Background The ultraviolet properties of textiles dyed with synthetic dyes have been widely reported in literature. However, no study has investigated the ultraviolet properties of natural fabrics dyed with natural colorants. This study reports the Ultraviolet Protection Factor (UPF) of cotton fabrics dyed with colorants of plant and insect origins.
Background
The ultraviolet properties of textiles dyed with synthetic dyes have been widely reported in literature. However, no study has investigated the ultraviolet properties of natural fabrics dyed with natural colorants. This study reports the Ultraviolet Protection Factor (UPF) of cotton fabrics dyed with colorants of plant and insect origins.Methods Three cotton fabrics were dyed with three natural colorants. Fabrics were characterized with respect to fabric construction, weight, thickness and thread count. Influence of fabric characteristics on Ultraviolet Protection Factor was studied. Role of colorant concentration on the ultraviolet protection factor was examined via color strength analysis.
Methods
Three cotton fabrics were dyed with three natural colorants. Fabrics were characterized with respect to fabric construction, weight, thickness and thread count. Influence of fabric characteristics on Ultraviolet Protection Factor was studied. Role of colorant concentration on the ultraviolet protection factor was examined via color strength analysis.Results A positive correlation was observed between the weight of the fabric and their UPF values. Similarly, thicker fabrics offered more protection from ultraviolet rays. Thread count appears to negatively correlate with UPF. Dyeing with natural colorants dramatically increased the protective abilities of all three fabric constructions. Additionally, within the same fabric type UPF values increased with higher depths of shade.
Results
A positive correlation was observed between the weight of the fabric and their UPF values. Similarly, thicker fabrics offered more protection from ultraviolet rays. Thread count appears to negatively correlate with UPF. Dyeing with natural colorants dramatically increased the protective abilities of all three fabric constructions. Additionally, within the same fabric type UPF values increased with higher depths of shade.Conclusion Dyeing cotton fabrics with natural colorants increases the ultraviolet protective abilities of the fabrics and can be considered as an effective protection against ultraviolet rays. The UPF is further enhanced with colorant of dark hues and with high concentration of the colorant in the fabric.
Conclusion
Dyeing cotton fabrics with natural colorants increases the ultraviolet protective abilities of the fabrics and can be considered as an effective protection against ultraviolet rays. The UPF is further enhanced with colorant of dark hues and with high concentration of the colorant in the fabric.

Background High, short-term exposure to ultraviolet radiation (UVR) from the sun causes sunburns and long-term exposure leads to skin cancer. The National Toxicology Program, U.S. Department of Health and Human Services has classified UVR as a known human carcinogen [ 1 ]. The American Cancer Society estimates that more than one million cases of skin cancer cases are diagnosed each year in the United States [ 2 ]. In 2002, an estimated 54,200 new cases of melanoma skin cancer alone were diagnosed [ 2 ]. A primary reason for the increased incidence of skin cancers is attributed to ozone depletion. Each one percent decrease in ozone concentration is predicted to increase the rate of skin cancer by two percent to five percent [ 3 ]. The United States Environmental Protection Agency estimates that ozone depletion will lead to between three and fifteen million new cases of skin cancer in the United States by the year 2075. Other reasons for the skin cancer epidemic can be traced to lifestyle changes such as excessive exposure to sunlight during leisure activities, for example, playing outdoors and swimming in the case of children and golfing and fishing in the case of adults. In the case of agricultural and other outdoor workers, exposure to the sun is an occupational hazard as they have no choice about the duration of their exposure to the sun [ 3 - 5 ]. The ultraviolet radiation (UVR) band consists of three regions: UV-A (320 to 400 nm), UV-B (290 to 320 nm), and UV-C (200 to 290 nm). UV-C is totally absorbed by the atmosphere and does not reach the earth. UV-A causes little visible reaction on the skin but has been shown to decrease the immunological response of skin cells [ 3 ]. UV-B is most responsible for the development of skin cancers [ 3 ]. Other than drastically reducing exposure to the sun, the most frequently recommended form of UV protection is the use of sunscreens, hats, and proper selection of clothing. Unfortunately, one cannot hold up a textile material to sunlight and determine how susceptible a textile is to UV rays. Even textiles which seem to be non-light transmitting may pass significant amounts of erythema-inducing UV irradiation [ 4 ]. Therefore, knowledge of the factors that contribute to the protective abilities of textiles is vital. Important factors include fiber composition, fabric construction and wet-processing history of the fabric such as color and other finishing chemicals that may have been applied to the textile material. To the author's knowledge, no study has investigated the ultraviolet properties of natural fabrics dyed with natural colorants. A plethora of previous studies have concluded that good UVR protection can be provided by synthetic fibers dyed with high concentrations of synthetic dyes. However, synthetic fibers such as polyester are hydrophobic and are generally not deemed to be comfortable for wear especially in warm weather. According to a report in America's Textile Industries [ 6 ] natural fibers are back in demand. The emergence and popularity of a more natural way of life as reflected in a return to organic farming and natural foods has now extended into textiles where the resurgence of natural fibers and natural dyes is on the increase [ 6 , 7 ]. It is hoped that data from the present study will be useful for dermatologists advising patients regarding the UV-protective properties of clothing made from natural fibers and dyed with natural colorants. In this study, cotton fabrics were dyed with three natural colorants of plant and insect origin. Fabrics were characterized with respect to fabric construction, weight, thickness and thread count. Ultraviolet Protection Factor (UPF) was measured using a labsphere ®  UV-100 F Ultraviolet Transmission Analyzer. The effect of colorant concentration on the ultraviolet protection factor was examined via color strength analysis using a HunterLab ColorQuest XE ®  spectrophotometer.
Background
High, short-term exposure to ultraviolet radiation (UVR) from the sun causes sunburns and long-term exposure leads to skin cancer. The National Toxicology Program, U.S. Department of Health and Human Services has classified UVR as a known human carcinogen [ 1 ]. The American Cancer Society estimates that more than one million cases of skin cancer cases are diagnosed each year in the United States [ 2 ]. In 2002, an estimated 54,200 new cases of melanoma skin cancer alone were diagnosed [ 2 ]. A primary reason for the increased incidence of skin cancers is attributed to ozone depletion. Each one percent decrease in ozone concentration is predicted to increase the rate of skin cancer by two percent to five percent [ 3 ]. The United States Environmental Protection Agency estimates that ozone depletion will lead to between three and fifteen million new cases of skin cancer in the United States by the year 2075. Other reasons for the skin cancer epidemic can be traced to lifestyle changes such as excessive exposure to sunlight during leisure activities, for example, playing outdoors and swimming in the case of children and golfing and fishing in the case of adults. In the case of agricultural and other outdoor workers, exposure to the sun is an occupational hazard as they have no choice about the duration of their exposure to the sun [ 3 - 5 ].The ultraviolet radiation (UVR) band consists of three regions: UV-A (320 to 400 nm), UV-B (290 to 320 nm), and UV-C (200 to 290 nm). UV-C is totally absorbed by the atmosphere and does not reach the earth. UV-A causes little visible reaction on the skin but has been shown to decrease the immunological response of skin cells [ 3 ]. UV-B is most responsible for the development of skin cancers [ 3 ].Other than drastically reducing exposure to the sun, the most frequently recommended form of UV protection is the use of sunscreens, hats, and proper selection of clothing. Unfortunately, one cannot hold up a textile material to sunlight and determine how susceptible a textile is to UV rays. Even textiles which seem to be non-light transmitting may pass significant amounts of erythema-inducing UV irradiation [ 4 ]. Therefore, knowledge of the factors that contribute to the protective abilities of textiles is vital. Important factors include fiber composition, fabric construction and wet-processing history of the fabric such as color and other finishing chemicals that may have been applied to the textile material.To the author's knowledge, no study has investigated the ultraviolet properties of natural fabrics dyed with natural colorants. A plethora of previous studies have concluded that good UVR protection can be provided by synthetic fibers dyed with high concentrations of synthetic dyes. However, synthetic fibers such as polyester are hydrophobic and are generally not deemed to be comfortable for wear especially in warm weather. According to a report in America's Textile Industries [ 6 ] natural fibers are back in demand. The emergence and popularity of a more natural way of life as reflected in a return to organic farming and natural foods has now extended into textiles where the resurgence of natural fibers and natural dyes is on the increase [ 6 , 7 ]. It is hoped that data from the present study will be useful for dermatologists advising patients regarding the UV-protective properties of clothing made from natural fibers and dyed with natural colorants.In this study, cotton fabrics were dyed with three natural colorants of plant and insect origin. Fabrics were characterized with respect to fabric construction, weight, thickness and thread count. Ultraviolet Protection Factor (UPF) was measured using a labsphere ®  UV-100 F Ultraviolet Transmission Analyzer. The effect of colorant concentration on the ultraviolet protection factor was examined via color strength analysis using a HunterLab ColorQuest XE ®  spectrophotometer.Methods Three fabrics were chosen to cover the gamut of basic weave constructions. They were a bleached, mercerized plain weave cotton fabric, a bleached mercerized cotton twill and a desized and bleached cotton sateen. Fabric weight was measured according to ASTM Test Method D3776-96 [ 8 ]. Fabric thickness was measured according to ASTM Test Method D1777-96 [ 8 ]. Thread counts were measured according to ASTM D3775-98 [ 8 ]. Natural plant colorants used were madder ( Rubia tinctorum ) and indigo ( Indigofera tinctoria ) and the natural colorant of insect origin was cochineal ( Dactylopius coccus ). Since natural dyes do not have affinity for cellulosic fibers an alum mordant was used to impart affinity. Fabrics were mordanted prior to dyeing by treating with alum at boil for 45 minutes. The liquor ratio was 1:40 and alum concentration was 10% on weight of the fabric. After mordanting, fabric was squeezed thoroughly and dyed. Madder and cochineal dyeings were done in stainless steel canisters of an Atlas launder-ometer using 2%, 4% and 6% dye on weight of fabric. The liquor-goods ratio was 40:1. Fabrics were introduced into the dyeing solutions at room temperature. Temperature was raised to the boil and dyeing continued at the boil for 60 minutes. After dyeing, fabrics were rinsed in deionized water, washed using a non-ionic detergent and air-dried. Three replications were done for each colorant and at each dye concentration. Dyeing with indigo was done in the following manner. Indigo dye was made into a paste and solubilized using sodium hydroxide and sodium hydrosulfite. Fabrics were introduced into dyebaths containing 2%, 4% and 6% dye on weight of fabric. The liquor-goods ratio was 40:1. After thirty minutes of dyeing the fabrics were removed and oxidized by drying in air. The fabrics were then rinsed in deionized water and washed using a non-ionic detergent and dried. Direct and diffuse UV transmittance through a fabric is the crucial factor determining the UV protection of textiles [ 9 ]. Ultraviolet protection factor (UPF) is the scientific term used to indicate the amount of Ultraviolet (UV) protection provided to skin by fabric. UPF values are analogous to SPF values the only distinction being that SPF values for sunscreens are determined through human testing whereas UPF values are based on instrumental measurements [ 10 ]. UPF is defined as the ratio of the average effective UV irradiance calculated for unprotected skin to the average UV irradiance calculated for skin protected by the test fabric [ 5 , 10 ]. The higher the value, the longer a person can stay in the sun until the area of skin under the fabric becomes red [ 5 , 10 ]. An effective UVR dose (ED) for unprotected skin is calculated by convolving the incident solar spectral power distribution with the relative spectral effectiveness function and summing over the wavelength range 290-400 nm. The calculation is repeated with the spectral transmission of the fabric as an additional weighting to get the effective dose (ED m ) for the skin when it is protected. The UPF is defined as the ratio of ED to ED m  and calculated as follows [ 11 ]: where: E λ  = erythemal spectral effectiveness S λ  = solar spectral irradiance in Wm -2 nm -1 T λ  = spectral transmittance of fabric Δ λ  = the bandwidth in nm λ  = the wavelength in nm UPF's were measured  in vitro  using a labsphere ®  UV-100 F Ultraviolet Transmission Analyzer according to standard AS/NZ 4399:1996 [ 12 ]. Fabrics with a UPF value in the range 15 – 24 were classified as having "Good UV Protection"; when the UPF values were between 25 and 39 fabrics were classified as having "Very Good UV Protection" and "Excellent UV Protection" classification was used when the UPF was 40 or greater [ 13 ]. In no event was a fabric assigned a UPF rating greater than 50. Measured UPF values were also correlated to the color strength of the dyed fabrics. Color strength was evaluated using K/S values generated by a HunterLab ColorQuest XE diffuse/8° spectrophotometer. K/S is a function of color depth and is represented by the equation of Kubelka and Munk (Equation 2). Higher the value of K/S greater is the color strength [ 14 , 15 ]. where R is the reflectance of the dyed fabric; K is the sorption coefficient, and S is the scattering coefficient. The spectrophotometer was standardized for a 1 inch diameter specimen viewing aperture in reflectance – specular included mode. Illuminant D65 and CIE 10-degree observer were used. During measurements, fabric samples were held flat and securely using a spring-loaded sample clamp. Three measurements were taken on each dyed fabric with the fabric rotated between measurements.
