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BOR - Papers in Press, published online ahead of print November 24, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.036087
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BIOLOGY OF REPRODUCTION 72, 796–804 (2005)
DOI: 10.1095/biolreprod.104.036087
© 2005 by the Society for the Study of Reproduction, Inc.

Relationship Between Low-Molecular-Weight Insulin-Like Growth Factor-Binding Proteins, Caspase-3 Activity, and Oocyte Quality1

B. Nicholas3,4, R. Alberio5, A.A. Fouladi-Nashta4, and R. Webb2,4

Division of Agricultural and Environmental Sciences4 Division of Animal Physiology,5 School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bovine follicular atresia is associated with the apoptosis of granulosa cells and the subsequent loss of oocyte competence through the reduction of cellular contact (e.g., gap junctions). Several components of the insulin-like growth factor (IGF) system are thought to affect follicular atresia. Whereas the IGF-binding proteins (IGFBPs) are present in varying quantities throughout follicular development, IGFBP-5 appears to be present only during atresia, in parallel with its regulation in other tissue remodeling systems. However, to our knowledge, no connection has yet been made between atresia, low-molecular-weight IGFBP content, and oocyte quality in the bovine ovary. Caspases are actively involved in ovarian follicular atresia, and apoptosis in antral follicles is caspase-3-dependent. Hence, the aim of the present study was to investigate the use of these factors in the assessment of oocyte quality and developmental potential. Oocytes were aspirated, morphologically classified, and individually matured in vitro. The follicular fluid and granulosa cells of these follicles were analyzed for IGFBP profile and caspase-3 activity, respectively. A significant correlation was found between the presence of low-molecular-weight IGFBPs in bovine follicular fluid and caspase-3 activity of granulosa cells isolated from individual follicles. The highest percentage of development to the blastocyst stage was observed in oocytes from slightly atretic follicles. This group of oocytes contained an equal proportion of oocytes at grades 1–3. These data demonstrate that low-molecular-weight IGFBP profile is a more reliable method than the traditional morphological assessment of oocytes and can be used as an effective marker of developmentally competent oocytes. Importantly, these results have implications for the use of noninvasive follicular fluid markers in the selection of competent oocytes to improve outcomes of in vitro fertilization.

apoptosis, follicle, oocyte development, ovary, ovum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many potential contributing factors determine the variation in quality of oocytes used during in vitro fertilization (IVF) procedures. These include in vitro manipulations, oocyte collection procedures [1], IVF and culture conditions [2], as well as the age and reproductive history of the donor [3]. In addition, cyclical growth and atresia of ovarian follicles result in changes in overall oocyte quality [4]. This might arise through changes in the granulosa cell support of the oocyte through changes in gap junctions [5] as well as through alterations in follicular fluid composition, such as changes in growth factor profiles that affect oocyte differentiation and survival [6].

Whatever the factors contributing to oocyte quality, useful markers of oocyte quality are sparse [7]. Time considerations have to be taken into account, because generally only a short period of time (~18 h in cattle) is available between in vitro maturation and fertilization, making lengthy marker quantitation unlikely. Traditional methods have relied on the morphological assessment of oocyte quality by scoring nuclear density and quality of cumulus-oocyte complexes (COCs) [8]. These assessments undoubtedly are popular, but they also are highly inaccurate forecasters of oocyte quality as measured by subsequent embryo development [9].

It is widely recognized that apoptotic cell death is an underlying mechanism of cell loss during follicular atresia [10, 11]. Throughout follicular development, the granulosa cells can communicate with each other and the oocyte via gap junctions [12]. These channels allow transfer of nutrients and regulatory factors. The cooperation of granulosa cells in oocyte metabolic processes probably is essential for normal oocyte development. In addition, signals from the granulosa cells regulate the progression of meiosis in oocytes [13]. Granulosa cell apoptosis may occur in healthy follicles during the luteal phase of the estrous cycle and/or very early during the process of atresia, before other morphological or biochemical changes are detected [14]. Several markers of atresia have been used to assess follicular status. These include caspases [15], Fas and Fas ligand [16], oxygen tension [17], TUNEL staining of granulosa cells [18], DNA condensation [10], and insulin-like growth factor-binding protein (IGFBP)-5 expression [19]. Several of these markers have been used to identify atretic follicles, but very little evidence exists for their subsequent use as markers of oocyte quality. Granulosa cells from healthy follicles possess almost exclusively the inactive (unprocessed) form of caspase-3, whereas granulosa cells from atretic follicles demonstrate increased concentrations of activated caspase-3. The active processing of caspase-3 is associated with the cleavage of poly-(ADP-ribose)-polymerase and actin and the formation of oligonucleosomes [20]. Unlike progression of apoptosis in oocytes that relies on caspase-2, the normal progression of apoptosis in granulosa cells from preantral to preovulatory follicles is dependent on the activity of caspase-3 and caspase-7 as determined from studies using caspase-3-knockout mice [21].

