Systemic distribution of progesterone receptor subtypes in human tissues.

Progesterone receptor (PR) is expressed in a wide variety of human tissues, including both reproductive and non-reproductive tissues. Upon binding to the PR, progesterone can display several non-reproductive functions, including neurosteroid activity in the central nervous system, inhibition of smooth muscle contractile activity in the gastrointestinal tract, and regulating the development and maturation of the lung. PR exists as two major isoforms, PRA and PRB. Differential expression of these PR isoforms reportedly contributes to different biological activities of the hormone. However, the distribution of the PR isoforms in human tissues has remained virtually unexplored. In this study, we immunolocalized PR expression in various human tissues using PR (1294) specific antibody, which is capable of detecting both PRA and PRB, and PRB (250H11) specific antibody. Tissues from the uterus, ovary, breast, placenta, prostate, testis, cerebrum, cerebellum, pituitary, spinal cord, esophagus, stomach, small intestine, colon, pancreas, liver, kidney, urinary bladder, lung, heart, aorta, thymus, adrenal gland, thyroid, spleen, skin, and bone were examined in four different age groups (fetal, pediatric, young, and old) in male and female subjects. PR and PRB were detected in the nuclei of cells in the female reproductive system, in both the nuclei and cytoplasm of pituitary gland and pancreatic acinar cells, and only in the cytoplasm of cells in the testis, stomach, small intestine, colon, liver, kidney, urinary bladder, lung, adrenal gland, and skin. Of particular interest, total PRB expression overlapped with that of total PR expression in most tissues but was negative in the female fetal reproductive system. The findings indicate that progesterone could affect diverse human organs differently than from reproductive organs. These findings provide new insights into the novel biological roles of progesterone in non-reproductive organs.

Safety of everolimus plus exemestane in patients with hormone-receptor-positive, HER2-negative locally advanced or metastatic breast cancer: results of phase IIIb BALLET trial in Spain

Background: Everolimus with exemestane has shown promising activity in patients with hormone-receptor (HR)-positive HER2-negative endocrine-resistant advanced breast cancer. It is necessary, therefore, to characterize the safety profile of this new combination in the real-world clinical setting and in the broadest possible population. Patients and methods: Post-menopausal women with HR-positive HER2-negative advanced breast cancer progressing after prior non-steroidal aromatase inhibitors (NSAIs) were included. The objectives of this analysis were to evaluate the safety profile of this combination in a subset of Spanish patients in the BALLET trial and to characterize grade 3 and 4 adverse events (AEs) in routine clinical practice in Spain. Results: Between September 2012 and July 2013, 429 patients (20% of the overall study population) were included in the BALLET study in 52 hospitals in Spain, of whom 100 (23%) were ≥ 70 years. The median treatment duration was 3.14 and 3.03 months for exemestane and everolimus, respectively. The most common reasons for discontinuation of treatment were local reimbursement of everolimus (43%), followed by disease progression (31%) and the incidence of AEs (15%). The most frequent AEs causing permanent discontinuation were pneumonitis (4%), asthenia (2%) and stomatitis (2%). Overall, 87% of patients experienced at least one AE of any grade, 30% of patients at least one grade 3 AE and 2% of patients a grade 4 AE. Conclusion: The safety profile in Spanish patients of the BALLET trial is consistent with the results obtained in the overall population of the trial, as well as in previous clinical trials

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Breast Cancer Subtype is Associated With Axillary Lymph Node Metastasis: A Retrospective Cohort Study.

The purpose of this study was to assess whether breast cancer subtype (BCS) as determined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 can predict the axillary lymph node metastasis in breast cancer. Patients who received breast conserving surgery or mastectomy and axillary lymph node dissection were identified from 2 cancer centers. The associations between clinicopathological variables and axillary lymph node involvement were evaluated in univariate and multivariate regression analyses. A total of 3471 patients met the inclusion criteria, and 53.0% had axillary lymph node metastases at diagnosis. Patients with hormone receptor (HR)-/human epidermal growth factor receptor 2 (HER2)- subtype had a higher grade disease and the lowest rate of lymphovascular invasion. Univariate and multivariable logistic regression analyses showed that BCS was significantly associated with lymph node involvement. Patients with the HR-/HER2- subtype had the lowest odds of having nodal positivity than those with other BCSs. HR+/HER2- (odds ratio [OR] 1.651, 95% confidence interval [CI]: 1.349-2.021, P < 0.001), HR+/HER2+ (OR 1.958, 95%CI 1.542-2.486, P < 0.001), and HR-/HER2+ (OR 1.525, 95%CI 1.181-1.970, P < 0.001) tumors had higher risk of nodal positivity than the HR-/HER2- subtype. The other independent predictors of nodal metastases included tumor size, tumor grade, and lymphovascular invasion. Breast cancer subtype can predict the presence of axillary lymph node metastasis in breast cancer. HR-/HER2- is associated with a reduced risk of axillary lymph node metastasis compared to other BCSs. Our findings may play an important role in guiding axillary treatment considerations if further confirmed in larger sample size studies.