Methods
Three fabrics were chosen to cover the gamut of basic weave constructions. They were a bleached, mercerized plain weave cotton fabric, a bleached mercerized cotton twill and a desized and bleached cotton sateen. Fabric weight was measured according to ASTM Test Method D3776-96 [ 8 ]. Fabric thickness was measured according to ASTM Test Method D1777-96 [ 8 ]. Thread counts were measured according to ASTM D3775-98 [ 8 ].Natural plant colorants used were madder ( Rubia tinctorum ) and indigo ( Indigofera tinctoria ) and the natural colorant of insect origin was cochineal ( Dactylopius coccus ). Since natural dyes do not have affinity for cellulosic fibers an alum mordant was used to impart affinity. Fabrics were mordanted prior to dyeing by treating with alum at boil for 45 minutes. The liquor ratio was 1:40 and alum concentration was 10% on weight of the fabric. After mordanting, fabric was squeezed thoroughly and dyed.Rubia tinctorumIndigofera tinctoriaDactylopius coccusMadder and cochineal dyeings were done in stainless steel canisters of an Atlas launder-ometer using 2%, 4% and 6% dye on weight of fabric. The liquor-goods ratio was 40:1. Fabrics were introduced into the dyeing solutions at room temperature. Temperature was raised to the boil and dyeing continued at the boil for 60 minutes. After dyeing, fabrics were rinsed in deionized water, washed using a non-ionic detergent and air-dried. Three replications were done for each colorant and at each dye concentration.Dyeing with indigo was done in the following manner. Indigo dye was made into a paste and solubilized using sodium hydroxide and sodium hydrosulfite. Fabrics were introduced into dyebaths containing 2%, 4% and 6% dye on weight of fabric. The liquor-goods ratio was 40:1. After thirty minutes of dyeing the fabrics were removed and oxidized by drying in air. The fabrics were then rinsed in deionized water and washed using a non-ionic detergent and dried.Direct and diffuse UV transmittance through a fabric is the crucial factor determining the UV protection of textiles [ 9 ]. Ultraviolet protection factor (UPF) is the scientific term used to indicate the amount of Ultraviolet (UV) protection provided to skin by fabric. UPF values are analogous to SPF values the only distinction being that SPF values for sunscreens are determined through human testing whereas UPF values are based on instrumental measurements [ 10 ]. UPF is defined as the ratio of the average effective UV irradiance calculated for unprotected skin to the average UV irradiance calculated for skin protected by the test fabric [ 5 , 10 ]. The higher the value, the longer a person can stay in the sun until the area of skin under the fabric becomes red [ 5 , 10 ]. An effective UVR dose (ED) for unprotected skin is calculated by convolving the incident solar spectral power distribution with the relative spectral effectiveness function and summing over the wavelength range 290-400 nm. The calculation is repeated with the spectral transmission of the fabric as an additional weighting to get the effective dose (ED m ) for the skin when it is protected. The UPF is defined as the ratio of ED to ED m  and calculated as follows [ 11 ]:where:E λ  = erythemal spectral effectivenessλS λ  = solar spectral irradiance in Wm -2 nm -1λT λ  = spectral transmittance of fabricλΔ λ  = the bandwidth in nmλλ  = the wavelength in nmλUPF's were measured  in vitro  using a labsphere ®  UV-100 F Ultraviolet Transmission Analyzer according to standard AS/NZ 4399:1996 [ 12 ]. Fabrics with a UPF value in the range 15 – 24 were classified as having "Good UV Protection"; when the UPF values were between 25 and 39 fabrics were classified as having "Very Good UV Protection" and "Excellent UV Protection" classification was used when the UPF was 40 or greater [ 13 ]. In no event was a fabric assigned a UPF rating greater than 50.in vitroMeasured UPF values were also correlated to the color strength of the dyed fabrics. Color strength was evaluated using K/S values generated by a HunterLab ColorQuest XE diffuse/8° spectrophotometer. K/S is a function of color depth and is represented by the equation of Kubelka and Munk (Equation 2). Higher the value of K/S greater is the color strength [ 14 , 15 ].where R is the reflectance of the dyed fabric; K is the sorption coefficient, and S is the scattering coefficient. The spectrophotometer was standardized for a 1 inch diameter specimen viewing aperture in reflectance – specular included mode. Illuminant D65 and CIE 10-degree observer were used. During measurements, fabric samples were held flat and securely using a spring-loaded sample clamp. Three measurements were taken on each dyed fabric with the fabric rotated between measurements.Results and discussion Fabric characterization parameters and UPF values prior to dyeing are listed in Table  1 . Based on the classification parameters referenced previously the plain weave fabric and the sateen weave fabric cannot be rated as offering any degree of protection since their UPF values were less than 15. The undyed twill weave fabric with a UPF of 19.2 is rated as having Good UV Protection. The UPF values of the undyed fabrics can be explained in terms of fiber composition and fabric construction. In terms of fiber composition it is known that undyed bleached cotton, linen, acetate, and rayon fabrics afford poor protection against UV radiation [ 16 ]. Fabric construction parameters of weight and thickness show a positive correlation with UPF values. Higher the weight and thicker the fabric, higher is the degree of protection afforded by the fabric. Accordingly, the twill weave fabric with a weight of 235 g/m 2  and a thickness of 0.069 centimeters has the highest UPF value followed by the sateen weave fabric which weighed 235 g/m 2  and was 0.061 centimeters thick. The plain weave fabric with a weight of 120 g/m 2  and a thickness of 0.035 centimeters offers no protection against transmittance of UV rays. The positive correlation between fabric weight and fabric thickness with UPF values can be explained with reference to porosity. Porosity is a measure of tightness of weave and is also called as Coverfactor. Cover factor is defined as the percentage area occupied by warp and filling yarns in a given fabric area [ 4 , 17 ]. The closer the weave, the more is the percentage area occupied by the yarns and more opaque is the fabric to UV radiation. Cover factor is increased by an increase in weight per unit area. Heavier fabric minimizes UV transmission by virtue of having smaller spaces between yarns thus blocking more radiation [ 3 , 17 ]. A related variable is thickness. Thicker, denser fabrics transmit less UV radiation and have a higher cover factor [ 10 ]. The data also reveals a negative correlation between thread count and UPF. Higher the thread count, lower is the degree of protection afforded by the fabric. The plain weave fabric with a thread count of 205 had a UPF of 3.2 whereas the twill weave fabric with a thread count of 81 had a UPF of 19.2 with the sateen weave between the two with a thread count of 106 and a UPF of 13.3. A possible explanation for the negative correlation between thread count and UPF is the fact that fabrics that are thinner tend to contain finer yarns and therefore have the highest thread counts [ 10 ]. In other words thickness and thread count are inversely correlated a point substantiated by the values in Table  1 . Table 1 Fabric characterization parameters and % UV transmittance of undyed fabrics Weight, g/m 2 Thickness, cm. Thread Count (per inch) UPF UV Protection Class Plain weave 120 0.035 205 3.8 No Class Twill weave 258 0.069 81 19.2 Good Sateen weave 235 0.061 106 13.3 No Class The percent UV transmittance data in the presence and absence of colorants for the plain weave fabric is shown in Figure  1 . It is noted that since the relative erythemal spectral effectiveness is higher in the UV-B region compared to the UV-A region, the UPF values depend primarily on transmission in the UV-B region. Undyed plain weave fabric had significant transmittance and consequently a very low UPF value of 3.8. UPF values and protection categories of the plain weave fabric dyed with the different colorants are listed in Table  2 . As is evident from the transmission data and the corresponding UPF values all colorants used in the study caused a dramatic reduction in UV radiation transmission through the plain weave fabric. The increase in UPF values in the presence of colorant was especially significant for the cochineal and indigo dyed samples which were classified as having Very Good (UPF values between 25 and 39) to Excellent UV Protection (UPF values 40 or greater). Madder dyed samples could be classified as having Good UV Protection (UPF values between 15 and 24) to Very Good UV Protection. Compared to cochineal and indigo, madder is a paler color and therefore these results agree with previous data reported by Reinert et al. [ 18 ] who showed that pale colored fabrics of cotton, silk, polyamide, and polyamide/elastan gave less protection against intense UV radiation. The results also show that UPF values for colorants applied at higher concentrations gave higher UPF values. For example, the UPF of the plain weave fabric at a 2% indigo on weight of fabric was 43.1 and that increased to greater than UPF 50 at an indigo concentration of 6%. We agree with Gies et al. [ 11 ] who indicated that dyeing fabrics in deeper shades and darker colors improves sun protection properties. Thus although the studies by Reinert at al. and Gies et al. were done with synthetic dyes their conclusions seem to hold with natural colorants as well. Figure 1 UV transmission of plain weave fabric in the absence and presence of colorants. Table 2 UPF values, protection class and K/S values of plain weave fabric dyed with natural colorants at different concentrations Colorant UPF UV Protection Class K/S Plain weave 2% Madder 11.1 No Class 0.20 4% Madder 15.8 Good 0.28 6% Madder 16.6 Good 0.38 2% Cochineal 28.5 Very Good 0.63 4% Cochineal 34 Very Good 0.79 6% Cochineal 36.6 Very Good 0.99 2% Indigo 43.1 Excellent 1.78 4% Indigo > 50 Excellent 2.56 6% Indigo > 50 Excellent 3.02 The K/S values of the dyed fabrics which are a measure of color depth seem to support the claim that higher color depths increases UPF values. For example, in the case of the madder dyed samples when the K/S value increased from 0.20 to 0.38 the UPF values rose from 11.1 to 16.6. However, the relationship of K/S with UPF is limited to the same fabric type and the results cannot be generalized across fabrics of different weave structures. A primary reason for this observation is the acknowledgement that UPF values are dependent on a multitude of fabric construction factors such as pores in the fabric, thickness, and weight in addition to processing parameters such as dyeing and finishing. Another probable reason is the dependence of K/S on the absorbing properties of colorants in the visible region of the spectrum and that may not influence the absorption characteristics of