Abstract
This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-D) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.
Keywords
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CLSM has been used primarily to study single cells and small tissue sections of minimal thickness. Applications of the technique have been made using human oocytes and embryos, but the tissues used were very early stages of development or isolated cells (Levy et al. 1997). Previous studies have combined confocal microscopy with various nucleic acid stains to visualize DNA and RNA in individual cells (Matsuzaki et al. 1997; Suzuki et al. 1997). The technique uses a specific laser line, appropriate barrier and dichroic filters, and a selected pinhole size to eliminate light that is out of focus in the image plane (Schotton and White 1989; Pawley 1995). The resulting image appears sharper and has a greater resolution than an image obtained with a standard fluorescence microscope. The laser light of the confocal microscope can penetrate deeper into the tissue than fluorescent light derived from a conventional fluorescent microscope. However, the light becomes scattered, reflected, refracted, and absorbed on entering, traversing, and exiting the tissue. The result of scanning into deep tissue is reduced light intensity with less optical quality. Therefore, depending on the light-gathering characteristics of the objective and the power of the laser light, the imaging of an ovary would typically be limited to a tissue depth of less than 100 μm. However, with proper sample preparation to make the tissue relatively transparent, it is possible to circumvent many of these obstacles in CLSM imaging (Zucker et al. 1998).
During mammalian oocyte development, oogonia that are formed from proliferating germ cells undergo a further meiotic division to reach an arrested diplotene stage. At the same time, each becomes surrounded by a structure of supporting cells, which together comprise a primordial follicle. Under hormonal influence, both the arrested oocyte and this immature follicle must progress through a number of further maturational stages before ovulation takes place (Hirshfield 1997). Comparisons between oocytic and follicular maturation in the rodent have shown that if the oocyte diameter is plotted against follicular indices, two phases of growth become evident (Brambell 1928; Arendsen de Wolff-Exalto and Groen-Klevant 1980; Gosden 1995). In the first, there is a rapid stimulation of oocyte growth, increasing the oocyte diameter by 3.5- to fourfold. After the oocyte reaches about 70-80% of its final size, this is followed by a second growth phase, which is characterized by a 10-fold progression in follicular volume that is primarily attributable to an accumulation of follicular fluid with the emergence of an internal antral cavity. The progression in development from a primordial follicle to a mature preovulatory follicle requires the proliferation and differentiation of granulosa cells surrounding the oocytes. Few follicles actually progress this far, and it is estimated that upwards of 95% undergo atresia during their antral period (Greenwald and Terranova 1988).
It has previously been demonstrated that programmed cell death (apoptosis) can be visualized in 8-9-day whole embryos treated in vitro with hydroxurea (Zucker et al. 1998). This study demonstrates that by using a similar sample preparation procedure, oocytes and follicles can be visualized in three dimensions in solid ovarian tissue. By combining LysoTracker stain with morphological criteria, atretic and regressing follicles were revealed in ovarian tissue. LysoTracker Red stained the dying granulosa cell layer in atretic follicles to a greater extent than in healthy antral follicles, as measured by relative fluorescent intensity.
In this study, to optimize the resolution of oocytes located in ovarian tissues, various dyes were incorporated into the ovarian tissue before confocal observation. Without the incorporation of fluorescent dyes, the tissue cannot be visualized. The dye specificity can indicate unique features of the developing follicle. LysoTracker Red stains lysosomes and acidic regions of cells/tissues (Zucker et al. 1998). The fluorescence analogue of actinomycin D, 7-aminoactinomycin D (7ADD), is an intercalating dye that binds to single-stranded DNA/RNA molecules, active chromatin, the nucleolus, and acidic environments (Wadkins and Jovin 1991; Linden et al. 1997). In the cell, hydroethidine (HE) is converted to ethidium bromide (EB), which stains intercellular nucleic acids. The amount of HE staining may be related to the metabolic activity of the cell and/or to the quantity of free radicals inside the cell (Robinson 1994). These fluorescent dyes were capable of fluorescence after the fixing, dehydration, and clearing process, making them very suitable for the studies described here (Poot et al. 1996; Zucker et al. 1998).