Correlation of hormone receptor status between circulating tumor cells, primary tumor, and metastasis in breast cancer patients

Purpose. Estrogen receptor (ER) and progesterone receptor (PR) status is prognostic and predictive in breast cancer. Because metastatic breast tumor biopsies are not routinely feasible, circulating tumor cells (CTCs) offer an alternative source of determining ER/PR tumor status. Methods/patients. Peripheral blood was collected prospectively from 36 patients with metastatic breast cancer. CTCs were isolated using the microfluidic OncoCEE™ platform. Detection was accomplished with an expanded anti-cytokeratin (CK) cocktail mixture and anti-CD45. ER/PR protein expression was assessed by immunocytochemistry (ICC) on the CK+ cells and compared to the primary and/or metastatic tumor by immunohistochemistry (IHC). Results. Among the 24 CK + CTC cases, a concordance of 68 % (15/22) in ER/PR status between primary breast tumor and CTCs and 83 % (10/12) between metastatic tumor and CTCs was observed. An overall concordance of 79 % (19/24) was achieved when assessing CTC and metastatic tumor (primary tumor substituted if metastatic breast biopsy not available). A test sensitivity of 72 % and specificity of 100 % was identified when comparing CTCs to tumor tissue. Of the 7 discordant cases between CTCs and primary tumor tissue, 2 were concordant with the metastatic biopsy. Conclusions. CTC ER/PR status using the OncoCEE™ platform is feasible, with high concordance in ER/PR status between tumor tissue (IHC) and CTCs (ICC). The prognostic and predictive significance of CTC ER/PR protein expression needs further evaluation in larger trials (AU)

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Purification of the goldfish membrane progestin receptor α (mPRα) expressed in yeast Pichia pastoris.

Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of progestins on plasma membranes. We established a procedure for the expression and purification of recombinant goldfish mPRα using the methylotropic yeast Pichia pastoris. In P. pastoris, the recombinant protein, which carried C-terminal histidine and c-Myc tags, was expressed in an active form as the receptor for maturation-inducing steroids of fish. Expressed proteins were bound reversibly with a high affinity (Kd = 9.4 nM) at a single binding site that could be saturated. After solubilization of mPRα with n-dodecyl-ß-D-maltoside (DDM) from yeast membranes, the recombinant protein was purified using three different columns: first it was affinity-purified over nickel-nitrilotriaceticacid (Ni-NTA), then bound to a cellulose resin with free amino groups and finally to a column with affinity for the c-Myc epitope. The identity of the purified protein was verified by MALDI-TOF/MS analysis and its capacity to bind progestin remained. Expression and purification of mPRα protein in its functional form will enable the screening of ligands and the determination of its three dimensional structure.

Biopsy of a 5 mm cystic lesion on the right heel of a 48-year-old woman.

An excisional biopsy of an asymptomatic cystic lesion that had been present for several years on the right heel of a 48-year-old woman revealed a subcutaneous cyst lined by ciliated columnar epithelium. On immunostaining, the epithelial cells were positive for Pan-cytokeratin (CK AE 1/3), estrogen receptor (ER) and progesterone receptor (PR), but negative for carcinoembryonic antigen (CEA), suggesting Mullerian type of epithelium. Cutaneous ciliated cyst of Mullerian type occurs almost exclusively on the lower extremity of premenopausal women. The lesion is benign and excision is curative.

Cloning, pharmacological characterization, and expression analysis of Atlantic salmon (Salmo salar L.) nuclear progesterone receptor.

