RESUMEN
The present study investigated the effect of birthweight on testicular development and spermatogenesis in boars. Twenty-four pairs of littermate boars were selected: one piglet with the highest birthweight (HW) and the other with the lowest birthweight (LW) within the litter. Two subsets of 12 pairs of male littermates from each birthweight group were obtained after selection: one subset was orchiectomised at 8 days and the other at 8 months of age. HW boars had higher body and testicular weights at both ages (P<0.05). Testosterone concentrations and the relative expression of 17α-hydroxylase in the testis were similar between birthweight groups. Birthweight affected somatic and germ cell numbers in the neonatal testis, which were higher in HW boars (P<0.05). Moreover, a significant reduction in the number of pachytene spermatocytes and round spermatids was observed in LW boars (P<0.05) at 8 months of age, which caused a decrease in the total number of elongated spermatids and daily sperm production (P<0.05). Hence, HW boars have the potential to produce more spermatozoa and consequently more semen doses per ejaculate, and would be very valuable to an industry that relies on AI.
Asunto(s)
Peso al Nacer/fisiología , Espermatogénesis/fisiología , Espermatozoides/citología , Testículo/anatomía & histología , Testículo/fisiología , Animales , Forma de la Célula/fisiología , Masculino , Tamaño de los Órganos/fisiología , Recuento de Espermatozoides , Espermátides/citología , Espermátides/fisiología , Espermatocitos/citología , Espermatocitos/fisiología , Espermatozoides/fisiología , Porcinos , Testosterona/metabolismoRESUMEN
Little is known about the involvement of microRNAs (miRNAs) in the follicular-luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles, (diameter, 4.0-5.5 âmm), pre-ovulatory follicles (6.0-7.0 âmm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs whose expression decreased in association with the follicular-luteal transition and eight miRNAs whose expression increased during this transition. Expression profiles were confirmed by northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in the levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identify a subset of miRNAs that are potentially important regulators of the follicular-luteal transition.
Asunto(s)
Fase Folicular/genética , Fase Luteínica/genética , MicroARNs/genética , Ovario/metabolismo , Rumiantes/genética , Animales , Bovinos , Diferenciación Celular/genética , Células Cultivadas , Cuerpo Lúteo/química , Cuerpo Lúteo/metabolismo , Femenino , Fase Folicular/metabolismo , Perfilación de la Expresión Génica , Células de la Granulosa/metabolismo , Células de la Granulosa/fisiología , Fase Luteínica/metabolismo , MicroARNs/aislamiento & purificación , MicroARNs/metabolismo , Folículo Ovárico/química , Folículo Ovárico/metabolismo , Folículo Ovárico/fisiología , Ovario/química , Progesterona/metabolismo , Rumiantes/metabolismo , Ovinos , Células Tecales/metabolismo , Células Tecales/fisiologíaRESUMEN
The response of follicles to IGF1 was compared between the transition into the ovulatory season (transitional period) and the ovulatory season (ovulatory period) in eight mares using a cross-over experimental design within periods. Granulosa cells were collected from follicles 15-24 or 25-34 mm and expression of IGF1R, IGF2R, FSHR, LHCGR and PAPPA was determined by qPCR. In addition, 10 mg IGF1 or vehicle were injected into the largest follicle (transitional period) or the second largest follicle (ovulatory period) of a follicular wave before the beginning of diameter deviation between the two largest follicles (mean diameters at injection 19.2 and 20.0 mm during transitional and ovulatory periods respectively). Follicular fluid was collected 24 h after injection for determination of free IGF1, IGFBP, inhibin A and oestradiol levels. Granulosa cells from follicles 25-34 mm, but not follicles 15-24 mm, expressed higher levels of IGF1R (P=0.01), FSHR (P<0.007) and LHCGR (P=0.09) during the ovulatory period than during the transitional period, whereas IGF2R expression was higher in transitional than ovulatory follicles (P=0.06). Follicular IGFBP2 levels were not different (P>0.1) between periods and treatments, whereas IGFBP5 levels were higher (P<0.05) during the ovulatory period. Finally, IGF1 injection before the beginning of deviation induced an approximately twofold increase (P=0.01) in follicular inhibin A levels during each period and did not affect oestradiol (P>0.1). These results suggest that, as during ovulatory waves, equine follicles during transitional waves are responsive to IGF1 before the beginning of deviation and that, therefore, inadequate IGF1 responsiveness before deviation may not underlie the deficient development of dominant follicles during transition.
