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Ovary |
Division of Basic Biomedical Sciences,3 University of South Dakota School of Medicine, Vermillion, South Dakota 57069
Laboratory of Reproduction and Lactation,4 IMBECU-CONICET, 5500 Mendoza, Argentina
Department of Physiology and Biophysics,5 University of Illinois, Chicago, Illinois 60612
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, corpus luteum, lactation, ovary, prolactin receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We recently demonstrated in pregnant rats that luteal regression and apoptosis can be accelerated by the administration of either the antigestagen RU486 or prostaglandin F2
[8], both of which induce abrupt declines in the capacity of the CL to produce progesterone. We have also shown that progesterone and androstenedione significantly interfere with apoptosis after parturition [6, 9]. Because in pregnant rats androstenedione is the main circulating androgen [10] and progesterone is the major steroid produced within the CL [11], one can speculate that these steroids could be responsible for protecting luteal cells from undergoing apoptosis during most of the pregnancy. CLs undergoing apoptosis after parturition were capable of being partially rescued by androgen or progestin [6, 9], suggesting that the gland, even during regression, maintains certain levels of function that can be rescued under appropriate stimuli. These findings indicate that the rat CL of pregnancy undergoes apoptosis during luteal regression and that this process can be hormonally regulated.
In the present study, we used an ex vivo approach to examine whether the ability of the CL to undergo apoptosis in vitro is developmentally regulated in pregnancy and after parturition. In addition, we examined the impact of the change in the in vivo hormonal environment on the susceptibility of the CL to apoptosis in vitro by inducing postpartum animals to begin lactation. By using a luteal cell line that undergoes apoptosis in culture upon serum deprivation [12, 13], we studied whether prolactin (one of the main luteotropic hormones in pregnant rats [11, 14]) protects the luteal cells from apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female rats (originally Wistar strain) weighing 180–220 g were housed under controlled light (lights-on from 0600 h to 2000 h) and temperature (22–24°C) conditions and had free access to standard rat chow (Cargill, Mendoza, Argentina) and tap water. To induce pregnancy, female rats were caged individually with fertile males beginning on the afternoon of proestrus. Mating was verified on the following morning by identifying sperm or copulation plugs in the vagina. This day was designated as Day 0 of pregnancy. In our laboratory, rats usually give birth on Day 22. Animals were killed by decapitation and handled in conformance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals [15]. The experimental protocol was approved by the Institutional Animal Care and Use Committee.
Experimental Procedure
In the first experiment, three rats per group were killed on Days 21 and 22 of pregnancy and on Days 1, 2, 3, and 4 postpartum. The CLs were isolated from the ovaries under a stereoscopic microscope, weighed, frozen in liquid nitrogen, and stored at -80°C until processed for isolation of genomic DNA. For the animals killed after parturition, only the CLs of the previous pregnancy were studied [6].
In the second experiment, three rats per group were killed on Days 7, 14, and 21 of pregnancy and on Day 4 after parturition. The CLs were isolated from the ovaries and used for in vitro incubation for various hours. At the end of the incubation, the CLs were either collected for DNA isolation and analysis of DNA fragmentation or embedded in paraffin for in situ evaluation of apoptosis. Only the CLs of the previous pregnancy were evaluated from ovaries of animals killed postpartum.
To determine the effect of lactation on luteal apoptosis, a third experiment was performed using two groups of postpartum rats (five or six rats per group). In the nonlactating group, the pups were removed from the mothers immediately after delivery, whereas in the lactating group the number of pups per mother was adjusted to eight, and these pups were maintained until the end of the experiment. The mothers were killed at 1300 h on Day 4 postpartum, and trunk blood was obtained to determine hormone concentrations. The ovaries were removed and fixed for 1 h at room temperature in a solution of 10% phosphate-buffered neutral formalin, dehydrated in an ethanol series, cleared in xylene, and embedded in paraffin for routine hematoxylin and eosin (H&E) staining. In two other groups of animals subjected to a similar experimental approach (six to eight rats per group), the CLs were isolated under a stereoscopic microscope, weighed, and used for in vitro incubation. Only CLs of the previous pregnancy were evaluated. The isolation of CLs of the previous pregnancy and the newly formed CLs after postpartum ovulation was performed under a stereoscopic microscope as previously reported [6].
