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BOR - Papers in Press, published online ahead of print May 11, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041152
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BIOLOGY OF REPRODUCTION 73, 443–451 (2005)
DOI: 10.1095/biolreprod.105.041152
© 2005 by the Society for the Study of Reproduction, Inc.

Cigarette Smoke Inhibits Hamster Oocyte Pickup by Increasing Adhesion Between the Oocyte Cumulus Complex and Oviductal Cilia1

Christine Gieseke , and Prue Talbot 2 

Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous research has shown that oocyte cumulus complex (OCC) pickup rate is inhibited in hamsters when oviducts and OCCs are simultaneously exposed to cigarette smoke solutions, independent of any effect on ciliary beat frequency. The purpose of this research was to determine whether smoke solutions caused a change in adhesion between the OCC and infundibulum of the oviduct and to determine whether a change in adhesion could account for decreased OCC pickup rate. OCC pickup rate and adhesion were measured before and after infundibula or OCCs recovered from acute in vitro exposures to mainstream and sidestream whole, gas, or particulate smoke solutions. Ciliary beat frequency was also measured on infundibula. Overall, smoke solutions decreased oocyte pickup rate 40% to 80% below control levels and increased adhesion 52% to 91% above control levels when infundibula were pretreated. A change in adhesion was observed in cases for which decreased OCC pickup rate could not be explained by a change in ciliary beat frequency. OCC pickup rate decreased 20% to 35% below control levels and adhesion increased 39% to 54% above control levels when OCCs were pretreated. These data show not only that cigarette smoke causes an increase in OCC adhesion to the oviduct, but also that there is a correlation between increased adhesion and decreased OCC pickup rate. The results also show that the both the OCC and oviduct are targets of cigarette smoke. The oviduct is more sensitive to the adverse effects of smoke; however, this may be caused by a combined impact on mechanisms involved in both adhesion and ciliary function.

cumulus cells, female reproductive tract, oviduct, ovum pickup/ transport, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A compact layer of cumulus cells surrounds the oocyte before ovulation in mammalian ovarian follicles [1–5]. Cumulus cells secrete a hyaluronan-rich extracellular matrix in response to the luteinizing hormone surge [6–13]. Secretion of the extracellular matrix causes the cumulus cells to separate in a process referred to as expansion [2, 5]. Proper secretion of the extracellular matrix and expansion of the oocyte cumulus complex (OCC) is necessary for ovulation and pickup [14–18]. Expanded OCCs are ovulated into the peritoneal space in humans or the bursal cavity in rodents and are picked up by the infundibular portion of the oviduct [19]. Pickup involves adhesion of the OCC to the infundibulum and movement of the OCC toward the ostium [14, 18, 20, 21]. The extracellular matrix between the cumulus cells adheres the OCC to the ciliated cells on the external surface of the infundibulum in a transient manner [18, 21]. Cilia on the surface of the infundibulum propel the OCC toward the ostium. Once the OCC enters the ostium, it is transported through the infundibulum to the ampulla, where fertilization occurs [19, 22]. After fertilization, the preimplantation embryo is transported to the uterus for implantation and further embryonic development. Proper timing throughout all stages of ovum pickup and transport is critical for successful fertilization and pregnancy [19, 23, 24].

OCC pickup is a complex process that involves both ciliary beating and adhesion between the oviductal cilia and OCC [14, 21, 25–28]. The OCC first attaches to the tips of the cilia and then is propelled to the ostium and into the lumen of the oviduct by ciliary forces. The cilia create currents that are strong enough to move small particles [21, 27], but the mass of the OCC is too great to be moved by ciliary beating alone [21]. Adhesion is necessary to transiently anchor the OCC to the infundibulum. This ensures constant contact between the beating cilia and the OCC as the cilia pass the OCC toward the ostium.

Factors that interfere with adhesion inhibit OCC pickup by the infundibulum. Treatment of rabbit oviducts with poly-l-lysine blocked pickup of surrogate and freshly ovulated OCCs without affecting ciliary beat frequency [27]. In vitro treatment of hamster oviducts with either poly-l-lysine or wheat germ agglutinin increased adhesion and decreased OCC pickup rate without affecting ciliary beat frequency [18]. Treatment of hamster OCCs with wheat germ agglutinin also increased adhesion and decreased OCC pickup rate. In contrast, treatment of hamster OCCs with poly-l-lysine decreased both adhesion and OCC pickup rate.

