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BOR - Papers in Press, published online ahead of print November 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.045641
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BIOLOGY OF REPRODUCTION 74, 501–510 (2006)
DOI: 10.1095/biolreprod.105.045641
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Expression and Glycosylation with Polylactosamine of CD44 Antigen on Macrophages During Follicular Atresia in Pig Ovaries1

Yuko Miyake 2 3, Hiromichi Matsumoto 3, Masaki Yokoo 3, Kohtaro Miyazawa 4, Naoko Kimura 3, Woro Anindito Sri Tunjung 3, Takashi Shimizu 3, Hiroshi Sasada 3, Hisashi Aso 4, Takahiro Yamaguchi 4, and Eimei Sato 3

Laboratories of Animal Reproduction3 and Functional Morphology,4 Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan

ABSTRACT

Macrophages are essential in cleaning up apoptotic debris during follicular atresia. However, the key factors of this process are still unclear. In the present study, we evaluated CD44 mRNA, CD44 protein, and CD44 antigen glycosylation on macrophages during follicular atresia in the pig. Atresia was classified into five stages: stage I, healthy follicles; stage II, early atretic follicles having apoptotic granulosa cells with an unclear basement membrane; stage III, progressing atretic follicles having apoptotic granulosa cells completely diffused from the basement membrane; stage IV, late atretic follicles with increasing lysosomal activity; and stage V, disintegrated atretic follicles having collapsed theca cells and strong lysosomal activity. Immunohistological analysis showed that macrophages expressing CD44 invaded the inside of stage III follicles, accompanied by a collapse of basement membrane. Semiquantitative RT-PCR showed that only mRNA of the CD44 standard isoform (CD44s) was present in inner cells of follicles, and not any CD44 variant isoform (CD44v) mRNAs. The amount of CD44s mRNA was increased at stage III. Western blot and lectin blot analyses showed that CD44 was markedly expressed at stage III and glycosylated with polylactosamine at the same time. After macrophages invaded atretic follicles at stages III–V, the CD44 expressed on macrophages was glycosylated with polylactosamine. The lysosomal activity began to increase at stage IV, and reached the highest level at stage V. Increased CD44s protein and posttranslational modification of CD44 with polylactosamine on macrophages from stage III could be involved in the cleaning up apoptotic granulosa cells.

apoptosis, granulosa cells, ovary

INTRODUCTION

In mammals, only a select few follicles—less than 1%—complete growth and development to ovulation; the majority (>99%) undergo a degenerative process known as atresia at some stage in their development [1, 2]. In reproductive physiology, follicular atresia is a key phenomenon by which the ovary eliminates those follicles that will not ovulate, and is apparently initiated by the apoptotic death of granulosa cells followed by degeneration of the oocyte at the last stages of atresia [3, 4]. Although studies have been made on the process of granulosa cell elimination in atretic follicles, the mechanism responsible for this phenomenon is still controversial. Macrophages can execute diverse functional activities, including phagocytosis and degradation of foreign antigens, matrix dissolution and tissue remodeling, and production and secretion of cytokines, chemokines, and growth factors [5]. In follicular atresia, it has been reported that macrophages are involved in cleaning up apoptotic debris (i.e., pyknotic granulosa cells and degenerated oocytes) in mouse, rat, guinea pig, rabbit, and cow [5–14]. In pig, the presence of macrophages containing phagocytosed cells in cytoplasm has also been reported in atretic follicles [15].

