Nonsteroidogenic Cells Research Articles

Characterization of Mitochondrial Heat Shock Protein 60 variants in HEK293 Cells Transformed into Steroidogenic

Introduction: During pregnancy, P4 is essential to maintain the maternal-fetal relationship. Maternal cholesterol is the main source of P4 production, a process that takes place in the syncytiotrophoblast mitochondrion. The mechanism and proteins involved in the cholesterol transport for the steroidogenic process are still unknown in detail. The STARD3 protein could be the substitute for its STARD1 equivalent localized in all acute response tissues. However, mutation or null STARD3 mice maintain their reproductive capacity, suggesting other proteins are involved in this process. Previously, we reported that the HSP60 participates in steroidogenesis in mitochondria isolated from the placental syncytiotrophoblast, mitochondrial contact sites or JEG-3. Also, take relevance that non-steroidogenic cells, such as the HEK293, which are human kidney embryo cells, when are transformed into steroidogenic by transfection of the steroidogenic machinery, they synthesize progesterone. To understand better the mechanism through which HSP60 participates in placental steroidogenesis, mutation of cysteine 442, which is essential in the active site for its activity, and deletion of 146 amino acid residues of the N-terminal of HSP60 were performed. The first was implemented to determine whether the protein structure is essential to support steroidogenesis, and the second was done to elucidate whether its activity occurs outside or inside the mitochondrion. Methods: Two mutants were obtained: a) cysteine 442 was replaced by alanine (HSP60C442A) and b) the HSP60-mature (HSP60M) without the mitochondrial-leading sequence. Human kidney cells HEK293 were transformed into steroidogenic by transfection with pECE-P450scc, pCMV-3βHSD-I. The transfected cells were transfected with the HSP60wt, HSP60C442A, or HSP60M plasmids. The transfection was validated by western blot and P4 was determined by an enzyme immunoassay kit. HSP60 without mutations was used as control (HSP60wt). Results: The synthesis of P4 was stimulated by the wild type HSP60 (HSP60wt). However, with both mutants, steroidogenesis occurred as in the control, suggesting that mutants do not support P4 synthesis. Discussion: The mechanism to transport cholesterol to steroidogenic mitochondria requires the full HSP60 to support P4 synthesis, which is necessary to maintain pregnancy. Highlights HSP60 participates in the steroidogenesis of transformed HEK293 cells. Cys442 mutant of HSP60 loses its activity in steroidogenesis. N-terminal deletion of HSP60 is not involved in steroidogenesis. Native HSP60 is critical for steroidogenesis.

A spatiotemporal steroidogenic regulatory network in human fetal adrenal glands and gonads.

Human fetal adrenal glands produce substantial amounts of dehydroepiandrosterone (DHEA), which is one of the most important precursors of sex hormones. However, the underlying biological mechanism remains largely unknown. Herein, we sequenced human fetal adrenal glands and gonads from 7 to 14 gestational weeks (GW) via 10× Genomics single-cell transcriptome techniques, reconstructed their location information by spatial transcriptomics. Relative to gonads, adrenal glands begin to synthesize steroids early. The coordination among steroidogenic cells and multiple non-steroidogenic cells promotes adrenal cortex construction and steroid synthesis. Notably, during the window of sexual differentiation (8-12 GW), key enzyme gene expression shifts to accelerate DHEA synthesis in males and cortisol synthesis in females. Our research highlights the robustness of the action of fetal adrenal glands on gonads to modify the process of sexual differentiation.

Differential Response of Transcription Factors to Activated Kinases in Steroidogenic and Non-Steroidogenic Cells.

Hormone-induced Leydig cell steroidogenesis requires rapid changes in gene expression in response to various hormones, cytokines, and growth factors. These proteins act by binding to their receptors on the surface of Leydig cells leading to activation of multiple intracellular signaling cascades, downstream of which are several kinases, including protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase I (CAMKI), and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). These kinases participate in hormone-induced steroidogenesis by phosphorylating numerous proteins including transcription factors leading to increased steroidogenic gene expression. How these various kinases and transcription factors come together to appropriately induce steroidogenic gene expression in response to specific stimuli remains poorly understood. In the present work, we compared the effect of PKA, CAMKI and ERK1/2 on the transactivation potential of 15 transcription factors belonging to 5 distinct families on the activity of the Star gene promoter. We not only validated known cooperation between kinases and transcription factors, but we also identified novel cooperations that have not yet been before reported. Some transcription factors were found to respond to all three kinases, whereas others were only activated by one specific kinase. Differential responses were also observed within a family of transcription factors. The diverse response to kinases provides flexibility to ensure proper genomic response of steroidogenic cells to different stimuli.