Additional intraovarian mechanisms also are involved in controlling folliculogenesis. A range of growth factor signaling systems act to regulate both granulosa and theca cell growth and differentiation, and one important system is that of the insulin-like growth factors (IGFs), which consists of two IGF ligands (IGF-I and IGF-II); a family of at least six IGFBPs (IGFBP-1 through IGFBP-6), which regulate the bioavailability of the IGF ligands; the IGF type 1 and type 2 receptors; and a range of proteases, which regulate the affinity of the IGFBPs for the IGF ligands [22]. The IGF system components are highly compartmentalized within the mammalian ovary. For example, in cattle, IGF-II ligand is produced by theca cells and IGFBP-4 by theca cells, whereas IGFBP-2 is produced by granulosa cells [23, 24].

The IGF ligands serve to regulate granulosa cell [22] and theca cell [25] growth and differentiation, and they also are thought to act as antiapoptotic factors [26, 27]. The array of IGFBPs alters throughout folliculogenesis such that follicular fluid from healthy dominant follicles contains mainly IGFBP-3, whereas follicular fluid from subordinate follicles contain increased amounts of the low-molecular-weight binding proteins (IGFBP-2, IGFBP-4, and IGFBP-5) [28]. The expression of the low-molecular-weight binding proteins also has been linked to follicular atresia in both the human [23] and cow [29]. The expression of one binding protein, follicular IGFBP-5, is particularly interesting in that it is not universally present during the period of follicular selection in cattle (between Day 0 and Day 2 of the first follicular wave) but is highly upregulated in the majority of subordinate follicles by Day 5. It therefore appears to be present in higher amounts only during the period when these follicles are starting to undergo atresia [30]. In addition, IGFBP-5 has been implicated in tissue remodeling and cell death processes in other tissues, including chondrocytes [31], osteoblasts [32], and mammary tissue [33]. It also has been associated with follicular atresia [34], and its function is thought to relate to the antiapoptotic abilities of the IGF ligands, a function that may well be linked to the strong affinity of IGFBP-5 for extracellular matrix [35, 36].

In summary, it has been demonstrated previously in cattle that follicular atresia is accompanied by a considerable increase in the low-molecular-weight IGFBPs [29, 37, 38]. Importantly, IGFBP-5 appears to be a particularly good marker of atresia, because other IGFBPs are expressed at different stages of follicular development whereas IGFBP-5 is exclusive to atresia [39].

Using morphological assessment or immunocytochemical staining, previous studies have shown that in isolated COCs, cumulus and granulosa cell apoptosis is related to oocyte viability as measured by embryo development post-IVF [40]. However, no studies have examined in detail the relationship between granulosa cell apoptosis, oocyte maturational capacity, and embryo viability in an experimental paradigm that would allow selection and subsequent implantation of the embryos following their evaluation. In these studies, we have used two rapid methods to assess atretic markers in follicular fluid and granulosa cells, both of which are by-products of the IVF process, to preselect oocytes for subsequent IVF. Furthermore, these parameters have been assessed on an individual-follicle basis to ensure accuracy of the selection procedure and to demonstrate that preselection is achievable within the desired time frame. Changes in caspase-3 protein have been described, but to our knowledge, no previous work has quantified changes in caspase-3 activity to identify follicular atresia. We present data regarding the use of low-molecular-weight IGFBPs, particularly IGFBP-4 and IGFBP-5, and caspase-3 activity as markers of both follicular atresia and oocyte quality as assessed by measuring development after IVM and IVF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Protein assay reagent; fluorescent caspase-3, caspase-7, and caspase-10 peptide substrate (Ac-DEVD-AFC); caspase-3 irreversible inhibitor (Z-DEVD-FMK); and protein assay reagent were purchased from Bio-Rad Laboratories Ltd. (Hemel Hempstead, U.K.). Recombinant bovine IGFBP-2 and IGF-II were purchased from Gropep Pty Ltd. (Adelaide, Australia). Biotinylated IGF-II was prepared as described previously [38]. The Enhanced Chemiluminescence (ECL) Plus reagent and polyvinylidene fluoride (PVDF) blotting membrane were purchased from Apbiotech. Extravidin-peroxidase and RIA-grade BSA were purchased from Sigma (Poole, Dorset, U.K.).

