Expression of progesteronereceptor membrane component-1 in bovine reproductive system duringestrous cycleA.M. Luciano,1 D. Corbani,1 V. Lodde,1

I. Tessaro,1 F. Franciosi,1 J.J. Peluso,2

S. Modina1

1Department of Animal Sciences, Facultyof Veterinary Medicine, University ofMilan, Italy; 2Department of Cell Biology,University of CT Health Center,Farmington, CT, USA

Abstract

Several reports suggest the participation ofprogesterone receptor membrane component1 (PGRMC1) in progesterone signaling in thereproductive system. This study aimed atinvestigating the presence and localization ofPGRMC1 in bovine ovary, oviduct and uterus,during the follicular and luteal phases of theestrous cycle. In the ovary, PGRMC1 has beendetected in surface germinal epithelium,granulosa cells, theca cells and in the germi-nal vesicle of the oocytes at all stages of fol-liculogenesis. In the corpus luteum theexpression of PGRMC1 was influenced by thestage of the estrous cycle. In the oviducts andin the uterus horns, PGRMC1 was immunolo-calized in the luminal epithelium, in the mus-cle layer cells and in the endothelial cells. Inthe uterus, PGRMC1 was intensely localizedalso in the glandular endometrium. However,in the oviducts and in the uterus horns, thelocalization of PGRMC1 was independent onthe stage of the estrous cycle and on whetherevaluating the ipsilateral or the contralateralorgan. In conclusion, the present immunohis-tochemical study showed that PGRMC1 islocated in various compartments of the bovinefemale reproductive organs. With the excep-tion of the corpora lutea, PGRMC1 localizationshowed similar pattern during different stagesof the estrous cycle.

Introduction

Progesterone receptor membrane compo-nent 1 (PGRMC1) belongs to the membrane-associated progesterone receptor (MAPR) pro-teins family that is widespread in eukaryotesand involved in regulating numerous biologi-cal functions.1 PGRMC1 is predominantly

located in intracellular membranes and co-localizes with the endoplasmic reticulum andGolgi apparatus in rat liver cells and hippocam-pal Purkinje cells.2,3 Several studies describeda nuclear or chromosomal localization.PGRMC1 was detected in nuclear extracts ofHeLa cells,4 among the protein of the humanmitotic spindles,5,6 and recently has been pro-posed in regulating microtubule stability inhuman and rat ovarian cell mitosis.7,8 Variousovarian cell types, including granulosa cellsand ovarian surface epithelial cells as well asovarian tumors, express PGRMC1 where itplays an essential role in promoting the sur-vival of both normal and cancerous ovariancells in vitro.9

Recently, PGRMC1 expression has beendemonstrated in several female reproductivetissues in different mammalian species.PGRMC1 has been detected in uterine and pla-cental tissues of the mouse and human10 and itis among the proteins that were differentiallyexpressed in the human endometrium duringmenstrual cycle.11

Other reports investigated the localizationand function of PGRMC1 in the ovary. In partic-ular immunohistochemical studies localizedPGRMC1 in granulosa cells, theca cells andovarian surface epithelial cells (germinalepithelium) in rat.12 Moreover the expressionof PGRMC1 mRNA was found in granulosa orluteal cells in humans,13 mice,14 pigs15 andcows.16 Several works suggest that proges-terone mediates its antiapoptotic actionthrough PGRMC1.17,18 PGRMC1 is alsoexpressed in bovine,19 rat12 and humanoocytes.20 Recently, PGRMC1 was associated tothe meiotic spindle suggesting a role in oocytematuration, which may be specifically relatedto the mechanism by which chromosomes seg-regate.19 However, with regard to localizationof the protein, a recent work did not detectPGRMC1 in the bovine oocyte.21 To date thereis little information on the localization ofPGRMC1 in the reproductive organs of the cow.Thus, the aim of the present study was toexamine the presence and localization ofPGRMC1 in a variety of cow reproductiveorgans, including ovary, oviduct and uterusduring the follicular and luteal phases of theestrous cycle using immunohistochemistry.