CD44 is a broadly distributed transmembrane glycoprotein that plays a critical role in a variety of cellular behaviors, including adhesion, migration, invasion, and survival [16–18]. Differential posttranslational modifications, including glycosylation and the attachment of glycosaminoglycans, generate additional structural diversity. Furthermore, CD44 mediates cell-cell and cell-matrix interactions, in large part through its affinity for hyaluronan (HA), a glycosaminoglycan of extracellular matrices (ECMs), but also potentially through its affinity for other ligands such as osteopontin, collagens, and matrix metalloproteinases (MMPs) [18]. The cell surface glycoprotein, CD44, is expressed in many cell types, including leukocytes, erythrocytes, fibroblasts, endothelial and epithelial cells, and a variety of tumor cells [19]. Recently, it has been reported that CD44 is present on human macrophages [20], and the ligation of macrophage surface CD44 by bivalent monoclonal antibodies rapidly and profoundly augments the capacity of macrophages to phagocytose apoptotic neutrophils in vitro [21]. In addition, CD44 has a critical role in resolving lung inflammation in mice [22]. However, it is still unknown whether CD44 on macrophages can enhance the macrophage-mediated clearance of apoptotic cells in the ovary.

Although macrophages are essential during follicular atresia, it is not clear whether key factors are implicated in macrophage migration and phagocytosis. In the present study, we examined the levels of CD44 mRNA, CD44 protein, and glycosylation of CD44 in porcine follicles during atresia. The CD44 mRNA was markedly upregulated in invading macrophages, and CD44 was glycosylated with polylactosamine during the phagocytic processing of apoptotic granulosa cells.

MATERIALS AND METHODS

Immunohistochemical Localization of CD44 and Macrophages

Porcine ovaries from prepubertal gilts were snap-frozen using Freez-It (–51°C, ITW Chemtronics) at the slaughterhouse. Immunolocalization was performed as previously described with slight modifications [23–26]. Frozen sections (12 µm thick) were mounted on Matsunami adhesive silane-coated glass slides (Matsunami), and fixed in cold acetone for 10 min before being washed in Ca2+ and Mg2+-free Dulbecco PBS (DPBS, Nissui Pharmaceutical Co.) for 10 min twice. For the blocking of nonspecific protein binding, sections were incubated with nonimmune sera derived from goat or rabbit.

For experiments on the colocalization of CD44 and macrophages, rat anti-mouse CD44 monoclonal antibody (IM7, BD Pharmingen) at a dilution of 1:3.125 and mouse anti-pig SWC3a (CD172a) antigen monoclonal antibody (Research Diagnostics), as swine macrophage marker, at a dilution of 1:50 were added for 1 h at room temperature [15, 27]. Before the start of the experiments, we confirmed that the rat anti-mouse CD44 monoclonal antibody (IM7) reacts with pig CD44, as guaranteed by BD Pharmingen. We also confirmed the specificity of anti-porcine CD172a antibody. Previously, we have reported that CD172a antibody did not react with CD8 positive cells in bovine dendritic cells [28]. Using the same methods, we confirmed that anti-porcine CD172a antibody had no cross-reaction with CD8 positive cells (Supplemental Figure 1; available online at http://www.biolreprod.org). After several washes with DPBS, the sections were incubated with rabbit anti-rat IgG conjugated with FITC (Sigma) at a dilution of 1:300 for 1 h at room temperature, and goat anti-mouse IgG conjugated with biotin (Zymed Laboratories) at a dilution of 1:500 for 30 min. After several washes with DPBS, the sections were incubated with streptavidin conjugated with Cy5 (Zymed Laboratories) at a dilution of 1:20 with DPBS containing 10% goat normal serum for 1 h at room temperature. For the staining of macrophages in sections of follicles, streptavidin conjugated with FITC (Vector Laboratories) at a dilution of 1:50 with DPBS was used instead of Cy5. After several washes with DPBS, nuclei were stained with propidium iodide (10 µg/ml; Sigma). The sections were mounted with fluoromount-G (Southern Biotechnology Associates Inc.). They were then viewed in a confocal scanning laser microscope (MRC-1024; Bio-Rad). Cy5 originally generates a red fluorescent signal (670 nm), which is represented in blue in the images shown here. This allows for distinction between Cy5 and the red signal of propidium iodide (615 nm) [29].


