Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print March 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.039008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/1/131    most recent
biolreprod.104.039008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hu, J.
Right arrow Articles by Spencer, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hu, J.
Right arrow Articles by Spencer, T. E.
Agricola
Right arrow Articles by Hu, J.
Right arrow Articles by Spencer, T. E.
BIOLOGY OF REPRODUCTION 73, 131–138 (2005)
DOI: 10.1095/biolreprod.104.039008
© 2005 by the Society for the Study of Reproduction, Inc.

Carbonic Anhydrase Regulate Endometrial Gland Development in the Neonatal Uterus1

Jianbo Hu , and Thomas E. Spencer 2 

Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carbonic anhydrases (CAs) are zinc metalloenyzmes that catalyze the reversible conversion of carbon dioxide to carbonic acid and are involved in respiration, calcification, acid-base balance, and formation of fluids. Transcriptional profiling of the developing neonatal mouse uterus detected expression of Car1, Car2, Car11, and Car13 between Postnatal Days (PNDs) 3 and 18. In the neonatal mouse uterus, Car2 and Car11 mRNAs were predominantly localized in endometrial epithelial and stromal cells, respectively, whereas Car13 mRNA was detected in both epithelia and stroma. CAR2 protein was detected primarily in the endometrial epithelia and from PND 3 to PND 18 in the uteri of neonatal mice. To determine whether CA regulated uterine development, neonatal mice were treated s.c. with acetazolamide, a CA inhibitor, from PND 3 to PND 18. Treatment with acetazolamide decreased CA activity in the uterus and the number of endometrial glands without apparent effects on differentiation of the stroma or myometrium. In the neonatal sheep uterus, CA2 mRNA was initially expressed at birth (PND 0) in the endometrial luminal epithelium and was predominantly expressed in the developing glandular epithelium from PND 7 to PND 56. These results support the hypothesis that CA has a functional role in endometrial gland development during postnatal uterine morphogenesis.

carbonic anhydrase, development, developmental biology, endometrium, female reproductive tract, mouse, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible hydration-dehydration of carbon dioxide and bicarbonate [1]. Among human CAs, several active isozymes, acatalytic isozymes, and CA-related proteins have been identified, and the CA gene family continues to increase in number [2]. At least 11 members of the CA family identified at present, with CA2 being the most abundant and most efficient enzyme [1]. The known enzymes differ in their kinetic properties, subcellular localization, and tissue-specific distribution [1]. CAs appear to have a role in diverse physiological and biological processes including respiration, acid-base balance, ion transport, bone absorption, renal acidification, gluconeogenesis, and ureagenesis as well as the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid [1, 3].

Carbonic anhydrases are involved in many aspects of reproduction and have a functional role in sperm maturation and capacitation by influencing bicarbonate secretion [4, 5]. Inhibition of CA activity with acetazolamide (ACTZ) inhibited acidification of epididymal fluid [6, 7], decreased fluid secretion in the testis, and impaired sperm function [8, 9]. In rabbits, CA activity in the uterus is related to plasma progesterone levels [10, 11]. Direct distillation of ACTZ into the rabbit uterine lumen prevented pregnancy [11], likely by preventing secretion of bicarbonate by the endometrial epithelia [12]. In the human uterus, CA12 is abundantly expressed in the endometrial epithelia [13]. In the neonatal mouse uterus, several members of the CA family are expressed during postnatal development [14]. However, the role of CAs in uterine development and morphogenesis has not been investigated.

The mature uterus consists of two functional compartments, the endometrium and myometrium [15]. The endometrium is the inner mucosal lining of the uterus, consisting of two epithelial cell types: luminal epithelium (LE) and glandular epithelium (GE), and stroma [16, 17]. The myometrium is the smooth muscle component of the uterine wall and has two layers, an inner circular and an outer longitudinal layer. Although uterine development begins in the fetus, it is not completed until after birth. Postnatal uterine morphogenesis involves the differentiation and development of the endometrial GE from the LE as well as the endometrial stroma and inner circular and outer longitudinal layers of the myometrium from the uterine mesenchyme [18, 19]. Endometrial gland development, also termed adenogenesis, is principally a postnatal process in all mammals. Endometrial glands and their secretions are critical regulators of peri-implantation embryo survival and implantation as well as establishment of uterine receptivity [20–23]. Therefore, it is important to understand the hormonal, cellular, and molecular mechanisms regulating postnatal development of the uterus. In mice, postnatal development of the endometrial glands is initiated between birth (Postnatal Day, or PND 0) and PND 5, and is essentially complete by PND 15 [14, 24]. In sheep, endometrial gland genesis is initiated between PND 0 and PND 7, when shallow epithelial invaginations appear along the LE in presumptive intercaruncular areas [25]. Nascent GE buds proliferate and invaginate into the stroma between PNDs 7 and 14, forming tubular structures that coil and branch by PND 21. By PND 56, the caruncular and intercaruncular endometrial areas are histoarchitecturally similar to those of the adult uterus. Little is known of the mechanisms regulating postnatal uterine morphogenesis and, in particular, endometrial gland development.