To better understand the role(s) of progestogens during early stages of spermatogenesis, we carried out studies on the nuclear progesterone receptor (Pgr) of the Atlantic salmon. Its open-reading frame shows the highest similarity with other piscine Pgr proteins. When expressed in mammalian cells, salmon Pgr exhibited progestogen-specific, dose-dependent induction of reporter gene expression, with 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) showing the highest potency. We then analyzed testicular pgr mRNA and DHP plasma levels in animals during the onset of spermatogenesis, which were exposed to natural light or to constant light, to induce significant differences in testis growth. Grouping of the animals according to their progress through spermatogenesis showed that testicular pgr mRNA levels as well as DHP plasma levels first increased when germ cells had reached the stage of late type B spermatogonia and further increased when entered meiosis, i.e. when spermatocytes were present. However, in situ hybridization studies revealed that pgr mRNA expression was restricted to Sertoli cells, with a strong signal in Sertoli cells contacting type A/early type B spermatogonia, while Sertoli cells contacting larger germ cell clones with further differentiated stages (e.g. late type B spermatogonia) were less intensely/not stained. We conclude that the increase in pgr mRNA levels per pair of testis reflects, at least in part, the increased number of Sertoli cells enveloping type A and early type B spermatogonia. We propose that Sertoli cell-expressed Pgr may mediate DHP-stimulated early steps in spermatogenesis in Atlantic salmon, such as an increase in the number of new spermatogonial cysts.

Immunohistochemical assessment of hormone receptor status of breast carcinoma: interobserver variation of the quick score.

BACKGROUND: Immunohistochemical (IHC) assessment of estrogen receptor (ER) and progesterone receptor (PR) status has become a routine practice to predict the likely outcome of Tamoxifen therapy. AIMS: To assess the interobserver variation in scoring hormone receptor status of breast carcinoma, using the Quick Score. MATERIALS AND METHODS: IHC-stained slides of breast carcinomas reported by the two authors during a 28-month period were included in the study. Both authors independently reassessed all the tumors. Both were blinded to each other's assessment. The Quick score with a 0-8 point scale was used to score the hormone receptor status. Weighted Kappa was calculated to assess the interobserver variation. RESULTS: A total of 210 breast carcinomas were included in this study. There was a substantial to almost perfect agreement between the two observers in scoring the hormone receptor status (kappa values; ER=0.856, PR=0.711). Both ER and PR showed an almost perfect agreement in assessing the intensity of staining (kappa value; ER=0.882, PR=0.840), while the scoring of proportion of cells gave lower Kappa values (kappa value; ER=0.778, PR=0.592). Interobserver agreement was less in scoring hormone receptor status of breast carcinomas after mastectomies compared with excision biopsies, wide local excisions and metastatic deposits in lymph nodes. Suboptimal fixation resulting in background staining has contributed to the variation. CONCLUSION: A substantial to almost perfect interobserver agreement was seen in assigning an overall Quick score. Detection of complete negative and strong expression had a moderate to substantial agreement.

Prognostic significance of molecular classification of breast invasive ductal carcinoma.

OBJECTIVE: Breast carcinoma classification has dramatically changed in recent years following application of molecular techniques. Immunohistochemistry can help select patients for different therapies. The objective of the present report is to determine the prognostic influence of the molecular classification of breast carcinoma with immunohistochemistry. MATERIALS AND METHODS: We have retrospectively selected a cohort of 257 patients with invasive ductal carcinoma NOS diagnosed and treated in the same hospital between 1997 and 2000. We have classified the cases in four tumor types according to the immunohistochemical expression of several markers, as luminal A tumors [estrogen and/or progesterone receptors (ER/PE) positive and Her-2 negative]; luminal B tumors (ER/PR positive and Her-2 positive); Her-2 positive tumors (ER/PR negative with Her-2 positive); and triple negative phenotype (all markers negative). RESULTS: In our series, 116 patients had tumors of luminal A type (47.93%); 67 (27.68%) were luminal type B; 33 (13.63%) were Her2 positive; and 26 (10.74%) were triple negative. The recurrence rate was 19% for luminal type A tumors, 25.4% for luminal type B, 39.4% for Her2 positive and 30.8% for triple negative lesions. The mean relapse free survival was 79.07, 73.07, 64.3 and 83,5 months for luminal A and B, Her-2 and triple negative lesions, respectively. Mortality rate reached 11.2% for luminal A tumors compared with 19.4, 33.3 and 26.9% for luminal B, Her2 and triple negative tumors, respectively. The mean overall survival for these groups was 88.42, 81.41, 77.62 and 93.6 months. CONCLUSION: Molecular classification with immunohistochemistry behaves as a significant prognosticator for breast invasive ductal carcinoma in our series of patients. The worse prognosis observed for Her2 expressing lesions may have changed after trastuzumab use.

