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Ovary |
Molecular Reproduction Research Laboratory,3 Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7
Faculté de Médecine,4 Université de Montréal, Montréal, Quebec, Canada H3C 3J7
Department of Physiology,5 McGill University, Montreal, Quebec, Canada H3G 1Y6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, follicle-stimulating hormone receptor, granulosa cells, oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) [4, 5], growth differentiation factor 9 (GDF-9) [6, 7], and bone morphogenetic protein 15 (BMP-15) [8–10].
The Kit-ligand expressed by GCs of growing follicles interacts with c-Kit, a tyrosine kinase receptor of the platelet-derived growth factor receptor family, produced by the oocytes and theca cells [3]. Kit-ligand together with c-Kit controls oocyte growth and theca cell differentiation and protects preantral follicles from apoptosis [11, 12]. FIG is a germ cell-specific basic helix-loop-helix transcription factor that has been implicated in coordinate expression of three zona pellucida (ZP) glycoproteins [4]. Consequently, mice that lack FIG are infertile because they fail to produce the three ZP transcripts [5]. GDF 9 [13, 14] and BMP-15 (also called GDF 9b) are members of the transforming growth factor-ß (TGFß) superfamily selectively expressed in the oocytes. Both these proteins induce GC proliferation and differentiation and are necessary for female fertility [6, 13]. BMP-15 has two functions: the inhibition of FSH-induced GC cytodifferentiation through the inhibition of FSH receptor (FSH-R) expression in GCs and stimulation of GC proliferation [9]. BMP-15 and Kit-ligand are all expressed in the early stages of follicular development and appear to be involved in GC mitosis [10].
The mouse ZP comprising three glycoproteins called ZP-1 (ZP-B), ZP-2 (ZP-A), and ZP-3 (ZP-C) is an extracellular matrix that surrounds the growing oocytes and remains associated with it after ovulation and formation of the early embryo [15]. Despite the fact that the zona matrix physically separates the oocyte and the surrounding somatic GCs, very close associations are continuously maintained throughout folliculogenesis by means of gap junctions that [16] allow interaction of paracrine factors noted above.
The major endocrine signal that is essential for normal folliculogenesis is FSH [17] that acts by binding to its receptor expressed exclusively on GCs. More recent reports, however, indicate its presence in oocytes, suggesting additional sites of action in the ovary [18, 19]. FSH interacts with its receptor isoforms, resulting in activation of a variety of signaling pathways to initiate follicle development and induce steroidogenesis [20, 21]. Targeted disruption of the mouse FSH-R gene [22] results in female sterility and induces a gene dose-related novel ovarian phenotype in the adult animal [23]. FSH-R gene disruption causes complete loss of ovarian estrogen production, creating steroid hormone imbalance [23–25]. The mutant females exhibit profound changes in ovarian structure and secondary sex-organ deficiencies. They are sterile because of a block in folliculogenesis before antral follicle formation. Interestingly, heterozygous female mice also undergo early ovarian senescence and lose fertility [24]. In consideration of such a phenotype, this haplo-insufficient animal has been dubbed the "Menopause mouse" [26]. The hormonal imbalances and other changes following loss of FSH-R signaling lead to follicular degeneration in both null and +/- mice.