Interesting similarities exist between endometrial gland development and tumor cell invasion. Numerous studies indicate that the environment of tumor cells generally is more acidic than that of normal cells [26]. Many tumor cell types specifically express different members of the CA family, suggesting their involvement in acidification and cell invasion [27, 28]. Indeed, inhibition of CA activity with ACTZ prevents tumor cell invasion [29]. Because CA activity is associated with endometrial gland proliferation in the rabbit [11] and Car2 null mice appear to lack endometrial glands [30], our working hypothesis is that CAs have a functional role in endometrial gland development in the postnatal uterus. Therefore, our objectives were as follows: 1) to determine the ontogeny of Car expression in the neonatal mouse uterus, 2) discern the role of CA in neonatal mouse uterine development, and 3) determine whether CA2 mRNA is expressed in the developing neonatal sheep uterus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mouse Uterine Tissue Collection and CodeLink Microarray Analysis

The University Laboratory Animal Care and Use Committee of Texas A&M University approved all experimental procedures. Adult virgin CD-1 female mice were obtained from Charles River Laboratories (Wilmington, MA) and mated with fertile males of the same strain to establish pregnancy. All mice were housed in a temperature-controlled room (21 to 22°C) in a 12L:12D cycle in the Kleberg Center Mouse Facility (Texas A&M University) and provided fresh reverse osmosis/deionized water and NIH-31 lab chow ad libitum.

In study 1, uteri were obtained from mice from different litters on PNDs 3, 6, 9, 12, or 15 (n ≥ 20 per day). Portions of each uterine horn were fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.2). The remainder of the uterine horns were snap-frozen in liquid nitrogen and stored at –80°C for RNA or protein extraction. After 24 h, fixed tissues were changed to 70% ethanol and then embedded in Paraplast Plus (Oxford Labware, St. Louis, MO).

In study 2, epithelial and stromal/myometrial cells were isolated from PND 3, 6, and 9 (n ≥ 10 per day) mouse uteri by enzymatic digestion as described previously [14]. Total RNA was extracted from either the entire uterus on each PND or separated epithelia and stroma/myometrium using Trizol reagent (Gibco-BRL, Bethesda, MD). Transcriptional profiling was conducted using total RNA and a CodeLink UniSet Mouse I Expression Bioarray (Amersham Biosciences, Piscataway, NJ) by the genomics core facility of the Texas A&M University National Institute of Environmental Health Sciences Center for Environmental and Rural Health as described previously [14].

Sheep Uterine Tissue Collection

In study 3, crossbred Spring-born Suffolk ewes (n = 45) were assigned randomly at birth (PND 0) to be hysterectomized on PND 0 (n = 6), 7 (n = 4), 14 (n = 5), 21 (n = 5), 28 (n = 5), 35 (n = 5), 42 (n = 5), 49 (n = 5), or 56 (n = 5). At hysterectomy, the entire reproductive tract was excised, and the uterus was trimmed free of the broad ligament, oviduct, and cervix. Each uterine horn was fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% ethanol and then embedded in Paraplast Plus.

Effect of ACTZ Treatment on Uterine CA Activity and Development in Neonatal Mice

Acetazolamide (Sigma Chemical Co., St. Louis, MO) was dissolved in dimethylsulfoxide (DMSO) and corn oil vehicle (final DMSO concentration was 10%). In study 4, female mice were randomly assigned to receive daily s.c. injections of ACTZ at a dose of either 0, 25, 50, or 250 mg/kg body weight in vehicle from PND 3 to PND 18 (n = 8 mice per treatment group). Mice were weighed daily before injections to adjust for growth. Treatments were administered in a final volume of ~20 µl from PND 3 to PND 9 and ~50 µl from PND 10 to PND 18. Uteri were collected 6 h after last injection on PND 18 and then frozen in liquid nitrogen and stored at –80°C for assay of CA activity.

In study 5, female mice were assigned randomly (n = 8 mice per treatment group) to remain uninjected as a control or to receive daily s.c. injections of ACTZ at a dose of either 0, 25, 50, or 250 mg/kg body weight from PND 3 to PND 18 as described in study 4. Uteri were collected 6 h after last injection on PND 18, and body weight and uterine wet weights were measured and recorded. Uteri from control or ACTZ-treated mice were fixed in 4% paraformaldehyde and embedded in Paraplast Plus. Cross-sections (n = 6) of each uterus were prepared (5 µm) and stained with hematoxylin. Endometrial gland number was determined by counting the total number of glands in a complete cross-section of the uterine horn using methods similar to those described previously [31]. Gland number estimates were generated for at least six nonsequential sections from each uterus. Data are presented as total gland number per uterine horn cross-section.