Factors that affect ciliary beat frequency have also been shown to alter OCC pickup rate. For example, in vitro experiments showed that increased viscosity of the culture medium decreased both ciliary beat frequency and OCC pickup rate [29] and cyanide, a component of cigarette smoke, decreased ciliary beat frequency and OCC pickup rate [30]. The oviduct has been shown to be a direct target of cigarette smoke during both in vivo [31, 32] and in vitro [30, 33] studies. During in vivo exposures, inhalation of mainstream or sidestream smoke by female hamsters caused blebs to form on the apical surfaces of ciliated cells and decreased the ratio of ciliated to secretory cells lining the oviduct [31]. During in vitro exposures of hamster infundibula to smoke solutions, ciliary beat frequency was decreased by mainstream whole smoke solutions and increased by sidestream whole smoke solutions [33]. Both types of smoke solutions decreased OCC pickup rate. When infundibula were allowed to recover from the exposure to both types of smoke solutions, ciliary beat frequency returned to control levels; however, OCC pickup rate continued to decrease [33]. Although ciliary beat frequency was altered during in vitro exposures to both mainstream and sidestream whole smoke solutions, the changes in ciliary beat frequency alone did not explain the observed decrease in OCC pickup rate. Because OCC pickup involves both ciliary beating and adhesion, it is likely that cigarette smoke solutions interfere with proper adhesion between the OCC and oviductal cilia.

The first purpose of this study was to test the hypothesis that cigarette smoke solutions alter adhesive strength between the hamster OCC and infundibulum, which leads to decreased OCC pickup rate. To test this hypothesis, experiments were designed so that adhesion, ciliary beat frequency, and OCC pickup rate could be measured simultaneously. A change in adhesive strength was expected to correlate with changes in OCC pickup rate. It was expected that decreased OCC pickup rate caused by changes in adhesion would be observed in cases when decreased OCC pickup rate could not be explained by changes in ciliary beat frequency. The second purpose of this study was to compare the relative sensitivity of the OCC and infundibulum to smoke exposure and to determine whether the OCC was a direct target of cigarette smoke.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Eight- to twelve-week-old female golden hamsters (Mesocricetus auratus) were purchased from Harlan (San Diego, CA) and kept in a 26°C room on a 14 h light:10 h dark cycle. All had free access to Purina rodent chow and water. Females were evaluated each morning for a vaginal discharge (Day 1 of their estrous cycle) and caged according to their cycles. No more than four females were caged together. Females were injected with 25 IU of hCG (Sigma, St. Louis, MO) at 2230 h on the evening of Day 3 of their estrous cycles to promote maturation of follicles and oocytes. Females were sacrificed at about 1000 h on Day 4, which is before the time of ovulation, and the ovary and oviduct were removed. Infundibula, with a part of the ampulla attached, were dissected from the rest of the oviduct and used in all assays. Expanded OCCs were released by poking the follicles with an insect pin and were used in the OCC pickup rate and adhesion assays. For the OCC dispersion experiments, superovulation was induced by injecting 25 IU of eCG (Sigma) on the morning of Day 1 of the estrous cycle followed by 25 IU of hCG on the evening of Day 3. Animal care and usage was approved by the campus Animal Care Committee.

Media

Earles Balanced Saline Solution (EBSS) was made fresh for each experiment from a 10x stock solution (2.0 g/L CaCl2 [anhyd.], 4.0 g/L KCL, 2.0 g/L MgSO4 · 7H2O, 54.0 g/L NaCl, 1.4 g/L NaH2 PO4 · H2O). HEPES (EM Science, Gibbstown, NJ) and sodium bicarbonate (Fisher Scientific, Fair Lawn, NJ) were added to 1x EBSS to make EBSS-H. EBSS-HA, EBSS-H plus 0.1% BSA, Fraction V (A-9418) (Sigma), was used as the control medium for all experiments. Sodium hydroxide (Fisher Scientific) was used to bring the pH of EBSS-HA up to 7.4.

All smoke solutions were made fresh before each experiment. Smoke solutions were prepared using a University of Kentucky (Lexington, KY) smoking system and 2R1 research-grade cigarettes. The method used for the preparation of mainstream whole (MSW), mainstream gas (MSG), mainstream particulate (MSP), sidestream whole (SSW), sidestream gas (SSG), and sidestream particulate (SSP) has been described in detail previously [33, 34]. All smoke solutions were first collected in EBSS-H, 0.1% BSA was added to the smoke solutions, pH was adjusted to 7.4, and the smoke solutions were sterilized by passage through a 0.2 µm filter. Absorbance was measured with a Hitachi Model 100–40 spectrophotometer (San Jose, CA) at a wavelength of 300 nm to confirm that the concentrations of smoke solutions used were consistent for all experiments. Absorbance was measured at full strength for MSW, MSG, SSW, and SSG solutions and at 0.1x concentration for MSP and SSP.