The development of follicles in the mammalian ovary involves a bidirectional communication system between the follicular cells and oocyte [16]. Our preceding article established the perinatal developmental changes in the somatic cells of the follicle and noted that structural alterations in the ovary of null females are apparent at or before 2 days of life [27]. In the present investigation, our aim was to characterize the developmental state of the oocytes during FSH-R deficiency and explore follicle relationship with known oocyte-specific gene products. Because poor oocyte quality is a major cause for the aging-related decline in fertility in middle-aged women [28] and increase in the incidence of aneuploidy [29], we have taken advantage of the strong impact of receptor haplo-insufficiency in inducing early reproductive senescence to examine some oocyte characteristics in this model. Our data reveals that FSH-R deletion produces major changes in oocyte structure (and function), disrupting intercellular communication in the follicle, creating opportunities for additional mechanistic investigations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experiments involving animals were performed according to institutionally approved and animal care guidelines. Mice were housed five mice per cage under standard and approved laboratory conditions with 12L:12D at 22°C, with unrestricted access to food and water. Mice were genotyped by polymerase chain reaction from DNA extracted from tailpieces or toes. For all experiments in this study, virgin 24-day-old and 3- and 7-mo-old female mice were used. All animals were killed at random, disregarding the stage of the estrous cycle with the exception that sections used for histology and immunohistochemistry from +/- and +/- are derived from mice killed on the morning of proestrus. In any case, this was not relevant for the 1-mo-old mice or the FORKO (any age) that do not cycle.
Antibodies
The following antibodies were used in our study to perform either immunohistochemistry or Western blot analysis. Well-characterized and specific ZP-A (ZP2), ZP-B (ZP1), and ZP-C (ZP3) antibodies were kindly donated by Dr. U. Eberspaecher (Schering AG Berlin, Germany) [30]. These investigators prepared the ZP-A rabbit antibody against synthetic peptides CGTRYKFEDDKVVVYE and NRDDPNIKLVLDDC that had no homology to ZP-B or ZP-C. Rabbit antisera against the latter two were prepared using highly purified recombinant proteins. It should be noted that the nomenclature of ZP glycoproteins has been changed [31] to reflect the proteins according to their length rather than apparent molecular weights. The old nomenclatures are indicated in parentheses for clarification. The BMP-15 antibody was the gift of Dr. S. Shimasaki (University of California, San Diego). Antibodies to c-Kit and Kit-ligand were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Histological Analysis of Follicles
Ovaries were removed and cleaned of fat and fixed in 10% buffered formalin for 24 h at 4°C, processed in a tissue processor for paraffin embedding. Then 5-µm sections were cut serially and stained by standard protocols with hematoxylin and eosin. Histological examination of the ovaries was performed by light microscopy and microphotographs were taken at the same time using a Carl Zeiss microscope (Jena, Germany).
Follicle Development and Zona Pellucida Thickness
Follicles were classified into six groups as described in the previous article [27]. In brief, a primordial is a follicle with an intact oocyte surrounded by a single layer of flattened GCs. Primary follicles consist of an intact enlarged oocyte and are surrounded by a single layer of mixed squamous and cuboidal or a single layer of cuboidal GCs, secondary is a small preantral follicle with two layers of GCs. A preantral follicle has more than two layers of GCs. An antral structure is a follicle with a fluid-filled antrum. A preovulatory follicle is one that is close to the stage of ovulation. As will be noted in the results, FORKO mice lack structures beyond the preantral stage.
The follicle development at different ages was analyzed by measuring the diameter of oocytes using a microscope coupled to a camera. More than 1500 follicles were examined to compile data on oocytes. There were 4–5 mice for each age and genotype and more than 20–80 follicles of each type were included in each case. Only those follicles sectioned through the oocyte nucleolus (the largest follicle cross-section) were analyzed. The longest and shortest follicular and oocyte diameter were recorded and their average was used [24]. The zona pellucida thickness was measured in four directions around the oocyte of each follicle. Data are plotted as mean oocyte, ZP, and follicle diameters ± SEM.