Asunto(s)
Caballos/fisiología , Factor I del Crecimiento Similar a la Insulina/farmacología , Folículo Ovárico/metabolismo , Estaciones del Año , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Estradiol/análisis , Femenino , Líquido Folicular/química , Células de la Granulosa/química , Células de la Granulosa/metabolismo , Immunoblotting , Inhibinas/análisis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/análisis , Microinyecciones , Folículo Ovárico/efectos de los fármacos , Reacción en Cadena de la Polimerasa/métodos , Proteína Plasmática A Asociada al Embarazo/genética , Proteína Plasmática A Asociada al Embarazo/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Receptores de HFE/genética , Receptores de HFE/metabolismo , Receptores de HL/genética , Receptores de HL/metabolismoRESUMEN
The insulin-like growth factors, IGF-I and -II, have been shown to play a key role in luteal function in some species. The IGF binding proteins, IGFBP-2 and -3, have been shown to inhibit binding of IGF-I and -II to bovine luteal cells and decrease progesterone production. We have recently shown that equine follicles have the genetic capacity to produce IGFBP-2, and that levels decrease in healthy preovulatory follicles. In the present study expression of mRNAs encoding IGFBP-2, as well as the rate-limiting steroidogenic enzyme, P450scc, were studied in equine corpora lutea to investigate whether IGFBP-2 might be involved in luteolysis. Corpora lutea were collected from mares in mid-luteal phase (day 10), at early regression (day 14), late regression (day 17), and 12 and 36 h after intramuscular administration of the PGF(2alpha) analogue, cloprostenol (0.5 microg/kg). During early natural regression, and 12 h after administration of cloprostenol on day 10, steady state levels of mRNAs encoding P450scc had decreased significantly compared with day 10 of dioestrus (P < 0.001). Levels of mRNA encoding IGFBP-2 increased significantly between mid-diestrus and early (P < 0.01) and late (P < 0.001) regression, and 36 h after cloprostenol administration (P < 0.001). We conclude that the genetic capacity for increased IGFBP-2 production in the early stages of natural luteolysis in the mare may act to sequester IGF-I in the CL, assisting in inhibition of progesterone production. However the delay in increase in mRNA encoding IGFBP-2 after cloprostenol administration, combined with the sharp fall in expression of P450scc mRNA, suggests that the luteolytic action of a pharmacological dose of cloprostenol may not be mediated via IGFBP-2 in the mare.
Asunto(s)
Cloprostenol/farmacología , Cuerpo Lúteo/química , Expresión Génica , Caballos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Luteólisis/metabolismo , ARN Mensajero/análisis , Animales , Femenino , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/fisiología , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/fisiología , Luteólisis/efectos de los fármacos , Progesterona/sangreRESUMEN
Many studies have highlighted the role of the insulin-like growth factor (IGF) system in the control of antral follicular growth. However, much less is known about the involvement of the IGF system in the regulation of preantral follicular development. In an attempt to address this lack of knowledge, the present study describes the spatial and temporal patterns of expression of mRNA encoding components of the IGF system in bovine follicles during preantral stages of development. mRNA was detected by in situ hybridization using frozen sections (14 microm) of bovine ovarian tissue. Serial sections were probed with 35S-labelled bovine riboprobes. Type 1 IGF receptor mRNA was detected in granulosa cells and in the oocyte of preantral follicles; however, in this study, as in previous studies, it was not possible to detect mRNA encoding either IGF-I or -II. IGF binding protein (IGFBP)-2 mRNA was present in granulosa cells and oocytes of preantral follicles, and immunoreactive IGFBP-2 was detected around granulosa cells during this early stage of development. Occasionally, preantral follicles were identified in which there was no expression of IGFBP-2 in granulosa cells or the oocyte. IGFBP-3 mRNA was detected in the oocyte of preantral follicles and in the surrounding stromal tissue. mRNAs encoding IGFBP-2 and -3, and type 1 IGF receptor were first detected in type 2 follicles. In conclusion, although the IGF ligands are not expressed in preantral follicles, mRNAs encoding the type 1 IGF receptor, and IGFBP-2 and -3 were present and showed unique spatial patterns of expression within preantral follicles.