In Situ Hybridization Analysis

Partial cDNAs for ovine CA2 and mouse Car1, Car2, Car11, and Car13 mRNAs were cloned by reverse transcription-polymerase chain reaction as described previously [25]. The amplified partial cDNAs were cloned into pCRII vector using a TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced to confirm identity. In situ hybridization was conducted on cross-sections of the mouse (study 1) and ovine uterus (study 3) as described previously [32]. Briefly, antisense and sense radiolabeled cRNA probes were generated using appropriate polymerases by in vitro transcription with [{alpha}-35S]UTP. Transcripts protected from RNase digestion were visualized by liquid emulsion autoradiography using NTB-2 (Kodak, Rochester, NY). Slides were stored at 4°C for several weeks as judged from autoradiographs, developed in Kodak D-19 developer, and counterstained with hematoxylin.

Western Blot Analysis

Total protein extracts of pooled mouse uteri (n = 3 separate pools per PND) from study 1 were prepared by homogenizing the uterus in extraction buffer (60 mM Tris pH 7.0, 1 mM Na3VO4, 10% glycerol, 2% SDS, and 1x protease inhibitor cocktail from Roche, Indianapolis, IN). Proteins extracted from adult uteri from mice in estrus were used as a positive control. Protein content of uterine extracts was determined using the DC protein assay (Bio-Rad, Hercules, CA) with BSA as the standard. Total protein (30 µg) from uterine extracts was denatured and separated by 10% SDS-PAGE, and Western blot analysis was conducted as described previously [33]. Immunoreactive CAR2 protein was detected using polyclonal rabbit antiserum generated against mouse CAR2 (kindly provided by Dr. William Sly, Washington University School of Medicine, St. Louis, MO) at a 1:10 000 final dilution. Negative control blots were performed in which primary antiserum was replaced by nonimmune rabbit serum at the same concentration. As an internal control to correct for differences in protein loading, blots were reprobed with mouse anticytochrome C immunoglobulin G (65981A; Pharmingen, San Diego, CA) at a 1:10 000 final dilution. Immunoreactive proteins were detected by chemiluminescence (SuperSignal West Pico, Pierce, Rockford, IL) according to the manufacturer's recommendations using X-OMAT AR x-ray film (Kodak). Western blots were quantified by scanning densitometry using a Bio-Rad GS-690 imaging densitometer and Multi-Analyst Software.

Immunohistochemistry

Immunohistochemical localization of CAR2 protein in the mouse uterus was performed as described previously [25] in mouse uteri from study 1. After antigen retrieval using a boiling citrate buffer, uterine sections (at least three sections per mouse) were washed and endogenous peroxidase activity was inactivated by incubating with 3% hydrogen peroxide in methanol. Rabbit anti-mouse CAR2 serum or nonimmune rabbit serum was used at a 1:1000 final dilution. The chromagen used for peroxidase localization was 3,3'-diaminobenzidine tetrahydrochloride (Sigma), and the slides were not counterstained.

Measurements of CA Activity

Mouse uteri from study 4 were pooled within treatment and then homogenized in 50 mM Hepes buffer containing 1x protease inhibitor cocktail (Roche) for determination of CA activity, which was performed essentially as described by Miyamoto et al. [34]. Protein content of uterine extracts was determined using the Bio-Rad DC protein assay with BSA as the standard. Briefly, 3 ml of ice-cold Veronal buffer (pH 8.3) was mixed with 150 µl of 1% phenol red and 20 µl of sample (~60 µg total protein) or buffer as a negative control in a glass tube with a stopper. After incubation on ice for 5 min, 2 ml of ice-cold CO2-saturated water was added to the mix, and the tube was quickly inverted. The time (in seconds) for the color to change from red to yellow (pH 6.3) was recorded and compared with the time for standards. All the samples were assayed in triplicate, and the entire assay was repeated at least three times. The intraassay coefficient of variation (CV) was 4.9%, and the interassay CV was 7.2%.

Photomicroscopy

Representative photomicrographs of uteri were taken using a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc., Lewisville, TX) under brightfield or darkfield illumination (or both) and captured using a Nikon DXM1200 digital camera.

