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Expression of a green fluorescence protein-carrier protein into mouse spermatozoa
BBRC Biochemical and Biophysical Research Communications 297 (2002) 841–846 www.academicpress.com
Expression of a green fluorescence protein-carrier protein into mouse spermatozoa Teresa Mogas,a Josep M. Fern andez-Novell,b Maria Jes us Palomo,a Pedro J. Otaegui,c Roger R. Gomis,b Joan Ballester,a Dolors Izquierdo,a Joan J. Guinovart,b Joan C. Ferrer,b Teresa Rigau,a and Joan E. Rodrıguez-Gila,* a
Unitat de Reproducci o, Departament de Medicina i Cirurgia Animals, Facultat de Veterin aria, Universitat Aut onoma de Barcelona, E-08193 Bellaterra, Spain b Departament de Bioquımica i Biologia Molecular, Universitat de Barcelona, C/. Martı i Franqu es, 1, E-08028 Barcelona, Spain c Departament de Bioquımica i Biologia Molecular, Universitat Aut onoma de Barcelona, E-08193 Bellaterra, Spain Received 29 July 2002
Abstract Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria green fluorescence protein/rat-liver glycogen synthase (GFP/LGS) resulted in the presence of GFP/GLS in spermatozoa from 7 days to, at least, 16 days after inoculation. The GFP/LGS was detected in the sperm heads after an ‘‘in vitro’’ fertilization procedure, either before or after the oocyte penetration. Our results indicate that spermatozoa carrying GFP/LGS protein conserved their fertilizing ability and were also detectable after oocyte penetration. This technique will allow to develop an easy system to follow the fate of mature sperm proteins. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Green fluorescence protein; Mature spermatozoa; Protein tracking
In recent years, there has been an increasing amount of information about the role that mammalian spermatozoa play in modulating fecundation and early embryonic development. Thus, the human sperm centrosome is responsible for normal syngamy and early embryonic development [1]. Moreover, spermatozoa introduce a factor of 33 kDa into the oocyte cytoplasm, which is needed to activate the egg [2]. However, the study and the identification of other sperm controlling factors are difficult, owing to the peculiar structure and functions of mammalian spermatozoa [3]. One of the most promising approaches to the study of sperm proteins is to perform a feasible technique to mark a specific sperm protein. This would permit following the fate of this protein from penetration to the first embryonic divisions. The use of chimeric genes, *
Corresponding author. Fax: +34-93-581-2006. E-mail address: [email protected] (J.E. RodrıguezGil).
which integrate the Aequorea victoria green fluorescence protein (GFP), can be a feasible approach to this technique. However, the application of this technique to spermatozoa is difficult, since mammalian sperm is unable to synthesize proteins [3]. In fact, the intra-testicular injection of a GFP-codifying DNA has been carried out only to achieve in vivo gene transfer, but not to generate an spermatic protein that could be tracked during the sperm life cycle [4–6]. The main goal of this work is to develop a system by which fully functional mammalian spermatozoa have a GFP-marked protein. This is developed by intra-testicular inoculation with an adenoviral vector containing the chimeric gene of the GFP and the rat-liver glycogen synthase DNA (GFP/LGS). The idea is that a massive testicular expression of the GFP/LGS DNA will result in the appearance of a specific protein marking in spermatozoa. This will allow to specifically study the fate of a determined spermatic protein during its life cycle, from ejaculation to the first stages of embryonic development.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 2 9 5 - 7
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T. Mogas et al. / Biochemical and Biophysical Research Communications 297 (2002) 841–846