Asunto(s)
Bovinos/metabolismo , Oocitos/química , Folículo Ovárico/fisiología , ARN Mensajero/análisis , Receptores de Somatomedina/genética , Somatomedinas/genética , Animales , Femenino , Células de la Granulosa/química , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Folículo Ovárico/química , Receptor IGF Tipo 1/genéticaRESUMEN
We have previously shown that the number of ovarian follicles <4 mm in diameter can be increased by enhanced dietary intake in heifers. This study investigated the effect of the same dietary treatment on superovulatory response. The estrous cycles of 24 mature Hereford x Friesian heifers were synchronized by a standard progesterone plus prostaglandin protocol. The animals were fed with either 100% (group M, n = 12) or 200% (group 2M, n = 12) maintenance requirements for a 3-week period. Starting from day 4 of the synchronized estrous cycle, all the animals were superovulated using a standard 4-day FSH regime followed by an injection of GnRH analogue (GnRHa) to induce ovulation. Rectal ultrasound scanning was carried out to assess ovarian follicular populations at the start of FSH treatment and on the day of GnRHa injection, and to determine the number of corpora lutea 5 days after GnRHa injection. The body weight (BW) and body condition score (BCS) were recorded weekly and plasma samples were collected throughout the experimental period. There were no differences in either BW or BCS between two groups at the start of the experiment. The BW and BCS were maintained during the experiment in the group M, whilst animals in the group 2M showed a non-significant (P > 0.05) increase in BW and BCS. Circulating concentrations of insulin were significantly (P < 0.01) higher in heifers from the group 2M throughout the controlled feeding period. The group 2M had significantly (P < 0.05) more follicles 2-4 mm in diameter at the start of FSH treatment and more (P < 0.01) follicles >9 mm in diameter on the day of GnRHa injection, when compared with the group M. Similarly, 5 days after GnRHa injection there were significantly (P < 0.01) more corpora lutea in the group 2M (18.1+/-2.2) than in the group M (10.6+/-3.0). In addition, plasma progesterone concentrations following GnRHa injection were significantly (P < 0.01) higher in heifers from the group 2M. In conclusion, these results confirm that increased dietary intake can enhance the recruitment of ovarian follicles in heifers. This treatment may provide a valuable approach to improving superovulatory response in cattle.
