Insights from two independent transcriptomic studies of the bovine corpus luteum during pregnancy.

Several recent studies have used transcriptomics to investigate luteal changes during the maternal recognition of the pregnancy period in ruminants. Although these studies have contributed to our understanding of luteal function during early pregnancy, few attempts have been made to integrate information across these studies and distinguish key luteal transcripts or functions that are repeatably identified across multiple studies. Therefore, in this study, two independent studies of the luteal transcriptome during early pregnancy were combined and compared. In the first study, corpora lutea (CL) from day 20 of pregnancy were compared with CL collected on day 14 of pregnancy, prior to embryonic signaling. The cattle were nonlactating. In the second study, CL from day 20 of pregnancy were compared with CL collected from day 20 cyclic cattle that had been confirmed as not yet undergoing luteal regression. These were lactating cattle. Three methods were used to compare these two datasets, to identify key luteal regulators. In the first method, all transcripts with Benjamini-Hochberg-adjusted P-value (Q value) < 0.05 in both datasets were considered. This yielded 22 transcripts, including several classical interferon-stimulated genes, as well as regulators of transforming growth factor-beta (TGFB) and latent TGFB-binding proteins (LTBP)1 and 2. In the second, less conservative method, all transcripts with P < 0.01 and changed in the same direction in both datasets were considered. This yielded an additional 20 transcripts that were not identified in the first analysis, for a total of 42 common transcripts. These transcripts were regulators of functions such as inflammatory balance and matrix remodeling. In the third method, transcripts with Q < 0.10 were subject to pathway analysis, and common pathways were identified. Retinoic acid signaling and classical interferon signaling pathways were identified with this method. Finally, regulation by interferon tau (IFNT) was investigated. Among the 42 transcripts identified, 32 were regulated by IFNT in cultured luteal cells (Q < 0.05). Among those not regulated by IFNT were LTBP1 and 2, which are TGFB-binding proteins. In summary, common transcripts from two studies of the luteal transcriptome during early pregnancy were combined and shared changes were identified. This not only generated a list of potential key luteal regulators, which were mostly IFNT regulated, but also included transcripts not regulated by IFNT, including LTBP1 and 2.

Reproductive efficiency is necessary for the financial and environmental sustainability of cattle production. A critical component of this efficiency is the maintenance of pregnancy. The corpus luteum (CL) is a transient ovarian endocrine gland that produces progesterone­the hormone that maintains pregnancy in all mammals. With each new reproductive cycle, a new CL is formed from the remnant of the ovulatory follicle, and at the end of each cycle, in the absence of a pregnancy, the CL regresses in response to prostaglandin F2α from the uterus. In contrast, in the presence of a pregnancy, the CL is rescued from regression by the embryo, in a process known as maternal recognition of pregnancy. The embryo is known to alter uterine function, but its effect on the CL has remained a mystery until recent years. In this study, we compared two independent studies of global changes in the CL of pregnancy, to identify the most important luteal changes that occur during early pregnancy. We confirmed evidence for embryonic signaling to the CL and generated a list of candidate genes that are the likeliest regulators of changes to luteal function in early pregnancy. This list includes regulators of tissue integrity and inflammation.

History, insights, and future perspectives on studies into luteal function in cattle.

The corpus luteum (CL) forms following ovulation from the remnant of the Graafian follicle. This transient tissue produces critical hormones to maintain pregnancy, including the steroid progesterone. In cattle and other ruminants, the presence of an embryo determines if the lifespan of the CL will be prolonged to ensure successful implantation and gestation, or if the tissue will undergo destruction in the process known as luteolysis. Infertility and subfertility in dairy and beef cattle results in substantial economic loss to producers each year. In addition, this has the potential to exacerbate climate change because more animals are needed to produce high-quality protein to feed the growing world population. Successful pregnancies require coordinated regulation of uterine and ovarian function by the developing embryo. These processes are often collectively termed "maternal recognition of pregnancy." Research into the formation, function, and destruction of the bovine CL by the Northeast Multistate Project, one of the oldest continuously funded Hatch projects by the USDA, has produced a large body of evidence increasing our knowledge of the contribution of ovarian processes to fertility in ruminants. This review presents some of the seminal research into the regulation of the ruminant CL, as well as identifying mechanisms that remain to be completely validated in the bovine CL. This review also contains a broad discussion of the roles of prostaglandins, immune cells, as well as mechanisms contributing to steroidogenesis in the ruminant CL. A triadic model of luteolysis is discussed wherein the interactions among immune cells, endothelial cells, and luteal cells dictate the ability of the ruminant CL to respond to a luteolytic stimulus, along with other novel hypotheses for future research.