Materials and Methods

Tissue collectionAll the chemicals used in this study were

purchased from Sigma Chemical Companyexcept for those specifically mentioned.Bovine ovary, oviduct and uterus were recov-ered at a local abattoir (INALCA JBS S.p.A.,

Ospedaletto Lodigiano, LO, IT 2270M CE, Italy)from non-pregnant dairy cows, 4 to 8 yrs old,subjected to routine veterinary inspection andin accordance to the specific health require-ments stated in Council Directive 89/556/ECCand subsequent modifications, and were trans-ported to the laboratory on ice. The stage of theestrous cycle was assessed by morphologicalobservations of the ovary.22 In particular, ani-mals in which one of the two ovaries present-ed an active corpus luteum were classified asbelonging to luteal phases of estrous cyclewhile animals in which one of the two ovariespresented the ovulatory follicle and a regressedcorpus luteum were classified as belonging tofollicular phases of estrous cycle.23,24

For each animal, both ovaries were trans-versely cut and 2-4 fragments of corpus luteumand 2-4 fragments of ovarian cortex were col-lected; from both oviducts samples of isthmus,ampulla and infundibulum were separated.Finally the apical third of each uterine hornswas isolated dissecting at about 5 cm from theuterus-isthmic junction.25 All samples werefixed in B5 (Formalin-Mercury II Chloride) fix-ative (Bio-Optica, Milan, Italy) for 12-24 h,dehydrated by a graded series of ethanol,cleared with xylene, paraffin embedded andsectioned at 5 µm on glass slides, previouslytreated with Vectabond (Vector Laboratories,Burlingame, CA, USA) to enhance the adher-ence of tissue.

European Journal of Histochemistry 2011; volume 55:e27

Correspondence: Alberto M. Luciano, Sezione diAnatomia ed Istologia Veterinaria, Dipartimentodi Scienze Animali, Facoltà di MedicinaVeterinaria, Università degli Studi di Milano, viaCeloria 10, 20133 Milano, Italy. Tel. +39.02.50317969 – Fax: +39.02.50317980. E-mail: alberto.luciano@unimi.it

Key words: Uterus, oviduct, ovary, corpus luteum,follicle, oocyte, progesterone receptor

Acknowledgements: the authors wish to thank Dr.Nadia Fiandanese for her excellent technical support.

Contributions: DC, VL, IT, FF laboratory experi-ments performing; AML, VL, SM results analysisand interpretation; AML, VL, JJP, SM, researchtheme defining, methods designing and manu-script writing. All authors approved the manu-script final version.

Received for publication: 28 July 2011.Accepted for publication: 24 August 2011.

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

©Copyright A.M. Luciano et al., 2011Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2011; 55:e27doi:10.4081/ejh.2011.e27

[European Journal of Histochemistry 2011; 55:e27] [page 145]

[page 146] [European Journal of Histochemistry 2011; 55:e27]

Western blot analysisWestern blot analysis was performed as pre-

viously described with minor modifications.19

Briefly, aliquots of 50 mg of ovarian cortex andcorpus luteum were homogenized in RIPAbuffer, which was supplemented with completeprotease and phosphatase inhibitor cocktails,incubated for 30 min on ice and then cen-trifuged at 14,000 g for 20 min at 4°C. Totalamount of protein was determined using theBio-Rad Protein Assay (Biorad) and 20 µg oftotal protein was used for western blot analy-sis. Equal protein loading was verified byPonceau staining. PGRMC1 immunodetectionwas conducted using different concentrations(1 µg/mL and 0.3 µg/mL) of a rabbit polyclonalantibody (Sigma Prestige, Cat. No.HPA002877)or a goat polyclonal antibody (AbCam, Cat. No.ab48012) overnight at 4°C. PGRMC1 wasrevealed using an anti-rabbit or anti-goat HRP-labelled antibodies (1:8000) and Super Signal®

West Pico Chemiluminescence Substrate(PIERCE Biotechnologies Inc., Rockford, IL,USA). Negative controls were conducted byomitting the primary antibodies.

