BIOLOGY OF REPRODUCTION 61, 1468–1479 (1999)
Luteal Regression in the Primate: Different Forms of Cell Death During Natural
and Gonadotropin-Releasing Hormone Antagonist or Prostaglandin AnalogueInduced Luteolysis
H.M. Fraser,1,2 S.F. Lunn,2 D.J. Harrison,3 and J.B. Kerr4
MRC Reproductive Biology Unit,2 Edinburgh, EH3 9ET, United Kingdom
Department of Pathology,3 University of Edinburgh, Edinburgh, EH3 9ET, United Kingdom
Department of Anatomy,4 Monash University, Clayton, Melbourne, Victoria 3168, Australia
ABSTRACT
Morphological changes in the corpus luteum following natural and induced luteolysis in the marmoset were investigated
by light and electron microscopy. Functional corpora lutea were
studied in the mid and late luteal phase, naturally regressed corpora lutea in the early and late follicular phase, and corpora
lutea induced to regress by administration of GnRH antagonist
or prostaglandin F2a 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.
INTRODUCTION
After ovulation in primates, the resultant corpus luteum
remains functional for only about 2 weeks unless rescued
by chorionic gonadotropin (CG). This process is essential
for the establishment and maintenance of early pregnancy
since, in the absence of the luteotrophic signal, the corpus
luteum undergoes spontaneous loss of cell function and
structural regression. The mechanisms involved in luteolysis in primates are poorly understood. The hormonal products of the corpus luteum are under the control of pituitary
LH in Old World primates [1, 2], New World primates [3],
and women [4], but luteal regression is not caused directly
by a decline in LH secretion [2]. This suggests that a specific luteal phase length is an inherent property of this tissue.
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
Accepted July 15, 1999.
Received February 2, 1999.
1
Correspondence: H.M. Fraser, MRC Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK.
FAX: 44 131 228 5571; e-mail: [email protected]
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 39
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
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LUTEAL CELL DEATH IN THE PRIMATE
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.
MATERIALS AND METHODS
Treatments and Collection of Tissue
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 2208C 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 5 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 mg prostaglandin F2a analogue [33]
(Planate, Coopers Animal Health Ltd., Crewe, Cheshire,
UK) i.m. or the GnRH antagonist [14] Antarelix ([N-Ac-DNal1,D-pCl-Phe2,D-Pal3,D-(Hic)6,-Lys(iPr)8,D-Ala10] GnRH,
500 mg/kg s.c; Europeptides, Argenteuil, France). Ovaries
were obtained 12 h or 24 h after treatment (n 5 3 animals
per group).
Fixation
The animals were sedated using 100 ml ketamine hydrochloride (Parke-Davis Veterinary, Pontypool, Gwent, UK)
i.m. and killed with an i.v. injection of 400 ml 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 1to 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 48C 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 mm) 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.
RESULTS
Plasma progesterone concentrations after ovulation in
the marmoset are some 30 times higher than in women and
macaques [32]. In two control midluteal phase animals,
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plasma progesterone concentrations were 186 and 124 ng/
ml; they were 20 and 12 ng/ml in the late luteal phase
marmosets. Values obtained during the early and midfollicular phase marmosets were , 4 ng/ml. Both GnRH and
prostaglandin treatment resulted in a dramatic fall in progesterone concentrations, such that even after 12 h, progesterone was , 4 ng/ml, i.e., at follicular phase levels.
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.
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
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LUTEAL CELL DEATH IN THE PRIMATE
1471
FIG. 2. Naturally regressing corpora lutea. a) Early phase of luteal regression showing condensed, intensely stained structures, probably lutein cells
(curved arrow), and smaller bodies (arrowhead) of uncertain identity. 3530. b) Another region of the same corpus luteum showing pyknotic-type bodies
(curved arrows) and fragmented structures (arrowheads). 3530. Both areas also exhibited lutein cells without apparent degenerative change. c) Corpus
luteum in advanced regression. The lutein cells were shrunken, often condensed, and surrounded by much connective tissue. 3530. d) Apoptotic
granulosa cells of an atretic follicle showing nuclear condensation, rounding-up of the degenerating cells, and fragmentation into apoptotic bodies.
3630.
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
b
FIG. 1. A) Normal midluteal phase corpus luteum showing lutein cells
with central nucleus, and abundant cytoplasm containing mitochondria
and lipid. Capillaries containing erythrocytes are evident. B) Luteal tissue
after GnRH antagonist treatment illustrating a complex mixture of recognizable lutein cells with small and large cytoplasmic inclusions (curved
arrows) and fragments of cells, vacuoles, and debris within the connective
tissue. When present, most lutein cell nuclei appeared morphologically
normal (arrowheads). C) Luteal tissue after prostaglandin treatment showing two types of structural alterations, with most lutein cells with or without a nucleus displaying many cytoplasmic vesicles while others contained mostly dense inclusions. Scattered cellular debris and necrotic-type
structures were noted in the connective tissue. 3920.
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.
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
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FIG. 3. A) A typical normal lutein cell with central nucleus (N), abundant mitochondria (M), and smooth endoplasmic reticulum (SER). 33500. B)
Lutein cell 24 h after GnRH antagonist treatment showing multiple myelin bodies (Mn) of various densities, forming membranous whorls at times in
association with areas of compact membranes. 33300. C) Lutein cells after prostaglandin treatment showing many vesicles of variable size. Nuclei (N)
were intact. 32800. D) Higher magnification of part of C showing small and large vesicles often closely associated or coalesced (arrows). The nucleus
(N) showed peripheral aggregations of heterochromatin. 38050.
LUTEAL CELL DEATH IN THE PRIMATE
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FIG. 4. A) Lutein cell, after prostaglandin administration, showing clumping of nuclear chromatin (C). The nucleolus (N), mitochondria (M) with
particulate morphology, and vesicles (V) are indicated. 37900. B) Lutein cell after prostaglandin, showing vesicle coalescence into giant intracellular
spaces. The nucleus (N) was involuted, and misshapen and mitochondria (M) were still observed. 33100.
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 mm 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.
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.
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
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FRASER ET AL.
FIG. 5. A) Early natural regression of the corpus luteum showing shrinkage of lutein cells into condensed, irregular fragments (fr) and small, dense
circular bodies (arrows) scattered within the extracellular space or within macrophages (Ma). Intact lutein cells (N), a neutrophil (Ne), and fibroblasts
(F) are indicated. 34000. B) Early regressing corpus luteum showing fragmentation of lutein cells into smooth membrane structures (S), mitochondriarich components (M), and condensed cellular material (arrow). Note densely stained clustered mitochondria in adjacent lutein cells. 34000.
LUTEAL CELL DEATH IN THE PRIMATE
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FIG. 6. A) Cytoplasm of a naturally regressing lutein cell with several condensed, particulate bodies (arrows). 37800. B) A highly shrunken lutein cell
fragment engulfed by a macrophage in the regressing corpus luteum showing a single large dense body, and mitochondria (M), lipid (L), and lysosomes
(Ls). 35800. C) Higher magnification showing the particulate nature of dense bodies and a membrane surrounding the degenerating cell (arrow), with
the macrophage membrane (m) surrounding the ingested cell fragment. 313 700.
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.
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.
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.
DISCUSSION
To our knowledge, this is the first report of ultrastructural
changes in the corpus luteum following GnRH antagonistinduced luteolysis. In addition, while extensive information
is available on the luteolytic effects of prostaglandin F2a
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 au-
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FRASER ET AL.
LUTEAL CELL DEATH IN THE PRIMATE
tophagocytosis 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 antagonisttreated 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
b
FIG. 7. Advanced naturally regressing corpus luteum. A) Lutein cells
were shrunken and contained lipid, lysosomes, and (extracted) cholesterol-rich crystalline inclusions (arrows). 32900. B) Fragments of a degenerated lutein cell (arrows) within a capillary; note endothelial cell nucleus
(EN) and cytoplasm (c). 310 360. C) Degenerating body within a macrophage (Ma). 34000. D) Higher magnification showing a degenerating
nucleus (N) and organelles (arrows) engulfed by macrophage cytoplasm
(Ma). 38400. E) Shrunken lutein cells containing lipid (L), lysosomes (Ly),
crystals (arrows), and morphologically normal nuclei (N). 34900.
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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 F2a 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 F2a 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 F2a 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
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FRASER ET AL.
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 39 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 39 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 39 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 39 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.
ACKNOWLEDGMENTS
We thank Prof. A.H. Wyllie for helpful discussions; K. Morris and
staff for animal management; S. McKenzie, M. Millar, and S. MacPherson
for preparation of the sections; R. McDougal, Department of Anatomy,
University of Edinburgh, for assistance with electron microscopy; and Dr.
R. Deghengi (Europeptides) for the gift of Antarelix.
REFERENCES
1. Fraser HM, Nestor JJ Jr, Vickery BH. Suppression of luteal function
by a luteinizing hormone-releasing hormone antagonist during the early luteal phase in the stumptailed macaque monkey and the effects of
subsequent administration of human chorionic gonadotropin. Endocrinology 1987; 121:612–618.
2. Zeleznik AJ, Benyo DF. Control of follicular development, corpus
luteum function, and the recognition of pregnancy in higher primates.
In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. New
York: Raven Press; 1994: 751–782.
3. Webley GE, Hodges JK, Given A, Hearn JP. Comparison of the luteolytic action of gonadotrophin-releasing hormone antagonist and
cloprostenol, and the ability of human chorionic gonadotrophin and
melatonin to override their luteolytic effects in the marmoset monkey.
J Endocrinol 1991; 128:121–129.
4. Dubourdieu S, Charbonnel B, Massai M, Marraoul J, Spitz I, Bouchard P. Suppression of corpus luteum function by the gonadotropin
releasing hormone antagonist Nal Glu: effect of the dose and timing
of human chorionic gonadotropin administration. Fertil Steril 1991;
56:440–445.
5. Green JA, Maquedo M. Ultrastructure of the human ovary. Am J
Obstet Gynecol 1965; 92:946–957.
6. Corner GW Jr. The histological dating of the human corpus luteum
of menstruation. Am J Anat 1956; 98:377–401.
LUTEAL CELL DEATH IN THE PRIMATE
7. Adams EC, Hertig AT. Studies on the human corpus luteum. I. Observations on the ultrastructure of development and regression of the
luteal cells during the menstrual cycle. J Cell Biol 1969; 41:696–715.
8. Clement PB. Histology of the ovary. In: Sternberg SS (ed.), Histology
for Pathologists. Philadelphia: Lippincott-Raven Press; 1997: 929–
959.
9. Lei ZM, Chegini N, Rao CV. Quantitative cell composition of human
and bovine corpora lutea from various reproductive states. Biol Reprod 1991; 44:1148–1156.
10. Joel RV, Foraker AG. Fate of the corpus albicans: a morphologic
approach. Am J Obstet Gynecol 1960; 80:314–316.
11. Hsueh AJW, Billig H, Tsafriri A. Ovarian follicle atresia: a hormonally
controlled apoptotic process. Endocr Rev 1994; 15:707–724.
12. Tilly JL. Apoptosis and ovarian function. Rev Reprod 1996; 1:162–
172.
13. Rodger FE, Fraser HM, Krajewski S, Illingworth PJ. Production of
the proto-oncogene BAX does not vary with changing in luteal function in women. Mol Hum Reprod 1998; 4:27–32.
14. Fraser HM, Lunn SF, Cowen GM, Illingworth PJ. Induced luteal regression in the primate: evidence for apoptosis and changes in c-myc
protein. J Endocrinol 1995; 147:131–137.
15. Rueda BR, Tilly KI, Botros IW, Jolly PD, Hansen TR, Hoyer PB,
Tilly JL. Increased bax and interleukin-1b-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine
corpus luteum during structural regression. Biol Reprod 1997; 56:
186–193.
16. Kenny N, Williams RE, Kelm LB. Spontaneous apoptosis of cells
prepared from the non-regressing corpus luteum. Biochem Cell Biol
1994; 72:531–536.
17. Juengel JL, Garverick A, Johnson AL, Youngquist RS, Smith MF.
Apoptosis during luteal regression in cattle. Endocrinology 1993; 132:
249–254.
18. Zheng J, Fricke PM, Reynolds LP, Redmer DA. Evaluation of growth,
cell proliferation, and cell death in bovine corpora lutea throughout
the estrous cycle. Biol Reprod 1994; 51:623–632.
19. Dharmarajan AM, Goodman SB, Tilly KI, Tilly JL. Apoptosis during
functional corpus luteum regression: evidence of a role for chorionic
gonadotropin in promoting luteal cell survival. Endocr J 1994; 2:295–
303.
20. Gaytán F, Bellido C, Morales C, Sánchez-Criado JE. Both prolactin
and progesterone in proestrus are necessary for the induction of apoptosis in the regressing corpus luteum of the rat. Biol Reprod 1998;
59:1200–1206.
21. McCormack JT, Friedrichs MG, Limback SD, Greenwald GS. Apoptosis during spontaneous luteolysis in the cyclic golden hamster: biochemical and morphological evidence. Biol Reprod 1998; 58:255–
260.
22. O’Shea JD, Nightingale MG, Chamley WA. Changes in small blood
vessels during cyclical luteal regression in sheep. Biol Reprod 1977;
17:162–177.
23. Azmi TI, O’Shea JD. Mechanism of deletion of endothelial cells during regression of the corpus luteum. Lab Invest 1984; 51:206–217.
24. Sawyer HR, Niswender KD, Braden TD, Niswender GD. Nuclear
changes in ovine luteal cells in response to PGF2a. Domest Anim
Endocrinol 1990; 72:229–238.
25. Behrman HR, Endo T, Aten RF, Musicki B. Corpus luteum function
and regression. Reprod Med Rev 1993; 2:153–180.
26. Quatacker JR. Formation of autophagic vacuoles during human corpus
luteum involution. Z Zellforsch 1971; 122:479–487.
27. Koering MJ, Wolf RC, Meyer RK. Morphological changes in the corpus luteum correlated with progestin levels in the rhesus monkey during early pregnancy. Biol Reprod 1973; 9:254–271.
28. Shikone T, Yamoto M, Kokawa K, Yamashita K, Nishimori K, Nakano R. Apoptosis of human corpora lutea during cyclic luteal regression and early pregnancy. J Clin Endocrinol Metab 1997; 81:2376–
2380.
29. Yuan W, Giudice LC. Programmed cell death in human ovary is a
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
1479
function of follicle and corpus luteum status. J Clin Endocrinol Metab
1997; 82:3148–3155.
Young FM, Illingworth PJ, Lunn SF, Harrison DJ, Fraser HM. Cell
death during luteal regression in the marmoset monkey (Callithrix
jacchus). J Reprod Fertil 1997; 111:109–119.
Kirton JT, Koering MJ. Prostaglandin F2a and primate corpus luteum:
a correlation of structure and function. Fertil Steril 1973; 24:926–934.
Smith KB, Lunn SF, Fraser HM. Inhibin secretion during the ovulatory cycle and pregnancy in the common marmoset monkey. J Endocrinol 1990; 126:489–495.
Summers PM, Wennink J, Hodges JK. Cloprostenol-induced luteolysis
in the marmoset monkey (Callithrix jacchus). J Reprod Fertil 1985;
73:133–138.
Webley GE, Richardson MC, Smith CA, Masson GM, Hearn JP. Size
distribution of luteal cells from pregnant and non-pregnant marmoset
monkeys and a comparison of the morphology of marmoset luteal
cells with those from the human corpus luteum. J Reprod Fertil 1990;
90:427–437.
Clarke PGH. Developmental cell death: morphological diversity and
multiple mechanisms. Anat Embryol 1990; 181:195–213.
Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980; 68:251–306.
Kressel M, Groscurth P. Distinction of apoptotic and necrotic cell
death by in situ labelling of fragmented DNA. Cell Tissue Res 1994;
278:549–556.
Christensen AK, Gillim SW. The correlation of fine structure and
function in steroid-secreting cells, with emphasis on those of the gonads. In: McKerns WW (ed.), The Gonads. New York: AppletonCentury-Crofts; 1972: 415–488.
Fraser HM, Sellar RE, Illingworth PJ, Eidne KA. GnRH receptor
mRNA expression by in-situ hybridization in the primate pituitary and
ovary. Mol Hum Reprod 1996; 2:117–121.
Michael AE, Abayasekara DRE, Webley GE. Cellular mechanisms of
luteolysis. Mol Cell Endocrinol 1994; 99:R1-R9.
Zeleznik AJ. In vivo responses of the primate corpus luteum to luteinizing hormone and chorionic gonadotropin. Proc Natl Acad Sci
USA 1998; 95:11002–11007.
Salazar H, Furr BJA, Smith GK, Bentley M, Gonzalez-Angulo A.
Luteolytic effects of a prostaglandin analogue, cloprostenol (ICI
80,996) in rats: ultrastructural and biochemical observations. Biol Reprod 1976; 14:458–472.
Stacy BD, Gemmell RT, Thorburn GD. Morphology of the corpus
luteum in the sheep during regression induced by prostaglandin F2a.
Biol Reprod 1976; 14:280–291.
McLellan MC, Abel JHJ, Niswender GD. Function of lysosomes during luteal regression in normally cycling and PGF2a-treated ewes. Biol
Reprod 1977; 16:499–512.
Young FM, Illingworth PJ, Fraser HM. Ubiquitin and apoptosis in the
corpus luteum of the marmoset monkey (Callithrix jacchus). J Reprod
Fertil 1998; 114:163–168.
Rueda BR, Wegner JA, Marion SL, Wahlen DD, Hoyer PB. Internucleosomal DNA fragmentation in ovine luteal tissue associated with
luteolysis: In vivo and in vitro analyses. Biol Reprod 1995; 52:305–
312.
Modlich U, Kaup FJ, Augustin HG. Cyclic angiogenesis and blood
vessel regression in the ovary: blood vessel regression during luteolysis involves endothelial cell detachment and vessel occlusion. Lab
Invest 1996; 74:771–780.
Welsh AO, Enders AC. Chorioallantoic placenta formation in the rat.
III. Granulated cells invade the uterine luminal epithelium at the time
of epithelial cell death. Biol Reprod 1993; 49:38–57.
Prince F, Mann DR, Fraser HM. Blockade of the pituitary testicular
axis by a gonadotrophin releasing hormone antagonist in the neonatal
marmoset: changes in Leydig cell ultrastructure. Cell Tissue Res 1998;
30:651–661.
Aoki MP, Maldonado CA, Aoki A. Apoptotic and non-apoptotic cell
death in hormone-dependent glands. Cell Tissue Res 1998; 291:571–
574.