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Department of Molecular and Cellular Biology (M.S., I. H.-G., I. G.-R., J.S.R.), Baylor College of Medicine, Houston, Texas 77030; Department of Applied Animal Science (M.S.), Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, 724-8528, Japan; and Departments of Biochemistry and Molecular Biology and Physiology (I.H.-G., I. G.-R.), Faculty of Sciences, University of Las Palmas, 35016 Las Palmas Grand Canaria, Spain
Address all correspondence and requests for reprints to: JoAnne S. Richards, Ph.D., Department of Molecular Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: joanner{at}bcm.tmc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B and increased expression of Il6 and Tnfa target genes, documenting that the TLR pathway is functional. Cumulus cells and granulosa cells also express the scavenger receptors CD36 and scavenger receptor type B1 and exhibited phagocytic uptake of fluorescently tagged bacterial particles. Collectively, these results provide novel evidence that cumulus cells as well as granulosa cells express innate immune related genes that may play critical roles in surveillance and cell survival during the ovulation process. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gene profiling of COCs collected at specific intervals after LH/hCG induction of ovulation revealed that genes thought to be associated selectively with immune cell function and innate immunity are expressed at high levels in cumulus cells before and after ovulation (10). Factors that are known to be critical for fertility include PTX3 and TNF
IP6, which act to stabilize the matrix (11, 12, 13) and CD147/basigin, which is a transmembrane glycoprotein with IgG-like domains (14, 15). Programmed cell death 1 (Pdcd1) and its transcriptional regulator Runx1 are involved autoimmune diseases (16). Activated leukocyte adhesion molecular (Alcam), CD34 (Cd34), and CD52 (Cd52) are cell surface markers of immune cells (10). Based on the highly uniform expression of these genes in cumulus cells (10) and the dearth of immune cells in ovulating follicles (17), we have proposed that cumulus cells of ovulating follicles acquire specific innate immune-like functions (10).
Further analyses of our COC gene profiling database allowed us to identify additional genes heretofore restricted to immune cells or cells with innate immune-like functions (10). Importantly, these include cell surface signaling molecules known as pattern recognition receptors (PRRs) that discriminate pathogen-coded molecular structures known as pathogen-associated molecular patterns (18, 19). PRRs act as sensors of the external environment to detect self from nonself or altered self. Among these receptors are CD14 and the Toll-like receptors (TLRs) 4, 8, and 9 that directly and selectively bind bacterial lipopolysaccharides (LPS), short-strand RNA, and unmethylated cytosine-phosphate-guanine dinucleotide (CpG) DNA. Also observed was the complement factor C1q that is critical for monitoring a variety of external factors and is expressed abundantly at sites of tissue damage. C1q also plays an initiating role in clearance of apoptotic cells (18, 20). Genes encoding the A- and B-type scavenger receptors including scavenger receptor BI and BII (Scarb1 and Scarb2) as well as Cd36 were identified. Although these latter receptors regulate cholesterol and lipid trafficking in steroidogenic cells (21, 22), they can also mediate phagocytic activity and the ingestion of bacteria and apoptotic cells (23). Based on these observations, we sought to analyze the expression of these immune cell receptor genes in ovarian cells, to determine whether PRR signaling cascades were operational, and to examine the phagocytic capabilities of cumulus cells and granulosa cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). TLR4, TLR8, and TLR9 recognize LPS, short-strand RNA, and unmethylated CpG DNA, respectively (18, 19, 25). Small fragments of HA, the backbone of the COC matrix, can also bind and activate TLR4 (26, 27, 28). TLR4 interacts with CD14 and possibly C1q to mediate many of its effects via activating the adaptor protein MYD88 (myeloid differentiation primary response gene 88) (18, 29). MYD88, in turn, leads to the activation of downstream signaling molecules such as ERK1/2, p38MAPK, and nuclear factor-B (NF-B) (19). In addition, TLR signaling mediated by the transcription factor IRF3 (interferon regulatory factor 3) leads to the expression of target genes such as Il6, Tnfa, and Ptgs2 (19, 29). In macrophages, TLR signaling also induces expression of members of the scavenger receptor family Scarb1 and Cd36 (20, 24).
