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BOR - Papers in Press, published online ahead of print March 19, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.065474
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BIOLOGY OF REPRODUCTION 79, 100–114 (2008)
DOI: 10.1095/biolreprod.107.065474
© 2008 by the Society for the Study of Reproduction, Inc.

Ovary; 

Changes in the Proteome of Functional and Regressing Corpus Luteum During Pregnancy and Lactation in the Rat1

Raquel González-Fernández 3, Emilia Martínez-Galisteo 3, Francisco Gaytán 4, José Antonio Bárcena 2 3, and José Eugenio Sánchez-Criado 4

Department of Biochemistry and Molecular Biology,3 and Department of Cell Biology, Physiology and Immunology,4 University of Córdoba, 14071 Córdoba, Spain

ABSTRACT

The corpus luteum (CL) is an exquisitely regulated transitory endocrine gland necessary for the onset and maintenance of pregnancy in mammals. Most of the data on the mechanisms of CL differentiation at the molecular level come from genomic studies, but direct protein data are scarce. Here we have undertaken a differential expression proteomic approach to identify, in an unbiased way, those proteins whose levels change significantly in the rat CL as it evolves from functionality during pregnancy to regression after parturition. Moreover, we have compared the regressing CL with the newly formed functional CL that coexist during lactation under the same endocrine environment. We have defined a "proteomic signature" of CL functionality, which is constituted by a set of 24 proteins with a few differences between pregnancy and lactation. Most of these markers are new and are involved in microtubule assembly, retinoic acid transport, and Raf kinase signaling cascade; 10 are enzymes that define a ketogenic metabolic landscape, demonstrating, for the first time, the prevalence of de novo cholesterol synthesis in luteal cells. The "proteomic signature of regression," on the other hand, is composed of nine proteins, one of which is 20alpha-hydroxysteroid dehydrogenase and two, ferritin and gamma-actin, are new. The discovery of unpredictable new actors in the differentiation process of CL reported here will contribute to new hypotheses that explain the complex female reproductive function at the protein level. It will also open new doors to research on each identified protein by relating them to cellular differentiation.

20{alpha}-hydroxysteroid dehydrogenase, acetyl-CoA, actin, annexin A5, biomarkers, cathepsin D, cholesterol synthesis, corpus luteum function, ferritin, ketogenesis, lactation, peroxiredoxin, phosphatidylethanolamine binding protein, pregnancy, progesterone, progesterone secretion, rat reproductive cycle, retinoic acid binding protein, steroidogenesis

INTRODUCTION

The corpus luteum (CL) is a unique endocrine gland. Several aspects of the gland make it unique, including its origin from the remnants of another organ, its transitory nature, and its sequential dependence on various trophic hormones, to which it responds as a function of its physiological state [1]. These characteristics make it a very good model to study the mechanisms of differentiation toward a functional organ and then its disappearance when its role is over.

The main function of CL is the production and secretion of progesterone (P4), necessary for the onset and maintenance of pregnancy in mammals. Because of its fundamental importance for reproductive function, it is exquisitely regulated and has been the subject of considerable research efforts to shed light on the mechanisms underlying the formation (luteinization of granulosa cells), maintenance, steroidogenesis, and regression of CL in many species [2–8].

The rodent CL is formed after ovulation and becomes fully functional during pregnancy; then, after functional luteolysis and parturition, it undergoes a prolactin (PRL)-dependent structural regression process during lactation. In parallel with the regressing process of this CL of pregnancy, a new CL is formed in the lactating rat, so that, during lactation, two opposing differentiating processes coexist under the same endocrine environment: one occurs in the newly formed CL from postpartum ovulation, which keeps it active in terms of P4 secretion; and the other takes place in the CL from pregnancy, which suffers structural regression. Both functional and regressing CL depend on PRL during lactation [9].

Most of the data available on the functional evolution of the CL at the molecular level come from genomic studies in which changes in the expression of genes are determined [10, 11]. In most cases, these consist of transcriptomic studies in which the levels of specific mRNA were measured. On the other hand, studies affording direct determination of protein levels are scarce due to the lower availability of protein tools, such as recombinant proteins and antibodies, compared with the easily obtained, specific nucleic acid probes. This trend is changing with the advent of proteomics approaches that allow for the analysis of a whole proteome in one single shot. Moreover, transcriptomic and proteomic approaches are not always 100% overlapping. Instead, they give complementary information as a result of posttranslational processing of a majority of proteins [12].

Gel-based differential expression proteomics is a powerful technique for the discovery and identification of new proteins, allowing for the determination of the proteomic signature of a specific cell's state [13]. It is a two-step procedure, involving separation of proteins by two-dimensional (2D) gel electrophoresis followed by analysis by mass spectrometry (MS). The identified proteins would aid in providing a deeper understanding of the underlying mechanisms or in the development of biomarkers for disease diagnosis or prognosis [14].

In the study reported here, we have applied this approach to functional and regressing CL of pregnant and lactating rats in order to determine the proteomic signature of CL under different functional states and physiological contexts.

MATERIALS AND METHODS

Animal Preparation and Sample Collection

Virgin adult female Wistar rats, weighing 190–210 g, were used. Rats were housed under a 14L:10D cycle (lights-on at 0500 h) and 22 ± 2°C ambient temperature, with ad libitum access to rat chow and tap water. Vaginal smears were taken daily, and only rats showing at least two consecutive 4-day estrous cycles were used. On the day of vaginal proestrus, females were mated with Wistar males of proven fertility. The day on which vaginal spermatozoa were detected (day of vaginal estrus) was arbitrarily designed as Day 1 of pregnancy. In our colony, parturition took place between Days 22 and 24; thus, pregnancy lasted 21–23 days. While all pregnant rats were used for the study of the corpora lutea of pregnancy, only rats delivering on Day 23 and ovulating the next day [15, 16] were used for the study of corpora lutea during lactation. Day of parturition was assigned as Day 0 of lactation [17]. On the day after parturition, litters were adjusted to 10 pups. Pregnant rats were killed by decapitation on Days 5, 10, 15, and 20 (n = 4), and lactating females on Days 1, 7, 13, and 19 (n = 5). A scheme of the experimental design is shown in Figure 1.