Materials and methods Obtainment of green fluorescence protein-marked spermatozoa. Thirty male B6 SJL-F1 mice (age: 2 months) were anesthetized and analgesized by a single intra-peritoneal injection of 0.05 mg/kg buprenorphine, 100 mg/kg ketamine, and 2.5 mg/kg azepromacin. Mice were inoculated in both testes in the following manner: Right testes were inoculated with 20 lL Dulbecco’s modified Eagle’s medium (DMEM), divided into two 10-lL injections directed to both poles of the testes. Left testes were similarly inoculated with 20 L adenoviral suspension in DMEM (viral stocks of 50 107 plaque-forming U/mL). The mice were placed to recover from the anesthesia and were left with water and food ad libitum. Animals were sacrified by cervical dislocation at 24 h, 7, 14, 16, and 19 days after inoculation. Dissected testes, epididymis, and spermatozoa collected from the epididymis and ductus deferens were either frozen in liquid N2 or fixed in a 2% paraformaldehyde solution with 150 mM ClNa (pH 7.4). Fixed tissues and spermatozoa were used for histological analysis as late as two weeks after the end of the experiment, whereas frozen samples were used for Western blot analysis as late as 4 weeks. As a negative control, 6 male mice of the same strain were kept throughout the entire experiment without manipulation and, at the end of this, they were sacrificed and processed as described above. Preparation of recombinant adenoviruses. The GFP/LGS carrier adenoviruses (AdCMV-GFPRLGS) contain an expression unit with the cytomegalovirus promoter, a 2.8-kb fragment coding for the fusion of the GFP with the LGS, and the SV40 polyadenylation signal. These adenoviruses were constructed as described in [7] and they were purified as described in [8]. In vitro fertilization. Freshly ovulated oocytes were obtained from 3 to 4-week-old B6 SJL-F1 hybrid female mice induced to superovulate by intra-peritoneal injection of 7.5 IU eCG (Intervet; Salamanca, Spain) and 5 IU hCG (Intervet) administered 48–52 h apart. The oviducts were dissected from mice killed 14–15 h after the hCG injection and cumulus mass was released into 1 mL drops of pregassed Whittingham’s medium [9] plus 30 mg/mL bovine serum albumin (BSA) under paraffin oil and kept at 37 °C in a 5% CO2 /95% air atmosphere (5% CO2 atmosphere). Sperm were obtained from the cauda epididymes of males, which were inoculated 12 days before the in vitro fertilization procedure as described above. The sperm were released into 500 lL drops of pregassed Whittingham’s medium plus 30 mg/mL BSA under paraffin oil. Spermatozoa were capacitated after 1.5 h at 37 °C in a 5% CO2 atmosphere. Oocytes were co-incubated with 1– 2 106 capacitated sperm/mL for 4 h at 37 °C in a 5% CO2 atmosphere and then they were removed, washed, and placed in M16 culture drops [9] containing 4 mg/mL BSA. Samples of oocytes were removed from insemination or culture drops at 1 h (n ¼ 42), 2 h (n ¼ 44), 3 h (n ¼ 34), 4 h (n ¼ 44), 5 h (n ¼ 35), 6 h (n ¼ 22), 8 h (n ¼ 32), and 24 h (n ¼ 31) post-insemination. The cumulus cells were removed from oocytes by a 3-min incubation at 37 °C with 300 lg/mL hyaluronidase and washed three times in phosphate-buffered saline (PBS). Oocytes were then fixed in 2% paraformaldehyde in PBS for 1 h at room temperature. Then, the cells were washed three times in PBS and incubated in 0.5 lg/mL bisbenzimide Hoechst 33342 in PBS for 3 min at 38.5 °C to simultaneously assess the nuclear stage and the presence of the sperm GFP/LGS. For confocal microscopy, oocytes were placed on poly-(L -lysine)-coated glass coverslips in mounting solution and mounted using a spacer between coverglass and slide. Detection of the GFP/LGS protein. GFP images of fluorescence were obtained with a Leica TCS 4D confocal scanning laser microscope (Leica Lasertechnik; Heidelberg, Germany), adapted to an inverted Leitz DMIRBE microscope and a 63 (NA 1.4 oil) Leitz Plan-Apo lens (Leitz; Stuttgart, Germany). The light source was an argon/krypton laser of 74 mW. Western blot analysis based on SDS-gel electrophoresis and further processing of nitrocellulose blots were performed as described in
[10–12]. The polyclonal anti-GFP antibody was from Clontech (Palo Alto, CA). Anti-LGS was generated as in [12]. All of the other reactives were of analytical grade and came from Sigma (St. Louis, MO), Merck (Darmstadt, Germany) or BioRad Laboratories (Hercules, CA).