Asunto(s)
Bovinos/fisiología , Dieta , Hormona Folículo Estimulante/farmacología , Superovulación , Animales , Composición Corporal , Peso Corporal , Buserelina/administración & dosificación , Cloprostenol/administración & dosificación , Cuerpo Lúteo/anatomía & histología , Dinoprost/administración & dosificación , Sincronización del Estro , Femenino , Insulina/sangre , Folículo Ovárico/anatomía & histología , Ovulación , Progesterona/administración & dosificación , Progesterona/sangreRESUMEN
The nutritional status of a cow is a key factor in the regulation of both follicle growth and oocyte quality. In this study, the effect of diets designed to increase circulating insulin and insulin-like growth factor I (IGF-I) concentrations on steroid production by granulosa cells in vitro was examined to analyse the mechanisms through which these changes occur. Hereford x Friesian heifers (n = 24) were offered maintenance or twice maintenance diets during the experimental period (17 days). Circulating concentrations of FSH did not differ between the two dietary groups, whereas insulin and IGF-I concentrations showed significant diet x day of oestrous cycle interactions. Ovaries were collected on day 3 of the first follicle wave after synchronization of oestrus. Granulosa cells were isolated from small (1-4 mm) and medium-sized (4-8 mm) follicles and cultured in the presence of long R3-IGF-I or bFSH or both. After 4 days in culture, granulosa cells isolated from small follicles, but not medium-sized follicles, collected from cattle offered the twice maintenance diet secreted significantly higher (P < 0.05) amounts of oestradiol compared with granulosa cells collected from cattle offered the maintenance diet. The effect was apparent in either the presence or absence of FSH and long R3-IGF-I. This nutritional effect on aromatase activity in granulosa cells was not apparent after day 6 of culture. There was no effect of diet on progesterone production by granulosa cells after 4 or 6 days of culture. These results support the hypothesis that dietary-induced changes in circulating insulin and IGF-I concentrations have a direct effect on the steroidogenic potential of bovine granulosa cells from small follicles. The dietary-induced increases in aromatase activity in small follicles combined with the increased concentration of metabolic hormones are possible mechanisms through which short-term changes in nutrition may affect follicle dynamics.
Asunto(s)
Fenómenos Fisiológicos Nutricionales de los Animales , Bovinos/metabolismo , Estradiol/biosíntesis , Estro/fisiología , Células de la Granulosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/análogos & derivados , Animales , Células Cultivadas , Femenino , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/farmacología , Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Progesterona/biosíntesis , Factores de TiempoRESUMEN
Heifers were assigned either low or high (HE) levels of energy intake and low or high concentrations of dietary crude protein. The effect of these diets on the plasma concentrations of insulin, insulin-like growth factor (IGF)-I, and urea on follicular growth and early embryo development is described. We propose that the observed dietary-induced changes in the ovarian IGF system increase bioavailability of intrafollicular IGF, thus increasing the sensitivity of follicles to FSH. These changes, in combination with increased peripheral concentrations of insulin and IGF-I in heifers offered the HE diet, contribute to the observed increase in growth rate of the dominant follicle. In contrast to follicular growth, increased nutrient supply decreased oocyte quality, due in part to increased plasma urea concentrations. Clearly a number of mechanisms are involved in mediating the effects of dietary energy and protein on ovarian function, and the formulation of diets designed to optimize cattle fertility must consider the divergent effects of nutrient supply on follicular growth and oocyte quality.
Asunto(s)
Bovinos/fisiología , Proteínas en la Dieta/farmacología , Desarrollo Embrionario y Fetal , Ingestión de Energía , Fertilización In Vitro , Folículo Ovárico/fisiología , Ovario/metabolismo , Somatomedinas/fisiología , Animales , Blastocisto/fisiología , Bovinos/embriología , Femenino , Fertilización , Hormona Folículo Estimulante/sangre , Hibridación in Situ , Insulina/sangre , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/fisiología , Factor II del Crecimiento Similar a la Insulina/genética , Cinética , Progesterona/sangre , ARN Mensajero/análisis , Urea/sangreRESUMEN
Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.
