Macaque Corpus Luteum Research Articles

Changes in immune cell distribution and their cytokine/chemokine production during regression of the rhesus macaque corpus luteum.

Our previous flow cytometry results demonstrated a significant increase in neutrophils, macrophages/monocytes, and natural killer (NK) cells in dispersed rhesus monkey corpora lutea (CL) after progesterone (P4) levels had fallen below 0.3 ng/ml for ≥3 days during the natural menstrual cycle. In this study, immunohistochemistry revealed the CD11b+ cells (neutrophils, macrophages/monocytes) present in the CL after luteal P4 synthesis ceased were distributed throughout the tissue. CD16+ cells (presumptive NK cells) were observed mainly near the vasculature in functional CL, until their numbers increased and they became widely distributed in regressing CL. To determine if the immune cells that enter luteal tissue during structural regression are functionally different from those that are present during peak function, CD11b+ or CD16+ populations were enriched from mid-late stage (functional) and regressing (days 1.8±0.3 postmenses) CL using antibody-conjugated magnetic microbeads. Flow cytometry analyses revealed the majority of CD11b+ cells expressed CD14, a protein mainly produced by macrophages/monocytes. The antibody-enriched and depleted fractions were cultured for 24 h, and the media then analyzed for the production of 29 cytokines/chemokines. From the mid-late CL, the CD11b+-enriched fraction produced three cytokines/chemokines, whereas CD16+-enriched cells only produced the chemokine CCL2. However, CD11b +-enriched cells isolated from regressed CL produced eight cytokines/chemokines. The CD16+-enriched cells isolated from regressing CL produced significant levels of only three cytokines. Thus, the CD11b+ cells that appear in the rhesus macaque CL after functional regression produce several cytokines/chemokines that likely play a role in orchestrating structural regression.

Dynamics of Immune Cell Types Within the Macaque Corpus Luteum During the Menstrual Cycle: Role of Progesterone.

The goal of the current study was to characterize the immune cell types within the primate corpus luteum (CL). Luteal tissue was collected from rhesus females at discrete intervals during the luteal phase of the natural menstrual cycle. Dispersed cells were incubated with fluorescently labeled antibodies specific for the immune cell surface proteins CD11b (neutrophils and monocytes/macrophages), CD14 (monocytes/macrophages), CD16 (natural killer [NK] cells), CD20 (B-lymphocytes), and CD3epsilon (T-lymphocytes) for analysis by flow cytometry. Numbers of CD11b-positive (CD11b(+)) and CD14(+) cells increased significantly 3 to 4 days after serum progesterone (P4) concentrations declined below 0.3 ng/ml. CD16(+) cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases and were 3-fold increased 3 to 4 days after serum P4 decreased to baseline levels. CD3epsilon(+) cells tended to increase 3 to 4 days after P4 decline. To determine whether immune cells were upregulated by the loss of luteotropic (LH) support or through loss of LH-dependent steroid milieu, monkeys were assigned to 4 groups: control (no treatment), the GnRH antagonist Antide, Antide plus synthetic progestin (R5020), or Antide plus the estrogen receptor agonists diarylpropionitrile (DPN)/propyl-pyrazole-triol (PPT) during the mid-late luteal phase. Antide treatment increased the numbers of CD11b(+) and CD14(+) cells, whereas progestin, but not estrogen, replacement suppressed the numbers of CD11b(+), CD14(+), and CD16(+) cells. Neither Antide nor steroid replacement altered numbers of CD3epsilon(+) cells. These data suggest that increased numbers of innate immune cells in primate CL after P4 synthesis declines play a role in onset of structural regression of primate CL.

Effects of steroid ablation and progestin replacement on the transcriptome of the primate corpus luteum during simulated early pregnancy