Immunohistochemistry
Mouse ovary sections (5 µm) were deparaffinized, rehydrated using an immunoCruz kit (Santa Cruz Biotechnology Inc.), incubated in peroxidase block solution in order to quench endogenous peroxidase activity. To avoid any nonspecific reactivity of the antibodies, the sections were pretreated with 3% normal rabbit serum for 1 h and incubated with primary antisera (antibodies ZP-A 1:400, ZP-B 1:250, ZP-C 1:1000, c-Kit 1:50, BMP-15 1:3000 in normal rabbit serum) overnight at 4°C. The sections were washed three times for 5 min each with PBS before the biotinylated secondary antibody (goat anti-rabbit 1:1000 in serum) was added for 1 h at room temperature. This was followed by incubation with horseradish peroxidase (HRP)-conjugated antibody. After a final wash in PBS, the immunoreactive sites were visualized with the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride dihydrate. The sections were counterstained with hematoxylin and mounted with Permount (Fisher Scientific Company (Fairlawn, NJ). Finally, slides were observed under a microscope and pictures were taken at the same time using a Carl Zeiss microscope and computer-aided Eclipse image analyzer (Northern Eclipse, Ontario, Canada). In these evaluations, sections processed by treating with normal serum instead of the primary antibody served as the negative control.
Western Blotting
Ovaries were homogenized in a lysis buffer (50 mM Tris-HCl, pH 6.8) with additives described previously [23]. Samples (60–100 µg protein) were diluted with equal volume of reducing loading buffer (187 mmol/L Tris, pH 6.8; 2% SDS; 2% ß-mercaptoethanol; 1% sucrose; 0.01% bromophenol blue) and boiled for 6 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis on a 6%–10% acrylamide gel in parallel with prestained protein molecular weight markers (BioRad, Richmond, CA) and blotted onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia, Buckinghamshire, U.K.) overnight using wet blot apparatus (Biorad). Membranes were then blocked for 2 h at room temperature in 0.02 mol/L TBS (pH 7.6) containing 5% weight/volume dry milk powder, and then washed in TBS with 0.1% Tween-20 (TBST) before being incubated for 1–2 h with primary antibody (anti-c-Kit ligand goat polyclonal, 1: 750, Santa Cruz) in TBST with 5% dry milk or the ZP-A antibody (1:2500) as appropriate. Bound antibody was detected using a rabbit anti-goat (1:6000) or goat anti-rabbit (1:15 000) HRP linked secondary antibody) and the enhanced chemiluminescence visualization system (Amersham Pharmacia Biotech) ECL+ Plus according to the manufacturer's instructions. Quantitative comparisons were done using Image-Quant software (Amersham Biosciences, Uppsala, Sweden).
Statistical Analysis
Data are presented as the mean ± SEM and were analyzed by Student t-test or ANOVA with a Fischer least square difference (LSD) post hoc test using P < 0.05 as the level of significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At the onset of these studies, there was no information on the potential influence of FSH-R signaling per se on oocyte growth and function. Therefore, ovaries from mice of all three genotypes of three different ages (24 days and 3 and 7 mo) were serially sectioned for morphometric analysis. Comparing data on oocyte diameter, we found differences for different types of follicles in the three genotypes (Fig. 1). In the immature +/+ mouse, the oocyte diameter steadily increased from 13 µm in the primordial, reaching about 62 µm in the antral stage. The growth patterns for the +/- were similar to the wild type except at the primary stage, where they were significantly larger. The size of -/- oocytes differed significantly from the wild type (WT) at the primary (showing a decrease) and preantral (increase) stages. It should be noted that there are no antral follicles in the -/- ovary at any age. At 3 mo of age, oocyte size was in general smaller in the FORKO ovary as compared with the wild type at all stages. For the heterozygous mice, significant differences were seen for the secondary and preantral follicles. At this age, the wild-type and +/- oocytes of antral follicles reached an average diameter of 108 µm. At 7 mo, there was no apparent difference among the groups and the maximum diameter in the wild type reached 74 µm in the antral stage, being considerably smaller than that seen at 3 mo.
