Seminiferous tubule transfection in vitro to define post-meiotic gene regulation
© Danner et al; licensee BioMed Central Ltd. 2009
Received: 08 April 2009
Accepted: 29 June 2009
Published: 29 June 2009
Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.
Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48 h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment.
Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals.
We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.
In contrast to earlier assumptions, it is now evident that a large number of genes are expressed in meiotic and post-meiotic, haploid male germ cells, in late spermatocytes and spermatids . This represents a special and discrete phase of transcription between the constraints of meiosis and the final replacement of most histones by the highly compact structure of transition proteins and subsequently protamines. At present there are only a few limited procedures available to examine the molecular details of gene regulation for such post-meiotically expressed genes, due to the lack of any suitable cell-lines for this stage of spermatogenesis. The majority of studies have employed heterologous transfection of promoter-reporter constructs into diploid somatic cells with co-transfection of expression constructs for various suspected transcription factors. This approach has the obvious tautological limitation that only factors can be characterized which are a priori suspected of being involved [2–5]. Hecht and colleagues attempted to overcome this problem by using an in vitro transcription assay comprising the promoter of the gene of interest linked to a G-free cassette, and using nuclear extracts from mature testes as the source of transcription factors . Whilst providing useful information, this method lacks the discrimination of using transcription factors from specific cell types, and has proven difficult to reproduce in many laboratories. Conventional gain-of-function transgenesis has also been used to assess promoter specificity for post-meiotic genes [7–10]. However, this approach is largely limited to mice, and the fact that only a single construct can be used per individual animal has severely restricted both statistical analysis and a more detailed molecular dissection of promoter regions. Another procedure, which has permitted the analysis of post-meiotic promoters, is to transfect isolated spermatogonia in vitro, and then to transplant these into the testes of prepubertal or azoospermic animals . Again, however, this method is restricted by the number of animals needed for reliable statistical analysis of individual constructs, by the inherent difficulty of germ cell transplantation itself, and the paucity of germ cells maturing through to post-meiotic stages. Finally, there are reports of direct in vivo transfection of gene constructs into the exposed seminiferous tubules of rodent testes, again with the limitation that a single testis is required for each construct, and that there are no appropriate means of quantification of the specific gene expression [12–16].
In order to redress this obvious methodological deficit, we have developed a procedure using explanted seminiferous tubules from rats, transfecting these with promoter-reporter constructs in vitro in a microtiter plate format, followed by short-term culture and quantitation. This new method allows many constructs to be characterized using tissues from a single animal, offers assessment of cell-type specificity, and provides a biologically relevant environment relatively free of the artefacts caused by using heterologous systems.
Preparation and culture of rat seminiferous tubules
All chemicals used were from Sigma-Aldrich (Deisenhofen, Germany), unless otherwise stated. Adult (7–8 months) male Wistar rats, which had been maintained under a 12 h/12 h light/dark regime with food and water ad libitum, were used for all experiments. Animals were killed by excessive CO2 anaesthesia, and testes immediately removed and further processed under sterile conditions. The tunica albuginea was slit to release seminiferous tubules, which were carefully teased apart, cut into approximately 10–15 mm lengths, transferred into a Petri dish containing TKM-medium (modified Tres – Kierszenbaum  medium: Minimal Essential Medium (without L-glutamine and phenol red; Gibco-Invitrogen; Karlsruhe, Germany), containing 5 μg/ml insulin (Invitrogen; Karlsruhe, Germany), 5 μg/ml human apo-transferrrin, 10 ng/ml epidermal growth factor (Upstate Biotechnology; Lake Placid, CA), 10 ng/ml insulin-like growth factor 1 (Upstate Biotechnology), 10 ng/ml human growth hormone (Eli Lilly; Bad Homburg, Germany), 10 ng/ml recombinant human follicle stimulating hormone (National Hormone and Peptide Program, Harbor – UCLA Medical Center, Torrance, CA), 5 μM retinol, 10 nM testosterone, 10 nM dihydrotestosterone, 4 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (Gibco-Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin) and then held in a tissue incubator at 32C (approximate scrotal temperature) and 5%CO2. All subsequent cultures were in 24-well plates containing 0.4 ml TKM medium per well.