Results Presence of the green fluorescence protein/liver glycogen synthase chimeric protein in mice testes and spermatozoa Inoculation of the GFP/LGS DNA induced an intense testicular protein expression. After 24 h, GFP/LGS protein was mainly present in the interstitial tissue (Fig. 1B). This presence was expanded to the spermatic tubules 7–16 days after inoculation (Figs. 1C and D). Afterwards, GFP/LGS protein presence began to decrease and it was only present in interstitial tissue after 19 days (Fig. 1E). These results were confirmed after Western blot analysis of testes (data not shown). Furthermore, GFP/LGS protein was also expressed in the not inoculated testes, although the signal intensity seemed to be less intense (data not shown). About 70% of the collected spermatozoa showed the presence of the protein 7–16 days after inoculation (Fig. 2). There were no apparent differences in the percentages of motility and morphological abnormalities and in the total number of spermatozoa obtained from both left and right testes nor from inoculated and control animals (data not shown). The GFP/LGS protein was located in the head, the midpiece, and the cranial segment of the principal piece (Fig. 2). The Western blot analysis using the anti-GFP antibody showed the presence of a specific 120-kDa band in both left and right testes 7–16 days after inoculation (Fig. 3). Western blot analysis against LGS showed results similar to that obtained with the anti-GFP antibody (data not shown). Spermatozoa from non-inoculated mice did not show any signal corresponding to the GFP/LGS protein (Fig. 3). Presence of the green fluorescence protein/liver glycogen synthase chimeric protein in oocytes subjected to in vitro fertilization Twenty-five out of the 42 oocytes recovered after 1 h of co-culture showed the presence of one more sperm carrying GFP/LGS (GFP-spz) attached to the zona pellucida (Figs. 4A and B). The GFP-spz showed the GFP signal only in the head (Fig. 4B). Very similar results were determined in oocytes after 2, 3, 4, 5, and 6 h of co-culture (data not shown). In the latter samples, there were some GFP-spz which initiated oocyte penetration and GFP was observed inside the oocyte (data not shown). After 8 h of co-culture, 17 out of 32 collected oocytes showed a clear signal of penetration with a GFP-spz (Figs. 4C and D). None of the 2–3-cell
T. Mogas et al. / Biochemical and Biophysical Research Communications 297 (2002) 841–846
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Fig. 1. Expression of GFP/LGS in mouse testes. (A) Testes from an untreated animal. (B)–(E) Testes from inoculated mice after 24 h (B), 7 days (C), 16 days (D), and 19 days (E). Magnification: 200. Arrows indicate the lumen of seminiferous tubules.
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T. Mogas et al. / Biochemical and Biophysical Research Communications 297 (2002) 841–846
Fig. 2. Expression of GFP/LGS in spermatozoa from epididymis and ductus deferens 7 days after inoculation (arrows indicate the positive signal). (A) A significant picture showing the high percentage of GFP/LGS spermatozoa. (B) Individual spermatozoon showing the head and midpiece location of GFP/LGS protein (arrows). (C) Spermatozoa from negative control mice observed. There were no signals of the cells. Magnification: 1000.
embryos observed after 42 h of co-culture showed significant signal for GFP/LGS (data not shown).
Discussion Our results show that intra-testicular inoculation of a GFP-containing chimeric gene resulted in the successful expression of the GFP-fused protein in spermatozoa developed from these testes. Moreover, the GFP-spz showed no apparent differences, in terms of motility or morphological abnormalities, than those obtained from non-inoculated animals. This results in fully functional cells, since GFP-spz showed penetrating ability tracking their presence into the oocytes. This is in accordance with the results shown in [4], where the presence of GFP
protein has been detected in spermatogenic cells after intra-testicular inoculation of the GFP gene [4]. These result will then allow the development of a feasible system to easily track a specific sperm protein during the entire sperm life cycle. It is worth noting that the GFP-linked protein has a more prolonged presence in the testes interstitial tissues than in germ cells and spermatozoa. This would be related to the different proliferative activities of these testicular fractions. Thus, since the most proliferative testicular cells were the germ-line ones [13], it is logical that GFP expression is more transient in these cells, since adenoviral vectors do not integrate into chromosomes efficiently, remaining episomal [14]. On the other hand, the majority of the sperm-linked GFP protein have to be synthesized in spermatogonia, since DNA
T. Mogas et al. / Biochemical and Biophysical Research Communications 297 (2002) 841–846
Fig. 3. Western blot analysis of spermatozoa extracts with an anti-GFP antibody. Spermatozoa were obtained from epididymis and ductus deferens of inoculated (I) or non-inoculated (NI) testes 7 days, 14 days or 16 days after inoculation. Another lane shows spermatozoa extracts obtained from non-inoculated mice (C-). M: Molecular weight marker. It has to be stressed that the total protein amount which was loaded in each lane was different, due to difficulties in the protein obtention from the scarce sperm samples. Thus, the information involving the Western blots has to be analysed only under a qualitative point of view.