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Thickness of Zona Pellucida Matrix
The ZP is first observed as extracellular patches that come together and form a uniform matrix surrounding oocytes in primary follicles. This structure increases in width to about 7 µm surrounding fully grown oocytes of early antral follicles in the mouse [32]. Thus, the thickness of the ZP reflects the local environment during folliculogenesis and serves as an indicator of oocyte development. Accordingly, ZP thickness was measured in all follicle types of 3-mo-old mice (Fig. 2) as the size of oocytes was maximal at this age. This matrix gradually thickens with increasing follicular growth, becoming maximal in the ovulatory follicle (8.3 ± 0.06 µm) in the wild-type ovary. For this genotype, differences were significant at each stage (see Fig. 2, inset). Structural changes were evident in the +/- ovaries at two stages: the zona matrix was thinner in the antral and ovulatory follicles (P < 0.005). In the FORKO mice, the average thickness of the ZP matrix in preantral follicles is smaller (4.57 ± 0.114 µm) than that of wild-type mice (5.43 ± 0.148 µm) (P < 0.001). But in secondary follicles, the ZP became thicker (4.58 ± 0.147 µm) than that of wild type (3.64 ± 0.284 µm) (P < 0.005). This result confirms that, in the FORKO mice, some oocytes developed earlier than surrounding GCs, indicating developmental imbalance (see Fig. 3F). As reported previously [23, 27], antral follicles are absent in FORKO ovaries.
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Histological Characterization of Zona Pellucida, Oocyte, and Follicle Growth in FORKO and Heterozygous Mice
The normal oocyte at various stages of its development in a healthy follicle is a perfectly rounded structure. Usually the ZP is present in the perivitelline space between the plasma membrane of the oocytes and the layer of surrounding GCs. However, during folliculogenesis, we noted that the periphery of the oocyte in FORKO mice was not round and smooth as in the wild type (see Fig. 3, A–C). Some irregularity from its normal shape was found in the ZP of most -/- oocytes at 3 mo. The ZP matrix that appeared uneven in null mutant was also apparent in +/- animals. A random sampling of ovarian sections from different mice revealed that about 5% of follicles contained GCs between the zona pellucida and the oolemma in the FORKO females (Fig. 3E). It may be noted that we have excluded such follicles and oocytes from our estimates of size or thickness (Fig. 2). This feature was unique to the null mutants because it was not seen in the +/- at any age examined. In the previous article [27], we have pointed out that this aberration of infiltration is already evident at 24 days of age in the FORKO ovary. In Figure 3E (see arrow), we depict the appearance of breaks, indicating discontinuity in the extracellular matrix that might allow the seepage of GCs (denoted by asterisks) into the oocyte. In contrast with this aberrant characteristic in null mice, the +/- ovaries showed a different feature: frequent appearance of two oocytes present in one follicle at the secondary or preantral stages. Although this was seen rarely in the control animals, their frequent presence in the +/- animals was unique to this genotype (Fig. 3D) and occurred at all ages (from 24 days to 7 mo) that we have examined. Figure 3F depicts an example of an enlarged oocyte in a secondary follicle in the FORKO ovary.
Expression of Oocyte Markers c-Kit, Kit-ligand, and BMP-15
In order to gain some mechanistic insights into the above perturbations, we evaluated some well-established candidate markers that participate in oocyte-GC communication. In the normal ovary, c-Kit is expressed in the oocyte and Kit-ligand expression is confined to GCs in the ovary [3]. In the 24-day-old mouse ovary, c-Kit was clearly detectable by immunohistochemistry. Clear positive staining was seen in the oocyte of +/+ ovary, but not in the FORKO ovary, where it was barely detectable (Fig. 4C). Ovarian sections from heterozygous mice showed positive staining that was intermediate; it was weaker than that of wild type but stronger than that of FORKO (Fig. 4, B and C). The antisera that were available to us only allowed quantitation by Western blotting of the Kit-ligand in the ovarian extracts. By this analysis, expression in the FORKO ovary was 47% of wild type, and in the heterozygous state, it was 85% of wild type (Fig. 5A). BMP-15 expression in the 24-day ovary of FORKO and heterozygous mice was also weaker than that of wild-type ovary (Fig. 4, D–F).
