| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Institute of Biochemical Sciences, College of Science, National Taiwan University, Taipei, Taiwan
b Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
estradiol, ovulatory cycle, pregnancy, progesterone, uterus
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The following materials were obtained from commercial sources: Sephadex G-100 and Full Range Rainbow molecular-weight markers (Amersham Pharmacia Biotech, Uppsala, Sweden); CM-Affi-Gel Blue (Bio-Rad Laboratories, Richmond, CA); Protein PAK SP 5PW column (Waters, Milford, MA); Nucleosil C18 column (Macherey-Nagel GmbH & Co., Düren, Germany); BCA protein assay kit (Pierce, Rockford, IL); E2, P4, diethylstilbestrol dipropionate (DES), nitroblue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), PMSF, and silanated glass slides (Sigma Chemical Co., St Louis, MO); goat anti-human Bf (Calbiochem, La Jolla, CA); goat anti-mouse C3 (ICN Biomedicals, Inc., Irvine, CA); biotin-conjugated rabbit anti-goat IgG, alkaline phosphatase-conjugated streptavidin, and horse radish peroxidase-conjugated rabbit anti-goat IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD); nuclear fast red (Vector Laboratories, Burlingame, CA); enhanced chemiluminescent (ECL) substrate and [
-32P]dATP (NEN Life Science Products, Boston, MA); Prime-a-Gene kit and pGEM-T-easy vector (Promega, Madison, WI); Ultraspec-II RNA isolation kit (Biotecx Laboratories, Inc., Houston, TX); ZAP cDNA synthesis kit (Stratagene, La Jolla, CA); and Oligotex mRNA kit (Qiagen, Chatsworth, CA). Mouse uterus stage blots designed to observe gene expression during the pregnancy period were purchased from Seegene, Inc. (Seoul, Korea). All other chemicals were reagent grade.
Animals and Steroid Hormone Treatment
Outbred ICR mice (Charles River Laboratories, Wilmington, MA) were supplied by the Animal Center, College of Medicine, National Taiwan University. Animals were kept under conditions following the institutional guidelines for the care and use of experimental animals. They were housed under controlled lighting (14L:10D), and both immature (21-day-old) and sexually mature (6- to 8-wk-old) female mice were used for the study. The estrous cycle was staged by examining vaginal smears. The presence of a vaginal plug after mating was designated as Day (D) 1 of pregnancy.
To investigate the effect of ovarian steroids, immature mice received a daily s.c. injection of DES in corn oil (30 ng/g body weight) for 3 consecutive days, and adult female mice, in which the ovaries had been removed by bilateral oophorectomy 1 wk before initiation of steroid treatment, received a daily s.c. injection of E2 (30 ng/g body weight) and/or P4 (150 µg/g body weight) in corn oil for 3 consecutive days. Control animals received corn oil only. Uteri were removed from animals 12 h after the last injection.
Protein Isolation and Characterization
The ULF was immediately collected from animals after they were killed between 0900 and 1000 h on a specified day by cervical dislocation. The ULF was mixed in a final concentration of 1.0 mM EDTA and 1.0 mM PMSF. The solution was centrifuged at 14 000 x g for 10 min to remove insoluble materials and stored at -70°C until use. According to the previously described procedure [2], ULF collected from uteri of DES-stimulated immature mice was resolved into 3 fractions by gel filtration (denoted as fractions I-III in Fig. 1). The fraction II sample was passed through a CM-Affi-Gel Blue column (1.5 x 10 cm) to remove serum albumin.
|
The protein solution was concentrated using a Centricon (Millipore, Bedford, MA). The amount of protein was determined using a BCA protein assay kit. A modification of the method of Van der Geer et al. [13] was generally followed to cleave a protein with 2.36 mM CNBr in 70% formic acid at room temperature (RT) for 18 h. Automated Edman degradation for the determination of protein sequence was carried out by a gas-phase sequenator (Applied Biosystems, Foster City, CA).