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translation to protein is low or absent in the germ cells, which initiate meiosis and onwards [3,13]. Hence, the presence of GFP-linked protein in spermatozoa is the end-point for a protein synthesis process, which is initiated in the first stages of the germ cell’s life, and it is translated in the presence of GFP-marked spermatozoa at 7–16 days after adenoviral inoculation. Adenoviral vectors were transported from the inoculated testes to the contra-lateral one, where they expressed the gene. We can speculate that this transport was caused by the capillary/ies bursting and additional extravasation, which was a consequence of testicular inoculation. We have not tested the possible expression of the GFP-marked gene in other tissues, since we did not store them. However, our results indicate that the technique can cause GFP-linked expression in extratesticular tissues via blood flux as a spreading system for adenoviral expression.
Fig. 4. (A) and (B), Significant surface images of the initial oocyte-spermatozoa interaction after 1 h of co-culture. (C) and (D), Significant threedimensional images of the oocyte-spermatozoon interaction after 8 h of co-culture. (A) and (C) show the image obtained with the Hoechst 33,342 stain, whereas (B) and (D) show the presence of the green fluorescence protein. The background of images (A) and (C) has been artificially increased to facilitate the observation of the structures. Arrows indicate sperm heads and asterisk indicates the nucleus of the oocyte. Magnification: 500.
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T. Mogas et al. / Biochemical and Biophysical Research Communications 297 (2002) 841–846
It is worth noting that the GFP signal was associated with the sperm heads, but not with tails, in both cells attached to the outer oocyte surface and those placed inside the oocyte. This result can be explained in two ways. First, the amount of GFP/LGS associated with the sperm tail was much lower than that of the head and this small amount was totally diluted in the image. We have to remember that the GFP-associated bioluminescence is not artificially strengthened, and thus, the signal is strictly related to the amount of luminescent protein [15]. The combination of the enormous size difference between the oocyte and spermatozoa and the lack of potency of the GFP-associated bioluminescence could render the loss of the tail signal. This possibility has to be taken into account to develop further refinements to intensify the fluorescence related to the protein in our conditions. A second explanation is that the GFP/LGS protein suffered a change of location from the tail to the head after the GFP-spz/oocyte interaction. This phenomenon has been described with different sperm proteins, such as PH-20 protein [16,17] or fertilin [18]. Unpublished results from our laboratory indicate that the SGS changes its sperm location when the environmental conditions change. It would be logical that the strongly related GFP/LGS has the same behaviour. However, more experiments are needed to confirm if intra-spermatic displacements play a leading role in the control of the sperm glycogen synthase.
Acknowledgments We thank Mr. Alejandro Pe~ na and Mrs. Anna Adrover for their skillful technical assistance, Mr. Chuck Simmons for assistance in preparing the English manuscript, and the Serveis Cientıfico-Tecnics of the University of Barcelona and the Servei de Microscopia Electr onica of the Autonomous University of Barcelona for the confocal microcopy facilities. This work was supported by Grant 97/2040 from FISSS (Spain).
References [1] G.D. Palermo, L.T. Colombero, Z. Rosenwaks, The human sperm centrosome is responsible for normal syngamy and early embryonic development, Rev. Reprod. 2 (1997) 19–27. [2] K. Swan, Soluble sperm factors and Ca2þ release in eggs at fertilization, Rev. Reprod. 1 (1996) 33–39.
