RESUMO
In pigs, luteolytic sensitivity to PGF-2α (=LS) is delayed until d 13 of the estrous cycle. While the control of LS is unknown, it is temporally associated with macrophage (MAC; which secretes tumor necrosis factor [TNF]-α) infiltration into the corpora lutea (CL), and previous studies have shown that TNF-α induces LS in porcine luteal cells (LCs) in culture. This study was designed to explore the control of LS by CL macrophage (CL MAC)/TNF-α by progesterone (P4), and to examine the hypothesis that P4 acting via the genomic P4 receptor (PGR) inhibits CL MAC TNF-α and thus plays a key role in regulating LS during the pig estrous cycle. In experiment 1, the effects of LCs on CL MAC cytokine/TNF-α mRNA expression in co-culture were examined (MID cycle; ~d 7-12; no LS); results showed that LC was inhibitory to cytokine/TNF-α. In experiment 2, the effects of P4 or R5020 (PGR-agonist) on CL MAC cytokine/TNF-α mRNA expression were examined (MID cycle; ~d 7-12; no LS); results showed that both P4 and R5020 dose-dependently inhibited TNF-α. In experiment 3, CL MACs were isolated from CL at MID (~d 7-12; no LS) and LATE (~d 13-18; + LS) cycle, and TNF-α/PGR mRNA measured. Results indicated that while TNF-α mRNA was 4.2-fold greater in CL MACs from LATE vs MID cycle, PGR mRNA was 4.5-fold greater in CL MACs from MID vs LATE cycle. These data support our hypothesis and suggest that progesterone, acting via PGR, plays a critical physiological role in the control of TNF-α production by CL MACs and LS during the pig estrous cycle.
Assuntos
Citocinas/metabolismo , Macrófagos/efeitos dos fármacos , Progesterona/farmacologia , Receptores de Progesterona/metabolismo , Suínos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Citocinas/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Genômica , Macrófagos/metabolismo , Progesterona/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genéticaRESUMO
Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll-like receptors (TLR) mediate innate immune mechanisms via the production of pro-inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30-50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll-like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN-G), and interleukin (IL)-12, mRNA expressions were greatest in regressing CL compared with earlier stages (p < .05), whereas no change was observed for IL-6 mRNA expression. Cytokine mRNA expression in cultured luteal cells was not altered by LPS. Based on these data, one or more of the TLRs found within the CL may play a role in luteolysis, perhaps via pro-inflammatory cytokine mRNA expression.
Assuntos
Corpo Lúteo/metabolismo , Citocinas/genética , Ciclo Estral/genética , Prenhez/genética , RNA Mensageiro/genética , Receptores Toll-Like/genética , Animais , Bovinos , Corpo Lúteo/efeitos dos fármacos , Ciclo Estral/metabolismo , Feminino , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Luteólise/genética , Gravidez , Prenhez/metabolismo , RNA Mensageiro/metabolismo , Receptores Toll-Like/metabolismoRESUMO
The porcine corpus luteum (CL) displays delayed sensitivity to PGF-2α (luteolytic sensitivity, [LS]) until days 12 to 13 of cycle. The control of LS is unknown, but it is temporally associated with macrophage (which secrete tumor necrosis factor-α; TNF-α) infiltration into the CL. Other studies showed that TNF-α induces LS in vitro and that prostaglandins (PGs) may be involved in this mechanism. In experiment 1, PGF-2α and PGE secretion by luteal cells (LCs) was measured on days 4 to 14 of the estrous cycle, and the expression of PTGFS/AKR1B1 and PTGES/mPGES-1, determined by Western blot, before (day 7) vs after (day 13) the onset of LS. Results showed that the PGF-2α:PGE ratio increased significantly (P < 0.05) from day 4 to 13-14, and PTGFS/AKR1B1 and PTGES/mPGES-1 were significantly increased (P < 0.05) on day 13 (vs day 7). In experiment 2, LCs were collected from porcine CL at early (â¼days 4-6) or mid (â¼days 7-12) stages of the estrous cycle and cultured with 0, 0.1, 1, or 10 ng/mL TNF-α. Results showed that TNF-α significantly increased (P < 0.05) messenger RNA (mRNA) expression of cyclooxygenase (COX)-2 and mPGES-1 but not AKR1B1. TNF-α had no significant effects on AKR1B1 or mPGES protein abundance. TNF-α significantly increased (P < 0.05) PGE-2 but had no effect on PGF-2α secretion or on the PGF-2α:PGE2 ratio. In conclusion, although TNF-α increased COX2 and mPGES-1 mRNA, and PGE-2 secretion in vitro, it did not increase the PGF-2α:PGE2 ratio. Studies are currently directed toward exploring other pathways (eg, FP receptor signaling) by which TNF-α induces LS in the porcine CL.
