Introduction
Spermatozoa from a variety of species have a spontaneous ability to take up exogenous DNA and internalize it in the nucleus (Anzar & Buhr, Reference Anzar and Buhr2006). During the fertilization process, this foreign DNA is transferred from the spermatozoa to oocytes and further propagated during the development of transgenic embryos (Arias et al., Reference Arias, Sanchez-Villalba, Delgado and Felmer2017). This technique is known as sperm-mediated gene transfer (SMGT) (Smith & Spadafora, Reference Smith and Spadafora2005).
CD4 (cluster of differentiation 4) is a transmembrane glycoprotein found in several mammal cells including spermatozoa (Gobert et al., Reference Gobert, Amiel, Tang, Barbarino, Bene and Faure1990; Dimitrova-Dikanarova et al., Reference Dimitrova-Dikanarova, Marinova and Fichorova1998). Lavitrano et al. (Reference Lavitrano, Maione, Forte, Francolini, Sperandio, Testi and Spadafora1997) demonstrated that membrane CD4-like molecules are responsible for the interaction and binding of exogenous DNA in mice sperm. As the exogenous DNA binding is a CD4-dependent process, the membrane redistribution of these molecules could affect the exogenous DNA uptake by the sperm. Our previous results showed that the sex-sorted sperm have a reduced capacity to interact with and internalize exogenous DNA in comparison with the unsorted ones. However, CD4-like molecule localization was not evaluated in those study (Campos et al., Reference Campos, de Leon, Komninou, Dellagostin, Deschamps, Seixas and Collares2011a, Reference Campos, Komninou, Urtiaga, de Leon, Seixas, Dellagostin, Deschamps and Collares2011b).
It has been demonstrated in other species that the sex-sorting process itself can lead to marked changes in the sorted cells (Suh et al., Reference Suh, Schenk and Seidel2005; Quan et al., Reference Quan, Ma, Li, Wu, Li, Ni, Lv, Zhu and Hong2015; Balao da Silva et al., Reference Balao da Silva, Ortega-Ferrusola, Morrell, Rodriguez Martinez and Pena2016), specifically in the sperm membrane, such as increase in phospholipid disorder (Balao da Silva et al., Reference Balao da Silva, Ortega-Ferrusola, Morillo Rodriguez, Gallardo Bolanos, Plaza Davila, Morrell, Rodriguez Martinez, Tapia, Aparicio and Pena2013) and protein redistribution (Spinaci et al., Reference Spinaci, Volpe, Bernardini, de Ambrogi, Tamanini, Seren and Galeati2006). However, there is no related report on the sex-sorted bovine sperm so far. In this sense, we hypostatized that sex sorting could lead to CD4 redistribution in bovine sperm cells leading to a reduced DNA binding in comparison with the unsorted sperm. Such reduction in the efficiency of SMGT can be overcome by lipofection. The use of liposomes to introduce exogenous DNA into spermatozoa has been widely demonstrated (Ball et al., Reference Ball, Sabeur and Allen2008; Campos et al., Reference Campos, de Leon, Komninou, Dellagostin, Deschamps, Seixas and Collares2011a; Xin et al., Reference Xin, Liu, Zhao, Wang, Liu, Zhang and Qi2014), but the reports of its application in bovine SMGT using sex-sorted cells are scarce.
Therefore, this study aimed to investigate whether the flow cytometry-based sex-sorting process leads to the membrane phospholipid disorder and redistribution of the CD4-like molecules. In addition, we also studied the effect of this redistribution in exogenous DNA binding and the efficiency of lipofection in sex-sorted bovine sperm.
Materials and Methods
Reagents and media
Opti-MEM I medium and DNase I of amplification grade were obtained from Invitrogen (Carlsbad, CA, USA); anti-bovine CD4 monoclonal antibody was procured from BioRad (Hercules, CA, USA). Merocyanine-540, Lectin from Arachis hypogaea (peanut) conjugated with FITC and Hoechst 33342 from Sigma-Aldrich (St. Louis, MO, USA) and Lipofectamine® 3000 Reagent were purchased from ThermoFisher Scientific (Waltham, MA, USA).