Protein Estimation in Cell Homogenates

Protein concentrations in granulosa cell homogenates were assessed using the Bio-Rad protein assay that is compatible with Hepes. Briefly, 100 µl of BSA protein standards (diluted in caspase assay lysis buffer) or samples in duplicate and diluted 1:10 (in caspase assay buffer) were prepared in a clear, flat-bottomed, 96-well microtiter plate. Twenty microliters of Bio-Rad protein assay reagent were added, and the plate was incubated for 30 min before the measurement of absorbance at 595 nm using a microplate reader (Titertek Multiskan II).

Analysis of IGFBPs in Follicular Fluid

The IGFBP profiles in follicular fluid of aspirated follicles were analyzed by nonradioactive Western ligand blot as described previously [38]. Essentially, 0.75 µl/well of bovine follicular fluid was fractionated on 12% (w/v) nonreducing SDS-PAGE gels and electroblotted onto a PVDF membrane. Nonspecific binding sites on the membrane were blocked using 3% (w/v) BSA in PBS-0.1%(v/v) Tween 20 (blocking solution).

The membrane was then incubated overnight at 4°C in blocking solution with agitation in the presence of 2 µl/blot of biotinylated IGF-II. The following day, the membrane was washed three times with PBS-Tween 20 and then incubated in blocking solution containing a 1:4000 dilution of extravidin-peroxidase for 2 h at room temperature with agitation. The membrane was again washed with PBS-Tween 20, and peroxidase signal was detected using the ECL Plus system on Kodak Biomax SR film (Eastman Kodak, Rochester, NY) with a typical exposure time of 10 sec to 1 min. Developed films were imaged using a Fluor-S Max imager (Bio-Rad Laboratories Ltd., Hemel Hempstead, U.K.), and the relative optical density of each IGFBP band (42–44, 33, 28–30, and 24 kDa) was quantified using Quantity One software (Bio-Rad). This procedure enabled the specific identification of small-molecular-weight IGFBPs, such as IGFBP-4 and IGFBP-5.

To confirm the IGFBP-2 and IGFBP-5 bands by immunoblotting, follicular fluid samples were fractionated using SDS-PAGE as previously described, but under reducing conditions, and semidry blotted onto PVDF. Membranes were blocked with 5% nonfat dried milk in PBS-0.1% Tween 20 for 1 h at room temperature and then incubated overnight at 4°C with blocking solution containing a 1:3000 dilution of rabbit anti-bovine IGFBP-2 or human IGFBP-5 (Upstate Biotechnology, Inc., NY). The membrane was then incubated with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (Bio-Rad) for 2 h at room temperature, washed with PBS-Tween as before, signal detected using ECL Plus, and captured on autoradiographic film.

Fluorometric Caspase Assay

Stock solutions of AC-DEVD-AFC peptide (4 mg/ml) and caspase-inhibitor Z-DEVD-FMK (100 mM) were prepared in dimethyl sulfoxide and stored in aliquots at –20°C until use. Just before performing the analysis, the fluorescent peptide was diluted 1:10 in water, and the inhibitor was diluted 1:1000 in water. A reaction mixture was then prepared containing 10 µl of granulosa cell homogenate and 4 µl of reaction buffer and made up to 90 µl with distilled water in black, flat-bottomed microtiter plates. The reaction was started by the addition of 10 µl of AC-DEVD-AFC working solution to all wells. Controls included wells containing no sample (buffer controls) and wells containing sample plus 5 µl of Z-DEVD-FMK, which were included for all samples. Those samples containing caspase inhibitor were incubated at 37°C for 15 min before the addition of the fluorescent substrate to allow the inhibitor to bind to the caspase-3 in each control sample. The plate was then incubated at 37°C, taking fluorescence readings (excitation wavelength, 390 nm; emission wavelength, 540 nm) every 30 min for 3 h using a Victor microplate fluorometer (Wallac, Turku, Finland). The change in fluorescence for samples (S) and inhibited controls (B) at each time point was determined (S(t) – B(t)), and the change in fluorescence units over time for each sample was calculated as {Delta}S = [S(ti) – B(ti)] – [S(t0) – B(t0)], where t0 is the start of the reaction and ti is the time point where the reaction is still linear. The change in absorbance over time in minutes per milligram of protein in the original sample was then calculated, and caspase activity was expressed as {Delta}S/min/mg.

Animals

All animal studies were performed in accordance with the U.K. Animals (Scientific Procedures) Act of 1986 and with the approval of the University of Nottingham Ethical Review Committee.