Statistical Analysis

All quantitative data were subjected to least-squares analysis of variance (LS-ANOVA) using the General Linear Models procedures of the Statistical Analysis System version 8.1 for Windows (SAS Institute, Cary, NC). For Western blot analyses of CAR2, integrated optical density measurements were analyzing using a model that incorporated PND and replicate as the sources of variation. The measurement of band optical density for cytochrome C was used as a covariate. The data for uterine CA activity measurements were analyzed by least squares regression analysis. In these analyses, time was considered a continuous, independent source of variation with replicate as a dependent source. Statistical models for analysis of morphometry data included main effects of treatment, mouse within treatment, tissue section, and the appropriate interactions. Initial analyses indicated that tissue section was not a significant source of variation. Data are presented as least square means with overall standard errors (SEM).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CA in the Mouse Uterus

The normalized expression level of different CA family members in uteri from neonatal mice (study 1) and separated uterine cell populations (epithelium and stroma/myometrium) from neonatal mice (study 2) are summarized in Tables 1 and 2, respectively, as determined by microarray analysis. Expression of Car1, Car2, Car11, and Car13 mRNAs was detected in all neonatal mouse uteri. Cell separation analyses indicated that Car2 and Car13 mRNAs were expressed most abundantly in the endometrial epithelium, whereas Car11 mRNA was expressed predominantly in the stroma and myometrium.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Expression of carbonic anhydrases (Car) in the postnatal mouse uterus


View this table:
[in this window]
[in a new window]
  		TABLE 2. Expression of carbonic anhydrases (Car) in separated cell populations of the postnatal mouse uterus

Localization of Car mRNA Expression in Neonatal Uteri from Mice and Sheep

In the uterus of neonatal mice, Car2 and Car11 mRNAs were detected predominantly in endometrial epithelium and stroma, respectively, but not in the myometrium (Fig. 1). However, Car13 mRNA was detected in all uterine cell types (Fig. 1B). Expression of Car1 mRNA was below the limits of detection by in situ hybridization (data not shown).



View larger version (152K):
[in this window]
[in a new window]
  		FIG. 1. Carbonic anhydrase (Car) mRNA expression in the neonatal mouse uterus. Representative photomicrographs are shown in brightfield and darkfield illumination. A) Car2 mRNA was detected predominantly in the endometrial epithelia. B) Car11 mRNA was detected predominantly in the stroma (PND 6 shown), whereas Car13 mRNA was expressed in the endometrial epithelia and stroma (PND 15 shown). LE, luminal epithelium; GE, glandular epithelium; S, stroma; M, myometrium. Bar = 20 µm

In the uterus of neonatal sheep (study 3), CA2 mRNA was detected in the endometrial LE on PND 0, but was present predominantly in the endometrial GE from PNDs 7 to 56 (Fig. 2). Expression of CA2 mRNA was not observed in the endometrial stroma or myometrium. The melanocytes in the PND 14 and PND 49 uteri are not positive for CA2 mRNA, but contain melanin pigment that appears white under darkfield illumination.



View larger version (119K):
[in this window]
[in a new window]
  		FIG. 2. CA2 mRNA expression in the neonatal ovine uterus. Representative photomicrographs are shown in brightfield and darkfield illumination. CA2 mRNA was detected predominantly in the endometrial luminal epithelium at birth (PND 0), but was expressed predominantly in the developing endometrial glands from PND 7 to PND 56. The pigmented melanocytes (Mel) appear white under darkfield illumination. GE, glandular epithelium; LE, luminal epithelium; M, myometrium; Mel, melanocyte; S, stroma. Bar = 20 µm

CAR2 in the Neonatal Mouse Uterus

Car2 was the most abundant member of the CA family expressed in the neonatal mouse uterus (Tables 1 and 2). A single immunoreactive CAR2 protein of ~30 kDa was observed in Western blot analyses of whole uterine extracts (Fig. 3A). The overall abundance of CAR2 protein in the mouse uterus increased (quadratic, P < 0.05) ~2-fold from PND 3 to PND 18 (Fig. 3B). The uteri from adult mice in estrus were used as a positive control.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3. CAR2 protein in the neonatal mouse uterus. A) Whole uterine extracts from neonatal mice or adult mice in estrus were analyzed by Western blotting as described in Materials and Methods. The blot presented is representative of three independent experiments. B) Densitometric data from Western blot analysis. CAR2 protein increased (quadratic, P < 0.05) in the neonatal mouse uterus from PND 3 to PND 18. Data are expressed as least square means relative optical density units with SEM

In the developing neonatal mouse uterus, immunoreactive CAR2 protein was observed predominantly in the cytoplasm of the endometrial epithelia of the endometrium with lower levels in the stroma, depending on the day (Fig. 4). On PND 3, CAR2 protein was most abundant in the endometrial LE based on comparison with negative controls in which nonimmune rabbit serum was used in place of the rabbit anti-mouse CAR2 serum. On PNDs 6 and 9, CAR2 protein remained most abundant in the endometrial LE and nascent GE, but was also observed in the stroma, albeit at lower abundance. Between PNDs 9 and 12, the abundance of CAR2 protein increased in the endometrial LE and GE, but not in the stroma. No immunoreactive CAR2 protein was observed in the myometrium on any PND.



