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1.
J Reprod Dev ; 70(5): 343-347, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39135241

RESUMO

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke's cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.


Assuntos
Diferenciação Celular , Fatores de Transcrição SOXB1 , Nicho de Células-Tronco , Tetraspanina 29 , Animais , Tetraspanina 29/metabolismo , Ratos , Fatores de Transcrição SOXB1/metabolismo , Adeno-Hipófise/metabolismo , Adeno-Hipófise/citologia , Masculino , Técnicas de Cultura de Células em Três Dimensões/métodos , Células Cultivadas , Proteoglicanas/metabolismo , Laminina/metabolismo , Colágeno , Combinação de Medicamentos
2.
Int J Mol Sci ; 25(20)2024 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-39457033

RESUMO

The intestinal tract is a typical radiosensitive tissue, and radiation rectal injury is a severe side effect that limits the prescribed dose in radiotherapy of the abdominal and pelvic region. Understanding the post-irradiation kinetics of Lgr5-positive stem cells is crucial in comprehending this adverse process. In this study, we utilized Lgr5-EGFP knock-in mice expressing EGFP and LGR5 antibody fluorescence staining of wild-type mice. At the state of radiation injury, the qPCR analysis showed a significant decrease in the expression level of Lgr5 in the rectal epithelial tissue. The dose-response relationship analysis showed that at low to moderate doses up to 10 gray (Gy), Lgr5-clustered populations were observed at the base of the crypt, whereas at sublethal doses (20 Gy and 29 Gy), the cells exhibited a dot-like scatter pattern, termed Lgr5-dotted populations. During recovery, 30 days post-irradiation, Lgr5-clustered populations gradually re-emerged while Lgr5-dotted populations declined, implying that some of the Lgr5-dotted stem cell populations re-clustered, aiding regenerations. Based on statistical analysis of the dose-response relationship using wild-type mice, the threshold dose for destroying these stem cell structures is 18 Gy. These findings may help set doses in mouse abdominal irradiation experiments for radiation intestinal injury and for understanding the histological process of injury development.


Assuntos
Proctite , Receptores Acoplados a Proteínas G , Células-Tronco , Animais , Proctite/etiologia , Proctite/patologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Camundongos , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação , Lesões por Radiação/patologia , Lesões por Radiação/metabolismo , Lesões por Radiação/etiologia , Reto/efeitos da radiação , Reto/patologia , Reto/metabolismo , Cinética , Células Epiteliais/efeitos da radiação , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Intestinal/efeitos da radiação , Mucosa Intestinal/patologia , Mucosa Intestinal/metabolismo , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Relação Dose-Resposta à Radiação , Técnicas de Introdução de Genes
3.
J Reprod Dev ; 69(6): 308-316, 2023 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-37778977

RESUMO

The adenohypophysis is comprised of the anterior and intermediate lobes (AL and IL, respectively). Cluster of differentiation 9 (CD9)- and sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor hormone-producing cells in the AL. They are located in the marginal cell layer (MCL) facing Rathke's cleft between the AL and IL (primary niche) and the parenchyma of the AL (secondary niche). We previously showed that, in rats, CD9/SOX2-positive cells in the IL side of the MCL (IL-side MCL) migrate to the AL side (AL-side MCL) and differentiate into prolactin-producing cells (PRL cells) in the AL parenchyma during pregnancy, lactation, and diethylstilbestrol treatment, all of which increase PRL cell turnover. This study examined the changes in CD9/SOX2-positive stem/progenitor cell niches and their proportions by manipulating the turnover of growth hormone (GH)- and thyroid-stimulating hormone (TSH)-producing cells (GH and TSH cells, respectively), which are Pit1 lineage cells, as well as PRL cells. After induction, the isolated CD9/SOX2-positive cells from the IL-side MCL formed spheres and differentiated into GH and TSH cells. We also observed an increased GH cell proportion upon treatment with GH-releasing hormone and recovery from continuous stress and an increased TSH cell proportion upon propylthiouracil treatment, concomitant with alterations in the proportion of CD9/SOX2-positive cells in the primary and secondary niches. These findings suggest that CD9/SOX2-positive cells have the potential to supply GH and TSH when an increase in GH and TSH cell populations is required in the adult pituitary gland.