Previous microarray analyses indicated that a portion of the transcriptome in the macaque corpus luteum (CL) of the menstrual cycle was regulated indirectly by luteinizing hormone via the local actions of steroid hormones, notably progesterone (P). The current study was designed to investigate this concept in the CL of early pregnancy by analyzing chorionic gonadotrophin (CG)-regulated genes that are dependent versus independent of local steroid action. Exogenous human chorionic gonadotropin treatment simulating early pregnancy (SEP) began on Day 9 of the luteal phase in female rhesus monkeys with and without concurrent administration of the 3-β-hydroxysteroid dehydrogenase inhibitor trilostane (TRL) with or without the synthetic progestin R5020. Compared with SEP treatment alone, TRL altered 50 mRNA transcripts on Day 10, rising to 95 on Day 15 (P<0.05, ≥2-fold change in gene expression). Steroid-sensitive genes were validated; notably effects of steroid ablation and P replacement varied by day. Expression of some genes previously identified as P-regulated in the macaque CL during the menstrual cycle were not significantly altered by steroid ablation and P replacement during CG exposure in SEP. These data indicate that the majority of CG-regulated luteal transcripts are differentially expressed independently of local steroid actions. However, the steroid-regulated genes in the macaque CL may be essential during early pregnancy, based on previous reports that TRL treatment initiates premature structural regression of the CL during SEP. These data reinforce the concept that the structure, function and regulation of the rescued CL in early pregnancy differs from the CL of the menstrual cycle in primates.

Luteinizing hormone regulates inhibin-α subunit expression through multiple signaling pathways involving steroidogenic factor-1 and beta-catenin in the macaque corpus luteum

We employed different experimental model systems to define the role of GATA4, beta-catenin, and steroidogenic factor (SF-1) transcriptional factors in the regulation of monkey luteal inhibin secretion. Reverse transcription polymerase chain reactions and western blotting analyses show high expression of inhibin-α, GATA4, and beta-catenin in corpus luteum (CL) of the mid-luteal phase. Gonadotropin-releasing hormone receptor antagonist-induced luteolysis model suggested the significance of luteinizing hormone (LH) in regulating these transcriptional factors. Inducible cyclic AMP early repressor mRNA expression was detected in the CL and no change was observed in different stages of CL. Following amino acid sequence analysis, interaction between SF-1 and beta-catenin in mid-stage CL was verified by reciprocal co-immunoprecipitation experiments coupled to immunoblot analysis. Electrophoretic mobility shift analysis support the role of SF-1 in regulating luteal inhibin-α expression. Our results suggest a possible multiple crosstalk of Wnt, cAMP, and SF-1 in the regulation of luteal inhibin secretion.

Liver X Receptor Modulation of Gene Expression Leading to Proluteolytic Effects in Primate Luteal Cells1

The expressions of genes involved in cholesterol efflux increase, whereas those involved in extracellular cholesterol uptake decrease, during spontaneous functional regression of the primate corpus luteum (CL). This may result from liver x receptor (LXR) alpha (official symbol NR1H3) and/or beta (official symbol NR1H2) control of luteal gene transcription, because these nuclear receptor superfamily members are key regulators of cellular cholesterol homeostasis. Therefore, studies were conducted to assess endogenous LXR ligands in the primate CL through the luteal phase, and to determine the effect of synthetic or natural LXR ligands on cholesterol efflux and uptake in functional primate luteal cells. Using high-performance liquid chromatography tandem mass spectrometry, three LXR ligands were identified and quantified in the rhesus macaque CL, including 22R-hydroxycholesterol (22ROH), 27-hydroxycholesterol (27OH), and desmosterol. Levels of 22ROH paralleled serum progesterone concentrations, whereas mean levels of 27OH tended to be higher following the loss of progesterone synthesis. Desmosterol was present throughout the luteal phase. Functional macaque luteal cells treated with the synthetic LXR agonist T0901317 or physiologically relevant concentrations of the endogenous luteal ligands 22ROH, 27OH, and desmosterol had increased expression of various known LXR target genes and greater cholesterol efflux. Additionally, T0901317 reduced low-density lipoprotein receptor protein and extracellular low-density lipoprotein uptake, whereas 27OH decreased low-density lipoprotein receptor protein, most likely via a posttranslational mechanism. Collectively, these data support the hypothesis that LXR activation causes increased cholesterol efflux and decreased extracellular cholesterol uptake. In theory, these effects could deplete the primate CL of cholesterol needed for steroidogenesis, ultimately contributing to functional regression.

Analysis of microarray data from the macaque corpus luteum; the search for common themes in primate luteal regression.

