RESUMO
Osteosarcoma is an aggressive bone cancer affecting both humans and dogs, often leading to pulmonary metastasis. Despite surgery and chemotherapy being the primary treatment modalities, survival rates remain low in both species, underscoring the urgent need for more efficacious therapeutic options. Accumulating evidence indicates numerous biological and clinical similarities between human and canine osteosarcoma, making it an ideal choice for comparative oncological research that should benefit both species. The EphA2 receptor has been implicated in controlling invasive responses across different human malignancies, and its expression is associated with poor prognosis. In this study, we utilized a comparative approach to match EphA2 functions in human and canine osteosarcoma models. Our objectives were to assess EphA2 levels and its pro-malignant action in osteosarcoma cells of both species. We found that EphA2 is overexpressed in most of both canine and human osteosarcoma cell lines, while its silencing significantly reduced cell viability, migration, and invasion. Moreover, EphA2 silencing enhanced the sensitivity of osteosarcoma cells to cisplatin, a drug commonly used for treating this cancer. Furthermore, inhibition of EphA2 expression led to a significant reduction in tumor development capability of canine osteosarcoma cells. Our data suggest that these EphA2 effects are likely mediated through various signaling mechanisms, including the SRC, AKT, and ERK-MAPK pathways. Collectively, our findings indicate that EphA2 promotes malignant behaviors in both human and canine osteosarcoma and that targeting EphA2, either alone or in combination with chemotherapy, could offer potential benefits to osteosarcoma patients.
Assuntos
Movimento Celular , Invasividade Neoplásica , Osteossarcoma , Receptor EphA2 , Animais , Cães , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Receptor EphA2/metabolismo , Receptor EphA2/genéticaRESUMO
Eph receptors and their ephrin ligands are viewed as promising targets for cancer treatment; however, targeting them is hindered by their context-dependent functionalities. To circumvent this, we explore molecular landscapes underlying their pro- and anti-malignant activities. Using unbiased bioinformatics approaches, we construct a cancer-related network of genetic interactions (GIs) of all Ephs and ephrins to assist in their therapeutic manipulation. We also apply genetic screening and BioID proteomics and integrate them with machine learning approaches to select the most relevant GIs of one Eph receptor, EPHB6. This identifies a crosstalk between EPHB6 and EGFR, and further experiments confirm the ability of EPHB6 to modulate EGFR signaling, enhancing the proliferation of cancer cells and tumor development. Taken together, our observations show EPHB6 involvement in EGFR action, suggesting its targeting might be beneficial in EGFR-dependent tumors, and confirm that the Eph family genetic interactome presented here can be effectively exploited in developing cancer treatment approaches.
Assuntos
Efrinas , Neoplasias , Efrinas/genética , Proteômica , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Transdução de Sinais , Receptores ErbB/genética , Neoplasias/genéticaRESUMO
PURPOSE: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. EXPERIMENTAL DESIGN: Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. RESULTS: Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab. CONCLUSIONS: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment. See related commentary by Kumar, p. 2570.