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Distribution of Zona Pellucida Proteins (ZP-A, B, C) in the Oocyte
Mouse ZP matrix is composed of three glycoproteins, ZP-A, ZP-B, and ZP-C [15]. These glycoproteins are secreted by the growing mouse oocytes. ZP-A and ZP-C assemble into organized filaments that are cross-linked by ZP-B. The resulting extracellular matrix, called the ZP, is unique to the oocyte and is a thick coat that surrounds oocytes and plays a crucial function in oogenesis, fertilization, and early embryogenesis [32]. In addition, ZP-A and ZP-C are believed to be essential for gamete recognition [33]. Thus, it was of interest to understand if the oocytes in the mutants would exhibit molecular changes that would affect their regulatory function. We were able to use formalin-fixed and paraffin-embedded sections of mouse ovaries to assess ZP protein expression during different ages in the three genotypes. Using the specific antibodies (see Materials and Methods), staining for all three ZP proteins was observed in the ovaries of normal and mutant mice. However, their expression patterns were greatly altered in a differential manner. For ZP-A and C proteins in the +/+, expression was confined to the adjacent extracellular area around the oocyte, with virtually no staining in GCs. However, with anti ZP-B, expression was intense and localized to the whole oocyte but some weakly positive reaction was evident in the GCs. In this case, GCs of mutant ovaries showed more intense staining than normal ovaries. In the 24-day-old FORKO female, the ZP-A (Fig. 6C) and ZP-B (Fig. 6F) expressions were lower. We also noted lower expression for these proteins in haplo-insufficient mice at this early age (Fig. 6, B and E). The pattern of ZP-C expression was also markedly different. In this case, it was stronger in both heterozygous and FORKO mice compared with the wild type (Fig. 6, G–I). Abundant expression in the FORKO ovary is clearly evident when compared with the +/+ or +/- state (compare Fig. 6, I with H). Overall, there was clear evidence for imbalance in the expression of these important glycoproteins that was gene-dosage dependent. We were able to quantify expression by Western blotting only for ZP-A (see Fig. 5B). ZP-A expression in the FORKO ovary was reduced to 49% of the wild type, whereas in the heterozygote, there was a 33% deficit. This confirmed the reduced staining that was seen by immunohistochemistry (Fig. 6, B and C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicle development depends on optimal communication between oocyte and the surrounding GCs [34, 35]. Such transcellular communication within the follicular compartment occurs as a result of direct physical contacts (intercellular junctions) and the local production of soluble factors that act in an autocrine or paracrine fashion [36]. This communication is bidirectional and occurs throughout follicular development [16, 37, 38]. These events must be tightly coordinated to ensure orderly development and completion of meiosis [34].
Our data of the present study reveals that this bidirectional communication is influenced in a quantitative manner by FSH-R signaling events, and oocyte contribution to this process is greatly perturbed in the ovaries of mutant mice during the peri/postnatal period. Thus, targeted disruption of the FSH-R gene adds critical information to study the role played by receptor signaling in maintaining the follicular milieu in a state conducive for development. The phenomenon of gene haplo-insufficiency in mutant animals derived from homologous recombination is of considerable experimental interest as shown by the present studies because such animals have allowed us to investigate gene-dose-related effects as they appear at different ages (data not shown). Even though the young +/- and -/- mutants were normal in outward appearance, functional deficits were already apparent at 24 days. This emphasizes the need for careful developmental assessments to understand quantitative changes that could become apparent later in life. In the previous communication, we compared the postnatal developmental pattern of the ovary and showed that follicles from FORKO mice are structurally defective [27].
Previous reports from this laboratory on adult FSH-R mutants pointed out differences in follicular growth patterns [23, 24]. Extending this further, we can now hypothesize that some of the molecular alterations detected in the current study might be a direct result of developmental asynchrony between the oocyte and GCs. However, additional investigations such as the transplantation of asynchronous and growth-advanced mutant oocytes into normal early-stage ovaries might help in establishing if their signals alter GC and folliculogenesis.