colorants in the UV region. The percent UV transmittance data in the presence and absence of colorants for the twill weave fabric is shown in Figure  2 . UPF values and protection categories for the dyed twill weave fabric are shown in Tables  3 . The twill weave fabric which prior to dyeing was rated as offering Good UV Protection moved to the Excellent UV Protection classification irrespective of the colorant and the concentration of the dye used. Again, it was found that dark colors within the same fabric type transmit less UV radiation than light colors and consequently have higher UPFs. Figure 2 UV transmission of twill weave fabric in the absence and presence of colorants. Table 3 UPF values, protection class and K/S values of twill weave fabric dyed with natural colorants at different concentrations Colorant UPF UV Protection Class K/S Twill weave 2% Madder > 50 Excellent 0.27 4% Madder > 50 Excellent 0.44 6% Madder > 50 Excellent 0.59 2 % Cochineal > 50 Excellent 0.82 4% Cochineal > 50 Excellent 1.70 6% Cochineal > 50 Excellent 1.89 2% Indigo > 50 Excellent 2.33 4% Indigo > 50 Excellent 3.76 6% Indigo > 50 Excellent 4.00 Table  4  shows the UPF values and protection categories for the dyed sateen weave fabric. The percent UV transmittance data in the presence and absence of colorants for the sateen weave fabric is shown in Figure  3 . The increase in UPF values of the sateen weave dyed fabrics was dramatic in the sense that the sateen which prior to dyeing could not be rated (UPF < 15) achieved the Excellent UV Protection classification by virtue of its UPF values increasing by more than a factor of four (UPF > 50). This result was true for all colorants and at all dye concentrations. Again, as was the case with the dyed plain weave fabric, the color strength (K/S) of the cochineal and indigo dyed twill and sateen fabrics were higher than the color strength of the madder dyed fabrics conclusively establishing that indigo and cochineal colorants resulted in deeper colors on the fabrics. Table 4 UPF values, protection class and K/S values of sateen weave fabric dyed with natural colorants at different concentrations Colorant UPF UV Protection Class K/S Sateen weave 2% Madder > 50 Excellent 0.25 4% Madder > 50 Excellent 0.36 6% Madder > 50 Excellent 0.59 2% Cochineal > 50 Excellent 1.78 4% Cochineal > 50 Excellent 1.87 6% Cochineal > 50 Excellent 2.42 2% Indigo > 50 Excellent 1.66 4%Indigo > 50 Excellent 2.05 6% Indigo > 50 Excellent 2.40 Figure 3 UV transmission of sateen weave fabric in the absence and presence of colorants.
Results and discussion
Fabric characterization parameters and UPF values prior to dyeing are listed in Table  1 . Based on the classification parameters referenced previously the plain weave fabric and the sateen weave fabric cannot be rated as offering any degree of protection since their UPF values were less than 15. The undyed twill weave fabric with a UPF of 19.2 is rated as having Good UV Protection. The UPF values of the undyed fabrics can be explained in terms of fiber composition and fabric construction. In terms of fiber composition it is known that undyed bleached cotton, linen, acetate, and rayon fabrics afford poor protection against UV radiation [ 16 ]. Fabric construction parameters of weight and thickness show a positive correlation with UPF values. Higher the weight and thicker the fabric, higher is the degree of protection afforded by the fabric. Accordingly, the twill weave fabric with a weight of 235 g/m 2  and a thickness of 0.069 centimeters has the highest UPF value followed by the sateen weave fabric which weighed 235 g/m 2  and was 0.061 centimeters thick. The plain weave fabric with a weight of 120 g/m 2  and a thickness of 0.035 centimeters offers no protection against transmittance of UV rays. The positive correlation between fabric weight and fabric thickness with UPF values can be explained with reference to porosity. Porosity is a measure of tightness of weave and is also called as Coverfactor. Cover factor is defined as the percentage area occupied by warp and filling yarns in a given fabric area [ 4 , 17 ]. The closer the weave, the more is the percentage area occupied by the yarns and more opaque is the fabric to UV radiation. Cover factor is increased by an increase in weight per unit area. Heavier fabric minimizes UV transmission by virtue of having smaller spaces between yarns thus blocking more radiation [ 3 , 17 ]. A related variable is thickness. Thicker, denser fabrics transmit less UV radiation and have a higher cover factor [ 10 ]. The data also reveals a negative correlation between thread count and UPF. Higher the thread count, lower is the degree of protection afforded by the fabric. The plain weave fabric with a thread count of 205 had a UPF of 3.2 whereas the twill weave fabric with a thread count of 81 had a UPF of 19.2 with the sateen weave between the two with a thread count of 106 and a UPF of 13.3. A possible explanation for the negative correlation between thread count and UPF is the fact that fabrics that are thinner tend to contain finer yarns and therefore have the highest thread counts [ 10 ]. In other words thickness and thread count are inversely correlated a point substantiated by the values in Table  1 .Fabric characterization parameters and % UV transmittance of undyed fabricsThe percent UV transmittance data in the presence and absence of colorants for the plain weave fabric is shown in Figure  1 . It is noted that since the relative erythemal spectral effectiveness is higher in the UV-B region compared to the UV-A region, the UPF values depend primarily on transmission in the UV-B region. Undyed plain weave fabric had significant transmittance and consequently a very low UPF value of 3.8. UPF values and protection categories of the plain weave fabric dyed with the different colorants are listed in Table  2 . As is evident from the transmission data and the corresponding UPF values all colorants used in the study caused a dramatic reduction in UV radiation transmission through the plain weave fabric. The increase in UPF values in the presence of colorant was especially significant for the cochineal and indigo dyed samples which were classified as having Very Good (UPF values between 25 and 39) to Excellent UV Protection (UPF values 40 or greater). Madder dyed samples could be classified as having Good UV Protection (UPF values between 15 and 24) to Very Good UV Protection. Compared to cochineal and indigo, madder is a paler color and therefore these results agree with previous data reported by Reinert et al. [ 18 ] who showed that pale colored fabrics of cotton, silk, polyamide, and polyamide/elastan gave less protection against intense UV radiation. The results also show that UPF values for colorants applied at higher concentrations gave higher UPF values. For example, the UPF of the plain weave fabric at a 2% indigo on weight of fabric was 43.1 and that increased to greater than UPF 50 at an indigo concentration of 6%. We agree with Gies et al. [ 11 ] who indicated that dyeing fabrics in deeper shades and darker colors improves sun protection properties. Thus although the studies by Reinert at al. and Gies et al. were done with synthetic dyes their conclusions seem to hold with natural colorants as well.UPF values, protection class and K/S values of plain weave fabric dyed with natural colorants at different concentrationsThe K/S values of the dyed fabrics which are a measure of color depth seem to support the claim that higher color depths increases UPF values. For example, in the case of the madder dyed samples when the K/S value increased from 0.20 to 0.38 the UPF values rose from 11.1 to 16.6. However, the relationship of K/S with UPF is limited to the same fabric type and the results cannot be generalized across fabrics of different weave structures. A primary reason for this observation is the acknowledgement that UPF values are dependent on a multitude of fabric construction factors such as pores in the fabric, thickness, and weight in addition to processing parameters such as dyeing and finishing. Another probable reason is the dependence of K/S on the absorbing properties of colorants in the visible region of the spectrum and that may not influence the absorption characteristics of colorants in the UV region.The percent UV transmittance data in the presence and absence of colorants for the twill weave fabric is shown in Figure  2 . UPF values and protection categories for the dyed twill weave fabric are shown in Tables  3 . The twill weave fabric which prior to dyeing was rated as offering Good UV Protection moved to the Excellent UV Protection classification irrespective of the colorant and the concentration of the dye used. Again, it was found that dark colors within the same fabric type transmit less UV radiation than light colors and consequently have higher UPFs.UPF values, protection class and K/S values of twill weave fabric dyed with natural colorants at different concentrationsTable  4  shows the UPF values and protection categories for the dyed sateen weave fabric. The percent UV transmittance data in the presence and absence of colorants for the sateen weave fabric is shown in Figure  3 . The increase in UPF values of the sateen weave dyed fabrics was dramatic in the sense that the sateen which prior to dyeing could not be rated (UPF < 15) achieved the Excellent UV Protection classification by virtue of its UPF values increasing by more than a factor of four (UPF > 50). This result was true for all colorants and at all dye concentrations. Again, as was the case with the dyed plain weave fabric, the color strength (K/S) of the cochineal and indigo dyed twill and sateen fabrics were higher than the color strength of the madder dyed fabrics conclusively establishing that indigo and cochineal colorants resulted in deeper colors on the fabrics.UPF values, protection class and K/S values of sateen weave fabric dyed with natural colorants at different concentrationsConclusions Fabric weight and thickness are important predictors of UPF values for undyed cotton fabrics. In general, it was found that increase in weight and thickness increased the UPF though the relationship was not linear. UPF of undyed fabrics was significantly enhanced by dyeing with natural colorants especially for fabrics such as the plain weave and the sateen weave fabrics that displayed no protective abilities in the undyed state. The degree of protection imparted after dyeing was a function of the concentration of the colorant in the fabric. Within the same fabric type, as the percentage depth of shade increased so did the UPF values. In addition, darker colors such as indigo provide better protection on account of higher UV absorption. Based on the results of this study it can be theorized that plain, twill or sateen weave cotton fabrics dyed with natural colorants can provide good protection against ultraviolet rays with the only condition being that either the color has to be a dark hue or the concentration of the colorant in the fabric has to be high.