Western Blot Analysis and Immunohistochemical Staining
Protein components in ULF were resolved by SDS-PAGE according to the method of Laemmli [14]. Proteins were transferred from the gel slab to a nitrocellulose membrane (Micron Separations, Inc., Westborough, MA) using an electroblotting method, which was conducted at 35 V and 4°C for 18 h in a solution containing 25 mM Tris-HCl, 197 mM glycine, and 13.3% methanol. Filters were blocked with 5.0% (w/v) skim milk and 0.1% (v/v) Tween 20 in PBS (blocking solution) for 2 h, then incubated with polyclonal goat anti-human Bf or goat anti-mouse C3 diluted to 1:15 000 (v/v) in the blocking solution for 1 h at RT. After filters were gently agitated in 4 changes of PBS containing 0.1% Tween 20 (washing solution) for 15 min each, they were immunoreacted with horse radish peroxidase-conjugated rabbit anti-goat IgG diluted to 1:15 000 (v/v) in the blocking solution for 1 h at RT. Immunoreactive bands were revealed using an ECL substrate according to the manufacturer's instructions.
Mouse uteri were fixed in Bouin solution and embedded in paraffin. Then, 8-µm serial cross-sections were mounted on silanated glass slides. Deparaffinized sections were blocked in the blocking solution for 1 h at RT, then incubated with polyclonal goat anti-human Bf or goat anti-mouse C3 diluted to 1:1000 in the blocking solution for 1 h at RT. After slides were gently agitated in 3 changes of the washing solution for 10 min each, they were treated with biotin-conjugated rabbit anti-goat IgG (1 µg/ml) in the blocking solution for 1 h at RT. Slides were washed as mentioned, then incubated with alkaline phosphatase-conjugated streptavidin (1 µg/ml) in the blocking solution for 1 h at RT. Protein signals on specimens were observed after slides were incubated for 8 min with 0.0375% NBT and 0.0188% BCIP in a solution containing 100 mM Tris-HCl, 100 mM NaCl, and 5 mM MgCl2 at pH 9.5. Slides were washed in 3 changes of water for 3 min each and counterstained with nuclear fast red for 3 min. Finally, slides were briefly washed with water and photographed under bright-field illumination using a microscope (AH3-RFCA; Olympus, Tokyo, Japan).
RNA Isolation and Northern Blot Analysis
Total RNA was extracted from uterine homogenates of mice using an Ultraspec-II RNA isolation kit. Following the standard procedures recommended for use of the ZAP cDNA synthesis kit, double-stranded cDNAs were prepared from the polyadenylated fraction, which was isolated from the total RNA using an Oligotex mRNA mini kit according to the manufacturer's instructions. We synthesized two primer pairs that were employed for polymerase chain reaction to amplify cDNAs to obtain DNA fragments of nucleotides 989–2064 in Bf cDNA [15] and nucleotides 4141 to 4586 in C3 cDNA [16]. One primer pair included the oligonucleotide 5'-TGATTGAGAAGGTGGCGAGTTACG-3', which represents 989–1012 nucleotide sequences of Bf cDNA, and the oligonucleotide 5'-GGCTTTCTTGTCCCCATTCTTGAT-3', which is complementary to nucleotides 2041 to 2064 of Bf cDNA. The other primer pair was the oligonucleotide 5'-ACCTCAGGGTCAGCATA-3', which represents 4141–4157 nucleotide sequences of C3 cDNA, and the oligonucleotide 5'-GCTGTGCCACAGTGAAATG-3', which is complementary to nucleotides 4568 to 4586 of C3 cDNA sequence. Polymerase chain reaction with Taq polymerase was carried out for 30 cycles as follows: 94°C for 1 min, 55°C for 30 sec, and 68°C for 3 min. These two amplified DNA fragments were separately ligated to pGEM-T-easy vector via TA cloning, and recombinant plasmids were introduced into Escherichia coli JM109 strain using a transformation technique [17]. Positive clones containing the Bf cDNA fragment (1076 base pairs [bp]) and the C3 cDNA fragment (446 bp) were sequenced to confirm 99% identify to their parental cDNA.