Sonic Hedgehog signaling pathway is expressed in normal human adrenal cortices and up-regulated in adrenocortical carcinomas

The Sonic Hedgehog (Shh) signaling pathway is involved in mouse adrenal growth and differentiation. However, it remains unclear whether this pathway plays a similar role in human especially because we previously showed that this organ displays a significant capacity for tissue regeneration from differentiated cells. The expression of the components of the pathway was examined in 38 normal adult adrenal glands and 23 adrenocortical carcinomas (ACCs). We found that Shh is widely expressed in steroidogenic cells. Shh-positive cells also expressed the Shh receptor Patched-1 as well as the receptor-like signal transducer Smoothened. Moreover, expression of the transcription factors Gli1, Gli2 and Gli3 was detected in the nucleus of the steroidogenic cells. Our data identified Shh pathway as a new autocrine/paracrine loop in human steroidogenic cells. However, we cannot exclude that a subpopulation of cells may correspond to stem/progenitor cells since non-steroidogenic cells located in the capsule express components of the Shh pathway. ACCs exhibited an increased expression of all components of Shh pathway with the exception of Gli2 indicating a possible involvement in the tumorigenic process. In addition, the Smoothened inhibitor NVP-LDE225 potently inhibited tumor growth and reduced cortisol and aldosterone secretion by H295R cells. We also showed that LDE225 acts by targeting mitochondria leading to a decrease in transmembrane potential and an increase in ROS production. Collectively, our findings show that Shh pathway is expressed in normal human steroidogenic cells and is activated in carcinomas. Shh may be a promising target for the treatment of this aggressive cancer.

The Intracellular Cholesterol Pool in Steroidogenic Cells Plays a Role in Autophagy, Basal Steroidogenesis and Mitochondrial Dynamics

In steroidogenesis, cholesterol‐ the precursor substrate molecule for all steroid hormones is central and the framework of steroidogenesis across steroidogenic cells is constructed around it— including its cellular uptake, storage in intracellular lipid droplets, mobilization upon steroidogenic stimulation, and finally, its transport to the mitochondria where cholesterol is converted to pregnenolone by the P450 side chain cleavage enzyme. Thus, cholesterol and the mitochondria are highly interconnected in all steroidogenic cells. Moreover, accruing evidence suggests that autophagy and mitochondrial dynamics are important cellular events in the regulation of trophic hormone‐induced cholesterol homeostasis and steroidogenesis. However, a potential role of cholesterol in the regulation of steroidogenic events and factors remain unexplored. We tested the hypothesis that cholesterol plays a role in the regulation of cell‐intrinsic factors and events involving steroidogenesis. We used an intermediary approach to investigate the role of cholesterol in the regulation of steroidogenesis in different steroidogenic cell types such as MA‐10 cells (a murine Leydig cell line), BeWo cells (placental cell line) producing different steroid hormones. Steroidogenic cells were cultured in a lipoprotein‐depleted medium that leads to the depletion of the intracellular cholesterol pool and lipid droplets. Furthermore, cholesterol deprivation induced steroidogenic events. To get a snapshot of cholesterol deprived (CD)‐induced changes in lipid droplets, autophagy, and mitochondrial dynamics at an ultrastructural level we analyzed steroidogenic cells using transmission electron microscopy. As expected, cholesterol deprivation resulted in the depletion or loss of intracellular lipid droplets and the cholesterol pool across steroidogenic cell lines. Moreover, a difference in autophagy‐related structural changes was observed between cells cultured under the normal and CD conditions. In general, the structural characteristics of autophagy were more apparent in the CD group when compared with the normal control group across different steroidogenic cell types. Moreover, a change in mitochondrial shape was observed between the cells from two groups. For instance, the elongated or tubular mitochondria showing signs of mitochondrial dynamics (fusion or fission) were more common in the CD experimental group. Importantly, an increase in lysosome size and numbers were consistently found in cells under CD conditions which inversely correlated with the depletion of lipid droplets and the cholesterol pool, suggesting a compensatory increase in cholesterol mobilization. Taken together, this data suggests that cholesterol indeed plays a role in the regulation of autophagy and mitochondrial dynamics across steroidogenic cells in a context‐dependent manner. Such effects of cholesterol deprivation on autophagy and mitochondrial dynamics were not observed in the non‐steroidogenic cells such as HepG2 (human hepatoma cells) and H4IIE (rat hepatoma cells), indicating that cholesterol insufficiency‐induced changes in steroidogenic cells are specific to steroidogenesis. Thus, we unraveled this previously unknown role of cholesterol in steroidogenesis beyond being a mere substrate for steroid hormones. The implications of our findings are broad and offer new insights into trophic hormone‐dependent and independent steroidogenesis during development, as well as in health and disease.

The intracellular cholesterol pool in steroidogenic cells plays a role in basal steroidogenesis

The framework of steroidogenesis across steroidogenic cells is constructed around cholesterol — the precursor substrate molecule for all steroid hormones — including its cellular uptake, storage in intracellular lipid droplets, mobilization upon steroidogenic stimulation, and finally, its transport to the mitochondria, where steroidogenesis begins. Thus, cholesterol and the mitochondria are highly interconnected in steroidogenic cells. Moreover, accruing evidence suggests that autophagy and mitochondrial dynamics are important cellular events in the regulation of trophic hormone-induced cholesterol homeostasis and steroidogenesis. However, a potential role of cholesterol in itself in the regulation of steroidogenic factors and events remain largely unexplored. We tested the hypothesis that cholesterol plays a role in the regulation of cell-intrinsic factors and events involving steroidogenesis. Here, we show that depleting the intracellular cholesterol pool in steroidogenic cells induces autophagy, affects mitochondrial dynamics, and upregulates steroidogenic factors and basal steroidogenesis in three different steroidogenic cell types producing different steroid hormones. Notably, the cholesterol insufficiency-induced changes in different steroidogenic cell types occur independent of pertinent hormone stimulation and work in a dynamic and temporal manner with or without hormonal stimulation. Such effects of cholesterol deprivation on autophagy and mitochondrial dynamics were not observed in the non-steroidogenic cells, indicating that cholesterol insufficiency-induced changes in steroidogenic cells are specific to steroidogenesis. Thus, our data suggests a role of cholesterol in steroidogenesis beyond being a mere substrate for steroid hormones. The implications of our findings are broad and offer new insights into trophic hormone-dependent and hormone-independent steroidogenesis during development, as well as in health and disease.

CXCL12 May Drive Inflammatory Potential in the Ovine Corpus Luteum During Implantation

Adequate corpus luteum (CL) function is paramount to successful pregnancy. Structural and functional CL integrity is controlled by diverse cell types that contribute and respond to the local cytokine milieu. The chemokine ligand 12 (CXCL12) and receptor, CXCR4, are modulators of inflammation and cell survival, but little is understood about CXCL12-CXCR4 axis and CL functional regulation. Corpora lutea from control nonpregnant ewes (n = 5; day 10 estrous cycle (D10C)) and pregnant ewes (n = 5/day) on days 20 (D20P) and 30 (D30P) post-breeding were analyzed for gene and protein expression of CXCL12, CXCR4, and select inflammatory cytokines. In separate cell culture studies, cytokine production was evaluated following CXCL12 treatment. Abundance of CXCL12 and CXCR4 increased (P < 0.05) in pregnant ewes compared to nonpregnant ewes, as determined by a combination of quantitative PCR, immunoblot, and immunofluorescence microscopy. CXCR4 was detected in steroidogenic and nonsteroidogenic cells in ovine CL, and select pro-inflammatory mediators were greater in CL from pregnant ewes. In vitro studies revealed greater abundance of tumor necrosis factor (TNF) following CXCL12 administration (P = 0.05), while P4 levels in cell media were unchanged. Fully functional CL of pregnant ewes is characterized by increased abundance of inflammatory cytokines which may function in a luteotropic manner. We report concurrent increases in CXCL12, CXCR4, and select inflammatory mediators in ovine CL as early pregnancy progresses. We propose CXCL12 stimulates production of select cytokines, rather than P4 in the CL to assist in CL establishment and survival.

Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction

Conditions of impaired adrenal function and tissue destruction, such as in Addison’s disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC50 values of 5.7 × 10−8, 8.1 × 10−7 and 2.1 × 10−8 M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.

Dog Steroidogenic Factor-1: Molecular cloning and analysis of epigenetic regulation.

Steroidogenic factor 1 (SF-1) is a nuclear receptor that is important in steroid hormone production, and adrenal and gonad development. The SF-1 gene is highly conserved among most vertebrates. However, dog SF-1 registered in public databases, such as CanFam3.1, lacks the 5’ end compared to other mammals including mouse, human, bovine, and cat. Whether this defect is due to species differences or database error is unclear. Here, we determined the full-length dog SF-1 cDNA sequence and identified the missing 5’ end sequence in the databases. The coding region of the dog SF-1 gene has 1,386 base pairs, and the protein has 461 amino acid residues. Sequence alignment analysis among vertebrates revealed that the 5’ end sequence of dog SF-1 cDNA is highly conserved compared to other vertebrates. The genomic position of the first exon was determined, and its promoter region sequence was analyzed. The DNA methylation state at the basal promoter and the expression of dog SF-1 in steroidogenic tissues and non-steroidogenic cells were examined. CpG sites at the basal promoter displayed methylation kinetics inversely correlated with gene expression. The promoter was hypomethylated and hypermethylated in SF-1 expressing and non-SF-1 expressing tissues, respectively. In conclusion, we identified the true full sequence of dog SF-1 cDNA and determined the genome sequence around the first exon. The gene is under the control of epigenetic regulation, such as DNA methylation, at the promoter.

Fetal Leydig cells dedifferentiate and serve as adult Leydig stem cells.

Previous studies have established that fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) show distinct functional characteristics. However, the lineage relationship between FLCs and ALCs has not been clarified yet. Here, we reveal that a subset of FLCs dedifferentiate at fetal stages to give rise to ALCs at the pubertal stage. Moreover, the dedifferentiated cells contribute to the peritubular myoid cell and vascular pericyte populations in the neonatal testis, and these non-steroidogenic cells serve as potential ALC stem cells. We generated FLC lineage-specific Nr5a1 (Ad4BP/SF-1) gene-disrupted mice and mice lacking the fetal Leydig enhancer (FLE) of the Nr5a1 gene. Phenotypes of these mice support the conclusion that most of the ALCs arise from dedifferentiated FLCs, and that the FLE of the Nr5a1 gene is essential for both initial FLC differentiation and pubertal ALC redifferentiation.

Prokineticins and their G protein-coupled receptors in health and disease.

Prokineticins are two conserved small proteins (~8kDa), prokineticin 1 (PROK1; also called EG-VEGF) and prokineticin 2 (PROK2; also called Bv8), with an N-terminal AVITGA sequence and 10 cysteines forming 5 disulfide bridges. PROK1 and PROK2 bind to two highly related G protein-coupled receptors (GPCRs), prokineticin receptor 1 (PROKR1) and prokineticin receptor 2 (PROKR2). Prokineticins and their receptors are widely expressed. PROK1 is predominantly expressed in peripheral tissues, especially steroidogenic organs, whereas PROK2 is mainly expressed in the central nervous system and nonsteroidogenic cells of the testes. Prokineticins signaling has been implicated in several important physiological functions, including gastrointestinal smooth muscle contraction, circadian rhythm regulation, neurogenesis, angiogenesis, pain perception, mood regulation, and reproduction. Dysregulation of prokineticins signaling has been observed in a variety of diseases, such as cancer, ischemia, and neurodegeneration, in which prokineticins signaling seems to be a promising therapeutic target. Based on the phenotypes of knockout mice, PROKR2 and PROK2 have recently been identified as causative genes for idiopathic hypogonadotropic hypogonadism, a developmental disorder characterized by impaired development of gonadotropin-releasing hormone neurons and infertility. In vitro functional studies with these disease-associated PROKR2 mutations uncovered some novel features for this receptor, such as biased signaling, which may be used to understand GPCR signaling regulation in general.

Expression of steroidogenic enzymes and metabolism of steroids in COS-7 cells known as non-steroidogenic cells

The COS-7 (CV-1 in Origin with SV40 genes) cells are known as non-steroidogenic cells because they are derived from kidney cells and the kidney is defined as a non-steroidogenic organ. Therefore, COS-7 cells are used for transfection experiments to analyze the actions of functional molecules including steroids. However, a preliminary study suggested that COS-7 cells metabolize [3H]testosterone to [3H]androstenedione. These results suggest that COS-7 cells are able to metabolize steroids. Therefore, the present study investigated the expression of steroidogenic enzymes and the metabolism of steroids in COS-7 cells. RT-PCR analyses demonstrated the expressions of several kinds of steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase, cytochrome P450 7α-hydroxylase, cytochrome P450 17α-hydroxylase/17,20-lyase, 17β-hydroxysteroid dehydrogenase, 5α-reductase, cytochrome P450 21-hydroxylase, cytochrome P450 11β-hydroxylase, and cytochrome P450 aromatase in COS-7 cells. In addition, steroidogenic enzymes 3β-HSD, P4507α, 5α-reductase, P450c17, P450c21, P450c11β, and 17β-HSD actively metabolized various steroids in cultured COS-7 cells. Finally, we demonstrated that 17β-HSD activity toward androstenedione formation was greater than other steroidogenic enzyme activities. Our results provide new evidence that COS-7 cells express a series of steroidogenic enzyme mRNAs and actively metabolize a variety of steroids.

Editorial: Adrenal Cortex: From Physiology to Disease

Editorial: Adrenal Cortex: From Physiology to Disease

Mapping lineage progression of somatic progenitor cells in the mouse fetal testis.

Testis morphogenesis is a highly orchestrated process involving lineage determination of male germ cells and somatic cell types. Although the origin and differentiation of germ cells are known, the developmental course specific for each somatic cell lineage has not been clearly defined. Here, we construct a comprehensive map of somatic cell lineage progression in the mouse testis. Both supporting and interstitial cell lineages arise from WT1+ somatic progenitor pools in the gonadal primordium. A subpopulation of WT1+ progenitor cells acquire SOX9 expression and become Sertoli cells that form testis cords, whereas the remaining WT1+ cells contribute to progenitor cells in the testis interstitium. Interstitial progenitor cells diversify through the acquisition of HES1, an indication of Notch activation, at the onset of sex determination. HES1+ interstitial progenitors, through the action of Sertoli cell-derived Hedgehog signals, become positive for GLI1. The GLI1+ interstitial cells eventually develop into two cell lineages: steroid-producing fetal Leydig cells and non-steroidogenic cells. The fetal Leydig cell population is restricted by Notch2 signaling from the neighboring somatic cells. The non-steroidogenic progenitor cells retain their undifferentiated state during fetal stage and become adult Leydig cells in post-pubertal testis. These results provide the first lineage progression map that illustrates the sequential establishment of somatic cell populations during testis morphogenesis.

Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells.

Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

Mitochondrial cholesterol: mechanisms of import and effects on mitochondrial function

Mitochondria require cholesterol for biogenesis and membrane maintenance, and for the synthesis of steroids, oxysterols and hepatic bile acids. Multiple pathways mediate the transport of cholesterol from different subcellular pools to mitochondria. In steroidogenic cells, the steroidogenic acute regulatory protein (StAR) interacts with a mitochondrial protein complex to mediate cholesterol delivery to the inner mitochondrial membrane for conversion to pregnenolone. In non-steroidogenic cells, several members of a protein family defined by the presence of a StAR-related lipid transfer (START) domain play key roles in the delivery of cholesterol to mitochondrial membranes. Subdomains of the endoplasmic reticulum (ER), termed mitochondria-associated ER membranes (MAM), form membrane contact sites with mitochondria and may contribute tothe transport ofER cholesterol to mitochondria, either independently or in conjunction with lipid-transfer proteins. Model systems of mitochondria enriched with cholesterol in vitro and mitochondria isolated from cells with (patho)physiological mitochondrial cholesterol accumulation clearly demonstrate that mitochondrial cholesterol levels affect mitochondrial function. Increased mitochondrial cholesterol levels have been observed in several diseases, including cancer, ischemia, steatohepatitis and neurodegenerative diseases, and influence disease pathology. Hence, a deeper understanding of the mechanisms maintaining mitochondrial cholesterol homeostasis may reveal additional targets for therapeutic intervention. Here we give a brief overview of mitochondrial cholesterol import in steroidogenic cells, and then focus on cholesterol trafficking pathways that deliver cholesterol to mitochondrial membranes in non-steroidogenic cells. We also briefly discuss the consequences of increased mitochondrial cholesterol levels on mitochondrial function and their potential role in disease pathology.

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, β3 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.

Ovarian granulosa cells utilize scavenger receptor SR-BI to evade cellular cholesterol homeostatic control for steroid synthesis

Cellular cholesterol is known to be under homeostatic control in nonsteroidogenic cells, and this intrigued us to understand how such control works in steroidogenic cells that additionally use cholesterol for steroid hormone synthesis. We employed primary culture of rat ovarian granulosa cells to study how steroidogenic cells adapt to acquire sufficient cholesterol to meet the demand of active steroidogenesis under the stimulation of gonadotropin follicle-stimulating hormone (FSH) and cytokine transforming growth factor (TGF)β1. We found that TGFβ1 potentiated FSH to upregulate scavenger receptor class B member I (SR-BI) and LDL receptor (LDLR), both functional in uptaking cholesterol as hHDL(3) and hLDL supplementation enhanced progesterone production, and the effect of each lipoprotein was completely or partially blocked by SR-BI selective inhibitor BLT-1. Uptaken cholesterol could also be stored in lipid droplets. Importantly, LDLR and SR-BI responded to sterol with different sensitivity. Giving cells lipoproteins or 25-hydroxycholesterol downregulated Ldlr but not Scarb1; Scarb1 was ultimately downregulated by excessive sterol accumulation under 25-hydroxycholesterol and aminoglutethimide (inhibitor of steroidogenesis) cotreatment. Furthermore, transcription factors sterol regulatory element-binding protein (SREBP)-2 and liver receptor homolog (LRH)-1 crucially mediated Ldlr and Scarb1 differential response to sterol challenge. This study reveals that ovarian granulosa cells retain the cholesterol homeostatic control machinery like nonsteroidogenic cells, although during active steroidogenesis, they utilize SR-BI to evade such feedback control.

Role of the cell cycle in regression of the corpus luteum

The corpus luteum contains differentiated steroidogenic cells that have exited the cell cycle of proliferation. In some tissues, deletion of quiescent, differentiated cells by apoptosis in response to injury or pathology is preceded by reentry into the cell cycle. We tested whether luteal cells reenter the cell cycle during the physiological process of luteolysis. Ovaries were obtained after injection of cows with a luteolytic dose of prostaglandin F(2)(α) (PGF). In luteal sections, cells co-staining for markers of cell proliferation (MKI67) and apoptosis (cPARP1) increased 24 h after PGF, indicating that cells that reenter the cell cycle undergo apoptosis. The percent of steroidogenic cells (CYP11A1-positive) co-staining for MKI67 increased after PGF, while co-staining of non-steroidogenic cells did not change. Dispersed luteal cells were stained with Nile Red to distinguish lipid-rich steroidogenic cells from nonsteroidogenic cells and co-stained for DNA. Flow cytometry showed that the percent of steroidogenic cells progressing through the cell cycle and undergoing apoptosis increased after PGF. Culturing luteal cells induced reentry of steroidogenic cells into the cell cycle, providing a model to test the influence of the cell cycle on susceptibility to apoptosis. Blocking cells early in the cell cycle using inhibitors reduced cell death in response to treatment with the apoptosis-inducing protein, Fas ligand (FASL). Progesterone treatment reduced progression through the cell cycle and decreased FASL-induced apoptosis. In summary, steroidogenic cells reenter the cell cycle upon induction of luteal regression. While quiescent cells are resistant to apoptosis, entry into the cell cycle promotes susceptibility to apoptosis.

Life Cycle of Feline Corpora lutea: Histological and Intraluteal Hormone Analysis

The corpus luteum (CL) is a transient hormone gland on the ovary that produces progesterone (P4) for the maintenance of pregnancy. It develops from residual follicular granulosa and theca cells after ovulation. Very little is known about the cellular and hormonal processes within CLs obtained from pregnant and pseudopregnant felids. Therefore, our aim was to review the luteal function in feline CLs of different reproductive stages in conjunction with our data obtained in domestic cats and Eurasian lynxes. Corpus luteum function in lynxes is of particular interest, as a post-partum luteal activity was suggested based on repeated ultrasonography and endocrine examinations. Histology of CL from pregnant and pseudopregnant domestic cats clearly reflects the luteal function. The formation of the CL after ovulation is characterized by transforming of theca and granulosa cells into steroidogenic luteal cells and is accompanied by increased intraluteal and circulating P4 levels. Luteal regression is steadily progressive; the first signs (coarsed vacuolization, increased proportion of non-steroidogenic cells) are visible already in CL from the second trimester of pregnancy.