Conclusions
Fabric weight and thickness are important predictors of UPF values for undyed cotton fabrics. In general, it was found that increase in weight and thickness increased the UPF though the relationship was not linear. UPF of undyed fabrics was significantly enhanced by dyeing with natural colorants especially for fabrics such as the plain weave and the sateen weave fabrics that displayed no protective abilities in the undyed state. The degree of protection imparted after dyeing was a function of the concentration of the colorant in the fabric. Within the same fabric type, as the percentage depth of shade increased so did the UPF values. In addition, darker colors such as indigo provide better protection on account of higher UV absorption. Based on the results of this study it can be theorized that plain, twill or sateen weave cotton fabrics dyed with natural colorants can provide good protection against ultraviolet rays with the only condition being that either the color has to be a dark hue or the concentration of the colorant in the fabric has to be high.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions AKS conceived the study, carried out the dyeing and testing and drafted the manuscript.
Authors' contributions
AKS conceived the study, carried out the dyeing and testing and drafted the manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background Carbonic anhydrase (CA) classically catalyses the reversible hydration of dissolved CO 2  to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems.
Background
Carbonic anhydrase (CA) classically catalyses the reversible hydration of dissolved CO 2  to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems.Methods The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract.
Methods
The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract.Results The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta.
Results
The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta.Conclusion The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.
Conclusion
The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.

Background Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that are responsible for the reversible hydration of carbon dioxide in a reaction CO 2  + H 2 O ↔ H +  + HCO 3 - . CAs are produced in a variety of tissues where they participate in several important biological processes such as acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and body fluid generation [ 1 , 2 ]. The mammalian α-CA gene family includes at least twelve enzymatically active isoforms with different structural and catalytic properties. CA I, II, III, VII and XIII are cytosolic enzymes [ 1 , 3 , 4 ]. CA VA and VB are mitochondrial proteins encoded by nuclear DNA [ 5 , 6 ]. CA VI is the only secretory form being present in saliva and milk [ 7 ]. The cluster of membrane-bound CAs includes four isozymes: CA IV, IX, XII, and XIV [ 8 - 11 ]. The other members of the CA gene family (CA VIII, X and XI) are inactive isoforms whose functions have not yet been described [ 3 , 12 , 13 ]. It has been previously suggested that CAs may play important roles in the uterine endometrium by maintaining the appropriate pH balance through the catalysis of the production of bicarbonate ions [ 14 ]. Indeed, the role of bicarbonate in fertilization has been demonstrated in a number of previous studies. It is functionally involved in some key processes such as sperm cell capacitation and regulation of sperm motility [ 15 - 17 ]. Similarly, CAs may have several functions also in the placenta. They can be active in intermediary metabolism and provide ions for exchange in transepithelial movement of ions and fluid [ 18 ]. CA activity has been studied in pig, horse, cow, mink, rat and human placentas, and the results show considerable heterogeneity among different species [ 18 ]. Previous immunochemical studies have shown evidence for expression of CA II but not CA I or III in the bovine placenta [ 19 ]. Both CA I and II are expressed in the human syncytiotrophoblasts [ 20 - 22 ] and, especially CA II, in the fetal villous endothelium of mature placenta [ 22 ]. CA IV-positive staining has been reported in the mouse placenta by Rosen and coauthors [ 23 ]. Their data showed strong CA IV immunoreactivity in the mouse trophoblasts and endodermal layer of the yolk sac. In the mouse genital tract, CA I, II and III have been reported by Ge and Spicer [ 24 ]. These isozymes were reported to be present in the theca interna cells in the mouse ovary, and CA I was found in the zona pellucida and cytoplasmic foci in follicular granulosa cells. In the mouse oviductal epithelium, CA II expression showed distinct variation. The reaction was absent in the infundibulum, whereas the ampulla and isthmus showed positive staining. CA XIII is the newest member of the CA enzyme family, which has been described in the mouse and human endometrium along with several other positive tissues [ 4 ]. As a cytosolic isozyme it may be one of the major proteins regulating the pH and bicarbonate homeostasis not only in the endometrial cells but also in the lumen of the uterus. These mechanisms are complex due to the presence of several isozymes, however, and may greatly differ between species. For example, the human endometrium contains CA II only in the capillaries, whereas this high activity isozyme is abundantly expressed in the epithelial cells of the mouse endometrium [ 4 , 24 ]. CA IX is expressed at the basolateral plasma membrane of the human, rat and mouse epithelial cells [ 25 , 26 ]. In a recent extensive study, Ivanov  et al  [ 27 ] analyzed a number of normal human tissues for the expression of CA IX. Among reproductive organs, they reported positive signal for CA IX mRNA and protein in the efferent ducts, rete testis, and rete ovarii. Human CA XII is expressed in several organs including colon, kidney, and pancreas [ 28 - 30 ]. In the human female reproductive tract, CA XII has been shown both in the glandular and surface epithelium of the endometrium, while it was only occasionally present in the cervix [ 14 ]. Ivanov  et al  [ 27 ] further confirmed CA XII expression in the glandular epithelium during the proliferative phase. In this report we studied the expression of CA II, IX, XII and XIII in mouse female genital organs including uterus, ovary and placenta. The studies were specially focused on CA IX and XII, which have been designated as tumor-associated isozymes [ 9 , 10 ]. In addition to some normal tissues, both isozymes are overexpressed in several carcinomas such as renal and colorectal cancers [ 9 , 27 , 31 , 32 ]. A previous study has also demonstrated CA IX and XII expression in a number of neoplasias derived from the female reproductive tract [ 27 ]. However, there have been no previous studies on these isozymes in the female murine reproductive organs. The conservation of CA XII expression in both mouse and human endometrium shown in the present paper suggests a role for this isozyme in reproductive physiology.