ImmunohistochemistryIndirect immunohistochemistry was carried

out to evaluate the expression and localizationof PGRMC1. Endogenous peroxidase activitywas eliminated by incubation with 3% (v/v)H2O2 in methanol for 30 min. Then sectionswere incubated with 10% (v/v) normal goatserum or normal rabbit serum, 0.3% (v/v)Triton X-100 and 3% (w/v) bovine serum albu-min (BSA) in phosphate buffered saline (PBS)for 30 min to block non-specific binding of sec-ondary antibody. In a preliminary study thesections were incubated overnight at 4°C withdifferent concentrations (0.96, 0.48, 0.24 or0.16 µg/mL) of polyclonal rabbit anti-PGRMC1(Sigma Prestige) or polyclonal goat anti-PGRMC1 (Abcam) diluted in PBS with 1%(w/v) BSA and 0.3% (v/v) Triton X-100. In allthe subsequent experiments the rabbit poly-clonal antibody was used at a concentration of0.48 µg/mL. Primary antibodies were detectedby using a biotinylated anti-rabbit or anti goatIgG (Vector Laboratories), diluted 1:400 in PBSwith 1% (w/v) of BSA and detected with theVectastain Elite ABC kit (Vectastain Elite ABCkit, Vector Laboratories). For color develop-ment, all sections were incubated with DABsubstrate (DAB substrate kit for peroxidase,Vector Laboratories) for 2 min. After staining,the samples were counterstained with hema-toxylin QS (Vector Laboratories). Negativecontrols were performed by omitting the pri-mary antibodies. Samples were analyzed on aNikon Eclipse microscope (Nikon Corp., Tokyo,Japan) in bright field at a magnification of200X-400X.

Results

Preliminary studies were conducted in orderto compare the specificity and the sensitivityof two antibodies (polyclonal rabbit anti-PGRMC1 and polyclonal goat anti-PGRMC1) bywestern blot and immunohistochemical analy-sis with different primary antibodies concen-trations. Western blot analysis confirmed that

PGRMC1 was specifically detected in ovariancortex as a ≈26 kDa protein (Figure 1A).However, only the polyclonal rabbit anti-PGRMC1 detected the protein, while under thesame experimental conditions the goat poly-clonal antibody did not. Conversely, the goatpolyclonal antibody only detected the 26 kDaprotein when higher amounts of total proteins(100 and 50 µg) were loaded on the gel (Figure1B). Moreover, antibody titration on immuno-

Original paper

Table 1. Immunohistochemical localization of PGRMC1 in the reproductive tracts dur-ing estrous cycle. PGRMC1 is indicated as intense (+) weak (±) or negative signal in thedifferent organs.

Estrous cycleFollicular phase Luteal phase

Tissue/cell type Cytoplasm Nucleus Cytoplasm Nucleus

Ovary follicleOocyte ± + ± +Granulosa cells + + + +Theca cells + + + +Endothelial cells + + + +

Stromal tissueStromal cells ± ± ± ±Endothelial cells + + + +Germinal epithelium + + + +

Corpus luteumLuteal cells ± ± + ±Endothelial cells + + + +

OviductEpithelial cells + + + +Stromal cells ± ± ± ±Muscle layer cells + + + +Endothelial cells + + + +

UterusLuminal epithelial cells + + + +Glandular epithielial cells + + + +Stromal cells ± ± ± ±Muscle layer cells + + + +Endothelial cells + + + +

Figure 1. Detection ofPGRMC1 in bovine ovariancortex and corpus luteum bywestern blot analysis. A)Western blot analysis showingPGRMC1 expression in bovineovarian cortex and corpusluteum (20 µg total protein/lane) using the rabbit poly-clonal (Sigma-Prestige) or thegoat polyclonal (Abcam) anti-bodies at different concentra-tions. Note that only the rabbitpolyclonal anti-PGRMC1 anti-body detected the protein,while under the same experi-mental conditions the goatpolyclonal antibody did not.B) Western blot analysis ofPGRMC1 expression in ovari-an cortex with the goat poly-clonal antibody using increas-ing amount of total protein (50and 100 µg).

