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Biology of Reproduction 65, 726-732 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Role of Gelatinase on Follicular Atresia in the Bovine Ovary1

M.A.M. Yahia Khandokera,b, Kei Imaia, Toru Takahashia, and Kazuyoshi Hashizume2,a

a National Institute of Agrobiological Sciences, Kukizaki, Ibaraki ken 305-0901, Japan b Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

ABSTRACT

Follicular atresia, like follicular growth and ovulation, is characterized by excessive tissue remodeling. It is hypothesized that probably one of the tissue-remodeling enzymes, such as the gelatinases, could be playing an important role in this process. The present study was undertaken to determine the role of gelatinase on follicular atresia in the cow. Follicles of 2–6 mm in diameter were dissected from ovaries, and follicular fluid was categorized according to the morphological appearance of the cumulus-oocyte complexes. Gelatinase activity within the follicular fluid was analyzed by gelatin zymography, and film in situ zymography was employed in order to localize gelatinase. TUNEL was performed on cryosectioned ovaries to understand follicular health. The concentrations of steroids in follicular fluid were also measured by solid phase fluoroimmunoassay. ProMMP-2 was detected in all normal and atretic categories of follicular fluid. The active form of MMP-2 and an additional band of proMMP-9 were detected only in atretic follicular fluid. Gelatinase activity was recorded in both granulosa cells (GCs) and theca cells (TCs) but were found in comparatively higher numbers in those follicles that exhibited a thinned and partially detached granulosa layer. TUNEL confirmed that apoptosis had commenced in the GCs of follicles of the latter category. The estradiol-17ß (E2):progesterone (P4) ratio was found to be significantly lower in atretic follicles than in normal follicles. These results suggest a plausible role for gelatinase in follicular health, especially the active form of MMP-2 and proMMP-9, and that bovine follicular fluid may be a key indicator of atresia.

apoptosis, follicle, ovary, steroid hormones

INTRODUCTION

The ovary is a heterogeneous organ consisting of follicles at different maturation stages. Each of two or three follicular waves emerges during the bovine estrous cycle [1, 2] and each wave of follicular development is characterized by the simultaneous emergence of medium-sized (>4 mm) growing follicles from a pool of small follicles. A group of these follicles rapidly emerges as the dominant (>7 to 9 mm) and continues to develop, whereas the others undergo atresia and regress. Despite the overwhelming occurrence of follicular atresia in the ovary [3], the cellular and molecular mechanisms underlying this phenomenon still remain poorly understood, although many studies on the onset and progression of follicle atresia have been performed [4–9]. Follicular cytology (granulosa and theca cells) and steroid (progesterone, androgens, and estrogens) concentrations in follicular fluid are the primary factors that have been used to classify follicular status [10,11]. Correlations between histological analysis and endocrine evaluation of follicular status were not found useful [7] and other factors may be involved in this process but, as far as we know, no attempts have been made to clarify this possibility.

Previous studies have suggested that degenerative changes associated with atresia appear initially in the granulosa cell (GC) layer. The death of GCs leads to almost complete destruction of the GC layer lining the inner follicular wall [12] and triggers follicular atresia [13]. Other studies have demonstrated that the death of GCs during follicular atresia in ewe, pig, chicken, cow, and rodent ovaries occurs by apoptosis, which is a physiological, active, and genetically governed process whereby the death of cells occurs in a controlled fashion that is triggered by changes in the level of specific physiological stimuli [13]. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that possess proteolytic activities against several components of the extracellular matrix (ECM) [14]. They are secreted as inactive proenzymes, which become active by the removal of an amino-terminal peptide. The function of the active enzyme is to digest the ECM [14]. MMPs are divided into collagenases, gelatinases, and stromelysins according to their substrate specificity, and various tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of these enzymes. MMPs play a critical role in ECM remodeling associated with follicular growth [15], ovulation [16–19], as well as the development and regression of the corpus luteum [20]. Data on differential distribution of MMPs and TIMPs in the ovary of a number of species has already been published [21–24]. It has been postulated that like follicular growth [15], ovulation [16–19], and corpus luteum formation and regression [20], follicular atresia could involve extensive tissue remodeling, and that gelatinase may play an important role in this process. Therefore, in this study, the role of gelatinase was investigated in bovine follicles by employing gelatin and film in situ zymographies. Although atresia occurs at all stages of follicle development [25], the majority of follicles undergo degeneration at the early antral stage rather than at the preantral and preovulatory stages [26]. Considering that atresia is a stage-dependent process, gelatin zymography in follicular fluid was performed on follicles of 2–6 mm in diameter.