Figure 01
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  		FIG. 1 Schematic description of the experimental design. The upper drawing shows the evolution of the CL from ovum fertilization throughout pregnancy ("functional CL of pregnancy" [FCLP]) to parturition and its regression process ("regressing CL of pregnancy" [RCLP]) as it takes place throughout lactation. Below is a row of numbers indicating the days of pregnancy and the days of lactation when CL were dissected for proteomic analysis. The lower drawing shows the functional CL ("functional CL of lactation" [FCLL]) newly formed after postpartum ovulation and the days of lactation, where CL were dissected for proteomic analysis.

All experimental protocols were approved by the bioethics committee of the University of Córdoba, and experiments were performed in agreement with the rules on laboratory animal care and international law on animal experimentation.

Dissection and Collection of Corpora Lutea

During pregnancy, CL could be easily recognized macroscopically as large, reddish structures [17]: these are the functional corpora lutea of pregnancy (CLP).

During lactation, two types of corpora lutea were found: regressing CL of the preceding pregnancy (regressing CLP), which can be recognized macroscopically as pale structures [17]; and newly formed functional CL of lactation (CLL), recognized as reddish structures.

Corpora lutea in each rat were dissected out, weighed on a torsion balance, and stored at –80°C until assayed. Thus, the samples available for analysis were: functional CLP on Days 5, 10, 15, and 20 of pregnancy; regressing CLP on Days 1, 7, and 13 of lactation; and functional CLL on Days 7, 13, and 19 of lactation.

Blood Sampling and Assay of Serum Progesterone

Trunk blood was allowed to clot, centrifuged at 4°C, and the serum stored at –20°C until assayed. Progesterone concentrations were measured by use of a commercially obtained kit (Diagnosis Products Corporation, Los Angeles, CA), as previously described [18]. The sensitivity of the assay was 10 pg/tube and the intra-assay coefficient of variation was 6%.

Sample Preparation

Dissected corpora lutea from all the animals of each group were pooled and processed together, because our interest was in the characteristics of the population for each group and not in the individuals that constituted the group [19, 20]. It should be stressed that we assume a rather low biological variability or noise, because the animals were kept under strictly controlled genetic and environmental conditions. Pooling of samples was also necessitated by the scarcity of the material for individual biological replicates [19]. As a consequence, stringent criteria were applied for the acceptance of true differences (see comment in the third subheading of the Results section).

Corpora lutea were collected in ice-cold and sterile PBS and stored at –80°C until further analysis. Tissues were homogenized with glass micro Dounce grinding tubes in 1/2 (w/v) lysis buffer: 8 M urea (Sigma), 2% CHAPS (GE Healthcare), 50 mM dithiothreitol (DTT; Sigma), 0.2% carrier ampholytes (GE Healthcare), 0.02% bromophenol blue (Sigma), 1 mM PMSF (Sigma). The suspension was centrifuged at 15 000 rpm for 15 min to remove debris and the supernatant was colleted. Protein concentrations were determined by the Bradford assay [21]; the crude extract was distributed in 200 µl aliquots and kept at –80°C until being used for 2D electrophoresis. For each sample, three separate experiments were run independently on three analytical 2D electrophoresis gels.

Two-Dimensional Gel Electrophoresis

Two-dimensional electrophoresis was performed with a Bio-Rad 2-D Electrophoresis System. For the first dimension, the samples were used to hydrate 11-cm, pH 3–10 and pH 5–8 linear IPG Strips (Bio-Rad) overnight with 100 mg of protein in a total volume of 185 ml. Isoelectric focusing was carried out with a Protean IEF Cell (Bio-Rad) at 20°C for a total of 35 kV·h. IEF strips were equilibrated for 10 min in a 2% (w/v) DTT equilibration buffer (6 M urea [Sigma], 20% SDS [Sigma], 1.5 M Tris/HCl [Sigma], pH 8.8, gel buffer, 50% glycerol [Panreac]) and for 10 min in a 2.5% (w/v) iodoacetamide (Sigma) equilibration buffer.

For the second dimension, strips were transferred to 12% T polyacrylamide Bis-Tris Criterion Precast Gels (Bio-Rad) for use with 11-cm IPG strips with Dodeca Criterium cells (Bio-Rad), and 12 gels were run simultaneously, with the IPG strips sealed on top of the gels with 0.5% agarose containing bromphenol blue. SDS-PAGE was run at constant current of 150 V/gel for 90 min with MOPS running buffer (Bio-Rad).

Gel Staining, Image Analysis, Data Normalization, and Statistical Analysis

The gels were stained with the fluorescent dye, Sypro Ruby (Sigma), which offers a good concentration dynamic range [22]. Gel images were captured and digitized with Molecular Imager FX Pro MultiImager System (Bio-Rad), and the images were analyzed with PDQuest 2D Image Analysis Software (v 7.3; Bio-Rad). Image analysis procedures included spot detection, spot editing, background subtraction, and spot matching. Spot detection was controlled by four parameters, including sensitivity, operator size, noise factor, and background. The same parameters were used to detect spots in all the gels in order to guarantee comparability between the gels. One of the gels in the control group was chosen as a reference gel before matching. The amount of a protein spot was expressed as the volume of the spot, which was defined as the sum of the intensities of all the pixels that made up the spot. In order to correct for variations and quantify protein spots, the individual spot volume was normalized as a percentage of the total volume of all the spots detected on the gel.