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Humanos , Receptores ErbB/metabolismo , Linhagem Celular Tumoral , Cetuximab/farmacologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Recently, several radiometalated peptides have been approved for clinical imaging and/or therapy (theranostics) of several types of cancer; nonetheless, the primary challenge that most of these peptides confront is significant renal uptake and retention, which is often dose limiting and can cause nephrotoxicity. In response to this, numerous methods have been employed to reduce the uptake of radiometalated peptides in the kidneys, and among these is adding a linker to modulate polarity and/or charge. To better understand the influence of net charge on the biodistribution of radiometalated peptides, we selected the clinically popular construct DOTA-TATE (NETSPOT/LUTATHERA) as a model system. We synthesized derivatives using manual solid-phase peptide synthesis methods including mechanical and ultrasonic agitation to effectively yield the gold standard DOTA-TATE and a series of derivatives with different net charges (+2, +1, 0, -1, -2). Dynamic PET imaging from 0 to 90 min in healthy female mice (CD1) revealed high accumulation and retention of activity in the kidneys for the net-neutral (0) charged [68Ga]Ga-DOTA-TATE and even higher for positively charged derivatives, whereas negatively charged derivatives exhibited low accumulation and fast renal excretion. Ex vivo biodistribution at 2 h post injection demonstrated a significant retention of [68Ga]Ga-DOTA-TATE (â¼74 %ID/g) in the kidneys, which increased as the net positive charge per molecule increased to +1 and +2 (â¼272 %ID/g and â¼333 %ID/g, respectively), but the -1 and -2 net charged molecules exhibited lower renal uptake (â¼15 %ID/g and 16 %ID/g, respectively). Interestingly, the net -2 charged [68Ga]Ga-DOTA-(Glu)2-PEG4-TATE was stable in blood serum but had much higher healthy organ uptake (lungs, liver, spleen) than the net -1 compound, suggesting instability in vivo. Although the [68Ga]Ga-DOTA-PEG4-TATE derivative with a net charge of 0 also showed a decrease in kidney uptake, it also showed instability in blood serum and in vivo. Despite the superior pharmacokinetics of the net -1 charged [68Ga]Ga-DOTA-Glu-PEG4-TATE in healthy mice with respect to kidney uptake and overall profile, dynamic PET images and ex vivo biodistribution in male mice (NSG) bearing AR42J (SSTR2 overexpressing) subcutaneous tumor xenografts showed significantly diminished tumor uptake when compared to the gold standard [68Ga]Ga-DOTA-TATE. Taken together, these findings indicate unambiguously that kidney uptake and retention are significantly influenced by the net charge of peptide-based radiotracers. In addition, it was illustrated that the negatively charged peptides had substantially decreased kidney uptake, but in this instantiation the tumor uptake was also impaired.
Assuntos
Radioisótopos de Gálio , Tomografia por Emissão de Pósitrons , Camundongos , Masculino , Humanos , Feminino , Animais , Radioisótopos de Gálio/química , Distribuição Tecidual , Tomografia por Emissão de Pósitrons/métodos , Compostos Heterocíclicos com 1 Anel , Baço , Compostos Radiofarmacêuticos/químicaRESUMO
Chromosomal rearrangements involving the mixed-lineage leukemia (MLL1) gene are common in a unique group of acute leukemias, with more than 100 fusion partners in this malignancy alone. However, do these fusions occur or have a role in solid tumors? We performed extensive network analyses of MLL1-fusion partners in patient datasets, revealing that multiple MLL1-fusion partners exhibited significant interactions with the androgen-receptor signaling pathway. Further exploration of tumor sequence data from TCGA predicts the presence of MLL1 fusions with truncated SET domain in prostate tumors. To investigate the physiological relevance of MLL1 fusions in solid tumors, we engineered a truncated version of MLL1 by fusing it with one of its known fusion partners, ZC3H13, to use as a model system. Functional characterization with cell-based assays revealed that MLL1-ZC3H13 fusion induced chromosomal instability, affected mitotic progression, and enhanced tumorsphere formation. The MLL1-ZC3H13 chimera consistently increased the expression of a cancer stem cell marker (CD44); in addition, we detected potential collateral lethality between DOT1L and MLL1 fusions. Our work reveals that MLL1 fusions are likely prevalent in solid tumors and exhibit a potential pro-tumorigenic role.
