Lipid Distribution Variations in Different Stages of Cyclic Corpus Luteum of Indian Buffalo

Abstract

The present study was conducted on corpus luteum of healthy buffaloes ovaries (n = 24) collected from local slaughter house and were categorized into early (stage I, 1 to 5 days, n=6), mid (stage II, 6 to 11 days, n = 6), late luteal phase (stage III, 12 to 16 days, n = 6) and regressing phase (stage IV, 17 to 20 days, n=6). In the earliest phase i.e., corpus haemorrhagicum, the distribution of total lipids was moderate. However, in the early luteal phase, most of the luteal cells had intense staining for presence of total lipids by Sudan Black B and Oil Red O and phospholipids by Acid Hematin. By the mid luteal phase, fewer luteal cells at this stage showed positive staining for presence of lipids. During the mid luteal phase, the less frequent presence of lipid droplets in luteal cells indicated that cholesterol and its esters present at this stage might have been utilized for active synthesis of progesterone. In late luteal phase, the distribution of lipids increased to depict very intense staining. Moreover, in the regressing phase i.e., corpus albicans, the distribution of lipids increased further and was observed to be both intracellular and extracellular depicting the higher accumulation of lipids in the regressing corpus luteum. The increase in the lipid droplets in luteal cells at this stage indicated the poor mobilization of lipids and thus decline in the progesterone synthesis.

Full text

Journal of Animal Research: v.8 n.3, p. 379-385. June 2018
DOI: 10.30954/2277-940X.06.2018.7
Lipid Distribution Variations in Different Stages of Cyclic Corpus Luteum of
Indian Buffalo
Kritima Kapoor*, Opinder Singh and Devendra Pathak
Department of Veterinary Anatomy, College of Veterinary Sciences, GADVASU, Ludhiana, Punjab, INDIA
*Corresponding author: K Kapoor; Email: kritimakapoor89@gmail.com
Received: 24 Jan., 2018 Revised: 03 May, 2018 Accepted: 16 May, 2018
ABSTRACT
The present study was conducted on corpus luteum of healthy buffaloes ovaries (n = 24) collected from local slaughter house
and were categorized into early (stage I, 1 to 5 days, n=6), mid (stage II, 6 to 11 days, n = 6), late luteal phase (stage III, 12
to 16 days, n = 6) and regressing phase (stage IV, 17 to 20 days, n=6). In the earliest phase i.e., corpus haemorrhagicum, the
distribution of total lipids was moderate. However, in the early luteal phase, most of the luteal cells had intense staining for
presence of total lipids by Sudan Black B and Oil Red O and phospholipids by Acid Hematin. By the mid luteal phase, fewer
luteal cells at this stage showed positive staining for presence of lipids. During the mid luteal phase, the less frequent presence
of lipid droplets in luteal cells indicated that cholesterol and its esters present at this stage might have been utilized for active
synthesis of progesterone. In late luteal phase, the distribution of lipids increased to depict very intense staining. Moreover, in
the regressing phase i.e., corpus albicans, the distribution of lipids increased further and was observed to be both intracellular
and extracellular depicting the higher accumulation of lipids in the regressing corpus luteum. The increase in the lipid droplets
in luteal cells at this stage indicated the poor mobilization of lipids and thus decline in the progesterone synthesis.
Keywords: Lipids, Phospholipids, 3β-HSD, Corpus Luteum, Buffalo
In bovine ovary, following ovulation under the inuence
of Luteinizing hormone (LH), the granulosa and thecal
cells of the ruptured follicles are luteinized and the follicle
is transformed into a corpus luteum. The corpus luteum
formed is a dynamic endocrine gland that is composed of
steroidogenic and non-steroidogenic cells histologically.
The main function of the corpus luteum is the production
of progesterone. The steroidogenic cells i.e., small and
large luteal cells are associated with the production of
steroid hormones principally progesterone (Batra and
Sharma, 2014). For any steroid producing cell including
luteal cells, the initial step for production of progesterone
is to obtain the precursor i.e., cholesterol. However, the
luteal cells can also produce cholesterol de novo. By
default, the major mechanisms for obtaining cholesterol
are either the endocytosis of cholesterol rich low density
lipoprotein (LDL) or the selective uptake of cholesterol
esters from high density lipoprotein (HDL) (Gwynne and
Strauss, 1982). Christenson and Devoto (2003) reported
that progesterone biosynthesis required two important
enzymatic steps and it included, rst the conversion of
cholesterol to pregnenolone, catalyzed by P450 side chain
cleavage (P450 scc) located on the inner mitochondrial
membrane. This conversion was followed by succeeding
conversion to progesterone that was catalyzed by
3β-hydroxysteroid dehydrogenase (3β-HSD) present in
the smooth endoplasmic reticulum (SER). Therefore, the
present study was designed to study the distribution of
lipid and the localization of the 3β-HSD enzyme in luteal
parenchyma and their correlation during different stages
of development of corpus luteum i.e., formation and
structural regression.
MATERIALS AND METHODS
Collection of samples
The samples of corpus luteum of healthy buffaloes ovaries

