1 Departments of Pathology and 2 Cell Biology, Physiology and Immunology, Faculty of Medicine, University of Córdoba, Córdoba, Spain
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Abstract |
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Key words: apoptosis/cell death/corpus albicans/corpus luteum/structural luteolysis
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Introduction |
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Structural luteolysis has received relatively little attention, although this process is important for ovarian tissue homeostasis. In the absence of structural luteolysis, non-functional tissue would accumulate within the ovaries. The mechanisms ultimately responsible for the structural regression of the CL are poorly understood. Recently, apoptosis has been considered as the mechanism responsible for structural luteolysis. However, previous reports on the abundance of apoptotic cells in regressing CL are controversial. The reported proportions of luteal cells undergoing apoptosis ranged from only scattered cells in only some specimens (Funayama et al., 1996) to all luteal cells (Yuan and Giudice, 1997
), and affected both granulosa- and theca-lutein cells (Shikone et al., 1996
) or only granulosa-lutein cells (Yuan and Giudice, 1997
). In addition, previous studies focusing on the role of apoptosis in CL regression (Rodger et al., 1995
; Funayama et al., 1996
; Shikone et al., 1996
; Yuan and Giudice, 1997
) have been limited to the late luteal phase and early regression. However, degenerating CL beyond ovulation in the next cycle have not been studied.
It is generally assumed that CL involution gives rise to corpora albicantia (CA) through fibrosis and hyalinization of luteal tissue (Balboni, 1983; Scully et al., 1998). However, observation of different ovaries indicates that the abundance of CA is highly variable among normally cycling women. This is difficult to explain if CA formation is the only way to eliminate non-functional luteal tissue.
Alterations of CL regression could be involved in different pathological conditions. About 10% of climacteric women showed Halban's disease (Bukovsky et al., 1996), characterized by persistence of the CL, i.e. a lack of CL regression. On the other hand, premature CL regression may account for some cases of CL insufficiency (i.e. luteal phase defect; Hinney et al., 1996). Knowledge of the mechanisms responsible for the elimination of luteal tissue would contribute to the understanding of these alterations of CL disappearance.
The objective of this study was to analyse the process of structural luteolysis in cycling women.
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Materials and methods |
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The standard cycle was considered to be 28 days. CL were classified in the following phases: (i) functional CL (FCL) from the date of ovulation to day 24 of the first cycle; (ii) regressing CL (RCL), from day 25 of the first cycle to the date of ovulation in the next (second) cycle; and (iii) involuting CL (ICL), from day 15 of the second cycle onward. For the purposes of this study the dating of only two specific cycle points was necessary. Days 2425 of the standard cycle (corresponding to the transition from FCL to RCL) were recognized by CL histology [general shrinkage of the granulosa-lutein layer, presence of scattered apoptotic nuclei and vacuolated granulosa-lutein cells (GLC) showing CD68 immunostaining], endometrial characteristics (presence of prominent spiral arteries and predecidualization around these arteries or beneath the epithelium, corresponding to days 2325 of the standard cycle) and a compatible menstrual history, corresponding to the late luteal phase. Days 1415 of the standard cycle (corresponding to the transition from RCL to ICL) were recognized by the presence in the ovary of a just-ruptured follicle or a newly formed, still unluteinized CL, together with compatible endometrial dating and menstrual history. CA were considered to be present when only hyalinized material lacking luteal tissue was visible.
The following tissue compartments were considered in the CL: (i) luteal tissue, corresponding to the granulosa-lutein layer, together with theca-lutein areas; (ii) inner boundary tissue (IBT), which corresponded to the connective tissue lining the inner aspect of the luteal tissue; and (iii) central cavity, that was filled with blood and follicular fluid remnants and surrounded by the IBT.
Sections of the CL were stained with haematoxylin and eosin, or the AFIP method for lipofuscin (Johnson, 1992).