Materials and Methods
Animals
Adult 4-5-month-old female Long-Evans hooded rats (Charles River Laboratories; Raleigh, NC) were monitored for estrous cyclicity by daily evaluation of vaginal cytology. Those exhibiting either 4- or 5-day cycles were sacrificed with CO2 on diestrus. Ovaries were quickly removed, trimmed of fat and bursal membranes, and cut into 2-4-mm-thick sections before staining and fixation.
Fixation
Paraformaldehyde (PF 20%; Electron Microscopy Sciences, Ft Washington, PA) was diluted to 4% using Dulbecco's PBS (GIBCO; Gaithersburg, MD) and the PF was stored frozen at −20C. Glutaraldehyde (25%; Electron Microscopy Sciences) was diluted to 2% in PBS and stored frozen at −20C. The 2-4-mm-thick sections of ovaries were washed and either fixed immediately at 4C for 18 hr or stained immediately for 30 min at 37C with either LysoTracker Red or HE.
Staining
To visualize the ovarian tissue using a fluorescence microscope, it is necessary to stain the tissue with a fluorescent dye. Dyes having specificity to nucleic acids and excitable using visible wavelengths (488 nm, 568 nm) were initially chosen to visualize the solid ovarian fixed tissues. The following nucleic acid binding dyes were purchased from Molecular Probes (Eugene, OR): YoPro-1 (Yo-Pro, Y3603) 7-aminoactinomycin-D (7AAD, A1310), ethidium homodimer-1 (EB, E-1169), and Bo-Pro-1 iodide (Bo-Pro, B3587). Dyes were diluted to a final concentration of either 5 μM solution (Yo-Pro, Bo-Pro, Sytox) or 5 μg/ml (EB, 7AAD) in PBS. EB and 7-AAD were first dissolved in 1 ml of DMSO (1 mg/ml) before their final staining concentrations. Two to four ovary quarters were washed in PBS. After the PBS was removed, 500 μl of a dye solution was added to each tube containing the fixed tissue. The tubes were sealed with para-film and incubated in a 37C water bath for approximately 24 hr.
To allow the dyes to penetrate into the tissue before fixation, two dyes [hydroethidine (HE, D1168); LysoTracker Red (L7528); Molecular Probes] were used on ovarian tissue that were proved to be effective using a similar procedure on embryonic tissue (Zucker et al. 1998). LysoTracker Red was chosen because of its ability to be fixed with aldehyde fixatives and its ability to withstand the dehydration and clearing procedures (Zucker et al. 1998,1999). LysoTracker Red concentrated in acidic compartments of lysosomes and apoptotic cells. An added benefit of this dye is that its background fluorescence was sufficient to reveal morphological structures of the embryos and follicles.
The rationale for using HE was that it readily penetrates into tissue and is metabolically converted into EB, which effectively binds nucleic acids. EB is located on the interior of the cell, where it is normally inhibited from penetration by the cell membrane. It was also shown that EB and the other nucleic acid dyes were able to withstand the fixation, dehydration, and clearing process.
Only LysoTracker Red and HE were successfully used in fresh tissue; all the other dyes were applied to aldehyde-fixed tissue. HE and LysoTracker Red were dissolved in DMSO at 100 μM and 500 μM, respectively. The dyes were diluted (100-fold) directly into Hepes-buffered 199 medium (GIBCO). Unfixed, quartered ovarian tissues were incubated with the dye for 30 min before being washed twice and fixed in 4% PF made in Hepes buffered 199 medium. Relatively high concentrations of dye were used on fresh tissue for short periods of time to enable adequate staining of the tissue while preventing degeneration of the tissue before fixation. The LysoTracker Red should be checked on a given tissue because the product delivered from Molecular Probes comes with different activities, dependent on the lot number, and it appears that newer lots are more active than the older lots.
Dehydration
After staining, the ovarian sections were washed with PBS to remove the fixative. They were then dehydrated in MEOH with successive 15-min washes of 50% (v/v), 70%, 95%, and 100%. A final 100% MEOH wash was performed to ensure that the ovary was dehydrated. MEOH solutions were made by diluting 100% MEOH with distilled water. After the 100% MEOH wash was completed, the tissue was transferred into a clean glass tube without MEOH.