380 Journal of Animal Research: v.8 n.3, June 2018
Kapoor et al.
(n=24) were collected from local slaughter house and
were categorized into early (stage I, 1 to 5 days, n=6), mid
(stage II, 6 to 11 days, n=6), late luteal phase (stage III,
12 to 16 days, n=6) and regressing phase (stage IV, 17 to
20 days, n=6), based on their gross morphology (Ireland
et al., 1980).
Cryosectioning
The fresh unxed ovaries from buffaloes having different
stages of cyclic corpus luteum were immediately collected
immediately after slaughter and stored in liquid nitrogen.
These corpus luteum tissues collected from the ovaries
was subjected to cryostat sectioning at -20°C with cryostat
microtome. The sections of 10-12 µm thickness were
obtained on clean glass slides and stained with Sudan Black
B, Oil Red O and Acid Hematin to study the distribution
pattern of lipids and phospholipids in different stages of
cyclic corpus luteum i.e., development and regression.
The sections obtained on glass slides were incubated with
substrate for 3 β-Hydroxy steroid dehydrogenase (3βHSD)
enzyme activity (Nitro BT method; Pearse, 1972) and to
study the variation in its distribution pattern according
to lipid distribution in different stages of cyclic corpus
luteum.
RESULTS AND DISCUSSION
The lipid distribution was analyzed histochemically in
different stages of development and regression of cyclic
corpus luteum. The sudanophilic lipid distribution was
observed to be in the form of few small droplets in the
luteal cells present at periphery only with central cavity
in the earliest part of cyclic corpus luteum i.e., corpus
haemorrhagicum. Further, when the corpus luteum
development progressed, the lipid distribution was
established to be moderate in the developing small and
large luteal cells that comprised the luteal parenchyma at
this stage (Fig. 1A). However, the distribution of lipids
was observed to be more in luteal cells present at periphery
as compared to centrally located luteal cells. At this stage,
the total lipid distribution was observed to have started
accumulating as minute droplets within the cytoplasm of
the developing luteal cells and was as intensely stained
by Oil Red O (Fig. 1B). Similarly, Al-zi abi et al. (2002)
observed the intense staining for lipid in the early luteal
phase by Oil Red O in mare’s corpus luteum. The lipid
accumulation indicated the beginning of synthesis of
cholesterol by the developing luteal cells as it was a
requisite precursor for progesterone production further.
Thus, this increased deposition of lipid that started at this
stage might be the result of biosynthesis of cholesterol
esters and triglycerides by the luteal cells or due to an
increased uptake of cholesterol and triglycerides from
the blood, which were not being utilized for progesterone
biosynthesis (Flint and Armstrong, 1972).
The activity of 3 β-Hydroxy steroid dehydrogenase
(3βHSD) enzyme was also observed to be weak to moderate
in the developing luteal cells (Fig. 1C). The moderate
activity of 3βHSD here indicated the fact that less enzyme
is available for conversion of precursor to progesterone
at this stage as the luteal cells were developing at this
stage. The lipid deposition therefore was observed to as
moderate accumulations in the cytoplasm of developing
luteal cells in the parenchyma. Similar correlating nding
was made by Chatterjee and Greenwald (1976) that the
highest concentration of 3βHSD was found on day 1 of
the cycle with its gradual decline over the next 3 days in
hamster corpus luteum.
The activity of phospholipids was observed to be strong in
this stage. It was observed as uniform, strong bluish black
deposition within the cytoplasm of the developing luteal
cells. However, the peripheral luteal cells (Fig. 1D) were
larger and had more phospholipids whereas the center
had a little lighter staining for phospholipids being bluish
within their cytoplasm (Fig. 2). Weinhouse and Brewer
(1942) stated that as the corpus luteum developed after
ovulation, the phospholipids increased gradually whereas
the cholesterol esters remained constant or decreased
slightly.
In the mid luteal period, the distribution of lipid was
observed to be present throughout the luteal parenchyma
uniformly. The sudanophilic lipid droplets were observed
to be present abundantly within the cytoplasm of most of
the luteal cells. However, the luteal cells observed within
the center had more lipid droplets within the cytoplasm as
compared to the luteal cells at periphery. Moreover, the
connective tissue septa had nil lipid accumulation (Fig.
3A). Similar pattern of ne lipid droplets accumulation
was observed within the cytoplasm of most of the luteal
cells by the Oil Red O. However, the overall distribution
of total lipids in the form of ne lipid droplets stained by

Lipid distribution in different stages of cyclic corpus luteum
Journal of Animal Research: v.8 n.3, June 2018 381
Oil Red O was observed to be in lesser luteal cells and was
mainly observed in the large luteal cells.
Fig. 2: Photomicrograph of corpus haemorrhagicum showing
phospholipids distribution in the luteal cells (arrow) in center of
the parenchyma. Acid Hematin X400.
Conversely, the connective tissue elements were devoid
of the lipid accumulations (Fig. 3B). Guraya (1966) stated
that when the amount of lipid was less, the release of
hormone progesterone was occurring.
The localization of 3βHSD enzyme was observed to be
strong within the cytoplasm of luteal cells at this stage.
The enzyme was observed to be present strongly within
the luteal cells located at periphery (Fig. 3C). However,
the luteal cells at the center had activity for this enzyme
but it was observed to be weak to moderate (Fig. 3D).
The expression of 3βHSD enzyme was inversely related
to the distribution of total lipids i.e., periphery had more
enzyme localization and less lipid distribution and center
had lesser enzyme expression and more lipid distribution.
This pattern of expression of 3βHSD and lipids was
correlated with its physiological role and thus indicated
that lipid distribution was lesser at periphery probably
because more 3βHSD enzyme present there converted
Fig. 1: Photomicrograph of corpus haemorrhagicum showing, (A) moderate lipid accumulations in cytoplasm of developing small and
large luteal cells (LCs, arrow). Sudan Black B X400; (B) accumulation of minute lipid droplets (arrow) within the cytoplasm of the
developing luteal cells and no lipid in connective tissue septa (Ct-S). Oil Red O X400; (C) weak to moderate 3βHSD activity (arrow)
in developing luteal cells (LCs) and weak in connective tissue septa (Ct-S). Nitro BT method X400; (D) bluish black phospholipids
deposition within the cytoplasm of the developing small (SLCs) and large luteal cells (LCs, arrow) at periphery. Acid Hematin X400.

382 Journal of Animal Research: v.8 n.3, June 2018
Kapoor et al.
the lipids in the form of cholesterol within luteal cells to
progesterone. The process of progesterone biosynthesis
required two enzymatic steps: rstly the conversion of
cholesterol to pregnenolone which was catalyzed by
P450 side chain cleavage (P450scc) located on the inner
mitochondrial membrane and subsequently the conversion
of pregnenolone to progesterone that was catalyzed by
3β-hydroxysteroid dehydrogenase (3β-HSD) present
in the smooth endoplasmic reticulum (Fig 4). Similar
observations were made by Christenson and Devoto
(2003).
The distribution of phospholipids that stained intensely
with Acid Hematin increased within the cytoplasm of the
luteal cells that constituted the parenchyma in the mid
luteal phase. It was observed to be present as strong bluish
black ne granules of phospholipids within the cytoplasm
of luteal cells present in this phase. However, the
phospholipid accumulation was strong both at periphery
and center as well (Fig. 5). Similarly, Guraya (1968)
reported that the cytoplasm of luteal cells in corpus luteum
of American opossum was shown to contain phospholipids
in the luteal phase and it was closely associated with the
synthesis of steroid hormones.
Fig. 3: Photomicrograph of mid luteal corpus luteum showing, (A) abundant sudanophilic lipid accumulated within the cytoplasm of
the luteal cells (LCs, arrow) in center as compared to the luteal cells at periphery and nil in septa (Ct-S). Sudan Black B X100. (B)
More abundant and ne lipid droplets (arrow) within the luteal cell (LCs) cytoplasm. Oil Red O X400; (C) strong 3βHSD within the
luteal cells (LCs, arrow) at periphery (P) and weak in center (C). Nitro BT method X400; (D) weak to moderate 3βHSD within the
luteal cells (LCs, arrow) at center. Nitro BT method X400.