Assuntos
Adeno-Hipófise , Animais , Feminino , Ratos , Hormônio do Crescimento , Hipófise/metabolismo , Adeno-Hipófise/metabolismo , Prolactina , Tireotropina , Tetraspanina 29/metabolismo , Fatores de Transcrição SOXB1/metabolismo
4.
Health Commun ; 38(9): 1904-1915, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35253531

RESUMO

This study examines whether using virtual reality (VR) with older adults with mild cognitive impairment (MCI) or mild to moderate dementia with a family member who lives at a distance can improve the quality of life of the older adult and the family member. Twenty-one older adults in a senior living community and a family member (who participated in the VR with the older adult from a distance) engaged in a baseline telephone call, followed by three weekly VR sessions. The VR was associated with improvements in older adults' affect and stress, relationship with their family member, and overall quality of life, compared to baseline. Family members' negative affect, depressive symptoms, and caregiver burden also decreased and their mental health improved after using the VR, compared to baseline. Using the VR, however, did not change their relationship with the older adult. In addition, older adults and family members who experienced the VR sessions as more socially engaging reported better psychological and relational well-being, with older adults also experiencing greater improvements in overall quality of life. Finally, preliminary results suggest that older adults with dementia and their family members might benefit even more from using the VR than older adults with MCI and their family members.


Assuntos
Disfunção Cognitiva , Demência , Realidade Virtual , Humanos , Idoso , Qualidade de Vida , Disfunção Cognitiva/psicologia , Família , Demência/psicologia
5.
Cell Tissue Res ; 388(3): 583-594, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35316373

RESUMO

The adenohypophysis consists of the anterior and intermediate lobes (AL and IL). The marginal cell layer (MCL), including the ventral region of the IL and the dorsal region of the AL lining the Rathke's cleft, acts as the primary stem/progenitor cell niches in adult adenohypophysis. The cells of the MCL on the IL side consisted of cluster of differentiation 9 (CD9)-positive stem/progenitor cells with or without motile cilia. However, any additional cellular properties of multiciliated CD9-positive cells are not known. The present study aimed to identify the character of the multiciliated cells in stem cell niche of the pituitary gland. We observed the fine structure of the multiciliated cells in the MCL of male Wistar rats at an early stage after birth and in adulthood (P60) using scanning electron microscopy. Since the previous study showed that the MCL cells of adult rats synthesize retinoic acid (RA), the present study determined whether the multiciliated cells are involved in RA regulation by the expression of retinal aldehyde dehydrogenase 1 (RALDH1) and CYP26A1, an enzyme synthesizing and degrading RA, respectively. Results showed that 96% of multiciliated cells in adult male rats expressed CYP26A1, while 60% expressed RALDH1. Furthermore, the isolated CD9-positive cells from the IL side MCL responded to RA and activated the degradation system of RA by increasing Cyp26a1 expression. These findings indicated that multiciliated cells are involved in RA metabolism in the MCL. Our observations provide novel insights regarding the stem cell niche of the adult pituitary.


Assuntos
Adeno-Hipófise , Tretinoína , Animais , Masculino , Hipófise/metabolismo , Adeno-Hipófise/metabolismo , Ratos , Ratos Wistar , Ácido Retinoico 4 Hidroxilase/metabolismo , Tretinoína/metabolismo , Tretinoína/farmacologia
6.
J Reprod Dev ; 68(4): 278-286, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35691820