The DNA fragments inserted in pGEM-T-easy and a cDNA fragment (1233 bp) of the mouse glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) inserted in pGEM3 vector were used as a template to prepare the 32P-labeled cDNA probe using a Promega random-priming kit. The RNA samples (20 µg) were subjected to denaturing 1.0% agarose-formaldehyde gel electrophoresis, then blotted onto nylon membranes by capillary transfer as previously described [18]. After incubation with the prehybridization buffer (50% deionized formamide, 6x SSC [single strength:0.15 M sodium chloride and 0.015 M sodium citrate], 5x Denhardt solution, 1.0% SDS, and 100 µg/ml of sheared salmon sperm DNA) for 2 h at 60°C, membranes were hybridized with 1 labeled probe overnight at 60°C. Following hybridization, membranes were washed using standard procedures. The RNA messages on 1 filter membrane were observed after autoradiography, and probes were removed from membranes as previously described [18]. The same membrane was then hybridized with another labeled probe. Thus, hybridization with Bf, C3, and GAPDH probes was performed on the same filter membrane.
Statistical Analysis
Bonferroni post-hoc test was followed by one-way ANOVA using Instat software (Graph Pad, San Diego, CA). Data are presented as mean ± SD. A P value <0.05 was considered to be significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The ULF appears only in the postpubertal period in proestrus and estrus, and immature mice can be induced to secrete ULF by estrogen stimulation. As shown in Figure 1, the ULF collected from DES-stimulated immature mice was resolved into three fractions by gel filtration chromatography. The fraction II sample was further resolved by ion-exchange high-performance liquid chromatography (HPLC) into peaks 1–6 as shown in Figure 2A. To identify Bf in mouse ULF, the peak 4 sample was cleaved with CNBr, and the reaction mixture was resolved by reverse-phase HPLC (Fig. 2B). Automated Edman degradation of either the peak a or peak b sample in Figure 2B for 10 cycles gave reliable data. A peptide sequence of EDLENVFYQ, which is identical to residues 456–464 of mouse Bf [15] in all positions, was determined from peak a. Two predominant amino acids could be detected at each cycle in the analysis of peak b. The actual yields of these two sequences in an individual cycle were such that both components appeared to be present in nearly equal amounts. Matching these sequences with the protein sequences of mouse Bf were interpreted as representing residues 478–487 and residues 510–519 of the protein. To identify C3 in mouse ULF, the fraction III sample of Figure 1 was concentrated and resolved into six main peaks by reverse-phase HPLC (Fig. 3A). The peak 6 sample was cleaved with CNBr, and the reaction mixture was subjected to reverse-phase HPLC (Fig. 3B). Automated Edman degradation of peak a as shown in Figure 3B gave a peptide sequence of NKLEEPYLGK, which is identical to residues 1200–1209 in the chain of mouse C3 [16]. Apparently, Bf and C3 coexisted in the ULF of the DES-stimulated immature mice.
|
|
Mouse Bf is a single polypeptide with a molecular mass of 95 kDa. Mouse C3 has a molecular mass of 180 kDa, comprising two polypeptide chains, an chain cross-linked with a ß chain by one disulfide bond [19]. In complement activation through the alternative pathway, C3 cleaves to C3b, and Bf cleaves to Ba and Bb, to form a C3bBb complex that serves as a C3 convertase. According to SDS-PAGE under nonreducing conditions, Bf, Ba, and Bb were identified as a protein band of 95, 63, and 33 kDa, and C3 and C3b were indistinguishable as a broad band of approximately 180–250 kDa. We detected these protein species by Western blot analysis (Fig. 4). Both C3 and Bf, but none of their degradative products, were immunodetected in normal serum. The C3/C3b and several bands of smaller molecular weight, which might be the unidentified degradation products of C3/C3b, were immunodetected in the ULF accumulated in either DES-stimulated immature mice or adult females in proestrus and estrus. The Bf, Ba, and Bb appeared in the ULF of DES-stimulated immature mice. In the ULF of adults in proestrus, Bf was predominant and Ba and Bb were in a trace, whereas in the ULF of adults in estrus, Bf, Ba, and Bb were all prominent. The simultaneous expression of Bf and C3 as well as the presence of their fragments suggested activation via the alternative pathway in the uterine lumen.
|
Uterine Expression of Bf and C3 after Ovarian Steroid Administration to Immature or Ovariectomized Mice
We examined the distribution of Bf mRNA and C3 mRNA in tissue homogenates of the genital tracts of adult mice using Northern blot analysis. The Bf mRNA appeared as a single RNA species of approximately 2.4 kilobases (kb). Consistent with a previous report [20], C3 mRNAs included a predominant species of 6.0 kb and three minor species of approximately 4.3, 2.3, and 1.9 kb. As shown in Figure 5, both the 2.4-kb Bf mRNA and the 6.0-kb C3 mRNA were abundant in uterine homogenates and in a trace in the vas deferens, but they were barely detectable in homogenates of other sexual glands, such as the ovary, vagina, testis, epididymis, prostate, seminal vesicle, and coagulating gland. Apparently, transcription of both RNA messages in the mouse reproductive tract was tissue specific. This prompted us to study how ovarian steroids regulate the expression of Bf, C3, and their RNA messages in mouse uteri.