The factors and processes involved in regression of the primate corpus luteum (CL) are complex and not fully understood. Systemic identification of those genes that are differentially expressed utilizing macaque model systems of luteal regression could help clarify some of the important molecular events involved in loss of primate luteal structure and function during luteolysis. In addition, examining gene pathways involved in luteal regression may help elucidate novel approaches for overcoming infertility or designing ovary-based contraceptives. This review provides an overview of the current published microarray experiments evaluating the transcriptome of the macaque CL, and compares and contrasts the data from spontaneous, GnRH antagonist and prostaglandin F2α-induced luteal regression. In addition, further uses of these databases are discussed, as well as limitations of both array technology and the rhesus macaque genome array.

The reverse cholesterol transport system as a potential mediator of luteolysis in the primate corpus luteum

The cessation of progesterone (P(4)) production (i.e. functional regression), arguably the key event in luteolysis of the primate corpus luteum (CL), is poorly understood. Previously, we found that genes encoding proteins involved in cholesterol uptake decreased, while those involved in cholesterol efflux (reverse cholesterol transport, RCT) increased in expression during spontaneous functional regression of the rhesus macaque CL, thereby potentially depleting the cholesterol reserves needed for steroidogenesis. Therefore, a comprehensive analysis of the components necessary for RCT was performed. RCT components were expressed (mRNA and/or protein) in the macaque CL including cholesterol sensors (liver X receptors alpha or NR1H3; and beta or NR1H2), efflux proteins (ATP-binding cassette subfamilies A1 (ABCA1) and G1), acceptors (apolipoproteins A1 or APOA1; and E or APOE), and plasma proteins facilitating high-density lipoprotein formation (lecithin:cholesterol acyltransferase or LCAT; phospholipid transfer protein or PLTP). ABCA1, APOE, PLTP, and NR1H3 increased, while lipoprotein receptors decreased, in expression (mRNA and/or protein) through the period of functional regression. The expression of APOA1 and APOE, as well as NR1H3, was greatest in the CL and tissues involved in regulating cholesterol homeostasis. Immunolocalization studies revealed that RCT proteins and lipoprotein receptors were expressed in large luteal cells, which possess intracellular cholesterol reserves during periods of P(4) synthesis. Lipid staining revealed changes in luteal cholesterol ester/lipid distribution that occurred following functional regression. These results indicate that decreased cholesterol uptake and increased RCT may be critical for the initiation of primate luteolysis by limiting intracellular cholesterol pools required for steroidogenesis.

Existence of the lymphatic system in the primate corpus luteum.

To date, there have been no detailed studies on the lymphatic system in the primate corpus luteum (CL); early reports suggested that the presence of this "secondary circulation" in luteal tissue is species-dependant. Therefore, studies were designed to determine if (a) lymphatic vessels exist, and (b) recently discovered lymphangiogenic factors and their receptor are expressed in the macaque CL during the menstrual cycle. Immunohistochemistry (IHC) detected the lymphatic endothelial cell marker, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), in some endothelial cells and vessels within the ovarian stroma and theca layer of preovulatory follicles and in the CL. Dual fluorescent IHC demonstrated that LYVE1 co-localized with another lymphatic endothelial cell marker D2-40, but a blood vascular endothelial cell marker (von Willebrand Factor, VWF) was in different cells. The numbers and staining intensity of LYVE1-positive cells in the CL appeared to increase from early to mid luteal phase, and remained elevated thereafter. RT-PCR detected cDNA fragments for mRNAs encoding VEGFC, FIGF, and their receptor FLT4 in CL. Real-time PCR analyses revealed similar patterns of VEGFC and FLT4 expression during the luteal lifespan; mRNA levels increased (p < 0.05) from early to mid luteal phase and decreased (p < 0.05) by late luteal phase. In contrast, FIGF levels were elevated initially, declined (p < 0.05) at mid luteal phase, and then increased (p < 0.05) to very late luteal phase. The data strongly suggest that lymphatic vessels are present in the primate CL, and that the VEGFC/FIGF-FLT4 system regulates lymphangiogenesis and luteal structure-function during the menstrual cycle.

Liver X Receptor Ligands in the Rhesus Macaque Corpus Luteum Throughout the Luteal Phase: Implications for Reverse Cholesterol Transport Activity.

Liver X Receptor Ligands in the Rhesus Macaque Corpus Luteum Throughout the Luteal Phase: Implications for Reverse Cholesterol Transport Activity.