Assuntos
Corpo Lúteo/metabolismo , Prostaglandinas/biossíntese , Sus scrofa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Aldeído Redutase/análise , Aldeído Redutase/genética , Animais , Células Cultivadas , Corpo Lúteo/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Dinoprosta/análise , Dinoprosta/biossíntese , Dinoprosta/farmacologia , Dinoprostona/análise , Dinoprostona/biossíntese , Dinoprostona/metabolismo , Feminino , Células Lúteas/metabolismo , Luteólise/efeitos dos fármacos , Prostaglandina-E Sintases/análise , Prostaglandina-E Sintases/genética , RNA Mensageiro/análiseRESUMO
The corpus luteum (CL) of the pig lacks luteolytic sensitivity (LS) to prostaglandin (PG) F-2α until after day 12 of the oestrous cycle, but the mechanisms underlying this phenomenon are poorly understood. As luteolysis involves apoptosis, we hypothesized that critical apoptotic proteins may be deficient in CLs that lack LS. The specific aim of these studies was to examine mRNA expression and protein levels of apoptosis genes/proteins (BAX/Bax, BCLX/Bcl-x, CASP3/Caspase-3, CASP8/Caspase-8, NFΚB1/NFκB, TP53/p53) in porcine CLs collected at different stages of the oestrous cycle. CLs were collected surgically, mRNA and protein extracted, and expression/levels analyzed by semi-quantitative (SQ) PCR and Western blots, respectively. At the mRNA expression level, only BAX (maximal on day 4) and TP53 (maximal on day 7) showed significant variations during the oestrous cycle. At the protein level, only Bcl-x and Caspase-3 showed significant changes during the cycle; Bcl-x decreased on day 13 and Caspase-3 increased on day 13. It is concluded that apoptosis-associated proteins (i.e. Bcl-x and Caspase 3) may play a critical role in luteolytic sensitivity in the pig.
Assuntos
Apoptose/fisiologia , Corpo Lúteo/metabolismo , Ciclo Estral/fisiologia , Regulação da Expressão Gênica/fisiologia , Suínos/fisiologia , Animais , Feminino , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Porcine corpora lutea (CL) fail to show a luteolytic response to prostaglandin-F-2alpha (PGF-2alpha) (ie, luteolytic sensitivity [LS]) until about day 12-13 of the estrous cycle. Although little is known of the control of LS in any species, endothelin-1 (EDN1) is believed to play a role in LS control in ruminants. Therefore, we measured mRNA and protein expression and examined the cellular localization of EDN1 precursor (pre-pro EDN1, or ppEDN1), EDN-converting enzyme-1 (ECE1), and EDN receptors (A, EDNRA and B, EDNRB) in porcine CLs collected on days 4, 7, 10, 13, and 15 of the estrous cycle to look for differences between CLs displaying (days 13-15) versus those lacking (days 4-10) LS. Abundance of ppEDN1 mRNA was greatest (and significant vs all other days) on day 7 of the cycle, whereas EDN1 protein expression did not vary during the cycle and was localized exclusively to endothelial cells (EC). Abundance of ECE1 mRNA was also greatest on day 7 (vs all other days), but ECE1 protein was significantly elevated on day 10 (vs day 4) and was immunolocalized to ECs and large luteal cells (LLC). Abundance of EDNRA mRNA was also maximal on day 7 (vs all other days) of the cycle, whereas EDNRA protein expression was not significantly changed during the cycle and was observed in LLCs, ECs, and small luteal cells (SLC). On day 13, EDNRB mRNA was significantly decreased (versus day 7). Expression of EDNRB protein was decreased on day 10 (versus all other days), and on days 13-15 (vs day 4), and was primarily localized to ECs. In conclusion, the observed elevation in ECE1 protein concentrations on day 10 and the presence of EDNRA on LLC suggests a possible role for EDN1 (resulting from the actions of ECE1) acting via EDNRA in the control of LS in the pig.
Assuntos
Ácido Aspártico Endopeptidases/análise , Corpo Lúteo/química , Endotelina-1/análise , Metaloendopeptidases/análise , Receptores de Endotelina/análise , Suínos , Animais , Ácido Aspártico Endopeptidases/genética , Dinoprosta/farmacologia , Células Endoteliais/química , Endotelina-1/genética , Endotelina-1/fisiologia , Enzimas Conversoras de Endotelina , Ciclo Estral , Feminino , Imunofluorescência , Expressão Gênica , Células Lúteas/química , Luteólise/efeitos dos fármacos , Luteólise/fisiologia , Metaloendopeptidases/genética , Precursores de Proteínas/análise , Precursores de Proteínas/genética , Receptor de Endotelina A/análise , Receptor de Endotelina A/genética , Receptor de Endotelina A/fisiologia , Receptores de Endotelina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Clinical and subclinical endometritis are common causes of infertility and subfertility in high producing dairy cattle, delaying the onset of ovarian cyclic activity after parturition, extending luteal phases and reducing conception rates. Escherichia coli and Arcanobacterium pyogenes cause endometrial damage and inflammation. Components of microbes, such as lipopolysaccharide (LPS), are detected by Toll-like receptors on endometrial cells, leading to secretion of cytokines, chemokines and antimicrobial peptides. Long luteal phases associated with endometritis are probably caused by a switch in endometrial prostaglandin production from prostaglandin F2a (PGF) to prostaglandin E2. In addition, LPS impairs the function of the hypothalamus and pituitary, and directly perturbs ovarian granulosa cells steroidogenesis, providing mechanisms to explain the association between uterine disease and anovulatory anoestrus. Cows with uterine disease that ovulate have lower peripheral plasma progesterone concentrations that may further reduce the chance of conception associated with endometritis.