MATERIALS AND METHODS

Collection of Ovaries and Follicular Fluid

Bovine ovaries were collected from a local slaughterhouse and transported to the laboratory within 3–5 h after collection. Antral follicles measuring 2–6 mm in diameter were dissected from the ovaries (n = 95) and the follicular materials were harvested by blunt dissection from the prior follicles. Follicular atresia is a complex process and depends on interaction between the theca, granulosa, and oocyte [9]. Cumulus-oocyte complexes (COCs) were observed with light microscopy and the following criteria were used to classify follicular fluid: 1) normal, having regular, partially regular, and compact COCs, 2) atretic, those having no COCs, or they were in an expanded state. This classification is usually used for collecting oocytes for in vitro maturation/in vitro fertilization experiments. The follicular material was then centrifuged at 1000 x g for 10 min at 4°C, after which the follicular fluid was collected and stored at -30°C until used.

Protein Estimation

The protein concentration of each batch of follicular fluid was determined by the Bradford method [27] with the principle of reduction of Coomassie brilliant blue G-250 dye (Bio-Rad Laboratories, Melville, NY) and using BSA (Sigma Chemical Company, St. Louis, MO) as the protein standard.

Zymography and Quantitative Measurement

Metalloproteinase activity in follicular fluid was performed with gelatin zymography as described by Nakamura et al. [28] but with the following modifications. Briefly, 10 µg of total protein per sample was subjected to SDS-PAGE using a 12.5% (w:v) acrylamide slab gel containing 0.6 mg/ml of gelatin under nonreducing conditions. After electrophoresis, the gel was extracted for 30 min at room temperature in 50 mM Tris-HCl, 5 mM CaCl2, 1 µM ZnCl2, and 0.02% (w:v) NaN3 pH 7.5 containing 2.5% (w:v) Triton X-100, and washed three times for 10 min each in distilled water. The gel was then incubated for 15 h at 37°C in the same buffer without 2.5% Triton X-100. After incubation the gel was again washed three times for 10 min each in distilled water and stained with 0.1% (w:v) Coomassie Brilliant Blue R-250 in 50% (v:v) methanol and 20% (v:v) acetic acid. Finally, the gel was destained in 45% (v:v) methanol and 9.2% (v:v) acetic acid. The proteolytic activity appeared as clear bands on a blue background. The relative molecular mass (Mr) of gelatinolytic MMPs was determined by comparison with SDS-PAGE molecular weight protein markers (Bio-Rad) in an adjacent lane.

In order to determine the type of gelatinase observed on the zymograms, three samples from each group were incubated with 10 mM aminophenylmercuric acetate (APMA) at a final concentration of 2 mM for 1 h at 37°C prior to SDS-PAGE. APMA is an organomercurial compound that is widely used to activate precursor or inactive forms of MMPs [29]. For more confirmation, in another experiment, 10 mM EDTA (Ca2+ chelator) was added to the buffer during the incubation period. All procedures were performed as they were for gels without EDTA in the incubation buffer. In the zymogram, the migration fashion of bovine MMP-2 and MMP-9 was also compared with human MMP-2 and MMP-9.

The quantitative measurement of MMPs in each follicular fluid was performed by densitometric analysis of the zymogram by using the National Institutes of Health 1.62 image program. The pooled follicular fluid collected from follicles 2–6 mm in diameter was used as the control and the intensity of MMP was presented as a densitometric concentration after standardization with control. All reagents for electrophoresis were purchased from Bio-Rad and others for zymography were obtained from Sigma.

Steroid Assay

Steroids (E2, P4, and testosterone) profiles of follicular fluids were measured in a solid phase fluoroimmunoassay based on competition between europium-labeled steroids and sample steroids for polyclonal antisteroid antibodies derived from rabbit. The DELFIA steroid reagents (Wallac Inc., Gaithersburg, MD) were used and the manufacturer's instructions were followed during the experiment.

Collection, Preparation, and Cryosectioning of the Ovary

After a brief survey of the surface morphology, five ovaries were selected at random and were embedded in Tissue-Tek OCT compound (Sakura Finetechnical Co., Ltd, Tokyo, Japan). These were frozen immediately in liquid nitrogen without fixation and stored at -80°C before sectioning. They were sectioned with the Microtome Cryostat HM 500 OM (Microm Laborgeräte, GmbH, Germany) until an oocyte appeared in any one of the follicles. Serial sections were taken for film in situ zymography (FIZ), TUNEL, and hematoxylin and eosin (H&E) staining.