Assuntos
Proteínas de Ciclo Celular/genética , Instabilidade Cromossômica/genética , Proteína de Leucina Linfoide-Mieloide/genética , Fusão Oncogênica , Proteínas de Fusão Oncogênica/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Bases , Biomarcadores Tumorais/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Células Clonais , Redes Reguladoras de Genes , Células HCT116 , Humanos , Modelos Biológicos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares , Proteínas de Fusão Oncogênica/metabolismo , Fenótipo , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/metabolismoRESUMO
One of the most promising approaches to improve recovery after spinal cord injury (SCI) is the augmentation of spontaneously occurring plasticity in uninjured neural pathways. Acute intermittent hypoxia (AIH, brief exposures to reduced O2 levels alternating with normal O2 levels) initiates plasticity in respiratory systems and has been shown to improve recovery in respiratory and non-respiratory spinal systems after SCI in experimental animals and humans. Although the mechanism by which AIH elicits its effects after SCI are not well understood, AIH is known to alter protein expression in spinal neurons in uninjured animals. Here, we examine hypoxia- and plasticity-related protein expression using immunofluorescence in spinal neurons in SCI rats that were treated with AIH combined with motor training, a protocol which has been demonstrated to improve recovery of forelimb function in this lesion model. Specifically, we assessed protein expression in spinal neurons from animals with incomplete cervical SCI which were exposed to AIH treatment + motor training either for 1 or 7 days. AIH treatment consisted of 10 episodes of AIH: (5 min 11% O2: 5 min 21% O2) for 7 days beginning at 4 weeks post-SCI. Both 1 or 7 days of AIH treatment + motor training resulted in significantly increased expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) relative to normoxia-treated controls, in neurons both proximal (cervical) and remote (lumbar) to the SCI. All other markers examined were significantly elevated in the 7 day AIH + motor training group only, at both cervical and lumbar levels. These markers included vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and phosphorylated and nonphosphorylated forms of the BDNF receptor tropomyosin-related kinase B (TrkB). In summary, AIH induces plasticity at the cellular level after SCI by altering the expression of major plasticity- and hypoxia-related proteins at spinal regions proximal and remote to the SCI. These changes occur under the same AIH protocol which resulted in recovery of limb function in this animal model. Thus AIH, which induces plasticity in spinal circuitry, could also be an effective therapy to restore motor function after nervous system injury.
Assuntos
Vértebras Cervicais/fisiopatologia , Hipóxia/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Neurônios/patologia , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/fisiopatologia , Doença Aguda , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Vértebras Cervicais/patologia , Colina O-Acetiltransferase/metabolismo , Substância Cinzenta/patologia , Substância Cinzenta/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Vértebras Lombares/patologia , Vértebras Lombares/fisiopatologia , Masculino , Atividade Motora , Ratos Endogâmicos Lew , Receptor trkB/metabolismo , Traumatismos da Medula Espinal/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Triple-negative breast cancer (TNBC) tumours that lack expression of oestrogen, and progesterone receptors, and do not overexpress the HER2 receptor represent the most aggressive breast cancer subtype, which is characterised by the resistance to therapy in frequently relapsing tumours and a high rate of patient mortality. This is likely due to the resistance of slowly proliferating tumour-initiating cells (TICs), and understanding molecular mechanisms that control TICs behaviour is crucial for the development of effective therapeutic approaches. Here, we present our novel findings, indicating that an intrinsically catalytically inactive member of the Eph group of receptor tyrosine kinases, EPHB6, partially suppresses the epithelial-mesenchymal transition in TNBC cells, while also promoting expansion of TICs. Our work reveals that EPHB6 interacts with the GRB2 adapter protein and that its effect on enhancing cell proliferation is mediated by the activation of the RAS-ERK pathway, which allows it to elevate the expression of the TIC-related transcription factor, OCT4. Consistent with this, suppression of either ERK or OCT4 activities blocks EPHB6-induced pro-proliferative responses. In line with its ability to trigger propagation of TICs, EPHB6 accelerates tumour growth, potentiates tumour initiation and increases TIC populations in xenograft models of TNBC. Remarkably, EPHB6 also suppresses tumour drug resistance to DNA-damaging therapy, probably by forcing TICs into a more proliferative, drug-sensitive state. In agreement, patients with higher EPHB6 expression in their tumours have a better chance for recurrence-free survival. These observations describe an entirely new mechanism that governs TNBC and suggest that it may be beneficial to enhance EPHB6 action concurrent with applying a conventional DNA-damaging treatment, as it would decrease drug resistance and improve tumour elimination.