In order to determine the analytical variability, 12 triplicates from 4 experimental points were selected, the image of each gel was divided into 8 equivalent sectors, and 20 spots were randomly selected from each sector. The volume of each spot was measured and normalized, and their coefficients of variance (%), were calculated. Finally, the average of all the coefficients of variance was calculated, and the figure obtained was the analytical or technical variability of the experiment, which happened to be 23.56%.

Selection of spots and statistical conditions are described in the Results section. To find spots that differed quantitatively between groups, the average intensities of resolved spots were compared by using the statistical and quantitative functions within the PDQuest software (t-test; P < 0.05). The pattern of variation along the different physiological states of each selected protein is presented in histograms in the figures.

Enzymatic In-Gel Digestion

Spot picking, tryptic digestion, and MS analysis were all performed in the Proteomics facility of the University of Córdoba, Servicio Central de Apoyo a la Investigacion (SCAI) which is part of the Andalusian Genomics, Proteomics and Bioinformatics Platform, and is also Node 6 of the Spanish ProteoRed Platform. Selected protein spots were excised from the gel with an automatic spot picker (ProPic; Genomics Solutions), and in-gel digestion with trypsin was performed according to the following procedure with an automatic digestor (ProGest; Genomic Solutions). The gel pieces were shrunk by dehydration in acetonitrile (ACN) and dried in a vacuum centrifuge (Eppendorf). The gel pieces were then swollen in a digestion buffer containing 40 mM ammonium bicarbonate, 10% ACN, and 20 µg/ml trypsin (proteomics sequencing grade; Sigma) in an ice-cold bath. After 30 min, the supernatant was removed and replaced by 30 µl of the same buffer, but devoid of trypsin. The gel pieces were kept wet during enzymatic cleavage (37°C, overnight). The supernatants were collected, and peptides were extracted twice by two changes of 5% trifluoroacetic acid (TFA)/50% ACN (15 min for each change) at room temperature and dried out.

MS Analysis and Database Searching

The peptide extracts were mixed 1:1 with matrix, saturated {alpha}-cyano-4-hydroxycinnamic acid (Applied Biosystems) in 50% ACN/0.05% TFA, and applied onto matrix-assisted laser desorption/ionization (MALDI) plates. The operation was done automatically with a MALDI automatic spotter (ProMS; Genomics Solutions). Mass analysis of peptide mixtures was performed with a 4700 MALDI-time of flight (TOF)/TOF mass analyzer (Applied Biosystems) with a 337-nm N2 ultraviolet laser. The mass list was loaded onto the MASCOT search engine (Matrix Science). For the protein search, the National Center for Biotechnology Information mammalian protein database (updated monthly) was used with the following parameters: with monoisotopic masses, a maximum ±0.3 Da mass tolerance, cysteine in carboxyamidomethyl form and methionine in oxidization form, and an allowance for up to one missed cleavage per peptide. Proteins with peptide mass fingerprint (PMF) matching more than four peptides, with a MASCOT score higher than 64 and validated by at least one significant peptide collision-induced dissociation fragmentation spectrum, were considered true identifications. In a few cases, peptide fragmentation could not be achieved; in these cases, the PMF was accepted if the protein coverage was ≥30% and theoretical and experimentally determined isoelectric point (pI) and relative mass (Mr) were similar. Moreover, the fact that the identified protein belonged to the rodent database was considered an additional identity factor.

RESULTS

CL Weight and Serum Progesterone Levels

CL weight during pregnancy (functional CLP) increased from more than 1.5 mg on Day 5 to 4 mg on Day 20 (Fig. 2). Moreover, the weight of CL of pregnancy (regressing CLP) decreased during lactation, from about 3 mg on Day 1 to 1 mg on Day 13 of lactation. On Day 19 of lactation, the regressing CLP was too small to be distinguished from the rest of the ovarian structures, and could not be dissected. On the other hand, the weight of newly formed CL during lactation (functional CLL) kept increasing throughout the lactation period, from 1 mg on Day 7 to 2.5 mg on Day 19.


Figure 02
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  		FIG. 2 Serum progesterone concentration and CL weight throughout the experiment. CL weight is represented in histogram form in mg, as indicated in the left axis; the data are the average of all the CL dissected in each state ± SD. Serum progesterone (P4) concentration in ng/ml, as indicated in the right axis, is represented by a black line. The days of sampling as the CL progresses throughout pregnancy and then from Day 1 after parturition throughout lactation, are shown in the bottom axis. Grey bars correspond to FCLP; black bars represent the weight of RCLP; dashed bars correspond to FCLL. See the Materials and Methods section for further explanation.

Progesterone levels in serum were in agreement with the evolution of functional CL: augmented during pregnancy until the day before parturition, dropping to basal levels the first day postpartum, and then increasing and stabilizing until the end of lactation. However, its levels never reached the maximum obtained in the pregnant rats (Fig. 2).

Expression Proteomics Analysis of Functional and Regressing CL at Day 7 of Lactation

Before undertaking the analysis of the whole experiment, a simpler analysis was performed to evaluate and optimize the conditions. This consisted of a comparative analysis between the functional CLL and the regressing CLP of lactating rats at Day 7 postpartum. One of the limitations of 2D gel proteomics is that membrane, acidic, and basic proteins are not resolved properly, so we decided to optimize the conditions for soluble proteins instead. Hence, the extract was treated with ReadyPrep Protein Extraction (Membrane I) kit (Bio-Rad) before extracting the soluble fraction. Also, a pH range of 5–8 instead of 3–10 for the first dimension and 12% precast acrylamide gels for the second dimension were judged to be the best conditions. In this way, conspicuous differential spots were selected and subjected to protein trypsin digestion followed by analysis by MALDI-TOF/TOF.