Film In Situ Zymography

For localization of gelatinase activity in follicles, FIZ was performed according to the method described by Tamura et al. [30]. We used GN film (Fuji Photo Film Co., Ltd, Tokyo, Japan) coated with a gelatin base emulsion to detect and localize the gelatinase activity in underlying tissue. Six-micron cryostat unfixed ovarian sections were mounted on the coated film, followed by incubation for 12 h at 37°C, and staining with biebrich scarlet (BS; Chroma-Gesellschaft, mbH & Co., Münster, Germany) and hematoxylin. Gelatinase activity was detected when a pattern of gelatin digestion was found that was in accordance with localization of gelatinases of the specimens as recorded by a white color due to the weaker staining of BS.

TdT-Mediated dUTP-Biotin Nick End Labeling

TUNEL was performed to confirm programmed cell death (PCD) or apoptosis. An in situ cell death detection kit (Boehringer-Mannheim, Mannheim, Germany) was used and the experiment was performed according to the kit instructions. Briefly, 6-micron serial sections of ovary were mounted on superfrost micro-slide glass (Matsunami Glass Industries, Ltd., Osaka, Japan) and fixed with 4% paraformaldehyde for 20 min at room temperature, and then washed for 30 min with PBS. The slides were rinsed twice again with PBS and the area around the sample was dried. Labeling was initiated by adding 50 µl of TUNEL reaction mixture and incubation in a humidified chamber for 1 h at 37°C. After incubation the slides were rinsed three times with PBS and observed under a fluorescence microscope at an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm (green). A positive control was included in each experiment by incubating the serial ovarian section with DNase (grade I, 20 µg/ml in 50 mM Tris-HCl pH 7.5, 1 mg/ml BSA) for 10 min at room temperature to induce DNA strand breaks prior to labeling. The dose of DNase was confirmed through an initial experiment. The negative control was also included by incubating serial sections with 50 µl of label solution instead of with TUNEL reaction mixture.

Statistical Analysis

The densitometric unit of MMPs and steroid concentrations in follicular fluid are presented as means ± SEM. Data were analyzed by the Student t-test for comparison of the means. P values of < 0.05 were considered significant.

RESULTS

A total of 95 follicles were collected from 12 ovaries, and among them, 70 were categorized as normal, whereas the remaining 25 were classified as atretic.

The MMPs (MMP-2 and MMP-9) in the follicular fluid were quantified by gelatin substrate zymography, and the results are shown in Figure 1. ProMMP-2 was detected in all follicular fluids regardless of their category. The active form of MMP-2 (n = 10) plus an additional band of proMMP-9 (n = 7) were detected only in the atretic category of follicular fluids (Fig. 1A). The intensity of MMPs in control follicular fluids did not vary significantly in the zymograms. The intensity of proMMP-2 was found to be significantly higher in atretic (P < 0.01) than in normal follicular fluid (Fig. 1B).



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  		FIG. 1. Gelatinase activity in bovine follicular fluid detected by gelatin zymography. Gel zymography was performed as described in Materials and Methods. A) Representative gelatin zymogram. Equal amounts of total protein (10 µg/lane) were loaded into the wells of 12.5% acrylamide gels. B) Relative densitometric concentrations of proMMP-2 (n = 70 and 25 normal and atretic follicles, respectively), MMP-2 (n = 10), and proMMP-9 (n = 7). ProMMP-2 levels in normal follicular fluid was considered as 100. **Differed significantly from normal (P < 0.01). Mr, Relative molecular mass; M, protein marker; ND, not detected

Incubation of both groups of follicular fluids with 2 mM APMA for 1 h at 37°C to activate the latent enzyme resulted in a shift in the degraded region (approximately 62 kDa) on the gel (Fig. 2). The gelatinase activities were also completely inhibited by EDTA (data not shown). In a related experiment, we confirmed that bovine MMP-2 and MMP-9 migrated in fashions similar to human MMP-2 and MMP-9 on the zymogram (data not included). These findings confirm that the clear bands on the zymogram in Figure 1A represent bona fide gelatinase.