In addition, several interesting features of the current report with respect to oocyte development merit attention. The incidence of multiple oocyte follicles appeared to be unique to the +/- ovary (Fig. 3D) because they were extremely rare or not present in the +/+ or -/- ovaries. Because this was already apparent in some animals at 1 mo (not shown), we believe that such an abnormality takes effect quite early in development. This type of abnormal follicles has also been noted in ovaries of mutant mice lacking GDF-9 or BMP-15, both of which are oocyte-secreted growth factors [39, 40]. Several other deletions of genes expressed in GCs also exhibit this aberration as in ovaries of mice lacking the Ahch [41], which encodes the transcription factor Dax-1 involved in sex determination, or the Ca2+/Calmodulin-dependent protein kinase IV knockout females that show reduced fertility [42] and in mice that overexpress the inhibin alpha gene [43]. In comparison with all the above null mutants, the aberrations observed in our study are unique in that the multiple oocyte follicles appear only in the haplo-insufficient state. Although the mechanisms underlying such abnormalities are complex, it is possible that early developmental events that lead to follicle organization are aberrant and incomplete in the +/- FSH-R ovaries. Further work would be necessary to unravel the delicate imbalance of factors that prevail in the +/- state.
In contrast with the state in +/- FSH-R mice, the oocytes of null ovaries showed an infiltration of GCs in some preantral stage follicles (Fig. 3E), an event that never occurred in the +/+ ovary at any age (see below). The initial increases in zona thickness, only in secondary follicles but not at other stages of FORKO mice, indicate faster progress and developmental imbalance in the interaction between the oocyte and GC. Since such follicles also appeared in the GDF-9 knockout mice [7], we might speculate a deficit in GDF-9 secretion in the FSH-R-deficient state pending further analysis of this important oocyte-specific gene. However, based on drastic changes in many other markers (see below), we can also suggest that oocyte structure and function is irreversibly altered in the ovaries of our mutants. That some of these impairments occur as early as 24 days [27] indicates the strong impact of early establishment of communication in preserving the structural integrity of the oocyte-GC as a functional unit.
The Kit-ligand is a product of GCs specifically in ovarian follicles, where its expression is hormonally regulated [12]. Its alteration as seen in the whole ovary (Fig. 5A) coupled with drastic curtailment of the c-Kit expression (Fig. 4, A–C), can be taken as evidence of impaired or lack of communication between the adjacent germ cell and somatic compartments. In addition to controlling oocyte growth and theca cell differentiation during early folliculogenesis in the normal state, the c-Kit/Kit-ligand also protects preantral follicles from apoptosis. Moreover, formation of an antral cavity also requires a functional c-Kit/Kit-ligand system [11, 12]. In the FSH-R mutant, the ovarian follicle never advanced to these later developmental stages. Thus, because many steps of follicular development are controlled by the interactions between c-Kit and Kit-ligand, the phenotypes observed in our study could be a direct result of its perturbation.
Drastic changes seen in BMP-15 protein expression in our mutants offer additional proof that oocyte development is impaired. BMP-15, the oocyte-derived factor, is a known physiological regulator of follicle cell proliferation and differentiation necessary for female fertility [8]. BMP-15 mRNA and protein are both coexpressed in oocytes throughout folliculogenesis and GCs are the first targets of BMP-15. Our observations on reduced (in +/- state) or virtual absence of the protein in the -/- ovary (Fig. 4) are consistent with the reported data on BMP-15 regulation of proliferation and differentiation of GCs, inhibition of FSH-R mRNA expression [9], as well as selective modulation of FSH action. This indicates that FSH-R and BMP-15 have a direct relationship at least at the level of translation of the latter.