Incubation of CLs
The CLs (four to six CLs/ml in each well of a 24-well tissue culture plate) were incubated in serum-free medium (McCoy 5A:Ham F12 1:1, v/v; Sigma Chemical Co., St. Louis, MO) containing 25 mM Hepes, 200 IU/ml penicillin G, 200 µg/ml of streptomycin, and 0.5 µg/ml amphotericin B at 37°C for various periods of time in an atmosphere of 95% air/5% CO2. After incubation, the CLs used for analysis of DNA fragmentation by gel electrophoresis were immediately frozen in liquid nitrogen and stored at -80°C until isolation of genomic DNA. CLs used for in situ detection of apoptosis were fixed for 1 h at room temperature in a solution of 10% phosphate-buffered neutral formalin, dehydrated in an ethanol series, cleared in xylene, and embedded in paraffin [6, 8].
Luteal Cell Line
The simian virus 40-transformed temperature-sensitive rat luteal cell line GG-CL stably transfected with the long form of the prolactin receptor was used [12, 13, 16]. These cells multiply at the permissive temperature of 33°C and form a multilayer. However, if they are cultured at the nonpermissive temperature of 39°C, they revert to a normal differentiated phenotype similar to that of primary luteal cells in culture [12]. In the present experiment, GG-CL cells were cultured at 33°C to 60% confluence, differentiated at 39°C over 48 h, and then subjected to conditions of serum starvation for various times. At the end of the incubation, cells were fixed in 4% paraformaldehyde and stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Roche Diagnostics GmbH, Mannheim, Germany). Alteration of the chromatin was observed using a Leica DM/IRB microscope (Leica Microsystems, Bannockburn, IL).
Detection of Apoptosis In Situ
Serial paraffin-embedded sections (5 µm thickness) were mounted on 3-aminopropyltriethoxy-silane (Sigma)-coated slides and used for routine H&E staining. The sections were observed and photographed with a Nikon Eclipse E 400 microscope (Fryer Company, Huntley, IL). Apoptotic cells were recognized in H&E-stained sections on the basis of morphological criteria following the procedure described by Van de Shepop et al. [17] with slight modifications [6, 8]. Only cells with advanced signs of apoptosis (i.e., containing multiple nuclear fragments) were counted. A microscope with a 100x objective was used, and a large number of fields were analyzed in each CL for the presence of fragmented nuclei. All the CLs in each section were studied, and an average number of apoptotic nuclei per high-power field was obtained. The expression of apoptosis per unit area rather than per CL more accurately reflects the dynamic of the apoptotic process within each CL in a size-independent manner at any given time during pregnancy and after parturition. When apoptosis was studied in lactating versus nonlactating controls, the results were expressed relative to the nonlactating control, in which the number of apoptotic cells was considered to be 100%. This morphometric method for the identification of apoptotic cells was previously validated in the CL by in situ 3' end labeling [8].
Nuclei exhibiting DNA fragmentation were also confirmed by using the DeadEnd Colorimetric Apoptosis System (Promega, Madison, WI), which end labels the fragmented DNA of apoptotic cells using a modified TUNEL assay in paraffin-embedded sections, as previously described [8].
DNA Fragmentation
The internucleosomal cleavage of DNA was analyzed as follows. GG-CL cells or CLs were digested overnight at 50°C in a buffer composed of 100 mM NaCl, 10 mM Tris HCl (pH 8.0), 25 mM EDTA (pH 8.0), 0.5% SDS, and 0.1 mg/ml proteinase K (Life Technologies, Rockville, MD). The DNA was extracted from the digested tissues with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v), precipitated, and digested for 1 h at 37°C in 1 µg/ml RNase (DNase free; Roche, Indianapolis, IN). After extraction and precipitation, an equal amount of DNA for each sample (5 µg) was separated by electrophoresis on a 1.8% agarose gel (FMC Corp., BioProducts, Rockland, ME) impregnated with ethidium bromide, examined using an ultraviolet transilluminator (UVP, Upland, CA), and photographed with a photodocumentation camera (Fisher Scientific, Pittsburgh, PA). A 100-base pair (bp) DNA ladder (Promega) was used for determining the size of the DNA fragments. NIH image software was used to semiquantitate the fragmented DNA. The densitometry of the DNA fragments below 1500 bp was recorded and normalized against the density of the total genomic DNA of the sample (to correct for DNA loading). To allow comparisons between different gels, the densitometric values were expressed for CL as relative to the fragmentation at time zero of incubation and for GG-CL as relative to the fragmentation obtained after 48 h of incubation in serum-free medium.