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) was expressed in ovarian cells during ovulation, mice were primed with eCG (equine chorionic gonadotropin) to stimulate growth of PO follicles and human CG (hCG) to initiate ovulation (10). COCs and granulosa cells were isolated from ovaries at selected time intervals, total RNA was extracted, and semiquantitative RT-PCR analyses were done. Tlr4, Tlr8, and Tlr9 mRNA levels were low in COCs isolated from PO follicles (0 h) but increased significantly 8–12 h after hCG, a time that just precedes ovulation (Fig. 1B; arrow). Expression of these receptors remained high in ovulated COCs collected from the oviducts at 16 and 24 h. By contrast, Tlr4, Tlr8, and Tlr9 mRNAs were at higher levels in granulosa cells than was observed in COCs at 0 h; Trl4 and, to a lesser extent, Tlr8 and Tlr9 mRNAs increased at 4–8 h post-hCG but declined by 12 and 16 h after hCG. Each of the other membrane components, Cd14, and C1q, as well as downstream molecules (Myd88 and Irf3) and the target gene Il6, were induced markedly in COCs before ovulation. In these same samples, Ptgs2 mRNA was highly induced in COCs and granulosa cells by 4 h. With the exception of Il6 and Ptgs2, none of these genes was markedly induced in granulosa cells (Fig. 1B). Cd36 and Scarb1 mRNAs were clearly detected in COCs and granulosa cells at 0 h (Fig. 1C). Although a transient increase in Cd36 mRNA was observed in COCs at 4 h, no significant changes were observed in granulosa cells, whereas Scarb1 mRNA increased in both COCs and granulosa cells at 4 h (Fig. 1C).
Western blot (WB) analyses using specific antibodies to TLR4, MYD88, IRF3, and SCARBI show increased expression these genes in COCs collected at 8 and 16 h after hCG compared with those collected at 0 h (Fig. 2A). This pattern contrasts with the lower expression of TLR4 and MYD88 and the relatively constant levels of IRF3 and SCARBI in granulosa cells (Fig. 2A). Although the protein expression patterns do not precisely mimic those of mRNA, the results confirm the presence of these proteins in COCs and granulosa cells. Immunofluorescent analyses using the same antibodies revealed intense staining for TLR4, MYD88, and IRF3 as well as SCARBI in cumulus cells collected from COCs 8 or 16 h after hCG (Fig. 2, B and C). It is important to note that essentially all cumulus cells at the 8 and 16 h time points are immunopositive for these proteins, indicating that these factors are derived from cumulus cells and not from invading immune cells. Although the oocytes also appear to be immunopostive, it is not clear whether or not this is specific because oocytes frequently exhibit nonspecific staining. Levels of TLR4, MYD88, IRF3, and SCARB1 immunoreactivity were low in nonexpanded COCs isolated from PO follicles at 0 h (insets in Fig. 2, B and C), supporting the lower abundance of these proteins at this stage. Cells positive for leukocyte markers (CD4, CD8, CD14, CD45, and CD163) have been reported present in ovulated human COCs (17), leading these investigators to conclude that immune cells were present in the ovulated complexes. However, based on the highly uniform expression of TLR-related genes, including CD14 in mouse cumulus cells (shown herein) and the relative dearth of clear evidence for specific immune cell types in ovulating human follicles (17), the results presented herein indicate that cumulus cells of ovulating follicles in the mouse express a vast array of innate immune-related genes, confirming previous observations (10).