Figure 1
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  		FIG. 1. Dynamic changing of apoptosis and lysosomal activity during atresia. The serial sections are stained for TUNEL (A, brown) and acid phosphatase activity (B, dark blue). Atretic stages I–V are classified as described in Table 1. g, Granulosa cells; b, basement membrane; t, theca cells; o, oocyte. Bar = 100 µm

Lectin Staining of Polylactosamine

For colocalization experiments with polylactosamine, the sections were incubated with FITC conjugated Datura stramonium agglutinin (DSA; Seikagaku) at a dilution of 1:200 for 1 h at room temperature. DSA is guaranteed by the manufacturer to react with polylactosamine (poly-N-acetyllactosamine, (Galß1->4GlcNAcß1->3)n-type carbohydrate chains) [30]. Mouse anti-pig CD44 monoclonal antibody (PORC24A; VMRD, Inc.) was used instead of IM7, at a dilution of 1:50. After several washes with DPBS, the sections were incubated with goat anti-mouse IgG conjugated with biotin (Zymed Laboratories) at a dilution of 1:500 for 30 min at room temperature. After several washes with DPBS, the sections were incubated with streptavidin conjugated with Cy5 (Zymed Laboratories) at a dilution of 1:20 with DPBS containing 10% goat normal serum for 1 h at room temperature.

In Situ Hybridization

In situ hybridization was performed as previously described [31]. Sense and antisense 35S-labeled cRNA probes were generated for in situ hybridization using appropriate polymerases from porcine CD44 partial cDNA [23]. Frozen sections (12 µm thick) were mounted onto poly-L-lysine-coated slides and fixed with a 4% paraformaldehyde solution in PBS at 4°C. After prehybridization, sections were hybridized at 45°C for 4 h in 50% formamide buffer containing 35S-labeled sense or antisense cRNA probes. After hybridization, sections were incubated with RNase A (20 µg/ml) at 37°C for 20 min, and RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion. Sections were poststained with hematoxylin-eosin. Sections hybridized with sense probes served as a negative control.

TUNEL Staining and Staining of Acid Phosphatase Activity

To visualize apoptotic cells, adjacent sections from the specimens used for immunohistochemical localization were stained by the TUNEL method with an ApopTag kit (Intergen). The following positive and negative controls were included in each experimental run. As the negative control, sections were incubated by omitting terminal deoxynucleotidyl transferase. As the positive control, sections were treated with DNase I (1000~10 000 U/ml, Sigma), 4 mM MgCl2, and 0.1 mM dithiothreitol in 30 mM Tris-HCl (pH 7.2) for 10 min at room temperature.

To visualize lysosomes, the staining of acid phosphatase activity was demonstrated on sections by the azo dye method using 0.5 M acetate buffer (pH 3.5), naphthol AS-MX phosphate (Sigma) as a substrate, and hexazotized Fast blue RR salt (Sigma) as diazonium salt [6, 13, 32, 33]. The sections were counterstained with methyl green, and mounted with 10% glycerol.

Separation of Inner Cells from Antral Follicles

Porcine ovaries from prepubertal gilts were transported to the laboratory from a slaughterhouse in a container kept at 37°C within 1 h. Individual antral follicles 2–6 mm in diameter were dissected from ovaries and classified as morphologically healthy or atretic. Healthy follicles were classified as round in shape with a clearly identifiable cumulus-oocyte complex (COC) (see Fig. 4A-I), early atretic follicles as having granulosa cells partly detached from the basement membrane (see Fig. 4A-II), and progressing atretic follicles as having granulosa cells suspended in the antrum (see Fig. 4A-III) [34]. For identification of the atretic stages in ovarian sections, each dissected follicle was embedded in CMC compound (FINETEC), and then rapidly frozen for immunodetection of macrophages and TUNEL staining, which were performed as described above. Each follicle was punctured in DPBS, the theca layer removed, and the inner cells washed three times with DPBS. The degree of apoptosis was examined by Hoechst staining and detection of a DNA ladder. Inner cells of follicles were fixed with 1% glutaraldehyde for 24 h at room temperature. Fixed cells were centrifuged at 150 x g for 30 min, and the supernatant was discarded. Inner cells of follicles were resuspended with DPBS and stained for 1 min at room temperature by adding Hoechst 33342 (5 µg/ml, Sigma). Granulosa cells containing fragments of condensed chromatin or cytoplasmic fragments containing condensed chromatin were considered apoptotic (pyknotic) [35]. An apoptosis Ladder Detection Kit (Wako Pure Chemical Industries Ltd.) was used for the detection of DNA fragmentation.