Assuntos
Receptores da Família Eph/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Dano ao DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Nus , Recidiva Local de Neoplasia/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas ras/metabolismoRESUMO
Metabolic alterations play an important role in cancer and yet, few metabolic cancer driver genes are known. Here we perform a combined genomic and metabolic modeling analysis searching for metabolic drivers of colorectal cancer. Our analysis predicts FUT9, which catalyzes the biosynthesis of Ley glycolipids, as a driver of advanced-stage colon cancer. Experimental testing reveals FUT9's complex dual role; while its knockdown enhances proliferation and migration in monolayers, it suppresses colon cancer cells expansion in tumorspheres and inhibits tumor development in a mouse xenograft models. These results suggest that FUT9's inhibition may attenuate tumor-initiating cells (TICs) that are known to dominate tumorspheres and early tumor growth, but promote bulk tumor cells. In agreement, we find that FUT9 silencing decreases the expression of the colorectal cancer TIC marker CD44 and the level of the OCT4 transcription factor, which is known to support cancer stemness. Beyond its current application, this work presents a novel genomic and metabolic modeling computational approach that can facilitate the systematic discovery of metabolic driver genes in other types of cancer.
Assuntos
Neoplasias Colorretais/metabolismo , Biologia Computacional/métodos , Fucosiltransferases/metabolismo , Algoritmos , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Fucosiltransferases/genética , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Genômica , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
Death Receptor 5 (DR5) is a promising target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells. However, the therapeutic usefulness of DR5 agonists is currently limited by the frequent resistance of malignant tumours to its activation. The identification of molecular mechanisms that determine outcomes of DR5 action is therefore crucial for improving the efficiency of DR5-activating reagents in cancer treatment. Here, we provide evidence that an intrinsically kinase-inactive member of the Eph group of receptor tyrosine kinases, EPHB6, induces marked fragmentation of the mitochondrial network in breast cancer cells of triple-negative origin, lacking expression of the estrogen, progesterone and HER2 receptors. Remarkably, this response renders cancer cells more susceptible to DR5-mediated apoptosis. EPHB6 action in mitochondrial fragmentation proved to depend on its ability to activate the ERK-DRP1 pathway, which increases the frequency of organelle fission. Moreover, DRP1 activity is also essential to the EPHB6-mediated pro-apoptotic response that we observe in the context of DR5 activation. These findings provide the first description of a member of the receptor tyrosine kinase family capable of producing a pro-apoptotic effect through the activation of ERK-DRP1 signaling and subsequent mitochondrial fragmentation. Our observations are of potential practical importance, as they imply that DR5-activating therapeutic approaches should be applied in a more personalized manner to primarily treat EPHB6-expressing tumours. Finally, our findings also suggest that the EPHB6 receptor itself may represent a promising target for cancer therapy, since EPHB6 and DR5 co-activation should support more efficient elimination of cancer cells.
Assuntos
Mitocôndrias/metabolismo , Receptores da Família Eph/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Two experiments were done to develop an effective superovulatory treatment protocol in wood bison for the purpose of embryo collection and transfer. In experiment 1, donor bison were assigned randomly to four treatment groups (N = 5 per group) to examine the effects of method of synchronization (follicular ablation vs. estradiol-progesterone treatment) and ovarian follicular superstimulation (single slow-release vs. split dose of FSH). Recipient bison were synchronized with donor bison by either follicular ablation (N = 8) or estradiol-progesterone treatment (N = 9). In experiment 2, bison were assigned randomly to four treatment groups (N = 5 per group) to examine the ovarian response to two versus four doses of FSH, and the effect of progesterone (ovarian superstimulation with or without an intravaginal progesterone-releasing device). Donor bison were inseminated with fresh chilled wood bison semen 12 and 24 hours after treatment with GnRH (experiment 1) or LH (experiment 2). The ovarian response was assessed using ultrasonography. In experiment 1, the number of large follicles (≥ 7 mm) increased in response to both FSH treatments, but the diameter of the largest follicle detected 4 and 5 days after the start of ovarian superstimulation was greater in bison treated with a single dose of FSH than in those treated with two doses (P < 0.05). A total of 10 ova and/or embryos were collected. One blastocyst was transferred to each of five recipient bison resulting in the birth of two live wood bison calves. In experiment 2, two doses of FSH resulted in a greater number of large follicles (≥ 9 mm) on Days 4, 5, and 6 (P < 0.05) after beginning of superstimulation (Day 0), and more ovulations than four doses of FSH (11.2 ± 2.4 vs. 6.4 ± 0.8; P < 0.05). Embryo collection was performed on only five donors, and a total of 19 ova and/or embryos were recovered. In summary, fewer FSH treatments were as good or better than multiple treatments, consistent with the notion that minimizing handling stress improves the superovulatory response in bison. Follicular ablation and estradiol plus progesterone treatment were effective for inducing ovarian synchronization in embryo donor and recipient bison, and an intravaginal progesterone-releasing device during superstimulatory treatment did not influence the superovulatory response or embryo collection. Delaying ovulation-inducing treatment (GnRH or LH) to 5 days after superstimulatory treatment resulted in a greater number of ovulations and improved embryo collection efficiency (experiment 2). Embryo collection and transfer resulted in live offspring from wild wood bison.