Where using fluorescently labelled probes, cultured seminiferous tubules were fixed for 1 h in phosphate-buffered saline (PBS) containing 4% paraformaldehyde and 10% sucrose. Three to 4 tubules in similar orientation were then frozen together in Tissue-Tek medium (Sakura Finetek; Zoeterwoude, Netherlands) at -20C and stored at -80C. Sections of ca. 15 μM were cut, transferred to SuperFrost Plus slides (Menzel-Glaeser; Braunschweig, Germany), and stored at -80C until use. Sections were then air-dried for 1 h and then rehydrated in PBS containing 10 mM CaCl2 and 5 mM MgCl2. Sections were fluorescently labelled using PBS containing 10 mg/ml AlexaFluor 568 labelled PNA (Molecular Probes; Eugene, OR), 1 μg/ml DAPI (4',6-diaminodino-2-phenylindol; Merck; Darmstadt, Germany) or 2 μM TO-PRO-3-iodide (Molecular Probes) for 30 minutes in a moist chamber, rinsed twice for 5 mins in PBS, and then embedded in Fluoromount G (Southern Biotech; Birmingham, AL), prior to examination using a confocal or conventional fluorescence microscope (Leica TCS SL or Nikon Diaphot) as indicated. For immunohistochemistry, sections were rehydrated in PBS, followed by incubation in PBS containing 5% normal goat serum (NGS) for 30 min at room temperature, then in PBS containing 2% NGS plus 500:1 diluted rabbit polyclonal anti-ELP antiserum  overnight also at room temperature. Sections were then washed 3 times in PBS followed by incubation for 30 mins at room temperature with the secondary antibody (Cy3-labelled goat anti-rabbit IgG; Jackson ImmunoResearch; Newmarket, UK) diluted 100:1 in PBS with 2% NGS, also containing 1 μg/ml DAPI. After a further three washes in PBS, sections were mounted and analysed as above.
Preparation of DNA constructs
Oligonucleotide sequences used for the cloning of diverse gene promoter fragments from rat genomic DNA.
Oligonucleotide sequence 5'-3'
TAT TAA TGT CTA GTA ATG TCC AAC AGC
AAC CTG TGA GCA GGT GGA ATT TTG
TAT TAA TGG GTA GGA GCA TTC TCA TCT CGT
CAG ATC TGC CTG CAA GCT GTG ACC TCA CAA
TAT TAA TCC TCC AAT CTT AGG ACT AAC CTC
TGG CAC ACT CAA GAG CTG AGA AGA AAC
TAT TAA TGC AGG GTG TCA ACT AG
TAT TAA TGT GCC ATC TCA GGC TGC
TAA GAT CTT CGT GCC ATC TCA GGC
TAA GAT CTT CAT TCG CTC GCG G
TTG TTG GAA AGG AGT ACG CGT G
TAT ACC AGA AGC CGT GCC TCT G
Summary of the amplified promoter sequences, the oligonucleotide PCR primers used, and their respective annealing temperatures.
-403 to +28
-263 to +48
-556 to +30
-652 to +51
-957 to +617
-957 to +51
-652 to +617
-652 to +51
-652 to +617
-380 to +617
Transfection and analysis of introduced DNA constructs
The procedure used to introduce DNA expression vectors into explanted seminiferous tubule fragments was largely based on methods developed for gene therapy in vivo e.g. [12, 13]. After considerable preliminary experimentation, varying a wide range of possible parameters, the following protocol was considered optimal for EYFP-N1 or EGFP-N1 constructs. DNA solution at a concentration of 1 μg/μl was microinjected laterally into an individual tubule fragment that had been arranged lengthwise on a microscope slide. Microinjection used an Eppendorf Transjektor 5426 attached to an Eppendorf Micromanipulator 5171, both mounted onto the stage of a Zeiss Axiophot inverse microscope, providing a high degree of sensitivity and control over the microinjection process. The DNA was injected using a glass microcapillary whereby the tip had been deliberately fractured to provide a cutting surface allowing good penetration of the tubule lamina propria, and thus release of DNA into the tubule lumen. DNA volume was regulated to fill ca. 30% of the tubule length. This represents an excess, since the amount transfected is determined not by the volume of DNA but by the lateral dimensions of the Tweezertrodes used for electroporation (see below). Following microinjection, tubule fragments are transferred to a 24-well plate containing 0.4 ml TKM medium per well. Tweezertrodes (Cyto-Pulse Inc.; Glen Burnie, MD) are inserted into a well, straddling a tubule fragment, and transfection achieved by electroporation using an electrode gap of 4 mm, a pulse number of 10, an individual pulse strength of 20 V, a pulse length of 20 ms, and a pulse interval of 0.5 s, using a Cyto-Pulse PA-4000S square wave electroporation system. These parameters were determined following extensive variation and optimization in preliminary experiments. Transfected tubules were then transferred to an incubator at 32C/5% CO2 and examined at various intervals up to 48 h.