Figure 4
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  		FIG. 4. The advance of apoptosis in inner cells collected from dissected porcine follicles (stages I–III). See Separation of Inner Cells from Antral Follicles in Materials and Methods. A) Dissected follicles from ovaries. Arrowhead, COC. The box (examination at high magnification) shows granulosa cells detached from the basement membrane. Bar = 0.5 mm. B) Invasion of macrophages into follicles (a, green) and apoptotic cells (b, brown). Arrows, Invaded macrophages; g, granulosa cells; t, theca cells. Bar = 50 µm. C) Pyknosis of inner cells. Different superscripts denote significant differences (P < 0.05). Each bar indicates the mean ± SEM. D) DNA fragmentation of inner cells. M, 100-base pair DNA ladder marker

Semiquantitative RT-PCR Analysis of CD44

Semiquantitative RT-PCR was performed as previously described with slight modifications [23]. Total RNA from the inner cells of follicles was isolated with the RNeasy Mini kit (Qiagen K.K.). Total RNA was treated with DNase I (Qiagen) on the spin column at room temperature for 15 min, and reverse-transcribed into cDNA using SUPERSCRIPT II Reverse Transcriptase (Invitrogen). RNAguard (Amersham Biosciences) was used for protection from RNase. Semiquantitative PCR was carried using a GeneAmp PCR System 9700 (PE Applied Biosystems). Primer pairs specific for partial cDNA sequences of CD44 were used: forward 5'-GTA CAT CAG TCA CAG ACC TAC-3' and reverse 5'-CAC CAT TTC CTT GAG ACT TGC T-3' [23]. These primers can detect variant exons 1–10. The expected size of the PCR product CD44s, the smallest isoform of CD44, was 603 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an intrinsic control, using as primers forward 5'-GAT GGT GAA GGT CGG AGT G-3' and reverse 5'-CCA AGT TGT CAT GGA TGA CC-3' [34]. The expected size of the PCR product was 500 bp. Each reaction mixture contained cDNA solution, 0.2 µM of each primer, 0.2 mM dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 10% Triton-X, and 1 U of rTaq polymerase (Promega). The PCR profile was as follows: 34 cycles (CD44) or 30 cycles (GAPDH) of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The final cycle included a further 7 min at 72°C for complete strand extension. All RT-PCRs were carried out at least three times for each individual RNA sample. The RT-PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. The bands were quantified by densitometry using NIH Image 1.62 (National Institutes of Health). The products were identified by DNA sequencing with the dye terminator method, and each sequence was identical with our previous data [23].

Western Blot Analysis of CD44

Protein extraction and Western blotting were performed as previously described [24]. The extracted proteins were treated with or without sialidase (neuraminidase, Sigma) overnight at 37°C, then separated by 8% SDS-PAGE under nonreducing conditions. After electroblotting, the membranes were blocked with 5% skim milk for 1 h at room temperature and washed with DPBS containing 0.5% Tween 20 (PBS-T) several times. The membranes were incubated with mouse anti-pig CD44 monoclonal antibody (PORC24A) overnight at 4°C and then reacted with horseradish peroxidase-labeled anti-mouse IgG (Sigma). After several washes with PBS-T, the peroxidase activity was visualized using the ECL Plus Western blotting detection system (Amersham Biosciences).

Lectin Blot Analysis of Polylactosamine

Immunoprepicipitation was performed as previously described [24]. The protein extract (200 µg) was added to anti-CD44 antibody (PORC24A)-precoupled protein G beads (Amersham Biosciences) and incubated for 24 h at 4°C. After electroblotting, the membranes were blocked with 1% bovine serum albumin (Sigma) in PBS-T for 1 h at room temperature and washed three times with PBS-T. The membranes were incubated with biotinylated DSA (Seikagaku) overnight at 4°C and then reacted with horseradish peroxidase conjugated with streptavidin (Amersham Biosciences) for 1 h at room temperature. After three washes with PBS-T, the peroxidase activity was visualized using the ECL Western blotting detection system (Amersham Biosciences).