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  		FIG. 2. Gelatin zymogram of follicular fluid without (-) and with (+) APMA. Gelatin zymography was performed as described in Materials and Methods. Equal amounts of total protein (10 µg/lane) were loaded into the wells of 12.5% acrylamide gels and samples denoting plus signs were incubated with 2 mM APMA for 1 h at 37°C to activate the enzymes prior to SDS-PAGE. MMPs were activated due to APMA treatment in both groups of samples. Mr, Relative molecular mass; M, protein marker

Serial ovarian sections from five ovaries were taken for FIZ and TUNEL assessments, and a total of 59 follicles were examined. The lysis of the gelatin on the film by gelatinase was expressed as a white color, whereas the red areas of BS stain represented as absence of gelatinase activity (Fig. 3, A and B). Use of hematoxylin and eosin (H&E) staining in the serial section of the follicles (Fig. 3C) allowed us to localize gelatinase activity [31, 32]. The gelatinase activity was recorded in the GC and TC zones from early antral to antral stages. Among them, 32 follicles expressed higher gelatinase activity (represented in follicle 2, Fig. 3B) and the remaining 27 follicles depicted less activity (represented in follicle 1, Fig. 3B). Gelatinase activity was always comparatively higher in those follicles that exhibited a thinned and partial detached GC layer from the basement membrane (represented in follicle 2, Fig. 3C), whereas the follicles that had a healthy granulosa layer showed less gelatinase activity (follicle 1 in Fig. 3C).



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  		FIG. 3. Gelatinase activity in the representative cryosection of bovine ovary A). Six-micron unfixed sections were mounted onto GN film, followed by incubating for 12 h at 37°C and staining with BS and hematoxylin. The lysis of the gelatin on the film by gelatinase is expressed as a white color by BS staining. B) Two follicles at higher magnification (x100) marked by the rectangle in A. The higher gelatinase activity is recorded in the granulosa layer of follicle 2 (arrows). C) H&E staining (x100) in a serial section of the same follicle. Note the intact and well-organized multilaminar GC in follicle 1, whereas follicle 2 shows a considerably thinned and partially detached GC layer from the basement membrane (arrows). Bar = 100 µm

TUNEL was performed on serial sections of ovary. Fifty-nine follicles were examined, and the results are shown in Figure 4. TUNEL differentially fluoresced the atretic follicles and regressed corpus luteum (not shown). Nuclei of GCs and scattered TCs within follicles fluoresced at different intensities. However, apoptosis was not recorded in cumulus cells, even in atretic follicles (Fig. 4b). TUNEL confirmed that DNA fragmentation had commenced in GCs (Fig. 4b) that appeared thin and had partially detached from the basement membrane. This is shown in the H&E staining of the serial section of follicle 2 in Figure 3C (n = 12), which had recorded higher gelatinase activity on FIZ film (Fig. 3B, follicle 2). The remaining 47 follicles were TUNEL-negative, although 13 of them showed higher gelatinase activity. Pretreatment with DNase I caused an intense staining of all nuclei in the preparation (positive control; Fig. 4c). Lack of staining was evident in the nontreated group (negative control; Fig. 4d), thereby confirming the feasibility of this method.



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  		FIG. 4. TUNEL in a serial section of bovine ovarian follicles. Panels a and b are the in situ 3' end-labeling; c is the positive control (pretreatment with DNase; 20 µg/ml) and d is the negative control. These two follicles are the serial observation of follicles 1 and 2 in Figure 3, B and C. Note the TUNEL-positive signals in GCs of follicle 2 that display a thin and partial detached GC from the basement membrane and higher gelatinase activity in Figure 3, C and B, respectively. Bar = 100 µm

The mean concentrations of steroids in follicular fluid are summarized in Figure 5. The E2 concentration was found to be minimal (54.2 ± 7.6 [n = 65] and 58.9 ± 24.5 [n = 17] pg/mg of protein in normal and atretic follicles, respectively) and the values were almost the same regardless of category. Concentrations of P4 and testosterone were found measurable in nanograms (P4, 2.2 ± 0.3 [n = 55] and 5.3 ± 0.8 [n = 20]; T, 7.0 ± 0.5 [n = 75] and 2.2 ± 0.2 [n = 20] ng/mg of protein in normal and atretic follicles, respectively). Progesterone concentration was significantly higher (P < 0.001) in atretic than in normal follicular fluid, whereas the testosterone concentration was significantly lower (P <0.001) in atretic than in normal follicular fluid. The ratio of E2 to P4 was found to be significantly lower (P < 0.05) in atretic follicles than in normal follicles; values were found to be less than 1.0.