[European Journal of Histochemistry 2011; 55:e27] [page 147]

Original paper

Figure 2. Titrations of different primary antibodiesto detect PGRMC1. A) Representative images ofPGRMC1 localization in a medium antral folliclewall using rabbit polyclonal and goat polyclonalprimary antibodies. The values on the left refer tothe concentrations (µg/mL) of the primary anti-bodies used. Note that rabbit polyclonal antibodyshows a specific staining for PGRMC1 in the gran-ulosa cells, in the theca layer and in the endothelial

cells of blood vessels at all concentration used, whereas weaker signal was detected by the goat polyclonal antibody under the same experimen-tal conditions. No immunoreactivity was seen in negative controls where incubation with the primary antibodies was omitted. Final magnifi-cation, 200X; scale bar, 50 µm. B) Representative images showing details of PGRMC1 localization in oocytes and medium antral follicle wallusing the two antibodies under the same experimental conditions at a concentration of 0.48 µg/mL. Note that rabbit polyclonal primary anti-body exhibits a specific staining for PGRMC1 in cumulus cells, germinal vesicle, granulosa cells, theca layer and endothelial cells (upperimages) when compared with the goat polyclonal primary antibody (lower images). Final magnification, 400X; scale bar, 50 µm.

A B

[page 148] [European Journal of Histochemistry 2011; 55:e27]

histochemistry showed a higher sensitivity ofthe polyclonal rabbit anti-PGRMC1 since thisantibody detected PGRMC1 in the granulosacells, in the theca layer and in the endothelialcells of blood vessels at all concentration test-ed, whereas the goat polyclonal antibodydetected a much weaker signal at the corre-sponding concentration (Figure 2A,B). In addi-tion the rabbit polyclonal detected a muchhigher PGRMC1 signal in cumulus cells andgerminal vesicle of the oocyte when comparedwith the goat polyclonal used at the same con-centration (Figure 2B). Therefore, the poly-clonal rabbit anti-PGRMC1 antibody, at a con-centration of 0.48 µg/mL was utilized in furtherexperiments to assess PGRMC1 localization inbovine ovary, oviduct and uterine horn.

A total of 8 animals (4 for each phase of theestrous cycle) were analyzed for PGRMC1localization. Data are shown in Figures 3, 4and 5 and summarized in Table 1.

In all experiments, negative controls wereconducted in the absence of the primary anti-body and did not show any staining.

In the ovary, immunohistochemical studiesshowed that PGRMC1 was expressed in the fol-licles during all the stages of folliculogenesis,in the oocyte and in its surrounding cumuluscells, both in the nucleus and inside the cyto-plasm (Figure 3). Oocyte showed a positivestaining in the germinal vesicle (GV), whilethe ooplasm displayed only a weak signal(Figure 3A,B). In the cumulus oophorus, gran-ulosa cells lying closer to the oocyte exhibiteda more intense signal than granulosa cells inthe outer layers (Figure 2B). PGRMC1 was alsodetected in theca layers (Figure 3C).Furthermore, PGRMC1 was immunolocalizedin the germinal epithelium of the ovarian sur-face (Figure 3D) and in the endothelial cells ofthe stromal vessels (arrows in Figure 3A,B,C).On the contrary, in ovarian stromal tissue cellsPGRMC1 was weakly detected. Immunolo -calization of PGRMC1 revealed a similarexpression pattern for the follicular and lutealphase of the estrous cycle and differences werenot observed between the two ovaries of thesame animal.