Dynamic Changes in Gene Expression that Occur during the Period of Spontaneous Functional Regression in the Rhesus Macaque Corpus Luteum

Luteolysis of the corpus luteum (CL) during nonfertile cycles involves a cessation of progesterone (P4) synthesis (functional regression) and subsequent structural remodeling. The molecular processes responsible for initiation of luteal regression in the primate CL are poorly defined. Therefore, a genomic approach was used to systematically identify differentially expressed genes in the rhesus macaque CL during spontaneous luteolysis. CL were collected before [d 10-11 after LH surge, mid-late (ML) stage] or during (d 14-16, late stage) functional regression. Based on P4 levels, late-stage CL were subdivided into functional-late (serum P4 > 1.5 ng/ml) and functionally regressed late (FRL) (serum P4 < 0.5 ng/ml) groups (n = 4 CL per group). Total RNA was isolated, labeled, and hybridized to Affymetrix genome microarrays that contain elements representing the entire rhesus macaque transcriptome. With the ML stage serving as the baseline, there were 681 differentially expressed transcripts (>2-fold change; P < 0.05) that could be categorized into three primary patterns of expression: 1) increasing from ML through FRL; 2) decreasing from ML through FRL; and 3) increasing ML to functional late, followed by a decrease in FRL. Ontology analysis revealed potential mechanisms and pathways associated with functional and/or structural regression of the macaque CL. Quantitative real-time PCR was used to validate microarray expression patterns of 13 genes with the results being consistent between the two methodologies. Protein levels were found to parallel mRNA profiles in four of five differentially expressed genes analyzed by Western blot. Thus, this database will facilitate the identification of mechanisms involved in primate luteal regression.

Expression and regulation of tumor necrosis factor (TNF) and TNF‐receptor family members in the macaque corpus luteum during the menstrual cycle

Members of the tumor necrosis factor (TNF)-receptor (R) family may be involved in the tissue remodeling that occurs in the primate corpus luteum (CL) during development and regression. As a first step towards addressing this issue, studies assessed TNF ligand-R expression and regulation in CL collected from monkeys during the early (ECL, Days 3-5), mid (MCL, Days 7-8), mid-late (MLCL, Days 10-11), late (LCL, Days 14-16), and very late (VLCL, menses) luteal phase of the menstrual cycle. CL were also collected after gonadotropin and/or steroid ablation and replacement (with hLH and the progestin R5020) for 3 days at mid-late luteal phase. TNF-alpha, -beta, FAS ligand (FASL), and TNF-R1 mRNA levels were two- to sixfold greater (P < 0.05) at the MLCL or LCL phase as compared to earlier (ECL, MCL). In contrast, TNF-R2 and FAS mRNA levels did not change during the luteal phase. Immunohistochemical staining for TNF-beta, TNF-R1, TNF-R2, FAS, and FASL was observed in luteal cells, whereas only TNF-beta staining was observed in endothelial cells. Several TNF-R components were influenced by LH and/or steroid ablation; notably, steroid ablation reduced (P < 0.05) luteal TNF-alpha, but not TNF-beta, mRNA levels, which was prevented by progestin treatment. In contrast, steroid ablation increased (P < 0.05) luteal cell immunostaining for FAS and FASL, which was reduced by progestin treatment. Thus, several members of the TNF R-ligand family are expressed in the primate CL in an LH- and/or progestin-dependent manner. Peak expression in the late luteal phase may signify a role for the TNF-R system in death receptor-mediated apoptosis during luteolysis.

Prostaglandin Synthesis, Metabolism, and Signaling Potential in the Rhesus Macaque Corpus Luteum throughout the Luteal Phase of the Menstrual Cycle