RESUMO

Sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor cells in the adenohypophysis, comprising the anterior and intermediate lobes (AL and IL, respectively). The cells are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). We previously demonstrated in vitro that the tetraspanin superfamily CD9 and SOX2 double-positive (CD9/SOX2-positive) cells in the IL-side MCL migrate to the AL side and differentiate into hormone-producing and endothelial cells in the AL parenchyma. Here, we performed in vivo studies to evaluate the role of IL-side CD9/SOX2-positive cells in pregnancy, lactation, and treatment with diethylstilbestrol (DES; an estrogen analog) when an increased population of prolactin (PRL) cells was observed in the AL of the rat pituitary. The proportions of CD9/SOX2-, CD9/Ki67-, and PRL/TUNEL-positive cells decreased in the primary and secondary niches during pregnancy and DES treatment. In contrast, the number of CD9/PRL-positive cells increased in the AL-side MCL and AL parenchyma during pregnancy and during DES treatment. The proportion of PRL/Ki67-positive cells increased in the AL-side MCL and AL parenchyma in response to DES treatment. Next, we isolated CD9-positive cells from the IL-side MCL using an anti-CD9 antibody. During cell culture, the cells formed free-floating three-dimensional clusters (pituispheres). Furthermore, CD9-positive cells in the pituisphere differentiated into PRL cells, and their differentiation potential was promoted by DES. These findings suggest that CD9/SOX2-positive cells in the IL-side MCL may act as adult stem cells in the AL parenchyma that supply PRL cells under the influence of estrogen.


Assuntos
Adeno-Hipófise , Prolactina , Animais , Diferenciação Celular/fisiologia , Células Endoteliais , Feminino , Antígeno Ki-67 , Hipófise , Gravidez , Ratos , Ratos Wistar , Fatores de Transcrição SOXB1/imunologia , Células-Tronco , Tetraspanina 29/imunologia
7.
J Reprod Dev ; 68(3): 225-231, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35418523

RESUMO

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100ß, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.


Assuntos
Proteínas de Junções Íntimas , Junções Íntimas , Animais , Ocludina/genética , Ocludina/metabolismo , Hipófise/metabolismo , Ratos , Nicho de Células-Tronco , Células-Tronco , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo
8.
Histochem Cell Biol ; 155(3): 391-404, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33221951

RESUMO

SOX2-positive cells are stem/progenitor cells that supply hormone-producing cells; they are found in the anterior lobe of the rodent pituitary gland. However, they are likely composed of several subpopulations. In rats, a SOX2-positive cell populations can be distinguished by the presence of S100ß. We identified the novel markers cluster of differentiation (CD) CD9 and CD81, members of the tetraspanin superfamily, for the identification of S100ß/SOX2-positive cells. Recently, CD9/CD81 double-knockout mice were generated. Although they grew normally until 3 weeks after birth, they exhibited atrophy of the pituitary gland. These findings suggested that CD9/CD81/S100ß/SOX2-positive cells in the mouse pituitary are adult stem/progenitor cells. To substantiate this hypothesis, we examined CD9 and CD81 expression in the adult and developing anterior lobe. Immunohistochemistry showed that CD9/CD81-positive cells began appearing from postnatal day 0 and settled in the stem cell niches (marginal cell layer and parenchyma) of the adult anterior lobe while expressing S100ß. We next isolated CD9 -positive cells from the adult anterior lobe, using the anti-CD9 antibody for cell characterisation. The cells in culture formed free-floating three-dimensional clusters (pituispheres); moreover, induction into all types of hormone-producing cells was successful. Furthermore, reduction of CD9 and CD81 mRNAs by siRNAs inhibited cell proliferation. These findings indicate that CD9/CD81/S100ß/SOX2-positive cells may play a role as adult stem/progenitor cells in SOX2-positive subpopulations, thus supplying hormone-producing cells in the postnatal anterior lobe. Furthermore, CD9 and CD81 are implicated in cell proliferation. The current findings provide novel insights into adult pituitary stem/progenitor cells.


Assuntos
Hipófise/citologia , Células-Tronco/citologia , Tetraspanina 29/imunologia , Animais , Anticorpos/imunologia , Diferenciação Celular , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos ICR , Hipófise/imunologia , Células-Tronco/imunologia
9.
Histochem Cell Biol ; 155(4): 503-512, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33398434