View larger version (124K):
[in this window]
[in a new window]
  		FIG. 4. Immunohistochemical localization of CAR2 protein in the mouse uterus. CAR2 protein was detected primarily in the endometrial epithelia of the neonatal mouse uterus. Negative controls were performed in which nonimmune serum was substituted for rabbit anti-mouse CAR2 antibody. LE, luminal epithelium; GE, glandular epithelium; S, stroma; M, myometrium. Bar = 100 µm

Effect of ACTZ on Uterine CA Activity and Development in the Neonatal Mouse

In study 4, treatment of neonatal mice with ACTZ, a specific inhibitor of CA activity [35], from PND 3 to PND 18, decreased uterine CA activity on PND 18 in a dose-dependent manner (linear, P < 0.01) as indicated by the increase in time required for the pH to decrease from 8.3 to 6.3 in the CA activity assay (Fig. 5). At a dose of 50 and 250 mg/kg, the time required was not different (P > 0.10) from that of adding buffer alone, signifying complete inhibition of uterine CA activity.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 5. Effect of ACTZ treatment on CA activity in the neonatal mouse uterus. Neonatal female mice were treated with ACTZ from PND 3 to PND 18. On PND 18, whole uterine extracts were prepared and analyzed for CA activity. ACTZ inhibited uterine CA activity in a dose-dependent manner (linear, P < 0.01). Uterine CA activity was reduced (P < 0.10) by treatment with 50 mg or 250 mg of ACTZ. Data are presented as least square means with SEM

In study 5, neonatal mice were treated with ACTZ from PND 3 to PND 18. No toxic effects of ACTZ were observed in the neonatal mice regardless of dose. Treatment with vehicle alone decreased (P < 0.05) body weight and uterine wet weight relative to untreated controls (data not shown). However, uterine wet weight was not different (P > 0.10) among the ACTZ treatment groups if body weight was used as a covariate in the statistical analyses (data not shown). Treatment of neonatal mice with ACTZ appeared to specifically retard development of the endometrial glands without affecting development of the stroma or myometrium (Fig. 6A). Morphometrical analyses indicated that endometrial gland number was decreased (P < 0.01) in mice treated with ACTZ (25, 50, and 250 mg doses) compared with that in mice receiving the vehicle alone (0 mg dose) or in those left untreated as a control (Fig. 6B).



View larger version (73K):
[in this window]
[in a new window]
  		FIG. 6. Effects of ACTZ treatment on development of the uterus. Neonatal female mice were treated with ACTZ from PND 3 to PND 18. A) Representative photomicrographs of the uterus from PND18 mice treated with vehicle alone (0 mg) or 250 mg ACTZ. ACTZ treatment retarded development of the endometrial glands, but did not affect development of other uterine tissues. LE, luminal epithelium; GE, glandular epithelium; S, stroma; M, myometrium. Bar = 20 µm. B) Graph illustrating effects of ACTZ treatment on endometrial gland number in neonatal mice. Treatment with ACTZ at a dose of 25, 50, or 250 mg per kg body weight per day reduced (P < 0.01) the number of endometrial glands on PND 18 relative to mice receiving vehicle (0 mg dose) or left untreated as a control. Data are presented as least square means with SEM


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although CA activity has been detected in uterine endometrium of various mammals [11], which members of CA exist in the uterus and their roles in uterine function have not been well investigated. Our microarray data indicated that Car1, Car2, Car11, and Car13 were the most abundant members of the CA family expressed in the neonatal mouse uterus. In situ hybridization analysis indicated that Car2 and Car11 mRNAs were predominantly localized in the mouse uterine epithelium and stroma, respectively. In the neonatal sheep uterus, CA2 mRNA was also expressed in developing endometrial glands. The temporal and spatial alterations in uterine CA2 expression suggested a biological role in endometrial gland development during postnatal uterine morphogenesis.

Microarray analyses found that Car2 was predominantly expressed in the endometrial LE of the neonatal mouse uterus, which was confirmed by in situ hybridization and immunohistochemical analyses. Although Car2 mRNA increased approximately 3-fold in the endometrial LE between PNDs 3 and 6, CAR2 protein abundance did not exhibit the same relative increase until between PNDs 9 and 12 as determined by immunohistochemical analyses. Further, relative levels of CAR2 in the whole uterus did not increase until after PND 9 as determined by Western blot analyses. The discordance in Car2 mRNA and CAR2 protein levels could be due to posttranscriptional regulation through effects on mRNA or protein stability.