Background
Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that are responsible for the reversible hydration of carbon dioxide in a reaction CO 2  + H 2 O ↔ H +  + HCO 3 - . CAs are produced in a variety of tissues where they participate in several important biological processes such as acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and body fluid generation [ 1 , 2 ]. The mammalian α-CA gene family includes at least twelve enzymatically active isoforms with different structural and catalytic properties. CA I, II, III, VII and XIII are cytosolic enzymes [ 1 , 3 , 4 ]. CA VA and VB are mitochondrial proteins encoded by nuclear DNA [ 5 , 6 ]. CA VI is the only secretory form being present in saliva and milk [ 7 ]. The cluster of membrane-bound CAs includes four isozymes: CA IV, IX, XII, and XIV [ 8 - 11 ]. The other members of the CA gene family (CA VIII, X and XI) are inactive isoforms whose functions have not yet been described [ 3 , 12 , 13 ].It has been previously suggested that CAs may play important roles in the uterine endometrium by maintaining the appropriate pH balance through the catalysis of the production of bicarbonate ions [ 14 ]. Indeed, the role of bicarbonate in fertilization has been demonstrated in a number of previous studies. It is functionally involved in some key processes such as sperm cell capacitation and regulation of sperm motility [ 15 - 17 ]. Similarly, CAs may have several functions also in the placenta. They can be active in intermediary metabolism and provide ions for exchange in transepithelial movement of ions and fluid [ 18 ].CA activity has been studied in pig, horse, cow, mink, rat and human placentas, and the results show considerable heterogeneity among different species [ 18 ]. Previous immunochemical studies have shown evidence for expression of CA II but not CA I or III in the bovine placenta [ 19 ]. Both CA I and II are expressed in the human syncytiotrophoblasts [ 20 - 22 ] and, especially CA II, in the fetal villous endothelium of mature placenta [ 22 ]. CA IV-positive staining has been reported in the mouse placenta by Rosen and coauthors [ 23 ]. Their data showed strong CA IV immunoreactivity in the mouse trophoblasts and endodermal layer of the yolk sac. In the mouse genital tract, CA I, II and III have been reported by Ge and Spicer [ 24 ]. These isozymes were reported to be present in the theca interna cells in the mouse ovary, and CA I was found in the zona pellucida and cytoplasmic foci in follicular granulosa cells. In the mouse oviductal epithelium, CA II expression showed distinct variation. The reaction was absent in the infundibulum, whereas the ampulla and isthmus showed positive staining. CA XIII is the newest member of the CA enzyme family, which has been described in the mouse and human endometrium along with several other positive tissues [ 4 ]. As a cytosolic isozyme it may be one of the major proteins regulating the pH and bicarbonate homeostasis not only in the endometrial cells but also in the lumen of the uterus. These mechanisms are complex due to the presence of several isozymes, however, and may greatly differ between species. For example, the human endometrium contains CA II only in the capillaries, whereas this high activity isozyme is abundantly expressed in the epithelial cells of the mouse endometrium [ 4 , 24 ].CA IX is expressed at the basolateral plasma membrane of the human, rat and mouse epithelial cells [ 25 , 26 ]. In a recent extensive study, Ivanov  et al  [ 27 ] analyzed a number of normal human tissues for the expression of CA IX. Among reproductive organs, they reported positive signal for CA IX mRNA and protein in the efferent ducts, rete testis, and rete ovarii.et alHuman CA XII is expressed in several organs including colon, kidney, and pancreas [ 28 - 30 ]. In the human female reproductive tract, CA XII has been shown both in the glandular and surface epithelium of the endometrium, while it was only occasionally present in the cervix [ 14 ]. Ivanov  et al  [ 27 ] further confirmed CA XII expression in the glandular epithelium during the proliferative phase.et alIn this report we studied the expression of CA II, IX, XII and XIII in mouse female genital organs including uterus, ovary and placenta. The studies were specially focused on CA IX and XII, which have been designated as tumor-associated isozymes [ 9 , 10 ]. In addition to some normal tissues, both isozymes are overexpressed in several carcinomas such as renal and colorectal cancers [ 9 , 27 , 31 , 32 ]. A previous study has also demonstrated CA IX and XII expression in a number of neoplasias derived from the female reproductive tract [ 27 ]. However, there have been no previous studies on these isozymes in the female murine reproductive organs. The conservation of CA XII expression in both mouse and human endometrium shown in the present paper suggests a role for this isozyme in reproductive physiology.Materials and methods Antibodies In the present study, we used the following antibodies which have been produced and characterized earlier: rabbit anti-mouse CA II [ 4 ], rabbit anti-mouse CA IX [ 26 ], rabbit anti-mouse CA XII [ 33 ], and rabbit anti-mouse CA XIII [ 4 ]. Collection of tissue samples Two adult Balb/c mice were sacrificed by CO 2  asphyxiation followed by decapitation. Uterus, ovary and placenta samples were collected from both animals. The samples were immersion-fixed overnight in Carnoy's fluid (ethanol, chloroform and acetic acid (6:3:1)). Then the specimens were treated with absolute ethanol for 30 min, with 1:1 mixture of ethanol and chloroform for 15 min, and finally with chloroform for 30 min. Paraffin embedding was performed in a vacuum oven for 2 h at +58°C. Paraffin wax was purchased from Fluka Chemie GmbH (Buchs, Schwitzerland). To collect a placenta sample, a mouse was sacrificed at 9 days of pregnancy. The ninth day was chosen since it represents the middle gestational phase. It is also the time when the most critical steps of organogenesis occur in mouse. For western blotting, uterus, kidney and colon were removed and rapidly frozen in liquid nitrogen. The tissue samples for western blot were homogenized with HEPES buffer. Total protein concentration was determined after homogenization using BCA Protein Assay Kit (Pierce, Rockford, IL) according to manufacturer's instructions. The study protocols were approved by the Animal Care Committee of Tampere University. Immunohistochemistry In the mouse tissues, the localization of CA IX and XII was examined by immunoperoxidase method. Antibodies against CA II and XIII were used as positive controls for the immunostaining. All experiments were performed in duplicate and included control staining with non-immune normal rabbit serum (NRS). NRS was obtained from a rabbit that was later immunized against mouse CA XIII. The tissue samples fixed in Carnoy's fluid and embedded in paraffin were cut at 5 μm sections and placed on microscope slides. The peroxidase-anti-peroxidase complex method included the following steps: a) pretreatment of the sections with undiluted cow colostral whey (Biotop, Oulu, Finland) for 40 min and rinsing in phosphate-buffered saline (PBS); b) incubation for 1 h with the primary antiserum (anti-mouse CA II, CA IX, CA XII or CA XIII) or NRS diluted 1:100 in PBS containing 1% bovine serum albumin (BSA) (BSA-PBS solution); c) treatment with undiluted cow colostral whey (40 min); d) incubation for 1 h with swine anti-rabbit IgG (Dakopatts, Copenhagen, Denmark) diluted 1:100 in 1% BSA-PBS; e) incubation for 30 min with peroxidase-anti-peroxidase rabbit conjugate (Dakopatts) diluted 1:500 in PBS; f) incubation for 2 min with 3,3'diaminobenzidine tetrahydrochloride (DAB) solution (6 mg DAB in 10 ml PBS plus 3.3 μl H 2 O 2 ) as chromogen. The sections were washed three times for 10 min in PBS after incubation steps b and d and four times for 5 min after incubation step e. All of the incubations and washings were carried out at room temperature. The sections were finally mounted in Neo-Mount (Merck, Darmstadt, Germany). The stained sections were examined and photographed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany). Western blot The samples containing 50 μg of protein from mouse uterus, kidney and colon were analyzed by SDS-PAGE under reducing conditions [ 34 ]. All of the reagents and the protein standard (BenchMark™ Prestained Protein Ladder) for SDS-PAGE were purchased from Invitrogen (Carlsbad, CA) except Laemmli sample buffer that was obtained from Sigma (St. Louis, MO). Electrophoresis (200 V for 40 min) was performed in a Novex Xcell II mini cell electrophoresis unit (Invitrogen) with a 10% Bis-Tris gel (Invitrogen). The separated proteins were transferred electrophoretically from the gel to a polyvinylidene fluoride (PVDF) membrane (Invitrogen) in a Novex Xcell II blot module (Invitrogen). The transfer buffer (NuPAGE Transfer Buffer™) was purchased from Invitrogen. The blot module was filled with the transfer buffer until the gel/membrane assembly was covered. The outer buffer chamber was filled with 650 ml deionized water. The protein transfer was performed using a constant voltage of 36 V for 1 h 20 min. After the transblotting, the sample lines were detected by ECL western blotting detection reagents and analysis system (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. First, the sample lines were incubated with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0,3 % Tween 20) containing 10 % cow colostral whey for 25 min and then the first antibodies diluted 1:2000 (anti-CA II, anti-CA IX, anti-CA XII, NRS) or 1:1000 (anti-CA XIII) in TBST buffer for 1 h. The PVDF membranes were washed five times for 5 min with TBST buffer and incubated for 1 h with peroxidase-linked ECL Anti-Rabbit IgG (Amersham Biosciences) diluted 1:25 000 in TBST buffer. After washing four times 5 min in TBST buffer, the polypeptides were visualized by a chemiluminescence substrate (ECL detection reagents 1 + 2, Amersham Biosciences). Kodak™ Biomax™ MS-1 films (Amersham Biosciences) were exposed to the chemiluminescence for 5 min (CA IX and XII) or 1 min (CA II and CA XIII). All the steps were carried out at room temperature. The western blotting experiments were performed in triplicate to confirm the reproducibility of the results.
Materials and methods
Antibodies In the present study, we used the following antibodies which have been produced and characterized earlier: rabbit anti-mouse CA II [ 4 ], rabbit anti-mouse CA IX [ 26 ], rabbit anti-mouse CA XII [ 33 ], and rabbit anti-mouse CA XIII [ 4 ].
Antibodies
In the present study, we used the following antibodies which have been produced and characterized earlier: rabbit anti-mouse CA II [ 4 ], rabbit anti-mouse CA IX [ 26 ], rabbit anti-mouse CA XII [ 33 ], and rabbit anti-mouse CA XIII [ 4 ].Collection of tissue samples Two adult Balb/c mice were sacrificed by CO 2  asphyxiation followed by decapitation. Uterus, ovary and placenta samples were collected from both animals. The samples were immersion-fixed overnight in Carnoy's fluid (ethanol, chloroform and acetic acid (6:3:1)). Then the specimens were treated with absolute ethanol for 30 min, with 1:1 mixture of ethanol and chloroform for 15 min, and finally with chloroform for 30 min. Paraffin embedding was performed in a vacuum oven for 2 h at +58°C. Paraffin wax was purchased from Fluka Chemie GmbH (Buchs, Schwitzerland). To collect a placenta sample, a mouse was sacrificed at 9 days of pregnancy. The ninth day was chosen since it represents the middle gestational phase. It is also the time when the most critical steps of organogenesis occur in mouse. For western blotting, uterus, kidney and colon were removed and rapidly frozen in liquid nitrogen. The tissue samples for western blot were homogenized with HEPES buffer. Total protein concentration was determined after homogenization using BCA Protein Assay Kit (Pierce, Rockford, IL) according to manufacturer's instructions. The study protocols were approved by the Animal Care Committee of Tampere University.
Collection of tissue samples
Two adult Balb/c mice were sacrificed by CO 2  asphyxiation followed by decapitation. Uterus, ovary and placenta samples were collected from both animals. The samples were immersion-fixed overnight in Carnoy's fluid (ethanol, chloroform and acetic acid (6:3:1)). Then the specimens were treated with absolute ethanol for 30 min, with 1:1 mixture of ethanol and chloroform for 15 min, and finally with chloroform for 30 min. Paraffin embedding was performed in a vacuum oven for 2 h at +58°C. Paraffin wax was purchased from Fluka Chemie GmbH (Buchs, Schwitzerland). To collect a placenta sample, a mouse was sacrificed at 9 days of pregnancy. The ninth day was chosen since it represents the middle gestational phase. It is also the time when the most critical steps of organogenesis occur in mouse. For western blotting, uterus, kidney and colon were removed and rapidly frozen in liquid nitrogen. The tissue samples for western blot were homogenized with HEPES buffer. Total protein concentration was determined after homogenization using BCA Protein Assay Kit (Pierce, Rockford, IL) according to manufacturer's instructions. The study protocols were approved by the Animal Care Committee of Tampere University.Immunohistochemistry In the mouse tissues, the localization of CA IX and XII was examined by immunoperoxidase method. Antibodies against CA II and XIII were used as positive controls for the immunostaining. All experiments were performed in duplicate and included control staining with non-immune normal rabbit serum (NRS). NRS was obtained from a rabbit that was later immunized against mouse CA XIII. The tissue samples fixed in Carnoy's fluid and embedded in paraffin were cut at 5 μm sections and placed on microscope slides. The peroxidase-anti-peroxidase complex method included the following steps: a) pretreatment of the sections with undiluted cow colostral whey (Biotop, Oulu, Finland) for 40 min and rinsing in phosphate-buffered saline (PBS); b) incubation for 1 h with the primary antiserum (anti-mouse CA II, CA IX, CA XII or CA XIII) or NRS diluted 1:100 in PBS containing 1% bovine serum albumin (BSA) (BSA-PBS solution); c) treatment with undiluted cow colostral whey (40 min); d) incubation for 1 h with swine anti-rabbit IgG (Dakopatts, Copenhagen, Denmark) diluted 1:100 in 1% BSA-PBS; e) incubation for 30 min with peroxidase-anti-peroxidase rabbit conjugate (Dakopatts) diluted 1:500 in PBS; f) incubation for 2 min with 3,3'diaminobenzidine tetrahydrochloride (DAB) solution (6 mg DAB in 10 ml PBS plus 3.3 μl H 2 O 2 ) as chromogen. The sections were washed three times for 10 min in PBS after incubation steps b and d and four times for 5 min after incubation step e. All of the incubations and washings were carried out at room temperature. The sections were finally mounted in Neo-Mount (Merck, Darmstadt, Germany). The stained sections were examined and photographed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany).