RESUMO

Hypertension leads to structural remodeling of cerebral blood vessels, which has been implicated in the pathophysiology of cerebrovascular diseases. The remodeling and progression of arteriolosclerosis under hypertension involve fibrosis along with the production of type I collagen around cerebral arterioles. However, the source and regulatory mechanisms of this collagen production remain elusive. In this study, we examined if perivascular macrophages (PVMs) are involved in collagen production around cerebral small vessels in hypertensive SHRSP/Izm rats. Immunoreactivity for type I collagen around cerebral small vessels in 12-week-old hypertensive rats tended to higher than those in 4-week-old hypertensive and 12-week-old control rats. In ultrastructural analyses using transmission electron microscopy, the substantial deposition of collagen fibers could be observed in the intercellular spaces around PVMs near the arterioles of rats with prolonged hypertension. In situ hybridization analyses revealed that cells positive for mRNA of Col1a1, which comprises type I collagen, were observed near cerebral small vessels. The Col1a1-positive cells around cerebral small vessels were colocalized with immunoreactivity for CD206, a marker for PVMs, but not with those for glial fibrillary acidic protein or desmin, markers for other perivascular cells such as astrocytes and vascular smooth muscle cells. These results demonstrated that enhanced production of type I collagen is observed around cerebral small vessels in rats with prolonged hypertension and Col1a1 is expressed by PVMs, and support the concept that PVMs are involved in collagen production and vascular fibrosis under hypertensive conditions.


Assuntos
Artérias Cerebrais/metabolismo , Colágeno Tipo I/biossíntese , Hipertensão/metabolismo , Macrófagos/metabolismo , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY
10.
Cell Tissue Res ; 385(3): 713-726, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33961126

RESUMO

A supply of hormone-producing cells from stem/progenitor cells is critical to sustain the endocrine activity of the pituitary gland. In the adenohypophysis composing the anterior and intermediate lobe (AL and IL, respectively), stem/progenitor cells expressing sex-determining region Y-box 2 (SOX2) and S100ß are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). Our previous studies using mice and rats indicated that the tetraspanin superfamily CD9 and CD81 are expressed in S100ß/SOX2-positive cells of primary and secondary niches (named CD9/CD81/S100ß/SOX2-positive cell), and the cells located in the AL-side niches exhibit plasticity and multipotency. However, it is unclear whether CD9/CD81/S100ß/SOX2-positive cells in the IL-side primary niche are stem/progenitor cells for the AL or IL. Here, we successfully isolated pure CD9/CD81/S100ß/SOX2-positive cells from the IL-side primary niche. They had a higher level of S100ß and SOX2 mRNA and a greater pituisphere forming capacity than those of CD9/CD81/S100ß/SOX2-positive cells isolated from the AL. They also had capacity to differentiate into all types of adenohypophyseal hormone-producing cells, concomitantly with the loss of CD9 expression. Loss of CD9 and CD81 function in CD9/CD81/S100ß/SOX2-positive cells by siRNA treatment impaired prolactin cell differentiation. Consistently, in the pituitary gland of CD9/CD81 double knockout mice, dysgenesis of the MCL and a lower population of prolactin cells were observed. These results suggest that the CD9/CD81/S100ß/SOX2-positive cells in the MCL of the IL-side are potential suppliers of adult core stem cells in the AL.


Assuntos
Hipófise/anatomia & histologia , Prolactina/metabolismo , Tetraspanina 29/metabolismo , Animais , Masculino , Camundongos , Ratos , Ratos Wistar
11.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445338

RESUMO

Recently, another new cell type was found in the perivascular space called a novel desmin-immunopositive perivascular (DIP) cell. However, the differences between this novel cell type and other nonhormone-producing cells have not been clarified. Therefore, we introduced several microscopic techniques to gain insight into the morphological characteristics of this novel DIP cell. We succeeded in identifying novel DIP cells under light microscopy using desmin immunocryosection, combining resin embedding blocks and immunoelectron microscopy. In conventional transmission electron microscopy, folliculostellate cells, capsular fibroblasts, macrophages, and pericytes presented a flat cisternae of rough endoplasmic reticulum, whereas those of novel DIP cells had a dilated pattern. The number of novel DIP cells was greatest in the intact rats, though nearly disappeared under prolactinoma conditions. Additionally, focused ion beam scanning electron microscopy showed that these novel DIP cells had multidirectional processes and some processes reached the capillary, but these processes did not tightly wrap the vessel, as is the case with pericytes. Interestingly, we found that the rough endoplasmic reticulum was globular and dispersed throughout the cytoplasmic processes after three-dimensional reconstruction. This study clearly confirms that novel DIP cells are a new cell type in the rat anterior pituitary gland, with unique characteristics.


