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Biology of Reproduction 65, 1655-1662 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Fetuin and Fetuin Messenger RNA in Granulosa Cells of the Rat Ovary1

Poul Erik Høyer2,a, Ole B.F. Terkelsena, Anne Grete Byskovc, and Henrik Nielsenb

a Centre for Experimental Histochemistry, Institute of Medical Anatomy, and b Department of Medical Biochemistry and Genetics, The Panum Institute, Copenhagen, DK-2200 N, Denmark c Laboratory of Reproductive Biology, Juliane Marie Centre, The Rigshospital, University Hospital of Copenhagen, Copenhagen, DK-2100 Ø, Denmark


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hardening reaction that occurs in the zona pellucida to block polyspermy can be overcome in oocyte cultures in the presence of fetal serum or the serum component fetuin. Fetuin may also prevent precocious zona hardening by inhibiting a ZP2 proteinase released spontaneously by cortical granules during maturation of the oocyte. We demonstrated fetuin mRNA in the rat ovary by reverse transcriptase-polymerase chain reaction and localized it by in situ hybridization. Fetuin mRNA was present in all granulosa cells of growing and large follicles. Immunohistochemical analysis revealed that the fetuin protein was only present in some of the small, growing follicles. In large, healthy follicles, fetuin protein was confined to cumulus cells and granulosa cells bordering the antrum. Fetuin was present in atretic follicles, but the staining pattern differed from that of healthy follicles. The follicular antrum contained a substantial amount of fetuin, but whether granulosa cells secreted it or it originated in the ovarian blood supply could not be confirmed. We concluded that at least a portion of the fetuin is produced by granulosa cells of growing and large follicles, suggesting that fetuin may function in a paracrine manner to maintain the zona pellucida in a penetrable state for fertilization.

fertilization, follicle, follicular development, granulosa cells, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The fetuin family consists of closely related proteins (or glycoproteins) that belong to the cystatin superfamily. All members of this superfamily apparently act as inhibitors of at least one type of protease [1–3]. Recently, Olivier et al. [4] discovered that the fetuin family comprises two homologous proteins that are encoded by duplicate genes, and they designated these proteins as fetuin-A and fetuin-B. Fetuin-A is also known as fetuin, {alpha}2-HS-glycoprotein, countertrypin, or pp63, depending on the species [5].

Fetuin has been identified as a major protein in fetal blood and body fluids [6]. Fetuin is unevenly expressed in most organ systems of the rat during fetal development, such as in the brain, gastrointestinal tract, liver, kidney, and skin [7]. Whereas fetuin is widespread in adult tissues, it is primarily synthesized in the liver [4, 8].

Fetuin is present in the ovarian follicular fluid of the horse [9] and human [10]. It has been proposed that the protease inhibitory activity of fetuin plays an important role in preventing the so-called zona pellucida (ZP) hardening [11] (for review, see [12]). The first spermatozoon that fuses with the oolemma triggers release of the protease-active content of the cortical granules (CG), thereby inducing ZP hardening and blocking polyspermy [13–15]. A similar ZP hardening also takes place when the oocyte is released and cultured in serum-free media [16, 17]. This reaction can be overcome by maturing oocytes in the presence of follicular fluid, serum, or a number of individual serum components [9, 10, 17–19]. Fetuin inhibits the conversion of ZP2 to ZP2f in a concentration-dependent manner, with complete inhibition at concentrations lower than those found in fetal or newborn serum. However, follicular fluid that has been immunodepleted of fetuin retains the inhibitory effect. Other serum or follicular fluid components, such as sulfated glycosaminoglycans, can also inhibit the ZP-hardening reaction [18].

Proteases are also released by spontaneous, low-level exocytosis of CG content during follicular oocyte development and maturation [15, 18]. This event does not lead to zona hardening of the follicle-enclosed oocyte. It has therefore been suggested that follicular fluid contains a ZP2 proteinase inhibitor that may inhibit the effects of proteases released by the low-level exocytosis of CG contents [18].

Fetuin has been demonstrated in human follicular fluid by immunoblotting [9]. This finding is not surprising. Follicular fluid is composed of secretions from follicle cells and exudates from plasma, and fetuin is a plasma protein (although only present at low concentrations in adult human blood). Whether granulosa cells produce fetuin, however, and whether it is already present when the ZP is synthesized (i.e., shortly after the small follicle begins to grow) are not known [20].