Asunto(s)
Bovinos/metabolismo , Cuerpo Lúteo/metabolismo , Estro/metabolismo , ARN Mensajero/análisis , Receptor IGF Tipo 1/genética , Somatomedinas/genética , Análisis de Varianza , Animales , Sincronización del Estro , Femenino , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ/métodos , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genéticaRESUMEN
IGFs regulate gonadotrophin-stimulated proliferation and differentiation of granulosa and theca cells in vitro. However, the detailed pattern of mRNA expression of IGFs in bovine follicles remains controversial. The objectives of this study were therefore to describe the temporal and spatial pattern of expression of mRNA encoding IGF-I, IGF-II and the type 1 IGF receptor in bovine follicles in vivo. The expression of mRNA encoding IGF-II was detected in theca tissue from around the time of antrum formation up to and during the development of dominance. No IGF-II mRNA expression was detected in granulosa cells. In the majority of follicles we were unable to detect mRNA encoding IGF-I in either granulosa or theca tissue from follicles at any stage of development. Occasionally low amounts of mRNA encoding IGF-I were detected in the theca externa and connective tissue surrounding some follicles. Type 1 IGF receptor mRNA was detected in both granulosa and theca cells of preantral and antral follicles. Expression was greater in granulosa tissue compared with theca tissue. We also measured IGF-I and -II mRNA in total RNA isolated from cultured granulosa and theca cells using reverse transcriptase PCR. In contrast to the in vivo results, IGF-II mRNA was detected in both granulosa and theca tissue. IGF-I mRNA was detected in theca tissue and in very low amounts in granulosa cells. Using a specific IGF-I RIA we were unable to detect IGF-I immunoreactivity in granulosa conditioned cell culture media. Using immunohistochemistry we detected IGF-I immunoreactivity in some blood vessels within the ovarian stroma. We conclude from these results that IGF-II is the principal intrafollicular IGF ligand regulating the growth of bovine antral follicles. In preantral follicles the expression of mRNA encoding type 1 IGF receptor but absence of endogenous IGF-I or -II mRNA expression, highlights a probable endocrine mechanism for the IGF regulation of preantral follicle growth.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Folículo Ovárico/metabolismo , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/metabolismo , Animales , Bovinos , Femenino , Perfilación de la Expresión Génica , Células de la Granulosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Folículo Ovárico/citología , ARN Mensajero/genética , Receptor IGF Tipo 1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tecales/metabolismoRESUMEN
This work is concerned with the role of insulin-like growth factor binding protein (IGFBP)-2 and -4 in the regulation of IGF bioactivity in bovine follicles during the development of dominance. We measured the expression of IGFBP-2 and -4 messenger RNA (mRNA) in small (1-4 mm) gonadotropin-sensitive follicles and medium (4-8 mm) and large (>8 mm) gonadotropin-dependent follicles using in situ hybridization. In healthy nonatretic bovine follicles, IGFBP-2 and -4 mRNA expression was confined to granulosa and theca tissue, respectively. Moreover, during the development of follicular atresia, there were distinct changes in the temporal and spatial expression of these genes. IGFBP-2 immunoactivity was localized in granulosa tissue and the basement membrane of healthy preantral follicles, whereas IGFBP-4 immunoactivity was localized in both theca and granulosa tissue. Of particular interest was the lack of IGFBP-2 mRNA expression in large (>8 mm) gonadotropin-dependent follicles, an observation that was confirmed by the lack of immunoreactive IGFBP-2 in these follicles. The regulation of IGFBP-2 and -4 mRNA expression in granulosa and theca cells was analyzed using a serum-free cell culture system. FSH inhibited the expression of IGFBP-2 mRNA in granulosa cells, whereas LH stimulated IGFBP-4 mRNA expression in theca cells. Our results provide evidence for the existence of different roles for IGFBP-2 and -4 in the developing follicle.
Asunto(s)
Expresión Génica , Gonadotropinas Hipofisarias/farmacología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Folículo Ovárico/fisiología , ARN Mensajero/análisis , Animales , Bovinos , Células Cultivadas , Femenino , Hormona Folículo Estimulante/farmacología , Atresia Folicular/fisiología , Expresión Génica/efectos de los fármacos , Células de la Granulosa/química , Inmunohistoquímica , Hibridación in Situ , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Hormona Luteinizante/farmacología , Folículo Ovárico/química , Folículo Ovárico/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Células Tecales/químicaRESUMEN
Insulin-like growth factor-binding protein (IGFBP) production by the ovine ovary was examined using Western ligand blots and immunoblots. During follicle development, the follicular fluid content of the 39- to 42-kDa binding protein (IGFBP-3) increased, whereas that of the 34-kDa (IGFBP-2), 32-kDa, 30-kDa, and 25-kDa (IGFBP-4) binding proteins decreased. The granulosa and theca cell cultures produced different complements of binding proteins. IGFBP-2 and the 30- and 32-kDa binding proteins were produced by granulosa cells, and there was no obvious effect of the size of follicle from which the cells were isolated. Both FSH and insulin-like growth factor (IGF-I) were necessary for maximum production of IGFBP-2. The main binding proteins detected in theca cell cultures were IGFBP-4 and -2. The IGFBP-4 concentration increased when the cells were exposed to LH. In contrast, IGFBP-2 concentration in theca cell-conditioned medium decreased when the cells were incubated with LH. Theca cell cultures produced more IGFBP-4 when the cells were isolated from small follicles as opposed to large follicles. Neither granulosa nor theca cell cultures produced significant quantities of IGFBP-3, and the source of this binding protein in the follicular fluid from large ovine follicles is probably the circulation. The results indicate that IGFBP production in the developing ovine ovarian follicle is dependent on both cell type and follicle size and is regulated by IGF-I and gonadotropins.