Briefly, for MSW smoke solutions, 60 puffs (10 puffs/cigarette) were generated by a puffer box and drawn through a vial containing 10 ml of EBSS-H by a peristaltic pump. A Cambridge filter (University of Kentucky), which was changed after 30 puffs, was placed in the tubing between the puffer box and vial to separate the gas and particulate phases. The gas phase was drawn into the vial, and the particulate phase was trapped on the filter. The particulate phase solution was made by extracting the water-soluble components from the filter with 10 ml of EBSS-H.

SSW smoke solutions were collected from the burning tip of the cigarette during the generation of 30 mainstream puffs. The mainstream smoke was exhausted into the hood and SSW was drawn continuously from a cylinder that enclosed the cigarette into a vial containing 10 ml of EBSS-H. A Cambridge filter, which was changed after 15 puffs, was placed between the cylinder and vial to separate the gas and particulate phases.

OCC Dispersion Assay

Fully expanded OCC were harvested and exposed to one of four treatments, which included MSW and SSW smoke solutions with or without BSA added. Controls included EBSS-HA and EBSS-H. Each OCC was placed in a 200 µl droplet of the control or test solution and then the droplets were covered with oil to prevent dehydration. The length and width of each OCC was measured at the beginning of the exposure. After incubating for 6 h, each OCC was pipetted briskly three times to remove loose matrix and cumulus cells and measured again. Measurements were converted to volume (4/3{pi}ab2, where a is the radius of the length and b is the radius of the width of the OCC) to determine the percent change from the starting volume.

Adhesion and Oocyte Pickup Rate Assays

Adhesive strength between OCCs and oviductal cilia was measured using a method previously described in detail [18]. The adhesion assay was used in conjunction with the OCC pickup rate assay. OCC pickup rate was measured using a protocol previously described [29] with the following modifications. OCCs were not stained with methylene blue and OCCs were not allowed to travel the entire length of the infundibulum because adhesion was greatly increased near the ostium. Briefly, an infundibulum was held in a pipette within a modified culture chamber and covered with EBSS-HA. The chamber with the infundibulum was viewed with a Nikon SMZ-10 stereomicroscope (A.G. Heinze, Irvine, CA). A 50 µl Microcap pipette attached with tubing to a reversible low-flow peristaltic pump (Model P720, Instech Laboratories, Inc., Plymouth Meeting, PA) was used to place an OCC on the external surface of the infundibulum. OCC pickup rate (µm/sec) was measured by recording the time it took an OCC to travel 400 µm. An ocular micrometer mounted in the eyepiece was used to measure distance. As soon as the OCC pickup rate measurement was taken, the tip of the pipette attached to the peristaltic pump was immediately placed over the OCC and the flow rate of the pump was slowly increased from zero until the OCC was completely released from the cilia. The lowest flow rate (ml/min) required to release an OCC from the infundibulum was used as the measurement of strength of adhesion. Six measurements were taken with each OCC. A different OCC was used for each treatment, and each infundibulum was used as its own control. All experiments were completed within 2.5 h of the initial dissection time.

Ciliary Beat Frequency Assay

Ciliary beating was videotaped using either a Hitachi KP-250 CCD color video camera (San Jose, CA) or a Sony DXC-390 3CCD color video camera (A.G. Heinze, Irvine, CA) attached to either a Nikon SMZ-10 or a Wild M5A dissecting stereoscope (MAX ERB Instrument Co., Burbank, CA) and recorded on a Sony S-VHS, SVO-9500MO videocassette recorder (A.G. Heinze). A fiber-optic lamp was used to illuminate the surface of the infundibula, and a change in the reflected light intensity as the cilia beat was used to determine ciliary beat frequency. To calculate ciliary beats per second, videotapes were played back frame by frame, and the number of frames required for the reflected light to cycle from its brightest intensity and back were counted. For each treatment, the number of frames/cycle was counted 10 times at four different spots on each infundibulum. The video system records 30 frames/sec. The number of frames per ciliary beat was converted to beats per second.