Candidates for membrane progestin receptors--past approaches and future challenges.

Progestins have a broad range of functions in reproductive biology. Many rapid nongenomic actions of progestins have been identified, including induction of oocyte maturation, modulation of reproductive signaling in the brain, rapid activation of breast cancer cell signaling, induction of the acrosomal reaction and hypermotility in mammalian sperm. Currently, there are three receptor candidates for mediating rapid progestin actions: (1) membrane progestin receptors (mPRs); (2) progestin receptor membrane components (PGRMCs); and (3) nuclear progestin receptors (nPRs). The recently-described mPR family of proteins has seven integral transmembrane domains and mediates signaling via G-protein coupled pathways. The PGRMCs have a single transmembrane with putative Src homology domains for potential activation of second messengers. The classical nPRs, in addition to having well defined transcriptional activity, can also mediate rapid activation of intracellular signaling pathways. However, details of the mechanisms by which these three classes of progestin receptors mediate rapid intracellular signaling and their subcellular localization remain unclear. In addition, mPRs, nPRs and PGRMCs exhibit overlapping expression and functions in multiple tissues, implying potential interactions during oocyte maturation, parturition, and breast cancer signaling in individual cells. However, the overwhelming majority of studies to date have focused on the functions of one of these groups of receptors in isolation. This review will summarize recent findings on the three major progestin receptor candidates, emphasizing the different approaches used, some experimental pitfalls, and current controversies. We will also review evidence for the involvement of mPRs and nPRs in one of the most well-characterized nongenomic steroid actions in basal vertebrates, oocyte maturation, and conclude by suggesting some future areas of research. Clarification of the controversies surrounding the identities and localization of membrane progestin receptors may help direct future research that could advance our understanding of rapid actions of steroids.

Systematic expression analysis and antibody screening do not support the existence of naturally occurring progesterone receptor (PR)-C, PR-M, or other truncated PR isoforms.

Functional progesterone withdrawal associated with human parturition has been ascribed to various mechanisms modulating the function of the classical progesterone receptors (PRs), B and A, in utero. These include up-regulation of the inhibitory PR-C isoform, described as a 60-kDa protein occurring from translation initiation at codon 595. Our initial attempts to detect PR-C yielded uninterpretable results. To systematically validate antibodies for immunodetection of PR isoforms, we generated expression vectors for PR variants originating from putative start codons AUG-289, -301, -595, -632, and -692 in addition to those for PR-B and PR-A, and for alternative splice variants PR-T, PR-S, and PR-M. All constructs were subjected to in vitro and in vivo translation and immunoblotting with a panel of 13 PR antibodies. Antibodies raised against full-length PR were generally not capable of detecting N-terminally truncated forms, whereas C-terminal antibodies did not or only weakly reacted with PR-B and PR-A but produced prominent nonspecific signals. Thus, immunodetection of N-terminally truncated PR isoforms is prone to artifacts. Proteins of about 64 kDa were expressed from PR-289 and -301, but no corresponding endogenous forms were observed. PR-T, PR-S, and PR-M cDNAs yielded no detectable translation products. No protein was translated from AUG-595 in our PR-C expression vector unless a Kozak sequence was introduced, and the product was not 60 but 38 kDa in size. Thus, the 60-kDa protein called PR-C does not originate from AUG-595 and is not a naturally occurring PR isoform.

Age-dependent expression and localization of the progesterone receptor in the boar testis.

Expression of the progesterone receptor (PR) was monitored in testes of groups of five boars aged 50, 100, 150, 200 and 250 days. The primary monoclonal antibody used for immunohistochemistry (IHC) was raised against a peptide mapping the amino acids 922-933 of the carboxy-terminus of the human PR, negative controls were set up using an irrelevant monoclonal isotype-specific antibody, porcine endometrium served as positive control tissue. In parallel, qualitative reverse transcription-polymerase chain reaction was applied. Based on the developmental status of spermatogenesis the 50- and 100-day-old boars were considered as immature, the boars aged 200 and 250 days as mature. Positive and negative controls confirmed specificity of IHC. In the 50-day-old boars 85.1% of the prespermatogonia that had reached the basal lamina and 18.2% of the centrally located prespermatogonia stained positive, while it was 92.1% respectively 2.1% in the 100-day-old boars. The effect of time and location was highly significant (p < 0.005 resp. 0.0001). In mature boars between 77 and 80% of the A and B spermatogonia stained positive, there was no effect of boar age and stage of spermatogenesis. In both groups also few peritubular myoid cells stained positive. It is hypothesized that Leydig cell-derived progesterone plays a functional role in spermatogoniogenesis in a synergistic manner with Leydig cell-derived oestrogens.