Prostaglandins in the corpus luteum (CL) reportedly serve as luteotropic and luteolytic agents. Based mainly on studies conducted in domesticated animals and rodents, prostaglandin E2 (PGE2) is generally considered a luteotropic factor, whereas uterine-derived prostaglandin F2alpha (PGF2alpha) initiates luteolysis. However, the role of prostaglandins in regulating primate luteal structure-function is poorly understood. Therefore, a comprehensive analysis of individual mRNA or proteins that are involved in PGE2 and PGF2alpha biosynthesis, metabolism, and signaling was performed using CL obtained at distinct stages of the luteal life span during the menstrual cycle in rhesus monkeys. Peak levels of proteins involved in PGE2 synthesis (prostaglandin-endoperoxide synthase 2, microsomal PGE2 synthase-1) and signaling (PGE2 receptor 3) occurred during periods corresponding to development and maintenance of the primate CL. Immunohistochemistry studies indicated that large luteal cells express PGE2 synthesizing and signaling proteins. Expression of PGE2 synthesizing and signaling proteins significantly decreased preceding the period of functional regression of the CL, which also coincided with increasing levels of PGF2alpha receptor protein expression within the large luteal cells. Moreover, significant levels of mRNA expression for several aldoketo reductase family members that synthesize PGF2alpha from other prostaglandins were observed throughout the rhesus macaque luteal phase, thus supporting the possibility of intraluteal PGF2alpha production. Collectively, our results indicate that there may be intraluteal synthesis and signaling of PGE2 during development and maintenance of the primate CL, followed by a shift to intraluteal PGF2alpha synthesis and signaling as the CL nears the time of luteolysis.

Systematic Determination of Differential Gene Expression in the Primate Corpus Luteum during the Luteal Phase of the Menstrual Cycle

The molecular and cellular processes required for development, function, and regression of the primate corpus luteum (CL) are poorly defined. We hypothesized that there are dynamic changes in gene expression occurring during the CL life span, which represent proteins and pathways critical to its regulation. Therefore, a genomic approach was utilized to systematically identify differentially expressed genes in the rhesus macaque CL during the luteal phase of natural menstrual cycles. CL were collected between d 3-5 (early stage), d 7-8 (mid), d 10-12 (mid-late), d 14-16 (late), or d 18-19 (very-late) after the midcycle LH surge. From the early through very-late stages, 3234 transcripts were differentially expressed, with 879 occurring from the early through late stages that encompass the processes of luteinization, maintenance, and functional regression. To characterize gene changes most relevant to these processes, ontology analysis was performed using the list of 879 differentially expressed transcripts. Four main groups of related genes were identified with relevance to luteal physiology including: 1) immune function; 2) hormone and growth factor signaling; 3) steroidogenesis; and 4) prostaglandin biosynthesis, metabolism, and signaling. A subset of genes representing each of the four major categories was selected for validation of microarray results by quantitative real-time PCR. Results in mRNA levels were similar between the two methodologies for 17 of 18 genes. Additionally, protein levels for three genes were determined by Western blot analysis to parallel mRNA levels. This database will facilitate the identification of many novel or previously underappreciated pathways that regulate the structure and function of the primate CL.

Expression of Prostasin Serine Protease and Protease Nexin-1 (PN-1) in Rhesus Monkey Ovary During Menstrual Cycle and Early Pregnancy

Serine proteases and their cognate serpin-class inhibitors are involved in the controlled proteolytic events during follicular development, ovulation, formation, and maintenance of the corpus luteum (CL). In this study, we investigated the expression patterns of prostasin serine protease and protease nexin-1 (PN-1), a serine protease inhibitor also called serpin-E2, in rhesus monkey ovaries during the menstrual cycle and early pregnancy, by using in situ hybridization and immunohistochemistry. Expression of prostasin was localized in oocyte, granulosa cells, and/or theca cells of early antral follicles and antral follicles, with high levels observed in preovulatory follicles. Prostasin was also localized at high levels of abundance in the CL during the menstrual cycle and early pregnancy. During the menstrual cycle, PN-1 was coordinately localized with prostasin in oocytes, granulosa cells, and theca cells of antral follicles and preovulatory follicles and in the CL. In addition, the PN-1 expression level in macaque CL during early pregnancy increased as pregnancy proceeded. We propose that prostasin may be involved in follicular development, ovulation, and CL formation, whereas PN-1 may be present to regulate the proteolysis in these processes.