The corpus luteum (CL) forms on the ovary from the cellular remnants of the follicle following ovulation. The function of the CL is to produce progesterone that is required for successful pregnancy. In the absence of an embryo or sufficient embryonic signaling, the uterus will release a prostaglandin that kills the CL in a process called luteolysis. Therefore, the CL and the embryo share a symbiotic relationship, each requiring the other to be healthy and functional for survival. The Northeast Multistate Project, one of the oldest in the nation, has produced a large body of evidence that has enhanced our understanding of how the CL functions, its regulation, and the impact of ovarian activity on fertility of cattle. This review highlights some of the important advances made in the understanding of the ruminant CL.

A Synopsis of the NE1727 Multistate Project Collection in the Journal of Animal Science.

This article provides a synopsis of the collection of papers provided by participants of the NE1727 Multistate project. Five reviews and thirteen primary research articles are included that provide contributions the group has made to understanding the role of the corpus luteum in reproduction, describing how the ovary influences fertility, delineating mechanisms controlling oocyte quality and early embryo development, and exploring new reproductive management schemes.

Form of Supplemental Selenium Affects the Expression of mRNA Transcripts Encoding Selenoproteins, and Proteins Regulating Cholesterol Uptake, in the Corpus Luteum of Grazing Beef Cows.

Selenium (Se)-deficient soils necessitate supplementation of this mineral to the diet of forage-grazing cattle. Functionally, Se is incorporated into selenoproteins, some of which function as important antioxidants. We have previously shown that the source of supplemental Se; inorganic (sodium selenite or sodium selenate; ISe), organic (selenomethionine or selenocysteine; OSe) or 1:1 mix of ISe and OSe (MIX), provided to Angus-cross cows affects concentrations of progesterone (P4) during the early luteal phase of the estrous cycle. In this study, we sought to investigate (1) the effect of form of Se on the expression of mRNA encoding selenoproteins in the corpus luteum (CL), and (2) whether this previously reported MIX-induced increase in P4 is the result of increased luteal expression of key steroidogenic transcripts. Following a Se depletion and repletion regimen, 3-year-old, non-lactating, Angus- cross cows were supplemented with either ISe as the industry standard, or MIX for at least 90 days, with the CL then retrieved on Day 7 post-estrus. Half of each CL was used for analysis of targeted mRNA transcripts and the remainder was dissociated for culture with select agonists. The expression of three selenoprotein transcripts and one selenoprotein P receptor was increased (p < 0.05), with an additional five transcripts tending to be increased (p < 0.10), in cows supplemented with MIX versus ISe. In cultures of luteal cells, hCG-induced increases in P4 (p < 0.05) were observed in CL obtained from ISe-supplemented cows. The abundance of steroidogenic transcripts in the CL was not affected by the form of Se, however, the abundance of mRNA encoding 2 key transcripts regulating cholesterol availability (Ldlr and Hsl) was increased (p < 0.05) in MIX-supplemented cows. Overall, the form of Se provided to cows is reported to affect the expression of mRNA encoding several selenoproteins in the CL, and that the form of Se-induced effects on luteal production of P4 appears to be the result of changes in cholesterol availability rather than a direct effect on the expression of steroidogenic enzymes within the CL.

Roadmap to pregnancy during the period of maternal recognition in the cow: Changes within the corpus luteum associated with luteal rescue.

A viable corpus luteum (CL) producing an adequate amount of progesterone is absolutely essential for establishment and maintenance of pregnancy. One function of embryonic signaling to the mother is to ensure that the CL is maintained. In ruminants, the secretion of uterine prostaglandin F2alpha (PGF2A) is the signal that initiates luteolysis. Despite many studies to determine if PGF2A secretion from the uterus is altered in early pregnancy, conflicting interpretations have led to controversy regarding the exact mechanisms by which maternal recognition of pregnancy is achieved. In addition to alteration of uterine prostaglandin metabolism, changes within the CL itself may facilitate the establishment of a successful pregnancy. These changes include alteration of luteal blood flow, prostaglandin metabolism, sensitivity to prostaglandin actions, increased steroidogenic capacity, significant intracellular molecular changes and modification of the immune cells that are within the CL. Whether these intraluteal changes are necessary to establish pregnancy is undetermined. The focus of this review will be to provide a brief historical perspective on the utero-ovarian relationships that regulate luteal lifespan and review current knowledge of the CL of pregnancy in sheep and cattle.