Asunto(s)
Gonadotropinas Hipofisarias/farmacología , Células de la Granulosa/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/farmacología , Folículo Ovárico/anatomía & histología , Células Tecales/metabolismo , Animales , Western Blotting , Células Cultivadas , Medios de Cultivo Condicionados , Femenino , Hormona Folículo Estimulante/farmacología , Líquido Folicular/metabolismo , Immunoblotting , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Hormona Luteinizante/farmacología , OvinosRESUMEN
Expression of genes encoding insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II) and type I insulin-like growth factor receptor (IGFr) was measured in theca and granulosa cells from the ovary of the laying hen, using an RNase protection assay. Expression of genes encoding IGF-I and -II was confined to theca tissue and expression was not detected in granulosa cells. In contrast, expression of genes encoding IGFr in granulosa cells was significantly greater than that in theca tissue. The 98 base IGF-II probe was similar to a region of the second coding exon of chicken IGF-II and produced multiple RNase-protected RNA hybrids. Theca RNA from follicles at all stages of development produced RNase-protected hybrids of size 98, 96 and 90 bases; however, an additional band (66 bases) was also observed in theca RNA from small yellow follicles. The stage of follicular development during which maximum amounts of the 66 base RNase-protected fragment was detected correlates with the stage at which small follicles are selected for recruitment into the follicular hierarchy. The results provide evidence for the involvement of IGFs in the intraovarian control of ovarian function in a non-mammalian species, and highlight the importance of IGF-II in this process.
Asunto(s)
Pollos/genética , Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Ovario/química , Oviposición/genética , Receptor IGF Tipo 1/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos/metabolismo , Sondas de ADN/genética , Femenino , Técnicas Genéticas , Células de la Granulosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Datos de Secuencia Molecular , Ovario/citología , ARN Mensajero/metabolismo , Células Tecales/metabolismoRESUMEN
The extraction of mRNA from cartilage samples is complicated by the presence of proteoglycans and the low cellular density of the tissue. We required a method that would enable mRNA to be extracted from small biopsy-sized samples of cartilage. The method had to produce consistent results and sufficient RNA for Northern and PCR analysis. Methods of total RNA extraction, previously shown to be effective for cartilage, were compared with a new technique in which an oligo (dT) conjugated to biotin hybridises to the mRNA. The hybrids are captured with covalently coupled streptavidin paramagnetic particles. Samples of growth plate cartilage, including those specifically from the upper (proliferative and transitional) and lower (fully hypertrophic) zones, were collected and some were frozen at -70 degrees C. Samples for extraction by the paramagnetic method weighed approximately 80 mg and approximately 12 micrograms of mRNA was extracted from fresh tissue samples. The yield from similar frozen samples of the same weight was about a seventh of that from the fresh tissues. 5 micrograms of the mRNA from each sample was run on a gel, and a Northern blot was prepared and probed with a [32P]-labelled antisense RNA probe to type II collagen cDNA. A distinct band of type II collagen mRNA was detected (5.3 Kb) in the samples from the upper (proliferative and transitional) zone. The traditional methods of extracting RNA from cartilage required far greater quantities of tissue and the RNA produced was frequently degraded. The results obtained using the paramagnetic bead method precluded further trials with modification of the traditional methods of mRNA extraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cartílago/metabolismo , Colágeno/genética , Colágeno/metabolismo , Hibridación de Ácido Nucleico/métodos , ARN/análisis , Animales , Biopsia , Northern Blotting , Cartílago/patología , Pollos , Hígado/metabolismo , Magnetismo , Microesferas , Reproducibilidad de los ResultadosRESUMEN
