Haruo Nogami

Fine-structural heterogeneity and morphologic changes in rat pituitary prolactin cells after estrogen and testosterone treatment

SummaryThis study was conducted to determine the functional and/or developmental relationships among three heterogeneous types of prolactin cells (I, II and III) in rats. Rats were injected subcutaneously daily with estradiol or testosterone propionate on days 10–20 after birth. Estradiol increased the proportion of cell types II and III, increased serum PRL levels 12-fold in males and 15-fold in females, and increased pituitary levels of prolactin 12-fold in males and 5-fold in females. Testosterone mainly increased the proportion of the Type-II cells, decreased serum levels of prolactin in males only, and did not change pituitary levels of prolactin. In a second experiment, treatment of rats with nafoxidine for five days after E2 treatment (days 10–20 after birth) increased the proportion of Type-I cells and decreased the proportion of Type-III cells and decreased serum and pituitary levels of prolactin by 50% in females and by 15 and 45% in males. In a third experiment utilizing adult male rats, estradiol and testosterone were found to modulate the relative ratios of the different types of PRL cells as they did in immature animals. The data taken as a whole suggest the possibility of an estrogen-stimulated conversion of one cell type to another, which may be a reflection of prolactin secretory activity.

Prolactin immunoreactivity of acidophils of the small granule type

SummaryThe fine structure of some oval anterior pituitary cells of the adult male rats immunostained with an antiserum to rat prolactin was investigated electron microscopically on the adjacent thin sections. Their fine structural appearance is identical with that of acidophils of the small granule type (Yoshimura et al. 1974) resembling the Kurosumi-Oota LH gonadotrophs. The secretory granules of the oval cells are spherical in shape, ranging from 130 to 200 nm in diameter. Large polymorphic granules, which are generally believed to be characteristic of prolactin cells, are absent from their cytoplasm. It is concluded that the acidophil of the small granule type with a similar fine structure to the Kurosumi-Oota LH gonadotroph is a prolactin secreting cell.

Studies on the development of growth hormone and prolactin cells in the rat pituitary gland by in situ hybridization

SummaryCytogenesis of growth hormone and prolactin cells in the rat pituitary gland was studied using in situ cDNA-mRNA hybridization and immunocytochemistry. Frozen or Paraplast sections of fetal and neonatal pituitaries were hybridized with 3H-cDNAs for rat prolactin or growth hormone, and were then processed for autoradiography. A number of growth hormone mRNA-positive cells were encountered throughout the anterior lobe on day 19 of gestation. Individual variaction in growth hormone gene expression was observed between fetuses at day 19 of gestation (6 out of 8 fetuses examined were positive for growth hormone mRNA). In contrast, growth hormone mRNA was detected in the all fetuses examined on day 20 or later. The autoradiographic signal (number of reduced silver grains) appeared to increase with later stages of development. Fetal growth hormone mRNA-positive cells were evenly scattered throughout the anterior lobe. Most of them were isolated, however, small clusters of several growth hormone cells were infrequently observed. Prolactin mRNApositive cells were found first on the 22nd day (the last day of gestation) in 3 of 6 fetuses examined, but were rarely observed on earlier gestational days. By postnatal day 8, prolactin mRNA-positive cells were numerous and the grain density over prolactin cells increased. Both growth hormone and prolactin cells were found as early as 18 days of gestation using immunocytochemistry, although the number of positive cells was very small at this stage. Immunoreactive growth hormone cells increased sharply in number during the next 24 h, while the number of prolactin cells remained scarce until birth. The results suggest that many growth hormone cells are still in an immature state at 20∶00 of day 18 and that many begin to synthesize growth hormone mRNA during next 14 h. On the other hand, no substantial prolactin gene expression appears to take place until after birth.

Fine structural criteria for identifying rat corticotrophs.

