Temperature variable and the efficiency of sperm mediated transfection of HPV16 DNA into cells

Center for Fertility and In Vitro Fertilization, Department of Gynecology and Obstetrics, and Department of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, California 92350, USA

Asian J Androl 2002 Sep; 4: 169-173         

Keywords: spermatozoa; human papillomavirus; gene transfer; cumulus; fertility

Abstract

Aim: To pretreat sperm at various temperatures before exposure to human papillomavirus (HPV) 16 DNA fragments and to assess the efficiency of HPV carrier sperm to transfect cumulus cells. Methods: Cumulus cells from follicular aspirates were obtained, pooled and divided into culture dishes containing Sybr Gold-stained HPV DNA carrying sperm that were either pretreated at 4, 37 or 40 (n = 5). The cells were incubated in 5 % CO₂ in air mixture at 37 for 24 hours. The efficiency of sperm to take up fluorescent HPV DNA was determined at hour 0. After incubation, cumulus cell viability was assessed using the eosin method and the percentages of fluorescent cumulus cells determined. Results: Over half of all the cumulus cells became fluorescent with the highest percentage in the 37 group. Sperm pretreated at 4 had the greatest amount of HPV DNA fragments. Total sperm motility was similar for the 3 pretreatment groups. There were no differences in cumulus viability among the groups. Conclusion: Sperm pretreated at 37 transferred the greatest amount of fluorescent HPV DNA fragments to the cumulus cells. The HPV DNA was observed in the nuclear and cytoplasmic compartments. The data suggestedthe possibility of sperm as a vector for the transmission of HPV DNA to the cumulus cells surrounding ovulated oocytes, which might lead to early implantation failures.

Studies demonstrating the capacity of sperm to deliver exogenous DNA into oocytes at the time of fertilization have been of considerable interest and controversy [1-5].The sites of the uptake of foreign DNA fragments have been shown to be the postacrosomal and equatorial regions of the sperm head [6]. Although some of the exogenous DNA fragments appear to be internalized into the compact sperm head, the majority of the fragments are localized externally on the membrane surface [6]. Hence, there is a potential for sperm cells to transfer the external surface bound DNA to any cells that come into contact with the sperm [7].

The presence of human papillomavirus (HPV) DNA in semen has been well-documented [7-8]. Studies that demonstrate the transmission of HPV by the sperm cells serving as carriers or vectors to reproductive cells such as cumulus cells are lacking. It is important to obtain more information concerning the window of opportunity for the absorption and transfer of viral DNA to reproductive cells. The two main hypotheses were that temperature did not affect the sperm capacity to take up exogenous DNA and that the exogenous DNA was not transferable to cumulus cells. The objectives were: (a) to pretreat human sperm at various temperatures before exposing them to DNA fragments from HPV type 16 and (b) to introduce motile sperm carrying HPV DNA fragments to cumulus cells in a co-culture environment and assess the transference efficiency. The HPV DNA was stained with a recently developed and very sensitive fluorescent dye, Sybr Gold, which permitted tracking of the HPV DNA from cell to cell. The data obtained will be important in delineating the role of sperm as vectors for viral DNA and provide information on implantation failures due to sperm-mediated viral transmission to cells of the reproductive system.

2.1 Preparation of sperm cells

Semen specimens from a known HPV-negative donor were washed using the discontinuous 2-layer 90: 45 % colloid (Isolate; Irvine Scientific, Santa Ana, USA) gradient centrifugation wash [10]. Briefly, the 2-layer colloid gradient wash consisted of layering a portion (0.5 to 1.0 mL) of each semen specimen on top of a 2 layer dis-continuous colloid gradient inside a tube. Each colloid gradient was prepared by pipetting 1.5 mL of the 90 % colloid solution to the bottom of the 15 mL centrifuge tube and gently layering 1.5 mL of the 45 % colloid solution on top of the 90 % layer. The tubes of layered semen on top of the colloid gradients were centrifuged for 20 min at 300 g. The supernatants were decanted and each pellet resuspended in 1 mL of modified HTF (HEPES-synthetic human tubal fluid, Irvine Scientific, Santa Ana, USA) supplemented with 0.5 mg/mL human albumin fraction V (Irvine Scientific, Santa Ana, USA). The tubes of sperm were recentrifuged for 10 min. Each resultant pellet was resuspended in 0.4 mL of modified HTF.