If fetuin plays a role in maintaining the ZP in a penetrable state for fertilization, it must be produced within the follicle and/or accumulate in it after exudation from the ovarian blood supply. Consequently, we studied the distribution of fetuin protein with immunohistochemistry and the localization of fetuin mRNA with in situ hybridization. To rule out the possibility that in situ hybridization might be due to cross-hybridization to a sequence-related mRNA species, fetuin mRNA in the rat ovary was analyzed with reverse transcriptase-polymerase chain reaction (RT-PCR).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fixation and Preparation of Tissues

The experiments were conducted in accordance with the ethical guidelines for animal use determined by the Danish Ministry of Justice. Female Wistar rats (8–9 wk old) were anesthetized with Brietal (5 mg per 100 g body weight) when they reached estrus as determined by vaginal smears (Testimplets; Boehringer Mannheim, Mannheim, Germany). The estrous stage was selected to avoid any variation during the rat estrous cycle. A midline abdominal incision was made, and the ovaries were exposed and removed. The tissues were fixed immediately at room temperature for 12–24 h in Bouin fixative. The specimens were dehydrated in graded alcohols, cleared in xylene, and embedded in paraffin wax (melting point, 52°C; Merck, Darmstadt, Germany). Serial sections (thickness, 3–5 µm) were cut and placed on aminoalkylsilane-coated slides. Representative sections from each series were stained with hematoxylin-and-eosin or with toluidine blue.

Immunohistochemistry

The purification and characterization of rat fetuin and the subsequent production and characterization of a specific polyclonal rabbit antiserum to rat fetuin is described elsewhere [7]. The sections were incubated with the polyclonal antibody and immunoperoxidase-stained as described previously [21]. The sections were lightly counterstained with hematoxylin.

Synthesis of Digoxigenin-Labeled Riboprobes

A PstI fragment spanning the positions 444–819 (in the middle of the coding region) of rat fetuin cDNA was subcloned from the original recombinant plasmid RF619 [22] into the PstI site of the vector pBluescript KS+ (Stratagene, La Jolla, CA). The resulting recombinant plasmid, RF04-10II, was linearized with BamHI or EcoRI to produce templates for the transcription of sense or antisense RNA using T3 or T7 RNA polymerase, respectively. Probes for in situ hybridization were synthesized by using a digoxigenin (DIG) RNA-labeling mixture with a final concentration of 0.35 mM DIG-UTP (Boehringer Mannheim) together with the Stratagene RNA transcription kit. The probes were purified by DNase digestion followed by phenol extraction and ethanol precipitation. Routine recombinant DNA techniques were performed according to the methods described by Sambrook et al. [23].

In Situ Hybridization

In situ hybridizations were carried out essentially as described by Angerer and Angerer [24]. Before the hybridization step, the sections were treated with 1 µg/ml of Proteinase K (Boehringer Mannheim) at 37°C for 30 min, which was followed by three rinses in Proteinase K buffer. Hybridization was carried out by adding 25 µl of hybridization buffer containing 10 ng/µl of DIG-labeled probe to the sections. In the hybridization buffer, carrier DNA was substituted by 2 mg/ml of partially hydrolyzed, whole-cell RNA isolated from the ciliate Tetrahymena [25]. This addition of heterologous RNA abolished unspecific binding of the DIG-labeled RNA to nuclei in the sections. Following addition of the probe, the sections were covered with a siliconized coverslip and placed in a humidified chamber at 60°C overnight. Nonhybridizing probe was removed by RNase digestion (20 µg/ml of pancreatic RNase in 0.5 M NaCl, 10 mM Tris-HCl, and 1 mM EDTA, pH 8.0, for 30 min at 37°C) and salt rinses, with the most stringent being 15 min in 0.1x standard saline citrate at 42°C. The hybridized probe was detected by incubating the sections with alkaline phosphatase-conjugated anti-DIG Fab fragments (dilution, 1:100 v/v; Boehringer Mannheim) overnight at 4°C in a humidified chamber. The sections were subsequently stained for alkaline phosphatase activity using the Boehringer Mannheim DIG nucleic acid detection kit according to the manufacturer's instructions.