Assuntos
Doenças dos Bovinos/etiologia , Endometrite/veterinária , Infertilidade Feminina/veterinária , Animais , Anovulação/veterinária , Infecções Bacterianas , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Endometrite/imunologia , Endometrite/microbiologia , Feminino , Infertilidade Feminina/etiologia , Infertilidade Feminina/imunologia , Lactação , Lipopolissacarídeos/imunologia , Fase Luteal/fisiologia , Progesterona/sangue , Receptores Toll-Like/imunologia , VirosesRESUMO
Porcine corpora lutea (CL) fail to show a luteolytic response to prostaglandin-F-2alpha (PGF-2alpha) (ie, luteolytic sensitivity, or LS) until approximately day 13 of the estrous cycle. In view of the importance of protein kinase C (PRKC) in PGF-2alpha signal transduction, it was hypothesized that limiting levels of 1 or more PRKC isoforms may explain the lack of LS before day 13. This hypothesis was tested by examining expression of mRNA and protein, and the cellular localization patterns of the 11 PRKC isoforms throughout the porcine estrous cycle, to determine whether PRKC expression correlates with and thus may be associated with the control of the acquisition of LS in the pig. The expression patterns show that for most PRKC isoforms (ie, PRKC alpha, beta 1, beta 2, delta, epsilon, theta, iota, and zeta), mRNA was maximally expressed on day 7 or day 10 (protein kinase D1 only) of the cycle, whereas PRKCs gamma, eta, and lambda were unchanged. At the protein level, only PRKC epsilon (PRKCE) significantly changed during the estrous cycle and was elevated on day 13 (versus days 4, 7, and 15; P<0.05). By immunofluoresence, most PRKC isoforms, including PRKCE, were localized to steroidogenic large luteal cells (LLC) and small (nonendothelial cell) luteal cell subtypes (SLC). In conclusion, since the increase in PRKCE protein expression (day 13) occurred coincidentally with the onset of LS (> or =day 12), these results support a potential role for PRKCE in control of the acquisition of LS in the pig.
Assuntos
Corpo Lúteo/enzimologia , Ciclo Estral/metabolismo , Células Lúteas/enzimologia , Proteína Quinase C/metabolismo , Animais , Corpo Lúteo/citologia , Feminino , Regulação da Expressão Gênica/fisiologia , Isoenzimas/genética , Isoenzimas/metabolismo , Luteinização/metabolismo , Proteína Quinase C/genética , RNA Mensageiro/análise , Transdução de Sinais/fisiologia , Estatísticas não Paramétricas , Suínos , Fatores de Tempo , Distribuição TecidualRESUMO
Expression and localization of mRNAs for vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt) and VEGF receptor 2 (KDR) (VEGFR-1 and VEGFR-2, respectively) were investigated in pig corpora lutea. Northern blot analysis of total RNA indicated hybridization of pig VEGF, VEGFR-1 and VEGFR-2 cDNA probes to mRNA transcripts of approximately 3.9, 7.0 and 5.0 kb, respectively. The expression of mRNAs for VEGF and its receptors during the luteal phase (days 4, 7, 10, 13 and 15 after the onset of oestrus) were assessed by northern blot analysis, and hybridization signals were normalized to expression of pig 18S rRNA. Relative hybridization signals of expression of VEGF mRNA appeared to be constant; however, expression of VEGFR-1 mRNA was low on day 4, increased on day 7, and was higher on days 10, 13 and 15 (P<0.05, compared with day 4). In contrast, no changes in expression of mRNA for VEGFR-2 were evident on days 4-13, but a decrease was detected (P<0.05) at day 15. In situ hybridization revealed that VEGF mRNA was localized predominantly in large luteal cells, whereas both VEGFR-1 and VEGFR-2 were localized to small cells. These data indicate that the VEGF system may be involved in the regulation of luteal vasculature throughout the lifespan of the corpus luteum. Although the expression of VEGF mRNA was unchanged during the luteal phase, variations in the expression of VEGFR-1 and VEGFR-2 mRNAs indicate that differential regulation of expression of the VEGF receptors may play a role in the control of VEGF-mediated vascular growth at different phases of development and maturation of the pig corpus luteum.