Statistical Analysis

All experiments were repeated at least three times. The densitometry ratios in expression of CD44 and GAPDH mRNAs between healthy, early atretic, and progressing atretic follicles were analyzed with a one-way factorial ANOVA, followed by Fisher's protected least significant differences test. P values less than 0.05 were considered significant.

RESULTS

Classification of Atresia Stages by Apoptosis and Lysosomal Activity

The degree of atresia was classified into stages II to V based on the morphological status of theca cells, granulosa cells, basement membrane, TUNEL (apoptosis), and acid phosphatase activity (lysosomal activity) (Table 1). Stage I is a healthy follicle without apoptotic granulosa cells, with a clear basement membrane (Fig. 1A-I). Stage II is an early atretic follicle having apoptotic granulosa cells with an unclear basement membrane (Fig. 1A-II). Stage III is a progressing atretic follicle having completely diffused apoptotic granulosa cells from the basement membrane (Fig. 1A-III). Stage IV is a late atretic follicle, the lysosomal activity of which has begun to increase (Fig. 1, A-IV and B-IV). Stage V is a disintegrated atretic follicle having collapsed theca cells and strong lysosomal activity in follicle cells and a decreased number of apoptotic granulosa cells (Fig. 1, A-V and B-V).


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  		TABLE 1. Classification of stages of atresia in atretic porcine follicles

Distribution of Macrophages and CD44 During Atresia

The result of lysosomal activity in Fig. 1 indicated phagocytosis by macrophages during atresia. Therefore, the distribution of macrophages was investigated (Fig. 2). Macrophages were not detected inside the basement membrane at stages I and II, but existed outside the basement membrane (Fig. 2, A-I and A-II). Macrophages invaded follicles at stage III (Fig. 2A-III). The number of apoptotic granulosa cells was decreased after the invasion by macrophages between stages IV and V (Fig. 1, A-IV and A-V). CD44 was detected in macrophages at all stages (Fig. 2B). Very low to undetectable levels of CD44 were observed in healthy follicles (Fig. 2B-I), whereas strong CD44 immunoreactivity was detected in late atretic follicles (Fig. 2B-IV). CD44 was also detected in completely disintegrated atretic follicles (Fig. 2B-V). Similar to the localization of CD44, very low to undetectable levels of CD44 mRNA were observed in healthy, early atretic, and progressing atretic follicles by in situ hybridization (Fig. 3, I, II, and III), whereas CD44 mRNA was detected inside of complete atretic follicles strongly (Fig. 3, IV and V).


Figure 2
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  		FIG. 2. Distribution of macrophages and CD44 in porcine healthy (stage I) and atretic (stages II–V) follicles. Macrophages (blue), CD44 (green), and a merged image (blue and green) are shown in A, B, and C, respectively. D) Merged C with nuclei (red). These are the same serial sections shown in Fig. 1. g, Granulosa cells; t, theca cells; o, oocyte. Bar = 100 µm


Figure 3
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  		FIG. 3. Distribution of CD44 mRNA in porcine healthy (stage I) and atretic (stages II–V) follicles using in situ hybridization. CD44 mRNA was detected as yellow dot signals using dark field microscopy. Bar = 100 µm

Expression of CD44 mRNA in Inner Cells Collected from Dissected Antral Follicles

Because the invasion by macrophages was observed at stage III, stages I-III follicles were separated from ovaries, and the inner cells of each follicle were analyzed. Individual antral follicles were dissected from ovaries and classified as morphologically healthy or atretic, stages I–III (Fig. 4A). Macrophages have invaded at stage III (Fig. 4Ba-III), and the advance of atresia was indicated by TUNEL (Fig. 4Bb). Pyknosis was detected in 3.7% of cells at stage I, 80.8% at stage II, and 99.7% at stage III (Fig. 4C). Significant differences were observed among each stage. Pyknosis increased suddenly between stages I and II. DNA fragments were detected at stages II and III (Fig. 4D). These results confirmed that apoptosis occurred at stages II and III.