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  		FIG. 5. Changes in steroids of follicular fluid collected from normal and atretic follicles 2–6 mm in diameter. E2, Estradiol in pg (n = 65 and 17 for normal and atretic follicles, respectively); P4, progesterone in ng (n = 55 and 20 for normal and atretic follicles, respectively); and T, testosterone in ng (n = 75 and 20 for normal and atretic follicles, respectively). All values represent protein in milligrams. ***Differed significantly from normal (P < 0.001)

DISCUSSION

This study demonstrates that in atretic follicular fluid, proMMP-2 is present at a higher intensity, an active form of MMP-2 is present, and that an additional band of proMMP-9 is present. MMPs are considered to be the most important agents of tissue degradation during growth and remodeling [33, 34]. Within the ovary, they may permit turnover and reconstruction of the follicle wall at the time of growth [15] and ovulation [16–19], and facilitate the remodeling of tissue during corpus luteum formation and development [20, 35]. Bovine antral follicles are composed of various cells such as granulosa and theca cells, fibroblasts, and endothelial cells [36]. The exact origin of gelatinase in follicular fluid is uncertain, although granulosa cells and their derived cells are believed to the source of the enzymes [37]. On the other hand, the expression of MMP genes appears to be up-regulated in the theca cells of large preovulatory and ovulating follicles [38]. As far as we know, our in situ zymography is the first report to localize gelatinase activity in bovine ovarian follicles and are parallel with the findings of other researchers. Using defined culture systems, gelatinase activity was detected in the conditioned medium of human GCs [39].

Our findings revealed that atretic follicular fluid had higher proMMP-2 concentrations. Because atresia is related to the regression of the follicle and involves extensive tissue remodeling, our results support the hypothesis that MMPs are the local factors that contribute to the structural and functional changes during follicular atresia. Collagenases, gelatinases, stromelysins, and TIMPs comprise the MMP enzyme system [33]. MMP-2 is a gelatinase, possessing the ability to degrade and denature basement membrane collagens [33]. The role of gelatinase has been reported during ovulation as contributing to degradation of the follicular wall [40]. The follicular wall ECM is composed mainly of type IV collagen, fibronectin, laminin, and heparan sulfate proteoglycan [41, 42]. Thus, the relatively higher amount of proMMP-2 enzyme may serve as an extracellular activator during follicular atresia in vivo.

MMP-9 is also a gelatinase and plays a role in various tissue remodeling processes [43, 44]. The detection of proMMP-9 only in atretic follicular fluid further suggests its role in follicular atresia. In the present experiment, proMMP-2 was by far the most abundant MMP, although active MMP-2 plus an additional band of proMMP-9 were detected only in atretic follicular fluid that exhibited a typical plasma MMP profile [45]. This finding collaborates well with earlier reports [46]. In both groups of follicular fluids, there was a concomitant increase in the activity of the 62-kDa region upon activation with APMA, a complete inhibition of gelatinase activities by EDTA, and a similar migration pattern of bovine MMP-2 and MMP-9 to that of human, thus indicating that the enzymatic activity was due to bona fide gelatinase.

The ovaries are the major sites of production of steroid hormones and the pattern of steroid production depends on follicle diameter [11] and health [47, 48]. In normal follicles, the presence of higher testosterone and low or undetectable concentrations of estradiol in follicular fluid suggest that early developing antral follicles may develop a competence to synthesize androgen before appreciable aromatase activity is expressed in GCs [49]. The theca interna from healthy follicles as well as atretic follicles is reported to have LH/hCG receptors and that the binding characteristics of these receptors do not change with increasing atresia [50]. It was further reported that as follicles degenerate from early to late stages, the theca internae lose their capability of secreting androgen in response to LH [50, 51]. The lower concentrations of testosterone in atretic follicles are consistent with the above reports. Because atretic follicles contained higher (P < 0.001) progesterone levels than normal follicles do, it seems reasonable to conclude that increased progesterone synthesis by GCs is a common event during atresia in bovine follicles [50, 52]. Higher concentrations of E2 were reported in normal follicles than in atretic follicles [5–9]; however, our findings contradict these reports. Failure to find an increased level of E2 in normal follicles may be due to the small size of the follicles (2- to 6-mm diameter) employed in this study, as most of the previous studies were conducted in follicles larger than 6 mm diameter. To our knowledge, this is the first documentation of follicular hormone concentrations in individual follicles of 2–6 mm in diameter. Our data suggest that healthy, 2- to 6-mm follicles are biochemically competent to produce testosterone, but at this stage, GCs lack the capability of metabolizing this androgen to estradiol [49]. Further studies are needed to confirm this. The level of E2 found in both categories of follicular fluid may be optimal for follicles that are 2–6 mm in diameter. The ratio of E2 to P4 or androgens could be used to differentiate normal follicles from atretic follicles and the ratios were reported to be less than 1.0 in the latter [5]. Our ratios support the above statement, and the values were found to be less than 1.0. These findings suggest that for a stated size of follicle, not E2 but P4, testosterone, and the E2:P4 ratio could be key tools for classifying follicular health.