Experimental Design for In Vitro Cigarette Smoke Exposure

For all experiments, OCC pickup rate, adhesion, and ciliary beat frequency measurements were taken at room temperature, which ranged from 19°C to 24°C. Temperature was recorded at the beginning of each treatment phase for all experiments. Control measurements were taken for each infundibulum 5 min after the infundibula were placed in the holding pipette. For some experiments, only OCC pickup rate and adhesion were measured. In some experiments, ciliary beat frequency was measured in conjunction with OCC pickup rate and adhesion.

In the first set of experiments, both infundibula and OCCs were exposed to the smoke solution. Control measurements were taken after infundibula had been placed in EBSS-HA for 5 min. Infundibula were then exposed to smoke solutions for 20 min. Preliminary experiments showed that a maximum effect on OCC pickup rate was achieved after a 20-min exposure. At 20 min, an OCC was placed on the infundibula, and six OCC pickup rate and adhesion measurements were taken. While OCC pickup rate and adhesion measurements were taken, the OCCs were exposed to the smoke solution for about 5 min.

In the second set of experiments, only OCCs were exposed to each of the six types of smoke solutions. Six control OCC pickup rate and adhesion measurements were taken with an untreated OCC placed on an untreated infundibulum. A new OCC was incubated in one of the six types of smoke solution for 10 min. The smoke solution was then washed from the OCC, and the treated OCC was allowed to recover for 5 min before six OCC pickup rate and adhesion measurements were taken on the same untreated infundibulum.

In the third set of experiments, only infundibula were exposed to one of the six types of smoke solutions. Ciliary beat frequency was measured in conjunction with OCC pickup rate and adhesion for this set of experiments. Six control OCC pickup rate and adhesion measurements were taken with an untreated OCC and untreated infundibulum. Control ciliary beating was videotaped before the control OCC pickup rate and adhesion measurements. An infundibulum was then exposed to a smoke solution for 20 min. Ciliary beating was videotaped just before completion of the exposure phase. The smoke solution was thoroughly washed from the infundibulum, and it was allowed to recover for 5 min in EBSS-HA. Ciliary beating was videotaped before the wash OCC pickup rate and adhesion measurements. A fresh OCC was used to measure OCC pickup rate and adhesion during each treatment phase. Ciliary beating was videotaped during the exposure phase to distinguish a recovery from the effect of smoke solutions on ciliary beat frequency from no effect on ciliary beat frequency.

Statistical Analysis

All statistical analyses were done using Prism v. 3.0 (GraphPad Software, Inc., San Diego, CA). A Student unpaired t-test was used to compare the means between two groups, either control versus treated or control versus washed, following OCC pickup rate and adhesion experiments. One-way ANOVA was used to compare the means of the three phases of treatment measured during ciliary beat frequency experiments. A Dunnett posttest was used to compare the treated groups to the control group if the one-way ANOVA showed a significant difference in the means between groups. Means were considered significantly different if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Coexposure of Infundibula and OCCs to Smoke Solutions Decreased OCC Pickup Rate

The purpose of this experiment was to compare the potency of six types of cigarette smoke solution on OCC pickup rate after a maximum effect had been reached. Both infundibula and OCCs were treated with each of the six types of smoke solutions (MSW, MSG, MSP, SSW, SSG, and SSP) to determine the effect of coexposure. All six types of smoke solutions significantly decreased OCC pickup rate (Fig. 1, A–F). OCC pickup rate was decreased in three ways: 1) OCCs did not adhere to infundibula, preventing OCC pickup; 2) OCCs adhered to infundibula, but did not move; or 3) OCCs adhered to infundibula and OCC pickup rate was slowed. On some infundibula, the OCCs did not move for any of the six measurements, while on other infundibula, the OCC moved for some of the six measurements. The percentage of times when OCC pickup was prevented because OCCs did not adhere or because OCCs adhered but did not move (OCC pickup rate = 0 µm/sec) was as follows for each type of smoke solution: MSW (6.6%), MSG (54.2%), MSP (38.9%), SSW (50.0%), SSG (75.0%), and SSP (48.4%) (Fig. 2). In general, sidestream solutions were more inhibitory than the mainstream solutions, and sidestream gas was the most effective of the six types of smoke.