2.2 Temperature Treatments and Exposure to HPV

The washed sperm cells were equally divided into 3 portions. Sperm cells in the first portion were incubated at 37 for 2 hours (37 treatment), while the second portion was added with an equal volume of refrigeration medium (Irvine Scientific, Santa Ana, USA) and kept at 4 for 2 hours (4 treatment). After incubation, the 4-treated sperm were warmed in a 37 water bath for 5 min, centrifuged at 300 g for 10 min, and the sperm pellet resuspended in 1 mL of modified HTF. The third portion of washed sperm was placed in a 40oC incubator for 2 hours (40 treatment).

The sperm cells from the 4, 37 and 40 treatment groups were exposed (15 min, 23, pH 7.6) to approximately 0.01 mg/mL Sybr Gold-stained (Molecular Probes Inc., Eugene, USA) HPV 16 DNA fragments previously synthesized by polymerase chain reaction (PCR) [11]. Sybr Gold was chosen for the study because it was a novel DNA stain with proven greater sensitivity when compared with traditional stains such as ethidium bromide. The PCR synthesis used primers targeting the E6-E7 open reading frames (ORFs) region of HPV type 16 (98 base-pairs, bp), a conserved region shown to remain intact during virus-host genome DNA integration [12]. The source was HPV-positive cervical condyloma tissues and the synthesized DNA fragments were verified using 5 % acrylamide gel electrophoresis.

After exposing the sperm cells to the HPV-16 DNA fragments, they were washed using the 2-layer discontinuous 90: 45 % colloid wash to remove extraneous HPV DNA fragments. Aliquots of the resuspended sperm carrying HPV 16 DNA in each of the 3 different treatment groups were pipetted out and sperm motility parameters assessed using the Hamilton Thorn sperm motility analyzer (HTM-C, Hamilton-Thorn Corporation, Danvers, USA). The presence of the HPV DNA fragments in the washed sperm was also determined using fluorescent microscopy to detect the Sybr Gold-stained DNA fragments. The fluorescent images of the sperm were recorded and the pixel intensities assessed after conversion to gray-scale followed by analyses using the Paint Shop Pro 7 software (Jasc Software, Inc., Eden Prairie, USA). A higher pixel intensity reflected greater adsorption and/or uptake of the HPV DNA fragments.

2.3 Transfection and Analysis of Cumulus Cells by HPV-Carrier Sperm

The washed HPV 16-carrying sperm from each of the 3 different temperature treatment groups were placed in the center wells of Falcon 3037 dishes in bicarbonate-based HTF medium. Cumulus cells, obtained from discarded aspirates after assisted reproductive technology (ART) procedures [13], were pooled, divided and pipetted into the culture dishes containing the HPV-carrier sperm. Other types of target cells were not available during the ART procedures and the cumulus cells served as their own control. The cumulus cells were incubated in 5 % CO₂ in air mixture at 37 for 24 hours.

After incubation, drops of cumulus cells from each of the 3 different treatment groups were pipetted on to glass slides. Each glass slide had vaseline drops at each corner serving as posts to maintain a space below the cover slip in order to avoid flattening and destroying the cumulus cells. The cumulus cells were stained in 0.5 % eosin viability stain [14,15] and the percentages of live cumulus cells noted. Separate glass slides with cumulus cells were examined using a fluorescent microscope at 250 magnification and the percentages of fluorescent cumulus cells from each of the 3 treatment groups were determined.

The pixel intensities of the fluorescent sperm heads were presented as meanSEM. The pixel intensities, sperm motility and the percentages of transfected fluorescent cumulus cell were analyzed using the Student's t-test. P<0.05 was considered significant.

3 Results

Over half of all the cumulus cells became fluorescent after 24 hours of co-incubation with sperm carrying fluorescent HPV type 16 DNA fragments (Table 1). Sperm that were pre-treated at 40 for 1 hour took up the least amount of HPV DNA fragments when compared with sperm pre-treated at the 4 temperature. There were no differences in the total sperm motility in the 3 pre-treatment temperature groups. Most noteworthy was the observation that sperm pre-treated with mild heat (40) transferred the least amount of HPV DNA to the cumulus cells after 24 hours of co-incubation. About half of the cumulus cells remained viable after 24 hours of culture and there were no differences in viability among the treatment groups.

Table 1. Temperature pretreatment of sperm affects efficiency of transmission of HPV 16 DNA fragments from the sperm to cumulus cells. meanSEM, ^bP<0.05, compared with 40 pretreatment group.