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). These results along with the induction of PTX3 by FSH and EGF in isolated COCs (Ref. 11 and our own observations) indicate that genes linked to innate immune functions are induced in isolated cumulus cells devoid of immune cells. Granulosa cells from PO follicles were also cultured 16 h in the presence of FSH, AREG, or FSH+AREG. Whole-cell extracts (WCEs) were prepared, and the levels of TLR4, MYD88, IRF3, and SCARB1 were analyzed by WB. As shown in Fig. 3B, the components of the TLR pathway were expressed at relatively constant levels whereas SCARB1 was clearly induced by either FSH or AREG alone. No additive effect was observed when both factors were present, supporting results observed in granulosa cells isolated at specific times in vivo (Fig. 2A).
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B and p38MAPK Pathways Leading to IL-6 Expression in Cumulus Cells and Granulosa Cells., A and B). Because mRNAs encoding the Tlrs, Cd14, C1q, and Myd88 were all elevated in COCs at 16 h post-hCG, we first analyzed the effects of LPS on COCs isolated from oviducts at this time interval. In COCs, LPS (100 ng/ml) stimulated marked phosphorylation of p38MAPK at 60–120 min and ERK1/2 and NF-B at 120 min (Fig. 4A). Because expression of Tlr mRNAs was high granolosa cells of PO follicles, granulosa cells from eCG-primed mice were isolated. In these granulosa cells, the basal level of phospho-p38MAPK was higher than that in COCs, but LPS also increased the phosphorylation status of each of these factors (Fig. 4B).
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), additional experiments were done to determine whether LPS activated expression of any of these genes in COCs in culture. Accordingly, COCs were isolated from PO follicles of eCG-primed mice (0 h) and from oviducts of mice16 h post-hCG and cultured with LPS (100 ng/ml). In 16 h COCs, expression levels of Il6 as well as Tnfa mRNA increased 7-fold and 4-fold, respectively, whereas Ptgs2 mRNA did not (Fig. 4C, black bars). In nonexpanded COCs (0 h) where levels of TLRs were lower, LPS nevertheless increased Il6, Tnfa, and Ptgs2 mRNAs (Fig. 4C, gray bars). Furthermore, FSH enhanced (2-fold) the ability of LPS to increase Il6 and Ptgs2 mRNA in PO COCs in culture (data not shown). These results indicate that the TLR receptor system is operative in cumulus cells even before the LH surge but that the effects of LPS are enhanced by the actions of gonadotropins. Likewise, granulosa cells isolated from PO follicles responded to LPS as indicated by the marked increases in Il6 and Ptgs2 (but not Tnfa) mRNAs within 4 h (Fig. 4D). HA fragments that are known to activate TLR4 also increased expression of Il6 mRNA in granulosa cells, but the response was not as great as that to LPS. Curiously, FSH mimicked some of the effects of LPS, i.e. increased expression of Il6 and Ptgs2 mRNAs. Because it is highly unlikely that FSH directly activates TLRs, the effects of FSH may be mediated, in part, by its rapid induction of Areg transcripts. AREG is a potent regulator of Ptgs2 expression in these cells (31) and, as shown in Fig. 1B, the marked induction of Ptgs2 mRNA 4 h after hCG precedes the increased expression TLR pathway components. Because phosphorylation of p38MAPK and induction of Il6 and Tnfa are downstream events triggered by TLR pathway activation (19), the data presented herein provide strong evidence that CD14, the TLRs 4 and 8, and/or C1q are functional in both cumulus cells and granulosa cells. Furthermore, the regulation and function of the TLR pathway in these cells appears to be closely linked to the actions of FSH and LH.