Sperm processing
Bovine sperm (X-sorted, Y-sorted, and unsorted) from three Senepol bulls were procured from CRV Lagoa Ltd. (São Paulo, SP, Brazil). In addition, for each bull, each straw was taken from different ejaculates to avoid self-replication experiments. Frozen semen straws were thawed in a water bath (36.5°C for 30 s) and the sperm cells were separated from the commercial cryopreservation medium by double centrifugation at 300 g for 5 min, and then were resuspended and maintained in Opti-MEM I medium (Invitrogen, Carlsbad, CA, USA). The spermatozoa concentration was adjusted to 1 × 106 cells per 200 µl to perform the subsequent procedures.
Experimental design
To characterize sperm quality, the kinetics parameters and acrosome integrity of X-sorted, Y-sorted and unsorted bovine sperm were evaluated immediately after thawing.
To determine the effects of sex-sorting procedure on bovine sperm membrane phospholipid disorder and CD4 redistribution, the sperm cells were incubated with anti-bovine CD4 monoclonal antibody followed by incubation with a goat-anti-mouse FITC-conjugated secondary antibody. The cells that were previously processed for CD4 immunostaining were also stained with Merocyanine-540.
To determine the effect of CD4 redistribution on the exogenous DNA binding to bovine sperm, the fluorescence-conjugated exogenous DNA was incubated with the sex-sorted sperm and with those pretreated with anti-bovine CD4 monoclonal antibody. Furthermore, the binding of exogenous DNA associated with the liposome was evaluated.
Each analysis was performed in triplicate (three different ejaculates from the same animal) and was repeated in three different bulls for each bovine sperm (X-sorted, Y-sorted and unsorted).
Analysis of the sperm motion parameters
Motion parameters were determined using a computer-assisted sperm analysis system (AndroVision®, Minitube, Germany) following the methodology previously described for bovine spermatozoa (Canovas et al., Reference Canovas, Gutierrez-Adan and Gadea2010). The CASA-derived kinematic parameters recorded for each spermatozoa were total motility (%), progressive motility (%), distance curved line (DCL, µm), distance average path (DAP, µm), distance straight line (DSL, µm) curvilinear velocity (VCL, µm/s), average path velocity (VAP, µm/s), straight-line velocity (VSL, µm/s), linearity of the curvilinear trajectory (LIN, ratio of VSL/VCL), straightness (STR, ratio of VSL/VAP), wobble of the curvilinear trajectory (WOB, ratio of VAP/VCL), amplitude of lateral head displacement (ALH, µm) and beat cross-frequency (BCF, Hz).
Images were obtained at ×200 magnification using a contrast phase microscope (Olympus BX53, Olympus GmbH, Germany). A minimum of 10 microscopic fields per sample was evaluated to include at least 200 spermatozoa per sample.
Acrosome integrity
The sperm acrosomal integrity was assessed using the fluorescent dye lectin from Arachis hypogaea (peanut) conjugated with FITC (FITC/PNA) in a final concentration of 10 µg/ ml. The sperm cells and the fluorescent dye were homogenized and incubated for 10 min at 38°C. Sperm were sorted by its acrosomes into intact (FITC/PNA −) or reacted (FITC/PNA+). This rate was calculated by the number of acrosome-reacted sperm/total number of sperm and multiplied by 100 to express the percentage.
Indirect immunofluorescence assay
For CD4 immunostaining, the sperm cells were fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (10× PBS, pH 7.4) without Ca2+ and Mg2+ (Invitrogen, Carlsbad, CA, USA) at 4°C for 1 h, washed with PBS and then blocked with 3% BSA in PBS. Antibody dilutions were prepared in the blocking solution (3% BSA in PBS). Anti-bovine CD4 monoclonal antibody was diluted to 1:100 fold and the incubation with spermatozoa was carried out overnight at 4°C. After extensive washing with PBS, the sperm cells were incubated with a goat-anti-mouse FITC-conjugated secondary antibody (1:200 dilution) for 50 min in the dark. The control cells were treated similarly, with the omission of primary antibody.
Staining for detection of the phospholipid disorder in the sperm membrane
The phospholipid disorder in X-sorted, Y-sorted and unsorted bovine sperm membrane was evaluated following the methodology previously described by Balao da Silva et al. (Reference Balao da Silva, Ortega-Ferrusola, Morillo Rodriguez, Gallardo Bolanos, Plaza Davila, Morrell, Rodriguez Martinez, Tapia, Aparicio and Pena2013), staining the cells with merocyanine-540 (M540). The cells that were previously processed through CD4 immunostaining were also stained with M540 at a final concentration of 2.7 μM and with 1 µl of 1.7 μM Hoechst 33342 (H33342) stock solution added to counterstain the nucleus, following incubation for 10 min at 37°C under dark conditions.
