Naoko Kanno

Coxsackievirus and adenovirus receptor-positive cells compose the putative stem/progenitor cell niches in the marginal cell layer and parenchyma of the rat anterior pituitary

The pituitary gland is a slow generative tissue but actively responds to demands by changing homeostasis. The marginal cell layer (MCL) facing the residual lumen has long been indicated as a stem/progenitor cell niche of the pituitary. On the other hand, the coxsackievirus and adenovirus receptor (CAR), which localizes at the tight-junction of the polarized epithelium, is known to participate in the development, differentiation and regeneration of specified tissues. The present study attempts to characterize the cells lining the MCL during pituitary development by immunohistochemistry of CAR. Consequently, we found that CAR localizes in an apical surface of the single cell layer facing the oral cavity in the invaginating oral epithelium on rat embryonic day (E) 11.5. On E13.5, when this single layer constructs the MCL in the pituitary primordium Rathke’s pouch, CAR-positive cells occupied the MCL and this localization pattern of CAR was persistently maintained throughout life. Moreover, clusters of CAR-positive cells were also found in the parenchyma. CAR-positive cells were positive for stem/progenitor cell markers sex-determining region Y-box 2 (SOX2) and epithelial calcium-dependent adhesion (E-cadherin). However, prior to the postnatal growth wave, cells positive for CAR in the basolateral surface constructed multiple cell layers beneath the MCL and cell-type transition to a putative migratory cell phenotype by fading of SOX2 and E-cadherin occurred, suggesting the composition of new putative niches in the parenchyma. These data, together with our previous reports, suggest that CAR-positive cells are pituitary stem/progenitor cells and compose putative stem/progenitor cell niches in the MCL and parenchyma.

Rapid transition of NESTIN-expressing dividing cells from PROP1-positive to PIT1-positive advances prenatal pituitary development.

We recently reported that the quantitative and qualitative transition of stem/progenitor cells occurs by the acquisition of a novel mechanism in the terminal differentiation during postnatal development of the anterior pituitary. We hypothesised that this novel mechanism is an alteration of a cell supply system accompanying proliferation of the progenitor cells. In the present study, we examined the proliferation activities of progenitor cells by indication of the expression of Nestin, a marker of rapidly dividing progenitor cells, aiming to verify our hypothesis and to resolve another outstanding issue regarding whether the Nestin gene is expressed in the pituitary. We found that NESTIN‐positive dividing cells certainly exist in the pituitary through all stages of development. Almost all of the PROP1‐positive progenitor cells express Nestin in early embryonic pituitary development. Thereafter, Nestin‐expressing dividing cells involved in the cell supply system transfer from PROP1‐positive progenitor cells to committed progenitor cells, such as PIT1‐positive cells, on neonatal pituitary development. Furthermore, our data, together with the findings of previous studies on cell lineage tracing analyses using Nestin‐Cre mice derived by the central nervous system (CNS)‐specific Nestin promoter, suggest that at least two regulation systems for Nestin‐expression exist in the pituitary, with the majority of these not being CNS‐specific.

Temporospatial gene expression of Prx1 and Prx2 is involved in morphogenesis of cranial placode-derived tissues through epithelio-mesenchymal interaction during rat embryogenesis

Paired-related homeobox transcription factors, PRX1 and PRX2, are verified to play essential roles in limb, heart and craniofacial development by analyses of knockout animals. Their gene expression in the embryonic primordia derived from the mesoderm and neural crest is confirmed by in situ hybridization. Nevertheless, a detailed localization of PRX1 and PRX2 was not carried out because of a lack of specific antibodies for each factor. We have previously confirmed the presence of PRX proteins in rat embryonic pituitary by using an antibody that recognizes both PRX1 and PRX2. However, the pituitary originates in the cranial placodes, not the mesoderm or neural crest. In this study, we analyze the temporospatial distribution of PRX1 and PRX2 with novel antibodies specific for each factor, together with a stem/progenitor marker SOX2 (sex-determining region Y-box 2) in the primordia formed by epithelio-mesenchymal interaction. We observe immunoreactive signals of both PRX proteins in rat embryo, showing a similar pattern to that obtained by in situ hybridization. In early embryogenesis, PRX proteins are not co-localized with SOX2 but PRX2 and/or PRX1-positive cells are present in the border or periphery of SOX2-positive primordia originating in the cranial placode. During advanced embryogenesis, either PRX2-positive cells become condensed in the border of SOX2-positive cells or PRX1 and/or PRX2 become co-localized with SOX2. Our results suggest that PRX proteins, especially PRX2, play a role in the morphogenesis of the primordial tissues formed by the epithelio-mesenchymal interaction and that neural crest cells contribute to the morphogenesis of tissues derived from the cranial placode.

Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland

S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100β-positive cells by means of their property of adhering to laminin.

Gene tracing analysis reveals the contribution of neural crest‐derived cells in pituitary development

The anterior pituitary originates from the adenohypophyseal placode. Both the preplacode region and neural crest (NC) derive from subdivision of the neural border region, and further individualization of the placode domain is established by a reciprocal interaction between placodal precursors and NC cells (NCCs). It has long been known that NCCs are present in the adenohypophysis as interstitial cells. A recent report demonstrated that NCCs also contribute to the formation of pericytes in the developing pituitary. Here, we attempt to further clarify the role of NCCs in pituitary development using P0‐Cre/EGFP reporter mice. Spatiotemporal analyses revealed that GFP‐positive NCCs invaded the adenohypophysis in a stepwise manner. The first wave was detected on mouse embryonic day 9.5 (E9.5), when the pituitary primordium begins to be formed by adenohypophyseal placode cells; the second wave occurred on E14.5, when vasculogenesis proceeds from Atwells recess. Finally, fate tracing of NCCs demonstrated that NC‐derived cells in the adenohypophysis terminally differentiate into all hormone‐producing cell lineages as well as pericytes. Our data suggest that NCCs contribute to pituitary organogenesis and vasculogenesis in conjunction with placode‐derived pituitary stem/progenitor cells.

