose and stored at -20 ºC.
For in vivo gene transfections, plasmid and PEI (ExGen
500, Fermentas, Burlington, Ontario, Canada) were mixed
at a PEI nitrogen: DNA phosphate ratio of 6, according
to the manufacturer¡¯s suggestion. The total volume of
10 µL (containing 3 µg plasmid) was injected into each
testis.
2.3 Intratesticular injection
Mice were anesthetized with urethane and testes were
exteriorized through a midline abdominal incision of
approximately 5 mm. Glass microneedles were produced
from capillary tubing (G-100; Narishige Instruments,
Setagaya-ku, Tokyo, Japan) using a vertical pipette puller
(model PB-7; Narishige Instruments, Setagaya-ku, Tokyo, Japan) and the tips were finely beveled with a
pipette grinder (model EG-4; Narishige Instruments,
Setagaya-ku, Tokyo, Japan) to a final diameter of 30
µm-50 µm. The intratesticular injection procedure
was previously described [13], through which 70 % - 85 % of
the seminiferous tubules could be introduced with
transfection materials [6, 13]. After injection, the testes were
put back into the abdomen and the incisions were sutured.
The mice were recovered and raised until analysis.
Initially, 0.2 % trypan blue was used as an indicator
to make sure that the microinjection was successful.
However, as the dye was found to form adverse
deposits with PEI, no trypan blue was used throughout the
gene transfection procedures.
2.4 PEI-mediated gene transfection analysis
The mice were divided into three groups: PEI (PEI
alone); pEGFP-NYD-SP12
(pEGFP-NYD-SP12 alone); and
PEI+pEGFP-NYD-SP12 (PEI-mediated gene transfection of
pEGFP-NYD-SP12). The former two groups served as controls. Each group contained three mice.
Three days after gene transfection, the mice were
killed by cervical dislocation and the testes were removed,
embedded in OCT (Frozen tissue matrix) and frozen-sectioned at a thickness of 5 µm. The slides were then
analyzed by fluorescent microscope (Zeiss Axioskop 2;
Carl Zeiss, Oberkochen, Germany), to determine the
transfection efficiency of PEI and subsequently
visualized by light microscopy after 4 g/L hematoxylin (Sigma,
Louis, MO, USA) staining. The corresponding
fluorescent and light images were then contraposed for the
identification of positively transfected cell types in the
seminiferous tubules.
2.5 Identification of NYD-SP12 functional role by
subcellular localization in different stages of spermatogenic
cells
PEI plus pEGFP-NYD-SP12 was injected into mice
seminiferous tubules as described previously [13].
The mice were killed on days 3, 6, 10, 15, 20, 30 and 60 after
gene transfection. PEI-mediated pEGFP gene transfec
tion was used as a control. Mice in the PEI+pEGFP
group were killed at day 10 after gene transfection. Three
mice were killed at each time point. The testes were
removed, embedded in OCT (Frozen tissue matrix) and
frozen-sectioned at a thickness of 5 µm. After 4 %
paraformaldehyde (PFA) fixation for 20 s, the
sections were stained for 5 s in 0.5 % propidium iodide (PI) for the
visualization of nuclei. The slides were then rinsed with
distilled water, alcohol dehydrated, and visualized under
fluorescent microscope for the detection of
GFP-NYD-SP12 gene expression. The images were then
superimposed to determine the subcellular localization of
GFP-NYD-SP12 gene expression.
Additionally, sperms were collected from the
epididymis of transfected mice and washed three times with
phosphate-buffered saline. After fixation in 4 %
PFA for 30 min, sperms were spread on slides and viewed by
fluorescence microscopy. Sperms from three mice were
examined separately.
3 Results
3.1 Intratesticular injection
Successful intratesticular injection was achieved, as
traced by trypan blue, shown in Figure 1.
3.2 PEI-mediated gene transfection efficiency
PEI-mediated pEGFP-NYD-SP12 gene transfection
efficiency was much higher than that of
pEGFP-NYD-SP12 alone (Figure 2).
pEGFP-NYD-SP12 was hardly transferred into the seminiferous tubules when PEI was
not applied (Figure 2).
3.3 PEI-mediated gene transfer was
spermatocyte-pre-ferential in seminiferous tubules
When we contraposed fluorescent and light microscopic images, we found that PEI-mediated transfection
of pEGFP-NYD-SP12 was achieved mostly in
spermatocytes (Figure 3A). Most
GFP-NYD-SP12 positive spermatocytes (white arrow, Figure 3A) also had a strongly
stained particle within its nucleus (dark arrow, Figure 3B),
which represented the condensed PEI [14]. However,
approximately 10 % of PEI positive spermatocytes were
GFP-NYD-SP12 negative (Figure 3, yellow circles).
Sertoli cells seemed to be neither
GFP-NYD-SP12 nor PEI positive (Figure 3, red arrows).
3.4 Identification of NYD-SP12 functional role by
subcellular localization in different stages of spermatogenic
cells
Three days after gene transfection,
GFP-NYD-SP12 gene expression could be detected in some spermatogonia,
mostly spermatocytes and a few round spermatids in the
seminiferous tubules. However, transgene expression in
elongated spermatids could not be detected until day 10.
At this point, round and elongated spermatids made up
the majority of the GFP-NYD-SP12 positive cells.
GFP-NYD-SP12 gene expression in the seminiferous tubules
could last for more than 1 month after transfection.