Immunohistochemistry
In the mouse tissues, the localization of CA IX and XII was examined by immunoperoxidase method. Antibodies against CA II and XIII were used as positive controls for the immunostaining. All experiments were performed in duplicate and included control staining with non-immune normal rabbit serum (NRS). NRS was obtained from a rabbit that was later immunized against mouse CA XIII. The tissue samples fixed in Carnoy's fluid and embedded in paraffin were cut at 5 μm sections and placed on microscope slides. The peroxidase-anti-peroxidase complex method included the following steps: a) pretreatment of the sections with undiluted cow colostral whey (Biotop, Oulu, Finland) for 40 min and rinsing in phosphate-buffered saline (PBS); b) incubation for 1 h with the primary antiserum (anti-mouse CA II, CA IX, CA XII or CA XIII) or NRS diluted 1:100 in PBS containing 1% bovine serum albumin (BSA) (BSA-PBS solution); c) treatment with undiluted cow colostral whey (40 min); d) incubation for 1 h with swine anti-rabbit IgG (Dakopatts, Copenhagen, Denmark) diluted 1:100 in 1% BSA-PBS; e) incubation for 30 min with peroxidase-anti-peroxidase rabbit conjugate (Dakopatts) diluted 1:500 in PBS; f) incubation for 2 min with 3,3'diaminobenzidine tetrahydrochloride (DAB) solution (6 mg DAB in 10 ml PBS plus 3.3 μl H 2 O 2 ) as chromogen. The sections were washed three times for 10 min in PBS after incubation steps b and d and four times for 5 min after incubation step e. All of the incubations and washings were carried out at room temperature. The sections were finally mounted in Neo-Mount (Merck, Darmstadt, Germany). The stained sections were examined and photographed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany).Western blot The samples containing 50 μg of protein from mouse uterus, kidney and colon were analyzed by SDS-PAGE under reducing conditions [ 34 ]. All of the reagents and the protein standard (BenchMark™ Prestained Protein Ladder) for SDS-PAGE were purchased from Invitrogen (Carlsbad, CA) except Laemmli sample buffer that was obtained from Sigma (St. Louis, MO). Electrophoresis (200 V for 40 min) was performed in a Novex Xcell II mini cell electrophoresis unit (Invitrogen) with a 10% Bis-Tris gel (Invitrogen). The separated proteins were transferred electrophoretically from the gel to a polyvinylidene fluoride (PVDF) membrane (Invitrogen) in a Novex Xcell II blot module (Invitrogen). The transfer buffer (NuPAGE Transfer Buffer™) was purchased from Invitrogen. The blot module was filled with the transfer buffer until the gel/membrane assembly was covered. The outer buffer chamber was filled with 650 ml deionized water. The protein transfer was performed using a constant voltage of 36 V for 1 h 20 min. After the transblotting, the sample lines were detected by ECL western blotting detection reagents and analysis system (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. First, the sample lines were incubated with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0,3 % Tween 20) containing 10 % cow colostral whey for 25 min and then the first antibodies diluted 1:2000 (anti-CA II, anti-CA IX, anti-CA XII, NRS) or 1:1000 (anti-CA XIII) in TBST buffer for 1 h. The PVDF membranes were washed five times for 5 min with TBST buffer and incubated for 1 h with peroxidase-linked ECL Anti-Rabbit IgG (Amersham Biosciences) diluted 1:25 000 in TBST buffer. After washing four times 5 min in TBST buffer, the polypeptides were visualized by a chemiluminescence substrate (ECL detection reagents 1 + 2, Amersham Biosciences). Kodak™ Biomax™ MS-1 films (Amersham Biosciences) were exposed to the chemiluminescence for 5 min (CA IX and XII) or 1 min (CA II and CA XIII). All the steps were carried out at room temperature. The western blotting experiments were performed in triplicate to confirm the reproducibility of the results.
Western blot
The samples containing 50 μg of protein from mouse uterus, kidney and colon were analyzed by SDS-PAGE under reducing conditions [ 34 ]. All of the reagents and the protein standard (BenchMark™ Prestained Protein Ladder) for SDS-PAGE were purchased from Invitrogen (Carlsbad, CA) except Laemmli sample buffer that was obtained from Sigma (St. Louis, MO). Electrophoresis (200 V for 40 min) was performed in a Novex Xcell II mini cell electrophoresis unit (Invitrogen) with a 10% Bis-Tris gel (Invitrogen). The separated proteins were transferred electrophoretically from the gel to a polyvinylidene fluoride (PVDF) membrane (Invitrogen) in a Novex Xcell II blot module (Invitrogen). The transfer buffer (NuPAGE Transfer Buffer™) was purchased from Invitrogen. The blot module was filled with the transfer buffer until the gel/membrane assembly was covered. The outer buffer chamber was filled with 650 ml deionized water. The protein transfer was performed using a constant voltage of 36 V for 1 h 20 min. After the transblotting, the sample lines were detected by ECL western blotting detection reagents and analysis system (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. First, the sample lines were incubated with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0,3 % Tween 20) containing 10 % cow colostral whey for 25 min and then the first antibodies diluted 1:2000 (anti-CA II, anti-CA IX, anti-CA XII, NRS) or 1:1000 (anti-CA XIII) in TBST buffer for 1 h. The PVDF membranes were washed five times for 5 min with TBST buffer and incubated for 1 h with peroxidase-linked ECL Anti-Rabbit IgG (Amersham Biosciences) diluted 1:25 000 in TBST buffer. After washing four times 5 min in TBST buffer, the polypeptides were visualized by a chemiluminescence substrate (ECL detection reagents 1 + 2, Amersham Biosciences). Kodak™ Biomax™ MS-1 films (Amersham Biosciences) were exposed to the chemiluminescence for 5 min (CA IX and XII) or 1 min (CA II and CA XIII). All the steps were carried out at room temperature. The western blotting experiments were performed in triplicate to confirm the reproducibility of the results.Results Immunohistochemistry All the studied CA isozymes showed positive immunostaining in the epithelial cells of the mouse endometrium (Fig.  1 ). CA II and XII showed a somewhat reciprocal distribution pattern in that CA II was confined to the surface epithelial cells (Fig.  1C ), while CA XII was more intensely stained in the deeper endometrial glands (Fig.  1A ). It is noteworthy, however, that CA XII was clearly expressed also in the surface epithelial cells, but the staining intensity was weaker compared to the glands. As expected, the strongest reaction for CA XII was associated with the basolateral plasma membrane, and unexpectedly, also CA II immunoreaction was most intense at the plasma membrane. CA IX and XIII showed weak reactions in both surface and glandular epithelia (Fig.  1B,1D ). The control immunostaining with NRS was negative (Fig.  4A ). Figure 1 Immunohistochemical staining of CA XII (A), CA IX (B), CA II (C), and CA XIII (D) in the mouse endometrium. All the studied CA isozymes show positive immuostaining, although the staining intensity varies between different isozymes. CA XII shows stronger reaction in the deep endometrial glands compared to the surface epithelium. This pattern is inversed with CA II showing high reaction in the surface epithelium. Insert in panel A shows that the CA XII immunostaining is most abundant in the basolateral plasma membrane of the epithelial cells. Insert in panel C demonstrates that CA II immunoreactivity is also closely associated with the plasma membrane. CA IX and XIII show faint immunoreactions in both the surface and glandular epithelia. Arrows = endometrial glands, arrowheads = surface epithelium. Original magnifications: × 400. Figure 4 Control immunostaining of mouse uterus, ovary and placenta with normal rabbit serum. No immunoreaction is seen. Oringinal magnifications: × 400. In the ovary, immunoreactions for different CA isozymes were negligible (Fig.  2 ). In fact, only CA XII showed occasional positive cells in the corpus luteum (Fig.  2A ). No staining for these isozymes was observed in the developing follicles. No immunoreaction was obtained with NRS (Fig.  4B ) Figure 2 Immunolocalization of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse corpus luteum (A,C,E,G) and follicle (B,D,F,H). Only faint positive reaction for CA XII can be seen in occasional cells of the corpus luteum that is indicated in the insert of the panel A (arrows). Original magnifications: × 200 (A,C,E,G), × 400 (B,D,F,H). In the 9-days-old mouse placenta, the immunostaining reactions for CA isozymes remained quite weak or absent (Fig.  3 ). CA II was located to the endothelium of the placenta blood vessels and erythrocytes (Fig.  3E ), and it was also present in the amnionic epithelium (Fig.  3F ). The amnionic epithelium showed no or very weak staining for CA XII, whereas the decidual glands were strongly labeled (Fig.  3B ). The control staining again showed no positive signal (Fig.  4C ). Figure 3 Immunohistochemical staining of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse placenta (A,C,E,G) and amnionic epithelium (B,D,F,H). CA II is located in the endothelium of the blood vessels and erythrocytes (arrows in the panel E). It is also expressed in the amnionic epithelium (arrows in the panel F). Insert of the panel B shows that CA XII is highly expressed in the decidual glands, while the amnionic epithelium is negative. DE = Decidua, P = placenta. Original magnifications: × 400. Western blot Western blotting was performed for the mouse uterine protein to evaluate the specificity of the immunoreactions. Mouse kidney and colon samples were used as positive control tissues, since they are known to express CA II, XII and XIII [ 4 , 33 , 35 ], and the colon contains CA IX [ 36 ]. CA II and XIII were positive in all tissue specimens (Fig.  5 ). Both CA IX and XII showed weak positive reactions in the mouse uterus. The molecular weights for these isozymes were 51 and 46 kDa, respectively. Based on the western blotting the expression of CA XII was weaker in the uterus than in the colon or kidney. On the other hand, CA IX showed the strongest signal in the colon. It is notable that anti-mouse CA XII serum cross-reacted with 30 kDa polypeptide in the western blotting. Previous immunostaining of gastric mucosa with the same anti-CA XII and anti-CA II antibodies has clearly indicated that anti-CA XII serum does not recognize CA II which has a molecular mass of 30 kDa in western blot [ 35 ]. Even though gastric epithelial cells contain high levels of CA II, no immunoreaction was obtained with anti-CA XII serum in those cells. Furthermore, no staining has been obtained by anti-CA XII antibody in the red cells which contain high levels of CA I and II, nor in the brain which expresses high levels of CA II and XIII (data not shown). Figure 5 Western blotting of total homogenate from mouse uterus, kidney and colon for CA II, IX, XII and XIII. Normal non-immune rabbit serum (NRS) was used instead of the first antibodies as a negative control. Both CA IX and XII show weak positive reactions for the uterine proteins (arrowheads). The molecular weights for these isozymes are 51 and 46 kDa, respectively. The signal for CA XII is weaker in the uterus than in the colon or kidney. Note that anti-CA XII serum cross-reacts with a 30-kDa polypeptide. This cross-reaction is evident only in western blotting conditions as pointed out in the Results section. CA IX shows the strongest signal in the colon. CA II and XIII are positive in all tissue specimens.