Next, we examined the expression of CD44 mRNA at stages I–III by semiquantitative RT-PCR (Fig. 5). CD44 is encoded by 20 exons, and by alternative splicing up to 10 variant exons can be inserted at a single site within the membrane-proximal portion of the extracellular region [17, 18]. As shown in Fig. 5, our result indicated that a single band of CD44s mRNA was detected at stages I–III. Specifically, no expression of any CD44v mRNAs was detected. The expression level of CD44s was increased suddenly between stages II and III. Accompanying the progress of follicular atresia, the expression of CD44s increased significantly between each stage (P < 0.05).


Figure 5
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  		FIG. 5. The expression of CD44 mRNA in inner cells collected from dissected porcine follicles (stages I–III) examined by semiquantitative RT-PCR. Different superscripts denote significant differences (P < 0.05). Each bar indicates the mean ± SEM. Note the expression of CD44s mRNA but not any CD44v mRNAs

CD44 Protein and Glycosylation of CD44 in Inner Cells of Dissected Antral Follicles

Western blot analysis of CD44 showed a band of 85 kDa during stages I and III, whereas a band of 90 kDa was also detected in stages II and III (Fig. 6A). As the atresia proceeded, CD44 of 85 kDa shifted to 90 kDa, and was detected strongly. It has been reported that the expression of ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1) mRNA is upregulated in porcine granulosa cells during atresia, and modifications by sialic acid play a key role during follicular atresia in pig ovaries [36]. To address whether glycosylated sialic acids bind to carbohydrate chains of CD44, extracted proteins were treated with neuraminidase that removes {alpha}-2,6-, 2,3-, and 2,8-linked sialic acid residues. The bands around 85 kDa at stage I and 90 kDa at stage II shifted to 80 kDa and 85 kDa respectively, whereas there was no difference at stage III. These results indicated that 85 kDa at stage I and 90 kDa at stage II were sialylated.


Figure 6
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  		FIG. 6. Western blot analysis for CD44 and glycosylation in inner cells collected from dissected porcine follicles (stages I–III). A) The expression of CD44 treated without (N) or with sialidase (S). B) Detection of polylactosamine with CD44 by lectin blotting using DSA. Inner cells of each follicle immunoprepicipated with anti-CD44 antibody were loaded

The mature CD44 molecules are heavily glycosylated with polylactosamine chains composed of repeating disaccharide units (polylactosamine, (Galß1 -> 4GlcNAcß1 -> 3)n-type carbohydrate chains) in the epidermis [37]. Lectin blotting was performed to address whether CD44 molecules are glycosylated with polylactosamine. DSA, a lectin known to preferentially bind to polylactosamine, was used (Fig. 6B). Lectin blotting showed that a band was detected at 90 kDa only at stage III. This result indicated that stage III was the turning point because the expression of 90 kDa CD44 was dramatically increased followed by its glycosylation with polylactosamine-type carbohydrates.

Colocalization of Polylactosamine and CD44/Macrophages During Atresia

The result of lectin blotting in Fig. 6B indicated that CD44 was glycosylated with polylactosamine at stage III after macrophage invasion. Therefore, the colocalization of polylactosamine and CD44 (Fig. 7) or polylactosamine and macrophages (Fig. 8) was investigated. Polylactosamine was detected in theca cells and granulosa cells at stages I and II (Figs. 7, A-I and A-II, and 8, A-I and A-II). Particularly, theca interna cells were glycosylated with polylactosamine strongly. In contrast, polylactosamine was also observed in theca externa cells of stage I and II follicles. These cells were not CD44-positive cells or macrophages (Fig. 7, C-I and C-II, and 8, C-I and C-II). At stages III-V, CD44-positive macrophages were glycosylated with polylactosamine inside of follicles (Fig. 7C and 8C). Polylactosamine was also detected in apoptotic granulosa cells at stage III (Fig. 7A-III and 8A-III). High-level glycosylation with polylactosamine on CD44-positive macrophages was observed in stage IV follicles (Fig. 7A-IV and 8A-IV). The glycosylation with polylactosamine was decreased at stage V (Fig. 7A-V and 8A-V).