Lipid distribution in different stages of cyclic corpus luteum
Journal of Animal Research: v.8 n.3, June 2018 383
In the late luteal phase, the luteal cells were observed to
be degenerating and the lipid accumulation in the form of
coarse sudanophillic lipid droplets increased within the
degenerating luteal cells (Fig. 6A). The intense staining
for sudanophillic lipid droplets was observed both
intracellularly and extracellularly in luteal parenchyma
at this stage. The intense lipid accumulation in was
observed in the increased vacoulations that developed
within the regressing luteal cells at this stage. However,
no lipid deposition was observed within the endothelial
cells of thicker capillaries with increased smooth muscle
around them (Fig. 6B). However, the connective tissue
septa present at this stage also had mild deposition of
lipids within the bers. The total lipid distribution stained
with Oil Red O increased signicantly and observed
to be present around the lumen of regressing capillaries
present at this stage (Fig. 6C). The progressive increase
in lipid droplets at this stage indicated that it might be due
to increase in cholesterol content that corresponds to its
reduced utilization for progesterone synthesis and thus
increased accumulation (Quirke et al., 2001 and Logan et
al., 2002).
However, the phospholipids distribution within the luteal
cells was mildly reduced in the parenchyma at this stage.
Also, the activity of 3β-HSD was observed to be reduced
considerably at this stage. Deane et al. (1966) reported
that there was a decline in of 3β-HSD enzyme activity that
could be responsible for elevation in lipid content at this
stage. This observation was correlated by the fact that the
absence of this enzyme at this stage consequently inhibited
the production of progesterone from cholesterol and thus
corresponds to the increased level of lipids.
In the corpus albicans phase, the parenchyma was
considerably shrunken and the cellularity was reduced.
Most of the parenchyma was occupied by intense staining
total lipids present at this stage in the form of coarse
lipid granule accumulations. The sudanophilic lipid
accumulations increased sharply at this stage to such an
extent that it formed several small nodular accumulations
within the parenchyma around regressing luteal cells
and mainly around regressing capillaries and thicker
blood vessels (Fig. 6D). Subsequently, moving further
the parenchyma was constituted of mainly deposits of
lipid accumulations and regressed thicker blood vessels.
Fig. 4: Schematic diagram showing utilization of lipids within the
luteal cell cytoplasm for biosynthesis of hormone progesterone
and involvement of the enzyme 3β-HSD in the process of its
conversion (*created by rst author).
Fig. 5: Photomicrograph of mid luteal phase corpus luteum
showing strong phospholipids distribution within the cytoplasm
of luteal cells (LCs, arrow) throughout the parenchyma. Acid
Hematin X400.

384 Journal of Animal Research: v.8 n.3, June 2018
Kapoor et al.
Fig. 6: Photomicrograph of the late luteal phase corpus luteum showing, (A) lipid (Li) accumulation in the form of coarse sudanophillic
lipid droplets within the degenerating luteal cells and capsule (C) devoid of it. Sudan Black B X100; (B) intense lipid accumulation
in the increased vacoulations (arrow) within the regressing luteal cells (RLCs) and no lipid in thicker capillaries (BV) with increased
smooth muscle around them. Sudan Black B X400; (C) abundant lipid droplets (Li, arrow) around the regressing luteal cells (RLCs),
lumen of regressing capillaries and mild in connective tissue septa. Oil Red O X400; (D) coarse lipid granules (Li) accumulations
around regressing luteal cells (RLCs), capillaries and thicker blood vessels. Sudan Black B X400; (E) Photomicrograph of corpus
albicans phase showing intense lipid accumulations (Li, arrow) around prominent blood vessels (BV). Oil Red O X400; (F) reduced
phospholipids content in the parenchyma around the regressing luteal cells and around regressing capillaries (BV). Acid Hematin
X400.

Lipid distribution in different stages of cyclic corpus luteum
Journal of Animal Research: v.8 n.3, June 2018 385
Moreover, blood vessels had lipid deposits around their
outer boundaries (Fig. 6E). Similar observations were
made in regressing corpus luteum of sparrow (Guraya and
Chalana, 1975), mare (Al-zi abi et al., 2002) and goats
(Batra and Sharma, 2014).
The 3β-HSD enzyme activity was observed to be nil in
the corpus albicans phase corresponding to its inverse
relationship with sharp increase in lipid distribution at this
phase. However, the distribution of phospholipids reduced
considerably at this stage and was observed to be present
in few regressing luteal cells and around few regressing
capillaries observed at this stage (Fig. 6F). Several authors
stated that with the regression of the corpus luteum, the
luteal cells begin to store lipid granules which consisted of
cholesterol and cholesterol esters, triglycerides and some
phospholipids. Similar distribution of lipids stored in the
regressing corpus luteum was reported in corpus luteum of
rat (Guraya, 1964) and hamster (Guraya and Greenwald,
1965). The signicant increase in accumulation of lipids
and decline in progesterone production in regressing
corpus luteum was attributed to degeneration in
mitochondria and smooth endoplasmic reticulum (SER)
(Umo, 1975 and Levine et al., 1979). Therefore, the
variations in distribution of total lipids and phospholipids
were observed within the cyclic corpus luteum during
its development and regression and it was inversely
related with the activity of 3β-HSD enzyme involved in
steroidogenesis.
ACKNOWLEDGEMENTS
The authors are thankful to Guru Angad Dev Veterinary
and Animal Sciences University (GADVASU), Ludhiana
for providing all type of facilities to carry out the study.
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