Luteal Lipids Regulate Progesterone Production and May Modulate Immune Cell Function During the Estrous Cycle and Pregnancy.

Although the corpus luteum (CL) contains high concentrations of lipid in the form of steroid hormone precursors and prostaglandins, little is known about the abundance or function of other luteal lipid mediators. To address this, 79 lipid mediators were measured in bovine CL, using ultra performance liquid chromatography-tandem mass spectrometry. CL from estrous cycle days 4, 11, and 18 were compared and, separately, CL from days 18 of the estrous cycle and pregnancy were compared. Twenty-three lipids increased as the estrous cycle progressed (P < 0.05), with nine increasing between days 4 and 11 and fourteen increasing between days 4 and 18. Overall, this indicated a general upregulation of lipid mediator synthesis as the estrous cycle progressed, including increases in oxylipins and endocannabinoids. Only 15-KETE was less abundant in the CL of early pregnancy (P < 0.05), with a tendency (P < 0.10) for four others to be less abundant. Notably, 15-KETE also increased between estrous cycle days 4 and 18. Ingenuity Pathway Analysis (IPA, Qiagen) indicated that functions associated with differentially abundant lipids during the estrous cycle included leukocyte activation, cell migration, and cell proliferation. To investigate changes in CL during maternal recognition of pregnancy, this lipid dataset was integrated with a published dataset from mRNA profiling during maternal recognition of pregnancy. This analysis indicated that lipids and mRNA that changed during maternal recognition of pregnancy may regulate some of the same functions, including immune cell chemotaxis and cell-cell communication. To assess effects of these lipid mediators, luteal cells were cultured with 5-KETE or 15-KETE. One ng/mL 5-KETE reduced luteal progesterone on day 1 of culture, only in the absence of luteinizing hormone (LH), while 1 ng/mL 15-KETE induced progesterone only in the presence of LH (10 ng/mL). On day 7 of culture, 0.1 ng/mL 15-KETE reduced prostaglandin (PG)F2A-induced inhibition of LH-stimulated progesterone production, while 1 ng/mL 15-KETE did not have this effect. Overall, these data suggest a role for lipid mediators during luteal development and early pregnancy, as regulators of steroidogenesis, immune cell activation and function, intracellular signaling, and cell survival and death.

Molecular profiling demonstrates modulation of immune cell function and matrix remodeling during luteal rescue†.

The corpus luteum (CL) is essential for maintenance of pregnancy in all mammals and luteal rescue, which occurs around day 16-19 in the cow, is necessary to maintain luteal progesterone production. Transcriptomic and proteomic profiling were performed to compare the day 17 bovine CL of the estrous cycle and pregnancy. Among mRNA and proteins measured, 140 differentially abundant mRNA and 24 differentially abundant proteins were identified. Pathway analysis was performed using four programs. Modulated pathways included T cell receptor signaling, vascular stability, cytokine signaling, and extracellular matrix remodeling. Two mRNA that were less in pregnancy were regulated by prostaglandin F2A in culture, while two mRNA that were greater in pregnancy were regulated by interferon tau. To identify mRNA that could be critical regulators of luteal fate, the mRNA that were differentially abundant during early pregnancy were compared to mRNA that were differentially abundant during luteal regression. Eight mRNA were common to both datasets, including mRNA related to regulation of steroidogenesis and gene transcription. A subset of differentially abundant mRNA and proteins, including those associated with extracellular matrix functions, were predicted targets of differentially abundant microRNA (miRNA). Integration of miRNA and protein data, using miRPath, revealed pathways such as extracellular matrix-receptor interactions, abundance of glutathione, and cellular metabolism and energy balance. Overall, this study has provided a comprehensive profile of molecular changes in the corpus luteum during maternal recognition of pregnancy and has indicated that some of these functions may be miRNA-regulated.

Applications of large-scale molecular profiling techniques to the study of the corpus luteum.