Figure 7
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  		FIG. 7. Distribution of polylactosamine and CD44 in porcine healthy (stage I) and atretic (stages II–V) follicles. Polylactosamine (green) and CD44 (blue) and a merged image (green and blue) are shown in A, B, and C, respectively. Nuclei, red. g, granulosa cells; t, theca cells; o, oocyte. Bar = 100 µm


Figure 8
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  		FIG. 8. Distribution of polylactosamine and macrophages in porcine healthy (stage I) and atretic (stages II–V) follicles. Polylactosamine (green) and Macrophages (blue) and a merged image (green and blue) are shown in A, B, and C, respectively. Nuclei, red. C) Merged image of polylactosamine and macrophages. These are the same serial sections shown in FIG. 7. g, granulosa cells; t, theca cells; o, oocyte. Bar = 100 µm

DISCUSSION

Macrophages have been implicated in cleaning up apoptotic debris (i.e., pyknotic granulosa cells and degenerated oocytes) during follicular atresia in several species [5–14]. However, the key molecule involved in this process during follicular atresia is not clearly known. In the present study, increased levels of glycosylated CD44 were present on macrophages in atretic follicles, suggesting that CD44 may be a candidate molecule for the cleaning-up of apoptotic granulosa cells by macrophages.

Immunohistochemical analyses showed that very low to undetectable levels of CD44 were expressed in granulosa cells in both healthy (stage I) and early atretic (stage II) follicles, whereas CD44-expressing macrophages were located within the theca cell layers. In contrast, a high level of CD44 was detected in granulosa cells of progressing atretic follicles (stage III). Although RT-PCR and Western blot analysis showed that levels of CD44 mRNA and CD44 protein were low in granulosa cells at stages I and II, levels increased significantly at stage III. In late (stage IV) and disintegrated (stage V) atretic follicles, immunohistochemical analysis and in situ hybridization results showed high levels of CD44 mRNA and CD44 protein. Immunohistochemical localization of CD44 was consistent with macrophages through stages I to V. Apoptotic granulosa cells were detected in stages II to V follicles, but not at stage I. Furthermore, stage III follicles possessed a partially collapsed basement membrane. The lysosomal activity begun to increase at stage IV, and reached the highest level at stage V. Although numerous TUNEL-positive apoptotic granulosa cells were detected at stage IV, the number of apoptotic cells was decreased at stage V. These results indicate that increased CD44 levels results from invaded macrophages and expression of CD44 on macrophages would be associated with porcine follicular atresia.

CD44 is a broadly distributed transmembrane glycoprotein that plays a critical role in a variety of cellular behaviors, including adhesion, migration, invasion, and survival [16–18]. Differential posttranslational modifications, including glycosylation and the attachment of glycosaminoglycans, generate additional structural diversity. Therefore, to address the function of CD44 in atretic follicles, we examined the glycosylation of CD44. Lectin staining showed that CD44-positive macrophages were glycosylated with polylactosamine at stages III–V, whereas macrophages were not modified with polylactosamine at stages I and II. Lectin blot analysis of immunoprepicipated inner cells collected from stages I–III follicles with anti-CD44 antibody showed that a DSA-positive band was detected at stage III, but not at stages I and II. These results indicate that glycosylation of CD44 with polylactosamine on macrophages occurred after the macrophages invaded atretic follicles. At stage IV, the lysosomal activity began to increase, and high levels of CD44 and polylactosamine were observed. The modification with polylactosamine was decreased at stage V, when the number of apoptotic cells was also decreased. Therefore, this glycosylation could be associated with macrophage phagocytosis during atresia. Polylactosamine is well known as a unique glycan having N-acetyllactosamine repeats (Galß1 -> 4GlcNAcß1 -> 3)n in one side chain, and is attached to N-glycan, O-glycan, and glycolipid [38]. Glycosylations with polylactosamine have been reported to increase cell motility in several kinds of cells [39–41]. In contrast, investigation for glycosylation of macrophages is very limited and still not clear, although the acquisition and regulation of macrophage functions has been widely studied. In the present study, our results showed that CD44 expressed on macrophages was glycosylated with polylactosamine. CD44 is well known as a cell adhesive molecule that regulates the motility of cells such as leukocytes and lymphocytes and tumor cell migration [18, 42, 43]. Therefore, glycosylation with polylactosamine on CD44 may be involved in macrophage migration.