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  		FIG. 1. Mainstream (A–C) and sidestream (D–F) smoke solutions decreased OPR when both the infundibulum and the OCC were exposed. Measurements were taken before (control) and during exposure (exposure) of infundibula. Each bar represents the mean ± SD from 5 or 6 infundibula. (6 measurements were taken on each infundibulum per treatment.) ***P < 0.001. OPR = OCC pickup rate



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  		FIG. 2. Percentage of times each type of mainstream or sidestream smoke solution completely stopped OPR, either because the OCC did not attach to an infundibulum or because the OCC attached but did not move. For each of these events, OPR = 0. OPR = OCC pickup rate

Mainstream and Sidestream Whole Smoke Degrade the Extracellular Matrix of the OCC

The extracellular matrix of the OCC is required for adhesion between the OCC and oviductal cilia [18]. The extracellular matrix is rich in hyaluronan (hyaluronic acid), which could be a target of reactive oxygen species in smoke solutions [35–40]. The purpose of this experiment was to determine whether mainstream and sidestream whole smoke solutions affect the extracellular matrix of the OCC. OCCs were incubated in control or smoke solutions for 6 h, then pipetted vigorously three times to remove loose cumulus cells. Volumes of the OCC before and after incubation were compared. A decrease in volume because of loss of cumulus cells was interpreted to have been caused by an effect on the extracellular matrix. In some groups, BSA was included in the medium to see if it could protect against reactive oxygen species.

Figure 3 shows the effect of EBSS-H, MSW, and SSW on OCC volume. Control OCCs were incubated in EBSS-H or EBSS-HA. Three OCCs treated with EBSS-H and two OCCs treated with EBSS-HA expanded in size after 6 h. One OCC treated with EBSS-HA showed no change in size. All other control OCCs exposed to EBSS-H or EBSS-HA for 6 h decreased in size. The average decrease in size was 37% for OCCs in EBSS-H and 39% for OCCs in EBSS-HA. All OCCs that were treated in smoke solutions (MSW, SSW, MSW-BSA, SSW-BSA) decreased in size, with the exception of one OCC that increased in size after treatment with SSW. The overall extent of matrix degradation was greater for all smoke-treated OCCs than for EBSS-H- or EBSS-HA-treated OCCs that exhibited degradation. Overall, there was a 55% to 75% decrease in OCC size after exposure to any smoke treatment. In the cases with the most degradation, only the oocyte and corona radiata remained. Cumulus cells tended to come off in clumps rather than as individual cells, as would be the case with hyaluronidase treatment. BSA did not appear to protect the matrix against the effects of mainstream or sidestream smoke solutions.



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  		FIG. 3. Effect of EBSS-H, MSW, and SSW smoke solutions, with or without BSA, on OCC size after a 6-h exposure. Data are shown as change in percentage from control OCC volumes. Each bar represents the mean ± SD, except in the group with only one OCC. Numbers above or below each bar represent total number of OCCs with a similar effect. n = 6 OCCs for MSW, MSW-BSA, SSW, and SSW-BSA. N = 12 OCCs for EBSS-H and EBSS-HA. MSW = mainstream whole; SSW = sidestream whole; BSA = bovine serum albumin; EBSS-H = Earles balanced saline solution; EBSS-HA= Earles balanced saline solution ± BSA

OCC Exposure to Smoke Solutions Increased Adhesion and Decreased OCC Pickup Rate

The above experiment indicated that the OCC extracellular matrix, which is necessary for adhesion to the oviduct [18], was affected by mainstream and sidestream smoke treatments. The purpose of this experiment was to determine whether smoke solutions affected OCC pickup rate and/or adhesion when only OCCs were pretreated with smoke solution. MSW, MSG, and MSP significantly decreased OCC pickup rate (Fig. 4, A–C) and significantly increased adhesion (Fig. 4, D–F). OCC pickup rate and adhesion did not recover from the initial exposure to any mainstream smoke solutions. SSW and SSG also significantly decreased OCC pickup rate (Fig. 5, A and B), and both significantly increased adhesion (Fig. 5, D and E). SSP did not decrease OCC pickup rate (Fig. 5C) nor significantly increase adhesion (Fig. 5F).



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  		FIG. 4. Effect of MSW (A and D), MSG (B and E), and MSP (C and F) smoke solutions on OPR and adhesion when OCCs were pretreated. OPR (A–C) and adhesion (D–F) measurements were taken before exposure (control) and after OCCs were allowed to recover from a 10-min exposure (wash). All three types of mainstream smoke solutions decreased OPR (A–C) and increased adhesion (D–F). Each bar represents the mean ± SD from 6 or 7 infundibula (6 measurements were taken on each infundibulum per treatment). *P < 0.05, **P < 0.01, ***P < 0.001. OPR = OCC pickup rate; Adh = adhesion