A set of proteins were unambiguously identified [23] that were repeated and confirmed afterwards as part of the full experiment. Notably, a striking group of six spots was present in the regressing CL, but not in functional CL. These spots were all identified as 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}-HSD), a well known marker of regression [24, 25]. We took these results as a categorical validation of our approach. The success of this testing trial prompted us to undertake the whole experiment at once under the same conditions.

Proteomics Analysis of CL at Different Functional States During the Rat Reproductive Cycle

The proteomic protocol was optimized for maximum resolution and sensitivity: pI range from 5 to 8 in the first dimension, and from 80 to 12 kDa in the second. An aliquot of 100 µg of protein was loaded per gel and fluorescence stained with Sypro Ruby.

Three representative gels, one for each physiological state, are shown in Figure 3. Approximately 1200 well-focused spots were separated, and were reduced to 700 spots coincident in all 30 gels. Before proceeding into the proteomic analysis, the relative and absolute abundance of these proteins was studied carefully in order to detect any significant pattern of variation. We defined a set of stringent criteria for the selection of spots in order to avoid ambiguities. Although the fluorescent stain has a broad dynamic range [22], we discarded spots with intensities below 400 volume arbitrary units, because even statistically significant differences could be due to methodological variations; as such, we sacrificed the possibility of detecting changes in low-abundance proteins for the sake of certainty. Only spots with variations of >2-fold or <0.5-fold between samples that were statistically meaningful (P < 0.05) were picked from the gel and subjected to MALDI-TOF/TOF analysis for identification. On the grounds of these criteria, 136 protein spots were selected, from which 63 were identified unambiguously, corresponding to 47 distinct proteins. Of these, 39 proteins are discussed individually in this article and are listed in Table 1 for reference, which includes any parameter of interest for reviewing the validity of the identification described in Materials and Methods.


Figure 03
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  		FIG. 3 Representative gels of CL from the three physiological states. A) FCLP at Day 20 of pregnancy; B) RCLP at Day 7 of lactation; C) FCLL at Day 7 of lactation. Numbered spots are those selected, unambiguously identified by MALDI-TOF/TOF, and discussed in this article; the numbers are the same as in Table 1, where the names of the proteins and other parameters are listed.


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  		TABLE 1 List of proteins identified in CL and selected in this report for discussion of their variation pattern along the different physiological states during the reproductive cycle of the rat.

We could distinguish several variation patterns of the identified proteins throughout the evolution of the CL. Two groups could be clearly assigned to either regression or functionality. The latter includes all the proteins that are present at higher levels in functional CL, both in pregnant (functional CLP) and in lactating (functional CLL) rats, than in regressing CL (regressing CLP). These are "true" markers of functionality. The proteome composition 1 day after parturition revealed a group of proteins displaying a slow response in the functional CLP to regressing CLP transition; that is, when the CL differentiates from functionality to regression. Finally, we could also distinguish two other groups of proteins associated with CL functionality, either in functional CLP or in functional CLL, but not in both conditions.

Markers of Functional CL

A group of abundant proteins, the profile of which shows a characteristic pattern in the histograms in Figure 4, with levels markedly higher in functional CLP and functional CLL than in regressing CLP, should constitute a true functional proteomic signature, independent of the physiological state. These proteins are: dihydropyrimidinase-related protein 2 (DPYSL2), also named collapsin response mediator protein 2 (CRMP-2); acetyl-coenzyme A (CoA) acetyltransferase (ACAT2), a cytosolic enzyme that catalyzes the condensation of two acetyl-CoA molecules in the first step of the long anabolic pathway leading to progesterone from acetyl-CoA via cholesterol; 3-hydroxyisobutyryl-CoA hydrolase (HIBCH), a mitochondrial enzyme that catalyzes the fifth step in the catabolism of valine; phosphatidylethanolamine-binding protein 1 (PEBP1), the most abundant differential protein found in this study—also termed Raf-1 kinase inhibitory protein (RKIP), it is an evolutionarily conserved protein that regulates growth and differentiation in a variety of species; and cellular retinoic acid-binding protein 2 (CRABP2), a protein that binds specifically to retinoic acid, a derivative of vitamin A.


Figure 04
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  		FIG. 4 Pattern of expression of marker proteins of functionality (FCLP and FCLL). The profile of proteins showing significantly lower levels in RCLP as compared to FCLP and FCLL are represented in histogram form, where the level of each protein is indicated in the y-axis as the normalized volume of the spot in the gel in parts per million (PPM) fluorescence counts (CNT), as explained in Materials and Methods. Data are the average of at least three measurements ± SD, which reflects the analytical precision. The days of sampling as the CL progresses through pregnancy and then from Day 1 after parturition throughout lactation are shown on the x-axis. Grey bars, FCLP; black bars, RCLP; dashed bars, FCLL. See Materials and Methods for further explanation.

It is worth mentioning that the levels of these marker proteins of functionality, with the exception of DPYSL2, are higher in the functional CL during pregnancy than during lactation, in parallel with the higher activity of CL during pregnancy, which results in higher levels of serum progesterone (see Figure 2). This fact reinforces the ascription of these proteins as markers of functionality.