The corpus luteum (CL) is vital for the establishment and maintenance of pregnancy. Throughout the history of luteal biology, cutting-edge technologies have been used to develop a thorough understanding of the functions of specific luteal cell types, the signaling pathways that result in luteal cell stimulation or demise, and the molecules that regulate specific functions of luteal cells. The advent of large- scale profiling technologies such as transcriptomics, proteomics, and metabolomics, has brought with it an interest in discovering novel regulatory molecules that may provide targets for manipulation of luteal function or lifespan. Although the work to date is limited, transcriptomics have been effectively used to provide a global picture of changes in mRNA that relate to luteal development, steroidogenesis, luteolysis or luteal rescue. Some studies have been reported that profile microRNA (miRNA) and proteins, and although not yet published, metabolomics analyses of the CL have been undertaken. Thus far, these profiling studies seem to largely confirm earlier findings using targeted approaches, although previously unstudied molecules have also come to light as important luteal regulators. These molecules can then be studied using traditional mechanistic techniques. Use of profiling technologies has presented physiologists with unique challenges associated with analyses of big data sets. An appropriate technique for balancing the risks associated with type I (false discoveries) and type II (overlooking a real change) statistical error has not yet been developed and many big data studies may have potentially important differences that are overlooked. Also, it is imperative that attempts be made to integrate information from the various -omics studies before drawing conclusions based on expression of only one class of molecule, to better reflect the interdependency of molecular networks in cells. Currently, few analysis programs exist for such integrations. Despite challenges associated with these techniques, they have already provided new information about the biology of the CL, notably allowing identification of a key regulator of acquisition of luteolytic capacity and providing a big-picture view of the subtle changes that occur in the CL during early pregnancy. As these technologies become more accurate and less expensive, and as analysis becomes more user- friendly, their use will become much more widespread and many new discoveries will be made. This review will focus only on relevant studies in which these technologies were used to study the CL of ruminants.

Effects of early pregnancy on uterine lymphocytes and endometrial expression of immune-regulatory molecules in dairy heifers.

Natural killer (NK) cells are essential for establishment of human and rodent pregnancies. The function of these and other cytotoxic T cells (CTL) is controlled by stimulatory and inhibitory signaling. A role for cytotoxic cells during early pregnancy in cattle has not been described, but regulation of their function at the fetal-maternal interface is thought to be critical for conceptus survival. The hypothesis that the relative abundance of CTL and expression of inhibitory signaling molecules is increased by the conceptus during early pregnancy was tested. The proportions of lymphoid lineage cells and expression of inhibitory signaling molecules in the endometrium during early pregnancy in dairy heifers were determined using flow cytometry, immunofluorescence, and real-time PCR on days 17 and 20 of pregnancy and day 17 of the estrous cycle. Results revealed an increased percentage of NKp46+ and CD8+ cells in the uterus of pregnant heifers. Furthermore, a large percentage of uterine immune cells coexpressed these proteins. Compared to cyclic heifers, CD45+ uterine cells from pregnant heifers exhibited greater degranulation. Endometrium from pregnant heifers had greater mRNA abundance for the inhibitory molecules, CD274 and lymphocyte activating gene 3 (LAG3), and greater cytotoxic T lymphocyte-associated protein 4 (CTLA4), molecules that can interact with receptors on antigen-presenting cells and induce lymphocyte tolerance. This study demonstrates a dynamic regulation of both cytotoxic immune cells and tolerogenic molecules during the peri-implantation period that may be required to support establishment of pregnancy and placentation.

Changes in Myeloid Lineage Cells in the Uterus and Peripheral Blood of Dairy Heifers During Early Pregnancy.

