Category Archives: Corpus Luteum

Susceptibility of the Corpus Luteum: DISCUSSION(6)

Because progesterone interferes with luteal cell death and because prolactin is a potent stimulator of luteal progesterone production, we examined whether prolactin is an antiapoptotic factor for luteal cells independent of its stimulatory effect on progesterone. The use of a prolactin-responsive rat luteal cell line that carries the long form of the prolactin receptor and is unable to produce progesterone allowed us to demonstrate an antiapoptotic role for prolactin that could not be mediated by progesterone.

Susceptibility of the Corpus Luteum: DISCUSSION(5)

DISCUSSION(5)

This result agrees with previous observations in primary and immortalized granulosa cells, in which organelles associated with steroidogenesis remained intact and highly organized during the first 24 h of induction of apoptosis and chromatin condensation and breakdown of the nuclear membrane occurred while high levels of progesterone were still being produced. Likewise, a large number of apoptotic cells can be found within the CL after parturition while the gland retains its steroidogenic capacity, which is reflected in the high output of 20a-dihydroprogesterone, a metabolite of progesterone. However, it is not known whether 20a-dihydroprogesterone plays a physiological role in luteal apoptosis.

Susceptibility of the Corpus Luteum: DISCUSSION(4)

All types of CLs subjected to incubation in vitro were still able to release progesterone, even while undergoing apoptosis (see Fig. 4B). Because the index of apoptosis in the CL is negatively correlated with the mass of the CL (Fig. 5, C and D), we expressed the amount of progesterone released to the medium on a per CL basis. However, CLs freshly isolated on Day 7 of pregnancy and Day 4 postpartum have an average weight of approximately 2 mg/unit, whereas those isolated on Days 14 and 21 of pregnancy are approximately two to three times heavier.

Susceptibility of the Corpus Luteum: DISCUSSION(3)

DISCUSSION(3)

The differential susceptibility of the CL to apoptosis probably depends on the in vivo hormonal environment and, in particular, on the protective effects of progesterone and prolactin. Evidence for this is the clear delay in the appearance of fragmented DNA observed during lactation, which induces an increase in both circulating prolactin and progesterone. The effect of lactation on the process of structural luteal regression was also demonstrated by the partial prevention of the decrease of luteal weight and of the increase in number of apoptotic cells observed in regressing CLs after parturition.

Susceptibility of the Corpus Luteum: DISCUSSION(2)

After observing the morphological features of apoptosis in regressed CLs in vivo and in CLs subjected to various hours of incubation in vitro (present study), it is clear that more advanced features of apoptosis are observed in vitro. In vivo, evidence of apoptosis includes single small, densely stained nuclei (pyknotic appearance), nuclei containing marginated chromatin, and cells containing multiple densely stained nuclear fragments. In vitro, all these features of apoptosis are also visible (see Figs. 2, B and C, and 3, B-E), but cells containing multiple smaller fragments of chromatin are also present (see Figs. 2, B and C, and 3, FI). This observation suggests that under conditions of starvation the apoptotic process may involve additional steps that could explain the further fragmentation of the chromatin.

Susceptibility of the Corpus Luteum: DISCUSSION(1)

DISCUSSION(1)

Our previous morphological studies have revealed that in the rat CL the index of apoptosis is very low throughout the long gestational luteal phase in pregnancy, but a marked increase in the number of luteal cells undergoing apoptosis occurs after parturition, accompanying the decrease in the mass of the gland. However, the result of the present study clearly show that breakdown of genomic DNA as analyzed by gel electrophoresis is not discernable in CLs undergoing luteal regression at the end of pregnancy and after parturition at a time when features of apoptosis can be clearly observed morphologically.

Susceptibility of the Corpus Luteum: RESULTS(4)

The lactating animals also displayed higher concentrations of progesterone in circulation compared with nonlactating controls (Fig. 5B). The establishment of lactation reduced significantly the number of nuclei undergoing apoptosis (Fig. 5C), whereas it partially prevented the decrease in the weight of CLs in non-lactating controls killed on Day 4 postpartum (Fig. 5D). When CLs from similar groups of animals were incubated under serum-free conditions, the induction of DNA fragmentation was significantly delayed in CLs harvested from lactating rats compared with those harvested from nonlac-tating controls (Fig. 6, A-C).

Susceptibility of the Corpus Luteum: RESULTS(3)

RESULTS(3)

There was a large difference in the number of cells undergoing apoptosis, depending on the developmental stage during pregnancy and after parturition at which the animals were killed (Fig. 4A). A relatively high number of cells undergoing apoptosis was found early in pregnancy on Day 7, whereas only a few cells with apoptotic features could be detected later in pregnancy, at either Day 14 or Day 21. When CLs obtained from animals killed on Day 4 postpartum were incubated for 8 h, severe apoptosis was observed (Fig. 4A). Because progesterone protects luteal cells from apoptosis, we examined the capacity of the CL to produce progesterone while undergoing apoptosis in vitro. The progesterone concentration in the medium increased with time of incubation (Fig. 4B). However, there were no significant differences in the amount of progesterone released to the medium by CLs from animals killed on different days of pregnancy and by CLs obtained after parturition.

Susceptibility of the Corpus Luteum: RESULTS(2)

However, after 6 h of incubation, a large increase in the number of nuclei displaying features of apoptosis was visible in the H&E-stained sections (Fig. 2B), an increase further confirmed by in situ 3′ end labeling of the fragmented DNA (i.e., TUNEL) (Fig. 2C). All features of apoptosis distinguished in vitro are summarized in Figure 3, beginning with a healthy nucleus with dispersed chromatin (Fig. 3A). Early in the process of apoptosis, condensation of the chromatin (Fig. 3B) and its alternative localization on the margin of the nucleus (Fig. 3C) are apparent. When the process of apoptosis advances, large pieces of fragmented chromatin appear (Fig. 3, D and E). Small pieces of fragmented chromatin can also be clearly observed (Fig. 3, F-I), denoting very advanced apoptosis.

Susceptibility of the Corpus Luteum: RESULTS(1)

RESULTS(1)Luteal DNA Fragmentation Is Induced under Serum-Free Conditions

Apoptosis, as determined by DNA laddering, was not observed in CLs isolated at either of the last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition (Fig. 1A). An increase in the number of cells undergoing apoptosis after parturition has been observed in situ, but the number of cells undergoing apoptosis at the same time may not be sufficient to allow for visualization of DNA breakdown. In sharp contrast, large scale DNA fragmentation was observed in cultured CLs isolated from rats on Day 21 of pregnancy (Fig. 1B) and 4 days postpartum (Fig. 1C). However, the time course of DNA breakdown differed depending on the gestational stage of the animal. canadian health and care mall

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