Assuntos
Bison/fisiologia , Transferência Embrionária/veterinária , Hormônio Foliculoestimulante/administração & dosagem , Folículo Ovariano/efeitos dos fármacos , Indução da Ovulação/veterinária , Superovulação , Animais , Cruzamento , Transferência Embrionária/métodos , Estradiol/administração & dosagem , Estradiol/farmacologia , Sincronização do Estro , Feminino , Hormônio Foliculoestimulante/farmacologia , Indução da Ovulação/métodos , Progesterona/administração & dosagem , Progesterona/farmacologiaRESUMO
Treatment of non-prolific western white-faced ewes with prostaglandin F(2α) (PGF(2α)) and medroxyprogesterone acetate (MAP) increases the ovulation rate as a result of ovulations from the penultimate wave in addition to the final wave of the cycle. The objective of the current study was to evaluate the expression of markers of vascularization/angiogenesis, a marker of intercellular communication, and cellular proliferation and apoptosis in follicles from the penultimate and final waves. On day 8 of the estrous cycle, 15 ewes were administered a single injection of PGF(2α) and an intravaginal MAP sponge, which remained in place for 6 days. Two days after sponge removal, ovaries which contained follicles from the penultimate and final waves were collected and processed for immunohistochemistry followed by image analysis, and for quantitative real-time RT-PCR. Expression of factor VIII (marker of vascularization), proliferating cell nuclear antigen, and GJA1 (Cx43; marker of gap junctional communication) was greater (P<0.05) in follicles from the final wave compared with follicles from the penultimate wave. For theca cells, mRNA expression for vascular endothelial growth factor (VEGF) was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for GJA1 and endothelial nitric oxide synthase in follicles from the final wave compared with follicles from the penultimate wave. For granulosa cells, the mRNA expression for GJA1 was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for VEGF in follicles from the final wave compared with follicles from the penultimate wave. In conclusion, extension of the lifespan of follicles in the penultimate wave reduces follicular viability in the ewe.
Assuntos
Ciclo Estral/fisiologia , Hormônio Foliculoestimulante/fisiologia , Folículo Ovariano/fisiologia , Ovulação/fisiologia , Ovinos/fisiologia , Animais , Conexina 43/genética , Conexina 43/fisiologia , Dinoprosta/farmacologia , Estradiol/análise , Fator VIII/genética , Fator VIII/fisiologia , Feminino , Células da Granulosa/fisiologia , Imuno-Histoquímica/veterinária , Acetato de Medroxiprogesterona/farmacologia , Progesterona/sangue , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/fisiologia , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tecais/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Large antral follicles grow in waves in the ewe, with each wave triggered by a peak in serum FSH concentrations. In this study, our objectives were to determine if the slope of the rise in the FSH peak affects the ability of the peak to trigger wave emergence (experiment 1), and whether increasing serum FSH concentrations and holding them at peak concentrations would provide a stimulus for constant emergence of large antral follicles (experiment 2). In experiment 1, cyclic ewes received ovine FSH (n = 6; 0.1 µg/kg, s.c.) or vehicle (n = 6; control) every 6 h for 42 h. This treatment created a peak in serum FSH concentrations (P < 0.05) during the early growth phase of the first follicular wave of the interovulatory interval and enhanced the growth of follicles in that wave (P < 0.05), but did not trigger emergence of a follicular wave. In experiment 2, cyclic ewes were infused constantly with oFSH (1.98 µg/h; n = 6) or vehicle (control; n = 6) for 60 h starting at the time of the second endogenously driven FSH peak of the interovulatory interval. Infusion of oFSH resulted in a super-stimulatory effect, with a peak in the mean number of large follicles (≥5 mm) on Day 2 after the start of FSH infusion (13 ± 1.2 large follicles per ewe, 1.8 ± 0.2 in control ewes; P < 0.001). In conclusion, exposing early growing antral follicles in a wave to a gradual increase in serum concentrations of FSH enhanced their growth, but did not trigger the expected new follicular wave, and infusion of a dose of oFSH within the physiological range caused a super-ovulatory response in cyclic ewes.
