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Progesterone secretion and mitochondrial size of aging porcine corpora lutea.

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THE ANATOMICAL RECORD 223:252-256 (1989)
Progesterone Secretion and Mitochondria1Size of
Aging Porcine Corpora Lutea
Department of Animal Science, Iowa State University, Ames, Iowa 50011
A functional dependency between the nongravid uterus and the
ovaries is essential to luteolysis and the return to estrus in the pig. After mating of
gilts, the corpora lutea develop, and they are required for the maintenance of
pregnancy t o a normal duration of about 114 days. Hysterectomy of luteal phase
(day 6) nongravid gilts results in persistence of the corpora lutea to 150 days. We
report that these corpora secrete greater quantities ( P < 0.025) of progesterone than
during the later half of gestation (days 54-108). Although aging corpora lutea
remain functional for at least an additional 35 days, an abrupt reduction by half in
progesterone secretion (16 ng/ml) occurs about day 114 in hysterectomized gilts that
coincides with the prepartum decrease to basal serum levels (<0.5 ng/ml) at parturition (day 114) and during lactation. Aging corpora lutea remain large (averaging
>450 mg) on days 124 and 136 in hysterectomized gilts, whereas they regress
(averaging < 75 mg) in the lactating dams. Mitochondria continue to increase in size
in aging corpora lutea of hysterectomized gilts until day 136; in contrast, they
decrease during the postpartum period in lactating dams. A precisely timed signal,
possibly of ovarian origin or from the CNS and pituitary gland, entrains in hysterectomized and pregnant pigs at day 113 that results in marked shifts in relaxin and
progesterone secretion. Progesterone secretion and mitochondria1 features suggest
that porcine corpora lutea seem genetically controlled and are preprogrammed at
estrus for the duration of pregnancy, regardless of the presence of conceptuses or
absence of the uterus.
Progesterone and relaxin are produced by porcine corpora lutea during pregnancy and after hysterectomy
(Belt et al., 1971; Anderson et al., 1973, 1983; Musah et
al., 1984). Although the normal duration of the estrous
cycle is about 21 days in the pig, the corpora lutea
secrete progesterone and small amounts of relaxin (Masuda et al., 1967; Anderson et al., 1973; Sherwood and
Rutherford, 1981). Pregnancy lasts about 114 days in
this species, and soon after mating progesterone secretion peaks early and remains elevated from days 8-108,
when it decreases before parturition. The corpora lutea
are required for the maintenance of pregnancy; ovariectomy any time results in abortion within 36 hr (du
Mesnil du Buisson and Dauzier, 1957). Although these
corpora in hysterectomized gilts are capable of remaining large ( > 450 mg) at least 35 days beyond the time of
normal regression at parturition, abrupt changes occur
in their capacity to secrete both progesterone and relaxin (Felder et al., 1986). The ephemeral nature of
mammalian corpora lutea can be characterized by their
autonomy of progesterone secretion, responsiveness to
the luteolytic effects of prostaglandins, and their ability
t o make prostaglandins (Anderson et al., 1969; Rothchild, 1981).We describe sequential profiles of progesterone secretion into peripheral blood during periods
exceeding the normal life span of the corpora lutea in
0 1989 ALAN R. LISS, INC.
hysterectomized gilts, as compared with pregnant and
lactating animals, as well as the fine structure of mitochondria in aging IuteaI cells.
Purebred Yorkshire gilts, approximately 125 kg body
weight, that had exhibited at least one estrous cycle (21
f 2 days; mean f S.E.)were either mated at estrus (day
0) or remained unmated and were hysterectomized at
day 6 by surgical procedures described previously (Anderson et al., 1961).Anesthesia was induced by intravenous injection of thiamylal sodium (0.8-1.0 gm Surital,
Parke-Davis, Morris Plains, NJ) and maintained by a
closed-circuit system of halothane (4-9%; Ayerst Laboratories, New York, NY) and 0 2 (500-1,000 cc/min).During hysterectomy, the corpora lutea on each ovary were
marked with a loop of silk suture for later identification.
Animals were laparotomized on days 100, 112, 124, and
136 to obtain luteal tissue for electron microscopy. During these laparotomies, samples of luteal tissue from
Received November 2, 1987; accepted August 22, 1988.