Preparation of Cy-3-labelled DNA
To prepare fluorescence-conjugated DNA, Cy-3-labelled primers (Alpha DNA, Montreal, QC, Canada) were used to amplify the 546 bp fragment of the pEGFP-N1 vector (Clontech Laboratories, Basingstoke, UK). The primers used for preparation of the Cy-3-labelled DNA fragment were 5´-ACGTAAACGGCCACAAGTTC and 5´-AGTCGTGCTGCTTCATGTG (Sim et al., Reference Sim, Cha, Song, Kim, Yoon, Choi, Jeong, Kim, Huh, Lee, Kim, Lee, Kim and Chang2013). PCR was carried out for 35 cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s. After confirmation of a single band by gel electrophoresis, the Cy-3-labelled DNA concentration and purity was determined using a UV spectrophotometer (NanoVue Plus, GE Healthcare Life Sciences, NJ, USA), and the samples were stored at –20°C until further use.
Exogenous Cy-3-labelled DNA transfection of the sex-sorted sperm
Lipofectamine 3000® Reagent was prepared according to the manufacturer's instructions with few modifications. Two solutions were prepared: Solution A: 3 µl lipofectamine/25 µl Opti-MEM I; and Solution B: 1000 ng of Cy-3-labelled DNA and 2 µl of P3000 Reagent/50 µl of Opti-MEM I. Solutions A and B (25 µl each) were mixed in 1.5 ml tubes for 15 min at ambient temperature to allow the formation of exogenous DNA–liposome complexes (lipoplexes). After the incubation, the lipoplexes were combined with a suspension of X-sorted, Y-sorted or unsorted sperm (1 × 106 cells/150 µl Opti-MEM I) for a total volume of 200 µl, followed by 1 h incubation at 37.5°C. To remove the DNA remained unattached to the sperm membrane, the cells were centrifuged at 700 g for 5 min and washed three times using Opti-MEM I medium followed by incubation with 6 U of DNase I for 1 h at 37°C and then washed three times using Opti-MEM I medium prior to the flow cytometry analysis.
The transfection of the exogenous DNA without liposome (naked DNA) was carried out as described above. Briefly, a solution containing 1000 ng of Cy-3-labelled DNA was mixed with 1 × 106 cells in a total volume of 200 µl in Opti-MEM I medium. The transfected sperm were subjected to the DNase I treatment to separate them from DNA that remained unattached to the sperm membrane, in the same manner as described above.
Exogenous DNA transfection with anti-bovine CD4 monoclonal antibody treated sperm
Considering that the interaction of exogenous DNA with sperm cells is a CD4-like molecule-dependent process, this experiment was performed using the same anti-bovine CD4 monoclonal antibody that was used in the indirect immunofluorescence assay, to establish whether CD4-like molecule blocking has a direct effect on binding of exogenous DNA to bovine sperm. Briefly, 1 × 106 sperm cells were preincubated overnight with anti-bovine CD4 monoclonal antibody at 1:100 dilution, washed three times by centrifugation, and then were resuspended and incubated in a solution of 1000 ng of Cy-3-labelled DNA for 60 min. The transfected sperm were submitted to the DNase I treatment to separate sperm from exogenous DNA remained unattached to sperm membrane prior to flow cytometry analysis.
Flow cytometry
The intensity of green (CD4), orange (M540 and Cy-3-labelled DNA), and blue fluorescence (H33342) was recorded using band pass filters of 530/30 nm, 574/26 nm, (laser 488 nm), and 450/40 nm (laser 405 nm), respectively, in a flow cytometer (Attune® Acoustic Focusing Cytometer, Life Technologies, Carlsbad, CA, USA).
To determine the phospholipid disorder of the sperm membrane, the samples were divided into subpopulations (low or high disorder of phospholipids of the sperm membrane), and the frequency of each subpopulation was quantified.
The percentage (0–100%) of the transfected X-sorted, Y-sorted, and unsorted sperm was determined by the proportion of the cells emitting orange fluorescence out of the total number of the cells analyzed.