Estrous-Synchronized Animals

Heifers (n = 5) were synchronized for estrus by two injections of prostaglandin F2{alpha} given 12 days apart. On Day 5 following estrus, the animals were slaughtered and ovaries collected in warm saline solution. Follicles (dominant, largest subordinate [S1], and second-largest subordinate [S2]) were dissected out of each marked ovary in pairs and were scored according to diameter (mm) and follicular health [41]. Follicular fluid was then aspirated gently from each follicle, centrifuged at 12 000 x g in a microcentrifuge for 5 min at 4°C, and then stored at –80°C until use. Granulosa cells were harvested from each aspirated follicle by hemisection of the follicle and immersion in PBS at 4°C in microfuge tubes. The follicular contents were then removed by gentle vortexing, and the hemisected pieces of follicle were removed and discarded. The collected granulosa cells were then harvested by centrifugation (400 x g for 5 min at 4°C) and resuspended in ice-cold caspase assay lysis solution (final concentrations: 10 mM Hepes [pH 7.4] containing 100 mM NaCl, 0.1% [w/v] 3[(3-cholamidopropyl) dimethylamino]-1-propane sulfonate, 5 mM dithiothreitol, 2 mM EDTA, 0.35 mg/ml of PMSF, 10 µg/ml of pepstatin A, 10 µg/ml of aprotinin, and 2 µg/ml of leupeptin). Next, the cells were freeze-thawed three times in a bath of isopropanol in dry ice followed by a water bath at 37°C and then centrifuged at 12 000 x g in a microcentrifuge at 4°C for 30 min. The supernatant (cytosolic fraction) was collected and stored at –80°C until use.

Nonsynchronized Animals

Ovaries were collected from local abattoirs and washed, and follicles were dissected from ovaries displaying a small corpus luteum within 1 h of collection. The follicles were scored for health as described previously [41] and then hemisected into a sterile Petri dish to enable aspiration of the oocyte. Follicular fluid was aspirated from the Petri dish and treated as described for follicular fluid from synchronized animals. Granulosa cells were harvested and used to make homogenates for the caspase assay as described previously. Collected oocytes (n = 174) were classified as grades 1–4 as described by de Loos et al. [42] and were subjected to in vitro maturation and artificial activation or IVF.

In Vitro Maturation, Fertilization, and Culture

Selected oocytes were washed three times in culture medium TCM 199 (Sigma) supplemented with Hepes buffer and 10% fetal calf serum. The oocytes were cultured individually in 96-well plates multiwell tissue-culture dishes (Nunc, Denmark) containing 50-µl droplets of maturation medium (TCM 199 supplemented with 10 µg/ml of FSH [Follitropin; Bioniche Animal Health, IR], 10 µg/ml of LH [Leutropin; Bioniche Animal Health], 1 µg/ml of estradiol [Sigma], 50µg/ml of gentamicin, and 10% fetal calf serum) under mineral oil in a humidified atmosphere of 5% CO2 in air at 39°C. In vitro-matured oocytes were fertilized as described previously with some modifications [43]. Briefly, motile sperm were prepared after 45 min of swimup in calcium-free medium followed by centrifugation at 300 x g at room temperature and resuspension of the pellet in fertilization medium. The COCs were gently pipetted to remove adhering granulosa cells and to break up aggregated COCs. Disaggregated COCs were then washed once in oocyte wash medium and transferred into 45-µl microdrops of fertilization medium containing sperm (1 x 106 sperm/ml) and cultured for 24 h at 39°C in a humidified incubator of 5% CO2 in air. After 24 h, all presumptive zygotes were denuded from cumulus cells and cultured in 5 µl/embryo of SOFaaci (synthetic oviductal fluid medium supplemented with amino acids, sodium citrate, and myo-inositol) [44] supplemented with 4 mg/ml of fatty acid-free BSA and cultured at 39°C in a humidified incubator with 5% O2, 5% CO2, and 90% N2. The culture was continued up to Day 8, and medium was renewed every 2 days.

Oocyte Activation

To test the capacity of selected oocytes to initiate development, artificial oocyte activation was carried out on in vitro-matured oocytes. After 24 h of maturation at 39°C in an atmosphere of 5% CO2 and maximum humidity, oocytes were individually exposed for 3 min to modified PBS (PBS plus 3 mg/ml of BSA) containing 3 mg/ml of hyaluronidase and stripped from the surrounding granulosa cells by gentle pipetting. Oocytes with a well-extruded polar body were activated by incubation for 5 min in 7% ethanol at room temperature, thoroughly washed, and cultured in cycloheximide (5 µg/ml) for 5 h at 39°C and 5% CO2 in air. After activation, individual oocytes were carefully washed and cultured for 18 h in 20 µl of SOF medium supplemented with 4 mg/ml of BSA under mineral oil. The results from two replicates are presented.

Nuclear Analysis

Activated oocytes were mounted on slides, fixed in ethanol:acetic acid (3:1) for 24 h, and stained with aceto-orcein (1%) for evaluation using a Leica phase-contrast microscope (100x) under immersion oil. Nuclear structures were evaluated as described previously [45]. Pronuclear formation was defined as the presence of a well-formed pronucleus with an evenly granulated nucleoplasm surrounded by a nuclear envelope.