CD44 is characterized by alternative RNA splicing and posttranslational modifications [18]. The smallest CD44 isoform, CD44s, is ubiquitously expressed in developing and adult organisms, whereas the larger variant isoforms (CD44v) are expressed in only a few epithelial tissues, mainly in proliferating cells, and in several cancers [43]. In SW620 cells (human colon cancer cell line not expressing CD44), the motility of cells transfected with CD44s was higher than that of cells transfected with CD44v [44]. In addition, the alteration of glycosylation on CD44s in migrated macrophages caused high cell motility [45]. In the present study, we examined the expression of CD44 mRNA using the primers that can detect variant exons 1–10. Our RT-PCR results showed that only CD44s mRNA was detected, and not CD44v mRNAs. These results suggest that only CD44s mRNA was expressed in macrophages during atresia.

It has been reported that sialic acids are involved in apoptosis and phagocytosis [46, 47]. In porcine granulosa cells, {alpha}-2,6-linked sialic acid residues were increased during atresia [36]. In the present study, our Western blot analysis, using neuraminidase to examine binding {alpha}-2,6-, 2,3-, and 2,8- sialic acid residues, showed that glycosylation of CD44 by sialic acid was not changed at the initiation of atresia. Therefore, the sialic acid residues on CD44 in porcine follicle macrophages are not increased during atresia. These results suggest that sialoglycoconjugates of proteins other than CD44 are associated with granulosa cell apoptosis and the advance of atresia.

Previously, we have reported that CD44 has a positive role in cumulus cells during porcine oocyte maturation in vitro [23–25, 48]. Particularly, CD44-hyaluronan interaction in cumulus cells induces disruption of the gap junction protein {alpha}1 (GJA1, connexin 43) in cumulus-oocyte complexes, inhibits the transport of cyclic AMP from cumulus cells into oocytes, and leads to meiotic resumption of oocytes [25, 48]. In addition, hyaluronan inhibits apoptosis in both cumulus and mural granulosa cells in human via CD44 during in vitro culture. Therefore, CD44-hyaluronan interaction would be involved in survival function in cultured cumulus and mural granulosa cells [49]. In contrast, the present results showed that CD44 on macrophages was observed in follicles only during porcine follicular atresia in vivo.

In conclusion, the present investigation offers several new observations, including CD44 mRNA and CD44 modification with polylactosamine in macrophages during porcine follicular atresia. These observations may help to elucidate the status and role of adhesion molecules and/or the extracellular matrix in macrophage migration and phagocytosis for apoptotic granulosa cells during atresia in the ovary.

FOOTNOTES

2 Correspondence: Yuko Miyake, Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1–1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan. FAX: 81 22 717 8687; yuko-okuy{at}bios.tohoku.ac.jp Back

1 Supported by Japan Society for the Promotion of Science Grant JSPS-16108003 (to E.S.), and in part by research grants from JSPS-17780207, the Inamori Foundation, and the Ito Foundation (to H.M.), and Joint Project of Japan-U.S. Cooperative Science Program (JSPS-AGR10002 to E.S.). Y.M. is a JSPS research fellow (Research Fellowships for Young Scientists Program, JSPS-17005038). Back

Received: 15 July 2005.

First decision: 16 August 2005.

Accepted: 17 November 2005.

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