SYSTEMATIC DETERMINATION OF DIFFERENTIAL GENE EXPRESSION IN THE PRIMATE CORPUS LUTEUM DURING THE LUTEAL PHASE OF THE MENSTRUAL CYCLE

The molecular and cellular processes required for development, function, and regression of the primate corpus luteum (CL) are poorly defined. We hypothesized that there are dynamic changes in gene expression occurring during the CL life span, which represent proteins and pathways critical to its regulation. Therefore, a genomic approach was utilized to systematically identify differentially expressed genes in the rhesus macaque CL during the luteal phase of natural menstrual cycles. CL were collected between d 3-5 (early stage), d 7-8 (mid), d 10-12 (mid-late), d 14-16 (late), or d 18-19 (very-late) after the midcycle LH surge. From the early through very-late stages, 3234 transcripts were differentially expressed, with 879 occurring from the early through late stages that encompass the processes of luteinization, maintenance, and functional regression. To characterize gene changes most relevant to these processes, ontology analysis was performed using the list of 879 differentially expressed transcripts. Four main groups of related genes were identified with relevance to luteal physiology including: 1) immune function; 2) hormone and growth factor signaling; 3) steroidogenesis; and 4) prostaglandin biosynthesis, metabolism, and signaling. A subset of genes representing each of the four major categories was selected for validation of microarray results by quantitative real-time PCR. Results in mRNA levels were similar between the two methodologies for 17 of 18 genes. Additionally, protein levels for three genes were determined by Western blot analysis to parallel mRNA levels. This database will facilitate the identification of many novel or previously underappreciated pathways that regulate the structure and function of the primate CL.

Dynamic expression of epoxyeicosatrienoic acid synthesizing and metabolizing enzymes in the primate corpus luteum

Epoxyeicosatrienoic acids (EpETrEs), produced from arachidonic acid via cytochrome P450 (CYP) epoxygenases, regulate inflammation, angiogenesis, cellular proliferation, ion transport and steroidogenesis. EpETrE actions are regulated through their metabolism to diols (dihydroxyeicosatrienoic acids; DiHETrE) via the enzyme soluble epoxide hydrolase (EPHX2). We set out to determine, therefore, whether EpETrE generating (epoxygenases CYP2C8, 2C9, 2C19, 2J2, 1A2 and 3A4) and metabolizing (EPHX2) enzymes are expressed in the primate corpus luteum (CL). CL were isolated from rhesus macaques during the early (day 3-5 post-LH surge), mid (day 6-8), mid-late (day 10-12), late (day 14-16) and very-late (day 17-19: menses) luteal phase of natural menstrual cycles. EPHX2 mRNA levels peaked in mid-late CL (5-fold when compared with early CL, P<0.05) and remained elevated in the late CL. Ablation of pituitary LH secretion and luteal steroid synthesis significantly reduced (P<0.05) EPHX2 mRNA levels in the mid-late CL, with progestin replacement being insufficient to restore its level of expression to control values. EPHX2 protein was localized to large and small luteal cells, as well as vascular endothelial cells. The EpETrE-generating CYP epoxygenase 2J2, 2C9 and 3A4 genes were also expressed in the macaque CL. While CYP2J2 mRNA levels did not significantly change through the luteal phase, CYP2C9 and CYP3A4 levels were significantly (P<0.05) higher in the mid-late phase when compared with the early phase. CYP2C9, 2J2 and 3A4 proteins were each localized to the large luteal cells, with 2C9 and 2J2 also being present in the small luteal, stromal and endothelial cells. These studies demonstrate for the first time that an EpETrE generating and metabolizing system exists in the primate CL, with the latter being regulated by LH and steroid hormone(s).

Dynamic Expression and Regulation of the Corticotropin-Releasing Hormone/Urocortin-Receptor-Binding Protein System in the Primate Ovary during the Menstrual Cycle

Microarray analysis discovered that mRNA for CRH-binding protein (CRHBP) increased significantly in the primate corpus luteum (CL) after LH withdrawal. Our objective was to determine whether other components of the CRH/urocortin-receptor-binding protein (UCN-R-BP) system are expressed in the CL during the menstrual cycle and regulated by LH. CL were collected from monkeys during the early (d 3-5 after the LH surge) to very late (d 18-19) luteal phase and from controls or animals receiving GnRH antagonist (Antide, 3 mg/kg body weight). CRH/UCN-R-BP system components were quantitated for mRNA levels by real-time PCR and analyzed for protein localization by immunohistochemistry. All genes encoding the CRH/UCN-R-BP system, except for UCN3, were expressed in the CL. CRH mRNA levels did not change during the luteal phase, whereas expression of UCN, UCN2, CRHR1, and CRHR2 was maximal at early or mid luteal phase before declining (P < 0.05) at the later stages. CRHBP mRNA levels were lowest at mid and increased (P < 0.05) in the late luteal phase. Suppressing gonadotropin secretion reduced UCN2 (P < 0.05) and increased CRHBP (P < 0.05) mRNA levels, without altering the expression of other ligands or receptors. CRH, UCN, UCN2 and their receptors were localized to the granulosa-lutein cells of the CL, whereas CRHBP was limited to the theca and theca-lutein cells of the preovulatory follicle and CL. A local CRH/UCN-R-BP system exists in the macaque CL that is dynamically expressed and LH regulated during the luteal phase of the menstrual cycle. Ligand-receptor activity may regulate luteal structure-function, at this point in an unknown manner, in primates.