[3] E.M. Eddy, The spermatozoon, in: E. Knobil, J.D. Neill (Eds.), The Physiology of Reproduction, vol. 1, Raven Press, New York, 1988, pp. 27–69. [4] Y. Yamazaki, T. Yagi, T. Ozaki, K. Imoto, In vivo gene transfer to mouse spermatogenic cells using green fluorescence protein as a marker, J. Exp. Zool. 286 (2000) 212–218. [5] T. Yonezawa, Y. Furuhata, K. Hirabayashi, M. Suzuki, M. Takahashi, M. Nishihara, Detection of transgene in progeny at different developmental stages following testis-mediated gene transfer, Mol. Reprod. Dev. 60 (2001) 196–201. [6] M. Sato, A. Ishikawa, M. Kimura, Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa, Mol. Reprod. Dev. 61 (2002) 49–56. [7] T. Becker, R. Noel, W.S. Coats, A.M. G omez-Foix, T. Alam, R.D. Gerard, C.B. Newgard, Use of recombinant adenovirus for metabolic engineering of mammalian cells, Methods Cell Biol. 43 (1994) 161–189. [8] T. Ragot, P. Opolon, M. Perricaudet, Adenoviral gene delivery, Methods Cell Biol. 52 (1997) 229–260. [9] B. Hogan, R. Beddington, F. Costantini, E. Lacy, Culture media for preimplantation-stage embryos, in: E. Hogan, R. Beddington, F. Constantini, E. Lacy (Eds.), Manipulating the Mouse Embryo: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, New York, 1994, pp. 389–398. [10] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [11] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Biotechnology 24 (1992) 145–149. [12] J. Ballester, J.M. Fernandez-Novell, J. Rutllant, M. GarcıaRocha, M.J. Palomo, T. Mogas, A. Pe~ na, T. Rigau, J.J. Guinovart, J.E. Rodrıguez-Gil, Evidence for a functional glycogen metabolism in mature mammalian spermatozoa, Mol. Reprod. Dev. 56 (2000) 207–219. [13] D.M. De Kretser, J.B. Kerr, The cytology of the testis, in: E. Knobil, J.D. Neill (Eds.), The Physiology of Reproduction, vol. 1, Raven Press, New York, 1988, pp. 837–933. [14] L.D. Stratford-Perricaudet, I. Makeh, M. Perricaudet, M. Briand, P. Widespread, Long-term gene transfer to mouse skeletal muscles and heart, J. Clin. Invest. 90 (1992) 626–630. [15] A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, R.Y. Tsien, Understanding, improving and using green fluorescence proteins, TIBS 20 (1995) 448–455. [16] A.E. Cowan, D.G. Myles, D.E. Koppel, Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm, J. Cell Biol. 104 (1987) 917–923. [17] A.E. Cowan, D.G. Myles, D.E. Koppel, Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism, Dev. Biol. 144 (1991) 189–198. [18] G.R. Hunnicutt, D.E. Koppel, D.G. Myles, Analysis of the process of localization of fertilin to the sperm posterior head plasma membrane domain during sperm maturation in the epididymis, Dev. Biol. 191 (1997) 146–159.
Expression of a green fluorescence protein-carrier protein into mouse spermatozoa Teresa Mogas,a Josep M. Fern andez-Novell,b Maria Jes us Palomo,a Pedro J. Otaegui,c Roger R. Gomis,b Joan Ballester,a Dolors Izquierdo,a Joan J. Guinovart,b Joan C. Ferrer,b Teresa Rigau,a and Joan E. Rodrıguez-Gila,* a
Unitat de Reproducci o, Departament de Medicina i Cirurgia Animals, Facultat de Veterin aria, Universitat Aut onoma de Barcelona, E-08193 Bellaterra, Spain b Departament de Bioquımica i Biologia Molecular, Universitat de Barcelona, C/. Martı i Franqu es, 1, E-08028 Barcelona, Spain c Departament de Bioquımica i Biologia Molecular, Universitat Aut onoma de Barcelona, E-08193 Bellaterra, Spain Received 29 July 2002
Abstract Intra-testicular inoculation of an adenoviral vector carrying the fusion gene Aequorea victoria green fluorescence protein/rat-liver glycogen synthase (GFP/LGS) resulted in the presence of GFP/GLS in spermatozoa from 7 days to, at least, 16 days after inoculation. The GFP/LGS was detected in the sperm heads after an ‘‘in vitro’’ fertilization procedure, either before or after the oocyte penetration. Our results indicate that spermatozoa carrying GFP/LGS protein conserved their fertilizing ability and were also detectable after oocyte penetration. This technique will allow to develop an easy system to follow the fate of mature sperm proteins. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Green fluorescence protein; Mature spermatozoa; Protein tracking
In recent years, there has been an increasing amount of information about the role that mammalian spermatozoa play in modulating fecundation and early embryonic development. Thus, the human sperm centrosome is responsible for normal syngamy and early embryonic development [1]. Moreover, spermatozoa introduce a factor of 33 kDa into the oocyte cytoplasm, which is needed to activate the egg [2]. However, the study and the identification of other sperm controlling factors are difficult, owing to the peculiar structure and functions of mammalian spermatozoa [3]. One of the most promising approaches to the study of sperm proteins is to perform a feasible technique to mark a specific sperm protein. This would permit following the fate of this protein from penetration to the first embryonic divisions. The use of chimeric genes, *
Corresponding author. Fax: +34-93-581-2006. E-mail address: [email protected] (J.E. RodrıguezGil).