In contrast, in the corpus luteum PGRMC1 wasdifferently immunolocalized depending on thestage of the estrous cycle (Figure 4). During theluteal phase (Figure 4A) most of the luteal cellsshowed a cytoplasmic staining for PGRMC1, withsignal intensity varying from intense to weak,while the signal was completely absent only in afew cells. During the follicular phase (Figure 4B)PGRMC1 was detected as a weak signal in thecytoplasm of most of the cells. In both phasesPGRMC1 was expressed in the endothelial cellsand was detected as an intense to weak signal inthe nuclei of the luteal cells.

Microscopic examination of PGRMC1 local-ization in the oviduct revealed a specific

nuclear and cytoplasmic staining for PGRMC1in the luminal epithelium (Figure 5A,B), in themuscle layers (Figure 5C), as well as in theendothelial cells. On the contrary PGRMC1 wasweakly expressed in the cells of lamina propriaand submucosa (stromal cells). No differenceswere observed between infundibulum (Figure5A), ampulla (Figure 5B), and isthmus (Figure5C), neither in follicular nor luteal phase,either between the two organs of the same ani-mal. In the uterus, PGRMC1 was observed as

an intense signal in the luminal epithelium(Figure 5E), glandular endometrium (Figure5F), as well in the muscle layers (Figure 5F)and in the vessels while a weak signal waspresent in the stromal compartment. As in theoviduct, a positive staining was present both inthe nucleus and in the cytoplasm of all the cellsand no difference was observed between theestrous stages and between the two horns.

Original paper

Figure 3. Immunohistochemical localization of PGRMC1 in bovine ovarian cortex. PGRMC1is localized in primordial, primary and secondary follicles (A), in early antral follicles (B) and inantral follicles (C). Note that the expression of PGRMC1 is more evident in the granulosa cellscloser to the oocyte, in the GV and in the internal theca layer. PGRMC1 was detected in theendothelial cells (arrows) and in the germinal epithelium at the ovarian surface (D, arrowhead).No immunoreactivity was seen in negative control where incubation with the primary antibodyhad been omitted (E). Final magnification, 200X; scale bar, 50 µm.

Figure 4. Immunohistochemical localization of PGRMC1 in bovine corpora lutea during theluteal and follicular phase of the estrous cycle. During the luteal phase (A) PGRMC1 is local-ized in the cytoplasm of the luteal cells displaying an heterogeneous signal, varying from intenseto weak to completely absent. During the follicular phase (B) PGRMC1 is detected as a weaksignal in the cytoplasm of most of the luteal cells. In both phases PGRMC1 is expressed in theendothelial cells and is weakly detected in the nucleus of the luteal cells. Final magnification,200X; scale bar, 50 µm.

[European Journal of Histochemistry 2011; 55:e27] [page 149]

Discussion

To the best of our knowledge, this is the firststudy that describes PGRMC1 immunolocaliza-tion in various parts of bovine reproductive sys-tem. In the ovary, PGRMC1 has been detected ingerminal epithelium, granulosa cells, thecacells, and oocyte in all stages of folliculogenesis,in agreement with previous studies conductedin rat.12 Data from the literature indicate that, inthe ovary, PGRMC1 is continuously and highlyexpressed during fetal development in rat andcow and it has been suggested that PGRMC1participates through a progesterone-dependentmechanism to the process of primordial follicleassembly.26,27

In this study, PGRMC1 was intensely localizedin the GV of oocytes. This confirms our previousimmunofluorescence study on the oocyte wherePGRMC1 was restricted in the GV after follicleisolation.19 In the oocyte, PGRMC1 has a role inoocyte maturation, which may be specificallyrelated to the mechanism of chromosomes seg-regation.19 However, other Authors were unableto detect PGRMC1 in the bovine oocyte,21 while itwas detectedin the surrounding granulosa cells.This difference may be due to the different sen-sitivity of the antibody used and to the shorterincubation time employed in the previous study(3 h). As indicated in our study, the polyclonalrabbit anti-PGRMC1 antibody showed a muchhigher sensitivity respect to the polyclonal goatanti-PGRMC1 antibody and the titration assaydemonstrated that only high concentrations ofthe polyclonal goat anti-PGRMC1 antibody wereable to detect a specific signal by western blotand immunohistochemistry under our experi-mental conditions.