First Evidence for the Dynamic Expression and Regulation of the Corticotropin Releasing Hormone-Receptor-Binding Protein (CRH-R-BP) System in the Primate Corpus Luteum (CL) of the Menstrual Cycle

Since recent microarray analyses (Mol Hum Reprod 11: 151-9, 2005) indicated that CRHBP mRNA was expressed and up-regulated in the macaque CL following gonadotropin withdrawal, further studies were designed to test the hypotheses that (a) other components of the CRH-R-BP system are dynamically expressed in the primate CL during the luteal lifespan in the menstrual cycle, and (b) their expression is regulated by luteinizing hormone (LH).

Dynamic Expression of Caspase-2, -3, -8, and -9 Proteins and Enzyme Activity, But Not Messenger Ribonucleic Acid, in the Monkey Corpus Luteum during the Menstrual Cycle

Studies were designed to determine whether: 1) changes in caspase expression or activity occur in the macaque corpus luteum (CL) during its lifespan in the menstrual cycle, and 2) LH acting directly or via ovarian steroids regulates luteal caspases. Caspase-2, -3, -8, and -9 mRNAs were detectable by semiquantitative RT- or real time-PCR in CL, but levels did not differ between the early, mid, mid-late, late, and very-late luteal phases. Immunostaining for caspase-2 and -3 proteins was observed in luteal cells and appeared to peak by mid to mid-late stage. Enzyme activity for caspase-2, -3, -8, and -9 increased (P < 0.05) by mid-late stage, and then declined by the very-late stage. Treatment with GnRH antagonist + LH at the mid-late stage increased caspase-2, -8, and -9, but not -3, activity, compared with controls. Coadministration of a steroid synthesis inhibitor (trilostane) with GnRH antagonist + LH reduced (P < 0.05) caspase-2, -8, and -9 activity. Progestin (R5020) replacement during trilostane treatment did not restore caspase activity. Thus, initiator and effector caspases are present during CL development and regression in the menstrual cycle. The increased caspase activity at mid-late stage suggests that apoptosis is involved in early luteolysis in primates. Gonadotropin, perhaps via local steroids, modulates initiator caspases in the primate CL.

Expression of gelatinases and tissue inhibitors of metalloproteinases in the rhesus monkey (Macaca mulatta) corpus luteum

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play important roles in the formation and regression of corpus luteum (CL). This study is to investigate the expression of gelatinases (MMP-2, -9) and TIMPs in the rhesus monkey CL in both early and late luteal phases and during the early stages of pregnancy. Ovaries were collected from regularly cycling rhesus monkey at D5 and D15 following ovulation and at D12, D18 and D26 of pregnancy.In situ hybridization revealed that in the CL MMP-2 mRNA was expressed during both formation and regression, while MMP-9 mRNA was mainly localized in the late luteal phase. Reduction of MMP-2, -9 transcripts in the CL was observed during pregnancy. MMP-2 mRNA in the CL reduced to an undetectable level at D26 of pregnancy. TIMP-1 mRNA was highly expressed in the CL in both early and late luteal phases and persisted throughout the early stages of pregnancy. Strong signal for TIMP-2 mRNA was also detected in both luteal phases, and the level of TIMP-2 mRNA gradually increased with the progresses of pregnancy. No TIMP-3 mRNA was detected in the macaque CL in this study. In conclusion, these results suggest that MMP-2, -9 and TIMP-1, -2 may have functional roles in rhesus monkey CL. Coordinated expression of MMP-2, -9 and TIMP-2 may play a role in the maintaining of luteal function during early pregnancy. The unchanged expression pattern of TIMP-1 indicates that it may have other functions in the primate CL than inhibition of MMPs.