Assuntos
Corpo Lúteo/química , Ciclo Estral/fisiologia , RNA Mensageiro/análise , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Suínos/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Northern Blotting/métodos , Corpo Lúteo/fisiologia , Feminino , Fase Folicular/fisiologia , Hibridização In Situ/métodos , Fase Luteal/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Changes in the expression and localization of luteal mRNA for PGF(2alpha) (FP) receptors may be critical in determining the luteolytic action of PGF(2alpha) in pig corpora lutea. In this study, a full-length FP receptor (FPr) cDNA was isolated and cloned from pig corpora lutea. This isolate (GenBank accession no. U91520) contains an open reading frame of 1086 bases coding for a protein of 362 amino acids with seven potential transmembrane domains. The predicted amino acid sequence of this isolate was 83% identical to the FPr amino acid sequence of other species including sheep, cattle and humans. Northern blot analysis showed the presence of an FPr message of about 5 kb in mRNA from pig corpora lutea. Relatively weak FPr mRNA expression was detected on day 4 and day 7 of the oestrous cycle. The expression was greater (P < 0.05) on days 10, 13 and 15 than on days 4 and 7. In situ hybridization analysis revealed that mRNA for FPr was expressed predominantly in the steroidogenic large luteal subtype of cell, although there was some expression in small luteal cells, with histological appearance of steroidogenic small cells. Localization of hybridization signals of FPr was observed in luteal tissue at all stages examined. These data demonstrate that FPr is expressed in pig corpora lutea throughout the oestrous cycle and that upregulation of the FPr mRNA occurs when the corpora lutea becomes sensitive to PGF(2alpha). Direct luteal targets of PGF(2alpha) appear to be primarily large steroidogenic cells in this species.
Assuntos
Corpo Lúteo/química , DNA Complementar/genética , Dinoprosta , RNA Mensageiro/análise , Receptores de Prostaglandina/genética , Suínos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting/métodos , Bovinos , Clonagem Molecular/métodos , Ciclo Estral , Feminino , Hibridização In Situ/métodos , Camundongos , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos , OvinosRESUMO
This study was designed to investigate the roles of insulin-like growth factor I (IGF-I), IGF-type I receptor (IGF-IR) and IGF-binding proteins (IGFBPs) in regulating progesterone secretion by pig corpora lutea during the oestrous cycle, and the signal transduction pathways involved in mediating the steroidogenic actions of IGF-I. Corpora lutea were collected on days 4, 7, 10, 13 and 15 or 16 of the oestrous cycle, enzyme dissociated and the luteal cells were cultured for 24 h in Medium 199 with IGF-I (0-100 ng ml(-1)), long R(3)-IGF-I (0-100 ng ml(-1)), anti-IGF-I (Sm 1.2B; 0-10 microg ml(-1)), anti-IGF-IR (alphaIR3; 0-2 microg ml(-1)), or IGF-I signal transduction pathway inhibitors (phosphatidylinositol (PI)-3-kinase: 100 nmol Wortmannin l(-1) and 10 micromol LY 294002 l(-1); MAP kinase: 50 micromol PD 98059 l(-1)) to investigate their effects on IGF-I (100 ng ml(-1)) stimulated progesterone secretion. Pig luteal cells displayed dose-dependent responses to IGF-I and long R(3)-IGF-I on days 4 and 7 of the oestrous cycle, but not on days 10-16. There was no difference in the ED(50) or V(max) (maximal response) values between IGF-I and long R(3)-IGF-I. Neither anti-IGF-I nor anti-IGF-IR had significant effects on progesterone secretion, at any dose or day. Wortmannin and LY 294002 blocked IGF-I stimulated progesterone secretion, but PD 98059 was without effect. Finally, IGF-I (6 microg) infused into the ovary on day 7 in vivo significantly increased progesterone secretion within 45 min of infusion. The conclusions of this study are: (1) IGF-I has steroidogenic actions only on 'young' (days 4-7) pig corpora lutea; (2) endogenous IGF-I and IGFBP are insufficient to modulate progesterone secretion in vitro; and (3) the steroidogenic actions of IGF-I are mediated via PI-3-kinase.