Clearing
A clearing solution consisting of benzyl alcohol and benzyl benzoate (1:2 by volume, BABB; Sigma, St Louis, MO) was used. The refractive index of this solution was similar to the refractive index of the dehydrated ovary sections. Ovarian sections of 2-4 mm were cleared by sequential placement in 50% BABB in methanol (0.5-2 hr), 70% BABB in MEOH, and finally in 100% BABB. Depending on the stain used, the tissues can be difficult to observe after the clearing procedure. After careful removal of the BABB medium, the ovarian sections could be observed by light refraction. The ovarian pieces were then transferred into either a 2-mm-thick depression slide (Fischer 12-565A) or a specially made aluminum slide (2.7-mm thick) containing a 12.7-mm hole. A 22 × 30-mm coverslip (#1.5) was sealed to the bottom of the chamber of the aluminum slide with three or four successive coats of nailpolish (Sally-Hard as Nails; Del Laboratories, Framingham, NY), creating a well for the thick ovarian quarters. The ovarian quarters were then placed in the glass or aluminum depression slide and fresh BABB was added into the depression slide and finally sealed with a 20 × 30-mm coverslip. It is essential to seal the depression slide because the BABB cocktail is very harmful to microscope components and objectives. Only dry microscope objectives were used in this study. BABB tends to dissolve nailpolish with time, and subsequent coatings may be necessary.
Confocal Microscopy
The ovarian tissues contained in the sealed depression slide were imaged with a Leica confocal microscope (TCS4D). This system consists of a Leica inverted IRMB microscope and an Omnichrome laser (Melles Griot; Irvine, CA) emitting three wavelengths (488 nm, 568 nm, and 647 nm). Depending on the size of the follicles, the following objectives were used: Zeiss X5 (0.25 NA), Leica X10 (0.3 NA), and Leica X20 (0.6 NA). The 488-nm line excited the Yo-Pro dye, and a specially installed barrier filter 532/22 (Chroma; Battleboro, VT) was used to measure the emitted light. EB, HE, and 7ADD were excited with the 488-nm line using the single dichroic filter (RSP500), and a BP-TRITC barrier filter was used for the emitted light. Bo-Pro and LysoTracker Red were excited with the 568-nm line using a triple dichroic excitation filter and a BP-TRITC bandpass filter. Images of complete follicles were obtained with a X10 objective (X100), while enlarged images of only the oocytes within the follicles were obtained with a X20 objective and a zoom between 1 and 3 (magnification X200-600). Laser light was focused on the oocyte nucleolus in the images of the oocytes.
Correct operation of the confocal microscope increased image quality. The aim was to maximize the emitted signal while decreasing reflection, decreasing noise, and lowering PMT settings (Zucker and Price 1999). The images were usually averaged between 8-32 frames to eliminate the noise. Bandpass filters were used instead of longpass filters because they tended to reduce the unwanted light reflected from the tissues. To prevent reflective laser light signals from entering the image, the following was done: a narrow bandpass filter (BP 522/32 or BP 605/50; Chroma) was used to eliminate higher wavelengths of fluorescent light.
In our 8-bit system, only 256 gray scale values (GSV) are used in an image. As a general rule, the image is acquired by having all the GSVs below the 256 level. However, if the whole dynamic range of 256 GSVs are not used, the image quality suffers and it is difficult to visualize low GSV values.
For linearity of the image intensities, we have chosen not to increase the GSV above the 256 level. This results in some images that contain low GSV values in the oocyte. These data, however, can be visualized with image processing using image analysis programs such as Image Pro Plus (Media Cybernetics; Silver Springs, MD), Thumbs Plus (Cerrious Software; Charlotte, NC), and Adobe Photoshop. The TIFF images represented in Figures 5–7 were enhanced with Adobe PhotoShop (Adobe Systems; San Jose, CA). The brightness/contrast was adjusted to enhance low GSV intensities in the oocyte. The relative intensities reported in Table 1 were made on the raw images.