Isolation of adult pituitary stem/progenitor cell clusters located in the parenchyma of the rat anterior lobe

Recent studies have demonstrated that Sox2-expressing stem/progenitor cells play roles in the pituitary cell turnover. Two types of niches have been proposed for stem/progenitor cells, the marginal cell layer (MCL) and the dense cell clusters in the parenchyma. Among them, the appearance of the parenchymal-niche only after birth indicates that this niche is involved in the cell turnover required for the postnatal pituitary. However, little is known about the roles of the parenchymal-niche and its regulation. The present study aimed to isolate pituitary stem/progenitor cells from the parenchymal-niche in the adult rat pituitary. Cell dispersion by stepwise treatment with proteases allowed the isolation of dense cell clusters. Immunocytochemistry demonstrated that clusters are universally composed of SOX2-positive cells, and most of them are positive for PROP1. Taken together with the anatomical analysis, we concluded that the isolated clusters are the parenchymal stem/progenitor cell (PS)-clusters, not the MCL-one. PS-clusters cultivated by serum-free overlay 3-dimensional culture maintained their stemness, and treatment with bFGF and EGF induced cyst-formation. Moreover, PS-clusters demonstrated some differentiation capacity with GSK3β-inhibitor treatment. Collectively, the present study demonstrates a simple method for isolating stem/progenitor cells from the parenchymal-niche, and provides tools to analyze the factors for regulating the pituitary niches.

Search for regulatory factors of the pituitary-specific transcription factor PROP1 gene.

Pituitary-specific transcription factor PROP1, a factor important for pituitary organogenesis, appears on rat embryonic day 11.5 (E11.5) in SOX2-expressing stem/progenitor cells and always coexists with SOX2 throughout life. PROP1-positive cells at one point occupy all cells in Rathke’s pouch, followed by a rapid decrease in their number. Their regulatory factors, except for RBP-J, have not yet been clarified. This study aimed to use the 3 kb upstream region and 1st intron of mouse prop1 to pinpoint a group of factors selected on the basis of expression in the early pituitary gland for expression of Prop1. Reporter assays for SOX2 and RBP-J showed that the stem/progenitor marker SOX2 has cell type-dependent inhibitory and activating functions through the proximal and distal upstream regions of Prop1, respectively, while RBP-J had small regulatory activity in some cell lines. Reporter assays for another 39 factors using the 3 kb upstream regions in CHO cells ultimately revealed that 8 factors, MSX2, PAX6, PIT1, PITX1, PITX2, RPF1, SOX8 and SOX11, but not RBP-J, regulate Prop1 expression. Furthermore, a synergy effect with SOX2 was observed for an additional 10 factors, FOXJ1, HES1, HEY1, HEY2, KLF6, MSX1, RUNX1, TEAD2, YBX2 and ZFP36Ll, which did not show substantial independent action. Thus, we demonstrated 19 candidates, including SOX2, to be regulatory factors of Prop1 expression.

Expression studies of neuronatin in prenatal and postnatal rat pituitary

The pituitary gland, an indispensable endocrine organ that synthesizes and secretes pituitary hormones, develops with the support of many factors. Among them, neuronatin (NNAT), which was discovered in the neonatal mouse brain as a factor involved in neural development, has subsequently been revealed to be coded by an abundantly expressing gene in the pituitary gland but its role remains elusive. We analyze the expression profile of Nnat and the localization of its product during rat pituitary development. The level of Nnat expression was high during the embryonic period but remarkably decreased after birth. Immunohistochemistry demonstrated that NNAT appeared in the SOX2-positive stem/progenitor cells in the developing pituitary primordium on rat embryonic day 11.5 (E11.5) and later in the majority of SOX2/PROP1 double-positive cells on E13.5. Thereafter, during pituitary embryonic development, Nnat expression was observed in some stem/progenitor cells, proliferating cells and terminally differentiating cells. In postnatal pituitaries, NNAT-positive cells decreased in number, with most coexpressing Sox2 or Pit1, suggesting a similar role for NNAT to that during the embryonic period. NNAT was widely localized in mitochondria, peroxisomes and lysosomes, in addition to the endoplasmic reticulum but not in the Golgi. The present study thus demonstrated the variability in expression of NNAT-positive cells in rat embryonic and postnatal pituitaries and the intracellular localization of NNAT. Further investigations to obtain functional evidence for NNAT are a prerequisite.

S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland

The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s) of these S100β-positive cells, which are not classic endocrine cells but instead comprise a heterogeneous cell population with plural roles, especially as stem/progenitor cells. To better understand the roles of these S100β-positive cells, we performed immunohistochemical analysis using several markers in S100β/GFP-TG rats, which express GFP in S100β-expressing cells under control of the S100β promoter. GFP-positive cells were present as mesenchymal cells surrounding the developing pituitary gland and at Atwells recess but were not present in the anterior lobe on embryonic day 15.5. These cells were negative for SOX2, a pituitary stem/progenitor marker, and PRRX1, a mesenchyme and pituitary stem/progenitor marker. However, three days later, GFP-positive and PRRX1-positive (but SOX2-negative) cells were observed in the parenchyma of the anterior lobe. Furthermore, some GFP-positive cells were positive for vimentin, p75, isolectin B4, DESMIN, and Ki67. These data suggest that S100β-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis.

Detection of Human Herpesviruses (HHVs) in Semen of Human Male Infertile Patients

Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21–49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.