I Y I t 171I I’?I
171I tl?t
ON DAY 6 (9)
cells representing different regions of the corpora lutea
were examined for mitochondrial size in each animal.
The number of mitochondria measured per photomicrograph ranged from 18 to 160. The statistical analysis of
data on mitochondrial size was based on animal as the
experimental unit, not on luteal cell or mitochondria
within cell.
For statistical analysis the experimental units in this
study were the individual gilts, and they were assigned
to treatments a t random. Data were analyzed as a splitplot by using the statistical analysis for the general
linear model and by Student’s t-test for comparisons
among treatment groups (Snedecor and Cochran, 1980).
I. l. t. t. l. l. l, t. l, l. l , l . t . l . l . l . l . l . l 1 1 1 1 1 l t 1
Fig. 1. Description of experimental groups indicating days for sequential collections of peripheral venous blood for radioimmunoassay
of progesterone and for sequential laparotomies to obtain luteal tissue
for electron microscopic examination. The number of gilts in each
group is indicated in parentheses.
both ovaries were removed for electron microscopy. Blood
samples were allowed to clot at 4°C and were then
centrifuged (2,OOOg; 4”C), and serum was stored at -20°C
for radioimmunoassay of progesterone. The description
of experimental groups and number of gilts in each
group are indicated in parentheses in Figure 1.
Progesterone was extracted from duplicate aliquots of
200 p1 serum with benzene:hexane (1:2 vol/vol) by modification of a procedure described previously. A third
replicate served as a recovery for determining procedural losses by addition of 5,000 cpm (1,2,6,7-[N]-3H)progesterone (97.0 Ci/mmol; New England Nuclear
Corp., Boston, MA). Serum extracts were assayed for
progesterone as described by Anderson et al. (1979) with
the fully characterized antibody GDN 337 (Niswender,
1973; Gibori et al., 1977). The sensitivity of the assay
was defined as the amount of progesterone that yielded
95% of cpm in buffer control tubes; this ranged from 50
to 80 pg. Intraassay and interassay coefficients of variance (CV) were 2.0 and 5.0%, respectively. Mean blanks
from ovariectomized-hysterectomized gilts were < 0.05
ngiml (N = 4). The precision and accuracy were evaluated by adding 0.05, 0.10, 0.25,0.50, 1.00,2.50, and 5.00
ng/ml progesterone to serum from ovariectomized-hysterectomized gilts, and the recoveries of six replicates
were (mean f S.E.) 0.07 f 0.01, 0.15 f 0.01, 0.39 f
0.01, 0.73 f 0.009, 1.41 0.01, 3.25 5 0.07, and 5.5 k
0.10 ng/ml, respectively.
Fixation and dehydration of corpora lutea were done
a t 2°C. Luteal tissue was cut into cubes <1 mm and
fixed 3 h r in 2.5% glutaraldehyde in Millonig’s phosphate buffer, pH 7.25, rinsed in Millonig’s buffer, and
transferred to 1% osmium tetroxide for 2 hr. During the
last step of the acetone dehydrations, the tissue was
allowed to warm (24°C) for infiltration and embedding
in a n Epon-Araldite resin. Silver sections (60-80 pm)
were cut on glass knives with a n LKB Ultrotome I11
microtome. Thin sections were double-stained (with 2%
uranyl acetate in methanol and then lead citrate) and
examined in a n RCA EMU-4 electron microscope. At
least four randomly selected photomicrographs of luteal
The experimental design and details for the collection
of sequential samples of blood (10 ml), hysterectomy, and
the sequential laparotomies for collection of luteal tissue
are presented in Figure 1. The concentrations of progesterone in peripheral blood serum were determined by
radioimmunoassay as described in Figure 2. In nongravid gilts hysterectomized a t day 6, progesterone
serum levels remained significantly greater ( P < 0.025)
on days 54 through 108 than during pregnancy (Fig. 2).
Parturition occurred a n average of 114 days after mating in the six pregnant gilts. Progesterone blood levels
decreased abruptly in hysterectomized gilts at the time
equivalent to that of parturition. By using a split-plot
analysis, circulating levels of progesterone during days
114 to 168 were lower (P < 0.001) than those during
days 54 to 108 in both hysterectomized and pregnant/
lactating gilts (Fig. 2). Hysterectomized group means
remained significantly greater (P < 0.0001) than those
of pregnantAactating animals during days 114 to 168.
By day 120, progesterone concentrations were < 1ngiml
and remained consistently low throughout lactation. A
postpartum estrous cycle occurred in two of the six lac-
Fig. 2. Progesterone concentrations in peripheral blood serum of six
Yorkshire gilts during pregnancy and early lactation ( 0 )compared
with those in nine unmated gilts hysterectomized (A)on day 6 (day 0
= estrus). During lactation a postpartum estrous cycle occurred in
Y2084 (0)
and Y2113 (U), whereas the other four animals remained
acyclic as indicated by basal blood levels of progesterone from days
Fig. 3.Mitochondria were prominent in luteal cells of hysterectomized and pregnant gilts, but they were significantly greater in diameter in the hysterectomized compared with pregnant gilts at day 100
(a, b). The mitochondria of luteal cells in both groups of gilts contained
fenestrated larnelliform cristae at this time (a,b). From days 100 to
136, the corpora lutea in hysterectomized gilts remained large, but
they regressed in the pregnant and lactating dams. By day 136, most
luteal cells in hysterectomized gilts contained numerous large mitochondria and little evidence of regression (c), whereas a few of them
contained mitochondria of different diameters and intracellular as well
as intercellular collagen fibers. In lactating dams at day 136, regressed
luteal tissue contained cells with collagen and few mitochondria (d).
Bar = 1 pm.
tating gilts, as indicated by increased progesterone levels after day 138 (Fig. 2).
On days 100 and 112, large corpora lutea (>450 mg)
Reproductive No. of
Perimeter (pm)of mitochondria’
typify hysterectomized and pregnant gilts. The corpora
gilts Day 100 Day 112 Day 124 Day 136 lutea persisted and remained larger (averaging >450
mg) on days 124 and 136 in the hysterectomized gilts,
whereas they regressed (averaging < 75 mg) in the laclactation
2.13* 2.43** 2,32** tating dams. Mitochondria were prominent in luteal
cells and contained fenestrated lamelliform cristae in
corpora lutea from both groups of hysterectomized and
‘Values are the group means for animals within reproductive status pregnantgilts (Fig. 3). At days 100 and 112, the mitoand day. Micrographs were randomly chosen from each sample. A
sheet of clear acetate with parallel lines 1 cm apart was randomly
were larger (‘<O.O1)
in hysterectomized as
placed on each micrograph. Mitochondria intercepted by the lines compared with those of pregnant gilts (S.E. difference =
were measured with a Bioquant I1 Image Analysis System (R & M 0.067; Table 1). Although they increased (P<0.0005) in
Biornetrics, Nashville, TN).
size in both groups from days 100 to 112 (S.E. difference
“ P < 0.01 compared with corresponding day of pregnancy and - 0.044),
the mitochondria remained significantly larger
* + P < 0.001 compared with corresponding day of pregnancy and in the hysterectomized gilts to day 136 (Fig. 3, Table 11,
whereas they decreased in size in the lactating animals
TABLE 1. Size of mitochondria in aging porcine
corpora lutea
by that time. Sequential changes in the fine structure of
aging luteal cells in hysterectomized gilts and mated
animals indicated marked shifts in populations of electron-dense granules, as has been observed in previous
studies (Belt et al., 1971; Anderson et al., 1983; Fields
and Fields, 1985; Anderson, 1987).
The results presented here indicate that circulating
levels of progesterone in hysterectomized gilts are consistently greater than corresponding stages throughout
normal pregnancy. Although the corpora lutea remain
large to day 150, there is a n abrupt decrease in progesterone secretion in these hysterectomized gilts, which
coincides with that found in normal animals at parturition. Estrone (El) and 176-estradiol (176-E~)are primarily of fetal-placental origin in this species, and
circulating levels of them peak just preceding parturition (Fevre et al., 1968; Anderson et al., 1983). After
hysterectomy, peripheral blood concentrations of E l and
170-Ez remain consistently low from days 6 to 168 (Anderson et al., 1983). Although exogenous estrogens are
luteotropic in this species, the mechanisms of their action are undefined (Gardner et al., 1963; Anderson et al.,
1973). In hysterectomized gilts given intramuscular injections of 17fl-E~to mimic blood levels of endogenous
estrogen during normal pregnancy, progesterone secretion is suppressed to levels similar to those of normal
pregnancy (Musah et al., 1984).Nevertheless, progesterone secretion decreases abruptly in hysterectomized gilts
on days 113-114 in the absence or presence of exogenous
estrogens. Furthermore, relaxin blood concentrations
peak at day 113 in hysterectomized gilts, the same day
as found during late pregnancy (Felder et al., 1986).