Relative staining intensities of small and large follicles (n = 3-4) a
aIntensities are normalized to the nucleolar intensity.
Image Processing and Measurement
The stack of TIFF images derived using the confocal microscope was imported into VoxBlast (Vaytek; Fairfield, IA) to reconstruct a 3D image of the follicle from 25 scanned sections (see Figure 4). Single TIFF images of the follicles were imported into Image-Pro Plus to measure the intensity of each part of the oocyte and the area of the follicle. The basic procedure entailed selecting an area of interest (AOI) and then using the histogram function to find the average intensity of the selected area. The irregular AOI feature was used to automatically trace the nucleolus. To obtain an accurate measure of the intensities of the nucleus and cytoplasm in the oocyte, four ROI (regions of interest, circles) were drawn around the object in the middle (i.e., nucleolus or nucleus). Drawing four representative small circles that covered the majority of the area in each structure and then averaging these intensity measurements obtained the intensity measurements for the nucleus and cytoplasm. The mean of the four intensities is reported in Table 1. Parts of the zona pellucida were traced using the irregular AOI and their intensities averaged as well. The cumulus was measured by circling four or five representative cells and averaging their intensities.
The area of the follicle or oocyte was determined by manually tracing the region using the irregular AOI feature and obtaining the area of the bounded region in pixels2 from the histogram. The objective size and zoom factor of each image were used to convert the area in pixels2 to the actual area in μm2. The data were plotted as a logarithmic function using the Microsoft Excel program.
Three-dimensional reconstruction of 25 sections of a Graafian follicle on the surface of an ovary. Yo-Pro stain. Magnification ×100.
Results
CLSM has been used to obtain images that are comparable to histological sections. Laser light can penetrate into the tissue to reveal oocyte and follicle morphology if the tissues are properly fixed and stained with fluorescent dyes. Using this technology, the antral follicle is usually located by observing a black signal that indicates a hollow space in the tissue. Figure 1 shows a follicle on the surface of the ovarian tissue. Smaller follicles are not as easy to locate but can be identified by their staining intensity and morphological shape in the tissue at low scanning power (X5 or X10). Once an oocyte in a follicle was identified, the plane of focus was adjusted to the nucleolus position and the PMT was adjusted to maximize the image intensity of the emitted fluorescent signal. Figure 2 shows a sequential progression of 25 individual TIFF images that comprise Figure 1. The power of this technique is illustrated in Figure 3, which shows the interior of an oocyte contained within a follicle. Figure 3A shows one section of the follicle at the plane in which the nucleolus is located; Figure 3B shows a magnified view (X600) of the same oocyte.
Twenty-five individual sections (approximately 20 μm apart) of the follicle stained with YoPro and shown in Figure 1.
An individual section of an ovarian follicle is stained with Yo-Pro. The focal plane in these pictures was optically sliced at the point of the oocyte'snucleolus (×100), and Figure 3B is the same oocyte magnified X600. The individual components of the follicle and oocyte are labeled. A X10 Plan Fluor (0.3 NA) was used for
An antral follicle is represented in three dimensions to illustrate the visualization power of this technique (Figure 4). This 3D image was constructed from the individual sections of Figure 2 using Voxblast software. Various sections of the follicle were sliced away, thus exposing the interior features of the follicle. The slicing was done so that the germinal vesicle and nucleolus of the oocyte were clearly visible. A few 3D rotations of the follicle are shown in Figure 4.
The large antral follicle represented in Figures 1–3 was located near the surface of the ovarian tissue. Interior preantral follicles located inside the ovarian tissue are harder to find but can also be visualized by this technique. Nine sequential sections of a preantral follicle stained with LysoTracker Red are shown in Figure 5. This image has an extremely bright nucleolus, necessitating the use of Adobe Photoshop to increase the low intensity values. This facilitated the observation of the oocyte details that had low GSV intensity values.
Three-dimensional rotations of the follicle shown in Figure 3. (
Nine individual sections (approximately 20 μm apart) of a preantral follicle stained with LysoTracker Red. The GSV of the image was manipulated using Adobe Photoshop so that the components of the follicle could be visualized. Without this modification the GSV of the oocyte would be very low, appearing dark, and little detail could be ascertained. Magnification X200.