Hormone Assays
Serum prolactin was determined by double antibody RIA with reagents provided by the National Institute of Diabetes, Digestive, and Kidney Diseases (Bethesda, MD). Results are expressed in terms of the rat prolactin RP-3 standard. The sensitivity of the assay was 0.5 ng/tube. Inter- and intra-assay coefficients of variation were <10%.
Progesterone concentrations were measured using a commercially obtained kit (Diagnostic Products Corporation, Los Angeles, CA). The sensitivity of the assay was 0.02 ng/ml, and the inter- and intra-assay coefficients of variation were 5% and 6%, respectively.
RNA Isolation and Reverse Transcription Polymerase Chain Reaction
Total RNA from CLs was purified using TRIzol (Life Technologies) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed using the Advantage RT for PCR kit (Clontech Laboratories, Palo Alto, CA). For amplification of the reverse transcription (RT) products, the reaction mixture consisted of 1x polymerase chain reaction (PCR) buffer (ExTaq buffer; Panvera, Madison, WI), 150 µM deoxynucleoside triphosphates, 50 pmol of specific oligonucleotide primers, and 0.8 U ExTaq (Panvera) in a final volume of 40 µl. The samples were overlaid with light mineral oil, and PCRs were carried out in a PTC-1000 thermal cycler (MJ Research, Walthan, MA). Oligonucleotide primer pairs were based on the sequence of the long form of the rat prolactin receptor (5'-AAAGTATCTTGTCCAGACTCGCTG-3' and 5'-AGCAGTTCTTCAGACTTGCCCTT-3') gene as previously used [18]. Primers for L19 (5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-GGACAGAGTCTTGATATCTC-3') ribosomal protein mRNA were included to normalize the data [19]. The predicted sizes of the PCR-amplified products were 279 and 120 bp for the prolactin receptor long form and L19, respectively. Reaction products were electrophoresed on 1.8% agarose gels, visualized with ethidium bromide, and examined by ultraviolet transillumination.
Statistical Analysis
Comparison between means of two groups was carried out using the Student t-test. For multiple comparisons, a one-way ANOVA was followed by either the Tukey or Dunnet multiple-comparison test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 [6], 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. Whereas fragmented DNA was visible only after 6 h of incubation in CLs obtained from Day 21 pregnant rats (Fig. 1B), visible DNA laddering was observed after only 1 h of incubation in CLs obtained from rats killed on Day 4 postpartum (Fig. 1C). Further analysis of apoptosis was performed 4 days after parturition on CLs either freshly isolated (Fig. 2A) or after incubation in serum-free medium for 6 h (Fig. 2, B and C). An H&E-stained section of a freshly isolated CL (Fig. 2A) contained only a few cells undergoing apoptosis. 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.
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CLs Obtained at Different Stages of Pregnancy and after Parturition Have Differential Sensitivity to Apoptosis In Vitro
By counting the number of apoptotic nuclei based on the previous analysis, we determined that the number of apoptotic figures always increased with time of incubation regardless of the developmental stage of the CL. Using this approach, we examined the total number of cells undergoing apoptosis in CLs obtained at different stages of pregnancy and after parturition, following 8 h of incubation under serum-free conditions. 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 [6, 20], 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.