Establishment of pregnancy requires interaction between the developing conceptus and the uterine mucosal immune system. Myeloid lineage cells (macrophages and dendritic cells) are key mediators of pregnancy in rodents and humans but relatively little is known regarding their role and distribution during early pregnancy in ruminants. We tested the hypothesis that myeloid lineage cell number, distribution, and function are altered during early pregnancy in dairy heifers. Dairy heifers were inseminated using sperm from a single bull (Day 0), and uteri and blood were collected at slaughter on Days 17 and 20 of pregnancy to investigate the response of myeloid lineage cells to the presence of a conceptus. Responses were compared to noninseminated heifers on Day 17 of the estrous cycle. Peripheral blood and uterine-derived immune cells were isolated magnetically and examined using flow cytometry. Immunohistochemical analysis was used to evaluate the spatial distribution of myeloid lineage cells in the endometrium and quantitative polymerase chain reaction was conducted to quantify abundance of mRNA transcripts associated with myeloid lineage cell function. Transcripts for major histocompatibility complex (MHC) II, cluster of differentiation (CD) 80, CD86, CD163, and indoleamine 2,3-dioxygenase (IDO) 1 were greater in endometrium of pregnant compared to cyclic heifers. Immunofluorescence analysis revealed increased labeling for MHCII and SIRPA in pregnant compared to cyclic heifers. There were approximately 50% more CD14+CD11c+ cells in the peripheral circulation of pregnant compared to cyclic heifers. A greater number of myeloid lineage cells were observed during early pregnancy, and this increase was most pronounced in and around the shallow glands. Furthermore, expression of molecules associated with a tolerogenic or alternatively activated phenotype of these cells also increased in pregnant heifers. The results support the hypothesis that myeloid lineage cells with a tolerogenic phenotype are involved in establishment of pregnancy in dairy heifers.

Changes in MicroRNA Expression During Maturation of the Bovine Corpus Luteum: Regulation of Luteal Cell Proliferation and Function by MicroRNA-34a.

The corpus luteum (CL) develops from the remnants of the ovulatory follicle and produces progesterone, required for maintenance of pregnancy in mammals. The differentiation of granulosal and thecal cells into luteal cells is accompanied by hypertrophy and hyperplasia of cells. As the CL matures, growth ceases and in ruminants, the tissue acquires the ability to undergo regression in response to prostaglandin F2alpha. The regulators of this transition are poorly understood. MicroRNA, which are posttranscriptional regulators of tissue development and function, are expressed in the CL. However, the pattern of their expression and their function during the transition from developing to functional CL is not known. The objectives of this study were to profile the expression of miRNA in developing versus mature bovine CL and determine effects of miRNA on bovine luteal cell survival and function. Knockdown of Drosha in midcycle (MC) luteal cells decreased progesterone and increased luteal cell apoptosis in the presence or absence of proinflammatory cytokines. Microarray analysis demonstrated that a greater number of miRNA were expressed in MC compared to D4 CL. Ingenuity pathway analysis (IPA) predicted that D4-specific miRNA regulate pathways related to carbohydrate metabolism, while MC-specific miRNA regulate pathways related to cell cycle and apoptosis signaling. Both predictions are consistent with a switch in the CL from a growing phase to a maintenance phase. One of the MC specific miRNA, miR-34a, was selected for further analysis. Increased concentrations of miR-34a in MC luteal cells resulted in decreased luteal cell proliferation, increased progesterone production, and inhibition of Notch1 and YY1 translation, but had no effect on luteal cell apoptosis. In conclusion, these data support a role for miRNA in general, and miR-34a in particular, in luteal formation and function.

Regulating life or death: potential role of microRNA in rescue of the corpus luteum.

The role of miRNA in tissue biology has added a new level of understanding of gene regulation and function. The corpus luteum (CL) is a transitory endocrine gland; the dynamic nature of the CL makes it a candidate for regulation by miRNA. Rescue of the CL from luteolysis is essential for the maintenance of pregnancy in all eutherian mammals. Using next generation sequencing, we profiled miRNA expression in the bovine CL during maternal recognition of pregnancy. We identified 590 luteal miRNA, of which 544 were known and 46 were novel miRNAs. Fifteen (including 3 novel) miRNAs were differentially expressed between CL of pregnant vs. cyclic animals. Target analysis of the differentially expressed miRNA resulted in genes involved in regulating apoptosis and immune response, providing evidence that miRNAs regulate the intracellular pathways that lead to either luteal regression or survival.

Luteal cells from functional and regressing bovine corpora lutea differentially alter the function of gamma delta T cells.

