Souichi Oomizu

Identification of Epidermal Growth Factor mRNA-Expressing Cells in the Mouse Anterior Pituitary

Epidermal growth factor (EGF) produced within the pituitary gland is associated with the growth of pituitary cells in rats. The aim of the present study was to localize EGF- and EGF receptor-expressing cells, and to clarify the involvement of EGF in DNA replication in 2-month-old male mouse pituitary cells. In situ hybridization of the pituitaries of these mice demonstrated that EGF mRNA was expressed in the anterior and intermediate lobes. Within the anterior pituitary, EGF mRNA-expressing cells were medium-sized and round, and made up 40% of the total number of secretory cells. EGF receptor mRNA was only detected in anterior pituitary cells. Forty-seven percent anterior pituitary cells expressed EGF receptor mRNA. An immunocytochemical study showed that most somatotropes and some mammotropes expressed EGF mRNA. When anterior pituitary cells were enzymatically dissociated and cultured in serum-free medium, RT-PCR demonstrated both EGF mRNA and EGF receptor mRNA expression. Treatment with EGF (1 and 10 ng/ml) for 5 days stimulated DNA replication in mammotropes and corticotropes. These results indicate that the DNA replication in mammotropes and corticotropes is regulated by the paracrine and/or autocrine activity of EGF produced at least in part by these cell types themselves.

Gene expression and the physiological role of transforming growth factor-α in the mouse pituitary

Abstract Transforming growth factor-α (TGF-α), a member of the epidermal growth factor (EGF) family, is produced within the mouse anterior pituitaries. However, the cell types of TGF-α-expressing cells and the physiological roles of TGF-α within mouse pituitary glands remain unclear. The aim of the present study was to localize TGF-α mRNA-expressing cells, and to clarify the involvement of TGF-α in estrogen-induced DNA replication in mouse anterior pituitary cells. Northern blot analysis demonstrated TGF-α mRNA expression in adult male and female mouse anterior pituitaries. In situ hybridization analysis of the pituitaries in these mice showed that TGF-α mRNA-expressing cells in the anterior pituitary are round, oval, and medium-sized. TGF-α mRNA was colocalized in most of the growth hormone (GH) mRNA-expressing cells, while only some of the prolactin (PRL) mRNA-expressing cells. DNA replication in the anterior pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine (BrdU) in a primary serum-free culture system. Estradiol-17β (E2) and TGF-α treatment increased the number of BrdU-labelled mammotrophs, indicating that E2 and TGF-α treatment stimulates the DNA replication in mammotrophs. Immunoneutralization of TGF-α with anti-TGF-α-antibodies nullified the E2-induced increase in DNA replication. RT-PCR analysis of TGF-α mRNA expression in ovariectomized female mice revealed that E2 increases TGF-α mRNA levels. These results indicate that the TGF-α produced primarily in the somatotrophs mediates the stimulatory effects of estrogen on the DNA replication of pituitary cells in a paracrine or autocrine manner.

Augmentation of Prolactin Release by α-Melanocyte Stimulating Hormone Is Possibly Mediated by Melanocortin 3-Receptors in the Mouse Anterior Pituitary Cells

Abstract Suckling- and estrogen-induced prolactin release from the anterior pituitary is mediated by α-melanocyte stimulating hormone (α-MSH) secreted by the intermediate lobe of the pituitary in the rat. Melanocortin 5-receptors are expressed in the anterior pituitary and probably mediate the α-MSH function. In contrast, the mouse anterior pituitary does not express the receptor. To examine whether or not α-MSH regulates prolactin release in mice, we performed cell immunoblot assay using anterior pituitary cells from adult female mice. We found that α-MSH acted on mammotrophs (prolactin-secreting cells) and stimulated prolactin release in a dose dependent manner. A series of RT-PCR using oligonucleotide primer pairs specific for each subtypes of melanocortin receptors revealed that the melanocortin 3-receptor is the sole receptor expressed in the mouse anterior pituitary. These results suggest that α-MSH-induced prolactin release is mediated by melanocortin 3-receptors in female mice.

Intrapituitary regulatory system of mammotrophs in the mouse.

Estrogen stimulates the proliferation of pituitary cells, in particular mammotrophs. The present study was designed to clarify involvement of transforming growth factor a (TGF-a) in the estrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary, serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-a (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-a binds to the epidermal growth factor (EGF)-receptor, this action may be exerted through this receptor. Estradiol-17β (E2, 10 -9 M) stimulated proliferation of mammotrophs. RG-13022, an EGF receptor inhibitor, reduced the cell proliferation induced by EGF or E2, showing that the EGF receptor was involved in this induction of mammotroph growth. Treatment with TGF-a antisense oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by E2, but treatment with EGF antisense ODN did not. Dual detection of TGF-a mRNA and growth hormone by in situ hybridization and fluorescence-immunocytochemistry demonstrated that TGF-a mRNA was detected in most somatotrophs. Our recent RT-PCR analysis revealed that E2 stimulated TGF-a-mRNA and EGF-receptor mRNA expression. These results indicate that TGF-a produced in somatotrophs mediates the stimulatory effect of estrogen on pituitary cell proliferation in a paracrine manner, and that EGF-receptor expression is stimulated by estrogen. These findings indicate that intrapituitary cell-to-cell interaction plays an important role in the control of pituitary secretory cells.