Assuntos
Corpo Lúteo/metabolismo , Estro/metabolismo , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/farmacologia , Progesterona/metabolismo , Transdução de Sinais , Sus scrofa/metabolismo , Androstadienos/farmacologia , Animais , Células Cultivadas , Cromonas/farmacologia , Corpo Lúteo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Flavonoides/farmacologia , Fase Folicular , Fator de Crescimento Insulin-Like I/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Receptor IGF Tipo 1/antagonistas & inibidores , WortmaninaRESUMO
Insulin-like growth factor I (IGF-I) is believed to play a luteotrophic role in the pig corpus luteum during the oestrous cycle. Since the actions of IGF-I in target tissues are mediated by the type I IGF receptor, the concentrations of IGF-I receptor mRNA and protein were examined in pig corpora lutea at different stages of the oestrous cycle. Corpora lutea were collected from normally cyclic gilts on days 4, 7, 10, 13, 15 and 16 of the oestrous cycle (n = 4 animals per day). Corpora lutea on days 7, 10 and 13 were dissociated with collagenase, and large and small luteal cell sub-populations were separated by elutriation. Northern and slot blots were used to examine mRNA, and western blots were used to measure the concentrations of IGF-I receptor protein in the pig corpus luteum. On northern blots, luteal IGF-I receptor mRNA was present as a single 11 kb transcript. The slot blots showed that the steady state expression of IGF-I receptor mRNA increased significantly (P < 0.05) from its lowest value on day 4, to reach a maximum on days 13-16. IGF-I receptor mRNA was also expressed to a greater extent in large compared with small luteal cells (P < 0.05). On western blots, IGF-I receptor appeared as a 95 kDa protein band (beta-subunit) and IGF-I receptor protein concentrations were significantly higher (P < 0.05) on days 4-10 than on days 13-16. Finally, large luteal cells appeared to contain more IGF-I receptor protein than the small luteal cells. In conclusion, since IGF-I receptor was detected in the pig corpus luteum, it is a likely target tissue for IGF-I, especially during the early luteal phase. Furthermore, IGF-I receptor was localized primarily on large luteal cells, thus it is hypothesized that IGF-I may play a paracrine role in the pig corpus luteum.
Assuntos
Corpo Lúteo/química , Estro/metabolismo , RNA Mensageiro/análise , Suínos/metabolismo , Análise de Variância , Animais , Northern Blotting/métodos , Western Blotting/métodos , Corpo Lúteo/citologia , Feminino , Comunicação Parácrina , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/análise , Receptor IGF Tipo 1/genéticaRESUMO
It has recently been shown that, in follicular fluid, as in the circulation, insulin-like growth factors (IGFs)-I and -II exist in a ternary complex with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). The current study was designed to determine whether ovarian follicular and luteal cells could synthesize IGFBP-3 and ALS. Ovaries were collected, during the follicular and early luteal phases, from mature pigs whose cycles were synchronized with PGF2alpha. We studied IGFBP-3 and ALS messenger RNA (mRNA) by in situ hybridization. These transcripts were colocalized with aromatase mRNA, a marker of healthy granulosa cells. IGFBP-3 mRNA was equally expressed in granulosa cells of all growing follicles. In contrast, granulosa cell ALS mRNA levels were higher (P < 0.05) in preantral and small antral follicles than in large antral follicles. In thecal cells, expression of mRNA for IGFBP-3, ALS and cyclin D1 (a marker of cell proliferation) was restricted to healthy (aromatase-expressing) follicles. In those follicles, thecal expression of IGFBP-3 mRNA was low or absent in preantral follicles but increased (P < 0.05) in antral follicles. Thecal cell ALS transcripts peaked in small antral follicles (P < 0.05) and then declined. In granulosa cells of atretic follicles, transcripts for aromatase were greatly reduced, whereas IGFBP-3 mRNA levels remained high. In contrast, ALS transcript levels were greatly reduced in both granulosa (P < 0.05) and thecal cells (P < 0.001) of atretic follicles. After ovulation, IGFBP-3 mRNA was moderately expressed in granulosa luteins but strongly detected in a few theca-derived cells and in vascular endothelial cells. This study demonstrates that follicular fluid IGFBP-3 and ALS, like the IGFs, originate (at least in part) from the ovary. The ability of follicular cells to synthesize, assemble, and store all components of the ternary complex may be critical in determining the bioavailability of follicular IGF-I and -II.
Assuntos
Proteínas de Transporte/genética , Fase Folicular/metabolismo , Glicoproteínas/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fase Luteal/metabolismo , Ovário/metabolismo , RNA Mensageiro/metabolismo , Animais , Feminino , Células da Granulosa/metabolismo , Hibridização In Situ , Ovário/citologia , Suínos , Células Tecais/metabolismoRESUMO
Cell proliferation, terminal differentiation, and angiogenesis occur during cycles of follicular and luteal development. In other paradigms, mac25, a potent tumor inhibitor is strongly induced in senescent epithelial cells, whereas CTGF stimulates angiogenesis and wound healing. Using in situ hybridization and immunohistochemistry, we have examined the possibilities that mac25 is inhibited, whereas CTGF is induced during active periods of follicular development and luteogenesis. Ovaries were collected during the follicular and early luteal phases from prostaglandin F2alpha-treated mature pigs and from slaughterhouse sows. CTGF transcripts were induced during the late preantral stage in granulosa and theca cells concomitantly with the appearance of endothelial cells in the theca. CTGF mRNA expression increased in granulosa cells to a maximum (P < 0.01) in mid-antral follicles but was down regulated (P < 0.01) in preovulatory follicles. In contrast, granulosa cell mac25 mRNA expression was undetectable between the preantral and mid-antral stage but was strongly induced in terminally differentiated granulosa cells of preovulatory follicles. CTGF mRNA and peptide were also detected in the theca externa/interstitium and in vascular endothelial cells of ovarian blood vessels, whereas mac25 transcripts, which were also abundant in ovarian blood vessels increased in the theca interna with follicular development. Transcripts of cyclin D 1, a marker of cell proliferation, appeared during the early antral stage and were moderate in granulosa cells but abundant in capillary endothelial cells in the theca interna, underneath the basement membrane. Following ovulation, CTGF and cyclin D1 mRNAs were associated with the migration of endothelial cells into the CL. Subsequently, there was a marked up-regulation of CTGF mRNA expression in granulosa luteins concomitantly with an increase in endothelial cell proliferation within the CL. We hypothesize that CTGF may promote ovarian cell growth and blood vessel formation during follicular and luteal development whereas mac25, a tumor inhibitor, may promote terminal differentiation of granulosa cells in preovulatory follicles.