Seminiferous tubules were analysed for the expression of EYFP and EGFP at the indicated time intervals by direct fluorescence following excitation at 488 nm. Where luciferase expression constructs were used, protein lysates were prepared from tubules by first shock-freezing tubules in liquid nitrogen, pulverizing these in a small pestle and mortar, then dissolving in 150 μl of Reporter Lysis Buffer (Luciferase Assay System; Promega). After brief (2 min) centrifugation at 13000 rpm, 20 μl of the lysate was supplemented with 100 μl of Luciferase Substrate Solution (Luciferase Assay System; Promega), with measurement for 10 sec in a single-tube luminometer (Berthold; Bad Wildbad, Germany).
In order to assess the effect of culture and transfection conditions on the viability of cells in the explanted seminiferous tubules the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes) was used. The assay measures both the integrity of the plasma membrane and intracellular esterase activity using a mixture of Calcein-AM and Ethidium-Homodimer-1 (EthD-1), which differ in their membrane permeability. Calcein-AM is able to penetrate most cell membranes and is then cleaved inside cells by the esterase activity of living cells to form fluorescent Calcein (excitation 495 nm/emission 515 nm). Eth-D1 only enters damaged cells, where it complexes with DNA to yield a high nuclear fluorescence in the red spectrum (excitation 495 nm/emission 635 nm). Tubules are incubated in a mixture of EthD-1 (4 μM) and Calcein-AM (2 μM) in PBS for 30 mins at room temperature with subsequent analysis by confocal microscopy. Additionally, tubules were assessed following culture and/or transfection by TUNEL (Terminal dUTP nick end labelling) as a measure of apoptotic cell death, using the In situ Cell Death Detection Kit (TMR red; Roche Diagnostics; Mannheim, Germany) on tubule cryosections exactly following the manufacturer's instructions. This assay can lead to spurious results when used on testicular tissue because of the natural phagocytic activity of the Sertoli cells. This can be avoided by masking the "eat me" signals of apoptotic cells with Annexin V (10 μM; Apotech; Epalinges, Switzerland), which is microinjected into the seminiferous tubules simultaneously with the DNA constructs and blocks phagocytosis.
Sertoli cell culture, transfection and analysis
The mouse SK11 Sertoli cell-line  was additionally used to test the activity and specificity of some of the promoter-reporter constructs. These adherent cells were grown at 32C/5% CO2 in 250 ml flasks in DMEM (Dulbecco's Minimal Essential Medium – High Glucose; Gibco-Invitrogen) containing 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin, with passaging every 3 to 4 days. For transfection, cells were transferred to either 24-well plates (for luciferase assay) or 4-well chamber slides (for microscopic fluorescence assessment), and cultured overnight at 32C/5%CO2. Medium was then replaced by DMEM lacking additives, and 1 mg per well of DNA transfected using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. After transfection, cells were incubated for 5 h as above, then medium was replaced by DMEM plus 10% FCS, and incubation continued for a further 24 h until measurement of reporter gene expression. For assessment of luciferase activity the Luciferase Assay System (Promega) was used as already described.
Establishment of culture conditions
Optimization of transfection parameters
Cell specificity of transfection
Expression of cell-specific promoter-reporter constructs
We describe here an assay system to evaluate the qualitative and quantitative attributes of specific testicular gene promoters. In particular, this system seems suitable to analyse the promoter regions of genes expressed in the natural context of post-meiotic germ cells, which until now could only be assessed through in vitro transcription assays, or through transgenesis or similar approaches. Optimal parameters were determined for the introduction of gene promoter-reporter constructs into isolated seminiferous tubules by microinjection followed by pulsed electroporation. The advantage of this method is that it potentially allows both a qualitative assessment of cell specificity of expression, by using reporter genes encoding enhanced fluorescent proteins, as well as a quantitative assessment using either these constructs or better a luciferase reporter construct. The method would also be amenable to using other quantifiable reporter genes, such as that for human growth hormone  or chloramphenicol acetyl transferase (CAT). The major advantage, however, is that different animal species can be used, and from a single organ many tubule pieces can be collected and arrayed in a microtiter plate format for analysis, allowing a large number of constructs with multiple repeats to be tested. Based on the expression patterns from both a general viral promoter-driven construct, as well from constructs using more specific promoters, it is evident that most germ cell types as well as Sertoli cells can be targeted. The obvious exception here is the mature spermatozoon, which within the "dark" tubule sections appears not to be able to incorporate expression constructs, or if transfection does occur to these cells, they are unable to express the construct through a lack of appropriate cellular machinery.