Thus, extrinsic (uterine) prostaglandins may switch on
intrinsic (luteal) prostaglandins to cause luteolysis a t
the termination of a n estrous cycle or pregnancy,
whereas after hysterectomy, the rate of luteal regression
is limited to the intrinsic process. Whether luteal demise
during late pregnancy results primarily from prostaglandins of uterine and luteal cell origin and during late
hysterectomy from luteal cells is not known. Prolactin
(PRL) secretion is tonically inhibited by the porcine hypothalamus, as indicated by elevated PRL blood levels
after hypophysial stalk transection (Anderson et al.,
1982). In mated gilts hypophysial-stalk transected at
days 30 and 50, the pregnancies fail, whereas in those
stalk-transected a t days 70 and 90, the pregnancies are
maintained to normal term (du Mesnil du Buisson and
Denamur, 1969). Exogenous PRL maintains pregnancy
and progesterone secretion a t least 10 days in gilts hypophysectomized at day 70, whereas luteinizing hormone (LH) is ineffective (du Mesnil du Buisson, 1973).
In hypophysectomized-hysterectomized gilts, LH or human chorionic gonadotropin (hCG) maintains luteal
function and progesterone secretion to day 30, whereas
the corpora lutea regress in those similarly treated but
with the uterus intact (Anderson et al., 1965, 1967).
Daily injections of purified porcine PRL from days 110120 in pregnant and hysterectomized gilts increased
circulating levels of progesterone and relaxin (Felder et
al., 1988). These results provide clear evidence that porcine PRL prolongs secretory function by aging corpora
lutea with sustained progesterone as well as relaxin
secretion in pregnant and hysterectomized gilts. Furthermore, PRL maintains aging corpora lutea and progesterone secretion from days 110-120 in hypophysectomized-hysterectomized gilts (Li et al., 1987).
The data in Table 1 indicate that, a t estrus, a n unknown process begins that results in increased luteal
mitochondria size. Mitochondria reached their maximum size in pregnant gilts at parturition and are maintained a t that size for about 10 days after parturition.
Because luteal mitochondrial size in hysterectomized
gilts peaked approximately 10 days later and regressed
only slightly by day 136, a signal from the uterus and/
or a hormonal shift associated with lactation may be
required for mitochondrial regression. The similar
amount of decline in serum progesterone levels in both
groups of animals suggests that mitochondrial size is
not directly related to progesterone synthesis. Larger
and more mitochondria may, however, be required to
meet the metabolic needs of the larger corpora lutea in
the hysterectomized gilts.
The evidence presented here indicates that the presence of the conceptuses or the absence of the uterus,
fetuses, and their associated placental estrogens are required for prolonged progesterone secretion in the pig.
As the corpus luteum grows older, the aging luteal cells
may be preprogrammed to have a n inherent life span of
approximately 113 days. Mechanisms initiating a decrease in progesterone secretion during pregnancy and
after hysterectomy may be related to aging of porcine
luteal cells. These abrupt and autonomous shifts in progesterone secretion on about days 113-114 in hysterectomized as well as pregnant pigs may be genetically
controlled. Progesterone secretion continues at halfmaximal values in hysterectomized gilts while the mitochondria continue to increase in size. Although exogenous PRL can maintain elevated progesterone secretion
by aging corpora lutea in hysterectomized gilts, a role
for endogenous PRL secretion in the abrupt shifts in
progesterone secretion is unknown.
We thank Professor D.F. Cox for statistical analyses
and M.E. Shell, C.R. Bohnker, A. Alcivar, R. Perezgrovas, J.R. Molina, and R. Poyner for assistance. This
work was supported by U.S. Department of Agriculture,
A R S , CSRS, and OGPS Competitive grant 85-CRCR-11862. Journal Paper 5-12077 of the Iowa Agriculture
and Home Economics Experiment Station, Ames, IA
(Projects 2443, 2444, and 2754).
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progesterone, corpora, lute, size, secretion, porcine, aging, mitochondria
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