Assuntos
Desmina/metabolismo , Pericitos , Adeno-Hipófise/diagnóstico por imagem , Animais , Desmina/análise , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Pericitos/citologia , Pericitos/metabolismo , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Ratos , Ratos Wistar
12.
Histochem Cell Biol ; 153(6): 385-396, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32152663

RESUMO

Approximately 8% of CD9-, S100ß- and SOX2-triple positive (CD9/S100ß/SOX2-positive) stem/progenitor cells in the anterior lobe of the rat pituitary gland have previously been shown to differentiate into endothelial cells in vitro, suggesting that they play a role in vascularisation as tissue-resident vascular precursor cells. In the present study, we focused on chemokine ligands to further characterise the CD9/S100ß/SOX2-positive cells and found that they distinctively express CX3C chemokine ligand 1 (Cx3cl1). Immunohistochemical analysis of the anterior lobe showed that CX3CL1-positive cells comprised 7.8% in CD9-positive cells. By cultivation of the CD9-positive cells on laminin-coated plates, we observed that the expression levels of Cx3cl1 decreased, while those of Sox18, an endothelial cell-progenitor marker, and Cx3cr1, a CX3CL1 receptor, increased. Furthermore, in a rat model of prolactinoma, the most common pituitary tumour, which is accompanied by frequent neo-vasculogenesis in the anterior lobe, we have confirmed a decrease in Cx3cl1 expression and an increase in Cx3cr1 expression, as well as a prominent increase in Sox18 expression. These findings suggest that CX3CL1/CX3CR1 signalling in CD9/S100ß/SOX2-positive cells plays an important role in resupplying endothelial cells for vascular remodelling in the anterior lobe.


Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Células Endoteliais/citologia , Hipófise/citologia , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo , Tetraspanina 29/metabolismo , Animais , Diferenciação Celular , Células Endoteliais/metabolismo , Masculino , Hipófise/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Transdução de Sinais/genética , Células-Tronco/citologia
13.
Cell Tissue Res ; 381(2): 273-284, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32418131

RESUMO

The anterior pituitary gland is composed of five types of hormone-producing cells and folliculo-stellate cells. Folliculo-stellate cells do not produce anterior pituitary hormones but they are thought to play important roles as stem cells, phagocytes, or supporting cells of hormone-producing cells in the anterior pituitary. S100ß protein has been used as a folliculo-stellate cell marker in some animals, including rats. However, since no reliable molecular marker for folliculo-stellate cells has been reported in mice, genetic approaches for the investigation of folliculo-stellate cells in mice are not yet available. Aldolase C/Zebrin II is a brain-type isozyme and is a fructose-1,6-bisphosphate aldolase. In the present study, we first used immunohistochemistry to verify that aldolase C was produced in the anterior pituitary of rats. Moreover, using transgenic rats expressing green fluorescent protein under the control of the S100ß gene promoter, we identified aldolase C-immunoreactive signals in folliculo-stellate cells and marginal cells located in the parenchyma of the anterior pituitary and around Rathke's cleft, respectively. We also identified aldolase C-expressing cells in the mouse pituitary using immunohistochemistry and in situ hybridization. Aldolase C was not produced in any pituitary hormone-producing cells, while aldolase C-immunopositive signal co-localized with E-cadherin- and SOX2-positive cells. Using post-embedding immunoelectron microscopy, aldolase C-immunoreactive products were observed in the cytoplasm of marginal cells and folliculo-stellate cells of the mouse pituitary. Taken together, aldolase C is a common folliculo-stellate cell marker in the anterior pituitary gland of rodents.