An RNase protection assay is described that allowed the quantitative analysis of chicken type-I IGF receptor mRNA transcripts. The transcripts were measured in extracts of total nucleic acid (TNA) and, under the hybridization conditions described, protected probes of the expected size were obtained. The RNA-RNA hybrids could be quantified in the presence of at least a 1000-fold molar excess of DNA containing sequences which were complimentary to the RNA probe. The amount of protected probe was linearly related to the amount of TNA in the hybridization reaction medium, and this allowed the results to be expressed in the form of mRNA molecules/cell. Type-I IGF receptor mRNA transcripts were detected in all the tissues examined from a 20-day-old chick embryo. Their amount ranged from 5 to 24 molecules/cell, in the order liver < breast muscle < leg muscle < heart < brain. The amount of receptor mRNA was 65- to 300-fold less than that of beta-actin mRNA. The quantity of type-I IGF receptor mRNA varied significantly throughout embryonic and post-hatch development. Maximum amounts were measured in 21-day-old embryos (a two- to fourfold increase relative to 16-day-old embryos). Thereafter the amount of receptor mRNA decreased, during the 4-week period after hatching, to levels which were significantly lower than that observed in 16-day-old embryos. Throughout the period of embryonic and post-hatch development described here the amount of beta-actin mRNA remained constant, indicating that the changes in the quantity of receptor mRNA were due to specific mechanisms acting directly on the steady-state levels of type-I IGF receptor mRNA. Selection for increased growth had no effect on the amount of type-I IGF receptor mRNA. The result was the same when expressed either as molecules/cell or as a percentage of beta-actin mRNA.
Asunto(s)
Pollos/crecimiento & desarrollo , Pollos/genética , Regulación de la Expresión Génica , Receptor IGF Tipo 1/genética , Actinas/genética , Análisis de Varianza , Animales , Embrión de Pollo , ARN Mensajero/análisis , Ribonucleasas , Transcripción GenéticaRESUMEN
The biological activity of insulin-like growth factor-I (IGF-I) is mediated by a transmembrane glycoprotein (type-1 IGF receptor or IGF-I receptor) that shows considerable sequence homology with the insulin receptor. In order to detect the expression of this gene in chicken liver tissue, a plasmid was constructed containing a fragment of chicken IGF-I receptor cDNA. The cDNA fragment corresponded to nucleotides 326-599 of the human IGF-I receptor cDNA and showed 86.1 and 69.3% homology at the nucleotide level and 96.7 and 80.2% homology at the amino acid level with the human IGF-I receptor and insulin receptor respectively. The construct was used to generate an antisense RNA probe for the detection of IGF-I receptor mRNA transcripts in 1- and 4-week-old chick liver tissue. IGF-I receptor gene expression was initially detected by the reverse transcriptase polymerase chain reaction using synthetic chicken IGF-I receptor oligonucleotides. Amplified fragments of the correct size were detected in both RNA samples. Northern blots were also used to detect IGF-I receptor mRNA transcripts in the liver RNA samples. The results indicated that the amount of receptor mRNA decreased significantly between 1 and 4 weeks after hatch. In contrast, chicken beta-actin gene expression remained constant over this period. A major IGF-I receptor RNA transcript (11 kb) was observed in blots from 1-week-old livers, less abundant transcripts were also observed ranging in size from 8 to 9 kb.(ABSTRACT TRUNCATED AT 250 WORDS)