|
Both Bf mRNA and C3 mRNA were detected in uterine homogenates of immature animals, indicating a basal transcription of both protein genes before animal maturity. Compared with the corresponding basal transcription, DES enhanced Bf and C3 mRNA expression 3.6- and 3.7-fold, respectively (data not shown). These mRNA levels were positively correlated with the immunohistochemical results shown in Figure 6. Neither Bf nor C3 was immunodetected in uterine cells of immature mice. However, DES stimulated the uterine growth of immature animals, and uterine cells, particularly the luminal epithelium, became hypertrophied compared with the cell morphology of control animals. An intensive immunohistochemical staining of both proteins appeared around the apical region in the cytoplasm of luminal and glandular epithelia. Meanwhile, a much lower immunoactivity in the stroma toward the luminal side was detected, but almost no immunoreactivity was noted in the stroma on the other side or in the myometrium. No immunoreactivity of Bf and C3 appeared on the luminal surface of the endometrium, suggesting no deposition of Bf and C3 on the cell surface. These data support the idea that, among uterine cells, DES-stimulated uterine expression of Bf mRNA and C3 mRNA and their translated proteins mainly occurs in the endometrial epithelia. Apparently, Bf and C3 in the ULF are a primary result of their secretion from the luminal epithelium.
|
Removal of ovaries by bilateral oophorectomy caused atrophy of adult uteri. Administration of E2 (30 ng/g body weight) alone to ovariectomized adults for 3 consecutive days restored uterine cells to a hypertrophied stage, whereas cells remained atrophied after injection of P4 alone (150 µg/g body weight) to animals. We examined how the hormonal treatment of ovariectomized adults affected the uterine expression of both Bf and C3. The levels of both Bf mRNA and C3 mRNA increased remarkably in E2-treated animals but tended to decrease in P4-treated animals compared with the basal transcription in controls. The E2-stimulated Bf mRNA expression was slightly enhanced, whereas the E2-stimulated C3 mRNA expression was considerably potentiated, as the animals were injected with E2 and P4 together (Fig. 7). Parallel to the change in mRNA level, immunohistochemical patterns of uterine sections (Fig. 8) revealed that the intensive immunoreactivity of both proteins in animals treated with E2 alone or with E2 and P4 together was similar to that in DES-stimulated immature mice, compared with no immunoreactivity in control and P4-treated animals.
|
|
Uterine Expression of Bf and C3 in Adult Mice
We compared levels of Bf and C3 RNA messages in uterine homogenates and examined the distribution of Bf and C3 in tissue sections at various stages of the estrous cycle and during the pregnancy period. As shown in Figure 9, both RNA messages were abundant during proestrus and estrus but declined rapidly during metestrus. It was of interest to note that Bf mRNA remained at an undetectable level, but that C3 mRNA increased, during diestrus. This characteristic change in mRNA levels was positively correlated with the immunohistochemical staining patterns of both proteins in tissue sections (Fig. 10). The immunoreactivity of both Bf and C3 was strong in glandular and luminal epithelial cells of the endometrium during proestrus and estrus, compared to no immunoreactivity in the stroma and myometrium. Immunostaining intensity for both Bf and C3 in metestrus was weakly positive. During diestrus, almost no cells immunoreactive to Bf antiserum were seen, but an intensive immunoreactivity to C3 appeared in glandular and luminal epithelia.