Assuntos
Proteínas de Transporte/genética , Corpo Lúteo/fisiologia , Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Peptídeos e Proteínas de Sinalização Intercelular , Folículo Ovariano/fisiologia , RNA Mensageiro/metabolismo , Animais , Biomarcadores , Vasos Sanguíneos/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Fator de Crescimento do Tecido Conjuntivo , Corpo Lúteo/citologia , Corpo Lúteo/metabolismo , Ciclina D1/genética , Feminino , Substâncias de Crescimento/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Neovascularização Fisiológica/fisiologia , Folículo Ovariano/metabolismo , Ovário/irrigação sanguínea , Ovário/citologia , SuínosRESUMO
Insulin-like growth factor-I (IGF-I) is produced within the porcine corpus luteum (CL) and is thought to play an autocrine/paracrine role in CL development/function during the early luteal phase. This study examines the hypotheses that the luteolytic actions of prostaglandin F(2alpha) (PGF(2alpha)) during the early luteal phase may involve either a decrease in IGF-I or IGF receptor (IGF-IR), or an increase in IGF binding protein (IGFBP)-3, expression, any of which could interfere with the luteotropic actions of IGF-I in this tissue. Cycling gilts were treated twice daily with PGF(2alpha) (or saline) on Days 5-9 of the cycle to induce premature luteolysis. CL were collected on Days 6-9, and RNA, protein, or progesterone was extracted. By slot blot analysis, steady-state levels of IGF-I and IGFBP-3 mRNA were not different in PGF(2alpha)-treated vs. control animals; however, IGF-IR mRNA was increased in treated animals on Day 9. No changes in IGF-I content (ng/CL measured by RIA) were observed with respect to treatment. According to ligand blot analysis, the levels of IGFBP-3 increased on Day 6 and decreased on Days 8-9, while IGFBP-2 was higher on Days 6-7 and decreased on Day 9 in treated animals. IGF-IR levels, determined from Western blots, were higher on Day 7 (P < 0.05) and lower on Day 9 in PGF(2alpha)-treated animals vs. control animals (P < 0.05). In conclusion, PGF(2alpha)-induced premature luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. However, since IGFBP-2 and -3 protein levels increased early in the treatment period (Days 6-7), it is possible that they may mediate the luteolytic actions of PGF(2alpha) by sequestering IGF-I and preventing its interaction with the IGF-IR.
Assuntos
Corpo Lúteo/metabolismo , Dinoprosta/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/metabolismo , Suínos/metabolismo , Animais , Northern Blotting , Western Blotting , Corpo Lúteo/anatomia & histologia , Corpo Lúteo/efeitos dos fármacos , Feminino , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/análise , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like I/genética , Tamanho do Órgão , Progesterona/metabolismo , Receptor IGF Tipo 1/genéticaRESUMO
In the pig, the corpus luteum (CL) can develop and function autonomous of pituitary gonadotropins for approximately 12 days. We hypothesized that the insulin-like growth factor (IGF) system may play an autocrine/paracrine luteotrophic role(s) during this period. In this study, we monitored the expression (i.e., steady-state levels of mRNAs) of IGF-I and IGF binding proteins (IGFBP)-2, -3, -4, -5, and -6 mRNAs in whole CL and in small and large luteal cells on Days 4-16 of the estrous cycle. CL were dissociated with collagenase, and large and small luteal cells were isolated by centrifugal elutriation. Whole CL and luteal cells were extracted to isolate total or poly(A)+ RNA, which was subjected to Northern and/or dot-blot analyses using [32P]-labeled cDNA probes for IGF-I and IGFBP-2, -3, -4, -5, and -6. Northern blots showed readily detectable transcripts for IGF-I (6.7 and 0.9 kb), IGFBP-2 (1.8 kb), IGFBP-3 (2.8 kb), IGFBP-4 (2.6 kb), and IGFBP-5 (6.0 kb), but not for IGFBP-6. IGFBP-3 and -5 transcripts were observed mainly in small luteal cells, while IGFBP-2 and -4 were seen in both cell types. Dot-blot analyses for IGF-I and IGFBP-3 mRNAs were performed on total RNA from small and large luteal cells; blots were counter-probed with 3-phosphoglyceraldehyde dehydrogenase (p-GAD) cDNA to assess RNA quantity and quality. IGF-I mRNA (ratio IGF-I:p-GAD mRNA) expression was approximately 2-fold greater in small than in large luteal cells on Days 4-10. However, steady-state levels of IGF-I mRNA in small, but not large, luteal cells decreased significantly on Days 12-16 (vs. Days 4-10). IGFBP-3 mRNA expression was significantly greater (approximately 3-fold) in small than in large luteal cells but did not vary significantly between Days 4-10 and 12-16 for either cell type. We conclude that porcine CL express mRNAs for IGF-I and IGFBP-2, -3, -4, and -5, and that while small luteal cells are the major sources of IGF-I and IGFBP-3 and -5, IGFBP-2 and -4 appear to be expressed to approximately the same extent in small and large luteal cells. These results further suggest that the IGF-I/IGF system may have autocrine/paracrine regulatory actions in CL development/function in the pig.