Cumulus Cells and Granulosa Cells Exhibit Phagocytic Activity
Scavenger receptors regulate cholesterol and lipid transport in endocrine cells. Recent evidence indicates that these receptors can also mediate/facilitate uptake (phagocytosis) of bacterial particles (Figs. 1A and 5A) (21, 22, 23). Therefore, we analyzed the phagocytic capabilities of cumulus cells and granulosa cells, by examining their ability to ingest fluorescent-tagged bacterial particles. COCs, isolated 6 h after hCG, were incubated with 5 µg/ml bacterial particles at 37C for 2 h. Intense fluorescence was observed in these intact COCs (Fig. 5A). However, because the resolution was insufficient to determine whether the particles were ingested, additional COCs were incubated with bacteria for 2 h at 37 C in medium alone or in the presence of 16 µM cytochalasin B (to disrupt microtubule function) or at 4C (control) (Fig. 5B, upper, middle, and lower panels, respectively). After incubation, the cumulus cells were dissociated from the complex, spun onto coverslips, fixed, and stained with propidium iodide (red) to reveal intracellular nuclei acids (Fig. 5B). Many cumulus cells were fluorescently tagged when incubated at 37 C (43%; 274 of 631 counted) whereas very few cells exhibited bacterial ingestion at 4 C (10%; 46 of 459 counted). Likewise, cells incubated with cytochalasin B had reduced numbers of fluorescent bacteria (26%; 110 of 424 counted) (Fig. 5A; quantitation of panels in B). When examined by confocal microscopy, apparent binding/uptake (diffuse staining) was observed in cells incubated at 37 C. This pattern was blocked, and only bright green particles were observed in cells incubated with cytochalasin B or at 4 C (Fig. 5B, upper, middle, and lower panels on the right side).
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, upper panels). At 4 h only a limited number of bacterial particles were attached to the granulosa cell surface, and none were observed within the cells. By contrast, at 24 h, bacteria were attached not only to the surface of all cells but were also present within the cells as noted by the diffuse green staining. Additional confocal imaging of single cells (Fig. 5C, bottom; panels a–d) localized fluorescent bacterial particles to the surface of the cells (bright green) and colocalized many to SCARB1 (yellow circles) (see Fig. 5C legend for details). Note that the immunostaining of SCARB1 not associated with bacterial particles is also punctate. These experiments have been repeated three times with similar staining and localization patterns.
To document whether or not any bacterial particles were inside the granulosa cells and associated with lysosomes (23), additional PO granulosa cells were cultured with or without bacteria for 24 h. At that time fresh media containing the lysomosal marker Lyso Tracker DND-99 (100 nM; red) was added to all cells for 1 h. The cells exposed to bacteria were then washed, fixed, stained with TOPO3 (DNA), and examined by confocal microscopy. Control cells (no bacteria) were processed in the same manner or were also stained with SCARB1 (green). As shown in Fig. 6A, lysosomes (discrete red dots) were clearly evident in granulosa cells not exposed to bacteria. Moreover, the lysosomes were not associated with regions of the cell positive for SCARB1 (Fig. 6B). However, in cells exposed to bacteria, many lysosomes appeared as yellow/orange organelles indicating that small bacterial particles had been ingested and were present in the lysosomes (Fig. 6C). Bacterial particles not ingested were present on or near the cell surface (brilliant green). Lysosomes not containing bacteria were red as in panels A and B. These experiments have been repeated three times, and essentially all cells give the same patterns of staining for lysosomes, SCARB1, and bacteria.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The selective induction of surveillance receptors (C1q, CD14, TLR4, TLR8, and TLR9) in COCs in response to LH in vivo as well as to either FSH or AREG in culture indicates that cumulus cells possess mechanisms by which they can respond to a variety of external cues (18, 20). Indeed, we document that LPS not only stimulated phosphorylation of p38MAPK and NF-B but also induced expression of innate immune-related genes, namely Il6 and Tnfa, in ovulated COCs (19, 29, 33) (Figs. 1–4). These observations combined with the coincident temporal induction of Tlr4, Myd88, Il6, and Irf3 mRNAs in COCs 8–12 h post-hCG in vivo provide additional novel evidence that this pathway is highly regulated and operative during ovulation. Activation of TLRs (most likely TLR4) at this time may be important for regulating ovarian cell survival and the associated inflammatory-like events (Fig. 1) (19, 20, 28 ). Our results indicate further that the TLR pathway is present and functional in COCs and granulosa cells isolated from PO follicles (i.e. before the onset of ovulation-related events). Specifically, LPS not only induced Il6 but also Tnfa and Ptgs2 mRNA expression in the PO COCs (before hCG). Moreover, not only LPS but also HA increased Il6 mRNA in PO granulosa cells, indicating that two distinct activators of TLR4 can induce expression of this cytokine. That FSH itself potently stimulated Il6 and Ptgs2, but not Tnfa expression, in PO granulosa cells, suggests that the effects of FSH may be mediated by its rapid induction of Areg in these cells (31). AREG is a potent inducer of Ptgs2 in granulosa cells and cumulus cells (31) and can also increase expression of TLR pathway components (Fig. 3). Thus, FSH may impact TLR signaling by indirect mechanisms that involve the actions of AREG and prostaglandins. Collectively, these pathways may provide granulosa cells with mechanisms that promote cell survival before and during the tissue repair process that occurs during ovulation.