Assuntos
Frutose-Bifosfato Aldolase/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Adeno-Hipófise , Animais , Biomarcadores/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Ratos , Ratos Transgênicos
14.
J Reprod Dev ; 66(6): 515-522, 2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-32830152

RESUMO

Cluster of differentiation (CD) 9 and CD81 are closely-related members of the tetraspanin family that consist of four-transmembrane domain proteins. Cd9 and Cd81 are highly expressed in breast cancer cells; however, their expression in healthy mammary glands is unclear. In this study, we performed quantitative real-time PCR to analyze the expression levels of Cd9 and Cd81. Histological techniques were employed to identify Cd9- and Cd81-expressing cells in rat mammary glands during pregnancy and lactation. It was observed that Cd9 and Cd81 were expressed in the mammary glands, and their expression levels correlated with mammary gland development. To identify cells expressing Cd9 and Cd81 in the mammary glands, we performed double immunohistochemical staining for CD9 and CD81, prolactin receptor long form, estrogen receptor alpha, or Ki67. The results showed that CD9 and CD81 were co-expressed in proliferating mammary epithelial cells. Next, we attempted to isolate CD9-positive epithelial cells from the mammary gland using pluriBead cell-separation technology based on antibody-mediated binding of cells to beads of different sizes, followed by isolation using sieves with different mesh sizes. We successfully isolated CD9-positive epithelial cells with 96.8% purity. In addition, we observed that small-interfering RNAs against Cd9 and Cd81 inhibited estrogen-induced proliferation of CD9-positive mammary epithelial cells. Our current findings may provide novel insights into the proliferation of mammary epithelial cells during pregnancy and lactation as well as in pathological processes associated with breast cancer.


Assuntos
Células Epiteliais/citologia , Perfilação da Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Tetraspanina 28/biossíntese , Tetraspanina 29/biossíntese , Animais , Diferenciação Celular , Proliferação de Células , Dietilestilbestrol , Receptor alfa de Estrogênio/biossíntese , Feminino , Antígeno Ki-67/biossíntese , Lactação , Gravidez , Prenhez , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
15.
Cell Tissue Res ; 376(2): 281-293, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30666536

RESUMO

TtT/GF is a mouse cell line derived from a thyrotropic pituitary tumor and has been used as a model of folliculostellate cells. Our previous microarray data indicate that TtT/GF possesses some properties of endothelial cells, pericytes and stem/progenitor cells, along with folliculostellate cells, suggesting its plasticity. We also found that transforming growth factor beta (TGFß) alters cell motility, increases pericyte marker transcripts and attenuates endothelial cell and stem/progenitor cell markers in TtT/GF cells. The present study explores the wide-range effect of TGFß on TtT/GF cells at the protein level and characterizes TGFß-induced proteins and their partnerships using stable isotope labeling of amino acids in cell culture (SILAC)-assisted quantitative mass spectrometry. Comparison between quantified proteins from TGFß-treated cells and those from SB431542 (a selective TGFß receptor I inhibitor)-treated cells revealed 51 upregulated and 112 downregulated proteins (|log2| > 0.6). Gene ontology and STRING analyses revealed that these are related to the actin cytoskeleton, cell adhesion, extracellular matrix and DNA replication. Consistently, TGFß-treated cells showed a distinct actin filament pattern and reduced proliferation compared to vehicle-treated cells; SB431542 blocked the effect of TGFß. Upregulation of many pericyte markers (CSPG4, NES, ACTA, TAGLN, COL1A1, THBS1, TIMP3 and FLNA) supports our previous hypothesis that TGFß reinforces pericyte properties. We also found downregulation of CTSB, EZR and LGALS3, which are induced in several pituitary adenomas. These data provide valuable information about pericyte differentiation as well as the pathological processes in pituitary adenomas.


Assuntos
Plasticidade Celular , Proteínas do Citoesqueleto/metabolismo , Adeno-Hipófise/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Marcação por Isótopo , Espectrometria de Massas , Camundongos , Complexos Multienzimáticos/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Adeno-Hipófise/metabolismo , Proteômica
16.
Cells Tissues Organs ; 207(3-4): 127-137, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31805566