Results
Immunohistochemistry All the studied CA isozymes showed positive immunostaining in the epithelial cells of the mouse endometrium (Fig.  1 ). CA II and XII showed a somewhat reciprocal distribution pattern in that CA II was confined to the surface epithelial cells (Fig.  1C ), while CA XII was more intensely stained in the deeper endometrial glands (Fig.  1A ). It is noteworthy, however, that CA XII was clearly expressed also in the surface epithelial cells, but the staining intensity was weaker compared to the glands. As expected, the strongest reaction for CA XII was associated with the basolateral plasma membrane, and unexpectedly, also CA II immunoreaction was most intense at the plasma membrane. CA IX and XIII showed weak reactions in both surface and glandular epithelia (Fig.  1B,1D ). The control immunostaining with NRS was negative (Fig.  4A ). Figure 1 Immunohistochemical staining of CA XII (A), CA IX (B), CA II (C), and CA XIII (D) in the mouse endometrium. All the studied CA isozymes show positive immuostaining, although the staining intensity varies between different isozymes. CA XII shows stronger reaction in the deep endometrial glands compared to the surface epithelium. This pattern is inversed with CA II showing high reaction in the surface epithelium. Insert in panel A shows that the CA XII immunostaining is most abundant in the basolateral plasma membrane of the epithelial cells. Insert in panel C demonstrates that CA II immunoreactivity is also closely associated with the plasma membrane. CA IX and XIII show faint immunoreactions in both the surface and glandular epithelia. Arrows = endometrial glands, arrowheads = surface epithelium. Original magnifications: × 400. Figure 4 Control immunostaining of mouse uterus, ovary and placenta with normal rabbit serum. No immunoreaction is seen. Oringinal magnifications: × 400. In the ovary, immunoreactions for different CA isozymes were negligible (Fig.  2 ). In fact, only CA XII showed occasional positive cells in the corpus luteum (Fig.  2A ). No staining for these isozymes was observed in the developing follicles. No immunoreaction was obtained with NRS (Fig.  4B ) Figure 2 Immunolocalization of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse corpus luteum (A,C,E,G) and follicle (B,D,F,H). Only faint positive reaction for CA XII can be seen in occasional cells of the corpus luteum that is indicated in the insert of the panel A (arrows). Original magnifications: × 200 (A,C,E,G), × 400 (B,D,F,H). In the 9-days-old mouse placenta, the immunostaining reactions for CA isozymes remained quite weak or absent (Fig.  3 ). CA II was located to the endothelium of the placenta blood vessels and erythrocytes (Fig.  3E ), and it was also present in the amnionic epithelium (Fig.  3F ). The amnionic epithelium showed no or very weak staining for CA XII, whereas the decidual glands were strongly labeled (Fig.  3B ). The control staining again showed no positive signal (Fig.  4C ). Figure 3 Immunohistochemical staining of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse placenta (A,C,E,G) and amnionic epithelium (B,D,F,H). CA II is located in the endothelium of the blood vessels and erythrocytes (arrows in the panel E). It is also expressed in the amnionic epithelium (arrows in the panel F). Insert of the panel B shows that CA XII is highly expressed in the decidual glands, while the amnionic epithelium is negative. DE = Decidua, P = placenta. Original magnifications: × 400.
Immunohistochemistry
All the studied CA isozymes showed positive immunostaining in the epithelial cells of the mouse endometrium (Fig.  1 ). CA II and XII showed a somewhat reciprocal distribution pattern in that CA II was confined to the surface epithelial cells (Fig.  1C ), while CA XII was more intensely stained in the deeper endometrial glands (Fig.  1A ). It is noteworthy, however, that CA XII was clearly expressed also in the surface epithelial cells, but the staining intensity was weaker compared to the glands. As expected, the strongest reaction for CA XII was associated with the basolateral plasma membrane, and unexpectedly, also CA II immunoreaction was most intense at the plasma membrane. CA IX and XIII showed weak reactions in both surface and glandular epithelia (Fig.  1B,1D ). The control immunostaining with NRS was negative (Fig.  4A ).In the ovary, immunoreactions for different CA isozymes were negligible (Fig.  2 ). In fact, only CA XII showed occasional positive cells in the corpus luteum (Fig.  2A ). No staining for these isozymes was observed in the developing follicles. No immunoreaction was obtained with NRS (Fig.  4B )In the 9-days-old mouse placenta, the immunostaining reactions for CA isozymes remained quite weak or absent (Fig.  3 ). CA II was located to the endothelium of the placenta blood vessels and erythrocytes (Fig.  3E ), and it was also present in the amnionic epithelium (Fig.  3F ). The amnionic epithelium showed no or very weak staining for CA XII, whereas the decidual glands were strongly labeled (Fig.  3B ). The control staining again showed no positive signal (Fig.  4C ).Western blot Western blotting was performed for the mouse uterine protein to evaluate the specificity of the immunoreactions. Mouse kidney and colon samples were used as positive control tissues, since they are known to express CA II, XII and XIII [ 4 , 33 , 35 ], and the colon contains CA IX [ 36 ]. CA II and XIII were positive in all tissue specimens (Fig.  5 ). Both CA IX and XII showed weak positive reactions in the mouse uterus. The molecular weights for these isozymes were 51 and 46 kDa, respectively. Based on the western blotting the expression of CA XII was weaker in the uterus than in the colon or kidney. On the other hand, CA IX showed the strongest signal in the colon. It is notable that anti-mouse CA XII serum cross-reacted with 30 kDa polypeptide in the western blotting. Previous immunostaining of gastric mucosa with the same anti-CA XII and anti-CA II antibodies has clearly indicated that anti-CA XII serum does not recognize CA II which has a molecular mass of 30 kDa in western blot [ 35 ]. Even though gastric epithelial cells contain high levels of CA II, no immunoreaction was obtained with anti-CA XII serum in those cells. Furthermore, no staining has been obtained by anti-CA XII antibody in the red cells which contain high levels of CA I and II, nor in the brain which expresses high levels of CA II and XIII (data not shown). Figure 5 Western blotting of total homogenate from mouse uterus, kidney and colon for CA II, IX, XII and XIII. Normal non-immune rabbit serum (NRS) was used instead of the first antibodies as a negative control. Both CA IX and XII show weak positive reactions for the uterine proteins (arrowheads). The molecular weights for these isozymes are 51 and 46 kDa, respectively. The signal for CA XII is weaker in the uterus than in the colon or kidney. Note that anti-CA XII serum cross-reacts with a 30-kDa polypeptide. This cross-reaction is evident only in western blotting conditions as pointed out in the Results section. CA IX shows the strongest signal in the colon. CA II and XIII are positive in all tissue specimens.
