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Original Studies |
Department of Gynecology and Obstetrics, Division of Reproductive Endocrinology and Infertility, Stanford University School of Medicine, Stanford, California 94305-5317
Address correspondence and requests for reprints to: Linda C. Giudice, Department of Gynecology and Obstetrics, Room HH-333, Stanford University Medical Center, Stanford, California 94305-5317. E-mail: giudice{at}stanford.edu
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Introduction |
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Materials and Methods |
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Ovarian tissue was collected from 27 subjects at the time of surgery for benign conditions through the Cooperative Human Tissue Network, at Ohio State University (Columbus, OH) or Stanford University Hospital (Stanford, CA). The study was approved by the Stanford University Committee on the Use of Human Subjects in Medical Research, and specimens were obtained after informed consent.
In situ apoptosis detection
Ovarian tissue for in situ apoptosis detection. Follicles (n = 63) were obtained from 12 subjects in the follicular phase of the menstrual cycle, 30 follicles were obtained from 4 subjects in the luteal phase, and 23 follicles were obtained from 5 subjects whose menstrual cycle stage was not known. Indications for surgery were uterine fibroids (40%), pelvic pain and adhesions (34%), endometriosis (13%), and adenomyosis (13%). The median age of the subjects was 39 yr (range 28–48 yr), and the average was 41 yr (± 6.8 yr SD). Follicle sizes were determined by calculating the average of two diameters measured in the greatest dimension of three serial sections of each follicle. Ovarian tissue was immediately frozen in liquid nitrogen and stored at -80 C. For cryosections, frozen tissue was cut into a series of 12-µm sections without changing the angle of the cryostat at -20 C, so that sections always came from the adjacent positions within the ovaries. The cryosections were stored -80 C until further use.
Apoptosis detection by in situ hybridization. DNA fragmentation was detected by ApopTag Plus In Situ Apoptosis Detection Kit (Oncor, Gaithersburg, MD). A terminal deoxynucleotidedyl transferase (TdT) was employed to tail residues of digoxigenin-nucleotides to nucleotide triphosphates of the 3'-OH ends of double or single strand DNA. Digoxigenin antibody conjugated to peroxidase was added to the reaction site. The peroxidase catalytically generates an intense signal (brown staining) from the chromogenic substrate. Cryosections were removed from -80 C and maintained at room temperature for 30 min. Sections were fixed in 4% formalin in 1 x PBS for 10 min and then washed with fresh PBS twice. Sections were fixed again in ethanol/acetic acid (2:1) for 5 min at -20 C and then washed again. Sections were then quenched in 2% hydrogen peroxide in PBS for 5 min at room temperature. After quenching, sections were equilibrated in buffer for 15 sec, then with 54 µL TdT at 37 C for 1 h, and then with 55 µl antidigoxigenin-peroxidase for 30 min and washed with PBS. After washing, 125 µL diaminobenzidine substrate was added to the sections, and the color reaction was allowed to occur for 3–6 min at room temperature. Cryosections were washed with distilled water and counterstained with methyl green for 10 min. Sections were viewed and pictures were taken under microscopy with brightfield illumination. Estimation of positive staining for DNA fragmentation within granulosa cells was made according to a method previously used to estimate silver grains for messenger RNA in human follicles (13) with some modifications, i.e. we identified stained nuclei in which DNA fragmentation occurred. We divided apoptosis into four stages according to the extent of stained nuclei within granulosa cell layers: stage I, positive stained nuclei were a few in number; stage II, positive stained nuclei were more prominent; stage III, positive stained nuclei were abundant and seemed to fuse together; and stage IV, positive stained nuclei were found in all granulosa cells.
Normal histological staining of healthy and atretic follicles. Adjacent cryosections of the same follicles for in situ detection of DNA fragmentation staining were also evaluated for pyknotic bodies by hematoxylin and eosin staining.
Follicle collection for DNA fragmentation ladder detection
Ovarian tissue was collected from four premenopausal women (2 in the follicular phase and 2 in the luteal phase). The median age of the subjects was 36 yr (range 33–38 yr, mean 36 ± 2.16 yr SD). Indications for surgery were adenomyosis (n = 2), menorrhagia and endometriosis (n = 1), and uterine fibroids (n = 1). Ten follicles were dissected from the ovaries. The diameter of each follicle was measured, and follicle fluid was collected. Because follicles were collapsed after removing the follicular fluid, no attempt was made to detect DNA fragmentation by in situ hybridization in these follicles. Follicular fluid and follicles were stored separately at -20 C until use.