Treatment of neonatal mice with ACTZ, a CA inhibitor, decreased pup growth, as indicated by a reduction in body weight, but ACTZ was not toxic. The reduction in pup growth was likely due to the DMSO vehicle, which is an organic solvent that can inhibit cell growth in vitro [36]. Alternatively, daily handling of the pups might also inhibit their growth. Systemic application of ACTZ at doses ranging from 5 to 50 mg/kg body weight specifically inhibited CA activity without side effects depending on the organ tested [37]. Chronic application of ACTZ for 2 wk in rats is required to affect the morphology of the renal collecting ducts, but it does not affect systemic and urinary biological parameters [38]. The acute effect of ACTZ is the loss of sodium and bicarbonate in urine, which becomes self-limiting after chronic administration of ACTZ [38].

In the present study, treatment of neonatal female mice from PND 3 to PND 18 inhibited uterine CA activity and decreased the total number of endometrial glands at PND 18, indicating that CAs have a functional role in endometrial gland development during postnatal uterine morphogenesis. Although a significant inhibition of uterine CA activity was not achieved at the 25 mg dose, treatment of neonatal mice with the 25 mg dose did inhibit endometrial gland development. One possibility is that the assay used to measure uterine CA activity was not sensitive enough to detect ACTZ effects on uterine CA activity at the 25 mg dose. Other possibilities include differences in threshold effects of ACTZ on CA activity as compared to a biological endpoint (e.g., uterine gland development). Finally, different CA family members may exhibit differential sensitivity to inhibition by ACTZ. However, the present data agree with a report that Car2 null mice lack endometrial glands [30]. Of interest, CAR2 was the most abundantly expressed member of the family in the neonatal mouse uterus and was specifically expressed in the endometrial epithelia of both the neonatal mouse and sheep uterus. These findings suggest a specific role for CA2 in uterine gland development and perhaps function across species.

The mechanism of how CAR2 regulates endometrial adenogenesis during postnatal uterine morphogenesis is not known. Many tumor cell types specifically express different members of the CA family, and the environment of tumor cells generally is more acidic [26], suggesting the involvement of CA in acidification and cell invasion [27, 28]. In support of this notion, a combination of ACTZ treatment with chemotherapy further delayed tumor growth in vivo compared with chemotherapy alone [39]. Inhibition of CA activity prevented tumor cell invasion in vitro by modifying the extracellular pH [29] or through disturbing E-cadherin-mediated cell adhesion [40]. Human melanoma cells cultured at acidic pH secreted more active matrix metalloproteinase 9 (MMP-9) into the medium and are more invasive than cells cultured in neutral pH [40]. Further, increased CA9 activity was associated with enhanced Matrigel invasion of human colon carcinoma cells [40]. Due to the similarities in the processes of tumor cell invasion and endometrial gland development, as well as expression of MMPs and E-cadherin in mouse uteri [41], it can be hypothesized that CA play a key role in regulating uterine adenogenesis by affecting GE cell migration, MMP activity, or both. Indeed, MMPs and their inhibitors are involved in postnatal uterine morphogenesis in the mouse [41–43] and increased MMP activity would facilitate reorganization of extracellular matrix necessary for endometrial gland development. Further, CAs are expressed in the endometrium of the human uterus [13], which undergoes cyclical shedding and regrowth of the endometrial glands during the menstrual cycle [44]. Thus, CAs may have a biological role in uterine morphogenesis and, in particular, endometrial gland development in a number of different mammals.


   ACKNOWLEDGMENTS
 
We thank Dr. William Sly (Washington University School of Medicine, St. Louis, MO) and Dr. Robert Burghardt (Image Analysis Laboratory, Texas A&M University) for kindly providing the anti-mouse CAR2 antiserum and monoclonal anticytochrome C antibody used in this study, respectively.


   FOOTNOTES
 
1 Supported by grants HD38274 and P30ES0106 from the National Institutes of Health. Back

2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 862 2662; tspencer{at}tamu.edu Back

Received: 12 December 2004.

First decision: 5 January 2005.

