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a MRC Reproductive Biology Unit, Edinburgh, EH3 9ET, United Kingdom
b Department of Pathology, University of Edinburgh, Edinburgh, EH3 9ET, United Kingdom
c Department of Anatomy, Monash University, Clayton, Melbourne, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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analogue in the midluteal phase. Natural luteolysis was associated with lutein cell atrophy, condensation of cytoplasmic inclusions and organelles, and accumulation of lipid. GnRH antagonist treatment resulted in aggregations of smooth membranes and myelin-like bodies in the cytoplasm of the lutein cells together with complex aggregations of degenerative cells. After prostaglandin treatment, the lutein cells contained numerous small and large vesicles; as the degenerative changes advanced, these vesicles coalesced into alveolar-type vacuoles, and nuclei involuted. These results show that in the marmoset, natural luteolysis and the two luteolytic treatments reveal different forms of luteal degeneration and cell death, none of which fit the ultrastructural criteria for apoptosis. More emphasis needs to be placed on understanding these predominant nonapoptotic forms of cell death in order to elucidate the process of luteolysis in the primate.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The demise of the corpus luteum, resulting in its transformation into the irregular connective tissue that constitutes the corpus albicans, involves degeneration of all luteal cells leading to their disappearance. Involution of the tissue is accompanied by progressive fibrosis and shrinkage. Thus, the architecture of the mature corpus luteum is dramatically altered so that the rich vascular supply, the supporting connective tissue cells, and the granulosa and theca lutein cells are replaced with bundles of collagen fibers, scattered fibroblasts, and occasional macrophages [5–9]. Most corpora albicantia are resorbed and replaced by ovarian stroma [10]. Morphological regression of the corpus luteum necessarily involves cell death, but the mechanisms by which each of the cell types within luteal tissue is destroyed remain unclear.
Developing follicles within the ovary are continually undergoing remodeling, with cell proliferation and death; follicular atresia involving degeneration of granulosa cells is an example of apoptosis [11, 12], a process in which individual cells die within healthy tissue. Since the discovery that apoptosis is regulated by a group of survival and death factors associated with specific genes, there has been extensive investigation into the control of cell death in the ovary, and many of these factors have been shown to be present in the corpus luteum [12–15]. Recently, several reports have described significant tissue apoptosis as indicated by DNA electrophoresis or the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling reaction for demonstrating DNA fragmentation in sheep [16], cow [17, 18], rabbit [19], rat [20], and hamster [21] corpora lutea. These results confirm earlier studies showing morphological evidence for apoptosis during luteal regression in some species [22–24].
Because of the important differences in the mechanisms of luteal regression between species [25], it is essential for our understanding of the physiology of the human corpus luteum that suitable primate models be investigated. Morphological features of cell death in the regressing human [5–7, 26] and primate corpus luteum [27] have been described. Although the evidence for apoptosis is less convincing, it has been tempting to assume that these reports describe apoptotic cells, at least in part. Recently, using 3' end-labeling, apoptosis in the corpus luteum has been reported in the human [28, 29] and marmoset [30].
However, our studies on natural and induced luteolysis in the marmoset demonstrated another form of cell death associated with pronounced cellular vacuolation [14, 30]. Ultrastructural descriptions of natural luteolysis have not been reported for primate tissue in association with recent advances in the knowledge of different forms of cell death, and we question whether any of the previous reports have demonstrated apoptosis convincingly. In addition, little is known about the ultrastructural features of the primate corpus luteum after induced luteolysis [31]. In the current study, histological and ultrastructural changes in luteal cell death in the marmoset after natural and induced luteolysis have been explored and compared. The marmoset has the advantage that induction of luteolysis can be reliably achieved subsequent to GnRH antagonist treatment (by removing LH support), or by a direct inhibitory effect of prostaglandin treatment on the hormone-producing lutein cells. It was postulated that differences in response to the treatments would reveal divergent pathways of luteal cell death. Finally, by compressing the time scale of luteal regression with such treatments, it was anticipated that the model might show changes in the corpus luteum more readily than by examination of the more protracted process of natural luteolysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female common marmosets (Callithrix jacchus) were housed as described previously [14]. Blood samples were collected 3 times per week by femoral venepuncture without anesthesia to confirm normal ovulatory cycles. Plasma was stored at -20°C until required for assay. Criteria for the occurrence of ovulation (Day 0) and normal luteal phase length (18–22 days) were based on determination of plasma progesterone concentrations as described previously [32]. The experiments were carried out in accordance with the Animals (Scientific Procedures) Act, 1986. Ovaries were collected from animals during the midluteal (Day 10), late luteal (Days 20–22, but prior to functional luteolysis), early follicular (1–3 days postfunctional luteolysis), and late follicular (5–7 days postfunctional luteolysis) phases of the cycle (n = 2 animals per group). To examine the effect of induced luteal regression, on Day 8/9 after the estimated time of ovulation, the animals were treated with either 1 µg prostaglandin F2 analogue [33] (Planate, Coopers Animal Health Ltd., Crewe, Cheshire, UK) i.m. or the GnRH antagonist [14] Antarelix ([N-Ac-D-Nal1,D-pCl-Phe2,D-Pal3,D-(Hic)6,-Lys(iPr)8,D-Ala10] GnRH, 500 µg/kg s.c; Europeptides, Argenteuil, France). Ovaries were obtained 12 h or 24 h after treatment (n = 3 animals per group).
Fixation
The animals were sedated using 100 µl ketamine hydrochloride (Parke-Davis Veterinary, Pontypool, Gwent, UK) i.m. and killed with an i.v. injection of 400 µl Euthetal (sodium pentobarbitone; Rhone Merieux, Dublin, Ireland). Ovaries were removed immediately and the corpora lutea of the cycle identified macroscopically. After bisection, half of each corpus luteum (up to 3 per animal) was cut into 1- to 2-mm cubes using a razor blade and was immersion fixed for 2 h in 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3. Specimens were then rinsed overnight at 4°C in the same buffer, postfixed in buffered 2% osmium tetroxide for 2 h, and embedded in Araldite (Ladd Research, Burlington, VT) after dehydration in ethanol and propylene oxalate.
Semithin (1 µm) sections were stained with toluidine blue for light microscope analysis. At least three blocks from each corpus luteum were studied in detail. Thin sections were stained with uranyl acetate and lead citrate and examined in a Philips EM 301 (The Netherlands) electron microscope. The remainder of the ovary was fixed in 4% paraformaldehyde for 24 h and used in studies to determine changes in other factors associated with control of luteal cell function described elsewhere.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Light Microscopic Observations
Normal corpus luteum In contrast to observations in most other primate species studied, distinct enfoldings of lutein cells derived from the theca layer and separate from regions of granulosa-derived lutein cells are not present in the marmoset corpus luteum [34]. While a small number of hormone-producing cells had morphological features and locations consistent with theca lutein cells, i.e., were distributed in occasional aggregations peripheral to the extensive areas of large granulosa lutein cells, for the purposes of this report differences in the origin of the lutein cells were not taken into consideration. The most prominent features in the normal corpora lutea collected during the midluteal phase were the large polyhedral lutein cells, characterized by circular nuclei in cross section with a nucleolus and large cytoplasmic volume (Fig. 1A). Dense bodies within the cytoplasm included mitochondria and lysosomes together with less basophilic inclusions typical of lipid droplets. In some tissue sections the lutein cells showed varying degrees of basophilia, although it was not possible to determine whether cytoplasmic density was correlated with distinct differences in organelle or inclusion content. The lutein cells were supported by connective tissue displaying fibroblasts, and there was an extensive blood supply characterized by the occurrence of endothelial cell nuclei and numerous lumina often containing erythrocytes.
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GnRH antagonist treatment After GnRH antagonist treatment, most of the hormone-producing (lutein) cells were dramatically altered (Fig. 1B). Within each corpus luteum, varying degrees of structural change indicative of degeneration were observed, so that for descriptive purposes, data for the 12- and 24-h posttreatment groups were combined. The lutein cells were characterized by the presence of numerous small and large aggregations of granular material in the cytoplasm; basophilia in some cells was associated with a range of intracellular inclusions, from heterogeneous aggregations of granular materials to very dense, condensed material suggestive of degeneration. When present in the plane of section, some lutein cell nuclei appeared normal whereas others showed evidence of extraction or dissolution of nuclear content, i.e., karyolysis. In the more advanced state, the cells exhibited condensed granular materials with only occasional nuclei present. In capillaries of the supporting tissue, erythrocytes were observed, and the connective tissue contained pale-staining elliptical or fusiform nuclei characteristic of fibroblasts and endothelial cells. Areas of advanced luteolysis comprised complex aggregations of degenerative cells and cellular debris, and it was not possible to distinguish accurately elements of the vascular system within the supporting tissue.
Prostaglandin analogue treatment After prostaglandin analogue treatment, luteal tissue from both 12- and 24-h treatment groups displayed a heterogeneous range of degenerative change, so again, results for the two time points were combined. Typically, some lutein cells exhibited degenerative alterations similar to those seen in the GnRH antagonist-treated group. However, most of the lutein cells showed combinations of dense granules and minute clear vacuoles in the cytoplasm (Fig. 1C). The nuclei, where present, were at times considerably smaller than in comparable cells in control tissues. Some lutein cells showed intact nuclei within heterogeneous clumps of granular materials in the cytoplasm. In the more advanced state, numerous lutein cells showed larger vacuoles that often gave the impression of coalescence into extensive clear areas of cytoplasm with a minor proportion of granular material at times surrounding a small central nucleus or condensed material. Nuclei of the supporting tissue were seen, but often no clear distinction could be determined between the capillary endothelial cells and the fibroblasts of the connective tissue.
Naturally regressed corpus luteum Corpora lutea from the late luteal phase, prior to functional luteolysis, showed no significant changes in histology compared to the midluteal control tissue (not shown). In the two animals from which tissue was collected during the early follicular phase, and whose corpora lutea had ceased to function, numerous condensed, intensely stained structures that had probably been lutein cells were observed together with smaller bodies and fragmented structures of unknown identity (Fig. 2, a and b). Often these dense bodies were associated with spaces or vacuoles that appeared to be empty (Fig. 2b), but whether these were intracellular or extracellular could not be determined with light microscopy. At this stage, the corpora lutea also exhibited lutein cells without apparent degenerative change.
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The corpora lutea from ovaries of the late follicular phase were markedly decreased in volume compared to those within the midluteal control tissues, and they were surrounded by an abundance of connective tissue. Numerous lipid inclusions, partly extracted, were present in most of these cells. Scattered at random throughout the luteal tissue were pyknotic elements, at times showing morphological features suggesting cell degeneration and/or cell death (Fig. 2c). Because of their small cross-sectional area, it was not possible to determine the nature of the degenerating cells. Small arterioles and venules were structurally normal, but the close aggregation of atrophic lutein cells and the supporting cells and matrix of the connective tissue made it difficult to determine the morphological status of the capillaries.
Since apoptosis had been expected in luteal tissue, an established source of such cells, the granulosa layer of an atretic follicle, was examined to demonstrate nuclear condensation (Fig. 2d). Degeneration of granulosa cells consistent with apoptosis was shown by the rounded cell contours, single or multiple dense aggregations of nuclear chromatin or condensed cytoplasmic inclusions/organelles, and numerous dense cell fragments 2–4 µm in diameter, commonly referred to as "apoptotic bodies."
Ultrastructural Observations
Normal corpus luteum In control and mid and late luteal phase corpora lutea, the lutein cells were characterized by a central circular nucleus in cross section and a single nucleolus. In the cytoplasm, the dominant components were vesicles or short anastomosing tubules of smooth endoplasmic reticulum (ER), variable numbers of small lipid droplets, and many mitochondria (Fig. 3A). The morphology of the mitochondria was variable between the hormone-producing cells, some cells having lamellar-type cristae and others displaying tubular cristae that filled the mitochondrial matrix.
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GnRH antagonist treatment The striking accumulation of a variety of dense bodies within lutein cells, as noted with light microscopy, was confirmed by ultrastructural analysis. Where present, the nuclei and mitochondria of lutein cells appeared normal, but the cytoplasm contained numerous large structures of variable form and density suggestive of phases of degeneration and condensation of membranes during formation of myelin-type inclusions (Fig. 3B). Similar myelin bodies, disrupted cytoplasm, and cellular debris occurred external to the identifiable lutein cells; but whether these structures were slender extensions or fragments of lutein cells, or of connective tissue origin, was not determined.
Prostaglandin analogue treatment After prostaglandin analogue treatment, the smooth ER of lutein cells had a vesicular appearance, and large dilated membrane-bound vesicles occupied much of the cytoplasmic volume (Fig. 3C). When apposed, the larger vesicles were often confluent, and in many lutein cells the coalescence of vesicles gave rise to extensive interconnected structures resembling an alveolar-type morphology (Fig. 3D). In contrast, the nuclei remained intact, but often the heterochromatin was condensed into many small irregular clumps subjacent to the nuclear membrane (Fig. 3, C and D), a feature not observed in lutein cells from the normal corpus luteum. As the lutein cells became shrunken, the nuclear heterochromatin in some cells aggregated into several clumps (Fig. 4A), the cytoplasm showing many vesicles together with abnormal mitochondria in which the cristae showed disruption into particulate matter. In the more degenerate cells, multiple vesicles occupied much of the cell volume, while in others, fewer vesicles were noted but their large size again made them the dominant feature (Fig. 4B). In these cells, the nuclei were involuted and often misshapen.
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Within the connective tissue, the morphology of the cells and the extracellular matrix was suggestive of tissue disruption with ruptured cell membranes and transformation of cellular organelles into fragments, particulate matter, and residual bodies.
Naturally regressed corpus luteum In corpora lutea of ovaries collected from the early follicular phase of the cycle, the tissue contained morphologically normal lutein cells in addition to degenerating lutein cells identified by their increased electron density, irregular contours, and fragmentation (Fig. 5A). Small, dense circular elements representing clumps of cellular debris were numerous, and most of these had been engulfed by macrophages located in the connective tissue between normal or degenerating lutein cells. The presence of neutrophils suggested an inflammatory-type response associated with the destruction and removal of dead and dying cells. The ultrastructural response of lutein cells was markedly variable; it included clustering and increased density of the mitochondria, or changes in the smooth ER to form innumerable small vesicles, or giant whorls of tubular smooth ER (Fig. 5B). Condensation of cytoplasm was a universal feature either within focal regions of lutein cells, or as distinct, often extracellular clumps of cellular debris.
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At higher magnification of recognizable lutein cells, or fragments derived from them, numerous particulate bodies were seen in the cytoplasm (Fig. 6A). With further condensation, portions of degenerating lutein cells were phagocytosed by macrophages and became fully or partly surrounded by the macrophage plasma membrane (Fig. 6, B and C), indicating enclosure by a vacuole or phagosome.
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By the late follicular phase, the corpora lutea were in an advanced stage of regression characterized by general cell atrophy, fragmentation, and increased proportion of extracellular matrix and collagen. In some of the atrophied lutein cells, lipid inclusions had accumulated together with extracted, oblong cytoplasmic structures resembling cholesterol-rich crystalline inclusions known to occur in other steroidogenic cells, particularly in association with a decline in metabolic or synthetic activity (Fig. 7A). Fragments of condensed lutein cell cytoplasm were occasionally noted within the lumina of capillaries (Fig. 7B) and often within macrophages (Fig. 7C). In the latter, whole condensed nuclei had been ingested by the macrophages, and the associated presence of lipid inclusions and crystalline-type bodies suggested their identity as degenerating lutein cells (Fig. 7D). Within the tissue examined, not every lutein cell was either fragmented or engulfed by phagocytes. Some were atrophied, often aggregated together, and their markedly shrunken cytoplasm contained secondary lysosomes, large lipid inclusions, and crystalline bodies (Fig. 7E). The nuclei of these cells retained a morphologically normal appearance.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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analogue in the nonprimate, this is the first description of such effects on luteal morphology in the primate corpus luteum. Recently, there has been an emphasis on apoptosis as representing the principal form of luteal cell death, primarily stemming from the localization of fragmented DNA [16–21]; and, in the case of nonprimates, there is also unequivocal evidence for apoptosis based upon ultrastructural studies. However, it is emphasized that our observations do not indicate rapid or widespread cell degeneration via apoptosis, as defined by accepted histological or ultrastructural criteria [35–37]. With the exception of a very small number of degenerating lutein cells, including some engulfed by macrophages (e.g., Figs. 4A and 7D), classic apoptotic-type nuclei of lutein cell identity were rare. Our findings indicate that, during luteolysis in the marmoset, death of the vast majority of lutein cells proceeds via nonapoptotic mechanisms. During natural luteolysis, this involves cell atrophy with phagocytosis of cytoplasmic debris. This contrasts with both GnRH antagonist and prostaglandin-induced luteolysis, which are associated with autophagocytosis and nonlysosomal cell disintegration, respectively.
Ultrastructural changes during natural luteolysis in the marmoset are consistent with earlier reports on luteal regression in the human [5–7, 26, 38] in that the atrophied lutein cells show persistence of an intact nucleus, with a cytoplasm that contains autophagocytic vacuoles and both lipid and crystalloid inclusions. Cellular debris is present in the involuting lutein cells and in adjacent macrophages. Furthermore, heterochromatin does not condense or marginate into aggregates or crescentic caps; the nucleus does not convolute into separate protuberances or "blebs"; and many of the cytoplasmic organelles do not remain intact but exhibit spontaneous autolysis and are degraded by lysosomes to form autophagosomes and structurally complex debris. As regression proceeds, with phagocytic ingestion of cell debris, the remaining lutein cells are considerably atrophied and accumulate lipid and crystalline-type inclusions indicating cessation of steroidogenesis. It is likely that these shrunken lutein cells are destroyed by macrophages as the previously highly cellular corpus luteum is converted into a corpus albicans. Taken together, these results suggest that natural regression of lutein cells in the human and marmoset is a form of nonapoptotic degeneration.
The absence of detectable degenerative changes in the late luteal phase, prior to functional luteolysis in the normal cycle of the marmoset, was somewhat surprising, since shrinkage of lutein cells at this period has been described in the human corpus luteum [6]. The luteal phase of the marmoset is slightly longer and of more variable length than in Old World primates and women, and perhaps this is associated with an extended phase of maintenance of structural integrity. This may be followed by a more rapid degenerative phase when functional luteolysis finally occurs.
Both GnRH antagonist and prostaglandin analogue treatments were associated with dramatic changes in the morphology of the lutein cells within 12 h. Degenerative changes in the smooth ER were more advanced than any observed in the mitochondria, suggesting that the early failure to secrete progesterone was the result of an acute sensitivity of the smooth ER. Interestingly, the changes to smooth ER in the lutein cells were markedly different according to the luteolytic treatment. In the GnRH antagonist-treated animals, the membranes of the smooth ER, and possibly segments of the redundant plasma membrane, became condensed into islands of concentric membranes that resulted in the formation of myelin bodies. This response suggests cell degeneration via autophagocytic mechanisms followed by heterophagocytosis. While this process occurs on a small scale during normal cellular reorganization of lutein cells [38], the appearance of the cells from the GnRH antagonist-treated animals indicated a process that would lead to cell death. After prostaglandin administration, a similar response was observed in a minority of lutein cells; but the remainder showed a completely different appearance, with smooth ER becoming markedly swollen and forming many vesicles such that in the advanced state, these vesicles coalesced into large vacuoles. This observation may be similar to the heavily vacuolated cells described in paraffin sections in the naturally regressing human corpus luteum [6], and implies the production of a fluid that is not released from the cell—an event indicating disrupted cell metabolism, synthesis, or secretion.
The primary action of GnRH antagonist treatment is to suppress LH secretion [3], direct effects on the corpus luteum being unlikely in the marmoset [39]. The fall in progesterone secretion after GnRH antagonist administration is dependent upon the prior decline in LH [3]. In contrast, the luteolytic action of prostaglandin F2 in the marmoset is directly upon the corpus luteum; in vivo treatment with prostaglandin analogue is followed by a more rapid suppression of plasma progesterone secretion without change in LH levels [3]; and in vitro, LH-stimulated progesterone production is inhibited at pre- and post-cAMP sites [3, 40]. This would agree with in vivo studies in which prostaglandin analogue treatment at or after the midluteal phase led to irreversible suppression of progesterone secretion in the marmoset [33]. While the suppressive effects of GnRH antagonist treatment can be overcome by concomitant administration of hCG [1, 3], the marmoset corpus luteum cannot be "rescued" when prostaglandin analogue and hCG are given together [3]. Since both treatments result in failure of LH stimulation of steroidogenesis, the ultrastructural differences indicate an additional effect of prostaglandin on the lutein cell as a result of its direct action. Further studies may help provide an explanation at the cellular level as to why these treatments are associated with differing outcomes in vivo.
It is surprising that neither GnRH antagonist nor prostaglandin treatment resulted in changes identical to those occurring during natural luteolysis. In the marmoset, the mechanism of natural luteolysis is not known; and if there is an endogenous luteolysin, there is no direct evidence that it acts rapidly. However, it is not unreasonable to expect that both treatments may mimic and accelerate naturally occurring phenomena. For example, it is known that the responsiveness of the LH receptor declines toward the end of the luteal phase [41], so removal of interference with the normal LH stimulus by luteolytic treatments may be expected to induce a rapid precipitation of events associated with loss of LH receptor activation. Although the demise of the luteal cells during natural luteolysis does not follow the same morphological pattern as for induced luteolysis in the marmoset, GnRH antagonist and prostaglandin treatments may still provide a valuable approach for further studies of the mechanisms responsible for the functional and morphological demise of the primate corpus luteum.
In contrast to our findings in the marmoset, prostaglandin F2 analogue treatment in nonprimate species, e.g., the rat [42], guinea pig [23], and sheep [24, 43, 44], results in changes apparently similar to those observed in natural regression. This species difference may originate from the fact that natural luteolysis in sheep and cattle is brought about primarily by prostaglandin F2 of uterine origin and that this process is advanced by the exogenous administration of the analogue. Lutein cells of the ovine corpus luteum were reported to be highly vacuolated with contracted cytoplasm, accumulation of lipid droplets, a desegregation of smooth ER into myelin bodies, and an increase in the number of autophagosomes [44]. Sawyer et al. [24] proposed that the initial site of action of prostaglandin is the lutein cell, where it causes an inhibition of steroidogenesis, together with an increase in oxytocin release that in turn stimulates prostaglandin secretion from the uterus. At the same time the lumina of small blood vessels within regressing corpora lutea become filled with cellular debris, most of which represents fragments of capillary endothelial cells [22, 24]. This cascade would result in ischemia and hypoxia leading to apoptosis of endothelial cells, followed later by the apoptosis of lutein cells.
In previous reports we described the occurrence of apoptosis following natural and induced luteolysis in the marmoset using light microscopy and 3' end-labeling to detect apoptotic cells [14, 30, 45]. The same protocols and study periods were employed in the current experiments, and some of the ovaries were employed in both approaches. Indeed, in the semithin sections examined, structures resembling apoptotic bodies were observed, although this classification could not be established with certainty, particularly in comparison with degenerating granulosa cells. However, under the electron microscope, these structures in the corpus luteum were considered to be degenerating cells showing either autophagocytosis or nonlysosomal disintegration according to the classifications of these types of cell death, and that of apoptosis, reviewed by Clarke [35]. In the case of autophagocytosis, the pyknosis or heterochromatin clumping phenomenon is not prevalent. The nucleus ultimately disintegrates and is digested by autolysosomes, which are the abundant and characteristic feature seen in this type of lutein cell degeneration. Ultimately the lutein cell debris or fragments are destroyed by macrophages, i.e., by heterophagocytosis. In the case of nonlysosomal disintegration, organelles swell, and empty spaces arise by fusion, with cell destruction achieved by fragmentation. The destruction of the nucleus is delayed (in contrast to what occurs in apoptosis), and it disintegrates in a manner similar to that for the cytoplasm. Cytoplasmic lysosomes do not contribute to autophagolysosomes, and there is no significant engulfment of cellular debris by macrophages.
In situ 3' end-labeling of DNA fragments in cells of the regressing corpus luteum, and demonstration of laddering patterns on gels of DNA isolated from regressing corpora lutea, have been shown in all species reported—adding weight to the argument that luteolysis involves apoptosis [16–21, 28, 30, 45, 46]. However, as pointed out in a number of such reports, the DNA laddering is accompanied by smear patterns indicating nonapoptotic degradation of nuclear chromatin [28, 45, 46]. This has led these workers to suggest that luteolytic cell death might be a combination of apoptosis-type and necrotic-type degeneration, a concept supported by the present study. In the studies on human and monkey corpora lutea that combined 3' end-labeling with morphological analysis, study of luteal tissue was limited to light microscopic examination [28–30]. The most likely explanation for these findings is that the studies under the light microscope (using larger cross-sectional-area histological sections compared with epoxy resin sections) detected both true apoptotic cells together with structures that appeared both morphologically and by 3' end-labeling to be apoptotic cells or bodies but that do not, in fact, fit the ultrastructural criteria for apoptosis. Although an important difference between primates and nonprimates appears to be related to the extent to which apoptosis occurs, such differences are also apparent between nonprimates. For example, the dramatic apoptosis observed in the hamster corpus luteum [21] accounts for the disappearance of these structures within one cycle, while in the rat, the luteal tissue of previous cycles remains in the ovary.
There is evidence that death of the endothelial cells by apoptosis, with attendant reduction in blood supply, could play an important part in the luteolytic process, at least in some species [23, 24]. There was little evidence of endothelial cell death by apoptosis in the present study, but this is likely to be underestimated because of the small size of the blood vessels and the attenuated nature of the endothelial lining. Dead and dying cells would exfoliate into the lumina of affected vessels, with the remaining viable endothelium and basal lamina normally forming a barrier between dead cells and other cell types [23]. The engulfment of the apoptotic cells would be limited to the adjacent (endothelial) cells or perhaps the pericytes, which have a low phagocytic capability. The dying cells may undergo further degeneration in the lumen, but most if not all of these fragments might be expected to be lost via the flushing effects of continued blood flow. Indeed, a study of the regressing bovine corpus luteum [47] indicated that most of the endothelial cells detach from the basement membrane before undergoing apoptosis so that apoptotic endothelial cells were not observed in tissue sections. In the current study, there is no evidence either for or against the concept that endothelial cell death leads to the degenerative changes observed in the lutein cells.
Finally, a review of the literature shows that degenerative changes in the cells of other endocrine target tissues or hormone-producing tissues, such as the rodent uterine epithelium [48], marmoset Leydig cells [49], rat, rabbit, and primate ovarian theca cells [11], and the prostate and pituitary gland [50], may also show nonapoptotic death. The current results suggest that as far as the marmoset is concerned, and probably other primates, more emphasis should be on understanding the nonapoptotic forms of cell death that we have shown to be predominant during luteal regression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: July 15, 1999.
Received: February 2, 1999.
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M. Myers, E. Gay, A. S. McNeilly, H. M. Fraser, and W. C. Duncan In Vitro Evidence Suggests Activin-A May Promote Tissue Remodeling Associated with Human Luteolysis Endocrinology, August 1, 2007; 148(8): 3730 - 3739. [Abstract] [Full Text] [PDF]
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J Aguilar, H M Fraser, H Wilson, E Clutton, D J Shaw, and E D Watson Temporal relationship between proliferating and apoptotic hormone-producing and endothelial cells in the equine corpus luteum Reproduction, July 1, 2006; 132(1): 111 - 118. [Abstract] [Full Text] [PDF]
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V. K. Yadav and R. Medhamurthy Dynamic Changes in Mitogen-Activated Protein Kinase (MAPK) Activities in the Corpus Luteum of the Bonnet Monkey (Macaca radiata) during Development, Induced Luteolysis, and Simulated Early Pregnancy: A Role for p38 MAPK in the Regulation of Luteal Function Endocrinology, April 1, 2006; 147(4): 2018 - 2027. [Abstract] [Full Text] [PDF]
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A. A Goyeneche, J. M Harmon, and C. M Telleria Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis Reproduction, January 1, 2006; 131(1): 103 - 111. [Abstract] [Full Text] [PDF]
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W. C. Duncan, S. G. Hillier, E. Gay, J. Bell, and H. M. Fraser Connective Tissue Growth Factor Expression in the Human Corpus Luteum: Paracrine Regulation by Human Chorionic Gonadotropin J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5366 - 5376. [Abstract] [Full Text] [PDF]
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M. C. Peluffo, K. A. Young, and R. L. Stouffer Dynamic Expression of Caspase-2, -3, -8, and -9 Proteins and Enzyme Activity, But Not Messenger Ribonucleic Acid, in the Monkey Corpus Luteum during the Menstrual Cycle J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2327 - 2335. [Abstract] [Full Text] [PDF]
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V. K. Yadav, P. Muraly, and R. Medhamurthy Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase Mol. Hum. Reprod., September 1, 2004; 10(9): 629 - 639. [Abstract] [Full Text] [PDF]
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K.A. Young, J.D. Hennebold, and R.L. Stouffer Dynamic expression of mRNAs and proteins for matrix metalloproteinases and their tissue inhibitors in the primate corpus luteum during the menstrual cycle Mol. Hum. Reprod., September 1, 2002; 8(9): 833 - 840. [Abstract] [Full Text] [PDF]
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 S.F. Lunn, H.M. Fraser, and H.D. Mason Structure of the corpus luteum in the ovulatory polycystic ovary Hum. Reprod., January 1, 2002; 17(1): 111 - 117. [Abstract] [Full Text] [PDF]
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C. Morales, L. Garcia-Pardo, C. Reymundo, C. Bellido, J. E. Sanchez-Criado, and F. Gaytan Different patterns of structural luteolysis in the human corpus luteum of menstruation Hum. Reprod., October 1, 2000; 15(10): 2119 - 2128. [Abstract] [Full Text] [PDF]
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 P. T.K. Saunders, M. R. Millar, K. Williams, S. Macpherson, D. Harkiss, R. A. Anderson, B. Orr, N. P. Groome, G. Scobie, and H. M. Fraser Differential Expression of Estrogen Receptor-{alpha} and -{beta} and Androgen Receptor in the Ovaries of Marmosets and Humans Biol Reprod, October 1, 2000; 63(4): 1098 - 1105. [Abstract] [Full Text]
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 A. Bulling, F. D. Berg, U. Berg, D. M. Duffy, R. L. Stouffer, S. R. Ojeda, M. Gratzl, and A. Mayerhofer Identification of an Ovarian Voltage-Activated Na+-Channel Type: Hints to Involvement in Luteolysis Mol. Endocrinol., July 1, 2000; 14(7): 1064 - 1074. [Abstract] [Full Text]
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S. E. Dickson and H. M. Fraser Inhibition of Early Luteal Angiogenesis by Gonadotropin-Releasing Hormone Antagonist Treatment in the Primate J. Clin. Endocrinol. Metab., June 1, 2000; 85(6): 2339 - 2344. [Abstract] [Full Text]
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H. M. Fraser, S. E. Dickson, S. F. Lunn, C. Wulff, K. D. Morris, V. A. Carroll, and R. Bicknell Suppression of Luteal Angiogenesis in the Primate after Neutralization of Vascular Endothelial Growth Factor Endocrinology, March 1, 2000; 141(3): 995 - 1000. [Abstract] [Full Text] [PDF]
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