As the follicle increased in size, the relative staining intensity of the nucleus and cytoplasm was reversed (Figures 6 and 7). The dye concentrated in the nucleus relative to the cytoplasm in the younger oocytes and reversed the staining pattern in the antral follicles. In a young oocyte, nucleic acids were scattered throughout the cytoplasm, suggesting the existence of RNA cytoplasmic structures. There was a ring of nucleic acids concentrated in the cytoplasm around the nucleus. This substructure in the early oocytes suggests that the cytoplasm of the younger oocytes is heterogeneous. As the follicle/oocyte matures, the cytoplasm becomes homogeneous. These data correlate with cytological changes previously described (Gosden et al. 1997) A differential staining pattern with all dyes was observed in oocytes, which was usually dependent on their maturational state. The staining pattern is illustrated in Figure 6 by the images of a young and mature follicle stained with LysoTracker Red. In the preantral follicles (Figure 6A), the nucleus stained brighter than the cytoplasm and a ring of concentrated fluorescence around the nucleus was usually observed in the cytoplasm. In the more mature follicles (Figure 6B), the reverse staining pattern (cytoplasm stained more than the nucleus) was observed. The other nucleic acid binding dyes (Yo-Pro, Bo-Pro, HE, EB, and 7ADD) showed a similar staining pattern as LysoTracker Red, which is represented in Figures 5 and 6.
Images of a young follicle (
Images of a young follicle (
LysoTracker Red, HE, 7ADD, and Yo-Pro all appeared to selectively bind to the acidic nucleolus containing single-stranded RNA and acidic proteins. LysoTracker Red stained the nucleolus most intensely. HE stained the interior of the cell very clearly. EB, which is identical to the converted HE, did not stain the oocyte or nucleus very well in fixed tissue relative to the staining of the cumulus cells. A comparison of this relative staining intensity of the cytoplasm and nucleus is shown in Figures 3, 5, 6, 7, 10, and in Table 1.
Developmental changes in follicle area relative to oocyte/nucleus area. The representative curves of the oocyte and nuclear area were generated with a logarithmic model using Microsoft Excel software. The early Stage One is preantral follicles; Stage Two is antral follicles (>50,000). The values may be smaller that those reported in the literature because there may be some shrinking of tissue due to dehydration and clearing procedures.
Ratio of the fluorescent intensity of the nucleus/cytoplasm of oocytes relative to the area of the follicles. The tissues were stained with LysoTracker Red (LT) or YoPro (YP).
With Image Pro Plus, it was possible to measure the diameter and area of the oocyte and follicles. The combination of the zoom factor and objective magnification located in the information file allows the relative calculation of the nucleus, oocyte, and follicle size measurements on different images. The area of the oocyte was measured by tracing everything within the zona pellucida. Image enhancement was necessary at times to detect the boundary between the cytoplasm and the zona of the oocyte. The area of the follicle was measured by tracing everything within the basal lamina.
Figure 8 plots the increase in oocyte area as the follicle increases in size. Although this is a continuous transition in maturation, it appears that the growth can be divided into two stages. The first stage consists of a rapid oocyte growth relative to follicle growth. During this period the oocyte increases almost four fold in area. After the antrum appears early in stage 2, the oocyte size increases slightly while the measurable follicle size increases approximately 10 fold. The nucleus follows the same pattern of growth as oocyte relative to the growth follicle. This confocal microscopy data correlates to the previous studies made by classical techniques (Brambell 1928; Arendsen de WolffExalto and Groen-Klevant 1980).
Accompanying the changes in oocyte growth was a change in the relative staining intensity of the nucleus and cytoplasm. Figure 9 shows the intensity of the nucleus/cytoplasm (N/C) increasing in the preantral stage and then decreasing as the follicle entered into the antral stage. This pattern was observed with all the dyes (YoPro, 7ADD, LysoTracker Red, HE, BoPro) tested.