Proteomic Signature of Regressing CL

Seventeen spots were clearly associated to the regression state that corresponded to different isoforms of nine distinct proteins. One of these is 20{alpha}-HSD (AKR1C18), a well-known marker of regression [24, 25] the expression pattern of which confirms and validates our approach. Here we have separated six isoforms of 20{alpha}-HSD, with two Mr differing by 1 kDa and three pI differing by a few hundredths of a pH unit (Fig. 5). Other proteins are recognized here for the first time associated to CL regression, and some of them (actin, ferritin, and cathepsin D [CTSD]) have been revealed as excellent markers.


Figure 05
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  		FIG. 5 Pattern of expression of 20{alpha}-HSD (AKR1C18) in CL during pregnancy and lactation in the rat. A) Cutouts of gels from CL at three different stages of differentiation showing spots corresponding to 20{alpha}-HSD; the types of CL and differentiation stages are indicated on the left; the six spots identified as 20{alpha}-HSD are conspicuous in the central panel, together with their assigned numbers. Arrows in the upper and lower panels point to the position of the absent spots; Mr and pI are indicated in italics at the right-hand side and at the bottom, respectively. B) Histograms as in Figure 4, showing the expression profiles of 20{alpha}-HSD isoforms. The number that follows the protein acronym in each graph indicates the arbitrary number assigned to each isoform. Data is presented as mean ± SD. Black bars, RCLP; dashed bars, FCLL.

Ferritins, both light (FTL1) and heavy (FTH1) chains, are present at much higher levels in the regressing CLP than in the functional counterpart, functional CLL (Fig. 6). Both β-actin and {gamma}-actin 1 are markedly elevated in the regressing CL (Fig. 6). The level of CTSD shows a marked increase in the transition from functional CLP to regressing CLP, which is already conspicuous the first day after parturition, and continues to increase throughout the regression process (Fig. 6). Glucose-6-phosphate dehydrogenase X-linked (G6PDX) shows a similar, increasing trend throughout lactation in both regressing and functional CL, but is higher in the regressing state; moreover, its levels are higher in functional CLL than in functional CLP. NG,NG-dimethylarginine dimethylaminohydrolase 1 hydrolyzes NG,NG-dimethylarginine (ADMA), an endogenous competitive inhibitor of NO synthesis, to citrulline and is present in tissues that synthesize NO [26]. Dihydrolipoyllysine-residue S-succinyltransferase (DLST) is the E2 subunit of the 2-oxoglutarate dehydrogenase complex. Glutathione peroxidase is a well-known antioxidant enzyme involved in the scavenging of peroxides.


Figure 06
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  		FIG. 6 Pattern of expression of other marker proteins of regression (RCLP). Histograms of proteins showing significantly higher levels in RCLP as compared with FCLP and FCLL. Two isoforms differing in their pI and Mr values (see Table 1) are shown. Data is presented as mean ± SD. Grey bars, FCLP; black bars, RCLP; dashed bars, FCLL.

Pregnancy-Specific Proteomic Signature of Functional CLP

A group of nine proteins are associated with functionality, but are only present in higher levels in functional CLP as compared to the levels in functional CLL (Fig. 7). They seem to be under the control of pregnancy-specific factors. Five of these proteins have typical antioxidative functions: alcohol dehydrogenase (AKR1A1); inducible carbonyl reductase (CBR1); peroxiredoxin (PRX) 1 and 2; and hemopexin (HPX). Other proteins fitting into this group are proteasome {alpha}-subunit 2 (PSMA2), endoplasmic reticulum protein 29 (ERp29), related to protein degradation and folding, and the glycolytic enzyme, phosphoglycerate mutase 1 (PGAM1).


Figure 07
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  		FIG. 7 Expression pattern of proteins characteristic of pregnancy (FCLP). Histograms of proteins showing significantly higher levels in FCLP than in FCLL. Two isoforms of PRX2 differing in their pI and Mr are shown. Data is presented as mean ± SD. Grey bars, FCLP; black bars, RCLP; dashed bars, FCLL.

The common pattern in this group is characterized by high levels in functional CLP during pregnancy followed by a gradual drop after transition to regressing CLP. The proteins of this group are also at low levels in functional CLL, but all of them demonstrated an increasing trend from Day 7 to Day 19 of lactation, except HPX, which remained very low at both states in the lactating rat.

Lactation Specific Proteomic Signature of CL

A small group of proteins was markedly present during lactation, and was present at lower levels or even absent during pregnancy. Of these, seven were at higher levels in functional CLL than in the regressing CLP; these are: PGAM1; haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2); glycerol-3-phosphate dehydrogenase (NAD+) (GPD1); malic enzyme 1 (ME1); isovaleryl-CoA dehydrogenase (IVD); apolipoprotein AI; and the dihydrolipoyllysine-residue acetyltransferase (DLAT) component of pyruvate dehydrogenase complex (Fig. 8A). Four proteins of this group were also present at similar levels in regressing CLP: acetyl-CoA synthetase 2-like (ACSS1); long-chain specific acyl-CoA dehydrogenase (ACADL); propionyl-CoA carboxylase β chain (PCCB); and annexin A5 (Fig. 8B). All these could be proteins, the genes of which are subjected to exogenous endocrine signals typical of the lactating state.


Figure 08
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  		FIG. 8 Expression pattern of proteins characteristic of CL during lactation. A) Histograms of proteins showing significantly higher levels in FCLL than in FCLP. B) Histograms of proteins showing high levels of both functional CLL and regressing CLL. Data is presented as mean ± SD. Grey bars, FCLP; black bars, RCLP; dashed bars, FCLL.

DISCUSSION

In this expression proteomics study, we detected and selected 136 spots differentially expressed from pregnancy to lactation, of which 63 have been unambiguously associated to unique proteins. Next we discuss the particular proteins identified and integrate the data to present a meaningful view of the CL proteome dynamics.