Immunohistochemistry
Immunohistochemistry was performed on routinely neutral-buffered, formaldehyde-fixed, paraffin-embedded tissues. Sections (5 µm) were placed on poly-L-lysine-coated slides and, after dewaxing and rehydration in a graded series of ethanol, were incubated in 2% hydrogen peroxide in methanol for 30 min, to inhibit endogenous peroxidase. Monoclonal antibodies against CD68 antigen (Dako Diagnostica, Seville, Spain) as a macrophage marker (Bukovsky et al., 1995, 1996
; Suzuki et al., 1998
), CD34 antigen (Novocastra Laboratories Ltd, Barcelona, Spain) as an endothelial cell marker (Ito et al., 1995
; Gaytán et al., 1999
), Ki-67 antigen (Concepta, Barcelona, Spain) as a marker of proliferating cells (Hall and Levison, 1990
) and anti-human muscle actin (HHF35, Enzo Diagnostic Inc., New York, USA; Tsukada et al., 1987) were used. Sections destined to be immunostained for Ki-67 were predigested in 0.1% (w/v) trypsin (Difco, Detroit, MI, USA) in phosphate-buffered saline (PBS) containing 0.1% (w/v) sodium chloride for 20 min at 37°C. After washing in distilled water, these sections, as well as those destined to be stained for the CD68 and CD34 antigens were immersed in 10 mmol/l citrate buffer and submitted to antigen retrieval in a microwave oven (2x5 min at 700 W). Afterwards, the sections were allowed to cool at room temperature, washed in PBS, blocked with normal rabbit serum, and incubated overnight with the primary antibodies (anti-CD68 1:400; anti-CD34 1:25; anti-Ki67 pre-diluted, anti-actin 1:300). The sections were then processed by the avidinbiotin peroxidase complex (ABC) method, following previously described procedures (Gaytán et al., 1997
).
Negative control sections were run by incubating sections with non-immune serum or PBS instead of the first antibody. Positive controls were provided by non-luteal ovarian tissues (i.e. atretic follicles for macrophages, medullary blood vessels for endothelium, proliferating cells in healthy growing follicles for Ki-67 and smooth muscle of the arterial wall for actin). Sections were counterstained with haematoxylin.
Quantitative studies
The total number of cases (i.e. 340 women, 654 ovaries) was used for morphological evaluation. For quantitative studies, 168 women (in which both ovaries and two blocks per ovary were available) were selected. At least five non-consecutive equatorial sections (at 50 µm intervals) from each block were scored and the ovaries were classified into the following types: (i) CA(), with absence of CA, and showing only involuting CL without or with minute hyalinized areas; (ii) CA(+), containing abundant large CA; or (iii) mixed, containing both CA and involuting CL without hyalinization or CL with intermediate features, showing an asymmetrical distribution of hyalinized and non-hyalinized areas. To test the hypothesis that folding of the CL was associated with the luteolytic pattern, 62 of these 168 women who were in the luteal phase and showing adequate sections of the CL of the current cycle were selected. The CL of the current cycle was classified as: (i) folded, when luteal tissue showed a festooned contour, limiting a usually small cavity; or (ii) unfolded or irregularly folded, when the luteal tissue showed a smooth contour in the whole or large areas of the CL section. In all cases, the CL was evaluated only when a central section of the CL was present. The zone of the CL adjacent to the point of rupture was not considered, because unfolding was common in this area in most CL.
Statistical analysis was performed by the 2 test with Yates' correction.
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Results |
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Changes in luteal tissue
Morphological changes during the luteal phase of the current cycle have been described elsewhere (Gaytán et al., 1998, 1999
). Briefly, steroidogenic luteal cells reached full luteinization by day 19 and abundant macrophages, showing dendritic features (Figures 1A and 2B
), were observed in the granulosa-lutein layer. Clear morphological signs of structural luteolysis were not evident before day 25 of the cycle. In regressing CL, increasing regressive changes were apparent. There was a general shrinkage of the luteal tissue, due to the decrease in the volume of the GLC, as previously reported (Gaytán et al., 1998
, 1999
). Apoptotic cells, characterized by condensed chromatin, shrunken eosinophilic cytoplasm and fragmentation into clusters of apoptotic bodies at advanced stages, were observed (Figure 2A
) in both theca-lutein and granulosa-lutein areas during the perimenstrual period, from a few days before to a few days after menstruation. Apoptotic cells were scarce during the late follicular phase of the second cycle, and were only occasionally found from day 14 of the second cycle onward.