Measurement of estradiol and androstenedione
Estradiol and androstenedione in follicular fluid were determined using RIA kits from Diagnostic Products Corporation (Los Angeles, CA). The cross-reactivity of estradiol with estriol was 10%. There was no cross-reactivity with progesterone, and there was 0.001% cross-reactivity with testosterone. The intra- and interassay coefficients of variation for estradiol at 22 ng/mL were 4.81% and 9.76%, respectively. There was no detectable cross-reactivity of androstenedione with estradiol. There was 0.001% cross-reactivity with progesterone and 1.49% with testosterone. The intra- and interassay coefficients of variation for androstenedione at 400 ng/mL were 7.81% and 10.76%, respectively. The ratio of androstenedione to estradiol was expressed as A/E. Follicles with ratios of A/E <4:1 were categorized as estrogen-dominant, and follicles with ratios of A/E >4:1 were categorized as androgen-dominant (14).
DNA extraction
Genomic DNA was extracted from individual follicles whose follicular fluid had been collected (see Follicle collection for DNA fragmentation ladder detection). The quantity and purity of each nucleic acid sample were determined by measuring optical densities at A260 and A280 nm.
Biochemical analysis of DNA fragmentation
A biochemical characteristic of apoptosis is the activation of a Ca2+/Mg2+-dependent endonuclease, which acts on linker DNA located between nucleosomal units spaced every 185 bp, resulting in a distinct "ladder pattern" following size separation by agarose gel electrophoresis (4). DNA (1 µg) from each follicle was labeled at 3'ends with [32P]dideoxy-ATP (3000 Ci/mmol) (Amersham, Arlington Heights, IL) by incubation for 60 min at 37 C in the presence of 25 µg terminal transferase (Boehringer Mannheim, Indianapolis, IN) and 25 mM CoCl2. Labeled DNA was purified by ethanol precipitation using 20 µg transfer RNA as a carrier (5, 6). The labeled DNA was then subjected to gel electrophoresis. Gels were dried in a slab-gel dryer without heat for 2 h and exposed to Kodak x-ray film (Eastman Kodak, Rochester, NY) for 40 min to 1 h at -80 C.
Determination of DNA fragmentation by in situ hybridization and biochemical analysis in corpus luteum
Corpora lutea were collected from ovarian tissue (n = 5 from 5 women). The median age of subjects was 37 yr (range 33–39 yr, average 37 ± 2.63 yr SD). Two of these subjects were described above in Follicle collection for DNA fragmentation ladder detection, and indications for surgery were uterine fibroids (n = 1) and dysmenorrhea (n = 1). Another two subjects had surgery for adenomyosis and uterine fibroids, respectively. The fifth subject was described above in Ovary collection for in situ apoptosis detection, and the indication for surgery was endometriosis. Of five corpora lutea, two were from the midluteal phase, two were from the late luteal phase, and one was from the early follicular phase. Half of each corpus luteum (from four corpora lutea) was used for in situ hybridization, and the other half was used for biochemical analysis of DNA fragmentation/laddering. One corpus luteum from the early follicular phase was only used for in situ hybridization.
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Results |
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One hundred and sixteen follicles from 21 women were used
to detect follicular DNA fragmentation by in situ
hybridization (Table 1
). Table 2 demonstrates the scoring system used.
Within the limits of detection of the assay, positive staining for DNA
fragmentation was not found in primordial, primary, and secondary
follicles (Table 1). In preantral follicles, positive staining for DNA
fragmentation was found only in a small number of granulosa cells.