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  		FIG. 5. Effect of SSW (A and D), SSG (B and E), and SSP (C and F) smoke on OPR and adhesion when OCCs were pretreated. OPR (A–C) and adhesion (D–F) measurements were taken before exposure (control) and after OCCs were allowed to recover from a 10-min exposure (wash). Both SSW and SSG decreased OPR (A and B) and increased adhesion (D and E) during the recovery phase. SSP had no significant effect on OPR (C) nor adhesion (F). Each bar represents the mean ± SD from 5 or 7 infundibula (6 measurements were taken on each infundibulum per treatment). *P < 0.05, **P < 0.01, ***P < 0.001. OPR = OCC pickup rate; Adh = adhesion

Effect of Infundibulum Exposure to Smoke Solutions on OCC Pickup Rate, Adhesion, and Ciliary Beat Frequency

The purpose of this experiment was to determine whether smoke solutions affected adhesion and/or ciliary beat frequency when only infundibula were treated and to determine whether adhesion and ciliary beat frequency were independent factors responsible for decreased OCC pickup rate. OCC pickup rate and adhesion were measured before (control) and after (wash) infundibula were exposed to each type of smoke solution. Ciliary beat frequency was measured before, during, and after each infundibulum was exposed to smoke solutions.

When infundibula were pretreated, all mainstream smoke solutions caused a statistically significant decrease in OCC pickup rate and a statistically significant increase in adhesion (Fig. 6, A–C). MSW caused adhesion to remain increased after exposure, but had no effect on ciliary beat frequency (Fig. 6A). MSG caused a significant increase in adhesion after exposure and caused a decrease in ciliary beat frequency during the exposure phase; however, ciliary beat frequency returned to control levels after exposure (Fig. 6B). MSP also caused adhesion to remain significantly increased following exposure (Fig. 6C). MSP caused ciliary beat frequency to decrease during the exposure phase, and ciliary beat frequency remained decreased following exposure. MSG caused the greatest increase in adhesion; both MSG and MSP smoke solutions had a greater effect on adhesion and ciliary beat frequency than MSW.



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  		FIG 6. Effect of MSW (A, MSG (B), and MSP (C) smoke solutions on OPR, adhesion, and CBF when infundibula were exposed in vitro for 20 min. OPR and adhesion measurements were taken before exposure (control) and after infundibula were allowed to recover from exposure (wash). CBF was measured before (control), during (exp), and after exposure (wash). (A) MSW solutions decreased OPR and increased adhesion, but had no effect on CBF. (B) MSG decreased OPR and increased adhesion. CBF decreased during exposure but recovered to control levels after exposure. (C) MSP decreased OPR and increased adhesion. CBF decreased during exposure and did not recover after exposure. Bars represent the means ± SD. n = 5 or 6 infundibula per treatment group for a total of 30 or 36 OPR and adhesion measurements. n = 8 to 11 infundibula per treatment group for a total of 32 to 44 mean CBF measurements. **P < 0.01, ***P < 0.001. OPR = OCC pickup rate; CBF = ciliary beat frequency

All sidestream smoke solutions produced a statistically significant decrease in OCC pickup rate and a statistically significant increase in adhesion (Fig. 7, A–C). Both SSW (Fig. 7A) and SSG (Fig. 7B) caused adhesion to remain increased after exposure and caused an increase in ciliary beat frequency during the exposure phase; however, ciliary beat frequency returned to control levels after exposure. SSP (Fig. 7C) also caused adhesion to remain increased after exposure and caused a significant increase in ciliary beat frequency during the exposure phase; however, ciliary beat frequency remained increased following exposure. SSW and SSG caused greater increases in ciliary beat frequency during exposure than SSP; however, SSP had an irreversible effect on ciliary beat frequency.



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  		FIG. 7. Effect of SSW (A), SSG (B), and SSP (C) smoke solutions on OPR, adhesion, and CBF when infundibula were exposed in vitro for 20 min. OPR and adhesion measurements were taken before exposure (control) and after infundibula were allowed to recover from exposure (wash). CBF) was measured before (control), during (exp), and after exposure (wash). (A) SSW decreased OPR and increased adhesion. CBF increased during exposure, but returned to control levels after exposure. (B) SSG decreased OPR and increased adhesion. CBF increased during exposure and returned to control levels after exposure. (C) SSP decreased OPR and increased adhesion. CBF increased during exposure, but did not return to control levels after exposure. n = 5 or 6 infundibula per treatment group for a total of 30 or 36 OPR and adhesion measurements. n = 7 to 10 infundibula per treatment group for a total of 28 to 40 mean CBF measurements. *P < 0.05, **P < 0.01, ***P < 0.001. OPR = OCC pickup rate; CBF = ciliary beat frequency