Although this preliminary study was intended only to establish working parameters and to develop proof of principle, it has already highlighted some very interesting observations. Namely, that several so-called specific promoter regions, do not appear to delineate the degree of cell specificity that had been expected from the literature. The 877 bp promoter sequence of the proacrosin gene had been shown in transgenic mice to target a CAT reporter specifically to spermatocytes and spermatids [7, 26]. The same promoter in the present study appears to target both spermatids and Sertoli cells. Similarly, the promoters for the post-meiotically expressed genes SP-10 and protamine-1 had been shown to target specifically spermatids in transgenic mice [8–10, 21] in a stage-specific manner, but additionally indicated promiscuous expression in Sertoli cells following in vitro transfection into seminiferous tubules. Application of these promoters for in vivo transfection into seminiferous tubules also indicated a similar stage-specific expression in haploid cells [12, 13]. However, more recent studies using in vivo transfected seminiferous tubules and the gene promoters for the haploid-expressed genes for phosphoglycerate kinase 2 (Pgk2)  as well as for hst70  also indicate a promiscuous expression of the reporter genes in Sertoli cells, in addition to germ cells. Our experiments here with the ELP gene promoter largely confirm these observations concerning lack of cell specificity.
There are several possible reasons for this Sertoli cell expression of supposedly germ cell specific gene promoters. Firstly, when compared to transgenic expression, episomally expressed genes may lack certain stringency criteria provided by the embedding of a gene in a conventional chromatin environment. This may be epigenetic in nature, or may arise from the existence of neighbouring isolator or silencer sequences in the genomic neighbourhood of the insertion sites, all of which are likely to be missing in episomally expressed genes. It is reported that the specificity of the SP-10 promoter is in part due to the presence of an isolator sequence within its natural genomic location . A second possibility is that Sertoli cells are notoriously able to phagocytose damaged cells, particularly germ cells , and may thus express fluorescent protein by dint of having incorporated a germ cell expressing the reporter gene. We and others [15, 29] have shown that the microinjection and electroporation procedure used, whilst having minimal effect on cell death nevertheless does indeed lead to an increase in apoptosis, as assessed by TUNEL. In future studies, it will be important to test the effect of using much larger regulatory DNA regions which include the proximal promoter within a larger natural context, as well as to test the effect of different blockers of apoptosis and phagocytosis on the apparent cell specificity of reporter gene expression. Furthermore, the addition of a cell-separation step to isolate different component cells of the seminiferous tubules prior to reporter quantification, as suggested in , might prove valuable. The apparent expression of the proacrosin and ELP promoters in the Sertoli cell line SK11 may be due in part to the illegitimate use of Sertoli cell transcription factors which may share DNA binding sites with germ cell factors, as well as to an absence of restricting silencer elements in the larger DNA context of the natural gene locus. In contrast, the lack of promiscuous expression of the protamine-1 and SP-10 promoters in the SK11 cells could mean that, unlike in the tubules, these Sertoli cells, being an immortalized cell-line growing under basal conditions, may simply not express the appropriate factors, which are produced by primary Sertoli cells growing in the more natural milieu of the seminiferous tubule with moderate hormonal stimulation.
In conclusion, we have demonstrated that isolated seminiferous tubule fragments cultured in vitro for up to 2 days can be transfected with specific testicular promoter-reporter constructs yielding both qualitative and quantitative output. Further refinement of this method should lead to a valuable routine assay system for the evaluation especially of haploid expressed gene promoters, which to date cannot be assessed simply and accurately by any other procedure. This initial study has already highlighted the very important observation that several promoters, which from transgenic studies appear to be absolutely cell-type specific, lose some of that specificity when introduced by transient transfection.
We gratefully acknowledge all of the expert help and advice from our colleagues in the former Institute for Hormone and Fertility Research at the University of Hamburg. We also wish to thank Professor W. Schulze (Hamburg) for his help and encouragement throughout this project. We should also like to acknowledge the Deutsche Forschungsgemeinschaft (DFG) for the generous financial support of this project (Iv 7/11).
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