Ovarian follicles, each containing an oocyte, are embedded in the cortex of the ovary. Of the few hundred thousand follicles present at birth, only a small fraction reach maturation; the remainder are lost through the process of atresia [7]. Programmed cell death is a selective process of physiological cell deletion and exerts a homeostatic function in relation to tissue dynamics, as the steady state of continuously renewing tissues is achieved by a balance between cell replication and cell death [53]. TUNEL is a useful tool for in situ identification of PCD and, based on the specific binding of terminal deoxynucleotidyl transferease (TdT) to 3'-OH ends of DNA, synthesis of a polydeoxynucleotide polymer ensues. To expose nuclear DNA on histological sections to proteolytic treatment, TdT was used to incorporate biotinylate at sites of DNA breaks, which gave the signals [54]. Differential staining of atretic follicles and regressed corpus luteum confirmed cell degeneration [54]. In bovine follicles, the initial structural changes associated with cell degeneration occur primarily in the granulosa layer [9] and DNA fragmentation is initiated at the nuclear periphery and progresses toward the center [54]. The present experiment revealed that DNA fragmentation commences in GCs that display considerable thinning and partial detachment from the basement membrane, confirming follicular atresia. Follicles showed higher gelatinase activity but were TUNEL-negative (not shown), and may suggest a role for gelatinase in follicular development [15], however, this deserves further investigation. Due to an unknown reason, a TUNEL-positive signal was not recorded in the cumulus cells, even in atretic follicles [55]. Intense and lack of TUNEL signals were found in positive and negative controls, respectively, which confirmed the acceptability of this technique (Fig. 4).

Although a direct comparison was not made between gelatinase activity and steroid levels, a positive correlation was found between MMPs and progesterone levels in atretic follicles. This finding contradicts previous reports that progesterone down-regulates the production of proMMP-9 and/or activation of MMP-9 in human [56, 57] and rabbit [58] uteri. Most of the above studies suggested that progesterone preserves the integrity of the tissues needed for the establishment and maintenance of pregnancy by limiting MMP activities. As far as we know there is no report in literature on the correlation between progesterone level and MMP activity in bovine follicles, and our findings suggest that the function of progesterone in follicles may differ from that in uterus and could explain the discrepancies found.

Higher gelatinase activity was found in atretic follicles than in normal follicles, and TUNEL confirms follicular atresia in the former. In conclusion, these results suggest a plausible role for gelatinase in follicular atresia, and that the active form MMP-2 and proMMP-9 come into existence in follicular fluid may be a key indicator of atresia.

ACKNOWLEDGMENTS

We thank Dr. Ryoichi Nemori (Ashigara Research Laboratories, Fuji Photo Film Co., Ltd) for providing in situ zymography film, Mrs. Sanae Hamanaka for hormone assay assistance, and Dr. O.V. Patel for a critical reading of the manuscript. M.A.M. Yahia Khandoker is grateful to the Vice Chancellor of Bangladesh Agricultural University for granting leave to carry out this research in Japan.

FOOTNOTES

First decision: 5 December 2000.

1 M.A.M.Y.K. was a recipient of a Science and Technology Agency (STA) fellowship from the Japan Science and Technology Agency (JST) to carry out research at the Laboratory of Reproductive Endocrinology, National Institute of Animal Industry, Tsukuba, Ibaraki, Japan. Back

2 Correspondence: Kazuyoshi Hashizume, Laboratory of Reproductive Biology and Technology, National Institute of Agrobiological Sciences, Kukizaki Campus, 2 Ikenodai, Kukizaki, Ibaraki 305-0901, Japan. FAX: 81 298 38 8606; kazuha{at}affrc.go.jp Back

Accepted: April 13, 2001.

Received: October 19, 2000.

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