Likewise, in 200-µm follicles, positive staining for DNA
fragmentation was only observed in a few granulosa cells (Fig. 1, A and B). In the same follicle not
treated terminal transferase (control), no DNA fragmentation staining
was found (Fig. 1C). Pyknotic bodies were not found by
hematoxylin-eosin staining (Fig. 1D). Within 1.1- to 2-mm follicles,
DNA fragmentation staining was more prominent in granulosa cells, and
scattered staining was also found in theca-interna cells (Fig. 1, E and
F: stage II). Morphologically, the granulosa cell layer in the follicle
shown was intact. Within 2.1- to 5-mm antral follicles, abundant
positive staining of DNA fragmentation was found (stage III and Table 2). Morphologically, some of the
granulosa cells were lost from the granulosa cell layer (Fig. 2, A and B). Positive staining for DNA
fragmentation was found in whole granulosa layers (stage IV) and more
granulosa cell layers were partially sloughed (Fig. 2, C and D) or
totally sloughed (Fig. 2, E and F). Positive staining of DNA
fragmentation was not found in follicles whose diameter was >10 mm.
The frequency of apoptosis in follicles was calculated to be 37% in
0.1- to 2-mm follicles, 50% in 2.1- to 5-mm follicles, and 27% in
5.1- to 9.9-mm follicles (Table 1). Because there were too few
follicles in the luteal phase to be able to draw conclusions on
cycle-stage rates of apoptosis in this phase vs. the
follicular phase, analysis has been limited to follicle size and A/E
ratios (see below).
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Estrogen-dominant follicles (5 and 6 mm, A/E <4:1) did not show
any DNA fragmentation/laddering (Fig. 3, lanes 3 and 4). However, five androgen-dominant follicles (3, 3.5, 4,
5, and 6 mm, A/E >4:1) had obvious DNA fragmentation/laddering (lanes
5–9). Lanes 1 and 2 were loaded with DNA from two 2-mm follicles, and
they did not have DNA fragmentation/laddering (without follicular fluid
analysis because follicular fluid could not be taken out with a needle
because of their size). In lane 10, DNA was isolated from rat ovarian
follicles incubated for 24 h in serum-free medium and served as a
positive control for DNA fragmentation/laddering (15). In an 11-mm
follicle without follicular fluid, no DNA fragmentation/laddering was
detected (data not shown).
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DNA fragmentation was analyzed in the same corpora lutea (n =
4) by both in situ hybridization and biochemical analysis
(Fig. 4). Two corpora lutea in the
midluteal phase did not show positive staining of DNA fragmentation
(Fig. 4, panels 1 and 2) and no DNA fragmentation/laddering (Fig. 4, lanes 1 and 2). Two other corpora lutea in the late luteal phase had
positive staining for DNA fragmentation (Fig. 4, panels 3 and 4), which
were primarily in large luteal cells (open arrows).
They also showed typical DNA fragmentation/laddering (Fig. 4, lanes 3
and 4). The corpus luteum with more positive staining for DNA
fragmentation had more low molecular weight DNA labeling (Fig. 4, panel
3 and lane 3, respectively) than that in the corpus luteum with less
positive staining of DNA fragmentation (Fig. 4, panel 4 and lane 4,
respectively). In a heavily regressing corpus luteum in the early
follicular phase (Fig. 5), luteal cells had lost their
morphology and only fibers were observed, and all nuclei exhibited
pyknotic bodies (Fig. 5C). A follicle adjacent to the corpus luteum did
not show any positive staining for DNA fragmentation. Positive staining
for DNA fragmentation in the corpus luteum was not beyond the stromal
tissue of the follicle (Fig. 5A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the limited numbers of follicles obtained from human ovaries, and the inherent limits of the methods used to detect apoptosis in this study, our data provide two unique perspectives on human follicular apoptosis. First, apoptosis occurs incrementally with increasing follicular size. In stage I, only a few of granulosa cells were found to be apoptotic. In stages II and III, increasing numbers of granulosa cells were apoptotic. In stage IV, even though some granulosa cells are sloughed or all granulosa cell layers are sloughed, all remaining granulosa cells were apoptotic. The apoptotic status of human follicles, as judged by pyknotic bodies (1), was compatible with our results by in situ hybridization, i.e. apoptosis occurs in follicles as small as 1 mm in size and also in antral follicles (1). However, hematoxylin-eosin staining detects condensed nuclei but not nuclei that are not condensed. The best example is in 5.1- to 9.9-mm follicles, in which all granulosa cells stained positively for DNA fragmentation, whereas only some of the granulosa cells had condensed nuclei. Thus, hematoxylin-eosin staining for apoptosis is not as sensitive as the molecular and biochemical approaches used in this study, but it is still useful as an adjunct in assessing follicular apoptosis. Second, follicles whose diameter is >10 mm appear to be entering a critical stage, the preovulatory phase. In follicles >10 mm in diameter examined in this study, there was no sign of apoptosis as determined either by in situ hybridization or by DNA fragmentation/laddering. These data generally support the model for follicular selection proposed by Gougeon (1).