Western blot
Western blotting was performed for the mouse uterine protein to evaluate the specificity of the immunoreactions. Mouse kidney and colon samples were used as positive control tissues, since they are known to express CA II, XII and XIII [ 4 , 33 , 35 ], and the colon contains CA IX [ 36 ]. CA II and XIII were positive in all tissue specimens (Fig.  5 ). Both CA IX and XII showed weak positive reactions in the mouse uterus. The molecular weights for these isozymes were 51 and 46 kDa, respectively. Based on the western blotting the expression of CA XII was weaker in the uterus than in the colon or kidney. On the other hand, CA IX showed the strongest signal in the colon. It is notable that anti-mouse CA XII serum cross-reacted with 30 kDa polypeptide in the western blotting. Previous immunostaining of gastric mucosa with the same anti-CA XII and anti-CA II antibodies has clearly indicated that anti-CA XII serum does not recognize CA II which has a molecular mass of 30 kDa in western blot [ 35 ]. Even though gastric epithelial cells contain high levels of CA II, no immunoreaction was obtained with anti-CA XII serum in those cells. Furthermore, no staining has been obtained by anti-CA XII antibody in the red cells which contain high levels of CA I and II, nor in the brain which expresses high levels of CA II and XIII (data not shown).Discussion This study describes the expression of CA II, IX, XII and XIII in mouse female genital organs including uterus, ovary and placenta. CA II showed a very limited distribution pattern in the mouse placenta, being present only in the erythrocytes, endothelium of the blood vessels and amnionic epithelium. In previous studies, CA II has been detected by immunohistochemistry in the human villous syncytiotrophoblasts and in varying amounts in fetal villous endothelium [ 21 , 22 ]. Using a histochemical staining method, Ridderstråle et al [ 18 ] showed in several species that the highest CA activity located in the maternal capillaries, and the membrane-bound CA activity varied among different species. To date, CA IV is the only membrane-bound CA isozyme which has been detected in the mouse placenta [ 23 ]. In our study, CA IX and XII were not found in the mouse placental tissue except that CA XII showed a very weak reaction in the amnionic epithelium. Concluding from the results of the present and previous studies, CA I and II appear to represent the enzyme forms that are most relevant for the placental function [ 22 ], while CA IX and XII may play a role in other reproductive organs such as the male excurrent duct and female uterus [ 14 , 37 ]. It is known that CA activity facilitates transport of CO 2  across biological membranes by converting it to bicarbonate and hydrogen ions. These ions are then translocated across the plasma membrane through specific carrier proteins in a coordinated manner. It is of considerable interest that CA isozymes II and IV have been recently described to form active metabolon systems with ion exchanger proteins such as anion exhanger isoform 1 (AE1) and Na + /H + -exhanger isoform 1 (NHE1) [ 38 - 40 ]. Even though these associations have not yet been described in the placental tissue, it is possible that such metabolons play a role in facilitating placental ion transport processes. Previous studies have shown CA activity in the endometrium of several mammalian species [ 41 , 42 ]. Until now the only established isozymes in the human endometrium are CA XII [ 14 ] and CA XIII [ 4 ]. Interestingly, the high activity isoenzyme, CA II, is not expressed in the human endometrial epithelium [ 4 ]. In the present study, all the examined CA isozymes – including CA II – showed positive immunostaining in the epithelial cells of the mouse endometrium. To our knowledge, there are only a few examples of clear species-specific difference in CA expression. These include e.g. CA XII expression in the kidney (human principal cells versus mouse intercalated cells) [ 33 , 43 ] and CA XIII in the human and mouse testis [ 4 ]. What would be the physiological consequence of such variation between different species? Of course, there are marked differences in human and rodent reproductive physiology. Mouse is characterized by tremendous reproductive potential. Females generally have 5–10 litters per year if conditions are suitable. Gestation period is 19–21 days. Litters consist of 3–12 (generally 5 or 6) offspring, and the mice reach sexual maturity at 5–7 weeks. Even though our observations do not provide any clues whether CA expression could contribute to some of the described characteristics, these differences may have fundamental physiological effects that should be addressed in future investigations. In the present study, CA XII and II showed more intense staining in the surface endometrial epithelia than CA IX and XIII. CA XII was more intensely stained in the deeper endometrial glands, while CA II was confined to the surface epithelial cells. Interestingly, CA II showed positive immunoreaction not only in the cytoplasmic compartment but also at the plasma membrane of the cells that is quite surprising for a cytosolic enzyme. The same phenomenon is detectable in some other tissues including the human gallbladder [ 25 ] and gut [ 44 ]. The cell membrane reactivity may reflect a possible physical association between CA II and ion transport proteins, which has been demonstrated in cell cultures [ 38 - 40 ]. When CA XII was first discovered in the normal human endometrium, it was suggested to play a role in reproductive functions [ 14 ]. In the endometrium, pH and ion balance has to be tightly regulated to ensure normal fertilization. For example, the bicarbonate concentration has been implicated in the regulation of sperm motility, capacitation, and acrosome reaction [ 15 , 17 , 45 ]. One major regulatory pathway includes a bicarbonate-sensitive adenylate cyclase that is present in the plasma membrane of the sperm cell [ 46 ]. In the female genital tract, the endometrial and oviductal epithelium may produce an alkaline – bicarbonate rich – environment for maintaining the sperm motility. This suggestion is in agreement with the observations by Guerin et al. [ 47 ], that the sperm motility is improved by co-culture of human spermatozoa with either endometrial or oviductal epithelial cells. In the future studies, it will be important to investigate whether the hormonal status regulates the expression of different CA isozymes – particularly CA XII – in the endometrium. Another interesting line of investigations would be to analyze the fertilization capacity of CA XII knockout mice as soon as they become available. One could hypothesize that endometrial CA isozymes are important factors, contributing to the appropriate bicarbonate concentration and pH balance in the cervical and endometrial mucus needed for normal fertilization process. Based on our recent studies, CA IX-deficient mice showed no apparent phenotypic changes linked to fertility [ 26 ]. Even more interesting from this point of view is that CA XII may be an important isozyme present in the endometrium, and therefore, CA XII knockout mice will be very attractive targets for reproductive physiological studies.
Discussion
This study describes the expression of CA II, IX, XII and XIII in mouse female genital organs including uterus, ovary and placenta. CA II showed a very limited distribution pattern in the mouse placenta, being present only in the erythrocytes, endothelium of the blood vessels and amnionic epithelium. In previous studies, CA II has been detected by immunohistochemistry in the human villous syncytiotrophoblasts and in varying amounts in fetal villous endothelium [ 21 , 22 ]. Using a histochemical staining method, Ridderstråle et al [ 18 ] showed in several species that the highest CA activity located in the maternal capillaries, and the membrane-bound CA activity varied among different species. To date, CA IV is the only membrane-bound CA isozyme which has been detected in the mouse placenta [ 23 ]. In our study, CA IX and XII were not found in the mouse placental tissue except that CA XII showed a very weak reaction in the amnionic epithelium. Concluding from the results of the present and previous studies, CA I and II appear to represent the enzyme forms that are most relevant for the placental function [ 22 ], while CA IX and XII may play a role in other reproductive organs such as the male excurrent duct and female uterus [ 14 , 37 ].It is known that CA activity facilitates transport of CO 2  across biological membranes by converting it to bicarbonate and hydrogen ions. These ions are then translocated across the plasma membrane through specific carrier proteins in a coordinated manner. It is of considerable interest that CA isozymes II and IV have been recently described to form active metabolon systems with ion exchanger proteins such as anion exhanger isoform 1 (AE1) and Na + /H + -exhanger isoform 1 (NHE1) [ 38 - 40 ]. Even though these associations have not yet been described in the placental tissue, it is possible that such metabolons play a role in facilitating placental ion transport processes.Previous studies have shown CA activity in the endometrium of several mammalian species [ 41 , 42 ]. Until now the only established isozymes in the human endometrium are CA XII [ 14 ] and CA XIII [ 4 ]. Interestingly, the high activity isoenzyme, CA II, is not expressed in the human endometrial epithelium [ 4 ]. In the present study, all the examined CA isozymes – including CA II – showed positive immunostaining in the epithelial cells of the mouse endometrium. To our knowledge, there are only a few examples of clear species-specific difference in CA expression. These include e.g. CA XII expression in the kidney (human principal cells versus mouse intercalated cells) [ 33 , 43 ] and CA XIII in the human and mouse testis [ 4 ]. What would be the physiological consequence of such variation between different species? Of course, there are marked differences in human and rodent reproductive physiology. Mouse is characterized by tremendous reproductive potential. Females generally have 5–10 litters per year if conditions are suitable. Gestation period is 19–21 days. Litters consist of 3–12 (generally 5 or 6) offspring, and the mice reach sexual maturity at 5–7 weeks. Even though our observations do not provide any clues whether CA expression could contribute to some of the described characteristics, these differences may have fundamental physiological effects that should be addressed in future investigations.In the present study, CA XII and II showed more intense staining in the surface endometrial epithelia than CA IX and XIII. CA XII was more intensely stained in the deeper endometrial glands, while CA II was confined to the surface epithelial cells. Interestingly, CA II showed positive immunoreaction not only in the cytoplasmic compartment but also at the plasma membrane of the cells that is quite surprising for a cytosolic enzyme. The same phenomenon is detectable in some other tissues including the human gallbladder [ 25 ] and gut [ 44 ]. The cell membrane reactivity may reflect a possible physical association between CA II and ion transport proteins, which has been demonstrated in cell cultures [ 38 - 40 ]. When CA XII was first discovered in the normal human endometrium, it was suggested to play a role in reproductive functions [ 14 ]. In the endometrium, pH and ion balance has to be tightly regulated to ensure normal fertilization. For example, the bicarbonate concentration has been implicated in the regulation of sperm motility, capacitation, and acrosome reaction [ 15 , 17 , 45 ]. One major regulatory pathway includes a bicarbonate-sensitive adenylate cyclase that is present in the plasma membrane of the sperm cell [ 46 ]. In the female genital tract, the endometrial and oviductal epithelium may produce an alkaline – bicarbonate rich – environment for maintaining the sperm motility. This suggestion is in agreement with the observations by Guerin et al. [ 47 ], that the sperm motility is improved by co-culture of human spermatozoa with either endometrial or oviductal epithelial cells.In the future studies, it will be important to investigate whether the hormonal status regulates the expression of different CA isozymes – particularly CA XII – in the endometrium. Another interesting line of investigations would be to analyze the fertilization capacity of CA XII knockout mice as soon as they become available. One could hypothesize that endometrial CA isozymes are important factors, contributing to the appropriate bicarbonate concentration and pH balance in the cervical and endometrial mucus needed for normal fertilization process. Based on our recent studies, CA IX-deficient mice showed no apparent phenotypic changes linked to fertility [ 26 ]. Even more interesting from this point of view is that CA XII may be an important isozyme present in the endometrium, and therefore, CA XII knockout mice will be very attractive targets for reproductive physiological studies.Conclusions The present paper demonstrates for the first time the expression of transmembrane carbonic anhydrase isozymes IX and XII in the female murine reproductive tract. The data indicates that the endometrial epithelium is a prominent site for CA XII expression. The conservation of CA XII expression in the endometrium of different species (mouse and human) suggests a role for this isozyme in reproductive physiology.
Conclusions
The present paper demonstrates for the first time the expression of transmembrane carbonic anhydrase isozymes IX and XII in the female murine reproductive tract. The data indicates that the endometrial epithelium is a prominent site for CA XII expression. The conservation of CA XII expression in the endometrium of different species (mouse and human) suggests a role for this isozyme in reproductive physiology.Authors' contributions All authors participated in the design of the study. PH, JL and SP collected the tissue samples. PH, JL, ET, PK and SP drafted the manuscript. PH, JL and SP performed the western blotting. SPas, JP, AW and WSS provided the antibodies. PH, JL and SP participated in the immunohistochemical staining. All authors read and approved the final manuscript.
Authors' contributions
All authors participated in the design of the study. PH, JL and SP collected the tissue samples. PH, JL, ET, PK and SP drafted the manuscript. PH, JL and SP performed the western blotting. SPas, JP, AW and WSS provided the antibodies. PH, JL and SP participated in the immunohistochemical staining. All authors read and approved the final manuscript.