For all analyzes, forward and sideway light scattering were recorded for, in total, 10,000 sperm events per sample (high fluorescence for H33342). Non-sperm events (debris) were identified and eliminated from the analysis (low fluorescence for H33342).
Analysis of sperm membrane and binding of exogenous DNA by scanning spectral confocal microscopy
The channel images of X-sorted, Y-sorted, and unsorted sperm were acquired sequentially using a TCS SP8 scanning spectral confocal laser microscope (Leica©, Germany) at 405 nm/PMT 420–470 nm (H33342), 488 nm/510–550 nm (FITC), 552 nm/560–590 nm (M540 and Cy-3-labelled DNA) (solid-state laser/spectral photomultiplier tube), respectively.
Statistical analysis
Phospholipid disorder, motion parameters, sperm acrosomal integrity and the percentage of sperm cell-bound exogenous DNA were compared using one-way analysis of variance (ANOVA), followed by Tukey's test for multiple comparisons. Pearson's correlation was used to test the phospholipid disorder and CD4 immunoreactivity association. Significance was considered at a P-value of <0.05 in all analyzes. All data were being reported as the mean ± the standard error of the mean (SEM).
Results
Sex-sorted bovine sperm kinetics
Computer-assisted sperm analysis revealed that the sex-sorting procedure alters bovine sperm kinetic parameters (Tables 1 and 2). We observed that both X-sorted and Y-sorted bovine sperm showed a significant decrease in motility (P < 0.05) and progressive motility (P < 0.05), in comparison with the unsorted sperm. However, some motion parameters like DCL, DAP, VCL and VAP were increased in both sex-sorted sperm (Table 2) when compared with the unsorted group, while the others motion parameters analyzed remained unchanged.
Table 1 Values for seminal parameters and acrosome-reacted sperm in sex-sorted bovine sperm groups
Data are expressed as mean ± standard error of the mean (SEM).
a,bNumbers within columns with different superscripts differ (P < 0.05).
Table 2 Values for motility parameters in sex-sorted bovine sperm groups
Data are expressed as mean ± standard error of the mean (SEM). a,b Numbers within columns with different superscripts differ (P < 0.05).
Sperm acrosome integrity
The effects of sex-sorting procedure on bovine sperm acrosome integrity are displayed in Table 1. The unsorted group showed a significantly (P < 0.05) reduced percentage of acrosome-reacted sperm (23.07 ± 3.7) when compared with both X-sorted and Y-sorted sperm groups (40.40 ± 4.50 and 44.5 ± 5.40, respectively).
CD4 redistribution on sex-sorted bovine sperm
The indirect immunofluorescence assay showed that the sex-sorting procedure induces the redistribution of CD4-like molecules on the sperm membrane (Fig. 1 a). In the majority of the unsorted sperm (92% of total sperm cells), CD4 immunoreactivity was detected as a uniform fluorescence pattern over the entire head. In the X-sorted sperm, CD4 was observed in the post acrosomal region and midpiece (87% of total sperm cells), while in Y-sorted sperm (85% of total sperm cells), the immunoreactivity was detected in the form of small spots throughout the entire sperm (head to tail). No CD4 immunoreactivity was detected when the second antibody alone was used in this experiment (data not shown).
Figure 1 Representative immunolocalization of CD4 and phospholipid disorder in unsorted, X-sorted, and Y-sorted bovine spermatozoa. (A, B) (A) Green fluorescence represents positive CD4 immunoreactivity, and (B) the intense red fluorescence represents the disorder of membrane phospholipid. (C) The two-dimensional flow cytometric dot-plots for Merocyanine-540 vs. CD4 immunoreactivity. Arrows indicate CD4 redistribution areas, arrowheads indicate high disorder of membrane phospholipids and the black rectangular area comprises sperm cells with high CD4 redistribution/high disorder of membrane phospholipid. Images were obtained at ×400 magnification. Bar = 10 µm.
Phospholipid disorder of the sperm membrane
The sex-sorting procedure significantly increased the membrane phospholipid disorder in both X-sorted and Y-sorted sperm (29.05 ±2.5 and 19.73 ±1.98, respectively) in comparison with the unsorted sperm (3.85 ±0.18) (Fig. 1 b).