Reverse Transcriptase-Polymerase Chain Reaction

The RNA was extracted from single ovaries, liver, and heart muscle using the guanidinium isothiocyanate/acid phenol method [26]. Then, 1/500 of the RNA obtained from a single ovary was reverse transcribed in 20 µl of reaction buffer (50 mM Tris-HCl [pH 8.3], 30 mM KCl, 6 mM MgCl2, 10 mM dithiothreitol, 1 pmol dN6 primers [Pharmacia, Uppsala, Sweden], and 0.2 mM deoxyribonucleoside triphosphate) with 5 U of avian myeloblastosis virus RT (Boehringer Mannheim) for 30 min at 37°C. Next, 1/30 of the cDNA was used as template DNA in standard PCR reactions [27] using pairs of fetuin-specific oligonucleotide primers. The resulting amplified products were then analyzed by gel electrophoresis on a 2% (w/v) agarose gel run in Tris-acetic acid/EDTA buffer [23].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of Figures 1–7 indicates that the staining patterns for fetuin protein and fetuin mRNA differed, especially in granulosa cells.



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  		FIG. 1.

Granulosa cells of three early, healthy secondary follicles (F1–F3) are heavily stained for fetuin. Note, however, that only the most centrally located granulosa cells are stained in the more developed follicles, especially in F3. In this follicle, the precipitated follicular fluid is stained. Only a few scattered granulosa cells exhibit staining in a large follicle with early atresia (F4). The corpus luteum (CL) is moderately stained, whereas the surface epithelium seen in this section is heavily stained (arrowhead). x125.

FIG. 2. The granulosa cells of four primary follicles (F1–F4) exhibit different staining intensities for fetuin, ranging from unstained (F3) to intensely stained (F1). Note that protrusions from granulosa cells toward the oocyte of a small primary follicle are moderately stained (F5). The group of interstitial cells (IC) is not stained. Surface epithelial cells are unevenly stained (arrowheads). The wall and the lumen of a blood vessel (BV) are conspicuously stained. x250.

FIG. 3. A) A large follicle in advanced atresia stained for fetuin by immunohistochemistry. Note that most of the granulosa cells still present are stained. Arrows indicate areas of the granulosa layer where the peripheral granulosa cells are stained, respectively unstained. This section was not counterstained with hematoxylin. x40. B) Neither the oocyte nor the follicle cells of a primordial follicle (arrow) stain for fetuin. x320. C) One follicle cell of a primordial follicle (arrow) stains moderately for fetuin. The oocyte is lightly stained. x320. D) The follicle cells of a (growing?) primordial follicle (arrow) stain conspicuously for fetuin. The oocyte is lightly stained. x320.

FIG. 4. Primary follicle stained for fetuin mRNA by in situ hybridization. All granulosa cells (GC) and theca cells (TI) as well as the oocyte exhibit reaction product. x84.

FIG. 5. Protrusions from the granulosa cells toward the oocyte and a rim at the surface of the oocyte of a primary follicle are intensely stained for fetuin (arrow). Apart from this, the granulosa cells and the oocyte are lightly stained. Cells of a corpus luteum (CL) are moderately stained. The surfaces of erythrocytes in a blood vessel (BV) are stained. x200.

FIG. 6. Part of a large, healthy secondary follicle immunoassayed for fetuin. All the granulosa cells facing the antrum are stained. The cumulus cells, including protrusions toward the oocyte and a rim at the surface of the oocyte, are also stained. Several granulosa cells (arrowheads) are in mitosis. Note that the oocyte is unstained. Most theca interna cells (TI) are unstained. x110.

Fetuin Is Present in Granulosa Cells

The precipitate in the follicular antrum was intensely stained, demonstrating the presence of fetuin in follicular fluid, which is in agreement with evidence obtained from previously reported Western blot experiments [8] (Figs. 1 and 7A). Intracellular staining of the granulosa cells was also observed. The granulosa cells of primordial follicles (Fig. 3, B–D) and of small primary follicles (Fig. 2) stained heavily, moderately, or not at all. In some growing follicles with two or three layers of granulosa cells, most granulosa cells were stained (Fig. 2), but in others, only faint staining was observed (Fig. 5). In transitory stages from primary to secondary follicles, the peripheral granulosa cells of healthy follicles were unstained, whereas the cells lining the follicular antrum and those close to the oocyte were intensely stained (Figs. 6 and 7A).

Antral follicles in early atresia (i.e., <5% pyknotic granulosa cells) presented relatively few stained cells scattered in the granulosa layer and among the cumulus cells (Figs. 1 and 8). For those in more advanced stages of atresia (i.e., >5% pyknotic granulosa cells), a large fraction of the remaining granulosa cells were stained, including some in the periphery (Fig. 3A).

Oocytes of large, healthy follicles were not stained. Some oocytes of small follicles (Figs. 2, 3, C and D, and 5) as well as oocytes of follicles in an advanced stage of atresia were lightly stained. In growing and large follicles, a thin layer of the ZP close to the oocyte and protrusions from the cumulus cells to the oocyte were stained (Figs. 5 and 7A).

A light to moderate staining was observed in cells of the corpora lutea (Figs. 1 and 5), whereas the lumina of blood vessels were heavily stained (Figs. 2 and 5). Most theca interna and interstitial cells were unstained, but a few were lightly stained (Figs. 1, 2, and 6). Cells of the surface epithelium varied from being intensely stained to unstained (Figs. 1, 2, and 7A). No staining was detected when the specific polyclonal rabbit antiserum was replaced by nonhyperimmune rabbit serum (Fig. 7C).



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  		FIG. 7. A) Secondary follicle stained for fetuin by immunohistochemistry. The granulosa cells surrounding the oocyte and follicular antrum are heavily stained, as is the precipitated follicular fluid. Note that thin protrusions from the innermost granulosa cells to the oocyte are stained. Granulosa cells located in the periphery are lightly stained or unstained. An arrowhead indicates a granulosa cell in mitosis. Most theca interna cells (TI) are unstained. Surface epithelial cells (arrows) are variably stained. B) Secondary follicle stained for fetuin mRNA by in situ hybridization. The oocyte, all granulosa cells (GC), all theca interna cells (TI), as well as protrusions from granulosa cells toward the oocyte are stained. The follicular antrum is unstained. All surface epithelial cells (arrowheads) and cells of a corpus luteum (CL) are also stained. C) Control immunohistochemical procedure using a nonhyperimmune rabbit serum. D) Control in situ hybridization using the sense probe. x250

Fetuin mRNA Is Present in Granulosa Cells

To determine whether the fetuin detected in granulosa cells was produced in these cells or came from the follicular fluid or blood plasma, we looked for fetuin mRNA in ovaries with in situ hybridization. In these experiments, the follicle cells of primordial follicles were either unstained or lightly stained (data not shown). In contrast to the immunohistochemical staining pattern for fetuin, all granulosa cells of growing and large follicles showed a significant cytoplasmic staining for fetuin mRNA (Figs. 4 and 7B). In growing follicles, a thin layer of ZP close to the oocyte as well as protrusions from the granulosa cells to the oocyte were stained (Figs. 4 and 7B). Light to moderate staining was observed in the cytoplasm of small, growing oocytes and of large oocytes (Figs. 4 and 7B).

Theca interna cells, interstitial cells, and cells of the corpora lutea were moderately stained. All surface epithelial cells were moderately stained (Fig. 7B). The follicular antrum, lumina of blood vessels, or surrounding stroma were unstained.

The hybridization technique was specific, because parallel sections hybridized with a sense riboprobe generated from the same plasmid construct were blank (Fig. 7D).

Identification of Fetuin mRNA in Rat Ovaries by RT-PCR

Fetuin mRNA in ovaries was demonstrated by biochemical methods to rule out the possibility that the observed in situ hybridization might be the result of cross-hybridization to a sequence-related mRNA species. Yang et al. [8] could not demonstrate any fetuin mRNA in mouse ovaries by Northern blot analysis. Furthermore, because the amount of RNA obtained from single ovaries is relatively small, we chose the more sensitive RT-PCR method, which also provides detailed information regarding the structure of the mRNA compared with that obtained using Northern blot analysis.

The map coordinates of the oligonucleotide primers used are shown in Figure 9, and the gel electrophoretic analysis of the PCR products is depicted in Figure 10. In all cases, the PCR products were of the expected size and similar to the RT-PCR products of liver mRNA. Amplification from chromosomal DNA templates was ruled out, because these products would be of a considerably larger size considering most of the primer combinations used due to the inclusion of intron sequences. All no-template control amplifications were blank, which ruled out any contamination of the solutions with PCR products from previous experiments (data not shown). Because the amplification products, taken together, span the whole of the coding region of the cDNA map derived from the analysis of fetuin liver cDNA, we conclude that the overall structure of fetuin mRNA derived from the ovary is similar to that of fetuin mRNA derived from the liver.



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  		FIG. 9. This map shows the structure of rat fetuin cDNA according to the sequence entry AC M29758 in the European Molecular Biology Laboratory database. The start and stop codons are shown (AUG and UAG, respectively). The numbers above the line indicate the size of the exons. The numbers below the line indicate the approximate size of the introns. The arrows represent the oligonucleotides used in the RT-PCR analysis. The map coordinates are as follows: C10, 853–833; C11, 263–285; C12, 625–649; C13, 768–746; C40, 54–74; C41, 350–330; C42, 901–921; and C43, 1131–1108



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  		FIG. 10. A gel electrophoretic analysis of RT-PCR products. M: HinfI digested plasmid pBR322 was used as a molecular weight marker (band sizes from the top: 1631, 517/506, 396, 344, 298, 221/220, 154, and 75 base pairs). Lanes 1–5: RT-PCR analysis of rat ovary cDNA using various combinations of primers (lane 1, C40–C41; lane 2, C11–C13 [arrow]; lane 3, C12–C10; lane 4, C12–C43 [arrow]; and lane 5, C42–C43). Lanes 6–10: Corresponding RT-PCR analysis of rat liver RNA (positive controls). Lanes 11–15: RT-PCR analysis of rat heart RNA (negative controls). In all three control experiments, the same set of primers as in the experimental samples was used. In all cases, RT-PCR products of the expected size were observed. The smeary DNA staining pattern in lanes 11 and 13 is frequently seen in PCR reactions in the absence of a specific template. The correctly sized band seen in the amplification using C42–C43 in the negative control (lane 15) is probably due to amplification from genomic DNA present in the RNA sample. This primer set amplifies from within a single exon, in contrast to the four other primer sets that all span at least one intron


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The distribution of cells that stain intensely for fetuin within the healthy follicle and the presence of fetuin mRNA in all granulosa cells strongly suggest that cells stained for fetuin actually synthesize fetuin. The heterogeneous distribution of fetuin in the granulosa cell layer of healthy, large follicles makes the idea of uptake from blood plasma unlikely. The methods employed in this study did not provide the opportunity to determine whether fetuin inside the granulosa cells is confined to the secretory pathway and gives rise to the fetuin content of the follicular fluid. Just how much fetuin in the follicular fluid, if any, is taken up from plasma might be settled in future experiments by assessing whether radioactively labeled fetuin injected into a systemic blood vessel diffuses into the antrum. Our finding of fetuin mRNA in the ovary contradicts that of Yang et al. [8], who failed to detect any ovarian fetuin mRNA in a Northern blot analysis of RNA from a number of tissues isolated from 2- to 3-mo-old mice. This might be attributed to a species difference or to differences in method sensitivity.

Fetuin is commonly known as a plasma protein synthesized in the liver, but it has been observed in other tissues. Fetuin synthesis has been reported in brain cells of fetal sheep [28] and in rat osteoblasts in primary culture [29]. Fetuin mRNA has also been detected in several extrahepatic tissues in the adult mouse, including the adrenal gland, stomach, and placenta [8], and in several tissues in the fetal mouse [8] and rat [7].

The staining pattern of fetuin differed from that of fetuin mRNA: all the granulosa cells of healthy follicles stained positive for mRNA, but only a subset of these cells stained positive for fetuin protein. This difference was also observed in the theca interna and surface epithelium cells, although there were fewer fetuin-positive cells and the staining for fetuin mRNA was weaker. Varying the experimental parameters excluded the possibility that this could be due to loss of antigen or to inaccessibility of the antibodies. A similar difference was observed in the staining patterns of fetuin protein and mRNA in numerous tissues in our previous study regarding the expression of fetuin during rat development [7]. In particular, all columnar epithelial cells in the stomach of 16-day-old embryos stained positive for fetuin mRNA, whereas only a subset stained positive for fetuin protein. Similarly, all the epithelial cells lining the crypts in the intestines of 16-day-old embryos stained positive for fetuin mRNA, in contrast to the few, intensely stained cells observed when the protein was detected. This discrepancy could be explained by differences in the fetuin mRNA and/or fetuin turnover in the cells, or it could reflect a posttranscriptional regulation mechanism operating at the level of translation of the mature mRNA. Numerous examples of posttranscriptional regulation at this level with regard to the expression of genes during development (e.g., maternal effect genes during Drosophila development) are known, as are several well-studied cases of adult tissues (e.g., the ferritin genes). The molecular mechanisms involved include stimulation of translation by cytoplasmic polyadenylation as well as stimulation or repression of translation by protein factors binding to the 5'-untranslated region (UTR) or 3'-UTR of the mature mRNA (for review, see [30]). It would be interesting to study the possible involvement of the UTR sequences in translational repression of the fetuin mRNA and to address the possible importance of such a regulatory mechanism in the observed regional specification of the tissues involved.