RESUMO

Neuronatin (NNAT) was first identified as a gene selectively and abundantly expressed in the cytoplasm of the newborn mouse brain, and involved in neonatal neurogenesis. However, the particular roles of NNAT in the developing prenatal brain have not been identified, especially in mid to late stages. In this study, we performed immunohistochemical analyses of NNAT and SOX2 proteins, a nuclear transcription factor and neural stem/progenitor marker, in the rat brain on embryonic days 13.5, E16.5, and E20.5. NNAT signals were broadly observed across the developing brain on E13.5 and gradually more localized in later stages, eventually concentrated in the alar and basal parts of the terminal hypothalamus, the alar plate of prosomere 2 of the thalamus, and the choroid plexus in the lateral and fourth ventricles on E20.5. In particular, the mammillary body in the basal part of the terminal hypothalamus, a region with a high number of SOX2-positive cells, evidenced intense NNAT signals on E20.5. The intracellular localization of NNAT showed diverse profiles, suggesting that NNAT was involved in various cellular functions, such as cell differentiation and functional maintenance, during prenatal neurogenesis in the rat brain. Thus, the present observations suggested diverse and active roles of the NNAT protein in neurogenesis. Determining the function of this molecule may assist in the elucidation of the mechanisms involved in brain development.


Assuntos
Encéfalo/embriologia , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Células-Tronco Neurais/citologia , Neurogênese , Animais , Encéfalo/citologia , Feminino , Gravidez , Ratos , Ratos Wistar , Fatores de Transcrição SOXB1/análise
17.
Cell Tissue Res ; 371(2): 339-350, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29274061

RESUMO

The non-endocrine TtT/GF mouse pituitary cell line was derived from radiothyroidectomy-induced pituitary adenoma. In addition to morphological characteristics, because the cells are S100ß-positive, they have been accepted as a model of folliculostellate cells. However, our recent microarray analysis indicated that, in contrast to folliculostellate cells, TtT/GF cells might not be terminally differentiated, as they share some properties with stem/progenitor cells, vascular endothelial cells and pericytes. The present study investigates whether transforming growth factor beta (TGFß) can elicit further differentiation of these cells. The results showed that canonical (Tgfbr1 and Tgfbr2) and non-canonical TGFß receptors (Tgfbr3) as well as all TGFß ligands (Tgfb1-3) were present in TtT/GF cells, based on reverse transcription PCR. SMAD2, an intercellular signaling molecule of the TGFß pathway, was localized in the nucleus upon TGFß signaling. Furthermore, TGFß induced cell colony formation, which was completely blocked by a TGFß receptor I inhibitor (SB431542). Real-time PCR analysis indicated that TGFß downregulated stem cell markers (Sox2 and Cd34) and upregulated pericyte markers (Nestin and Ng2). Double immunohistochemistry using mouse pituitary tissue confirmed the presence of NESTIN/NG2 double-positive cells in perivascular areas where pericytes are localized. Our results suggest that TtT/GF cells are responsive to TGFß signaling, which is associated with cell colony formation and pericyte differentiation. As pericytes have been shown to regulate angiogenesis, tumorigenesis and stem/progenitor cells in other tissues, TtT/GF cells could be a useful model to study the role of pituitary pericytes in physiological and pathological processes.


Assuntos
Pericitos/metabolismo , Hipófise/citologia , Hipófise/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Antígenos/metabolismo , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Forma Celular , Humanos , Ligantes , Camundongos , Nestina/metabolismo , Isoformas de Proteínas/metabolismo , Proteoglicanas/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/genética
18.
J Reprod Dev ; 64(5): 417-422, 2018 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-30033984

RESUMO

Studies on mouse and rat pituitaries reported that Sox2-expressing cells play roles as stem/progenitor cells in the adult pituitary gland. The presence of cells with stem cell-like properties in the pituitary adenoma and SOX2-positive cells has been demonstrated in the human pituitary. However, considering the difficulty in fully examining the stem/progenitor cell properties in the human pituitary, in the present study, we analyzed the SOX2-positive cells in the pituitary of the adult common marmoset (Callithrix jacchus), which is used as a non-human primate model. Immunohistochemistry demonstrated that localization pattern of SOX2-positive cells in the common marmoset pituitary was similar to that observed in the rodent pituitary, i.e., in the two types of niches (marginal cell layer and parenchymal-niche) and as scattered single cells in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells express S100 and were located in the center or interior of LAMININ-positive micro-lobular structures. Collectively, the present study reveals properties of SOX2-positive cells in the common marmoset pituitary and suggests that the common marmoset proves to be a useful tool for analyzing pituitary stem/progenitor cells in a non-human primate model.