Proteomic Signature of Functional CL

Proteins included in this group are present both in functional CLP and functional CLL, common characteristics of which are their ability to synthesize and secrete P4, their repression of 20{alpha}-HSD, and their absence of or downregulation in regressing CLP.

DPYSL2 is a member of a family of cytoplasmic proteins that were originally identified as mediators of semaphorin-induced growth cone collapse in neurons—hence its other name: CRMP-2. It interacts with and promotes tubulin polymerization, and is involved in the configuration of the microtubule lattice [27]. DPYSL2 has been related to CL functionality for the first time in this article, and its precise role during the luteinization process is worthy of further, specific studies.

ACAT2 and HIBCH are discussed subsequently here in the context of acetyl-CoA synthesis.

PEBP1 functions in mammalian cells as an inhibitor of the mitogen-activated protein kinase (MAPK) signalling module comprised of Raf-1/MAP kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2, hence its other name, RKIP [28]. This is the first time that a relationship between the important protein PEBP1-RKIP and CL functionality has been revealed. It has been shown previously that MAPK elements play a role in progesterone synthesis and secretion, but the reports appear to be contradictory. On one side, MEK inhibition has been reported to enhance steroid biosynthesis in FSH-stimulated porcine granulosa cells, and inhibition of ERK increased gonadotrophin-induced progesterone secretion [29]. On the other side, several MAPKs have been found in the active state in CL from primates under different model functional status induced by pseudopregnancy or GnRH antagonists [30], as well as in isolated human granulosa cells in culture treated with kinase inhibitors and activators [31]. Our finding, that PEBP1-RKIP is upregulated in rat functional CL, will help to focus the debate and will surely add to both the knowledge of RKIP functions and the mechanisms of CL differentiation.

It has long been known that vitamin A is related to fertility, and that its abundance in CL gives this gland its yellowish appearance in mares, hence its name (from the latin, luteus, meaning "yellow"). The ancestral role of retinoic acid is critical during embryogenesis and organ regeneration in adult vertebrates [32, 33]. CRABP2 is a cytosolic protein that moves to the nucleus upon binding to retinoic acid. Its expression is much greater in rat CL than in any other tissue or organ examined [34]. Retinoic acid, acting on retinoic acid receptors, is involved in the signaling pathway leading to suppression of FSH receptors during luteinization triggered by LH [35]. Although little is known about which cells produce retinoic acid, it has been proposed that the function of CRABP2 consists of aiding in the production and/or secretion of retinoic acid by certain ovarian cells for action as a paracrine signal [36]. Our results then suggest that functional CL actively produces retinoic acid upon LH stimulation to act as a paracrine factor for FSH-R gene silencing, but nothing can be added to the possible dispensability of these protein as suggested from results with CRABP2-null mutant mice [37], particularly under vitamin A deficiency [38].

Influence of Pregnancy or Lactation on the CL Proteome

Aside from the ability of CL to make P4, regulation by trophic hormones differ. Thus, CL of pregnancy depends on PRL during the first week, but on decidual luteal tissue PRL [39] and LH-dependent E2 during the second week. Finally, during the third week, placental lactogens and aromatizable placental androgens support functional CLP secretion [40]. In contrast, the functional CLL depends exclusively on suckling-induced PRL secretion. Analyzing the proteome of CL from conception to the end of lactation, we have been able to discern two groups of proteins that are affected by the physiological state in different manners. The first group is formed by proteins associated with functionality, but present at higher levels in functional CLP (see Fig. 7) than in functional CLL, whereas the second is formed by proteins that are very conspicuous only during lactation, either in functional CLL (Fig. 8A) or in both functional CLL and regressing CLP (Fig. 8B).

Proteins of the first group (unique to functional CLP) are either under positive control of pregnancy-specific factors or repressed by lactation-specific signals. Five of these proteins have typical antioxidative functions: AKR1A1 and CBR1 should participate in removing carbonylic compounds while PRX1 and PRX2 should be involved in the elimination of peroxides and peroxinitrites [41]. HPX has also been related to antioxidative and protective mechanisms [42]. The PSMA2 degrades proteins through the ubiquitin pathway [43], but also seems to hydrolyze oxidatively damaged proteins [44]. ERp29 is an unusual stress-inducible redox-inactive member of the thioredoxin family, helping in the correct folding of proteins in the early secretory pathway [45]. Finally, PGAM1 catalyzes a glycolytic reaction, traditionally considered as trivial. However, recent studies have revealed the presence of several isoforms of the enzyme resulting from alternative splicing, which are sensitive to members of stress response networks, such as p53 [46], the redox-regulated Keap1-dependent ubiquitin ligase complex, and the antiapoptotic protein BCL2L1 (also known as Bcl-XL) [47].

Considered together, these proteins point to intense antioxidant defensive activity in the CL during pregnancy. The reason behind this could be an elevated production of reactive oxygen species as a consequence of the steroidogenic activity of luteal cells and the necessity to protect biomolecules from oxidative damage.

The second group (Fig. 8) is formed by two subsets of proteins, the levels of which are markedly higher during lactation than during pregnancy. In the first subset (Fig. 8A), we found seven proteins: phosphoglucomutase 1, acting in the direction of glucose-1-P formation, leads to uridine diphosphate-glucose, a precursor of glycogen and glycoprotein synthesis. It has been shown to be regulated positively through phosphorylation by p21-activated kinase 1 [48]. Perhaps its increase in functional CL during lactation has to do with synthesis of glycoproteins, a process likely to be important in this secretory organ. HDHD2 could fit well into this glycoprotein synthesis picture on the basis of its involvement in sugar phosphate phosphotransferase activity [49]. GPD1 and cytosolic malate dehydrogenase (NADP+), or ME1, together with IVD and subunit 2 of the pyruvate dehydrogenase complex, DLAT, will be discussed subsequently here in the context of acetyl-CoA synthesis.