Our observations in the ovary and corpusluteum support previous studies where theexpression of PGRMC1 was found in granulosaand luteal cells in human,13 mouse,14 rat,12 pig15

and cow.16,28 PGRMC1 positivity in endothelialcells of the corpus luteum reported in previousstudy on PGRMC1 expression in cow28 was alsoconfirmed by our observations. Furthermore, inthe present work, the expression of PGRMC1 inthe corpus luteum was found to be influenced bythe stage of the estrous cycle. In the lutealphase, PGRMC1 was intensely localized in thecytoplasm while in the follicular phase a weakPGRMC1 signal was detected in the cytoplasm ofmost of the cells. This stage-specific localizationof PGRMC1 could be explained through the func-tional status of the corpus luteum. Beside itspossible involvement in steroid synthesis in theactive corpus luteum, the ability of PGRMC1 inpromoting luteal cell viability by mediating prog-esterone anti-apoptotic action was demonstrat-ed.13

PGRMC1 was detected among the proteinsthat were differentially expressed in the human

endometrium during the menstrual cycle.11 Thisstudy showed that PGRMC1 protein was morehighly present in the proliferative (luteal) thanthe secretory (follicular) phase and immunos-taining data revealed that it was confined to thestromal component of proliferative phaseendometrium. Analogously, microarray profilingof progesterone-regulated endometrial genesduring the rhesus monkey secretory phaseshowed a down regulation of PGRMC1.29 Thesefindings were confirmed also in mice,10 wherePGRMC1 expression was up-regulated in thestromal compartment of the uterus from estrusto metestrus and reduced during diestrus. In thebovine, our study showed that PGRMC1 localiza-tion in the oviducts and in the uterus horns, wassimilar in the ipsilateral or the contralateralorgans and was irrespective of the stage of theestrous cycle. This difference between speciesmay be explained by the different technique

adopted since the immunochemical detection ofthe protein must be confirmed by a quantitativeanalysis. Moreover, the level of the transcriptmust be determined in order to assess whetheror not the phase-independent localization ofPGRMC1 observed in this study, in bovineoviduct and uterus, is accompanied by a phase-independent PGRMC1 transcript level.

In the present study we did not observe differ-ences in PGRMC1 localization in the uterusbetween the luteal and follicular phases.However, we do not exclude that these differ-ences could arise during pregnancy. In fact, adecrease in PGRMC1 has been observed inhuman myometrium during term or pretermlabor, where PGRMC1 down-regulation mightcontribute to the functional withdrawal of prog-esterone’s action and shift the balance to a stateof increased uterine contractility.30 Moreover,PGRMC1 was described in the human and

Original paper

Figure 5. Immunohistochemical localization of PGRMC1 in bovine oviduct and uterus.PGRMC1 is expressed in the different tracts of the oviduct, infundibulum (A), ampulla (B) andisthmus (C) in luminal epithelium, in the muscle layers (ml) and in the vessels (arrowheads).In the uterus PGRMC1 is localized in luminal (E) and glandular (F) epithelium, in the musclelayers (ml) and in the endothelial cells (arrowheads). Note that the nuclear staining of epithe-lial cells is intense both in oviduct and uterus. No immunoreactivity was seen in negative con-trol where incubation with the primary antibody had been omitted(D). Final magnification,200X; scale bar, 50 µm.

[page 150] [European Journal of Histochemistry 2011; 55:e27]

mouse placenta and associated membranes withthe most abundant expression in smooth muscleof the placental vasculature, villous capillariesand the syncytiotrophoblast suggesting that thisprotein functions in events important to earlypregnancy, including cellular differentiation,modulation of apoptosis and steroidogenesis.10

Therefore, the study of PGRMC1 localization andexpression during pregnancy in the cow wouldbe of particular interest since dissimilaritiesbetween species could be due to the differentinvolvement of the endometrium in the processof placentation in these species. Rodents andprimates are characterized by an haemochorialplacenta while cows have a synepitheliochorialplacenta. Further studies are needed in order toconfirm this hypothesis.