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Differential Sensitivity of Rat CLs to Apoptosis In Vitro Depends on the In Vivo Hormonal Environment
Because the rat CL is subjected to different hormonal environments during pregnancy and after parturition [10, 11, 14], we hypothesized that changes in the circulating levels of various hormones result in changes in the sensitivity of the CL to apoptosis. We obtained CLs from nonlactating and lactating mothers that were killed on Day 4 postpartum. At the time of death, the circulating concentration of prolactin was higher in lactating rats than in mothers whose pups where removed immediately after parturition (nonlactating controls, Fig. 5A). 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 nonlactating 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 nonlactating controls (Fig. 6, A–C).
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Prolactin Interferes with Luteal Cell Death
Because circulating concentrations of progesterone and prolactin are both elevated in lactating rats and because prolactin is a strong stimulus for the production of progesterone at this reproductive stage [21], we examined whether prolactin could be a survival factor for the CL by eliciting a direct effect not mediated by progesterone. We first determined the expression of prolactin receptor mRNA in the CL before, during, and after parturition. After a marked reduction in prolactin receptor mRNA expression at the end of pregnancy, a total recovery in expression was observed 4 days after parturition, reaching levels similar to those found during pregnancy before Day 21 (Fig. 7). The expression of the prolactin receptor postpartum indicates that at this reproductive stage the CL can be a target for prolactin. To examine whether prolactin directly interferes with apoptosis in luteal cells, we used an in vitro experimental approach. Prolactin injected into rats postpartum induces an increase in the production of progesterone [21], which per se is a survival factor for the CL [6, 22]. Hence, we used a luteal cell line that expresses the prolactin receptor but does not produce progesterone [12, 16]. Time course analysis indicated that these luteal cells undergo apoptosis in serum-free medium within 24 h, as demonstrated by chromatin condensation (Fig. 8) and DNA fragmentation (Fig. 9A). Addition of prolactin to the medium significantly reduced the extent of DNA fragmentation in these cells (Fig. 9, B and C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After observing the morphological features of apoptosis in regressed CLs in vivo [8] 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 [8]. 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, F–I). This observation suggests that under conditions of starvation the apoptotic process may involve additional steps that could explain the further fragmentation of the chromatin.
Although apoptosis occurred in vitro in CLs obtained at all stages of pregnancy and after parturition, the time necessary for the appearance of DNA breakdown was markedly different in CLs at different developmental stages. CLs obtained during the second half of pregnancy were more resistant to apoptosis than were those of early pregnancy and postpartum, i.e., more time elapsed before the appearance of signs of DNA fragmentation in vitro. This finding indicates that the CL has differential sensitivity to apoptosis in vitro. This difference in sensitivity does not appear to be due to different levels of progesterone to which the CL is subjected in vitro because the concentration of progesterone measured in the CL incubation medium at different stages during development was similar in all groups. 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. The capacity of the postpartum CL to react to changes in the circulating hormonal environment of the animal was also recently shown; postpartum treatment with progestin or androgen provoked a delay in DNA fragmentation induced by in vitro starvation of the CL [6, 9]. These findings indicate that different hormonal environments are responsible for determining the life span of the CL, which can be maintained in a functional state for a longer time, e.g., during pregnancy, or a shorter time, e.g., during the estrous cycle. In these two reproductive stages, the hormonal regulation of luteal function is very different and the CLs are exposed to dissimilar hormonal environments [10, 11, 26, 27]. Another possible explanation for the higher resistance to apoptosis of CLs during the second half of pregnancy is their exposure to placental lactogens, which have a longer half-life than does prolactin and may continue to protect the CL after isolation.
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 [6] (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 [6]. Therefore, even though there was no difference in the total amount of progesterone accumulated in the culture medium among the groups (Fig. 5), heavier CLs, such as those from Days 14 and 21 of pregnancy, probably produce less progesterone than do the small CLs of Day 7 of pregnancy and Day 4 postpartum. Smaller CLs also display more apoptosis, suggesting that when the apoptotic machinery is fully activated, the gland appears to release more progesterone during at least the 17 h of incubation in the experiment. This result agrees with previous observations in primary and immortalized granulosa cells [28, 29], 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 [6] while the gland retains its steroidogenic capacity, which is reflected in the high output of 20-dihydroprogesterone, a metabolite of progesterone [18]. However, it is not known whether 20-dihydroprogesterone plays a physiological role in luteal apoptosis.