Estradiol Measurements

Concentrations of estradiol in the follicular fluids were measured by RIA [46]. Estradiol assay sensitivity was 0.6 pg/ml, and the inter- and intraassay coefficients of variation were 9.4% and 7.5%, respectively.

Statistical Analysis

The quantities of IGFBP-4 and IGFBP-5 and activity of caspase-3 were compared by scatter plot and nonparametric Kendall tau-b correlation test. The correlation coefficients were calculated using the SPSS statistical package (SPSS Inc., Chicago, IL). The differences in activity of caspase-3 and concentrations of estradiol and IGFBP between follicles were analyzed by one-way ANOVA. Blastocyst development was compared between groups by chi-square analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IGFBP Profile of Follicles from Synchronized Animals

Figure 1 shows the IGFBP content of follicular fluid and caspase-3 activity of the granulosa cells from the dominant and two largest subordinate follicles (S1 and S2) dissected from the ovaries of synchronized cattle on Day 5 after estrus. The dominant follicle contained primarily IGFBP-3 (Fig. 1a, i), whereas the largest subordinate follicle (S1) contained both IGFBP-3 and IGFBP-2. The second-largest (S2) and subsequent subordinate follicles (data not shown) contained increased levels of the low-molecular-weight IGFBP band at 29–31 kDa as well as increased levels of caspase-3 activity, indicating that they are atretic. Both IGFBP-2 (Fig. 1a, ii) and IGFBP-5 (Fig. 1a, iii) could be identified on duplicate blots by Western immunoblot analysis; however, antibodies for the other IGFBPs were not sufficiently sensitive. The IGFBP-5 was confirmed to be a component of the 29- to 31-kDa IGFBP band.



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  		FIG. 1. Measurement of granulosa cell caspase-3 activity and IGFBPs and estradiol-17ß in follicular fluid from ovarian follicles from synchronized heifers on Day 5 after estrus. Heifers (n = 5) were synchronized for estrus and slaughtered on Day 5 following estrus. Follicular fluid and granulosa cells were collected from the individual follicles, representing the dominant and two largest subordinate follicles (S1 and S2, respectively), and were compared for total IGFBP content by Western ligand blot analysis using biotinylated IGF-II (a, i). Duplicate Western blots confirmed the presence of IGFBP-2 (a, ii) and IGFBP-5 (a, iii), particularly in S1 and S2 follicular fluid. The estradiol-17ß content of these same follicles was quantified by RIA (b). The diameter of the dominant, S1, and S2 follicles was compared between all five animals (b), as was the respective caspase-3 activity in granulosa cell homogenates and the 29- to 31-kDa IGFBP content in follicular fluid. mmOD, millimeter optical density; DF, change in fluorescence unit. * vs., **P < 0.05, * vs. ***P < 0.01

Three individual groupings of follicles could be made based purely on IGFBP profile in their follicular fluid when analyzed by Western ligand blot. These were as follows: group A, those containing only IGFBP-3 and negligible IGFBP-2; group B, those containing IGFBP-3 and elevated IGFBP-2 and/or 24-kDa IGFBP-4; and group C, those containing IGFBP-2 and IGFBP-3 plus the 29- to 31-kDa IGFBP band. These groups related to dominant (group A), healthy subordinate (group B), and atretic subordinate (croup C) follicles.

Figure 1b shows a graphical summary of follicular diameter as well as the quantities of the 29- to 31-kDa IGFBP band and caspase-3 activities found in follicular fluid and granulosa cells, respectively, of the three largest follicles from the ovaries of the five heifers. As expected, follicular diameter is seen to decrease sequentially from the dominant to the first (S1) and second-largest (S2) subordinate follicles. A significant, concomitant rise was observed in caspase-3 activity in granulosa cells (dominant vs. S1, P < 0.05; dominant vs. S2, P < 0.01) and low-molecular-weight IGFBP (29–31 kDa) content in their follicular fluids (dominant vs. S1, P < 0.01; dominant vs. S2, P < 0.05) in subordinate versus dominant follicles. Quantitation of estradiol in the follicular fluid of these three groupings confirmed that follicles in group A were dominant follicles and that those in groups B and C were subordinate follicles (Fig. 1b). In addition, the concentrations of estradiol in the dominant follicles were significantly higher than those in the subordinate follicles (P < 0.05).