In conclusion, the present immunohisto-chemical study showed that PGRMC1 is locat-ed in various compartments of the bovinefemale reproductive organs. Despite some dif-ference in PGRMC1 expression have beenreported in human and mouse, our data sug-gest that with the exception of the corporalutea, PGRMC1 localization does not seem tobe differentially regulated during differentstages of the estrous cycle. However, a quanti-tative study must be conducted in order to clar-ify the phase-independency of PGRMC1expression in this species.

References

1. Cahill MA. Progesterone receptor membranecomponent 1: an integrative review. J SteroidBiochem Mol Biol 2007;105:16-36.

2. Nolte I, Jeckel D, Wieland FT, Sohn K.Localization and topology of ratp28, a mem-ber of a novel family of putative steroid-bind-ing proteins. Biochim Biophys Acta 2000;1543:123-30.

3. Sakamoto H, Ukena K, Takemori H, OkamotoM, Kawata M, Tsutsui K. Expression andlocalization of 25-Dx, a membrane-associat-ed putative progesterone-binding protein, inthe developing Purkinje cell. Neuroscience2004;126:325-34.

4. Beausoleil SA, Jedrychowski M, Schwartz D,Elias JE, Villen J, Li J, et al. Large-scale char-acterization of HeLa cell nuclear phosphopro-teins. Proc Natl Acad Sci USA 2004;101:12130-5.

5. Sauer G, Korner R, Hanisch A, Ries A, NiggEA, Sillje HH. Proteome analysis of thehuman mitotic spindle. Mol Cell Proteomics2005;4:35-43.

6. Nousiainen M, Sillje HH, Sauer G, Nigg EA,Korner R. Phosphoproteome analysis of thehuman mitotic spindle. Proc Natl Acad SciUSA 2006;103:5391-6.

7. Lodde V, Peluso JJ. A novel role for proges-

terone and progesterone receptor membranecomponent 1 in regulating spindle micro-tubule stability during rat and human ovari-an cell mitosis. Biol Reprod 2011;84:715-22.

8. Lodde V, Peluso JJ, Franciosi F, Luciano AM. Anovel role for progesteronereceptor mem-brane component-1 (PGRMC1) in regulatingmitosis and meiosis. Proc. 34th NationalCongress of the Italian Society of Histo -chemistry, San Benedetto del Tronto, Italy,June 7-9, 2011. Eur J Histochem 2011;55(s1):4.

9. Peluso JJ. Progesterone Signaling MediatedThrough Progesterone Receptor MembraneComponent-1 in Ovarian Cells with SpecialEmphasis on Ovarian Cancer. Steroids 2011;76:903-9.

10. Zhang L, Kanda Y, Roberts DJ, Ecker JL, LoselR, Wehling M, et al. Expression of proges-terone receptor membrane component 1 andits partner serpine 1 mRNA binding proteinin uterine and placental tissues of the mouseand human. Mol Cell Endocrinol 2008;287:81-9.

11. Chen J, Hannan N, Mak Y, Nicholls P, ZhangJ, Rainczuk A, et al. Proteomic Characteri-zation of Mid-Proliferative and Mid-SecretoryHuman Endometrium. J Proteome Res 2009;8:2032-2044.

12. Peluso JJ, Pappalardo A, Losel R, Wehling M.Progesterone membrane receptor compo-nent 1 expression in the immature rat ovaryand its role in mediating progesterone's anti-apoptotic action. Endocrinology 2006;147:3133-40.

13. Engmann L, Losel R, Wehling M, Peluso JJ.Progesterone regulation of human granu-losa/luteal cell viability by an RU486-inde-pendent mechanism. J Clin EndocrinolMetab 2006;91:4962-8.