That COCs occupy a unique niche within the follicle and undergo a fate distinct from granulosa cells is reflected by the selective regulation of specific factors in COCs compared with granulosa cells (Fig. 1B) (10). These include components of the TLR pathway, CD14, IRF3, and Cq1. GPI-anchored CD14 is an LPS coreceptor, whereas IRF3 mediates Tnfa gene activation (29). The COC-related expression of these genes mimics that of other innate immune-regulatory factors Pdcd1 and Cd52, which are induced in COCs but not granulosa cells of ovulating follicles (10, 11). The complement factor C1q, which is critical for monitoring a variety of external factors, is expressed abundantly at sites of tissue damage and plays an initiating role in clearance of apoptotic cells (18). The coordinate induction of C1qa in a pattern similar to that of Cd14 and the Tlrs suggests that this pathway may also play a specific role other than, or in addition to, its ability to recognize IgG complexes. In fact, recent studies indicate that C1q recognizes myelin basic protein and serum amyloid protein [both of which are expressed in cumulus cells (10, 18, 34)] and has been shown to bind DNA, LPS, and some bacterial membranes (18). Curiously, mice null for C1q, like mice null for Pdcd1 and Runx1, two other genes induced in COCs (10), develop autoimmune responses with lupus-like disease characteristics (35). However, to date, no overt fertility defects have been reported in these mutant mouse models, perhaps because of redundant and/or overlapping functions of TLR family members.
Although in macrophages and other cells, activation of the TLR pathway is essential for induction of scavenger receptors (20, 24), this does not seem to be the major role of the TLRs in the ovary. SCARBI and CD36 are present and distributed in a punctate manner on the surface of essentially all cumulus cells and granulosa cells before full induction of the TLR pathway and its components (Figs. 2 and 5). However, activation of the TLR pathway may facilitate the functions of the SCARB pathway. Because the process of ovulation releases COCs into the external environment of the oviduct, the presence of CD36 and SCARBI in cumulus cells may provide a mechanism by which the cumulus cells can respond rapidly to any "toxic" or "foreign" material should this type of surveillance response be required. For example, one morphological study showed a cumulus cell within the oviduct phagocytosing a sperm (36), providing a possible role (in addition to regulation of cholesterol transport) for the scavenger receptors on these cells. That cumulus cells (and granulosa cells) have phagocytic activity was documented for the first time herein by their ability to ingest bacterial particles, a process sensitive to temperature (occurring at 37 C but not 4 C) and cytochalasin B. These data support a recent study showing that CD36, SCARBI, and SCARBII have phagocytic capabilities when expressed exogenously in a nonphagocytic cell type and that this process is sensitive to temperature (23, 24). Moreover, testicular Sertoli cells (analogous to granulosa cells) that phagocytose degenerating/apoptotic sperm (37, 38) express functional CD36 and SCARBI providing additional evidence for this kind of activity in gonadal cells (39, 40, 41). Mice null for Scarb1 are infertile and ovulate dysfunctional oocytes, apparently a consequence of abnormal lipoprotein metabolism (42).