|
|
Figure 11 shows the levels of Bf mRNA and C3 mRNA in uterine homogenates from D1 to D19 after copulatory plug formation. The postimplantation uterine samples did not contain the placenta or the conceptus tissues, but some decidual samples may be contaminated during sampling of the uterus. The Bf mRNA was abundant on D1, increased to a slightly higher level on D2, and declined to an almost undetectable level on D3. From D10 on, Bf mRNA increased to a fairly constant, low level until delivery. The C3 mRNA was at a high level on D1, declined rapidly from D2 to D9 to a low level, remarkably increased from D10 to reach a maximum level on D18, and quickly dropped near delivery. The change in mRNA levels during the preimplantation period was in accord with the immunoreactivity of Bf and C3 as detected in the uterine section (Fig. 12). Immunohistochemical staining of either protein was intensive in glandular and luminal epithelia on D1. Very weakly immunoreactive cells were seen on D3 and D4. Immunoreactivity of Bf appeared in the luminal and glandular epithelium, whereas C3 was immunodetected only in the glandular epithelium on D2.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Because the response of endometrial epithelial cells to E2 and P4 is rarely maintained under in vitro conditions [21, 22], our analyses of Bf and C3 gene expression in mouse uterus during the natural estrous cycle and the pregnancy period remain essential for investigating how ovarian steroids regulate their expression in the uterus in vivo. In response to physiological changes in the reproductive tract, serum ovarian steroid concentrations in mouse and rat during the estrous cycle and pregnancy period have been measured [23–25]. Correlating the endometrial expression of Bf and C3 to serum concentrations of ovarian steroids may shed some light on the in vivo hormonal regulation of uterine Bf and C3 synthesis and secretion. The stimulation of Bf and C3 gene expression occurs in proestrus and estrus, during which time a large E2 surge is accompanied by a basal P4 concentration. Their expressions sharply decline in metestrus, when both serum E2 and P4 are at a basal level, suggesting that the concentration of ovarian steroids in endometrial cells might not reach a threshold sufficient to exert their effects on the expression of these two genes. In diestrus, when serum E2 is at a low level and serum P4 increases to a high level, C3 gene, but not Bf gene, is expressed, suggesting that the low serum E2 level is enough for C3 gene expression but is insufficient for Bf gene expression; furthermore, E2-stimulated C3 gene expression is enhanced by P4. The endometrial expression of Bf and C3 during D1 and D2 of the preimplantation period may reflect preovulatory estrogen stimulation. Both Bf and C3 gene are barely expressed from D3 to D9, during which time serum E2 is maintained at a basal level and serum P4 increases. During late pregnancy, from D10 to D20, serum E2 rises to a level as high as that in proestrus and estrus, and serum P4 increases to a level much higher than that in the estrous cycle. Expression of C3 gene increases remarkably, whereas expression of Bf gene is not as prominent as that in proestrus and estrus. Again, P4 potentiates E2-stimulated C3 gene expression, but some unknown factor other than P4 may suppress the stimulation of Bf gene expression by E2. Apparently, the hormone-regulated endometrial expression of these two genes during the estrous cycle and the pregnancy period is congruent with the characteristics of their expression in the uteri of ovariectomized adult mice after hormonal treatments (Figs. 7 and 8).
Hasty et al. [8] immunodetected relatively large amounts of C3 and Bf in the glandular epithelium of human endometrium in the P4-dominant luteal phase, but only small amounts of these two proteins in tissue in the E2-dominant proliferative phase, during the menstrual cycle. They raised the possibility that synthesis of these two proteins in human endometrium may be regulated by P4. This concept seems to be supported by an increase in their syntheses in patients receiving exogenous P4 treatments [7]. On the other hand, a summary of our data in this work clearly illustrates that P4 alone produces almost no effect, but that E2 stimulates the gene expression of Bf and C3 in mouse endometrium. A similar situation has been previously illustrated for C3 mRNA expression in the uterus of immature rats receiving exogenous E2 treatment [11, 12]. The apparent P4 regulation in human endometrium and E2 regulation in rodent endometrium may suggest different molecular machinery between orders of mammals in the control of endometrial C3 and Bf gene expression. Even in the same family, P4 produces an inhibitory effect on E2-stimulated C3 gene expression in rat endometrium [12], but it potentiates E2-stimulated C3 gene expression in mouse endometrium (Figs. 7 and 8). Many lines of evidence indicate that the primary action of steroid hormones at the level of transcription involves the participation of a complex formed by a steroid and its receptor. Such a receptor complex modulates cooperative interactions of hormone response elements, such as estrogen response element (ERE), progesterone response element (PRE), and glucocorticoid response element (GRE), and other accessory response elements in the steroid-responsive genes. The C3 gene promoter has been used as a standard promoter in studying estrogen response. Fan et al. [26] has identified three functional EREs in the human C3 gene promoter. Vollmer and Schneider [27] have assayed the expression of C3 as an E2-induced protein to assess the estrogenic and antiestrogenic potency of antiestrogens. As yet, no functional steroid hormone response elements have been demonstrated in the mouse/rat C3 and Bf gene promoters. We applied the method of Quandt et al. [28] to predict consensus matches in the mouse C3 and Bf gene promoter [29, 30]: No potential PRE/GRE, but three and five potential EREs, are respectively predicted in the mouse C3 and Bf gene promoter. Possibly, P4 regulation may represent an indirect action through stimulation of various cytokines. In fact, functional response elements of interleukin 1 and interferon-
are present in the mouse and human Bf gene [29], and functional response elements of interleukin 1 and interleukin 6 are present in the mouse C3 gene [30]. The study of De et al. [31] indicates that interleukin 1, interleukin 6, and tumor necrosis factor- are produced in the mouse uterus during the estrous cycle and are induced by estrogen and P4. The RNA messages of these cytokines are also present in the mouse uterus at early preimplantation [32, 33]. Dissimilar to the C3 and Bf gene expressions described, E2-stimulated expressions of lactoferrin, a major ULF protein [2, 34], and of 24p3 protein, a lipocalin [3, 4, 35], in mouse endometrium are inhibited by P4. Further studies are needed to unravel the molecular mechanism underlying how P4 functions as an enhancer or silencer to regulate E2-stimulated gene expression.
Once C3 and Bf are simultaneously synthesized in the endometrium, they secrete to the uterine lumen to participate in the initiation and amplification of alternative pathways (Fig. 4). In addition to the inflammatory host defense against the invasion of viruses and bacteria, an exact role, if any, for these complement proteins in reproductive processes has yet to be determined.
|
FOOTNOTES |
|---|
1 Supported in part by grants NSC 89-2311-B-001-193 and NSC 89-2311-B-002-104 from the National Science Council, Taiwan. 
2 Correspondence: Yee-Hsiung Chen, Institute of Biochemical Sciences, College of Science, National Taiwan University, P.O. Box 23-106, Taipei 106, Taiwan, Republic of China. FAX: 886 2 2363 5038;bc304{at}gate.sinica.edu.tw
Accepted: September 17, 2001.
Received: May 10, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
,25-dihydroxyvitamin D3 in mouse marrow-derived stromal cells (ST2) and primary osteoblastic cells. Endocrinology 1991; 129::2774-2779[Abstract]This article has been cited by other articles:
|
|
 |
|
  P.-K. Tse, Y.-L. Lee, W.-N. Chow, J. M. C. Luk, K.-F. Lee, and W. S. B. Yeung Preimplantation Embryos Cooperate with Oviductal Cells to Produce Embryotrophic Inactivated Complement-3b Endocrinology, March 1, 2008; 149(3): 1268 - 1276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Simms and A. E. Jerse In Vivo Selection for Neisseria gonorrhoeae Opacity Protein Expression in the Absence of Human Carcinoembryonic Antigen Cell Adhesion Molecules. Infect. Immun., May 1, 2006; 74(5): 2965 - 2974. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Rhen and J. A. Cidlowski Estrogens and Glucocorticoids Have Opposing Effects on the Amount and Latent Activity of Complement Proteins in the Rat Uterus Biol Reprod, February 1, 2006; 74(2): 265 - 274. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. von Schalburg, M. L. Rise, G. D. Brown, W. S. Davidson, and B. F. Koop A Comprehensive Survey of the Genes Involved in Maturation and Development of the Rainbow Trout Ovary Biol Reprod, March 1, 2005; 72(3): 687 - 699. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-L. Lee, K.-F. Lee, J.-S. Xu, Q.-Y. He, J.-F. Chiu, W. M. Lee, J. M. Luk, and W. S. B. Yeung The Embryotrophic Activity of Oviductal Cell-derived Complement C3b and iC3b, a Novel Function of Complement Protein in Reproduction J. Biol. Chem., March 26, 2004; 279(13): 12763 - 12768. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and C3 mRNA (
) in the total RNA (20 µg) prepared from the tissue homogenates were determined by the same procedures as described in 