14. McRae RS, Johnston HM, Mihm M,O'Shaughnessy PJ. Changes in mouse gran-ulosa cell gene expression during earlyluteinization. Endocrinology 2005;146:309-17.

15. Jiang H, Whitworth KM, Bivens NJ, Ries JE,Woods RJ, Forrester LJ, et al. Large-scale gen-eration and analysis of expressed sequencetags from porcine ovary. Biol Reprod 2004;71:1991-2002.

16. Kowalik MK, Kotwica J. Progesterone recep-tor membrane component 1 (PGRMC1) geneexpression in corpus luteum during theestrous cycle in cows. Reprod Biol 2008;8:291-7.

17. Peluso JJ, Liu X, Gawkowska A, Lodde V, WuCA. Progesterone inhibits apoptosis in partby PGRMC1-regulated gene expression. MolCell Endocrinol 2010;320:153-61.

18. Peluso JJ, Romak J, Liu X. Progesteronereceptor membrane component-1 (PGRMC1)is the mediator of progesterone's antiapop-totic action in spontaneously immortalized

granulosa cells as revealed by PGRMC1 smallinterfering ribonucleic acid treatment andfunctional analysis of PGRMC1 mutations.Endocrinology 2008;149:534-43.

19. Luciano AM, Lodde V, Franciosi F, Ceciliani F,Peluso JJ. Progesterone receptor membranecomponent 1 expression and putative func-tion in bovine oocyte maturation, fertiliza-tion, and early embryonic development.Reproduction 2010;140:663-72.

20. Wood JR, Dumesic DA, Abbott DH, Strauss JF,3rd. Molecular abnormalities in oocytes fromwomen with polycystic ovary syndromerevealed by microarray analysis. J ClinEndocrinol Metab 2007;92:705-13.

21. Aparicio IM, Garcia-Herreros M, O'Shea LC,Hensey C, Lonergan P, Fair T. Expression,regulation, and function of progesteronereceptors in bovine cumulus oocyte complex-es during in vitro maturation. Biol Reprod2011;84:910-21.

22. Ireland JJ, Murphee RL, Coulson PB.Accuracy of predicting stages of bovineestrous cycle by gross appearance of the cor-pus luteum. J Dairy Sci 1980;63:155-60.

23. Fields MJ, Fields PA. Morphological charac-teristics of the bovine corpus luteum duringthe estrous cycle and pregnancy. Therio -genology 1996;45:1295-325.

24. Miyamoto Y, Skarzynski DJ, Okuda K. Istumor necrosis factor alpha a trigger for theinitiation of endometrial prostaglandinF(2alpha) release at luteolysis in cattle? BiolReprod 2000;62:1109-15.

25. Bauersachs S, Ulbrich SE, Gross K, SchmidtSE, Meyer HH, Einspanier R, et al. Geneexpression profiling of bovine endometriumduring the oestrous cycle: detection of molec-ular pathways involved in functionalchanges. J Mol Endocrinol 2005;34:889-908.

26. Nilsson EE, Skinner MK. Progesterone regu-lation of primordial follicle assembly inbovine fetal ovaries. Mol Cell Endocrinol2009;313:9-16.

27. Nilsson EE, Stanfield J, Skinner MK.Interactions between progesterone andtumor necrosis factor-alpha in the regulationof primordial follicle assembly. Reproduction2006;132:877-86.

28. Rae MT, Menzies GS, Bramley TA. Bovineovarian non-genomic progesterone bindingsites: presence in follicular and luteal cellmembranes. J Endocrinol 1998;159:413-27.

29. Ace CI, Okulicz WC. Microarray profiling ofprogesterone-regulated endometrial genesduring the rhesus monkey secretory phase.Reprod Biol Endocrinol 2004;2:54.

30. Wu W, Shi SQ, Huang HJ, Balducci J, GarfieldRE. Changes in PGRMC1, a potential proges-terone receptor, in human myometrium dur-ing pregnancy and labour at term andpreterm. Mol Hum Reprod 2011;17:233-42.

Original paper