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General evolution of the CL during structural luteolysis
The data obtained from this study suggested that the different luteolytic patterns observed were dependent on the existence of structural differences among different CL. Under this hypothesis, the final fate of an involuting CL seemed to be dependent on the presence or absence of a large, blood-filled cavity. In those CL displaying a small cavity with minimal haemorrhage, the cavity was usually filled with loose connective tissue derived from the IBT from opposite foldings. Involution of these CL seemed to give rise to scattered remnants of pigment-filled GLC (Figures 3AC and 6A), or to small scars with some entrapped pigment-filled luteal cells, but typical CA seemed not to develop.
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Involuting CL displaying intermediate features between the above-described modes of involution were also found (Figure 6C). In these cases asymmetrical CA (consisting of zones of pigment-filled cells) as well as areas of hyalinization were observed. In some cases, these involuting CL showed an empty cavity containing fresh blood and haemosiderin-containing macrophages.
Large variations with respect to the number and size of CA were found among the ovaries from regularly cycling women. From the quantitative study of a subset of 168 cases, we determined that some women (corresponding to 28.5%; n = 48), had CA() ovaries showing involuting CL and absence of CA, and only small scars or clusters of pigment-filled cells remaining after CL effacement. In contrast, some women (corresponding to 25.1%; n = 42) had CA(+) ovaries, showing high numbers of large CA at different stages of involution. In these ovaries, considerable distortion of large antral follicles and newly formed CL was observed, and a large proportion of the ovarian volume was occupied by CA. Finally, the majority of women (corresponding to 46.4%; n = 78) had mixed ovaries that showed a mixture of both types of CL involution, or involuting CL with intermediate features, with wide hyalinized areas together with areas lacking hyalinization. No differences were found between the two ovaries in individual women for CA(+) and CA() types.
The relationship between the degree of folding of the CL of the current cycle (Figure 7) and the luteolytic pattern for women with CA(+) and CA() ovaries is shown in Table I
. There was a significant (P < 0.001) association between the degree of folding of the new CL and the luteolytic pattern. In women lacking CA [CA()], the CL of the current cycle was folded in 84.2% of cases, whereas in women containing abundant CA [CA(+)], the CL of the current cycle was unfolded or irregularly folded in 83.3% of cases.
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Discussion |
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In this study, dying luteal cells showing the characteristic morphological features of apoptosis were almost exclusively observed at the beginning of CL regression, and were very scarce in involuting CL, when luteal cells were still abundant. Apoptotic cells were recognized on the basis of their morphological features. The morphological characteristics of apoptotic cells are highly specific to this type of cell death (Searle et al., 1982; Wyllie, 1986
). Previous studies comparing detection of apoptotic cells by morphological criteria and 3'-end-labelling methods demonstrated that cells exhibiting apoptotic morphological features contained fragmented DNA (Young et al., 1997
; Gaytán et al., 1998
). The amount of apoptosis evaluated by the two different techniques was equivalent in both rat (Gaytán et al., 1998
) and primate (Young et al., 1997
) species. This indicates that careful morphological evaluation is a useful, simple and rapid method for the detection of apoptosis.
Large differences in the number of apoptotic cells during structural luteolysis have been reported, ranging from only scattered apoptotic nuclei (Funayama et al., 1996) to the practical totality of luteal cells (Shikone et al., 1996
; Yuan and Giudice, 1997
). Although the reasons for these discrepancies are not known, it is clear that most luteal cells cannot undergo apoptosis at early stages of structural luteolysis, since luteal tissue persisted for several cycles. Studies in the marmoset monkey (Young et al., 1997
) indicated that apoptosis was relatively abundant at the beginning of the follicular phase of the next cycle, and decreased thereafter, which agrees with data obtained in the present study.