A subset of four proteins in this group includes those that are absent in pregnancy and are elevated in lactation, irrespective of the functional state (Fig. 8B). Annexin A5 is member of the annexin family of proteins, the finding of which at high levels in CL is not new, as it has previously been related to apoptosis in luteal cells [50]. Annexin A5 mRNA has been shown to be under strong negative control by PRL in a model of the pseudopregnant rat [50] and in hypophysectomized pregnant rats [11]. The pattern of annexin A5 protein observed in our experiment agrees with those observations in that it is much lower in pregnant than in lactating rats. However, it is high in both regressing and functional CL during lactation when PRL dominates the endocrine environment. Further studies are needed to explain these apparently contradictory results. The other three proteins of this subset are involved in ketogenic metabolism and will be discussed in the following text.

Proteomic Signature of Regression

Two isoforms of 20{alpha}-HSD, differing in their kinetic properties, have been described previously [51, 52]. Here we have separated six isoforms (see Figure 5), the differences in which could be due to their degree of glycosylation and/or phosphorylation [51]. The discerning of these posttranslational modifications could not have been achieved without a proteomic approach like the one used here.

The 20{alpha}-HSD enzyme is strongly repressed by PRL and induced by prostaglandin F2{alpha} (PGF2{alpha}) [11, 53]. It is worth noting that, under lactating conditions where PRL prevails in the endocrine environment, the enzyme is nevertheless at very high levels in the regressing CLP. The explanation may lie in the negative effect of PGF2{alpha} on the expression of the PRL receptor at the end of pregnancy that makes the cell insensitive to the hormone [54]. On the other hand, cells in the newly formed functional CL, functional CLL, are sensitive to PRL repression of 20{alpha}-HSD during the first days after delivery, likely owing to the absence of the PGF2{alpha} action in these cells. However, repression of 20{alpha}-HSD in functional CLL is relieved with time, showing high levels of most of the enzymes in the late phases of lactation. The presence there of 20{alpha}-HSD suggests that these CL, albeit functional, undergo regression to some extent which is in agreement with previous findings [55, 56]. The levels of all the isoforms in the regressing CLP increase steadily from Day 1 to the end of lactation, which does not exclude an explosive initial induction, as suggested by genomic data [11].

Ferritin, as an assembly of FTL1 and FTH1 chains, encoded by different genes [57, 58], plays a key role in maintaining iron homeostasis [59]. Actually, ferritin can be viewed not only as part of a group of iron regulatory proteins that includes transferrin and the transferrin receptor, but also as a member of the protein family that orchestrates the cellular defense against stress and inflammation [60]. Our results demonstrate, for the first time, that both FTL1 and FTH1 chains are excellent markers of CL regression.

Reorganization of the cytoskeleton triggered by redox changes has been associated with nuclear fragmentation during apoptosis [61]. The increase in actins in regressing CL could be related to the oxidative stress and apoptotic process known to prevail under these conditions [62].

It has been demonstrated that CTSD is translocated from the lysosome to the cytosol under oxidative stress and induces apoptosis acting upstream of the caspase cascade [63]. It is thus tempting to suggest that CTSD-increased levels in regressing CL has to do with the redox events that lead to cell death, which would imply a caspase-independent apoptotic pathway [64]. Under identical exogenous conditions, the functional CLL does not show any increase in CTSD, which speaks in favor of endogenous oxidative stress induction of this protease.

A well-orchestrated response to oxidative stress, such as apoptosis [65], would require some control over the extent of the damage inflicted [66]; otherwise, undesired responses and possibly necrosis could take place. The increased levels of G6PDX could be associated with the oxidative cellular environment in regressing luteal cells [62], in which it would provide NADPH for antioxidative defense [67]. The E2 subunit of the 2-oxoglutarate dehydrogenase complex, DLST, has covalently bound lipoamide as a cofactor that could channel reducing power to the glutathione system [68, 69], especially now that the intense production of succinyl-CoA from ketogenic amionacids catabolism (see below) might block its canonical catalytic cycle [70, 71]. The enhancement of glutathione peroxidase would be consistent with a temperate response to the oxidative conditions prevailing during the regression process [62].

The increase in dimethylarginine dimethylaminohydrolase 1 (DDHA1) may be an indication of the involvement of signaling through protein nitrosylation on the regression of CL. It should be noted that estrogens regulate the concentration of ADMA through changes in DDHA1 [72].

Remarks on the Proteomic Changes During the Transition Period from Functional CLP to Regressing CLP

When the functional CLP changes to regressing CLP around the end of pregnancy and the beginning of lactation, several endocrine events take place: a preovulatory LH and E2 surges; antepartum elevation of ovarian relaxin [73, 74] and PGF2{alpha} [75] levels in serum; and a drop in P4 level [76]. We have now detected the rather anomalous behavior of a group of proteins associated with functionality of the CL, which are still present at high levels at least 1 day after parturition, when the CL has entered regression, and then decrease drastically over the following sample days (Table 2). It is worth noting that among them are dihydropyrimydinase-related protein 2 and antioxidant enzymes, such as alcohol dehydrogenase, carbonyl reductase, or peroxiredoxins 1 and 2.


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  		TABLE 2 Proteins displaying a slow response in the transition from functionality (functional CLP) to regression (regressing CLP).