which integrate the Aequorea victoria green fluorescence protein (GFP), can be a feasible approach to this technique. However, the application of this technique to spermatozoa is difficult, since mammalian sperm is unable to synthesize proteins [3]. In fact, the intra-testicular injection of a GFP-codifying DNA has been carried out only to achieve in vivo gene transfer, but not to generate an spermatic protein that could be tracked during the sperm life cycle [4–6]. The main goal of this work is to develop a system by which fully functional mammalian spermatozoa have a GFP-marked protein. This is developed by intra-testicular inoculation with an adenoviral vector containing the chimeric gene of the GFP and the rat-liver glycogen synthase DNA (GFP/LGS). The idea is that a massive testicular expression of the GFP/LGS DNA will result in the appearance of a specific protein marking in spermatozoa. This will allow to specifically study the fate of a determined spermatic protein during its life cycle, from ejaculation to the first stages of embryonic development.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 2 9 5 - 7
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T. Mogas et al. / Biochemical and Biophysical Research Communications 297 (2002) 841–846
Materials and methods Obtainment of green fluorescence protein-marked spermatozoa. Thirty male B6 SJL-F1 mice (age: 2 months) were anesthetized and analgesized by a single intra-peritoneal injection of 0.05 mg/kg buprenorphine, 100 mg/kg ketamine, and 2.5 mg/kg azepromacin. Mice were inoculated in both testes in the following manner: Right testes were inoculated with 20 lL Dulbecco’s modified Eagle’s medium (DMEM), divided into two 10-lL injections directed to both poles of the testes. Left testes were similarly inoculated with 20 L adenoviral suspension in DMEM (viral stocks of 50 107 plaque-forming U/mL). The mice were placed to recover from the anesthesia and were left with water and food ad libitum. Animals were sacrified by cervical dislocation at 24 h, 7, 14, 16, and 19 days after inoculation. Dissected testes, epididymis, and spermatozoa collected from the epididymis and ductus deferens were either frozen in liquid N2 or fixed in a 2% paraformaldehyde solution with 150 mM ClNa (pH 7.4). Fixed tissues and spermatozoa were used for histological analysis as late as two weeks after the end of the experiment, whereas frozen samples were used for Western blot analysis as late as 4 weeks. As a negative control, 6 male mice of the same strain were kept throughout the entire experiment without manipulation and, at the end of this, they were sacrificed and processed as described above. Preparation of recombinant adenoviruses. The GFP/LGS carrier adenoviruses (AdCMV-GFPRLGS) contain an expression unit with the cytomegalovirus promoter, a 2.8-kb fragment coding for the fusion of the GFP with the LGS, and the SV40 polyadenylation signal. These adenoviruses were constructed as described in [7] and they were purified as described in [8]. In vitro fertilization. Freshly ovulated oocytes were obtained from 3 to 4-week-old B6 SJL-F1 hybrid female mice induced to superovulate by intra-peritoneal injection of 7.5 IU eCG (Intervet; Salamanca, Spain) and 5 IU hCG (Intervet) administered 48–52 h apart. The oviducts were dissected from mice killed 14–15 h after the hCG injection and cumulus mass was released into 1 mL drops of pregassed Whittingham’s medium [9] plus 30 mg/mL bovine serum albumin (BSA) under paraffin oil and kept at 37 °C in a 5% CO2 /95% air atmosphere (5% CO2 atmosphere). Sperm were obtained from the cauda epididymes of males, which were inoculated 12 days before the in vitro fertilization procedure as described above. The sperm were released into 500 lL drops of pregassed Whittingham’s medium plus 30 mg/mL BSA under paraffin oil. Spermatozoa were capacitated after 1.5 h at 37 °C in a 5% CO2 atmosphere. Oocytes were co-incubated with 1– 2 106 capacitated sperm/mL for 4 h at 37 °C in a 5% CO2 atmosphere and then they were removed, washed, and placed in M16 culture drops [9] containing 4 mg/mL BSA. Samples of oocytes were removed from insemination or culture drops at 1 h (n ¼ 42), 2 h (n ¼ 44), 3 h (n ¼ 34), 4 h (n ¼ 44), 5 h (n ¼ 35), 6 h (n ¼ 22), 8 h (n ¼ 32), and 24 h (n ¼ 31) post-insemination. The cumulus cells were removed from oocytes by a 3-min incubation at 37 °C with 300 lg/mL hyaluronidase and washed three times in phosphate-buffered saline (PBS). Oocytes were then fixed in 2% paraformaldehyde in PBS for 1 h at room temperature. Then, the cells were washed three times in PBS and incubated in 0.5 lg/mL bisbenzimide Hoechst 33342 in PBS for 3 min at 38.5 °C to simultaneously assess the nuclear stage and the presence of the sperm GFP/LGS. For confocal microscopy, oocytes were placed on poly-(L -lysine)-coated glass coverslips in mounting solution and mounted using a spacer between coverglass and slide. Detection of the GFP/LGS protein. GFP images of fluorescence were obtained with a Leica TCS 4D confocal scanning laser microscope (Leica Lasertechnik; Heidelberg, Germany), adapted to an inverted Leitz DMIRBE microscope and a 63 (NA 1.4 oil) Leitz Plan-Apo lens (Leitz; Stuttgart, Germany). The light source was an argon/krypton laser of 74 mW. Western blot analysis based on SDS-gel electrophoresis and further processing of nitrocellulose blots were performed as described in
[10–12]. The polyclonal anti-GFP antibody was from Clontech (Palo Alto, CA). Anti-LGS was generated as in [12]. All of the other reactives were of analytical grade and came from Sigma (St. Louis, MO), Merck (Darmstadt, Germany) or BioRad Laboratories (Hercules, CA).