An unexpected and remarkable oocyte defect noted in the FSH-R mutants is the state of the ZP that is normally formed during follicular development in the ovary. In the normal animal, this thick extracellular coat surrounding the growing oocytes, ovulated eggs, and preimplantation embryos provides for oocyte survival, binding and activation of sperm cells leading to fertilization and early embryogenesis. It also prevents polyspermy [38]. The ZP matrix consisting of three proteins (ZP-A, -B, and -C) physically separates the oocyte and GCs but maintains close associations throughout follicular development via paracrine factors and cellular processes. Several of the changes we have observed in these proteins (Fig. 6) are reminiscent of alterations reported for mouse mutants lacking these individual proteins. For example, in ZP-B-lacking mice, the ZP appears thinner than in normal mice and there is an ectopic accumulation of GCs in the perivitelline space [44], as also seen in FORKO mice (Fig. 3). Mice that lack ZP-A also produce a thin zona matrix but the abnormal zona matrix does not seem to affect the initial stages of folliculogenesis. However, the numbers of antral follicles significantly decrease in these mice [45]. Mice without ZP-C do not form a ZP matrix at all, even early in oogenesis [45]. The overexpression of this protein in both the +/- and -/- FSH-R mice is apparently insufficient to compensate for the loss in the other two because reproductive deficits occur in both states. This indicates that there is a different rate of change of the proteins that make up the ZP matrix in the FSH-R mutants. The FORKO mice develop an uneven ZP surrounding the growing oocyte and some cumulus cells even cross the ZP matrix to cover the oocyte (Fig. 3E). The matrix in the zona is visualized as a structure organized by filaments of heterodimeric repeats of ZP-A and ZP-C cross-linked by ZP-B [30, 46]. Based on this, pathology similar to that seen in our mutants in the ZP-B knockout mice was explained by the fragility and weakness of the ZP due to the mutation [44]. Thus, the ZP becomes leaky in the FSH-R ovary, facilitating the entry of somatic cells that are themselves not organized very compactly. Indeed, visual appearance of breaks in such oocytes in our study (Fig. 3E) confirms that the extracellular matrix is not continuous, creating a dysfunctional assembly that lacks the required filament structure [46]. A more detailed study of these features is likely to reveal the regulatory aspects of ZP genes/proteins.
Normally, ZP thickness increases gradually during follicular development. The measurement of ZP thickness in the different genotypes of 3-mo-old mice shows that the ZP thickness is less in the FORKO mice in the preantral follicles but thicker in the secondary follicles compared with the wild-type mice. The thickness of secondary follicle ZP in FORKO mice might be related to the advanced development of "secondary" oocytes. From the present study, we cannot say whether the thinness of the ZP in our model simply reflects the different ratio of ZP-A, ZP-B, and ZP-C proteins or other unknown perturbations. Nevertheless, these data imply that infertility in the FORKO mice must also be related in part to changes in zona pellucida protein expression that will in turn influence the status of the entire follicle. However, it remains unclear where the initial molecular association of the three ZP proteins occurs—at the secretion level or gene level—and through what precise mechanism FSH-R signaling controls ZP protein expression. We conclude that reduced fertility and early reproductive senescence previously observed in haplo-insufficient females [23, 24] must also be a consequence of the loss of the delicate balance of intercellular communication.
In conclusion, our studies report for the first time that oocyte integrity and perhaps functional status are directly impaired in the absence of FSH-R signaling. Evidence emerging from haplo-insufficient mice also suggests the existence of a clear gene dose-related effect on the oocytes. These are related to alteration in many growth factor-related genes and structural glycoproteins produced by the oocytes. Thus, collectively, these data draw attention to the possible link between embryonic and/or immediate postnatal defects during FSH-R haplo-insufficiency that need to be explored in future investigations. We believe that these phenomena may have relevance to premature ovarian failure and lower reproductive success in middle-age women.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: M. Ram Sairam, Molecular Reproduction Research Laboratory, Clinical Research Institute of Montréal, 110, avenue des Pins West, Montréal, PQ, Canada H2W 1R7. FAX: 514 987 5585; sairamm{at}ircm.qc.ca
Received: 17 January 2003.
First decision: 22 February 2003.
Accepted: 15 May 2003.
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0.005
