RESUMEN
Vasoinhibins are a family of peptides that act on endothelial cells to suppress angiogenesis and promote apoptosis-mediated vascular regression. Vasoinhibins include the N-terminal fragments from prolactin (PRL), GH, and placental lactogen. One of the vasoinhibins, the N-terminal PRL fragment of 16âkDa, is generated by the lysosomal representative protease cathepsin D (Cath D). Because the normal growth and involution of the mammary gland (MG) are profoundly affected by the expansion and regression of blood vessels and also because PRL stimulates the growth and differentiation of MG, we proposed that intact PRL produced during lactation contributes to MG angiogenesis and increased blood flow, whereas during involution, the N-terminal PRL fragment would have proapoptotic effects on mammary epithelial cells (MECs). Therefore, we investigated the production of the N-terminal PRL fragment and its direct effect on the MG. Mouse PRL (mPRL) was proteolytically cleaved by Cath D between amino acids 148 and 149. N-terminal PRL fragment and Cath D expression increased during MG involution. Furthermore, incubation of MG fragments and MCF7 with recombinant 16âkDa mPRL revealed a proapoptotic effect in MECs. Ectopic mPRL in MECs was cleaved to 16âkDa PRL by Cath D in the MG lysosomal fraction. The majority of PRL derived from pituitary gland was cleaved to 16âkDa PRL in culture medium. Therefore, N-terminal PRL fragment increases during the involution period, has a proapoptotic effect on MECs, and is mainly generated by secreted Cath D in the extracellular space of MG.
Asunto(s)
Catepsina D/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Glándulas Mamarias Animales/fisiología , Prolactina/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Catepsina D/biosíntesis , Catepsina D/genética , Diferenciación Celular , Línea Celular Tumoral , Femenino , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Células MCF-7 , Glándulas Mamarias Animales/irrigación sanguínea , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Neovascularización Fisiológica , Prolactina/biosíntesis , Prolactina/genética , ARN Mensajero/biosíntesis , Receptores de Prolactina/biosíntesis , Receptores de Prolactina/genética , Análisis de Secuencia de ProteínaRESUMEN
Prolactin (PRL) has long been known to be a hormone responsible for mammary gland development and lactation in females, whereas its role in males is still unclear. Thus, we investigated male mouse (m) PRL protein and mRNA expression in spermatozoa at various differentiation stages in the testes. Quantitative RT-PCR and in situ hybridization detected the expression of PRL not only in Leydig cells but also in germ cells, in particular in spermatogonia. The nucleotide sequence of testis PRL mRNA was the same as that in the pituitary. The mPRL was detected in Leydig cells and in round and elongated spermatids of the testes by immunohistochemistry. Immunoblotting detected 2 forms of mPRL in the testes, one form was 23-kDa PRL, and the other form was smaller than full-length PRL. Based on these results, we focused on N-terminal cleaved PRL to determine its involvement in spermatogenesis. Immunohistochemistry using two sets of antibodies, one that recognized full-length PRL and N-terminal cleaved PRL and another that recognized full-length PRL and C-terminal cleaved PRL, suggested that intact PRL was localized in the nucleus of round spermatids, while N-terminal cleaved PRL variants were localized in the Golgi apparatus of the sperrmatid nuclei of round spermatids, cytoplasms of elongated spermatids and in the spermatozoa tails. These findings suggest that PRL is ectopically expressed in the spermiogenesis and spermatogenesis and that cleaved PRL variants were localized in the Golgi apparatus of spermatids and in spermatozoa tails.
Asunto(s)
Expresión Génica , Fragmentos de Péptidos/metabolismo , Prolactina/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Animales , Núcleo Celular/metabolismo , Aparato de Golgi/metabolismo , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Especificidad de Órganos , Prolactina/química , Prolactina/genética , Transporte de Proteínas , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Cola del Espermatozoide/metabolismo , Espermátides/citología , Espermátides/metabolismo , Espermatogénesis , Espermatogonias/citología , Espermatogonias/crecimiento & desarrollo , Espermatogonias/metabolismo , Espermatozoides/crecimiento & desarrollo , Testículo/citología , Testículo/crecimiento & desarrolloRESUMEN
Phosphorylated prolactin (PPRL) is considered to be the most quantitatively important post-translationally modified form of prolactin (PRL) in rodents. We recently detected two different types of PPRL in the mouse pituitary gland; one was phosphorylated at serine and the other was phosphorylated at serine/threonine. Furthermore, we showed that there are obvious differences in the ratios between PPRLs and non-phosphorylated PRL in the pituitary gland based on age and sex and that estrogen influences PRL phosphorylation at serine in female mice. In the present study, we examined whether estradiol (E2) increases serine PPRL in the male pituitary gland in the same manner as in the female pituitary gland and examined whether PPRL is released into serum. We first determined the relative amounts of intrapituitary PPRLs in male mice under different pharmacological conditions that increased PRL secretion. The results indicated that treatment with E2 increases serine PPRL. We then performed two-dimensional electrophoresis and immunoblotting analysis after immunoprecipitation with anti-mouse PRL antibody using male and female sera under different pharmacological conditions that increased PRL secretion. The results of this experiment indicated that there were PRLs phosphorylated at serine and serine/threonine in the female serum but not in the male serum. The levels of PPRLs in sera were greatly increased with the E2 treatment for both male and female sera. Furthermore, we examined the effect of E2 on PPRL synthesis in cultured male pituitary glands. In this experiment, we observed increased serine PPRL synthesis and stronger immunohistochemical staining of PRL cells with E2 treatment. These findings suggested that serine PPRL synthesis and secretion were influenced by estrogen.