After parturition, the fate of the CLs of pregnancy is uncertain. Between 36 and 48 h after parturition, a postpartum ovulatory process takes place, allowing the formation of a new cohort of CLs [30]. These newly formed CLs, also known as CLs of lactation, are thought to be responsible for producing progesterone during lactation under the tropic control of prolactin [4, 21, 31], which is secreted upon the stimulus of suckling by the pups [32–34]. However, the role (if any) of the old CLs of pregnancy during lactation is totally unknown. In the present study, we clearly showed that mRNA encoding the long form of the prolactin receptor is highly expressed in the CLs after parturition, after being strongly downregulated just before parturition [18] (Fig. 7). The expression of the prolactin receptor in old CLs of pregnancy and in newly formed CLs after postpartum ovulation (unpublished data) demonstrates that both CL subtypes can be targets of prolactin action during the first stages of lactation.
Because progesterone interferes with luteal cell death [6] and because prolactin is a potent stimulator of luteal progesterone production [10, 14], 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 [12, 16] and is unable to produce progesterone [12] allowed us to demonstrate an antiapoptotic role for prolactin that could not be mediated by progesterone.
The GG-CL luteal cell line expresses the glucocorticoid receptor and the estrogen receptor ß [12] and responds to the action of glucocorticoid [22], progestin [22], and estrogen [16]. Therefore, the induction of cell death in this cell line when subjected to conditions of serum starvation not only opens a new avenue for the study of intracellular mechanisms governing luteal cell death but also provides a compelling model for study of the effects of various hormones on luteal cell function and survival. The fact that prolactin prevented cell death induced by starvation makes this cell line a powerful model for study of the molecular mechanisms whereby prolactin prevents apoptosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: C.M. Telleria, Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, 414 East Clark St., Vermillion, SD 57069. FAX: 605 677 6381; ctelleri{at}usd.edu
Received: 15 November 2002.
First decision: 9 December 2002.
Accepted: 28 January 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens. Endocrinology 1998 139:2432-2442-hydroxysteroid dehydrogenase expression in the rat corpus luteum through the glucocorticoid receptor. Endocrinology 1997 138:4497-4500-hydroxypregn-4-en-3-one levels in ovarian vein blood of the rat throughout lactation. J Endocrinol 1969 44:101-106This article has been cited by other articles:
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  M. C. Peluffo, R. L. Stouffer, and M. Tesone Activity and expression of different members of the caspase family in the rat corpus luteum during pregnancy and postpartum Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1215 - E1223. [Abstract] [Full Text] [PDF]
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 C. Stocco, C. Telleria, and G. Gibori The Molecular Control of Corpus Luteum Formation, Function, and Regression Endocr. Rev., February 1, 2007; 28(1): 117 - 149. [Abstract] [Full Text] [PDF]
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 M. C Peluffo, L. Bussmann, R. L Stouffer, and M. Tesone Expression of caspase-2, -3, -8 and -9 proteins and enzyme activity in the corpus luteum of the rat at different stages during the natural estrous cycle. Reproduction, September 1, 2006; 132(3): 465 - 475. [Abstract] [Full Text] [PDF]
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A. A Goyeneche, J. M Harmon, and C. M Telleria Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis Reproduction, January 1, 2006; 131(1): 103 - 111. [Abstract] [Full Text] [PDF]
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 J. J. Peluso, A. Pappalardo, R. Losel, and M. Wehling Expression and Function of PAIRBP1 Within Gonadotropin-Primed Immature Rat Ovaries: PAIRBP1 Regulation of Granulosa and Luteal Cell Viability Biol Reprod, August 1, 2005; 73(2): 261 - 270. [Abstract] [Full Text] [PDF]
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S. Takiguchi, N. Sugino, K. Esato, A. Karube-Harada, A. Sakata, Y. Nakamura, H. Ishikawa, and H. Kato Differential Regulation of Apoptosis in the Corpus Luteum of Pregnancy and Newly Formed Corpus Luteum after Parturition in Rats Biol Reprod, February 1, 2004; 70(2): 313 - 318. [Abstract] [Full Text] [PDF]
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