The relative staining intensities of the various components of the oocyte are described in Table 1. As the follicle/oocyte develops, the staining intensities of the nucleolus, nucleus, cytoplasm, zona pellucida, and cumulus varies. The amount of dye incorporated into the oocytes during maturation is dependent on the physiological and maturational state of the oocyte.
Follicles stained with LysoTracker Red showing normal development (
During normal follicle development, more follicles are stimulated than reach final maturity. The follicles that do not grow to final maturity apparently regress during the second stage of follicle development. During their death and atresia, the granulosa layer of the follicles undergoes apoptosis. Figure 10 shows four follicles stained with LysoTracker, two of which are normal (Figures 10A and 10B) and two of which show signs of degenerative activity (Figures 10C and 10D). The increased staining of the granulosa cells in the degenerating follicles is observed with the LysoTracker dye. The follicle in Figure 10C is in an early stage of degeneration, whereas that in Figure 10D has degenerated considerably.
Discussion
The confocal microscope using laser light has the capability of penetrating into millimeter thick tissue and visualizing oocytes located inside follicles. In this study, optical sections of the follicle were obtained using CLSM instead of using microtone-derived physical sections from the paraffin-embedded tissue blocks and using either conventional light or EM microscopy. These CLSM optical sections of the follicle were then easily reconstructed into a 3D morphological structure using various software programs.
Follicular development is a continuous process and not a series of discrete stages as shown in the illustrations. However, oocyte and follicle development can be considered to consist of a biphasic process during which the oocytes and follicles grow at different rates (Brambell 1928; Arendsen de Wolff-Exalto and Groen-Klevant 1980; Gosden 1995; Hirshfield 1997). Initially, the oocytes in preantral follicles grow in size more rapidly than the follicles. In antral follicles, the reverse occurs, in which the follicle increases in size more rapidly than the oocyte. The confocal microscopy data derived in this study using optical sections support the data published using classical stained sections of the ovary (Brambell 1928; Arendsen de Wolff-Exalto and Groen-Klevant 1980). Corresponding to these size changes are biochemical and dye-binding changes in the oocyte. In the preantral stages of follicular development, there is an increase in cytoplasmic protein and RNA synthesis that can be detected in the cytoplasm by dyes that bind nucleic acids (i.e., HE). Many organelles are clustered around the nucleus to form a yolk nucleus or Balbiani body (Figures 5–7; Gosden et al. 1997). In the antral stage, there is less cytoplasmic biochemical activity and nuclear condensation occurs (Moore and Lintern-Moore 1974). Corresponding to these size and biochemical changes in antral follicles, the nucleus was shown to absorb less dye than the cytoplasm and the cytoplasm showed a homogeneous staining pattern (Table 1; Figures 3, 5–7, and 10). This may be due in part to the fact that the cytoplasm of an antral stage oocyte contains more nucleic acids than a Stage One oocyte, and therefore will stain more intensely (Chouinard 1975; Palombi and Viron 1977; Antoine et al. 1988; Plancha and Albertini 1992).
Before meiotic division in the preovulatory follicle, there is a marked reduction in biochemical activity in the cytoplasm and a condensation of DNA in the nucleus (Figures 6 and 7; Albertini 1992; Moore and Lintern-Moore 1974). The heterochromatin or euchromatin state of DNA in oocytes will affect the amount of dye binding and therefore the fluorescence intensity observed using CLSM. In the case of nucleic acids, as the nucleus condenses there is less binding of fluorochromes to the nucleic acids (Darzynkiewicz et al. 1984). In the oocyte, there was a reversal of the nuclear/cytoplasmic staining pattern that corresponded to the shift in oocyte and follicle size changes (Chouinard 1975). It can be hypothesized that, as the nucleus prepares for meiotic division in older follicles, there is less binding of the dyes in the nucleus because the nuclear conformation becomes more condensed and thus provides fewer available binding sites.