Spectroscopic and biochemical characterization of heme binding to yeast Dap1p and mouse PGRMC1p.

Yeast damage-associated response protein (Dap1p) and mouse progesterone receptor membrane component-1 protein (mPGRMC1p) belong to a highly conserved class of putative membrane-associated progesterone binding proteins (MAPR), with Dap1p and inner zone antigen (IZA), the rat homologue of mPGRMC1p, recently being reported to bind heme. While primary structure analysis reveals similarities to the cytochrome b(5) motif, neither of the two axial histidines responsible for ligation to the heme is present in any of the MAPR proteins. In this paper, EPR, MCD, CD, UV-vis, and general biochemical methods have been used to characterize the nature of heme binding in both Dap1p and a His-tagged, membrane anchor-truncated mPGRMC1p. As isolated, Dap1p is a tetramer which can be converted to a dimer upon addition of 150 mM salt. The heme is noncovalently attached, with a maximal, in vitro, heme loading of approximately 30%, for both proteins. CD and fluorescence spectroscopies indicate a well-ordered structure, suggesting the low level of heme loading is probably not due to improperly folded protein. EPR confirmed a five-coordinate, high-spin, ferric resting state for both proteins, indicating one axial amino acid ligand, in contrast to the six-coordinate, low-spin, ferric state of cytochrome b(5). The MCD spectrum confirmed this conclusion for Dap1p and indicated the axial ligand is most likely a tyrosine and not a histidine, or a cysteine; however, an aspartic acid residue could not be conclusively ruled out. Potential axial ligands, which are conserved in all MAPRs, were mutated (Y78F, D118A, and Y138F) and purified to homogeneity. The Y78F and D118A mutants were found to bind heme; however, Y138F did not. This result is consistent with the MCD data and indicates that Tyr138 is most likely the axial ligand to the heme in Dap1p.

Self-association energetics of an intact, full-length nuclear receptor: the B-isoform of human progesterone receptor dimerizes in the micromolar range.

We are focused on understanding the mechanisms underlying eukaryotic gene regulation, using the human progesterone receptor (PR) and its interactions with its DNA response elements as a model system. An understanding of PR function is complicated by the presence of two transcriptionally distinct isoforms, an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The two isoforms are identical except the B-receptor contains an additional 164 residues at its N-terminus. As a first step toward understanding the principles by which the two isoforms assemble at complex promoters, we examined the energetics of PR-B self-association using sedimentation velocity and sedimentation equilibrium methods. Full-length human PR-B was purified to 95% homogeneity from baculovirus-infected insect cells. Using a fluorescence hormone binding assay, we determined the purified protein to be highly active in its ability to bind ligand. Sedimentation velocity studies of hormone-bound PR-B at pH 8.0, 4 degrees C, and 50 mM NaCl demonstrate that it undergoes a concentration-dependent change in its sedimentation coefficient, existing as a 4.0S species at submicromolar concentrations but forming a 5.7S species at higher concentrations. These results strongly suggest that PR-B undergoes self-association in the micromolar range. This hypothesis was examined rigorously using sedimentation equilibrium. Global analysis of the sedimentation equilibrium data demonstrated that PR-B self-association was well described by a monomer-dimer model with a dimerization free energy of -7.2 +/- 0.7 kcal/mol. The role of NaCl in regulating PR-B dimerization was examined by carrying out sedimentation velocity and equilibrium studies under high salt conditions. At 300 mM NaCl, PR-B is exclusively monomeric in the micromolar range, thus revealing a significant ionic contribution to the assembly energetics. Further, the monomer sediments as a structurally homogeneous, but highly asymmetric, 4.0S species. Limited proteolysis of PR-B demonstrated that the hydrodynamic asymmetry is due in part to an extended, nonglobular conformation localized to the N-terminal region of PR-B. In contrast, the DNA binding domain (DBD) and hormone binding domain (HBD) exist as independent structural units, and the activation function N-terminal to the DBD (AF-1) shows moderate structure. These results represent the first rigorous analysis of the self-assembly energetics of an intact nuclear receptor and suggest that PR function is more complex than envisioned by traditional models.