Correlation Between IGFBP-4 and IGFBP-5 Levels and Caspase-3 Activity

Based on the profile of IGFBPs, the relationship between IGFBP-4 and IGFBP-5 levels, which are increased in atretic follicles, with caspase-3 activity of granulosa cell homogenates derived from these follicles was investigated. Figure 2 shows scatter plots (with regression lines) of the caspase-3 activity in granulosa cell homogenates compared to the 29- to 31-kDa IGFBP content in follicular fluid in both synchronized (Fig. 2a) and nonsynchronized (Fig. 2b), cycling heifers. When analyzed by Kendall tau-b correlation analysis, a highly significant correlation was found between the two parameters (P < 0.001) for both the synchronized and nonsynchronized groups.



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  		FIG. 2. Scatter plots showing the relationship between caspase-3 activity in granulosa cell homogenates and IGFBP-4 and IGFBP-5 levels in follicular fluid. Caspase-3 activities (change in fluorescence unit [{Delta}F]/min/mg) and IGFBP-4 and IGFBP-5 (29–31 kDa) quantities (mmOD, millimeter optical density) were determined for all isolated follicles from both synchronized heifers (a; n = 51 follicles) and nonsynchronized heifers (b; n = 66 follicles). The results were compared by scatter plot, showing a regression line of best fit. The correlation coefficients of these scatter plots was determined by Kendall tau-b analysis and shown to be highly significant (P < 0.001) and is displayed next to the relevant graph

Grouping of Follicles from Nonsynchronized Animals by IGFBP Profile

Follicular fluid samples from individual follicles were analyzed by Western ligand blot. Follicles were grouped according to the three IGFBP groupings identified in the synchronized animals (groups A, B, and C). Figure 3a shows an example of the Western ligand blot for individual follicles allocated to one of the three groups. When the diameters, caspase-3 activities, and 29- to 31-kDa quantities were compared between the three groups of follicles (Fig. 3b), results were very similar to those seen in follicles from synchronized animals (Fig. 1b) (caspase-3 activity: group A vs. group C, P < 0.05; group B vs. group C, P < 0.01; IGFBP-4 and IGFBP-5 content: group A vs. group B and group A vs. group B, P < 0.01).



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  		FIG. 3. Comparison of follicles obtained from nonsynchronized animals grouped according to IGFBP profile. a) Individual follicles from slaughterhouse-derived ovaries were analyzed by Western ligand blot using biotinylated IGF-II and placed into one of three groups (A, B, or C) depending on their IGFBP profile as described in Materials and Methods, b) The follicles within these groups were then compared for mean diameter and caspase-3 activity of their granulosa cell homogenates. mmOD, millimeter optical density; DF, change in fluorescence unit. * vs. **P < 0.05, * vs. ***P < 0.01

Initial experiments using in vitro-matured and artificially activated oocytes derived from individual follicles were performed to test the potential of oocytes to initiate development after grouping into the three categories. Oocytes (n = 44) in group A (100%) had the greatest rate of polar body formation compared those in group B (69%) and group C (57%). However, oocytes derived from group B showed the greatest ability to form pronuclei (91%) compared with those from group A (50%) and from group C (56%). These results suggest that the level of caspase-3 activity is not directly related to oocyte viability as measured by in vitro activation and pronuclear formation. Furthermore, polar body formation cannot be used to predict the potential of the oocyte to form a pronucleus.

Use of Preselection to Identify Viable Oocytes

Based on the results obtained by artificial activation, oocytes were aspirated from slaughterhouse-derived ovaries (three separate experiments), and follicular fluid from each individual follicle was analyzed for IGFBP profile to allocate oocytes into either group A, B, or C as described previously. The oocytes from these follicles (n = 174) were in vitro matured, fertilized, and cultured to examine blastocyst development.

The distribution of oocytes in groups based on either oocyte quality, as defined in the Materials and Methods, or IGFBP expression pattern is shown in Figure 4a. These profiles were very similar to those observed in the previous experiment within the present study also using follicles from nonsynchronized animals. The distribution of oocyte quality between the IGFBP groups (Fig. 4b) indicated that oocytes at grades 1 and 2 were mainly represented within the IGFBP group A and that oocytes at grades 3 and 4 were mainly classified within IGFBP group C. However, it should be noted that a small number of oocytes at grade 4 (n = 6) were also classified into IGFBP group A.