Merocyanine-540 versus CD4 immunoreactivity signal plots of unsorted, X-sorted and Y-sorted bovine sperm are shown in Fig. 1(c). The sex-sorted sperm showed a positive correlation (r = 0.97, P < 0.001) between high phospholipid disorder and CD4 redistribution (Fig. 2).
Figure 2 CD4 redistribution and phospholipid disorder of the sex-sorted sperm membrane. Pearson's correlation analysis shows a significant positive correlation of CD4 redistribution versus phospholipid disorder of X-sorted and Y-sorted bovine sperm (Pearson r = 0.97; P < 0.0001, n = 12). Line represents linear regression of data (Y = 0.9517x + 1.162; r 2 = 0.95). No other regions had any significant correlation between CD4 redistribution and phospholipid disorder.
Effects of CD4 redistribution on exogenous DNA binding on sperm cells
The percentage of unsorted bovine sperm that have bound Cy-3-labelled DNA (45.65 ±1.76) was greater than that observed for the X-sorted and Y-sorted sperm (37.80 ± 2.57 and 34.20 ± 0.98, respectively) (Fig 4). In addition, our results demonstrated that both X-sorted and Y-sorted are equally able of binding to exogenous DNA within 1 h of incubation (Fig. 3 a).
Figure 3 Transfection of exogenous DNA on X-sorted and Y-sorted bovine sperm. (A) Transfection of exogenous DNA associated with liposome in both X-sorted and Y-sorted sperm. (B) Representative images of exogenous Cy-3-labelled DNA bound to bovine sperm cells. Asterisk indicates significant difference between naked DNA and lipoplex groups (P < 0.05). Images were obtained at ×400 magnification. Scale bar = 10 µm. Data are expressed as means ±standard error of the mean (SEM) (N = 3).
The ability of the sex-sorted spermatozoa to interact with exogenous DNA was significantly (P < 0.05) increased with the addition of liposomes in comparison with the naked DNA for the same incubation period (Fig. 3 a), in which X-sorted and Y-sorted showed a transfection efficiency of 59.47 ±12.85% and 72.17 ±0.63%, respectively.
The binding of Cy-3-labelled DNA demonstrating the ability of exogenous DNA uptake by bovine sex-sorted sperm is shown in Fig. 3(b). In the control spermatozoa, which were not incubated with exogenous DNA, no Cy-3-labelled DNA was detected (data not shown).
Preincubation with anti-bovine CD4 monoclonal antibody had a clear effect on the percentage of sperm cells able to interact with exogenous DNA (P < 0.05), resulting in a reduction of 75.44, 64.52 and 95.21% in X-sorted, Y-sorted and unsorted sperm, respectively (Fig. 4).
Figure 4 Exogenous DNA bound to pretreated anti-bovine CD4 monoclonal antibody sperm. Asterisk indicates significant difference between non-treated and treated sperm with anti-bovine CD4 monoclonal antibody (P < 0.05). Data are expressed as means ±standard error of the mean (SEM) (N = 3).
Discussion
To the best of our knowledge, this is the first report demonstrating the redistribution of the CD4-like molecules on bovine sperm cells subjected to sex-sorting procedure. This indicates that sorted cells have reduced transfection potential in comparison with unsorted cells. In addition, our results indicated that CD4 redistribution has a direct effect in the ability to bind exogenous DNA to sex-sorted bovine sperm, which can be overcome by the use of liposomes.
A powerful tool for sperm viability examination is computer-assisted kinetics analysis. Motility and progressive motility data can be used to assess any effects that may be upon sperm cells, such as stress generated by mechanical forces or environmental influence (Ibanescu et al., Reference Ibanescu, Leiding, Ciornei, Rosca, Sfartz and Drugociu2016). In this study, we observed a reduced motility and progressive motility in both sex-sorted sperm groups. These changes in the sex-sorted sperm, could have been caused by exposure to Hoechst 33342 stain, the laser light, or exposure in the droplets to electric charges during the flow cytometric sex-sorting procedure (Garner, Reference Garner2006). According to (Rath et al., Reference Rath, Barcikowski, de Graaf, Garrels, Grossfeld, Klein, Knabe, Knorr, Kues, Meyer, Michl, Moench-Tegeder, Rehbock, Taylor and Washausen2013), the effect of exposure to dye and then, the laser, may reduce mitochondrial activity, causing a decrease in the production of ATP.