Apoptosis is present during follicular degeneration and atresia (Hsueh et al. 1994). In this study, abnormal follicle growth was characterized by increased staining intensity by LysoTracker Red of the oocyte/follicle and by morphological changes. LysoTracker Red may be a specific fluorochrome stain that reveals a pattern that is correlated with abnormal follicular morphology and physiology. The dying granulosa cell layer of the degenerating follicles in Figure 10 stained more intensely with LysoTracker Red. This indicates a lower pH and possible phagocytosis of degenerating cells. This observation in ovaries correlates with our data using LysoTracker Red to reveal dying cell regions in both normal and hydroxyurea-treated embryos (Zucker et al. 1998).
The accumulation of LysoTracker Red in the nucleolus may be due to preference of the dye for acidic regions (Antoine et al. 1988; Zucker et al. 1998), which comprise the nucleolus. The selectivity of other nucleic acid binding dyes (EB, 7ADD, YoPro) for the nucleolus may be due to the acidic environment of this structure and/or to the abundance of single-stranded RNA in the nucleolus (Wadkins and Jovin 1991). Moreover, because LysoTracker Red stains only acidic structures, it can be inferred that if the dye does not stain a structure, as is the case with the zona pellucida, then that structure is probably basic. These dyes can reveal information on the biochemistry and physiology of the tissues.
Technical Issues Involved in Ovarian CLSM
To remedy the problem that is caused by the opaqueness of the tissue, a clearing procedure that has been previously applied to histological sections and embryos was used (Klymkowsky and Hanken, 1991; Gard 1993; Zucker et al. 1998). The procedure involves extracting liquid from the tissue and then replacing it with a solution that has a similar refractive index as the tissue. In choosing a clearing agent to render the tissue transparent, one must consider its compatibility with the staining reagents used, its ability to be immersed in aqueous solutions, and its toxicity to the fixed tissue. Previous clearing agents used include the following: potassium hydroxide/glycerol, methyl salicylate (artificial oil of wintergreen), carbon disulfide, glycerol, and xylene. The BABB mixture used in this study was derived by Andrew Murray and Marc Kirschner (Klymkowsky and Hanken 1991; Gard 1993; Gard and Kropf 1993) for use with amphibian eggs and embryos. This BABB mixture also appears to render mammalian ovaries more transparent, allowing the laser light to penetrate hundreds of micrometers into the tissue to visualize follicles.
The correct choice of objectives is critical to examine whole-mount ovarian tissue. To image parts of ovaries, it is necessary to use low-magnification objectives (X5, X10, and X20) because they increase both the field of view and the depth of field. Although an oil lens yields better resolution than an air lens, it introduces a series of problems that include shallow depth of focus (usually less than 100 μm), possible contamination caused by clearing agent residues on the coverglass surface, and possible mechanical stresses that may dislodge the coverslip. Dry objectives with high numerical apertures have been used in this series of experiments with thick tissues.
In summary, fluorescent dyes can be used to stain ovarian tissue to reveal structures in the developing oocytes. The most consistent pattern was observed with dyes that were used before fixation of the tissue, because the interaction of dyes on fresh tissue appears to enhance the dye specificity. Staining fixed tissue with YoPro or 7ADD yielded results that were not as reproducible as those for the dyes (HE, LysoTracker Red) used before fixation. HE yielded the greatest cytoplasmic detail, and LysoTracker Red yielded nucleolus specificity, atretic follicles, and some background fluorescence so that the subtle features of the follicle and oocyte development could be determined. From these studies, it appears that the best procedure is to cut the fresh ovaries into small 2-4-mm pieces followed by incubation with fluorescent dyes for 30 min at 37C. This is followed by fixation in 4% PF at 4C. After 24 hr, the tissues are dehydrated in MEOH and cleared in BABB and then transferred to well slides for observation using CLSM technology.
Footnotes
Acknowledgements
We would like to thank Joan Hedge and Mike Narotsky (Reproductive Toxicology Division NHEERL, USEPA) for their excellent technical assistance in acquiring ovarian tissue. We would also like to thank Earl Puckett of the USEPA machine shop for providing us with high-quality aluminum slides necessary to contain the ovarian samples for confocal microscopic evaluation. We thank Keith Tarpley of OAO Corporation for the Adobe Photoshop manipulation of the TIFF images and subsequent printing.