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  		FIG. 4. Outcome of IVF on preselected oocytes from nonsynchronized animals. Follicles were collected from nonsynchronized cycling heifers in three individual experiments. Western ligand blot analysis was performed on their follicular fluid using biotinylated IGF-II to determine their IGFBP profiles, and follicles were assigned to a particular group (A, B, or C) as described in Materials and Methods. The corresponding oocytes from these follicles were scored for COC quality (score of 1–4 as described in Materials and Methods) and then in-vitro matured overnight and placed into their respective IGFBP groups (A, B, or C) before fertilization. The percentage of oocytes in each group (A, B, or C) is shown (a), as is the percentage of COCs of grade 1–4 within each IGFBP group (b). The development of these oocytes was then monitored over 7 days to record cleavage rate, progression at least to the 4-cell stage, and blastocyst formation as described in Materials and Methods (c). * vs. **P < 0.05, * vs. ***P < 0.01

These oocytes were then assessed for developmental potential (Fig. 4c). The number of oocytes undergoing cleavage after IVF was not significantly different between the three groups, whereas progression of the fertilized oocytes to the 4-cell stage was greatest in oocytes from group A and least in oocyets from group C when measured as a percentage of cleaved embryos (P < 0.05). However, oocytes from group B were not significantly different from those of groups A and C. Interestingly, blastocyst formation was significantly greater (P < 0.01) in group B than in group A and group C. No significant differences were found between oocytes from group A and those from group C.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present results demonstrate that the IGFBP expression profile of follicular fluid can be used to better predict oocyte developmental competence. Western ligand blot analysis was used to examine the relationship between the 29- to 31-kDa IGFBP expression profile, of which IGFBP-5 is a component, and the caspase-3 activity in follicles from both synchronized (Fig. 1a, iii) and nonsynchronized (Fig. 3b) heifers. A distinctive IGFBP profile in follicular fluid of ovarian follicles has been demonstrated: The dominant follicle contains only IGFBP-3 and has a high estradiol content, whereas the two largest subordinate follicles contain increasing levels of IGFBP-2 through IGFBP-4 and the 29- to 31-kDa IGFBP band concomitant with reduced estradiol content (Fig. 1). Furthermore, using scatter plots and correlation analysis, an extremely significant correlation between 29- to 31-kDa IGFBP expression in follicular fluid and caspase-3 activity in the granulosa cells, measured in the same follicles, has been demonstrated (Fig. 2). This correlation is similar for follicles obtained from either synchronized or nonsynchronized animals and demonstrates that IGFBP expression patterns can be used to group follicles into healthy, early atretic, and late atretic follicles (group A, group B, and group C, respectively). This appears to hold true in animals at any stage of the estrous cycle. Furthermore, a higher proportion of oocytes derived from follicles in early atresia (group B) progressed to the blastocyst stage after IVF.

Traditionally, measuring follicular atresia has involved a number of approaches. These include scoring either the intact follicle for health markers, such as color and vascularization (wherein the follicular contents are preserved) [41], or, more usually, an analysis of the follicular cells for signs of apoptosis by either nuclear morphology [47], fluorescence-activated cell sorting analysis of propidium iodide-stained nuclei [48], ethidium bromide-stained agarose gels for intranucleosomal fragmentation [49], or in more sophisticated studies, TUNEL staining of either isolated follicular cells or sectioned whole follicles [18]. Biochemical markers of atresia include reduced estradiol concentration and oxygen tension [17]. These are limited, however, because they are not independent of stage of development. The results of the present study support the concept that caspase-3 also is involved in the atresia of bovine follicles (Figs. 1–3). Recently, immunohistochemical localization of active caspase-3 in the mouse ovary indicated that at least in this species, atresia was accompanied by an upregulation of caspase-3 activity in both granulosa cells and oocytes [15], although other reports indicated that oocyte apoptosis occurs via caspase-2 rather than caspase-3 activation [50].

A number of reports have implicated several other biochemical markers of atresia, including Fas antigen, interferon-{gamma}, LH-receptor expression, and tumor necrosis factor {alpha}. Those factors attenuating follicular atresia include the IGF-I and IGF-II ligands, transforming growth factor {alpha}, serum, and insulin. Furthermore, in previous studies, we have found, in agreement with other groups, that the expression of the IGFBPs changes with respect to the stage of follicular development [24]. For example, the expression of the low-molecular-weight IGFBPs in particular appears to be altered, especially during atresia [51]. In both cattle and sheep, these changes have been attributed to an increase in IGFBP-5 and, possibly, IGFBP-4 using two-dimensional Western ligand blot analysis, but we also have shown that several other IGFBP isoforms that likely represent other IGFBPs are present in follicular fluid [38]. For example, IGFBP-1 and IGFBP-6, which were not previously thought to be important in follicular atresia, also are increased (upregulated) in the follicular fluid during atresia [38]. However, to our knowledge, the source of these IGFBPs has yet to be determined.

Because the IGF ligands promote cellular survival and proliferation [52], the upregulation of proteins that bind to these ligands might reasonably be thought to provide a simple attenuation of IGF action, although closer examination in vitro has shown that binding of the ligand and binding proteins can both promote and inhibit cellular proliferation and steroidogenesis in granulosa cells. However, in other cellular models, IGFBP-5 has been implicated in tissue remodeling and apoptosis [35], namely in the regressing mammary gland, which suggests that IGFBP-5 in ovarian tissue is a good candidate for an atretic marker.