Other proteins, however, behave the opposite way; that is, being clearly characteristic of regression or lactation, they are still absent or at excessively low levels 1 day after parturition. Most of them increase progressively afterward throughout lactation in the regressing CL, like 20{alpha}-HSD, the mRNA of which has been reported to start being expressed just before parturition [77]. It may be that multiple regulatory signals are acting in a complex equilibrium that is sensitive to minor fluctuations. For instance, 20{alpha}-HSD may be tightly repressed at the beginning of lactation by a sudden rise of PRL and LH aimed at regulating the exceptional estrous cycle taking place immediately after parturition. On the other hand, the simultaneous controlling action of PGF2{alpha} just before parturition has to be taken into account. Moreover, in this study, we did not sample between Days 1 and 7 postpartum, or on the 2 days immediately preceding parturition.

Enzymes of Acetyl-CoA Production and Cholesterol Synthesis

During this proteomic study, a set of 10 enzymes appeared to be clearly associated with the functional state of the CL—most of them in the lactating rat, but some also during pregnancy. They were apparently disjointed at first sight, but, when pieced together, a coherent metabolic picture resulted, showing that, in the functional CL, the metabolism is committed to the massive synthesis of acetyl-CoA, likely oriented toward de novo synthesis of cholesterol (Fig. 9).


Figure 09
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  		FIG. 9 Concerted functioning of the metabolism for the massive production of acetyl-CoA in the functional corpus luteum. This is a summary of catabolic pathways for the conversion of three ketogenic amino acids and cytosolic triacylglycerols into acetyl-CoA in the mitochondria. The export of the acetyl-CoA produced therein to the cytosol for the synthesis of cholesterol as a precursor of progesterone in the luteal cells is also depicted. The pathways have been schematized to highlight the position of 10 enzymes, the protein levels of which increase in the FCLL. The thickness of the arrows varies in direct relationship to the tentative metabolic flow, which must increase as the pathways converge. A legend is included to correlate the acronyms with the extended names and with the figures where the expression profiles of each protein is included, as well as with the physiological state of which it is characteristic. See the text for a more in-depth discussion.

According to our results, mitochondrial acetyl-CoA has three origins in CL cells: 1) the catabolism of ketogenic aminoacids, leucine, valine, and isoleucine, with the mitochondrial enzymes IVD (Fig. 8), HIBCH (Fig. 4) and PCCB (Fig. 8) being induced; 2) triacylclycerol (TAG) catabolism involving the increase in the levels of the cytosolic enzymes, GPD1 (Fig. 8) and the glycolytic PGAM (Fig. 7), as well as two mitochondrial enzymes: long-chain-specific ACADL (Fig. 8), the first step of fatty acids β-oxidation and DLAT (Fig. 8), which is subunit 2 of the pyruvate dehydrogenase complex (the findings that ovariectomy represses and estrogens stimulate the synthesis of TAG by the liver [78] agree with these results); 3) acetate through the ACSS1 (Fig. 8), known to be induced under ketogenic conditions [79, 80].

Another two cytosolic enzymes are also highly elevated in functional CL: ME1 (Fig. 8), which is necessary for the export of acetyl-CoA by the citrate-pyruvate shuttle, and ACAT2 (Fig. 4), also named β-ketothiolase, which catalyzes the very first step in the long route from acetyl-CoA to cholesterol. The presence of these two proteins indicate that the Krebs cycle is not committed to ATP production, since the flow out of citrate would leave the stretch of the cycle spanning from citrate to succinyl-CoA almost devoid of intermediates, thus stressing the amphibolic nature of the cycle.

On the basis of much evidence, it has been widely accepted that cholesterol for steroid hormones synthesis in steroidogenic cells has an exogenous origin from low-density or high-density lipoprotein in the rat [81]. It has been suggested that endogenous de novo synthesis of cholesterol from acetyl-CoA is only prevailing when the supply from the blood is restricted [82]. However, apparent disagreement with the exogenous cholesterol supply paradigm has been raised in two cases [83, 84].

In the proteomic study reported here, 10 out of 48 proteins identified are enzymes of ketogenic metabolism that increase markedly in the functional CL, especially during lactation, thus convincingly arguing in favor of the functioning of de novo cholesterol synthesis in steroidogenic cells. This is a warning call that attention should be paid to the process of endogenous cholesterogenesis, although the simultaneous exogenous supply from high-density lipoprotein cannot be discarded.

The aim of a differential expression proteomics strategy—the search for biomarkers—has been categorically achieved in the study reported here. By this unbiased approach, we have found novel proteins differentially expressed at high levels, but which have hitherto remained unnoticed in other studies. The identification of certain proteins as markers of functionality, regression, pregnancy, or lactation, reported here for the first time, will certainly prompt researchers to validate new hypotheses that will be raised in the field of reproduction toward a better understanding of the molecular mechanisms governing the complex female reproductive function. It will also open new doors to research on each identified protein by relating them to cellular differentiation.

ACKNOWLEDGMENTS

The dedication of Dr. Consuelo Gómez from the UCO-SCAI proteomics facility is appreciated.

FOOTNOTES

1Supported by Spanish Ministry of Science and Education grants BFI2002-0755 to J.A.B. and BFU2005-01443 to J.E.S.-C. and by grant CVI-216 to J.A.B. from the Regional Andalusian Government. R.G.F. was recipient of a doctoral FPU fellowship from the Spanish Ministry of Science and Education. Back

Correspondence: 2José Antonio Bárcena, Department of Biochemistry and Molecular Biology, Campus de Rabanales, Ed. "Severo Ochoa," Pl. 1, University of Córdoba, 14071-Córdoba, Spain. FAX: 34 957 218592; e-mail: bb1barua{at}uco.es

Received: 12 November 2007.

First decision: 7 December 2007.

Accepted: 28 February 2008.

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