In summary, the expression of innate immune cell-like genes in cumulus cells of ovulating COCs, as well as in granulosa cells, provides strong evidence that ovulation not only involves the generation of an extracellular HA-rich matrix but also the acquisition of specific surveillance functions used by immune cells (Fig. 1A). Furthermore, the matrix via HA (and its fragments) may serve to regulate TLR signaling in ovulating COCs and granulosa cells. Toll receptor signaling could therefore mediate cell survival during the wound-healing process that occurs at the site of follicle rupture during ovulation. Additionally, TLRs and scavenger receptors provide mechanisms by which cumulus cells recognize and remove altered self, including apoptotic cells. Thus, cumulus cells and granulosa cells posses and acquire novel, heretofore unexpected, and potentially critical surveillance role(s) during the ovulation process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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150 kDa) were obtained from Hyalose (Oklahoma City, OK). Oligonucleotide poly-(dT) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ); AMV reverse transcriptase and Taq polymerase were from Promega Corp. (Madison, WI); [P32]dCTP was from ICN (Los Angeles, CA); oligonucleotide primers for RT-PCR reactions were from Sigma-Genosys (Houston, TX); routine chemicals were from Fisher Scientific (Pittsburgh, PA) or Sigma (St. Louis, MO). Antibodies used for WB, IF, and uptake studies are as indicated: SCARB1 (SR-B1; Novus Biologicals, Littleton, CO): WB, 1:2000; IF, 1:500; Uptake, 1:500. TLR4 (Cell Signaling Technology, Beverly, MA): WB, 1:1000; IF, 1:100; Uptake, 1:100. MYD88 (eBiosciences, San Diego, CA): WB, 1:1000; IF, 1:100; Uptake, 1:100. IRF3 (Cell Signaling Technology): WB, 1:1000; IF, 1:100; Uptake, 1:100. Phospho-p38MAPK: WB, 1:2000. Phospho-ERK1/2: WB, 1:2000. Phospho-NFB: WB, 1:1000. NF-kB: WB, 1:1000 (Cell Signaling Technology). Secondary antibodies tagged with Alexa Fluor 594 (red) and Alexa Fluor 488 (green) as well as Lyso Tracker (red) DND-99 were purchased from Invitrogen (Carlsbad, CA).
Animals
Immature female C57BL/6 mice were obtained from Harlan, Inc. (Indianapolis, IN). On d 23 of age, female mice were injected ip with 4 IU of eCG (Pregnyl; Organon, West Orange, NJ) to stimulate follicular growth, followed 48 h later with 5 IU hCG (Gestyl; Diosynth, Oss, The Netherlands) to stimulate ovulation (43). Animals were housed under a 16-h light/8-h dark schedule in the Center for Comparative Medicine at Baylor College of Medicine and provided food and water ad libitum. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Baylor College of Medicine.
Isolation of COCs and Granulosa Cells
Ovaries of immature mice primed with eCG for 48 h contain multiple PO follicles. COC cells were isolated from these follicles by needle puncture, collected by pipette, pooled, frozen, and stored at –80 C for RNA or protein extraction (10, 13). Granulosa cells that were released by needle puncture of the follicles were also pooled, collected by centrifugation, and frozen at –80 C for protein and RNA analyses. COCs and granulosa cells were also isolated from ovaries of eCG-primed mice after hCG treatment for 4, 8, and 12 h. During this time, COCs expand but have not yet ovulated. Ovulated (fully expanded) COCs were collected by needle puncture of the oviducts of mice 16 h or 24 h after hCG. Collection of each pool of COCs and granulosa cells (15 mice) at each time point was repeated two times (i.e. two separate experiments) (10). For culture, nonexpanded COCs (20–30) and granulosa cells were collected from the ovaries of eCG-treated mice, plated in separate wells of a Falcon 24-well plate (Becton Dickinson, Franklin Lakes, NJ) in 0.5 ml of defined medium (13) containing 1% fetal bovine seum without or with either FSH (100 ng/ml), Amphiregulin (AREG; 250 ng/ml) (13), LPS (100 ng/ml), or HA (100 µg/ml). After specific time intervals, as designated in the figure legends, total RNA or protein was extracted from the COCs. In other experiments, ovulated COCs were collected from the oviduct after 16 or 24 h post-hCG injection. Each experiment was repeated two times.