In the present study, we also observed that the flow cytometric sex-sorting procedure affected the sperm acrosome integrity, which can substantially impair the ability of sperm cells to fertilize the oocyte as an acrosome intact is necessary to bind to the zona pellucida and fertilize the oocyte (Anifandis et al., Reference Anifandis, Messini, Dafopoulos, Sotiriou and Messinis2014).
Similar to our data, other authors have also shown redistribution of other proteins, such as Heat Shock Protein 70 (HSP70) in boar spermatozoa (Spinaci et al., Reference Spinaci, Volpe, Bernardini, de Ambrogi, Tamanini, Seren and Galeati2006), induced by sex-sorting procedure. While the different isoforms of glucose transporters (GLUTs) did not show any redistribution after flow cytometry based sex sorting in boars (Bucci et al., Reference Bucci, Galeati, Giaretta, Tamanini and Spinaci2013). In the light of these studies, we conclude that protein redistribution induced by sex sorting depends on species and proteins.
Balao da Silva et al. (Reference Balao da Silva, Ortega-Ferrusola, Morillo Rodriguez, Gallardo Bolanos, Plaza Davila, Morrell, Rodriguez Martinez, Tapia, Aparicio and Pena2013) have recently demonstrated that the sex-sorting process in stallion sperm results in an increase in the membrane phospholipid disorder. Although similar studies in bovine are not available, we believe that this increase in disorder is indicative of membrane destabilization, similar to that occurring during the first steps of sperm capacitation (Flesch et al., Reference Flesch, Brouwers, Nievelstein, Verkleij, van Golde, Colenbrander and Gadella2001). One of the first changes that occur in capacitating mammalian spermatozoa is the loss of cholesterol from the sperm plasma membrane (Macias-Garcia et al., Reference Macias-Garcia, Gonzalez-Fernandez, Loux, Rocha, Guimaraes, Pena, Varner and Hinrichs2015). The fatty acids required for the esterification of cholesterol are released after their enzymatic cleavage from the membrane phospholipids. The result of this process is the creation of highly unstable phospholipids that increase membrane fluidity, which promotes sperm capacitation (Aitken & Nixon, Reference Aitken and Nixon2013).
The changes in motion parameters, acrosome integrity, redistribution of membrane proteins and the increase in fluidity (phospholipid disorder) are considered as the hallmark of capacitation status in semen (Gadella et al., Reference Gadella, Tsai, Boerke and Brewis2008; Ashrafzadeh et al., Reference Ashrafzadeh, Karsani and Nathan2013; Balao da Silva et al., Reference Balao da Silva, Ortega-Ferrusola, Morillo Rodriguez, Gallardo Bolanos, Plaza Davila, Morrell, Rodriguez Martinez, Tapia, Aparicio and Pena2013; Gangwar & Atreja, Reference Gangwar and Atreja2015), allowing the sperm to bind to the zona pellucida and immediately thereafter to realize the acrosome reaction (Parrish, Reference Parrish2014). Our data corroborating with these findings demonstrate that the sex-sorting process compromises membrane stability and initiates bovine sperm capacitation-like event. As demonstrated by Trigal et al. (Reference Trigal, Gomez, Caamano, Munoz, Moreno, Carrocera, Martin and Diez2012), male and female embryos produced with sex-sorted bovine sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. However, the early capacitation induced by sex-sorting procedure results in the reduced lifespan of sperm and available period for fertilization (Leahy & Gadella, Reference Leahy and Gadella2011). Thus, this implies that sex-sorted sperm need to be used as soon as possible.