Results Presence of the green fluorescence protein/liver glycogen synthase chimeric protein in mice testes and spermatozoa Inoculation of the GFP/LGS DNA induced an intense testicular protein expression. After 24 h, GFP/LGS protein was mainly present in the interstitial tissue (Fig. 1B). This presence was expanded to the spermatic tubules 7–16 days after inoculation (Figs. 1C and D). Afterwards, GFP/LGS protein presence began to decrease and it was only present in interstitial tissue after 19 days (Fig. 1E). These results were confirmed after Western blot analysis of testes (data not shown). Furthermore, GFP/LGS protein was also expressed in the not inoculated testes, although the signal intensity seemed to be less intense (data not shown). About 70% of the collected spermatozoa showed the presence of the protein 7–16 days after inoculation (Fig. 2). There were no apparent differences in the percentages of motility and morphological abnormalities and in the total number of spermatozoa obtained from both left and right testes nor from inoculated and control animals (data not shown). The GFP/LGS protein was located in the head, the midpiece, and the cranial segment of the principal piece (Fig. 2). The Western blot analysis using the anti-GFP antibody showed the presence of a specific 120-kDa band in both left and right testes 7–16 days after inoculation (Fig. 3). Western blot analysis against LGS showed results similar to that obtained with the anti-GFP antibody (data not shown). Spermatozoa from non-inoculated mice did not show any signal corresponding to the GFP/LGS protein (Fig. 3). Presence of the green fluorescence protein/liver glycogen synthase chimeric protein in oocytes subjected to in vitro fertilization Twenty-five out of the 42 oocytes recovered after 1 h of co-culture showed the presence of one more sperm carrying GFP/LGS (GFP-spz) attached to the zona pellucida (Figs. 4A and B). The GFP-spz showed the GFP signal only in the head (Fig. 4B). Very similar results were determined in oocytes after 2, 3, 4, 5, and 6 h of co-culture (data not shown). In the latter samples, there were some GFP-spz which initiated oocyte penetration and GFP was observed inside the oocyte (data not shown). After 8 h of co-culture, 17 out of 32 collected oocytes showed a clear signal of penetration with a GFP-spz (Figs. 4C and D). None of the 2–3-cell
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Fig. 1. Expression of GFP/LGS in mouse testes. (A) Testes from an untreated animal. (B)–(E) Testes from inoculated mice after 24 h (B), 7 days (C), 16 days (D), and 19 days (E). Magnification: 200. Arrows indicate the lumen of seminiferous tubules.
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Fig. 2. Expression of GFP/LGS in spermatozoa from epididymis and ductus deferens 7 days after inoculation (arrows indicate the positive signal). (A) A significant picture showing the high percentage of GFP/LGS spermatozoa. (B) Individual spermatozoon showing the head and midpiece location of GFP/LGS protein (arrows). (C) Spermatozoa from negative control mice observed. There were no signals of the cells. Magnification: 1000.
embryos observed after 42 h of co-culture showed significant signal for GFP/LGS (data not shown).