Accepted: 10 March 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Ann Rev Biochem 1995 64:375-401[CrossRef][Medline]
  2. Hewett-Emmett D, Tashian RE. Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families. Mol Phylogenet Evol 1996 5:50-77[CrossRef][Medline]
  3. Henry RP. Multiple roles of carbonic anhydrase in cellular transport and metabolism. Ann Rev Physiol 1996 58:523-538[CrossRef][Medline]
  4. Okamura N, Tajima Y, Soejima A, Masuda H, Sugita Y. Sodium bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa through direct activation of adenylate cyclase. J Biol Chem 1985 260:9699-9705[Abstract/Free Full Text]
  5. Boatman DE. Responses of gametes to the oviductal environment. Hum Reprod 1997 12:133-149[Abstract]
  6. Au CL, Wong PY. Luminal acidification by the perfused rat cauda epididymidis. J Physiol 1980 309:419-427[Abstract/Free Full Text]
  7. Breton S, Hammar K, Smith PJ, Brown D. Proton secretion in the male reproductive tract: involvement of Cl–independent HCO-3 transport. Am J Physiol 1998 275:C1134-1142
  8. Harris GC Jr, Goto K. Carbonic anhydrase activity of the reproductive tract tissues of aged male fowls and its relationship to semen production. J Reprod Fertil 1984 70:25-30
  9. Yu HM, Sun BM, Bai Q, Koide SS, Li XJ. Influence of acetazolamide on AQP1 gene expression in testis and on sperm count/motility in epididymis of rats. Arch Androl 2002 48:281-294[CrossRef][Medline]
  10. Lutwak-Mann C. Carbonic anhydrase in the female reproductive tract; occurrence, distribution and hormonal dependence. J Endocrinol 1955 13:26-38[Medline]
  11. Pincus G, Bialy G. Carbonic anhydrase in steroid-responsive tissues. Rec Prog Horm Res 1963 19:201-250
  12. Fong SK, Liu CQ, Chan HC. Cellular mechanisms of adrenaline-stimulated anion secretion by the mouse endometrial epithelium. Biol Reprod 1998 59:1342-1348[Abstract/Free Full Text]
  13. Karhumaa P, Parkkila S, Tureci O, Waheed A, Grubb JH, Shah G, Parkkila A, Kaunisto K, Tapanainen J, Sly WS, Rajaniemi H. Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium. Mol Hum Reprod 2000 6:68-74[Abstract/Free Full Text]
  14. Hu J, Gray CA, Spencer TE. Gene expression profiling of neonatal mouse uterine development. Biol Reprod 2004 70:1870-1876[Abstract/Free Full Text]
  15. Mossman HA. Vertebrate Fetal Membranes. New Brunswick, NJ: Rutgers University Press; 1987
  16. Cunha GR. Stromal induction and specification of morphogenesis and cytodifferentiation of the epithelia of the Mullerian ducts and urogenital sinus during development of the uterus and vagina in mice. J Exp Zool 1976 196:361-370[CrossRef][Medline]
  17. Cunha GR, Battle E, Young P, Brody J, Donjacour A, Hayashi N, Kinbara H. Role of epithelial-mesenchymal interactions in the differentiation and spatial organization of visceral smooth muscle. Epithelial Cell Biol 1992 1:76-83[Medline]
  18. Bartol FF, Wiley AA, Floyd JG, Ott TL, Bazer FW, Gray CA, Spencer TE. Uterine differentiation as a foundation for subsequent fertility. J Reprod Fertil Suppl 1999 54:287-302[Medline]
  19. Gray CA, Bartol FF, Tarleton BJ, Wiley AA, Johnson GA, Bazer FW, Spencer TE. Developmental biology of uterine glands. Biol Reprod 2001 65:1311-1323[Abstract/Free Full Text]
  20. Carson DD, Bagchi I, Dey SK, Enders AC, Fazleabas AT, Lessey BA, Yoshinaga K. Embryo implantation. Dev Biol 2000 223:217-237[CrossRef][Medline]
  21. Gray CA, Burghardt RC, Johnson GA, Bazer FW, Spencer TE. Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation. Reproduction 2002 124:289-300[Abstract]
  22. Burton GJ, Watson AL, Hempstock J, Skepper JN, Jauniaux E. Uterine glands provide histiotrophic nutrition for the human fetus during the first trimester of pregnancy. J Clin Endocrinol Metab 2002 87:2954-2959[Abstract/Free Full Text]
  23. Bagchi IC, Cheon YP, Li Q, Bagchi MK. Progesterone receptor-regulated gene networks in implantation. Front Biosci 2003 8:S852-S861
  24. Brody JR, Cunha GR. Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: I. Normal development. Am J Anat 1989 186:1-20[CrossRef][Medline]
  25. Taylor KM, Gray CA, Joyce MM, Stewart MD, Bazer FW, Spencer TE. Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin. Biol Reprod 2000 63:1192-1204[Abstract/Free Full Text]
  26. Griffiths JR. Are cancer cells acidic?. Br J Cancer 1991 64:425-427[Medline]