SummaryThe fine structural characteristics of normal rat corticotrophs stained with anti-porcine ACTH1–39 serum were studied. At the ultrastructure level immunoreactive corticotrophs appear to comprise four distinct cell types: (1) large stellate cells (Siperstein cells) containing granules (170–250 nm in diameter) arranged in a peripheral row and usually embracing an acidophil; (2) elongate spindle-shaped cells (Moriarty cells) in which the secretory granules (170–250 nm in diameter) are distributed in a row or in small clusters in the peripheral cytoplasm; (3) oval or polygonal cells filled only with small secretory granules (130–170 nm in diameter), resembling the “acidophil of small granules type” (Yoshimura et al. 1974); and (4) polygonal or stellate cells filled with secretory granules of varying diameters (180–300 nm in diameter) and occasionally embracing an acidophil. The first type is the most common, but the others are infrequent. It is concluded that the criteria of Siperstein and Miller (1970) do not necessarily include all categories of rat corticotrophs.

Single secretory granules contain both GH and prolactin in pituitary mixed type of adenoma

A mixed type of pituitary adenoma is described consisting of heavily and sparsely granulated cells. It produces GH and prolactin (PRL) and has been examined by immunocytochemistry. The superimposition immunocytochemical procedure reveals that single cells contain both GH and PRL. Furthermore, electron immunocytochemistry using adjacent sections reveals that single secretory granules contain both GH and PRL simultaneously.

A whole range of fine structural criteria for immunohistochemically identified LH cells in rats

SummaryPituitaries from normal, young and adult male rats were fixed either in sublimate-formalin or in glutaraldehyde-osmium. In adjacent Paraplast sections, almost all the gonadotrophs were immunostained with both LH and FSH antisera. The rat LHβ and FSH antisera used were shown to be highly specific by the absorption test and by double antibody radioimmunoassay. Thin and thick adjacent Epon sections were prepared for EM and immunohistochemical examination. Cells stained with the rat LHβ antiserum were identified by LM, and then observed in detail by EM. On the basis of these observations we suggest that the LH cells are arranged in a sequence of basophils, i.e., Types II/III, III, III/IV and IV: Type II/III basophils are elongate with a cytoplasmic process and less vesiculated. They have morphological features of Type II (classical thyrotrophs) and also of Type III basophils. Type III basophils are oval in shape and moderately vesiculated. Both Types II/III and III basophils can be divided into two classes of cell characterized mainly by the existence of only small secretory granules (150–220 nm in diameter) (Type A) or by the coexistence of small and large (350–500 nm) (Type B). Type III/IV basophils are cells intermediate between types III and IV basophils, and moderately vesiculated with an abundance of secretory granules (150–300 nm in diameter). Type IV basophils are large, spherical or oval cells whose RER cisternae are conspicuously dilated; they contain less numerous secretory granules (150–300 nm in diameter). It is concluded that LH cells are not a single cell type, but include a wide range of subtypes.

Increased Population of Nonhormone-Proclucing Cells Suggests the Presence of Dysfunctional Growth Hormone Cells in the Anterior Pituitary Gland of the Spontaneous Dwarf Rat

Anterior pituitary gland of the spontaneous dwarf rat (SDR) with isolated growth hormone (GH) deficiency was studied using immunocytochemistry, cell count and in situ hybridization. The standard immunocytochemistry of five anterior pituitary hormones [adrenocorticotropic hormone (ACTH), luteinizing hormone, thyroid-stimulating hormone, prolactin (PRL) and GH] and S-100 protein failed to detect any cytological difference between normal rats and SDRs, except for the size of different types of cells which were smaller in SDR than in normal rats, and GH cells which were undetectable in the SDR. The cell count study again showed lack of immunoreactive GH cells in the SDR. The population of PRL cells was significantly reduced in the SDR by 40% in male and 30% in female when compared to those of the control. The population of ACTH cells was larger in the male SDR. The population of the immunonegative cells was calculated by subtracting the sum of the percentages of immunopositive cells from 100, and it was found to be remarkably increased in the SDR. The population of immunonegative cells was about 55% in both male and female SDRs, whereas it was calculated to be 18.7% (male) or 10.2% (female) in the control rats. In situ hybridization study using GH cRNA indicated the presence of a considerable number of cells which express GH mRNA in the SDR as well as in the control rats. These results taken as a whole suggest the presence of a number of dysfunctional GH cells in the SDR.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical study of the postnatal development of pituitary thyrotrophs in the rat, with special reference to cluster formation