Discussion Our results show that intra-testicular inoculation of a GFP-containing chimeric gene resulted in the successful expression of the GFP-fused protein in spermatozoa developed from these testes. Moreover, the GFP-spz showed no apparent differences, in terms of motility or morphological abnormalities, than those obtained from non-inoculated animals. This results in fully functional cells, since GFP-spz showed penetrating ability tracking their presence into the oocytes. This is in accordance with the results shown in [4], where the presence of GFP
protein has been detected in spermatogenic cells after intra-testicular inoculation of the GFP gene [4]. These result will then allow the development of a feasible system to easily track a specific sperm protein during the entire sperm life cycle. It is worth noting that the GFP-linked protein has a more prolonged presence in the testes interstitial tissues than in germ cells and spermatozoa. This would be related to the different proliferative activities of these testicular fractions. Thus, since the most proliferative testicular cells were the germ-line ones [13], it is logical that GFP expression is more transient in these cells, since adenoviral vectors do not integrate into chromosomes efficiently, remaining episomal [14]. On the other hand, the majority of the sperm-linked GFP protein have to be synthesized in spermatogonia, since DNA
T. Mogas et al. / Biochemical and Biophysical Research Communications 297 (2002) 841–846
Fig. 3. Western blot analysis of spermatozoa extracts with an anti-GFP antibody. Spermatozoa were obtained from epididymis and ductus deferens of inoculated (I) or non-inoculated (NI) testes 7 days, 14 days or 16 days after inoculation. Another lane shows spermatozoa extracts obtained from non-inoculated mice (C-). M: Molecular weight marker. It has to be stressed that the total protein amount which was loaded in each lane was different, due to difficulties in the protein obtention from the scarce sperm samples. Thus, the information involving the Western blots has to be analysed only under a qualitative point of view.
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translation to protein is low or absent in the germ cells, which initiate meiosis and onwards [3,13]. Hence, the presence of GFP-linked protein in spermatozoa is the end-point for a protein synthesis process, which is initiated in the first stages of the germ cell’s life, and it is translated in the presence of GFP-marked spermatozoa at 7–16 days after adenoviral inoculation. Adenoviral vectors were transported from the inoculated testes to the contra-lateral one, where they expressed the gene. We can speculate that this transport was caused by the capillary/ies bursting and additional extravasation, which was a consequence of testicular inoculation. We have not tested the possible expression of the GFP-marked gene in other tissues, since we did not store them. However, our results indicate that the technique can cause GFP-linked expression in extratesticular tissues via blood flux as a spreading system for adenoviral expression.
Fig. 4. (A) and (B), Significant surface images of the initial oocyte-spermatozoa interaction after 1 h of co-culture. (C) and (D), Significant threedimensional images of the oocyte-spermatozoon interaction after 8 h of co-culture. (A) and (C) show the image obtained with the Hoechst 33,342 stain, whereas (B) and (D) show the presence of the green fluorescence protein. The background of images (A) and (C) has been artificially increased to facilitate the observation of the structures. Arrows indicate sperm heads and asterisk indicates the nucleus of the oocyte. Magnification: 500.
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It is worth noting that the GFP signal was associated with the sperm heads, but not with tails, in both cells attached to the outer oocyte surface and those placed inside the oocyte. This result can be explained in two ways. First, the amount of GFP/LGS associated with the sperm tail was much lower than that of the head and this small amount was totally diluted in the image. We have to remember that the GFP-associated bioluminescence is not artificially strengthened, and thus, the signal is strictly related to the amount of luminescent protein [15]. The combination of the enormous size difference between the oocyte and spermatozoa and the lack of potency of the GFP-associated bioluminescence could render the loss of the tail signal. This possibility has to be taken into account to develop further refinements to intensify the fluorescence related to the protein in our conditions. A second explanation is that the GFP/LGS protein suffered a change of location from the tail to the head after the GFP-spz/oocyte interaction. This phenomenon has been described with different sperm proteins, such as PH-20 protein [16,17] or fertilin [18]. Unpublished results from our laboratory indicate that the SGS changes its sperm location when the environmental conditions change. It would be logical that the strongly related GFP/LGS has the same behaviour. However, more experiments are needed to confirm if intra-spermatic displacements play a leading role in the control of the sperm glycogen synthase.
Acknowledgments We thank Mr. Alejandro Pe~ na and Mrs. Anna Adrover for their skillful technical assistance, Mr. Chuck Simmons for assistance in preparing the English manuscript, and the Serveis Cientıfico-Tecnics of the University of Barcelona and the Servei de Microscopia Electr onica of the Autonomous University of Barcelona for the confocal microcopy facilities. This work was supported by Grant 97/2040 from FISSS (Spain).
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