Unlike the fetuin staining pattern seen in healthy follicles, fetuin-positive granulosa cells of early atretic follicles were randomly scattered in the mural granulosa layer. This may indicate that some local control mechanisms cease to function in early atresia, a phenomenon that is also evident in late atretic stages. It may be of interest to determine whether oocytes of healthy follicles produce a regulator of fetuin mRNA translation in the granulosa cells, and whether this regulator ceases to operate in atresia. It has been reported that fetuin may stimulate the action of macrophages [31], and it is known that healthy granulosa cells may behave in a macrophage-like manner in follicular atresia in the mouse [32, 33], guinea pig [34], and cow [35]. That the proportion of remaining granulosa cells that stain for fetuin increases as atresia advances may support the notion that fetuin stimulates phagocytosis.

The varying expression of fetuin in granulosa cells of primordial and early primary follicles is striking. With the methods used in this study, we could not judge whether the presence or lack of staining might be related to growth, differentiation, or perhaps, atresia.

Based on structural considerations, Kellermann et al. [1] grouped the fetuins as a separate family within the cystatin superfamily that includes cysteine proteinase inhibitors. However, subsequent studies failed to demonstrate any cysteine proteinase inhibition by fetuin [36]. Yamamoto and Sinohara [37] isolated a trypsin inhibitor, countertrypin, from mouse plasma that subsequently turned out to be identical to mouse fetuin, and those authors also noted a similarity between the fetuin sequence and the reactive center of the Kunitz-type trypsin inhibitors. In addition, they subsequently demonstrated that bovine and human fetuin acted as a trypsin inhibitor in an assay similar to the one applied to countertrypin. The most direct demonstration that fetuin is a protease inhibitor is, however, its ability to inhibit the so-called ZP2 proteinase [14]. Unfortunately, this protease has not been characterized in detail, and a mechanistic model of how it is inhibited by fetuin is not yet available.

In the mouse follicle, a low precocious release of CG content from the oocyte takes place during follicular development [4]. Interestingly, we found that fetuin is present around the growing and mature oocyte, which may indicate that fetuin is involved in preventing premature hardening of the ZP.

We conclude that cumulus cells and granulosa cells facing the antrum of growing, healthy follicles produce fetuin. Fetuin may prevent premature zona hardening by inhibiting a protease (e.g., the ZP2 proteinase) released spontaneously by low-level exocytosis of CG content during the development and maturation of the oocyte.



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  		FIG. 8. Large secondary follicle in early atresia stained for fetuin. The cumulus cells and a few of the granulosa cells facing the antrum are stained moderately. Note that some granulosa cells with punctuate staining are scattered randomly in the granulosa layer. Several granulosa cells are pyknotic (arrows). x100


   ACKNOWLEDGMENTS
 
We would like to thank P. Nawratil and W. Müller-Esterl for the donation of the anti-rat fetuin antibody. S. Forchhammer, F. Frenzel, and K. Ottesen are acknowledged for excellent technical assistance and M. Ibba for correcting the language of the manuscript.


   FOOTNOTES
 
First decision: 26 November 2000.

1 Supported by a grant to H.N. and O.T. from the Vera and Carl Johan Michaelsen Foundation and by a fellowship to O.T. from the M.D. Sofus Carl Emil and Olga Doris Friis Foundation Back

2 Correspondence: Poul Erik Høyer, Institute of Medical Anatomy, The Panum Institute, Blegdamsvej 3, Copenhagen, DK-2200 N, Denmark. FAX: 45 35 32 72 85;p.e.hoyer{at}mai.ku.dk Back

Accepted: July 5, 2001.

Received: October 2, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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