SummaryThe postnatal development of rat pituitary thyrotrophs was investigated immunohistochemically on days 1, 3, 5, 10, 15 and 25. Fetal thyrotrophs are strongly immunoreactive. In the postnatal period, however, weakly immunoreactive thyrotrophs increase in number to constitute clusters on days 3–5. The numbers and dimensions of the clusters reach a maximum on day 10. Thereafter the clusters break down to give rise to single, scattered neogenic thyrotrophs. Thyrotrophs in clusters on day 10 were investigated by electron microscopy in adjacent sections. They can be characterized as an immature type of basophil, according to the classification of Yoshimura et al. (1977): 1) Type I basophils, which are irregularly shaped with elongate processes, and characterized by rows of secretory granules about 100 nm in diameter. 2) Type I/II basophils, i.e., forms intermediate between Types I and II, containing less numerous secretory granules about 100–150 nm in diameter. Type II basophils which correspond to the classical thyrotrophs are not fully developed on day 10. Thus, most thyrotrophs develop from the clusters in the neonatal period. Such neogenic thyrotrophs retain the immature characteristics of Type I and I/II cells and may develop into Type II cells during subsequent maturation.

Electron-microscopic study on the anterior pituitary gland of spontaneous dwarf rats

SummaryThe spontaneous dwarf rat is a novel experimental model animal on the study of pituitary dwarfism. The fine structure of the anterior pituitary cells was studied in the immature and mature dwarf rats. Pituitary glands were removed from 5-, 10-, 20-day-old immature dwarfs, adult (45 days-16 weeks) dwarfs and normal 3-month-old rats and processed for electron-microscopic observation. In the control animals, growth hormone cells were readily identified by their ultrastructural characteristics, such as the presence of numerous electron-dense secretory granules, 300–350 nm in diameter, well developed rough endoplasmic reticulum and a prominent Golgi complex. In contrast, growth hormone cells were not found in the anterior pituitary gland of the spontaneous dwarf rat at any age examined. Other pituitary cell types, i.e., luteinizing hormone/ follicle stimulating hormone, thyroid stimulating hormone, adrenocorticotropic hormone and prolactin cells, appeared similar in their fine structure to those found in the control rats. In the pituitary gland of dwarf rats, a number of polygonal cells were observed either with no or relatively few secretory granules. The rough endoplasmic reticulum was arranged in parallel cisternae and the Golgi complex was generally prominent in these cells. In addition, many were found to have abundant lysosomes. A few minute secretory granules were occasionally observed; however, the immunogold technique failed to localize growth hormone or prolactin in the granules. The nature of these cells remained obscure in this study. Since their incidence and fine structural features, other than the secretory granules, were quite similar to those of the growth hormone cells in normal rats, we postulate that these cells are dysfunctional growth hormone cells. These results suggest that the cause of the growth impairment in the spontaneous dwarf rat is due to a defect in the functional growth hormone cells in the pituitary gland, and since other pituitary cell types appeared normal, the disorder seems to be analogous to the isolated growth hormone deficiency in the human.

Intrauterine Growth Retardation Due to Growth Hormone Deficiency in Rats

The effect of growth hormone (GH) on fetal growth was examined in the spontaneous dwarf rat (SDR) with the isolated GH deficiency. A 328-bp GH gene fragment containing the site of SDR mutation was amplified from fetal liver genomic DNA using PCR and digested with restriction enzyme SauI to distinguish the fetal genotype. When heterozygote (dr/+) males and females were crossed, a significant reduction in body weight of dr/dr fetuses was observed on days 20 and 21 (92 and 89% that of +/+ fetuses, respectively). Body weight reduction was also noticed on day 21 in dr/dr fetuses (80%) in comparison with that of dr/+ fetuses crossed from dr/dr females and dr/+ males. Unexpectedly, the body weight of dr/+ fetuses was found to be significantly less than that of +/+ fetuses on days 19 (94%) and 20 (97%), which reached the level of +/+ fetuses by day 21 of gestation. The fetal tail length was not affected by the GH status. These results provide direct evidence for the involvement of pituitary GH in the regulation of fetal body weight during late gestation, but not in fetal skeletal growth.