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A major purpose of this study was to determine whether cigarette smoke solutions alter adhesive strength between the hamster OCC and the infundibulum and whether a change in adhesion correlates with changes in OCC pickup rate. Our results showed that smoke solutions increased adhesion between the OCC and the infundibulum when either the OCC or the infundibulum was pretreated and allowed to recover. A corresponding decrease in OCC pickup rate was observed for each significant increase in adhesion. All six types of smoke solutions increased adhesion and decreased OCC pickup rate when just the infundibulum was exposed. All smoke solutions, except sidestream particulate, also increased adhesion and decreased OCC pickup rate when just OCCs were pretreated. Overall, OCC pickup rate remained decreased 40% to 80% below control levels and adhesion increased 52% to 91% above control levels after infundibula recovered from smoke exposure. When OCCs were pretreated, OCC pickup rate decreased 20% to 35% below control levels and adhesion increased 39% to 54% above control levels. These results show that both the OCC and the infundibulum are targets of cigarette smoke, but that the infundibulum is more sensitive to the effects of cigarette smoke solutions on OCC pickup rate and adhesion.

The results also confirmed the hypothesis that changes in adhesion would be observed in cases for which decreased OCC pickup rate could not be explained by changes in ciliary beat frequency. Ciliary beat frequency returned to control levels after infundibula were exposed to all smoke solutions except mainstream and sidestream particulate. These results clearly showed that changes in adhesive strength could be responsible for the observed decreases in OCC pickup rate, independent of any changes in ciliary beat frequency. The conclusion that increased adhesion caused by smoke treatment retards OCC pickup rate is further supported by our observation that treatment of only the OCC increased adhesion and slowed pickup rate.

However, our data do not completely eliminate the possibility that cigarette smoke solutions slow pickup rate by altering parameters of ciliary beating that were not measured in this study. For example, smoke solutions could have altered the power or amplitude of the effective stroke without altering beat frequency. It is also possible that metachrony was disturbed or that the direction of beating of some cilia was altered. Reactive oxygen species, which are present in cigarette smoke, alter both ciliary beat amplitude and ciliary beat frequency of human respiratory epithelial cells during acute in vitro exposure [41]. Our system allowed analysis only of ciliary beat frequency, but when videotapes of infundibula exposed to sidestream smoke solutions were evaluated, the cilia often appeared to be quivering rather than spanning the distance of normal strokes. The possibility that smoke treatment affected ciliary parameters other than beat frequency is further supported by our observation that changes in the pattern of pickup occurred during some experiments. These changes included the OCC spinning in place, pausing or stopping during pickup, and moving backward. Spinning, pausing, or stopping could occur if ciliary amplitude was decreased while frequency was normal or if metachrony was lost during treatment. Backward movement could occur if some cilia changed their direction of beating.

The mechanism that caused an increase in adhesion after exposure to smoke solutions is not known; however, it is known that the proper amount of adhesion is critical for OCC pickup [14, 18, 20, 27, 42]. We previously showed that slight modulations in adhesion above or below control values after treatment with the lectin wheat germ agglutinin or the polycation poly-L-lysine can prevent OCC pickup or cause decreased OCC pickup rate, even when ciliary beat frequency is normal [18]. Some researchers have suggested that nonspecific factors such as surface charge are involved in adhesion [20, 27]. Others have rejected this idea because both the tips of the cilia and the OCC carry negative charges that would repulse each other [14]. These same researchers suggested that the adhesion was neither random nor highly specific, but was caused by the interaction with a class of materials, such as glucosaminoglycans, instead of a specific receptor. It is possible that both nonspecific and specific interactions are involved. Because both attachment and release are required for normal OCC pickup, it is possible that surface charges on the tips of the cilia are involved in detachment and more specific interactions are required for attachment.

Increased adhesion has been observed in other types of epithelial cells after exposure to cigarette smoke. A significant increase in the binding of respiratory pathogens, such as Bordetella pertussis and Staphylococcus aureaus, has been observed on buccal epithelial cells of active smokers compared to nonsmokers [43]. Interestingly, buccal epithelial cells of nonsmokers that were treated with diluted smoke solutions also showed increased binding of several pathogens. It was suggested that increased binding could be due to components in cigarette smoke that altered nonspecific factors such as surface charge, because no increase in the expression of host cell receptors was observed. In vitro exposure of bovine bronchial epithelial cells to sublethal doses of cigarette smoke extract increased cell attachment to matrix proteins and decreased cell migration after a 24-h exposure [44]. Cigarette smoke also increased uptake and retention of exogenous particles in epithelial cells [45]. Exposure of rat tracheal explants to cigarette smoke before exposure to asbestos increased the surface adhesion of fibers, compared to explants that were treated with only asbestos. The authors suggested that reactive oxygen species in the smoke interacted with iron on the surface of the asbestos to increase adhesion. This idea was supported by the observation that mannitol, a hydroxyl radical scavenger, and catalase, a hydrogen peroxide scavenger, both partially decreased adhesion of asbestos to the cells. From the preceding examples, it appears that cigarette smoke constituents have the ability to increase adhesion to epithelial surfaces in diverse biological interactions.