It was previously demonstrated that CD4-like molecules are mainly responsible for binding of exogenous DNA to mice sperm cells during the SMGT technique (Lavitrano et al., Reference Lavitrano, Maione, Forte, Francolini, Sperandio, Testi and Spadafora1997). Our previous results showed that both X-sorted and Y-sorted bovine sperm have a reduced exogenous DNA uptake in comparison with the unsorted sperm (Campos et al., Reference Campos, Komninou, Urtiaga, de Leon, Seixas, Dellagostin, Deschamps and Collares2011b). In this study, we have shown that redistribution of CD4-like molecules in sex-sorted bovine sperm has a direct effect on the ability of the sorted spermatozoa to interact with exogenous DNA. The unsorted sperm have homogeneous CD4 arrangement over the entire head, which could facilitate the interaction with the exogenous DNA in contrast with the sex-sorted sperm. In a study by Zaniboni et al. (Reference Zaniboni, Spinaci, Zannoni, Bernardini, Forni and Bacci2016), both X-sorted and Y-sorted swine spermatozoa showed the same binding capacity and internalization of exogenous DNA. This finding is in accordance with our results, in which no difference in the ability to interact with exogenous DNA is observed in sex-sorted bovine cells during the SMGT procedure, despite the fact that the localization of CD4 on the membrane of X- and Y-sorted bovine sperm is different. A previous study detected 42 proteins differentially expressed in X- and Y-sorted bull spermatozoa. These proteins are associated with energy metabolism, stress resistance, cytoskeletal structure and activity of serine proteases (Chen et al., Reference Chen, Zhu, Wu, Han, Hao, Zhao, Du, Qin, Liu and Wang2012). It can be gleaned from our results that the sex-sorting process does not affect the expression of CD4-like molecules, but only induces the redistribution of these molecules over the sperm membrane.
The significant reduction in exogenous DNA transfection in pretreated anti-bovine CD4 monoclonal antibody sperm confirms our hypothesis that the CD4-like molecules indeed play a crucial role in the process of binding of exogenous DNA to bovine sperm cells as previously observed similarly by Lavitrano et al. (Reference Lavitrano, Maione, Forte, Francolini, Sperandio, Testi and Spadafora1997). In line with this observation, (Wang et al., Reference Wang, Fan, Yu, Zheng and Zhao2011) described that CD4 polymorphisms are highly associated with the ability of the exogenous DNA uptake by goat sperm; and this molecular marker supported the idea that the interaction of sperm cells with exogenous DNA is a CD4-dependent process.
Liposomes are one of the most widely used strategies to improve the efficiency of SMGT. Several studies have demonstrated that endocytosis is the major pathway through which exogenous DNA interacts with and enters the cell when associated with liposomes (Cornelis et al., Reference Cornelis, Vandenbranden, Ruysschaert and Elouahabi2002; Magalhaes et al., Reference Magalhaes, Duarte, Monteiro and Fernandes2014; Rajaganapathy et al., Reference Rajaganapathy, Chancellor, Nirmal, Dang and Tyagi2015). These statements fit perfectly with our results about transfection of exogenous DNA associated with commercially cationic liposomes, as this transfection system improved the binding of exogenous DNA to both X-sorted and Y-sorted sperm. Liposomes have been in use for a long time to introduce a variety of molecules into living cells (Sercombe et al., Reference Sercombe, Veerati, Moheimani, Wu, Sood and Hua2015; Hoelker et al., Reference Hoelker, Mekchay, Schneider, Bracket, Tesfaye, Jennen, Tholen, Gilles, Rings, Griese and Schellander2007; Mo et al., Reference Mo, Zaro, Ou and Shen2012). However, no reports of this liposome application in bovine SMGT using sex-sorted sperm have been published previously.
In conclusion, our results demonstrated that flow cytometry based sex-sorting causes a redistribution of CD4 that could be induced by an increased phospholipid disorder in the membrane of sex-sorted sperm. In addition, these membrane modifications directly affect the process of binding of exogenous DNA to both X-sorted and Y-sorted bovine sperm. The delineation of the factors that affect the efficiency of SMGT in bovines remains a continuous challenge for the successful in vitro production of sex-selected transgenic bovine embryos.
Acknowledgements
This work was supported by the Brazilian CAPES, CNPq and FAPERGS. W.B. Domingues, T.L.R. Silveira and M.H. Remião are students of the Graduate Program in Biotechnology at Universidade Federal de Pelotas and are supported by Brazilian CAPES. O.A. Dellagostin, C.D. Corcini, A.S. Varela Junior, F.K. Seixas, T. Collares and V.F. Campos are research fellows from CNPq. We thank the undergraduate students Bruna Barreto, Ingrid Lessa and Lucas Santos for help in setting up the samples and assays in our laboratory.
Funding provided for this research
Funding for this research was provided by Brazilian CNPq (grant number: 472210/2013–0); and Brazilian CAPES (grant number: AUXPE 2900/2014).
Conflict of interest statement
There are no conflicts of interest.