Endometriosis umbilical con estudio de receptores hormonales / Umbilical Endometrosis with Study of Hormonal Receptors

La endometriosis es la presencia de tejido endometrial extrauterino, su localización habitual es intrapélvica, sin embargo puede localizarse a nivel cutáneo de forma espontánea o secundaria. La localización umbilical es la más frecuente de las endometriosis espontáneas alcanzando al 1 por ciento del total de las endometriosis. Se presenta el caso de una paciente con endometriosis umbilical espontánea, en la que se realiza un estudio inmunohistoquímico para receptores hormonales de estrógeno y de progesterona resultando ambos positivos (AU)

Serum p53 antibodies in correlation to other biological parameters of breast cancer.

Breast cancer is the second most frequent cancer of Thai women. Mutation of p53 is a common event in breast cancer. This alteration can result in cellular accumulation of p53 and may also found in serum p53 antibodies (p53-Abs). To clarify prognostic significance of these antibodies, we evaluated p53-Abs in 158 sera of patients with breast cancer. Thirty (19%) patients were found to have p53-Abs. The incidence of p53-Abs tended to be higher in patients with advanced disease group (stages III and IV) than patients with early disease group (stages I and II) (P=0.055). Strong correlations were found between the presence of p53-Abs and p53 protein expression (P<0.001) and lymph node status (P=0.021). The presence of p53-Abs was associated with lack of estrogen (ER) receptor expression (P=0.035) but was not related to progesterone receptor (PR) (P=0.567). In addition, there was a statistically significant correlation between p53-Abs and proliferation associated antigen Ki-67 (P=0.006), but no relation between c-erbB2 oncoprotein and p53-Abs was observed (P=0.112). Additionally, no correlation was noted between the presence of p53-Abs and serum carcinoembryonic antigen (CEA) or carbohydrate antigen (CA15-3). Our findings indicate that p53-Abs appears to be a promising new parameter to evaluate the cellular biology and prognosis of breast cancer.

The effects of endometrial scarification on uterine steroid receptors, bacterial flora and histological structure in the bitch.

Following laparotomy, the endometrium of six nulliparous Beagle bitches was scarified at the base of one uterine horn during early metoestrus, when the peripheral plasma P(4) concentration was >10 ng/ml; intrauterine swabs were taken at the same time for bacteriological culture. Twenty-one days later, a bilateral ovariohysterectomy was performed and segments of the scarified and non-scarified parts of the tubular genital tract removed; at the same time, swabs were taken from the uterine lumen. Tissue samples were collected and examined for histopathological structure, and the presence of nuclear oestrogen (ER) and progesterone (PR) receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score). All uterine swabs were sterile, and in three of the six bitches there were noticeable changes with distension of the uterine lumen with secretions and debris and distension of the endometrial gland ducts of the scarified uterine segment. There were no statistically significant differences in the H-scores of ER or PR between scarified and non-scarified segments, except for PR H-scores in the glandular epithelium where the values for the scarified were significantly higher than for the non-scarified endometrium (mean+/-S.E.M. is 129.9+/-22.8 versus 59.5+/-12.6; P<0.05). Thus, trauma can modify the structure of the endometrium and the characteristics of the PR. Whether changes in PR expression are involved in the pathogenesis of CEH/pyometra in the bitch could not be ascertained from this study.

Immunohistochemical localization of progesterone receptor isoforms in the chick pre-follicular ovary.

The distribution of progesterone receptor A and B isoforms in different cell types of the chick pre-follicular ovary was studied by immunohistochemistry. Newly hatched chicks were killed and the left ovary was removed, fixed and embedded in paraplast. Sections (5 microns thick) were made for the detection of progesterone receptor isoforms, using a technique of indirect immunoperoxidase. The results indicate that progesterone receptors were localized in the nuclei of germinal epithelium and germ cells of the ovarian cortex and in the interstitial and epithelial cells of the lacunar channels of the ovarian medulla. Undifferentiated cells did not present progesterone receptors. In all cell subpopulations progesterone receptor B was the predominantly expressed isoform. These data suggest that progesterone receptor isoforms are differentially expressed in the chick pre-follicular ovary and that progesterone effects in this tissue are mediated by the progesterone receptor B isoform.

Identification of the rat adrenal zona fasciculata/reticularis specific protein, inner zone antigen (IZAg), as the putative membrane progesterone receptor.

Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55-60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism.