Modulating the Mesenchymal Stromal Cell Microenvironment Alters Exosome RNA Content and Ligament Healing Capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by two inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Exosome-educated macrophages and exosomes differentially improve ligament healing.

Recently, our group used exosomes from mesenchymal stromal/stem cells (MSCs) to simulate an M2 macrophage phenotype, that is, exosome-educated macrophages (EEMs). These EEMs, when delivered in vivo, accelerated healing in a mouse Achilles tendon injury model. For the current study, we first tested the ability of EEMs to reproduce the beneficial healing effects in a different rodent model, that is, a rat medial collateral ligament (MCL) injury model. We hypothesized that treatment with EEMs would reduce inflammation and accelerate ligament healing, similar to our previous tendon results. Second, because of the translational advantages of a cell-free therapy, exosomes alone were also examined to promote MCL healing. We hypothesized that MSC-derived exosomes could also alter ligament healing to reduce scar formation. Similar to our previous Achilles tendon results, EEMs improved mechanical properties in the healing ligament and reduced inflammation, as indicated via a decreased endogenous M1/M2 macrophage ratio. We also showed that exosomes improved ligament remodeling as indicated by changes in collagen production and organization, and reduced scar formation but without improved mechanical behavior in healing tissue. Overall, our findings suggest EEMs and MSC-derived exosomes improve healing but via different mechanisms. EEMs and exosomes each have attractive characteristics as therapeutics. EEMs as a cell therapy are terminally differentiated and will not proliferate or differentiate. Alternatively, exosome therapy can be used as a cell free, shelf-stable therapeutic to deliver biologically active components. Results herein further support using EEMs and/or exosomes to improve ligament healing by modulating inflammation and promoting more advantageous tissue remodeling.

TimeMeter assesses temporal gene expression similarity and identifies differentially progressing genes.

Comparative time series transcriptome analysis is a powerful tool to study development, evolution, aging, disease progression and cancer prognosis. We develop TimeMeter, a statistical method and tool to assess temporal gene expression similarity, and identify differentially progressing genes where one pattern is more temporally advanced than the other. We apply TimeMeter to several datasets, and show that TimeMeter is capable of characterizing complicated temporal gene expression associations. Interestingly, we find: (i) the measurement of differential progression provides a novel feature in addition to pattern similarity that can characterize early developmental divergence between two species; (ii) genes exhibiting similar temporal patterns between human and mouse during neural differentiation are under strong negative (purifying) selection during evolution; (iii) analysis of genes with similar temporal patterns in mouse digit regeneration and axolotl blastema differentiation reveals common gene groups for appendage regeneration with potential implications in regenerative medicine.

Tendon-to-Bone Healing in a Rat Extra-articular Bone Tunnel Model: A Comparison of Fresh Autologous Bone Marrow and Bone Marrow-Derived Mesenchymal Stem Cells.

BACKGROUND: Despite widespread acceptance of fresh autologous bone marrow (BM) for use in clinical practice, limited information exists to analyze if tendon-to-bone healing could be accelerated with local use of fresh autologous BM. PURPOSE: To investigate the effect of fresh autologous BM on tendon-to-bone healing with a novel rat model. STUDY DESIGN: Controlled laboratory study. METHODS: An extra-articular bone tunnel was created and filled with an autologous tendon graft in skeletally mature Sprague-Dawley rats (N = 60). They were then randomly divided into 3 groups: BM group (injection of fresh autologous BM into the tendon-bone interface, n = 20), BM-derived mesenchymal stem cell (BMSC) group (injection of allogenic cultured BMSCs, n = 20), and the control group (tendon-bone interface without injection of BM or BMSCs, n = 20). Biomechanical, histological, and immunohistochemical analyses were performed at 2 and 6 weeks after surgery. RESULTS: The BM group showed a relatively well-organized and dense connective tissue interface with better orientation of collagen fibers as compared with the BMSC group. At 2 weeks, the tendon-bone interface tissue thickness of the BMSC group was 140 ± 25 µm (mean ± SEM), which was significantly greater than the BM group (58 ± 15 µm). The BM group showed fewer M1 macrophages at the tendon-bone interface at 2 and 6 weeks (P < .001). In contrast, there were more M2 macrophages at the interface in the BM group 2 and 6 weeks postoperatively when compared with controls and the BMSC group (P < .001). Biomechanical tests revealed significantly higher stiffness in the BM group versus the control and BMSC groups at 2 and 6 weeks after surgery (P < .05). Load to failure showed similar trends to stiffness. CONCLUSION: These findings indicate that local delivery of fresh autologous BM enhances tendon-to-bone healing better than the alternative treatments in this study. This effect may be partially due to the observed modulation of inflammatory processes, especially in M2 macrophage polarization. CLINICAL RELEVANCE: Fresh autologous BM could be a treatment option for this disorder.

Proinflammatory polarization of stifle synovial macrophages in dogs with cruciate ligament rupture.

OBJECTIVE: To determine polarization of synovial macrophages during development of cruciate ligament rupture (CR) and determine whether differences in synovial macrophage polarization in CR, osteoarthritis (OA), and healthy joints exist. STUDY DESIGN: Prospective case-controlled study. ANIMALS: Client-owned dogs with unstable stifles with CR (n = 22), paired stable contralateral stifles with partial CR (pCR; n = 7), joints with OA not related to CR (n = 6), and clinically normal (Normal; n = 7) joints. METHODS: Synovial fluid samples were collected. Smears were made for differential cytology counts and estimated total nucleated cell counts. Cytospin preparations were made, and immunocytochemical staining was performed with the pan-macrophage marker CD68, M1 macrophage markers inducible nitric oxide synthase (iNOS) and chemokine (C-C motif) receptor 7 (CCR7), and M2 macrophage markers arginase 1 and CD163. Positively stained cells were counted. RESULTS: Numbers of lymphocytes were increased in the CR group compared with the OA and Normal groups (P < .05). Numbers of CD68+ , CCR7+ , and iNOS+ cells in the CR and OA groups were increased compared with the Normal group (P < .05). Globally, the ratio of positively stained M1 polarized CD68+ cells to M2 polarized CD68+ cells was highest for the OA group (2.49), followed by the pCR (2.1), CR (1.63), and Normal (0.7) groups. CONCLUSION: Polarization of synovial macrophages toward an M1 proinflammatory phenotype is an early event in the development of canine CR. CLINICAL SIGNIFICANCE: M1 polarization in pCR stifles provides evidence of a possible role for macrophages in progressive development of cruciate ligament fiber damage. Lymphocytes may play a role in the synovitis found in CR joints. Our findings provide evidence that these cells are therapeutic targets.

Extracellular Vesicle-Educated Macrophages Promote Early Achilles Tendon Healing.

Tendon healing follows a complex series of coordinated events, which ultimately produces a mechanically inferior tissue more scar-like than native tendon. More regenerative healing occurs when anti-inflammatory M2 macrophages play a more dominant role. Mesenchymal stromal/stem cells (MSCs) are able to polarize macrophages to an M2 immunophenotype via paracrine mechanisms. We previously reported that coculture of CD14+ macrophages (MQs) with MSCs resulted in a unique M2-like macrophage. More recently, we generated M2-like macrophages using only extracellular vesicles (EVs) isolated from MSCs creating "EV-educated macrophages" (also called exosome-educated macrophages [EEMs]), thereby foregoing direct use of MSCs. For the current study, we hypothesized that cell therapy with EEMs would improve in vivo tendon healing by modulating tissue inflammation and endogenous macrophage immunophenotypes. We evaluated effects of EEMs using a mouse Achilles tendon rupture model and compared results to normal tendon healing (without any biologic intervention), MSCs, MQs, or EVs. We found that exogenous administration of EEMs directly into the wound promoted a healing response that was significantly more functional and more regenerative. Injured tendons treated with exogenous EEMs exhibited (a) improved mechanical properties, (b) reduced inflammation, and (c) earlier angiogenesis. Treatment with MSC-derived EVs alone were less effective functionally but stimulated a biological response as evidenced by an increased number of endothelial cells and decreased M1/M2 ratio. Because of their regenerative and immunomodulatory effects, EEM treament could provide a novel strategy to promote wound healing in this and various other musculoskeletal injuries or pathologies where inflammation and inadequate healing is problematic. Stem Cells 2019;37:652-662.

Immune modulation with primed mesenchymal stem cells delivered via biodegradable scaffold to repair an Achilles tendon segmental defect.

Tendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN-γ, TNF-α, and IL-1ß may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF-α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC-seeded PLG scaffold, or TNF-α-primed MSC-seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post-injury. In vivo, MSCs, regardless of priming, increased IL-10 production and reduced the inflammatory factor, IL-1α. Primed MSCs reduced IL-12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti-inflammatory factor IL-4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post-injury. Taken together delivery of TNF-α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC-induced healing response. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:269-280, 2017.

Primed Mesenchymal Stem Cells Alter and Improve Rat Medial Collateral Ligament Healing.

Cell therapy with mesenchymal stem cells (MSCs) can improve tissue healing. It is possible, however, that priming MSCs prior to implantation can further enhance their therapeutic benefit. This study was then performed to test whether priming MSCs to be more anti-inflammatory would enhance healing in a rat ligament model, i.e. a medial collateral ligament (MCL). MSCs were primed for 48 h using polyinosinic acid and polycytidylic acid (Poly (I:C)) at a concentration of 1 µg/ml. Rat MCLs were surgically transected and administered 1 × 10(6) cells in a carrier solution at the time of injury. A series of healing metrics were analyzed at days 4 and 14 post-injury in the ligaments that received primed MSCs, unprimed MSCs, or no cells (controls). Applying primed MSCs beneficially altered healing by affecting endothelialization, type 2 macrophage presence, apoptosis, procollagen 1α, and IL-1Ra levels. When analyzing MSC localization, both primed and unprimed MSCs co-localized with endothelial cells and pericytes suggesting a supportive role in angiogenesis. Priming MSCs prior to implantation altered key ligament healing events, resulted in a more anti-inflammatory environment, and improved healing.

Endogenously produced Indian Hedgehog regulates TGFß-driven chondrogenesis of human bone marrow stromal/stem cells.

Human bone marrow stromal/stem cells (hBMSCs) have an inherent tendency to undergo hypertrophy when induced into the chondrogenic lineage using transforming growth factor-beta 1 (TGFß) in vitro, reminiscent of what occurs during endochondral ossification. Surprisingly, Indian Hedgehog (IHH) has received little attention for its role during hBMSC chondrogenesis despite being considered a master regulator of endochondral ossification. In this study, we investigated the role that endogenously produced IHH plays during hBMSC chondrogenesis. We began by analyzing the expression of IHH throughout differentiation using quantitative polymerase chain reaction and found that IHH expression was upregulated dramatically upon chondrogenic induction and peaked from days 9 to 12 of differentiation, which coincided with a concomitant increase in the expression of chondrogenesis- and hypertrophy-related markers, suggesting a potential role for endogenously produced IHH in driving hBMSC chondrogenesis. More importantly, pharmacological inhibition of Hedgehog signaling with cyclopamine or knockdown of IHH almost completely blocked TGFß1-induced chondrogenesis in hBMSCs, demonstrating that endogenously produced IHH is necessary for hBMSC chondrogenesis. Furthermore, overexpression of IHH was sufficient to drive chondrogenic differentiation, even when TGFß signaling was inhibited. Finally, stimulation with TGFß1 induced a significant and sustained upregulation of IHH expression within 3 h that preceded an upregulation in all cartilage-related genes analyzed, and knockdown of IHH blocked the effects of TGFß1 entirely, suggesting that the effects of TGFß1 are being mediated through endogenously produced IHH. Together, our findings demonstrate that endogenously produced IHH is playing a critical role in regulating hBMSC chondrogenesis.

Interleukin-1 receptor antagonist modulates inflammation and scarring after ligament injury.

Ligaments have limited regenerative potential and as a consequence, repair is protracted and results in a mechanically inferior tissue more scar-like than native ligament. We previously reported that a single injection of interleukin-1 receptor antagonist (IL-1Ra) delivered at the time of injury, decreased the number of M2 macrophage-associated inflammatory cytokines. Based on these results, we hypothesized that IL-1Ra administered after injury and closer to peak inflammation (as would occur clinically), would more effectively decrease inflammation and thereby improve healing. Since IL-1Ra has a short half-life, we also investigated the effect of multiple injections. The objective of this study was to elucidate healing of a medial collateral ligament (MCL) with either a single IL-1Ra injection delivered one day after injury or with multiple injections of IL-1Ra on days 1, 2, 3, and 4. One day after MCL injury, rats received either single or multiple injections of IL-1Ra or PBS. Tissue was then collected at days 5 and 11. Both single and multiple IL-1Ra injections reduced inflammatory cytokines, but did not change mechanical behavior. A single injection of IL-1Ra also reduced the number of myofibroblasts and increased type I procollagen. Multiple IL-1Ra doses provided no additive response and, in fact, reduced the M2 macrophages. Based on these results, a single dose of IL-1Ra was better at reducing the MCL-derived inflammatory cytokines compared to multiple injections. The changes in type I procollagen and myofibroblasts further suggest a single injection of IL-1Ra enhanced repair of the ligament but not sufficiently to improve functional behavior.

Enhanced medial collateral ligament healing using mesenchymal stem cells: dosage effects on cellular response and cytokine profile.

Mesenchymal stem cells (MSCs) have potential therapeutic applications for musculoskeletal injuries due to their ability to differentiate into several tissue cell types and modulate immune and inflammatory responses. These immune-modulatory properties were examined in vivo during early stage rat medial collateral ligament healing. Two different cell doses (low dose 1 × 10(6) or high dose 4 × 10(6) MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. At both times, the high dose MSC group demonstrated a significant decrease in M2 macrophages compared to controls. At day 14, fewer M1 macrophages were detected in the low dose group compared to the high dose group. These results, along with significant changes in procollagen I, proliferating cells, and endothelialization suggest that MSCs can alter the cellular response during healing in a dose-dependent manner. The higher dose ligaments also had increased expression of several pro-inflammatory cytokines at day 5 (IL-1ß, IFNγ, IL-2) and increased expression of IL-12 at day 14. Mechanical testing at day 14 revealed increased failure strength and stiffness in low dose ligaments compared to controls. Based on these improved mechanical properties, MSCs enhanced functional healing when applied at a lower dose. Different doses of MSCs uniquely affected the cellular response and cytokine expression in healing ligaments. Interestingly, the lower dose of cells proved to be most effective in improving functional properties.

Interleukin expression after injury and the effects of interleukin-1 receptor antagonist.

Ligament healing follows a series of complex coordinated events involving various cell types, cytokines, as well as other factors, producing a mechanically inferior tissue more scar-like than native tissue. Macrophages provide an ongoing source of cytokines to modulate inflammatory cell adhesion and migration as well as fibroblast proliferation. Studying interleukins inherent to ligament healing during peak macrophage activation and angiogenesis may elucidate inflammatory mediators involved in subsequent scar formation. Herein, we used a rat healing model assayed after surgical transection of their medial collateral ligaments (MCLs). On days 3 and 7 post-injury, ligaments were collected and used for microarray analysis. Of the 12 significantly modified interleukins, components of the interleukin-1 family were significantly up-regulated. We therefore examined the influence of interleukin-1 receptor antagonist (IL-1Ra) on MCL healing. Transected rat MCLs received PBS or IL-1Ra at the time of surgery. Inhibition of IL-1 activation decreased pro-inflammatory cytokines (IL-1α, IL-1ß, IL-12, IL-2, and IFN-γ), myofibroblasts, and proliferating cells, as well as increased anti-inflammatory cytokines (IL-10), endothelial cells/blood vessel lumen, M2 macrophages, and granulation tissue size without compromising the mechanical properties. These results support the concept that IL-1Ra modulates MCL-localized granulation tissue components and cytokine production to create a transient environment that is less inflammatory. Overall, IL-1Ra may have therapeutic potential early in the healing cascade by stimulating the M2 macrophages and altering the granulation tissue components. However, the single dose of IL-1Ra used in this study was insufficient to maintain the more regenerative early response. Due to the transient influence on most of the healing components tested, IL-1Ra may have greater therapeutic potential with sustained delivery.

Tracking injectable microspheres in dynamic tissues with encapsulated superparamagnetic iron oxide nanoparticles.

Trackable spheres of similar size to those typically used for sustained protein delivery are prepared by incorporating superparamagnetic iron oxide (SPIO) nanoparticles into the core of poly(lactide-co-glycolide) microspheres. The visibility of injections in static and temporally in dynamic tissue systems is demonstrated. This method improves upon other, less sensitive imaging modalities in their ability to track injectable delivery systems. The results obtained confirm the localization of microspheres to the injected target area and highlight the novelty of tracking delivery vehicles for other applications.

The influence of macrophage depletion on ligament healing.

Despite a complex cascade of cellular events to reconstruct damaged extracellular matrix (ECM), ligament healing results in a mechanically inferior, scar-like tissue. During normal healing, the number of macrophages significantly increases within the wound site. Then, granulation tissue expands into any residual, normal ligamentous tissue (creeping substitution), resulting in a larger region of healing, greater mechanical compromise, and an inefficient repair process. To study the effects of macrophages on the repair process, bilateral, surgical rupture of their medial collateral ligaments (MCLs) was done on rats. Treatment animals received liposome-encapsulated clodronate, 2 days before rupture to ablate phagocytosing macrophages. Ligaments were then collected at days 5, 11, and 28 for immunohistochemistry (IHC) and/or mechanical testing. Clodronate treatment reduced both the M1 and M2 macrophages at day 5 and altered early healing. However, the macrophages effectively returned to control levels after day 5 and reinitiated a wound-healing response. Our results suggest that an early macrophage response, which is necessary for debridement of damaged tissue in the wound, is also important for cytokine release to mediate normal repair processes. Additionally, nonspecific inhibition of macrophages (without regard to specific macrophage populations) can control excessive granulation tissue formation but is detrimental to early matrix formation and ligament strength.

The spatio-temporal dynamics of ligament healing.

Ligament injury commonly occurs with no effective treatment to restore its original state. Numerous studies have examined wound healing after injury, reporting a provisional matrix and scar formation within the wound. Few studies however report the inflammatory, proliferative, and remodeling process during ligament healing in a spatio-temporal manner. Our goal was then to more completely elucidate this process in a rat medial collateral ligament (MCL) healing model. In this study, medial collateral ligaments were surgically transected and allowed to heal. At 1, 3, 5, 7, 9, 11, 14, and 28 days postinjury ligaments were collected and examined with microangiography or immunohistochemistry. We demonstrate that neutrophils and mitotic cells peak between 1 and 5 days postinjury. The majority of factors crest between 5 and 9 days postinjury, including circulating macrophages, resident macrophages, T lymphocytes, hematopoietic cells, vascular endothelial growth factor, and blood vessels. The apoptotic cells predominate from day 9 to the end of the study (day 28). Initially, most assayed markers localize to the epiligament and to granulation tissue at the site of damage. Later, the healing region with its granulation tissue and cells continues to expand into the uninjured tissue. From these results, we have expanded current descriptions of ligament healing and offer a more complete representation of the healing process.

Estradiol and luteinizing hormone regulation of insulin-like growth factor binding protein production by bovine granulosa and thecal cells.

To determine the effects of estradiol and luteinizing hormone (LH) on insulin-like growth factor-binding protein (IGFBP) production by bovine granulosa and thecal cells, both cell types were collected and cultured in serum-free medium with various hormone treatments, arranged in three experiments. In thecal cells, insulin stimulated (p < 0.05) production of IGFBP-2 and IGFBP-5, but had no effect (p > 0.10) on IGFBP-3 and IGFBP-4 production; LH stimulated (p < 0.05) production of IGFBP-2 and IGFBP-3 but had no effect (p > 0.05) on IGFBP-4 and IGFBP-5. Estradiol had no effect (p > 0.10) on IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 production by thecal cells. Production of IGFBP-2/-5 by granulosa cells from small follicles was inhibited (p < 0.05) by insulin, but estradiol and LH did not influence (p > 0.10) insulin's inhibitory effect on basal IGFBP-2/-5 production. Insulin, LH, and estradiol each inhibited IGFBP-4 production by small-follicle granulosa cells, but their effects were not additive. IGFBP-3 was not produced by small-follicle granulosa cells. In large-follicle granulosa cells, insulin and LH inhibited (p < 0.05) production of IGFBP-2/-5 and IGFBP-3, whereas estradiol had no effect. Insulin alone had no effect (p > 0.10) on production of IGFBP-4, but estradiol and LH inhibited (p < 0.05) production by large-follicle granulosa cells, and their effects were not additive. These results suggest that production of IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 by granulosa and thecal cells is differentially affected by hormonal stimuli.

Influence of gonadotropins on insulin- and insulin-like growth factor-I (IGF-I)-induced steroid production by bovine granulosa cells.

To determine the effect of gonadotropins on insulin- and insulin-like growth factor (IGF-I)-induced bovine granulosa cell functions, granulosa cells from bovine ovarian follicles were cultured for 2 days in the presence of 10% fetal calf serum (FCS), and then cultured for an additional 2 days in serum-free medium with added hormones. In the presence of 0 or 1 ng/mL of insulin or IGF-I, FSH had little or no effect (P > 0.05) on estradiol production by granulosa cells from both small (1-5mm) and large (> or = 8mm) follicles. However, in the presence of > or = 3 ng/mL of insulin, FSH increased (P < 0.05) estradiol production by granulosa cells from small and large follicles such that the estimated dose (ED(50)) of insulin necessary to stimulate 50% of the maximum estradiol production was decreased by 2- to 3-fold from 22 to 28 ng/mL in the absence of FSH to 7-14 ng/mL in the presence of FSH. Similarly, in the presence of > or = 3 ng/mL of IGF-I, FSH increased (P< 0.05) estradiol production by granulosa cells from small and large follicles such that the ED(50) of IGF-I for estradiol production was decreased by 4- to 5-fold from 25 to 36 ng/mL in the absence of FSH to 5-6 ng/mL in the presence of FSH. In the presence of FSH, the maximal effect of insulin on estradiol production was much greater than that of IGF-I (137- versus 12-fold increase) and were not additive; when combined, 100 ng/mL of IGF-I completely blocked the stimulatory effect of 100 ng/mL of insulin. In the absence of FSH, the maximal effect of insulin and IGF-I on estradiol production was similar. Concomitant treatment with 30 ng/mL of LH reduced (P<0.05) insulin-stimulated estradiol production by 52% on day 1 and 19% on day 2 of treatment. Insulin, IGF-I and FSH also increased (P<0.05) granulosa cell numbers and progesterone production but their maximal effects were less (i.e., < 4-fold increase) than their effects on estradiol production. In conclusion, insulin and IGF-I synergize with FSH to directly regulate ovarian follicular function in cattle, particularly granulosa cell aromatase activity.

Effect of follicle size on in vitro production of steroids and insulin-like growth factor (IGF)-I, IGF-II, and the IGF-binding proteins by equine ovarian granulosa cells.

Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medium (16-25 mm), and large (25-48 mm) follicles were collected from cyclic mares (n = 14), cultured for 2 days in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 days in serum-free medium with or without added hormones. In experiment 1, large-follicle granulosa cells produced less progesterone and more estradiol than did medium- and/or small-follicle granulosa cells (P < 0.05). Progesterone production was inhibited (P < 0.05) by FSH and insulin in small- and medium- but not in large-follicle granulosa cells; estradiol was without effect. Insulin increased (P < 0.05) estradiol production in small- and medium-follicle granulosa cells but had no effect in large-follicle granulosa cells. In experiment 2, IGF-I production was inhibited (P < 0.05) by insulin across all follicle sizes but was not affected by estradiol or FSH. Granulosa cells of medium and large follicles produced more IGF-II than did granulosa cells of small follicles (P < 0.05). Insulin and FSH inhibited (P < 0.05) IGF-II production by granulosa cells of large and medium but not of small follicles; estradiol was without effect. Only IGFBP-2 and -5 were produced by equine granulosa cells. Production of IGFBP-2 was less (P < 0.10) in granulosa cells of large versus those of small and medium follicles, whereas medium-follicle granulosa cells produced more (P < 0.05) IGFBP-5 than did small- or large-follicle granulosa cells. Averaged across follicle sizes, estradiol increased (P < 0.05) IGFBP-2 production, FSH increased (P < 0.10) IGFBP-2 and -5 production, and insulin was without effect. These results indicate that IGF-I, IGF-II, IGFBP-2, and IGFBP-5 are produced by equine granulosa cells and that insulin, FSH, and estradiol play a role in the regulation of steroidogenesis and the IGF system of equine granulosa cells.