RT-PCR Analyses
Total RNA was obtained from COCs and granulosa cells using the RNAeasy Mini kit (QIAGEN Sciences, Germantown, MD) according to the manufacturer’s instructions, and semiquantitative RT-PCR analyses were performed as described previously (44, 45). Briefly, total RNA was reverse transcribed using 500 ng poly-dT and 0.25 U avian myeloblastosis virus-reverse transcriptase at 42 C for 75 min and 95 C for 5 min. For the amplification of the cDNA products, specific primer pairs were selected as indicated in Table 1. All PCRs were done in the linear range of amplification and contained [32P]dCTP, Taq Polymerase, and Thermocycle buffer. cDNA products were resolved on 5% polyacrylamide gels that were dried and exposed to x-ray film. The radioactive PCR product bands were quantified using a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The authenticity of the PCR products was verified by subcloning and sequencing. Total RNA was obtained from COCs and granulosa cells using the RNAeasy Mini kit (QIAGEN Sciences) according to the manufacturer’s instructions.
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In Vitro Samples.
Nonexpanded COCs and granulosa cells were isolated from PO follicles of eCG-primed mice and cultured in defined medium plated in separate wells of a Falcon 24-well plate in 0.5 ml of defined medium (13) without or with LPS (100 ng/ml) for 0, 60, and 120 min. The COC and granulosa cell samples were collected by centrifugation and extracted in SDS boiling buffer as described previously (10). WCE (3 µg protein) and SDS extracts (30 µl) were resolved by SDS-PAGE (10%) and transferred to Immobilon-P nylon membranes (Millipore Corp., Bedford, MA). Membranes were blocked in Tris-buffered saline and Tween 20 [TBST; 10 mM Tris (pH 7.5), 150 mM NaCl and 0.05% Tween 20] containing 5% nonfat Carnation instant milk (Nestle Co., Solon, OH). Blots were incubated with selected primary antibodies overnight at 4 C. After washing in TBST, Enhanced chemiluminescence (ECL) detection was performed using Pierce Super Signal according to the manufacturer’s specifications (Pierce) and appropriate exposure of the blots to Kodak x-ray film. Specific bands were quantified by densitometric analyses using a Molecular Dynamics Personal Densitometer.
IF
COCs were immobilized on polylysine-coated coverslips, fixed with 4% paraformaldehyde, washed with PBS, and used directly or stored in 1% paraformaldehyde. COCs were permeabilized with 0.5% Nonidet P-40 (NP-40), washed, blocked with 5% BSA or Vector M.O.M. immunodetection reagents (Vector Laboratories, Burlingame, CA), and incubated overnight at 4 C with selected primary antibodies as previously (10). Antibody localization was visualized with fluorescein isothiocyanate-labeled antirabbit IgG and Streptavidine AlexaFluor 568 (Molecular Probes). Nuclei were visualized by 4',6-diamidino-2-phenylindole present in the VECTASHIELD D mounting medium (Vector Laboratories), propidium iodide, or PR-TOPO-3 (Molecular Probes). Digital images were captured using an Axiphot microscope with x10–40 objectives or a Zeiss Laser Scanning Confocal Microscope (LSM 510; Carl Zeiss, Thornwood, NY).
Phagocytosis Studies
COCs were isolated from ovaries of eCG-primed mice 6 h after hCG, a time selected based on the induction of TLRs, CD14, and MYD88 between 4 and 8 h. COCs were cultured for 4 h in a 12-well culture dish (1 ml defined medium containing 5% serum) with 5 µg/ml of sonicated bacterial (Escherichia coli strain K12) particles in the Vybrant Phagocytosis Assay Kit (Invitrogen) (23). The COCs and bacteria were cultured either at 37 C in medium alone, at 37 C in the presence of cytochalasin B (16 µM to inhibit cytoskeletal uptake mechanisms), or at 4 C (control). After 2 h culture, bacterial particles, COCs were washed twice (using a glass pipette) with medium containing 5% serum and then treated with hyalurondase for 1 min. After a 30-sec vortex (medium speed), the dispersed cells were collected by centrifugation at 10,000 rpm for 1 min. The supernatant was removed, 100 µl of trypan blue quenching solution was added, and the cells were collected again by centrifugation. The cells were washed in serum-free medium twice and then fixed in 2% paraformaldehye for 30 min. Cells were then spun onto (3-aminopropyl) triethoxysilane (Sigma A3648)-coated slides. The immobilized cells were washed in PBS, permeabilized in 0.5% NP-40, and then immunostained as described above using specific antibodies and DNA staining reagents. Granulosa cells obtained from ovaries of eCG-primed mice were plated on serum-coated coverslips in 12-well dishes and cultured in serum-free defined medium at 37 C for 24 h with bacterial particles (5 µg/ml) without or with FSH and additional 1 h in the presence of the lysosomal marker (Lyso Tracker; 100 nM). Granulosa cells were also cultured 24 h on coverslips and then incubated for 4 h with bacterial particles at 37 C. Cells were then washed, fixed in 2% paraformaldehyde 30 min, washed in PBS, permeabilized with 0.5% NP-40, and immunostained as described above and in the figure legends.
Statistics
The semiquantitative RT-PCR data are represented as mean ± SD. Data were analyzed by using GraphPad Prism Programs (ANOVA or t test and Neuman-Keuls Multiple Comparison Tests; GraphPad Prism, San Diego, CA) to determine significance. Values were considered significantly different if *P < 0.05 or **P < 0.01 (Figs. 1, 3, and 4).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online August 24, 2006
Abbreviations: AREG, Amphiregulin; CG, chorionic gonadotropin; COC, cumulus oocyte complex; Cq1, complement factor q1; EGF, epidermal growth factor; HA, hyaluronan; IF, immunofluorescence; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; PRR, pattern recognition receptor; MYD88, myeloid differentiation primary response gene 88; NF-B, nuclear factor-B; NP-40, Nonidet P-40; PO, preovulatory; PTGS2, prostaglandin synthase 2; SCARBI/ SCARBII, scavenger receptor type B1 and BII; TLR, Toll-like receptor; WB, Western blot; WCE, whole-cell extract.
Received for publication May 8, 2006. Accepted for publication August 18, 2006.
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REFERENCES |
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B as key regulators. Immunity 23:319–329[CrossRef][Medline] induces serum amyloid A3 in mouse granulosa cells. Endocrinology 145:2245–2252This article has been cited by other articles:
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R. G Lea and O. Sandra Immunoendocrine aspects of endometrial function and implantation Reproduction, September 1, 2007; 134(3): 389 - 404. [Abstract] [Full Text] [PDF]
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 D. C. Woods, M. J. Haugen, and A.L. Johnson Actions of Epidermal Growth Factor Receptor/Mitogen-Activated Protein Kinase and Protein Kinase C Signaling in Granulosa Cells from Gallus gallus Are Dependent upon Stage of Differentiation Biol Reprod, July 1, 2007; 77(1): 61 - 70. [Abstract] [Full Text] [PDF]
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