Assuntos
Adeno-Hipófise/citologia , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/citologia , Animais , Callithrix , Diferenciação Celular , Feminino , Imuno-Histoquímica , Laminina/metabolismo , Masculino , Ratos , Ratos Wistar , Nicho de Células-Tronco , Temperatura
19.
Med Mol Morphol ; 51(3): 147-155, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29344720

RESUMO

Prolactinomas are the most common tumor of the human pituitary. They result in excessive prolactin secretion and important changes in the vasculature. Pericytes are perivascular cells associated with capillaries and have crucial roles in physiological and pathological neovascularization. We previously reported that pericytes produce type I and III collagens in the anterior pituitary of adult rats. In addition, pituitary pericytes contained well-developed cell organelles and actively synthesized collagens during early postnatal development. However, the characteristics of pericytes in pituitary tumors are unclear. In this study, we used diethylstilbestrol (DES)-treated rats as an animal model of prolactinoma. Using five common pericyte markers, more pericytes were observed in rats treated with DES for 3 months (prolactinoma) compared to the control. Transmission electron microscopy revealed that attached and semidetached pericytes exhibited active cell organelles. Moreover, we identified pericyte migration between capillaries. Although the fine structure of pituitary pericytes was active in prolactinoma, expressions of type I and III collagen mRNAs were greatly diminished. In sum, the characteristics and functions of pericytes were altered in pituitary tumors. This study is the first to clarify fine structural changes of pericytes in rat prolactinomas and improves our understanding of the function of pericytes under pathological conditions.


Assuntos
Pericitos/patologia , Hipófise/citologia , Neoplasias Hipofisárias/patologia , Prolactinoma/patologia , Animais , Capilares/citologia , Capilares/ultraestrutura , Colágeno/metabolismo , Dietilestilbestrol/toxicidade , Feminino , Humanos , Microscopia Eletrônica de Transmissão , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Pericitos/ultraestrutura , Hipófise/irrigação sanguínea , Hipófise/patologia , Neoplasias Hipofisárias/induzido quimicamente , Prolactinoma/induzido quimicamente , Ratos , Ratos Endogâmicos F344
20.
Cell Tissue Res ; 368(2): 371-378, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28120110

RESUMO

Macrophages are present throughout the anterior pituitary gland. However, the features and function of macrophages in the gland are poorly understood. Recent studies have indicated that there are two main macrophage classes: M1 (classically activated) and M2 (alternatively activated). In this study, we examine whether both M1 and M2 macrophages are present in the anterior pituitary gland of rats. Our findings indicate that macrophages that are positive for CD68 (a pan-macrophage marker) were localized near capillaries in rat anterior pituitary gland. These macrophages were positive for iNOS or mannose receptor (MR), which are markers of M1 and M2 macrophages, respectively. To determine the morphological characteristics of M2 macrophages under pathological conditions, diethylstilbestrol (DES)-treated rats were used as an animal model of prolactinoma. After 2 weeks of DES treatment, a number of MR-immunopositive cells were present in the gland. Immunoelectron microscopy revealed that MR-immunopositive M2 macrophages had many small vesicles and moderately large vacuoles in cytoplasm. Phagosomes were sometimes present in cytoplasm. Interestingly, M2 macrophages in prolactinoma tissues did not usually exhibit distinct changes or differences during the normal, hyperplasia and adenoma stages. This study is the first to confirm that both M1 and M2 macrophages are present in the anterior pituitary gland of rats. Moreover, the number of M2 macrophages was greatly increased in rats with DES-induced prolactinoma. Future studies should attempt to characterize the functional role of M2 macrophages in the gland.


Assuntos
Polaridade Celular , Estrogênios/efeitos adversos , Macrófagos/patologia , Adeno-Hipófise/patologia , Prolactinoma/patologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Dietilestilbestrol , Imuno-Histoquímica , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Adeno-Hipófise/metabolismo , Adeno-Hipófise/ultraestrutura , Prolactinoma/metabolismo , Prolactinoma/ultraestrutura , Ratos Wistar , Receptores de Superfície Celular/metabolismo
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