Furthermore, the subcellular localization of
GFP-NYD-SP12 revealed that the fusion protein appeared to
shift from Golgi to acrosome (Figure 4A-G), whereas
GFP did not exhibit such a translocation profile (Figure
4H, I). Twenty days after transfection, GFP-NYD-SP12
protein could be detected in the acrosomes of sperms in
the epididymis (Figure 4G), however, the efficiency was
rather low, approximately 1:10 000.
4 Discussion
Functional research of spermatogenic cell-specific genes
is important for the understanding of spermatogenesis and
would facilitate the treatment of male infertility. Previous
studies using in vivo gene transfer methods [3-6]
circumvented the gene targeting and spermatogenic cell
in vitro culture. However, these newly-developed methods
preferentially affected Sertoli cells but not germ cells. In this
study, we showed that the PEI-mediated in
vivo gene transfection method using intratesticular injection was
efficient and convenient, and that this method was
spermatogenic cell preferential. This offered a new method
for the functional research of spermatogenic
cell-specific genes. However, it needs to be further investigated
whether the method was a selective transfer into
spermatogenic cells.
Despite the relatively low transfection efficiency as
compared with viral vectors, cationic vectors retain a
high attractiveness in gene transfer due to their
theoretically excellent safety profile. As a new member of the
cationic vector family, PEI performed more efficiently in
mediating gene transfection and expression in mammalian
cells as compared with other cationic vectors [15]. The
transfer activity of PEI is related to its ability to condense
DNA, interact with anionic proteoglycans of the cell mem
brane [10,16,17], protect DNA [18], and induce endosomal
swelling and rupture prior to DNA degradation [19]. PEI
or PEI/DNA complexes could be transported to the nucleus
and exhibited as distinct structures [12], which could be
visualized by light microscopy after hematoxylin staining.
By contraposing the fluorescent and light images we
found that the positive cells were mainly spermatocytes.
Transfection into Sertoli cells was not observed.
Because the CMV promoter is well known for its
ubiquitous activity of initiating transcription in various
eukaryotic systems, the possibility of a successful
GFP-NYD-SP12 gene transfection into Sertoli cells without
transcription/expression under a CMV promoter is very slim.
Furthermore, in order to make clear whether this was
caused by the difference between a
spermatogenic-specific gene (like GFP-NYD-SP12
) and a non-spermatogenic-specific gene, a GFP vector (without the fusion
with the NYD-SP12 gene) was used as a control. The
results were the same as that for
GFP-NYD-SP12 (data not shown). Thus, the most reasonable conclusion is
that PEI-mediated gene transfection is spermatogenic cell
selective. However, the detailed mechanisms of this
process need to be investigated further.
PEI-mediated gene transfection in the seminiferous
tubules occurred in a restricted population of
spermatogenic cells, mainly the spermatocytes. The efficiency
was approximately five cells per tubule. Approximately
10 % of PEI positive cells did not show a GFP signal,
which might have resulted from the sole transfection of
PEI (excessive PEI might be applied for PEI/DNA interaction), or the expression silence of the unlinearized
plasmid DNA. The lower transfection efficiency in
spermatogonia was most likely due to their location in the
basal compartment of the seminiferous tubules where
the PEI/DNA complexes were less accessible. We also
noticed that much more GFP-NYD-SP12 positive round
and elongated spermatids could be found on day 10
compared with day 3. We thought that GFP-NYD-SP12
protein in round and elongated spermatids might not be the
result of direct transfection but that of inheritance from
the positively transfected spermatocytes.
GFP-NYD-SP12 protein detected in spermatogozoa on day 20 might
be explained in the same way. This could also explain
why there were fewer GFP positive sperms and weaker
GFP signals in sperms as compared with
spermatocytes. After meiosis, a diploid spermatocyte divides into four
haploid spermatids, so the transgene
(GFP-NYD-SP12) and the GFP signal are also distributed into four daughter
cells. PEI-mediated gene transfection is also transient.
Nevertheless, more efficient transfection could be
achieved if this method was modified and developed.
The NYD-SP12 gene was cloned from human testis
through the microarray technique in our previous
work [1]. It was specifically expressed in spermatogenic cells, and
its product was proved to localize in Golgi apparatus [1].
As Golgi is an important organelle in the formation of
sperm acrosome, the NYD-SP12 gene was assumed to
relate to this process. In the present study, this proposal
was confirmed by the PEI-mediated in vivo gene
transfection method. By the subcellular localization of
exogenous GFP-NYD-SP12 in different stages of
spermatogenic cells, the functional role of the
NYD-SP12 gene in spermatogenesis was revealed to be involved in the
process of acrosome formation. In brief, along with
sperma-togenesis, the GFP-NYD-SP12 fusion protein dispersed
in the cytoplasm of spermatocytes at the beginning of
transfection, and subsequently congregated at one pole
in round spermatids where it further formed the acrosome
vehicle-like structure. It then elongated along one side
of the nucleus in elongated spermatids, and finally
located in the acrosome of spermatozoon. All of this
implicated that NYD-SP12 protein might play important roles
in acrosome formation, spermatogenesis and, probably,
in the later sperm-egg fusion.
PEI-mediated in vivo gene transfection offered a
convenient and efficient method for the functional
research of genes related to the intrinsic regulation of
spermatogenesis. By this method, the functional role of
the NYD-SP12 gene related to Golgi apparatus/acrosome
formation was visually identified, which provided
important information for further research.
Acknowledgment
This work was supported by the Major State Basic
Research Development Program of China ( 973 Program),
the National Natural Science Foundation of China (No.
30400248, No. 30425006), and the Ministry of Science
and Techonology of the People¡¯s Repubic of China
(MOST) Fund (No. 2004CCA06800).
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