Assuntos
Ciclo Estral/sangue , Hormônio Foliculoestimulante/administração & dosagem , Hormônio Foliculoestimulante/sangue , Folículo Ovariano/metabolismo , Ovinos/sangue , Animais , Estradiol/sangue , Ciclo Estral/efeitos dos fármacos , Feminino , Folículo Ovariano/diagnóstico por imagem , Folículo Ovariano/efeitos dos fármacos , Progesterona/sangue , Distribuição Aleatória , UltrassonografiaRESUMO
The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 x 0.47 or 5 x 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe.
Assuntos
Anestro , Hormônio Luteinizante/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/fisiologia , Ovinos/metabolismo , Ovinos/fisiologia , Algoritmos , Anestro/sangue , Anestro/metabolismo , Anestro/fisiologia , Animais , Tamanho Celular/efeitos dos fármacos , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/metabolismo , Implantes de Medicamento , Estradiol/administração & dosagem , Feminino , Hormônio Foliculoestimulante/sangue , Infusões Subcutâneas , Hormônio Luteinizante/sangue , Folículo Ovariano/citologia , Ovulação/sangue , Ovulação/efeitos dos fármacos , Fluxo Pulsátil , Ovinos/sangueRESUMO
In ewes, immunization against GnRH blocks LH pulses but mean serum FSH concentrations are only partly reduced; the fate of the FSH peaks that precede ovarian follicular waves has not been studied. In this study, we used immunization against GnRH to examine the need for pulsed GnRH secretion in the genesis of FSH peaks in the anestrous ewe. Six anestrous ewes were given a GnRH immunogen on Day 0 and a booster injection on Day 28. Control ewes (n=6) received adjuvant only. Transrectal ultrasonography was performed daily for 2 days prior to and 10 days following both the primary (Days -2 to 10) and booster (Days 26-38) injections and for a 13-day period beginning 26 days after booster injection (Days 54-66). Blood samples were collected daily. Intensive bleeding (every 12min for 7h) was performed on Days 9, 37, and 65 of the experimental period to characterize the pulsatile pattern of LH secretion. GnRH antibody titers were increased and LH pulses were abolished immediately after booster immunization (P<0.05). The number of FSH peaks, FSH peak concentration and amplitude and basal FSH concentrations were only decreased in immunized ewes in the period of observations starting 26 days after booster immunization (P<0.05); however, some peaks were still seen. The number of follicular waves was decreased in the period around booster immunization and no follicular waves were seen during the period starting 26 days after booster immunization in immunized ewes (P<0.05). In summary, in anestrous ewes, when pulsed LH secretion was abolished by immunization against GnRH, the peaks in serum concentrations of FSH that trigger ovarian follicular waves continued for a period of time. We concluded that although blocking the effects of GnRH gradually causes a diminution of FSH secretion, there is no acute requirement for GnRH in the regulation of FSH peaks. The existence of FSH peaks in the absence of follicular waves, and pulsed LH secretion, suggests that some endogenous rhythm may drive the occurrence of FSH peaks, independent of both changes in negative feedback by secretory products from ovarian antral follicles and GnRH.