Indeed, the data of this study suggested that in the human CL, apoptosis represents an initial and probably important step in structural luteolysis. However, most steroidogenic luteal cells seemed to survive to the perimenstrual apoptotic wave. Thereafter, the remaining luteal cells underwent slow degenerative changes [consisting of extreme cytoplasmic vacuolation, expression of lysosome-associated antigens (CD68) and accumulation of lipofuscin pigments and, in some cases, apparent cytoplasmic membrane disruption and syncytia formation] and persisted for long periods of time. Changes similar to those described in this study have been reported recently (Young et al., 1997; Fraser et al., 1999
) during luteolysis in the marmoset monkey. Vacuolation and apparent syncytia formation have been also reported in the regressing CL of sheep (Sawyer, 1995
), while cytoplasmic vacuolation and autophagia have also been described in developing tissues (Clarke, 1990
). The expression of the lysosome marker CD68 reported here, in accordance with previous studies (Bukovsky et al., 1995
, 1996
), as well as the increased numbers of phagolysosomes reported in steroidogenic cells during luteolysis (Sawyer, 1995
), are suggestive of the existence of autophagic activity. Furthermore, the accumulation of lipofuscin pigments in the remaining luteal cells also agrees with an autophagic model of cell degeneration. Lipofuscin is an insoluble pigment that represents the accumulation of indigestible material in lysosomes after autophagy (Yeldandi et al., 1996
). A previous study (Quatacker, 1971
), also suggested that cells within the regressing CL corresponded to granulosa cells with autophagocytic capabilities. Whether pigment-filled cells are finally removed by apoptosis is unknown. If apoptosis is the mechanism responsible for the disappearance of luteal cell remnants, it should happen at a very low rate, probably below the limits of detection by tissue section assessment.
Expression of CD68 was observed in isolated GLC at the late luteal stage, and GLC expressing CD68 became more abundant during the follicular phase of the next cycle. Expression of several macrophage markers by GLC has been reported previously (Best et al., 1996; Bukovsky et al., 1996
; Gaytán et al., 1998
) in the regressing CL. In this study, generalized expression of CD68 in the whole granulosa-lutein layer was coincident with generalized vacuolation of GLC and with the expected date of ovulation in the second cycle. In fact, when a CL of the previous cycle showing generalized vacuolation and expression of CD68 was present, a newly formed CL was found in the same or in the contralateral ovary. This suggests that pre-ovulatory hormone surges are involved in the regressive changes observed at this time in the CL of the previous cycle. In cycling rats, the pre-ovulatory hormone surges on the evening of prooestrus links ovulation to the induction of regressive changes in the CL of the previous cycle (Gaytán et al,. 1997
). Although regressive changes in the human CL were already present from the late luteal phase, generalized vacuolation and CD68 expression seemed to represent a specific step in structural luteolysis. Changes in the CL associated with the next ovulation are characteristic enough to recognize this stage of the cycle by observation of the CL of the previous cycle.
It is generally agreed that the final fate of the CL is the formation of a CA, and that the CA arises through hyalinization of collagen produced by the connective tissue that replaces luteal tissue and invades the central cavity during luteolysis (Balboni, 1983; Scully et al., 1996
). However, the observations of this study did not support this view. First, the absence of CA in about 28% of regularly cycling women indicated that CA formation is not the only luteolytic pathway. Second, only a small part of the CA seemed to be formed by hyalinization of the IBT and in some cases, by hyalinization of the interstitial areas of the luteal tissue. The bulk of the CA seemed to be formed through hyalinization of the central blood clot. Hyaline is a descriptive term applied to any homogeneous, eosinophilic material that is produced by a multitude of mechanisms. In some cases, extracellular hyaline is composed of precipitated plasma proteins (Cotran et al., 1989
), which agrees with the proposed origin of CA hyaline in this study. Under this hypothesis, only CL displaying a large blood-filled cavity give rise to large typical CA. Several lines of evidence support this view: (i) Resorption of the central blood clot was not observed at any stage of CL involution; macrophages that would be necessary to eliminate the central coagulum, and become haemosiderin-containing cells, were rarely observed in involuting CL. (ii) Replacement of the central blood clot by connective (collagen-synthesizing) tissue was not observed; connective tissue cells were almost exclusively found in involuting CL lacking a central cavity and seemed to correspond to the IBT from opposite foldings, and proliferative activity was extremely scarce in the IBT throughout CL involution. (iii) Hyalinization-like changes, such as intense eosinophilia and the acquisition of a homogeneous appearance, were observed in the central blood clot during CL involution. (iv) The existence in young CA of an external zone containing actin-immunostained cells (i.e. myofibroblasts), probably derived from the IBT, surrounding a central hyalinized area lacking cells. The presence of a band of actin-containing cells surrounding the CA suggested that progressive contraction of the CA could be due to the presence of this outer band of contractile cells. These data suggest that only CL displaying a large blood-filled cavity give rise to CA, whereas if there was no central blood-filled cavity or it was minimal, typical CA did not develop. Mixed involuting CL were probably derived from CL showing a small blood-filled cavity. Since resorption of CA seems to take years, CA are over-represented in ovarian sections with respect to non-hyalinized involuting CL.
Large variations among CL within the same ovary, as well as among women, with respect to the presence and numbers of CA, were observed in regularly cycling women of similar age. These differences ranged from ovaries lacking CA and containing only involuting CL without (or with minimal) hyalinization, to ovaries containing large numbers of CA of different sizes, corresponding to different stages of evolution. This indicated that alternative luteolytic patterns (involving development of CA or not) may account for the elimination of non-functional luteal tissue in the human ovary. The existence of differences among different corpora lutea within the same ovary (mixed ovaries that were present in 46.4% of women) indicated that the luteolytic pattern was dependent on the characteristics of each particular CL. On the other hand, the existence of women showing exclusively a particular luteolytic pattern [25% CA(+) and 28.5% CA()] indicated the existence of an individual tendency to follow a specific luteolytic pathway. Only CL displaying a large blood-filled cavity seemed to give rise to large CA. However, the cause for the presence of a large blood-filled cavity in some CL is not clear. A possibility is that variations in the collapse of the post-ovulatory follicle are responsible for the accumulation of blood. This was also suggested by the existence of a statistically significant association between unfolding of the newly formed CL and the luteolytic pattern. However, the presence of unfolded luteal tissue could be either a cause or a consequence of the presence of a large central blood clot. Therefore, a cause-and-effect relationship cannot be established from the data presented in this study.
The unfolded/irregularly folded CL considered in this study, and characterized by a well-luteinized wall surrounding a blood-filled cavity and a prominent IBT, are usually referred to as CL cysts (Clement, 1989; Scully et al., 1996
). Most of them contained folded and unfolded areas, and the site of follicular rupture was observed. The existence of luteinized unruptured follicles (LUF) has been reported, mostly associated with ovarian stimulation (Coetsier and Dhont, 1996
) or endometriosis (Kaya and Oral, 1999
). The presence of LUF in normally cycling women seems to be a sporadic and infrequent phenomenon (Kerin et al., 1983
), that cannot account for the abundance of CA reported in this study [CA(+) 25% of women and mixed ovaries 46.5% of women]. However, it cannot be ruled out that some blood-containing unfolded CL corresponded to LUF. Furthermore, the lack of collapse of the post-ovulatory follicle has been achieved experimentally in ewes after immunization against inhibin-related peptides (Russell et al., 1995
), even in the presence of follicle rupture. This suggests that follicle collapse is not only due to follicle rupture, but also depends on complex tissue remodelling processes.
In summary, luteal cells surviving to the perimenstrual apoptotic wave underwent progressive degenerative changes, characterized by extreme vacuolation, expression of macrophage markers and accumulation of lipofuscin pigments. The pattern of structural luteolysis (formation of a CA or not) seemed to be determined shortly after ovulation, depending on the presence or absence of a large, blood-filled central cavity.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 10, 2000; accepted on June 22, 2000.