  27. Liao SY, Aurelio ON, Jan K, Zavada J, Stanbridge EJ. Identification of the MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney. Cancer Res 1997 57:2827-2831[Abstract/Free Full Text]
  28. Ivanov SV, Kuzmin I, Wei MH, Pack S, Geil L, Johnson BE, Stanbridge EJ, Lerman MI. Downregulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes. Proc Natl Acad Sci U S A 1998 95:12596-12601[Abstract/Free Full Text]
  29. Parkkila S, Rajaniemi H, Parkkila AK, Kivela J, Waheed A, Pastorekova S, Pastorek J, Sly WS. Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro. Proc Natl Acad Sci U S A 2000 97:2220-2224[Abstract/Free Full Text]
  30. Spicer SS, Lewis SE, Tashian RE, Schulte BA. Mice carrying a CAR-2 null allele lack carbonic anhydrase II immunohistochemically and show vascular calcification. Am J Pathol 1989 134:947-954[Abstract]
  31. Carpenter KD, Gray CA, Noel S, Gertler A, Bazer FW, Spencer TE. Prolactin regulation of neonatal ovine uterine gland morphogenesis. Endocrinology 2003 144:110-120[Abstract/Free Full Text]
  32. Spencer TE, Stagg AG, Joyce MM, Jenster G, Wood CG, Bazer FW, Wiley AA, Bartol FF. Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model. Endocrinology 1999 140:4070-4080[Abstract/Free Full Text]
  33. Stewart DM, Johnson GA, Vyhlidal CA, Burghardt RC, Safe SH, Yu-Lee LY, Bazer FW, Spencer TE. Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells. Endocrinology 2001 142:98-107[Abstract/Free Full Text]
  34. Miyamoto H, Miyashita T, Okushima M, Nakano S, Morita T, Matsushiro A. A carbonic anhydrase from the nacreous layer in oyster pearls. Proc Natl Acad Sci U S A 1996 93:9657-9660[Abstract/Free Full Text]
  35. Innocenti A, Firnges MA, Antel J, Wurl M, Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors. Inhibition of the membrane-bound human and bovine isozymes IV with sulfonamides. Bioorg Med Chem Lett 2005 15:1149-1154[CrossRef][Medline]
  36. Bilir A, Guneri AD, Altinoz MA. Acetaminophen and DMSO modulate growth and gemcitabine cytotoxicity in FM3A breast cancer cells in vitro. Neoplasma 2004 51:460-464[Medline]
  37. Maren TH. Use of inhibitors in physiological studies of carbonic anhydrase. Am J Physiol 1977 232:F291-F297
  38. Bagnis C, Marshansky V, Breton S, Brown D. Remodeling the cellular profile of collecting ducts by chronic carbonic anhydrase inhibition. Am J Physiol Renal Physiol 2001 280:F437-F448[Abstract/Free Full Text]
  39. Teicher BA, Liu SD, Liu JT, Holden SA, Herman TS. A carbonic anhydrase inhibitor as a potential modulator of cancer therapies. Anticancer Res 1993 13:1549-1556[Medline]
  40. Bartosova M, Parkkila S, Pohlodek K, Karttunen TJ, Galbavy S, Mucha V, Harris AL, Pastorek J, Pastorekova S. Expression of carbonic anhydrase IX in breast is associated with malignant tissues and is related to overexpression of c-erbB2. J Pathol 2002 197:314-321[CrossRef][Medline]
  41. Hu J, Zhang X, Nothnick WB, Spencer TE. Matrix metalloproteinases and their tissue inhibitors in the developing neonatal mouse uterus. Biol Reprod 2004 71:1598-1604[Abstract/Free Full Text]
  42. Nothnick WB. Disruption of the tissue inhibitor of metalloproteinase-1 gene in reproductive-age female mice is associated with estrous cycle stage-specific increases in stromelysin messenger RNA expression and activity. Biol Reprod 2001 65:1780-1788[Abstract/Free Full Text]
  43. Zhou HE, Zhang X, Nothnick WB. Disruption of the TIMP-1 gene product is associated with accelerated endometrial gland formation during early postnatal uterine development. Biol Reprod 2004 71:534-539[Abstract/Free Full Text]
  44. Spencer TE, Carpenter KD, Hayashi K, Hu J. Uterine glands. In: Davies JA (ed.), Branching Morphogenesis. Georgetown, TX: Landes Biosciences; 2005



This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
J. C. Kwekel, L. D. Burgoon, J. W. Burt, J. R. Harkema, and T. R. Zacharewski
A cross-species analysis of the rodent uterotrophic program: elucidation of conserved responses and targets of estrogen signaling
Physiol Genomics, November 17, 2005; 23(3): 327 - 342.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/1/131    most recent
biolreprod.104.039008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hu, J.
Right arrow Articles by Spencer, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hu, J.
Right arrow Articles by Spencer, T. E.
Agricola
Right arrow Articles by Hu, J.
Right arrow Articles by Spencer, T. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS