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 Analogue-
Induced 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 nat-
ural 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 cor-
pora 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 degen-
erative cells. After prostaglandin treatment, the lutein cells con-
tained 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 predomi-
nant 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 luteol-
ysis in primates are poorly understood. The hormonal prod-
ucts 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 spe-
cific luteal phase length is an inherent property of this tis-
sue.
The demise of the corpus luteum, resulting in its trans-
formation into the irregular connective tissue that consti-
tutes 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 con-
nective 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, Cen-
tre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK.
FAX: 44 131 228 5571; e-mail: h.fraser@ed-rbu.mrc.ac.uk
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are replaced with bundles of collagen fibers, scattered fi-
broblasts, 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 un-
clear.
Developing follicles within the ovary are continually un-
dergoing remodeling, with cell proliferation and death; fol-
licular atresia involving degeneration of granulosa cells is
an example of apoptosis [11, 12], a process in which indi-
vidual 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 ex-
tensive 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 re-
ports have described significant tissue apoptosis as indicat-
ed by DNA electrophoresis or the in situ terminal deoxynu-
cleotidyl transferase-mediated dUTP nick end-labeling re-
action 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 mor-
phological 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. Mor-
phological features of cell death in the regressing human
[5–7, 26] and primate corpus luteum [27] have been de-
scribed. Although the evidence for apoptosis is less con-
vincing, 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 re-
ported 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 cor-
pus 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 re-
moving LH support), or by a direct inhibitory effect of
 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 re-
gression with such treatments, it was anticipated that the
model might show changes in the corpus luteum more read-
ily 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. Cri-
teria for the occurrence of ovulation (Day 0) and normal
luteal phase length (18–22 days) were based on determi-
nation of plasma progesterone concentrations as described
previously [32]. The experiments were carried out in ac-
cordance with the Animals (Scientific Procedures) Act,
1986. Ovaries were collected from animals during the mid-
luteal (Day 10), late luteal (Days 20–22, but prior to func-
tional luteolysis), early follicular (1–3 days postfunctional
luteolysis), and late follicular (5–7 days postfunctional lu-
teolysis) 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-D-
Nal1,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 hydro-
chloride (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 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 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 sec-
tions 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 midfolli-
cular phase marmosets were , 4 ng/ml. Both GnRH and
prostaglandin treatment resulted in a dramatic fall in pro-
gesterone concentrations, such that even after 12 h, pro-
gesterone 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
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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 dis-
tributed in occasional aggregations peripheral to the exten-
sive 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 fea-
tures in the normal corpora lutea collected during the mid-
luteal phase were the large polyhedral lutein cells, charac-
terized 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 dis-
playing 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 lu-
teum, varying degrees of structural change indicative of
degeneration were observed, so that for descriptive purpos-
es, data for the 12- and 24-h posttreatment groups were
combined. The lutein cells were characterized by the pres-
ence of numerous small and large aggregations of granular
material in the cytoplasm; basophilia in some cells was as-
sociated with a range of intracellular inclusions, from het-
erogeneous aggregations of granular materials to very
dense, condensed material suggestive of degeneration.
When present in the plane of section, some lutein cell nu-
clei 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 ob-
served, 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 de-
bris, and it was not possible to distinguish accurately ele-
ments 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 de-
generative change, so again, results for the two time points
were combined. Typically, some lutein cells exhibited de-
generative alterations similar to those seen in the GnRH
antagonist-treated group. However, most of the lutein cells
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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 com-
parable cells in control tissues. Some lutein cells showed
intact nuclei within heterogeneous clumps of granular ma-
terials in the cytoplasm. In the more advanced state, nu-
merous 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 ma-
terial. Nuclei of the supporting tissue were seen, but often
no clear distinction could be determined between the cap-
illary 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 ous lipid inclusions, partly extracted, were present in most
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 rec-
ognizable 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 show-
ing two types of structural alterations, with most lutein cells with or with-
out a nucleus displaying many cytoplasmic vesicles while others con-
tained 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 mid-
luteal 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 bod-
ies 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 cor-
pora lutea also exhibited lutein cells without apparent de-
generative 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. Numer-
of these cells. Scattered at random throughout the luteal
tissue were pyknotic elements, at times showing morpho-
logical 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 degen-
erating 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.
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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 con-
densation (Fig. 2d). Degeneration of granulosa cells con-
sistent with apoptosis was shown by the rounded cell con-
tours, single or multiple dense aggregations of nuclear chro-
matin or condensed cytoplasmic inclusions/organelles, and
numerous dense cell fragments 2–4 mm in diameter, com-
monly referred to as ‘‘apoptotic bodies.’’
Ultrastructural Observations
Normal corpus luteum. In control and mid and late lu-
teal 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 endo-
plasmic reticulum (ER), variable numbers of small lipid
droplets, and many mitochondria (Fig. 3A). The morphol-
ogy of the mitochondria was variable between the hor-
mone-producing cells, some cells having lamellar-type cris-
tae and others displaying tubular cristae that filled the mi-
tochondrial 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 lu-
tein cells appeared normal, but the cytoplasm contained nu-
merous large structures of variable form and density sug-
gestive 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 conflu-
ent, 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 nu-
clei remained intact, but often the heterochromatin was con-
densed 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 dis-
ruption 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 cy-
cle, 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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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), mitochondria-
rich components (M), and condensed cellular material (arrow). Note densely stained clustered mitochondria in adjacent lutein cells. 34000.
 LUTEAL CELL DEATH IN THE PRIMATE
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 in-
flammatory-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 ves-
icles, or giant whorls of tubular smooth ER (Fig. 5B). Con-
densation of cytoplasm was a universal feature either within
focal regions of lutein cells, or as distinct, often extracel-
lular 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 con-
densation, portions of degenerating lutein cells were phago-
cytosed by macrophages and became fully or partly sur-
rounded 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 extra-
cellular matrix and collagen. In some of the atrophied lutein
cells, lipid inclusions had accumulated together with ex-
tracted, oblong cytoplasmic structures resembling choles-
terol-rich crystalline inclusions known to occur in other ste-
roidogenic 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 nu-
clei had been ingested by the macrophages, and the asso-
ciated presence of lipid inclusions and crystalline-type bod-
ies suggested their identity as degenerating lutein cells (Fig.
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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 appear-
ance.
DISCUSSION
To our knowledge, this is the first report of ultrastructural
changes in the corpus luteum following GnRH antagonist-
induced 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, primar-
ily stemming from the localization of fragmented DNA
[16–21]; and, in the case of nonprimates, there is also un-
equivocal evidence for apoptosis based upon ultrastructural
studies. However, it is emphasized that our observations do
not indicate rapid or widespread cell degeneration via ap-
optosis, as defined by accepted histological or ultrastruc-
tural criteria [35–37]. With the exception of a very small
number of degenerating lutein cells, including some en-
gulfed by macrophages (e.g., Figs. 4A and 7D), classic ap-
optotic-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 non-
apoptotic mechanisms. During natural luteolysis, this in-
volves cell atrophy with phagocytosis of cytoplasmic de-
bris. This contrasts with both GnRH antagonist and pros-
taglandin-induced luteolysis, which are associated with au-
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 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 re-
gression 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 mar-
ginate 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 ly-
sosomes 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 inclu-
sions 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 mar-
moset 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 oc-
curs.
Both GnRH antagonist and prostaglandin analogue treat-
ments were associated with dramatic changes in the mor-
phology of the lutein cells within 12 h. Degenerative chang-
es in the smooth ER were more advanced than any ob-
served 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 ani-
mals, the membranes of the smooth ER, and possibly seg-
ments of the redundant plasma membrane, became con-
densed 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 re-
sponse was observed in a minority of lutein cells; but the
remainder showed a completely different appearance, with
b fact that natural luteolysis in sheep and cattle is brought
FIG. 7. Advanced naturally regressing corpus luteum. A) Lutein cells
were shrunken and contained lipid, lysosomes, and (extracted) cholester-
ol-rich crystalline inclusions (arrows). 32900. B) Fragments of a degen-
erated lutein cell (arrows) within a capillary; note endothelial cell nucleus
(EN) and cytoplasm (c). 310 360. C) Degenerating body within a mac-
rophage (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.
1477
smooth ER becoming markedly swollen and forming many
vesicles such that in the advanced state, these vesicles co-
alesced into large vacuoles. This observation may be sim-
ilar 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 metabo-
lism, synthesis, or secretion.
The primary action of GnRH antagonist treatment is to
suppress LH secretion [3], direct effects on the corpus lu-
teum being unlikely in the marmoset [39]. The fall in pro-
gesterone 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
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directly upon the corpus luteum; in vivo treatment with
prostaglandin analogue is followed by a more rapid sup-
pression 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 prostaglan-
din 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 an-
tagonist treatment can be overcome by concomitant admin-
istration 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 dif-
ferences 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 out-
comes in vivo.
It is surprising that neither GnRH antagonist nor pros-
taglandin 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 ex-
pected 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 treat-
ments 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, prostaglan-
din 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 re-
gression. This species difference may originate from the
about primarily by prostaglandin F2a of uterine origin and
that this process is advanced by the exogenous administra-
tion of the analogue. Lutein cells of the ovine corpus lu-
teum 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] pro-
posed that the initial site of action of prostaglandin is the
1478 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 regress-
ing 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 ap-
optosis following natural and induced luteolysis in the mar-
moset 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 re-
sembling apoptotic bodies were observed, although this
classification could not be established with certainty, par-
ticularly 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 disinte-
gration 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 heterochro-
matin 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 disin-
tegration, organelles swell, and empty spaces arise by fu-
sion, 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 signifi-
cant 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 num-
ber of such reports, the DNA laddering is accompanied by
smear patterns indicating nonapoptotic degradation of nu-
clear 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 lim-
ited 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 his-
tological sections compared with epoxy resin sections) de-
tected 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 dif-
ferences are also apparent between nonprimates. For ex-
ample, the dramatic apoptosis observed in the hamster cor-
pus 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 endo-
thelial 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 endothe-
lial lining. Dead and dying cells would exfoliate into the
lumina of affected vessels, with the remaining viable en-
dothelium and basal lamina normally forming a barrier be-
tween dead cells and other cell types [23]. The engulfment
of the apoptotic cells would be limited to the adjacent (en-
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dothelial) 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 frag-
ments 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 en-
dothelial 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 ob-
served in the lutein cells.
Finally, a review of the literature shows that degenera-
tive 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 pi-
tuitary gland [50], may also show nonapoptotic death. The
current results suggest that as far as the marmoset is con-
cerned, 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 re-
gression.
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.
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