Asunto(s)
Estradiol/farmacología , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Prolactina/sangre , Animales , Electroforesis en Gel Bidimensional , Femenino , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos ICR , Técnicas de Cultivo de Órganos , Fosforilación , Embarazo , Prolactina/metabolismoRESUMEN
Several studies have indicated that prolactin (PRL) assumes oligomeric, proteolytically cleaved, phosphorylated and glycosylated forms. Phosphorylated PRL (PPRL) is considered to be the most important posttranslationally modified form in the rat. In the present study, we examined whether or not PRL is present in the mouse pituitary gland in the phosphorylated form. Mouse pituitary PRL was digested with acid phosphatase, resolved by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue, and then immunoblotted against the anti-PRL, anti-phosphoserine and anti-phosphothreonine antibodies. We also examined whether PRL is phosphorylated by protein kinases and semi-quantified the ratios of PPRL to PRL in the pituitary gland. The results indicated that three types of PRL are present in the pituitary glands of both male and female mice. One was non-phosphorylated (isoform 1), and the other two were immunoreactive to anti-phosphoserine (isoform 2) and/or anti-phosphothreonine (isoform 3) antibodies. The ratio between isoforms 2 and 1 of the 30-day-old female mice was higher than that of the 20-day-old female mice. However, the ratios among the three isoforms in the male pituitary glands did not differ with age. The ratio of PPRL to isoform 1 was obviously reduced after ovariectomy (OVX), and it recovered with estrogen replacement. These results suggest that estrogen influences PRL phosphorylation in female mice.
Asunto(s)
Estrógenos/metabolismo , Hipófisis/metabolismo , Prolactina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Densitometría , Electroforesis en Gel Bidimensional , Femenino , Immunoblotting , Masculino , Ratones , Ratones Endogámicos ICR , Fosforilación , Embarazo , Isoformas de Proteínas/metabolismoRESUMEN
The placenta is a highly differentiated organ essential for embryonic growth and development. In order to search for key molecules that are associated with mouse placental lactogen II (mPL-II) gene expression, we applied mouse cDNA microarray analysis to RNAs extracted from placentae on days 10, 12, 14, 16 and 18 of pregnancy. Changes in gene expression were categorized between days 10 and 12, 12 and 14, 14 and 16 and 16 and 18 of pregnancy. After microarray analysis, which had a minimum detectable fold change for differential expression of 2, we selected 10 genes, Apoa2, Apoc2, Ceacam14, Creg1, Fmo1, Igf2, Slc2a1, Spink3, Spi1-1 and Tpbpa, exhibiting a expression pattern similar to the mPL-II gene. Furthermore, we performed real-time PCR analysis and in situ hybridization (ISH) to find correlative expression genes for the mPL-II gene. From these results, we identified a resemblance in gene expression between mPL-II and Igf2 and selected these genes for performance of double-fluorescence immunohistochemical staining. We colocalized these proteins in labyrinthine trophoblast cells. These results strongly suggest that the expression of mPL-II and Igf2 is highly related to placental development in mice. This large-scale identification of genes regulated during placentogenesis assists in further elucidation of the molecular basis of extraembryonic development and function.
Asunto(s)
Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Placenta/fisiología , Lactógeno Placentario/genética , Animales , Apolipoproteína A-II/genética , Apolipoproteína C-II/genética , Moléculas de Adhesión Celular/genética , Femenino , Transportador de Glucosa de Tipo 1/genética , Glicoproteínas/genética , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos ICR , Neovascularización Fisiológica/fisiología , Oxigenasas/genética , Placenta/irrigación sanguínea , Lactógeno Placentario/metabolismo , Embarazo , Proteínas de Secreción Prostática/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Transactivadores/genética , Inhibidor de Tripsina Pancreática de KazalRESUMEN
Gene expressions and their interaction are complex and have not been definitely clarified in the placenta. To identify interactions of gene products previously not studied, we applied cDNA subtraction analyses to the placenta between days 12 and 16, days 12 and 14, days 14 and 16 of pregnancy. Among subtracted cDNAs cathepsin M, Q and R in PECs were specifically identified on days 14 and 16 pregnancy. All of these gene expressions exhibited a similar pattern to the mPL-II gene expression determined by northern blot and RT-PCR analyses. By means of in situ hybridization, these mRNAs were localized in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Double staining studies of cathepsin Q or cathepsin R mRNA by in situ hybridization followed by immunohistochemical staining of mPL-II in the same section revealed that signals for cathepsin Q and cathepsin R mRNAs were colocalized in mPL-II immunopositive trophoblast cells in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Possible association of cathepsins with mPL-II may play important roles in placental functions during the latter half of pregnancy in mice.