Assuntos
Anestro/metabolismo , Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Folículo Ovariano/fisiologia , Ovinos , Algoritmos , Anestro/sangue , Animais , Ritmo Circadiano/fisiologia , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Liberador de Gonadotropina/sangue , Hormônio Liberador de Gonadotropina/imunologia , Imunização Secundária/veterinária , Folículo Ovariano/metabolismo , Ovulação/sangue , Ovulação/metabolismo , Ovulação/fisiologia , Fluxo Pulsátil/fisiologia , Ovinos/sangue , Ovinos/metabolismo , Ovinos/fisiologia , Titulometria , Vacinas Anticoncepcionais/sangue , Vacinas Anticoncepcionais/imunologiaRESUMO
Large antral follicles grow in waves in the ewe, and each wave is triggered by a peak in serum concentrations of FSH. The existence of follicular dominance in the ewe is unclear. The objective of experiment 1 was to determine if an endogenously driven follicular wave could emerge during the growth phase of a wave induced by injection of ovine FSH (oFSH). Cyclic ewes (n = 7) were given oFSH (two injections of 0.5 microg/kg; s.c.; 8 h apart) on 2 separate days equally spaced in the interval between the first two endogenously driven follicular waves of an estrous cycle. Injection of oFSH induced two follicular waves in the interval between the first two endogenously driven waves of the cycle. The second endogenously driven wave of the estrous cycle emerged in the midgrowth phase of a follicular wave induced by injection of oFSH and its day of emergence, and growth pattern did not differ from that of the equivalent wave in control ewes (emerging 5.4 +/- 0.2 and 4.8 +/- 0.5 days after ovulation, respectively; P > 0.05). Experiment 2 was designed to determine if emergence of follicular waves could be induced on a daily basis. Six anestrus ewes were given oFSH (two injections of 0.35 microg/kg; s.c.; 8 h apart) on each of 4 days, starting 24 h after the expected time of an endogenously driven FSH peak. Each injection of oFSH resulted in a discrete peak in serum FSH concentrations and the emergence of a new follicular wave. The present findings provide evidence for the lack of follicular dominance in the ewe.
Assuntos
Hormônio Foliculoestimulante/sangue , Folículo Ovariano/efeitos dos fármacos , Ovinos/sangue , Animais , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/administração & dosagemRESUMO
BACKGROUND: In the ewe, ovarian antral follicles emerge or grow from a pool of 2-3 mm follicles in a wave like pattern, reaching greater than or equal to 5 mm in diameter before regression or ovulation. There are 3 or 4 such follicular waves during each estrous cycle. Each wave is preceded by a peak in serum FSH concentrations. The role of pulsatile LH in ovarian antral follicular emergence and growth is unclear; therefore, the purpose of the present study was to further define this role. METHODS: Ewes (n = 7) were given 200 ng of GnRH (IV) every hour for 96 h from Day 7 of the estrous cycle, to increase LH pulse frequency. Controls (n = 6) received saline. In a second study, ewes (n = 6) received subcutaneous progesterone-releasing implants for 10 days starting on Day 4 of the cycle, to decrease LH pulse frequency. Controls (n = 6) underwent sham surgery. Daily transrectal ovarian ultrasonography and blood sampling was performed on all ewes from the day of estrus to the day of ovulation at the end of the cycle of the study. At appropriate times, additional blood samples were taken every 12 minutes for 6 h and 36 min or 6 h in studies 1 and 2 respectively. RESULTS: The largest follicle of the follicular wave growing when GnRH treatment started, grew to a larger diameter than the equivalent wave in control ewes (P < 0.05). Mean serum estradiol and progesterone concentrations were higher but mean serum FSH concentrations were lower during GnRH treatment compared to control ewes (P < 0.05). The increased serum concentrations of estradiol and progesterone, in GnRH treated ewes, suppressed a peak in serum concentrations of FSH, causing a follicular wave to be missed. Treatment with progesterone decreased the frequency of LH pulses but did not have any influence on serum FSH concentrations or follicular waves. CONCLUSION: We concluded that waves of ovarian follicular growth can occur at LH pulse frequencies lower than those seen in the luteal phase of the estrous cycle but frequencies seen in the follicular phase, when applied during the mid-luteal phase, in the presence of progesterone, do enhance follicular growth to resemble an ovulatory follicle, blocking the emergence of the next wave.