In the present study, we have shown, using a range of procedures, that gross morphological assessment of COCs is not an accurate method of determining whether oocytes have been derived from healthy or atretic follicles (Fig. 3) whereas in vitro activation studies have indicated a trend in favor of the developmental competence of oocytes in group B compared with those in groups A and C. These results are exciting. However, the technical challenges involved prevented us from performing sufficient replicates to prove statistical significance, because tests were performed on individually in vitro-matured and artificially activated oocytes (IVF embryos develop better when cultured in groups rather than as single embryos) [40, 53]. Hence, a preselection procedure was developed whereby Western ligand blot analysis was performed during the time of in vitro maturation (usually 24 h for bovine oocytes) to preselect oocytes of different quality before IVF and subsequent culture. This approach allowed oocytes in the same category to be cultured in groups after fertilization, resulting in higher blastocyst rates. The IGFBP profiles, rather than caspase-3 activities, were used as the selection marker, because this procedure is more rapid, does not require such sophisticated or expensive equipment, and has the potential to be refined for dot-blot purposes should an appropriate antibody become available for cattle.

Cleavage rates of the three groupings did not differ significantly, indicating that fertilization rates were identical between the three groups (Fig. 4). This is important, because cumulus coverage potentially could have impeded fertilization in oocytes derived from the less atretic follicles. However, oocyte progression, at least to the 4-cell stage, was significantly reduced in oocytes derived from group C, indicating that highly atretic follicles contain oocytes with reduced developmental competence.

Interestingly, blastocyst formation was highest in group B rather than in group A, where theoretically oocytes are derived from healthier follicles. These data agree with those of previous studies that indicated oocytes derived from early atretic follicles had improved embryo viability [54, 55]. Furthermore, in the human, oocytes obtained from follicles with decreasing aromatase activity show a higher developmental capacity [56]. It is hypothesized that the oocyte of an early atretic follicle may go through changes similar to oocytes from preovulatory follicles [54]. We are unsure, however, whether follicles in group B represent early atretic follicles or simply follicles with reduced growth. Despite their slightly raised caspase-3 activity, these follicles showed low levels of IGFBP-5 expression, indicating that they may not be atretic in any meaningful sense but, rather, may have a lower level of cell proliferation. The absence of IGFBPs other than IGFBP-3 in bovine preovulatory follicles may allow for increased bioavailability of IGF-I, which is important for oocyte maturation and ovulation [51, 57, 58]. However, raising IGF-I concentrations, either by gonadotropins during ovulation induction or by implanting slow-release pumps containing IGF-I, adversely affects developmental progression to the blastocyst stage in mice with a larger percentage of degenerated embryos [59]. Women with polycystic ovarian syndrome exhibit hyperinsulinemia, leading to a decrease in IGFBPs. These women also experience significantly higher rates of pregnancy loss [60]. Therefore, low levels of IGFPBs, as observed in follicles of group B, may modulate IGF and insulin concentration in follicular fluid, resulting in oocytes with a higher rate of developmental potential. This may explain why a lower blastocyst rate is achieved by oocytes of group A, because these oocytes were derived from follicles with negligible IGFBP expression (Figs. 1 and 3).

In conclusion, the present study demonstrated that it is possible to select oocytes from follicles of defined quality using a procedure for a noninvasive marker (i.e., IGFBP expression patterns) that can be performed rapidly enough to preselect the oocytes before IVF. Selection of good-quality oocytes is highly desirable for procedures requiring extensive manipulations, such as pronuclear microinjection, intracytoplasmic sperm injection, and nuclear transplantation, that need to ensure a high developmental competence after embryo transfer. Initial studies by our group indicate that these markers can be quantified in human reproductive tissues and hold the potential for a biochemically based oocyte selection procedure for the benefit of patients undergoing IVF, although further tests regarding in vivo development of oocytes categorized according to IGFBP profiles are needed. These results may enable an improvement in IVF pregnancy rates from the present success rate of approximately 30% by preselection of oocytes on a more biochemically sound basis than simple morphological evaluation, which is currently used and is a rather nonempirical method of assessment.


   FOOTNOTES
 
1 B.N. was funded, in part, by the BBSRC (U.K.), and A.A.F.-N. was supported by Defra (U.K.). Back

2 Correspondence: R. Webb, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, U.K. FAX: 00 44 115 951 6060; bob.webb{at}nottingham.ac.uk Back

3 Current address: Division of Infection, Inflammation and Repair, Mailpoint 810, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, U.K Back

Received: 6 September 2004.

First decision: 13 October 2004.

Accepted: 8 November 2004.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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