In addition to large dominant follicles not displaying DNA fragmentation/laddering, small estrogen-dominant follicles likewise do not display apoptosis. Conversely, androgen-dominant follicles uniformly display characteristics of apoptosis. In this study, limited numbers of follicles in the luteal phase prevented analysis of cycle-stage-specific rates of apoptosis. However, apoptosis was found to be a function of follicular androgen dominance. Whether the onset of apoptosis occurs at a critical A/E ratio, or whether it is a continuous process with increasing A/E ratios cannot be determined from the data presented herein, because of the limited numbers of follicles investigated, and is worthy of further investigation. What initiates apoptosis and what suppresses it are not well understood, although steroid hormones themselves, as well as intraovarian growth factors and gonadotropins, may influence these processes. For example, androgen can induce follicular apoptosis, whereas estradiol and insulin-like growth factor I (IGF-I) inhibit it (5, 6). In androgen-dominant human ovarian follicles, we have determined that IGF-II levels are lower and that IGF binding proteins (IGFBPs) are higher than in estrogen-dominant follicles (16, 17). It is likely that the level of bioavailable intrafollicular IGFs contributes, in part, to whether apoptosis is initiated. In addition, IGFBPs can inhibit follicular function through sequestering IGFs, inhibiting estradiol production, or FSH-induced estradiol secretion by follicular cells (18, 19, 20). Thus high concentrations of androgens and IGFBPs in androgen-dominant follicles could be signals for follicle apoptosis in normal ovaries of premenopausal women. Furthermore, follicular apoptosis could delete androgen-dominant follicles and keep estrogen-dominant follicles for further development and ovulation.
The apoptotic status of corpora lutea in human ovary is similar to that reported by others (21). In the current study, DNA fragmentation was present in corpora lutea that had positive staining for DNA fragmentation but not in corpora lutea without positive staining for DNA fragmentation. Using two different techniques, we found that only degenerating corpora lutea have typical DNA fragmentation/laddering. We found that apoptosis primarily occurs in large luteal cells. This is in contrast to a report by Shikone et al. (21) in which apoptosis was found in both large and small luteal cells. It is possible that sample variability may contribute to this discrepancy. It is possible that corpora lutea described in our study were early in their development of apoptosis, because some of the nuclei were still intact and did not exhibit typical pyknotic bodies. In contrast, in a heavily degenerated corpus luteum, all nuclei exhibit typical pyknotic bodies by either in situ hybridization or by normal hematoxylin-eosin stain. This corpus luteum was in the late luteal phase. Interestingly, apoptosis is strictly limited to the area of the degenerating corpus luteum and never was found beyond the stroma of adjacent follicles. These observations suggest that apoptosis of the corpus luteum (and other follicles) promotes deletion of atretic follicles or corpora lutea, likely to benefit subsequent growth of new follicles and corpora lutea.
In summary, apoptosis occurs in preantral and antral follicles and
primarily in the granulosa cell layer in the human ovary. However,
apoptosis does not occur in the dominant follicles (
10 mm). Typical
DNA fragmentation/laddering is only present in androgen-dominant
follicles but not in estrogen-dominant follicles. The results suggest
that human follicular atresia is a normal process driven by programmed
cell death, primarily in the granulosa cell layer, and that apoptosis
could delete androgen-dominant follicles and preserve estrogen-dominant
follicles for further development, maintaining homeostasis within the
ovary. The fact that apoptosis occurs within 200-µm follicles,
suggests that follicular selection could begin during the early
formation of the antrum. Degeneration of the corpus luteum is also
likely a consequence of apoptosis.
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Acknowledgments |
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Footnotes |
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Received January 6, 1997.
Revised April 23, 1997.
Accepted May 20, 1997.
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