Our prior work on OCC pickup was done by coexposure of infundibula and OCCs to cigarette smoke solutions [33]. Our current data confirm our prior findings using coexposure and are the first to show that both the OCC and the infundibulum are targets of chemicals in cigarette smoke. It was previously shown that the extracellular matrix between cumulus cells of the OCC adheres to the tips of the cilia on the infundibulum during pickup [18]. The current experiments in which OCCs were exposed to smoke solutions for an extended time showed that smoke solutions disrupted the extracellular matrix of the OCC. The extracellular matrix of the expanded OCC is rich in hyaluronan [5, 6, 8–10, 13, 46] and is organized into a three-dimensional network with other matrix components [13]. Research suggests that this network is maintained by a ternary complex formed by TSG-6 (the secreted product of tumor necrosis factor [TNF]-stimulated gene 6, also known as TNFAIP6) and inter(alpha)trypsin inhibitor binding to hyaluronan [47]. Hyaluronan is a target of reactive oxygen species [38–40, 48, 49] that degrade hyaluronan in a manner similar to hyaluronidase [35]. Cigarette smoke generates several reactive oxygen species [50–53] and targets hyaluronan in the extracellular matrix of the chick chorioallantoic membrane [54]. We hypothesized that smoke solutions would target hyaluronan in the matrix of the OCC and cause the matrix to completely degrade and disperse away from the oocyte, as it does when treated with hyaluronidase [46]. When OCCs were treated with mainstream and sidestream whole smoke solutions, the cumulus mass came off in clumps and degradation occurred from the outer areas of the cumulus mass. This suggested that hyaluronan was not the target or that only a small amount of hyaluronan was degraded. If reactive oxygen species caused the cumulus mass to degrade, then it is possible that hyaluronan was protected by inter(alpha)trypsin inhibitor, which has been shown to provide some protection against reactive oxygen species [55]. This could explain why only the periphery of the cumulus mass was affected and why cells came off in clumps rather than individually. Mainstream and sidestream smoke solutions could also be targeting matrix proteins. Inter(alpha)trypsin inhibitor has serine protease inhibitor activity, and this activity is increased by its interaction with TSG-6 [56]. It has been suggested that the TSG-6/inter(alpha)trypsin inhibitor complex localizes active serine protease inhibitors along hyaluronan and protects proteins in the matrix from degradation [57]. Because the matrices of the smoke-treated solutions were degraded faster than the control OCCs, it is possible that the smoke solutions targeted serine protease inhibitors or increased the activity of serine proteases such as plasminogen activator. In either case, an increase in proteolytic enzymes would speed up matrix degradation.

This study has successfully shown that cigarette smoke solutions alter adhesion between the OCC and hamster infundibulum and that changes in adhesion correlate well with changes in OCC pickup rate. In addition, the observed increases in adhesion explain the decreases in OCC pickup rate that could not be explained by changes in ciliary beat frequency. We have also successfully shown that the OCC is a target of cigarette smoke during in vitro exposures. However, cigarette smoke has a greater impact on the oviduct than on the OCC, which could be attributable to an impact on mechanisms involved in both ciliary beating and adhesion.


   ACKNOWLEDGMENTS
 
The authors would like to thank John Chen for his help in administering hormones to the hamsters and aiding in the initial analysis of ciliary beat frequency. We would also like to thank Rochelle Pederson for her assistance in making smoke solutions and confirming the analysis of ciliary beat frequency.


   FOOTNOTES
 
1 Supported by Tobacco-Related Disease Research Program 10RT-029 and 13RT-0068. Back

2 Correspondence: Prue Talbot, Department of Cell Biology and Neuroscience, University of California, Riverside CA 92521. FAX: 951-827-4286; talbot{at}ucr.edu Back

Received: 18 February 2005.

First decision: 9 March 2005.

Accepted: 27 April 2005.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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