Assuntos
Corpo Lúteo/metabolismo , Expressão Gênica , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like I/genética , RNA Mensageiro/metabolismo , Suínos , Animais , Northern Blotting , Feminino , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genéticaRESUMO
The mechanism by which multiple injections of PGF2 alpha result in premature luteolysis in pigs is unknown. In the present study we evaluated whether PGF2 alpha receptor concentrations on large luteal cells changed when gilts were injected IM with 12.5 mg of PGF2 alpha every 12 hours from the morning of day 5 of an estrous cycle (estrus = day 0) until ovariectomy on day 6, 7, 8, or 9. Luteal PGF2 alpha receptor concentrations remained constant from day 6 through 9 in the PGF2 alpha-treated group, but increased linearly (P > 0.05) in control gilts from day 6 to 9. Receptor affinity for PGF2 alpha did not change throughout the study in either PGF2 alpha-treated or control gilts. Luteal progesterone concentrations were significantly lower in PGF2 alpha-treated gilts than in control gilts only on day 9. Histological examination of luteal tissue obtained from PGF2 alpha-treated gilts revealed definite evidence of luteolysis by day 8. We conclude that PGF2 alpha-induced premature luteolysis is not mediated by an increase in luteal PGF2 alpha receptor concentrations and, based on luteal progesterone concentrations and histological features, that the PGF2 alpha-based protocol used to shorten the estrous cycle is accompanied by premature functional and structural luteal regression.
Assuntos
Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/fisiologia , Diestro/fisiologia , Dinoprosta/farmacologia , Receptores de Prostaglandina/fisiologia , Suínos/fisiologia , Animais , Dinoprosta/metabolismo , Estro/efeitos dos fármacos , Feminino , Luteólise/efeitos dos fármacos , Ovariectomia , Progesterona/metabolismo , Fatores de TempoRESUMO
This study examined the effects of LH and PGE2 on progesterone secretion by small and large porcine luteal cells with or without low-density lipoproteins. Corpora lutea were isolated from gilts 13-14 days after administering gonadotrophins; enzymatically dissociated and small and large cells were isolated by elutriation. Culture plates, 24-well, were then seeded with 150,000 small or 30,000 large luteal cells suspended in 1 ml M199 medium supplemented with 5 micrograms insulin ml-1, 40 ng hydrocortisone ml-1 and with or without low-density lipoproteins (50 micrograms cholesterol ml-1) or PGE2. Cells were cultured for up to 24 h in a humidified incubator at 37 degrees C under 5% CO2 in air. The low-density lipoproteins stimulated (P < 0.05) progesterone secretion by large, but not small, luteal cells. Prostaglandin E2 stimulated (P < 0.05) progesterone production by large luteal cells in a dose-dependent manner, and the stimulatory effects of PGE2 were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. Progesterone secretion by small luteal cells was not significantly affected by PGE2. Progesterone production by small luteal cells was enhanced (P < 0.05) by LH, and the stimulatory effects of LH were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. In the absence of these lipoproteins, LH had no effect on progesterone secretion by large luteal cells; however, in the presence of low-density lipoproteins, LH increased (P < 0.05) progesterone secretion by large cells, though to a lesser (P < 0.05) extent than the effect of LH on small cells. These data demonstrate that progesterone secretion by porcine luteal cells is stimulated differentially by LH and PGE2 and that small luteal cells are more responsive to LH and PGE2 acts primarily on large luteal cells.