The luteal microenvironment is thought to direct the function of resident immune cells to facilitate either luteal function or regression. To determine if luteal cells from functional (Days 10-12) and regressing (8 h after administration of prostaglandin F2alpha) corpora lutea (CL) induce different responses in γδ T cells, luteal cells were cocultured with autologous γδ T cells isolated from peripheral blood. Proliferation, functional phenotypes, and cytokine synthesis were analyzed by flow cytometry. To determine if the luteal cells from functional CL induce hyporesponsiveness in γδ T cells, γδ(+) cells were cocultured with midcycle luteal cells and further stimulated with concanavalin A. Coculture of γδ(+) cells with midcycle luteal cells did not inhibit concanavalin A-induced proliferation. In a proliferation assay, luteal cells from midcycle CL predominantly induced proliferation of γδ(+) WC1(-) cells (P < 0.05), while luteal cells from regressing CL predominantly induced proliferation of γδ(+)WC1(+) cells (P < 0.05). Analysis of intracellular cytokines indicated that midcycle luteal cells increased the proportion of γδ(+) cells containing interleukin 10 (P < 0.05), but reduced the proportion of γδ(+) cells containing interferon gamma (IFNG; P < 0.05). There were no changes in the proportions of γδ(+) cells synthesizing interleukin 4 or tumor necrosis factor. Unexpectedly, coculture of γδ(+) cells with luteal cells from regressing CL had no effect on any of the cytokines analyzed. These data support the hypothesis that the function of resident T cells is differentially modulated depending on the status of the CL.

Expression and regulation of secreted phosphoprotein 1 in the bovine corpus luteum and effects on T lymphocyte chemotaxis.

Secreted phosphoprotein 1 (SPP1) in the bovine corpus luteum (CL) regulates cell function during the transitional periods of luteinization and luteal regression. The objectives were to i) characterize SPP1 expression in the CL throughout the estrous cycle, ii) determine factors that regulate SPP1 expression in luteal cells, and iii) examine the role of SPP1 in lymphocyte chemotaxis, proliferation, and function. SPP1 mRNA was greater in fully functional (d10) CL and late cycle (d18) CL compared with developing (d4) CL. Additionally, SPP1 mRNA increased within 1 h and remained elevated 4 and 8 h following induction of luteolysis with prostaglandin (PG)F2α. Expression of the SPP1 receptor, ß3 integrin, was not different throughout the estrous cycle but decreased following induction of luteolysis. Expression of CD44 increased during the estrous cycle but did not change during luteal regression. In cultured luteal cells, SPP1 mRNA was upregulated by PGF2α and/or tumor necrosis factor α. Western blots revealed the presence of both full-length SPP1 and multiple cleavage products in cultured luteal cells and luteal tissue. Depletion of endogenous SPP1 did not hinder luteal cell-induced lymphocyte proliferation or lymphocyte phenotype but did inhibit lymphocyte migration toward luteal cells. Based on these data, it is concluded that SPP1 is initially activated to establish and maintain cellular interactions between steroidogenic and nonsteroidogenic cells during the development of the CL. Upon induction of luteolysis, SPP1 serves as a signaling molecule to recruit or activate immune cells to facilitate luteal regression and tissue degradation.

Progesterone effects on lymphocytes may be mediated by membrane progesterone receptors.

Luteal cell-induced proliferation of T lymphocytes devoid of the nuclear progesterone receptor (PGR) is inhibited by progesterone. Functional effects of progesterone on bovine lymphocytes and the expression of membrane progesterone receptors (mPRs) alpha (PAQR7), beta (PAQR8), gamma (PAQR5), and progesterone receptor membrane component 1 (PGRMC1) mRNA were analyzed in corpus luteum (CL) and lymphocytes. Progesterone and a cell-impermeable progesterone conjugate caused a dose-dependent decrease in IL2 receptor α-subunit (IL2RA) mRNA and an increase in interleukin 2 (IL2) mRNA concentrations in cultured PBMCs. In luteal tissues, concentrations of PAQR7 and PAQR8 mRNA were lower in CL collected on day 11 compared with day 18, whereas PGRMC1 and PGR mRNA were greater on day 11 than on day 18. The mRNA of all three PAQRs and PGRMC1 were detected in bovine T lymphocytes, but not in B cells/monocytes. Progesterone increased intracellular Ca(++) and reduced the phosphorylation of zeta-chain-associated protein kinase 70 (Zap70). A specific, saturable, and single progesterone binding site with a steroid specificity characteristic of mPRs was demonstrated by saturation and competitive binding assays using T lymphocyte membranes, and PAQR7 receptors were localized on the plasma membranes by immunofluorescence. Thus, progesterone induces specific and rapid functional effects on T lymphocytes in the absence of PGR. The mPRs are potential intermediaries of the cell-surface actions of progesterone because they are expressed in lymphocytes, the actions of progesterone are mimicked by a cell-impermeable form of progesterone, and specific, saturable progesterone binding, which is characteristic of mPRs, is present on lymphocyte membranes.