Figure 1. Gray scale images of cultured cumulus cells showing fluorescence after transfection by sperm carrying fluorescent HPV 16 DNA fragments. The incubation was at 37 in 5 % CO₂ in air mixture for 24 hours. The sperm cells were pretreated by incubating at either (a) 37 (b) 40 or (c) 4 for 2 hours before HPV DNA pickup and subsequent exposure to the cumulus cells. Arrows in the figure indicate unincorporated sperm outside the cumulus cells. Dense fluorescence in some cumulus cells suggests multiple sperm contact with those cells.

4 Discussion

Cumulus cells (12-15 mm) are larger in size when compared with the length (5-6 mm) of the sperm head [16] and they can be easily distinguished when viewed using the phase contrast microscope. Cumulus cells, derived from follicular granulosa cells, are found around each ovulated oocyte with the innermost layer forming the corona radiata. In this study, the cumulus cells that were bombarded with sperm carrying fluorescent HPV DNA also became fluorescent. The Sybr Gold fluorescent stain used in this study was a new type of very sensitive stain recently developed to detect very small DNA fragments and track internalized DNA movements [17]. The fluorescence in each cumulus cell was evenly distributed throughout the nucleus and in the cytoplasm. The smaller sized fluorescent sperm could also be seen randomly attached to the outside surface membrane of the cumulus cell.

The results suggested the transference of exogenous HPV DNA from the carrier sperm into at least 52 % and up to 68 % of the cumulus cells. Furthermore, the group of HPV-carrier sperm pre-treated at 37 was associated with the highest percentage of fluorescent cumulus cells when compared with the 40oC pre-treatment group suggesting that temperature affected the efficiency of transmitting exogenous DNA to target cells. The rationale for studying the temperature variable was based on previous reports demonstrating temperature effects on gene transfer efficiency [18-20]. Kim and colleagues [20] were successful in transferring lacZ plasmids into primary rat fibroblasts only when the temperature was 37oC and not at room temperature. The mechanism of the sperm uptake of exogenous DNA fragments involved temperature-dependent binding kinetics of receptors identified on the sperm membrane. A class of 30-35 kDa proteins related to the major histocompatibility complex (MHC) class II genes has been implicated as the binding proteins for exogenous DNA on the sperm surface [18,21-22]. The exogenous DNA has been localized to the equatorial and postacrosomal regions of the sperm head [23]. Interestingly, although sperm pretreated at 4oC had the greatest amount of HPV DNA fragments as detected by the high fluorescent intensity at the sperm head, this was not significantly different from sperm pretreated at 37.

In this study, viral DNA from HPV was used as the exogenous DNA fragment because of the relevance of HPV infection in the cervical canal and the site of sperm deposition during coitus. HPV is considered an epithe-liotropic DNA virus because certain types cause warts or papillomas. In addition, HPV, particularly type 16, has been implicated as the cause of some spontaneous abortions [24]. Low-risk HPV types include 6 and 11 while high-risk types include 16, 18, 31, 33, 35, 39, 52, 56 and 58 [25]. Rigorous sperm washing procedures that separate sperm cells from the seminal plasma are not effective in removing this virus in infected specimens as shown by sensitive PCR assays [26]. DNA fragments (80-98 base pairs) rather than oncoproteins from the transforming gene region, E6-E7, were utilized for transfection because of previous studies demonstrating the capacity of sperm to deliver the exogenous DNA [7].

The exact mechanism of transference from sperm to the cumulus cells remains unknown. It has been reported that somatic cells have the capacity to engulf sperm cells [27] and it is possible that the cumulus cells took up the exogenous HPV DNA through an endocytosis mecha-nism. A recent report corroborates the capacity of sperm to transfer exogenous DNA of HPV types 16 and 18 to cells lining the uterine cavity [7]. Another mechanism to consider would simply be the exchange of membrane particles including the adsorbed HPV DNA between the sperm membrane and cumulus cell membrane at the time of physical contact. This would be followed by internalization of the HPV DNA through CD4 molecules on the cumulus plasma membrane [22]. The status of the sperm, whether it was capacitated and/or acrosome reacted, was not a consideration due to the fact that the exogenous DNA was localized to the postacrosomal region of the sperm head [23].

In summary, the results showed the transmission of fluorescent HPV DNA fragments from the sperm head to the cumulus cells. The HPV DNA was observed in the nuclear and cytoplasmic compartments. The data sug-gestthe possibility of sperm as a vector for the transmission of HPV DNA to the remnant cumulus cells surrounding each ovulated oocyte, which might lead to early implantation failures.

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Correspondence to: Philip J. Chan, Ph.D., Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, Loma Linda, California 92350, USA.

Tel: +1-909-558 2851, Fax: +1-909-558 2450

E-mail: pchann@yahoo.com

Received 2002-05-08 Accepted 2002-08-06