Assuntos
Corpo Lúteo/metabolismo , Dinoprostona/farmacologia , Hormônio Luteinizante/farmacologia , Progesterona/metabolismo , Suínos/fisiologia , Animais , Tamanho Celular/fisiologia , Células Cultivadas , Corpo Lúteo/citologia , Corpo Lúteo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Hidrocortisona/farmacologia , Insulina/farmacologia , Lipoproteínas LDL/farmacologia , Estimulação QuímicaRESUMO
In this study we examined the stimulatory effects of PGF2 alpha on progesterone secretion by porcine luteal cells on different days of the estrous cycle, and the effects of PGF2 alpha, A23187 and PMA on progesterone secretion by isolated large and small luteal cells, in vitro. Corpora lutea were obtained from cycling pigs (days 6-16), collagenase dispersed and luteal cells incubated in medium 199 in the absence or presence of increasing doses of PGF2 alpha, A23187, and PMA. Progesterone concentrations in spent media were measured by RIA. PGF2 alpha stimulation of progesterone secretion by mixed luteal cells did not vary significantly throughout the estrous cycle. Progesterone secretion by large, but not small, luteal cells was increased (p < 0.05) in a dose-dependent fashion by PGF2 alpha. A23187 also caused a dose-dependent increase in progesterone secretion by large luteal cells but inhibited small luteal cells. Progesterone secretion by both large and small luteal cells was significantly increased by increasing doses of PMA. We conclude that the stimulatory response of luteal cells to PGF2 alpha in vitro did not correlate with PGF2 alpha receptor concentrations (not measured in this study), and we speculate that calcium/protein kinase C may be involved in mediating the stimulatory action of PGF2 alpha on luteal cell progesterone secretion.
Assuntos
Corpo Lúteo/efeitos dos fármacos , Dinoprosta/farmacologia , Estro/metabolismo , Progesterona/metabolismo , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Animais , Calcimicina/farmacologia , Células Cultivadas , Corpo Lúteo/citologia , Di-Hidrotestosterona/análogos & derivados , Di-Hidrotestosterona/farmacologia , Relação Dose-Resposta a Droga , Feminino , Gravidez , Radioimunoensaio , Suínos , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
The objective of this study was to measure and compare the concentrations of PGF 2 alpha receptors on luteal cells taken from cycling, pregnant, and pseudopregnant pigs. Corpora lutea were removed surgically from cycling, pregnant, and pseudopregnant (induced with 5 mg estradiol valerate/day i.m. beginning on Day 11) pigs on Days 12, 13, and 14 (postestrus) and were subjected to collagenase dissociation. Dissociated luteal cells (approximately 100,000 large viable cells per tube) were assayed for specific PGF 2 alpha binding by Scatchard analysis, using [3H]PGF 2 alpha and varying doses (0-5 microM) of unlabeled PGF 2 alpha. Luteal cells from all three types of pigs were shown to possess two specific PGF 2 alpha binding sites (high affinity, Kd = 9-47 nM; low affinity, Kd = 243-1359 nM). The concentrations of the high-affinity PGF 2 alpha binding site (PGF 2 alpha "receptor") on Days 12 and 13 were not significantly different (NS) between cycling (1.7 and 1.1 x 10(6) receptors per large luteal cell, respectively), pregnant (1.3 and 1.2 x 10(6)), and pseudopregnant (1.1 and 0.8 x 10(6)) pigs. However, on Day 14, luteal PGF 2 alpha receptor concentrations were significantly higher (p < 0.05) in cycling (4.2 x 10(6)) compared with pregnant (1.3 x 10(6)) and pseudopregnant (1.4 x 10(6)) pigs. We speculate that reduced luteal PGF 2 alpha receptor concentrations on Day 14 in pregnant and pseudopregnant compared with cycling pigs may lead to decreased luteal sensitivity to PGF 2 alpha in these animals, and that this mechanism may play a role in the maternal recognition of pregnancy in this species.
Assuntos
Corpo Lúteo/metabolismo , Estro/fisiologia , Prenhez/metabolismo , Pseudogravidez/metabolismo , Receptores de Prostaglandina/metabolismo , Suínos/metabolismo , Animais , Dinoprosta/metabolismo , Feminino , GravidezRESUMO
Previous investigators considered pig corpora lutea refractory to the luteolytic effects of prostaglandin (PG) F2 alpha before Day 12 of the estrous cycle. This study was designed to determine whether multiple injections of PGF2 alpha would result in a sustained reduction of serum progesterone and luteolysis, leading to significant shortening of the estrous cycle and interestrous interval. On Days 5-10 of an estrous cycle, gilts (n = 4) received injections of 12.5 mg PGF2 alpha (dinoprost tromethamine) i.m. every 12 h, or vehicle (PBS; n = 4) according to the same schedule. Mean interestrous interval in PGF2 alpha-treated gilts was reduced (p < 0.001) to 13.3 +/- 0.5 days compared with 19.8 +/- 0.6 days for control gilts. Serum progesterone declined below 1 ng/ml by Day 10.5 in PGF2 alpha-treated gilts compared to Day 17.5 in control animals. Serum concentrations of estradiol-17 beta (E2) reached maximal levels in PGF2 alpha-treated gilts earlier (Day 12.5) in the cycle than in control gilts (Day 19.5). Peak E2 and LH concentrations coincided with the periestrous period, suggesting that PGF2 alpha-induced estrus is accompanied by normal follicular development and ovulation. These results demonstrate that the pig is susceptible to the luteolytic effects of PGF2 alpha before Day 12 if repeated injections are given from Day 5 through Day 10.