Luteal microenvironment directs resident T lymphocyte function in cows.

The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture of cells. Functional interactions exist between peripheral T cells and luteal cells in vitro; however, the precise role of resident T cells (RTC) remains unknown. The goals of the present study were to isolate RTC from within the CL and determine if alteration of luteal function resulted in changes in RTC phenotypes. Functional lymphocyte phenotypes identified in the bovine CL by using quantitative flow cytometric analysis were clearly different from those in the peripheral circulation. The proportion of CD8(+) RTC was greater than CD4(+) RTC. These proportions were opposite in peripheral blood. The proportion of γδ(+) lymphocytes was not different in the CL compared to that in peripheral blood nor was it altered during luteal regression. There was a significant increase in CD8αα(+) and γδ(+)CD8αα(+) RTC during luteal regression. The proportion of FOXP3(+) lymphocytes in the CL was greater than that isolated from peripheral blood, and this proportion of lymphocytes was dramatically reduced by induction of luteolysis. Within the CL of early pregnancy, there was an increase in the CD8αß(+) and γδ(+)CD8αß(+) populations compared to those in the CL of nonpregnant animals. Based on these data, we concluded that the functional state of the CL creates a microenvironment that regulates the recruitment of or differentiation into specific lymphocyte types. Understanding the interactions between steroidogenic cells and ovarian lymphocytes within CL will not only enhance understanding of reproductive function but may provide vital clues to lymphocyte regulation within tissues.

A role for cysteine-rich 61 in the angiogenic switch during the estrous cycle in cows: regulation by prostaglandin F2alpha.

The development and demise of the corpus luteum (CL) are accompanied by angiogenic and angioregressive processes; however, the mediators of these processes have not been fully identified and characterized. Transcriptional profiling studies revealed the upregulation of cysteine-rich 61 (CYR61) in the CL, about which nothing was previously known. In the present study, we found that over a 12-h period following a single injection of prostaglandin F(2alpha) (PGF(2alpha)), RT-PCR revealed the upregulation of CYR61 at 0.5 and 1 h, after which it declined. We also determined that luteal-derived endothelial cells as well as luteal steroidogenic cells are sources of CYR61. Treatment with PGF(2alpha) in vitro had no effect on CYR61 expression in luteal-derived endothelial cells, but it increased CYR61 expression in luteal steroidogenic cells. During the estrous cycle, CYR61/CYR61 (transcript/protein) was increased in the Day 4 but not in the Day 10 and Day 16 CL, suggesting that it may be associated with the switch to the angiogenic phenotype. In addition, the specific but transient upregulation of CYR61 by PGF(2alpha) in vivo, and in luteal steroidogenic cells but not endothelial cells in vitro, may be part of the mechanism underlying the previously reported transient increase in blood flow during the early onset of luteolysis. This is supported by our preliminary finding that CYR61 transiently inhibited endothelial cell expression of endothelin-converting enzyme 1 mRNA but not endothelin 1. Collectively, the increased expression of CYR61 in the Day 4 CL and its transient increase by PGF(2alpha) in Day 6, Day 10, and Day 16 CL indicate that CYR61 may play a role in regulating angiogenesis over the life span of the CL.

The interface of the immune and reproductive systems in the ovary: lessons learned from the corpus luteum of domestic animal models.

The dynamic changes that characterize the female reproductive system are regulated by hormones. However, local cell-to-cell interactions may mediate responsiveness of tissues to hormonal signals. The corpus luteum (CL) is an excellent model for understanding how immune cells are recruited into tissues and the role played by those cells in regulating tissue homeostasis or demise. Leukocytes are recruited into the CL throughout its lifespan, and leukocyte-derived cytokines have been found in corpora lutea of all species examined. The proinflammatory cytokines inhibit gonadotropin-stimulated steroidogenesis, profoundly stimulate prostaglandin synthesis by luteal cells, and promote apoptosis. However, there is mounting evidence that leukocytes and luteal cells communicate in different ways to maintain homeostasis within the functional CL. Domestic animals have provided important information regarding the presence and role of immune cells in the CL.