Linkage between growth retardation and pituitary cell morphology in a dystrophin-deficient pig model of Duchenne muscular dystrophy.

OBJECTIVE: Human patients with Duchenne muscular dystrophy (DMD) commonly exhibit a short stature, but the pathogenesis of this growth retardation is not completely understood. Due to the suspected involvement of the growth hormone/insulin-like growth factor 1 (GH/IGF1) system, controversial therapeutic approaches have been developed, including both GH- administration, as well as GH-inhibition. In the present study, we examined relevant histomorphological and ultrastructural features of adenohypophyseal GH-producing somatotroph cells in a porcine DMD model. METHODS: The numbers and volumes of immunohistochemically labelled somatotroph cells were determined in consecutive semi-thin sections of plastic resin embedded adenohypophyseal tissue samples using unbiased state-of-the-art quantitative stereological analysis methods. RESULTS: DMD pigs displayed a significant growth retardation, accounting for a 55% reduction of body weight, accompanied by a significant 50% reduction of the number of somatotroph cells, as compared to controls. However, the mean volumes of somatotroph cells and the volume of GH-granules per cell were not altered. Western blot analyses of the adenohypophyseal protein samples showed no differences in the relative adenohypophyseal GH-abundance between DMD pigs and controls. CONCLUSION: The findings of this study do not provide evidence for involvement of somatotroph cells in the pathogenesis of growth retardation of DMD pigs. These results are in contrast with previous findings in other dystrophin-deficient animal models, such as the golden retriever model of Duchenne muscular dystrophy, where increased mean somatotroph cell volumes and elevated volumes of intracellular GH-granules were reported and associated with DMD-related growth retardation. Possible reasons for the differences of somatotroph morphology observed in different DMD models are discussed.

Protective mechanisms involving enhanced mitochondrial functions and mitophagy against T-2 toxin-induced toxicities in GH3 cells.

T-2 toxin is the most toxic member of trichothecene mycotoxin. So far, the mechanism of mitochondrial toxicity and protective mechanism in mammalian cells against T-2 toxin are not fully understood. In this study, we aimed to investigate the cellular and mitochondrial toxicity of T-2 toxin, and the cellular protective mechanisms in rat pituitary GH3 cells. We showed that T-2 toxin significantly increased reactive oxygen species (ROS) and DNA damage and caused apoptosis in GH3 cells. T-2 toxin induced abnormal cell morphology, cytoplasm and nuclear shrinkage, nuclear fragmentation and formation of apoptotic bodies and autophagosomes. The mitochondrial degradative morphologies included local or total cristae collapse and small condensed mitochondria. T-2 toxin decreased the mitochondrial membrane potential. However, T-2 toxin significantly increased the superoxide dismutase (SOD) activity and expression of antioxidant genes glutathione peroxidase 1 (GPx-1), catalase (CAT), mitochondria-specific SOD-2 and mitochondrial uncoupling protein-1, -2 and -3 (UCP-1, 2 and 3). Interestingly, T-2 toxin increased adenosine triphosphate (ATP) levels and mitochondrial complex I activity, and increased the expression of most of mitochondrial electron transport chain subunits tested and critical transcription factors controlling mitochondrial biogenesis and mitochondrial DNA transcription and replication. T-2 toxin increased mitophagic activity by increasing the expression of mitophagy-specific proteins NIP-like protein X (NIX), PTEN-induced putative kinase protein 1 (PINK1) and E3 ubiquitin ligase Parkin. T-2 toxin activated the protective protein kinase A (PKA) signaling pathway, which activated the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/PINK1/Parkin pathway to mediate mitophagy. Taken together, our results suggested that the mammalian cells could increase their resistance against T-2 toxin by increasing the antioxidant activity, mitophagy and mitochondrial function.

Endoplasmic reticulum stress and apoptosis contribute to the pathogenesis of dominantly inherited isolated GH deficiency due to GH1 gene splice site mutations.

Dominantly inherited isolated GH deficiency is mainly caused by a heterozygous donor site mutation of intron 3 in the GH1 gene. An exon 3 deletion in GH (del32-71 GH) is produced from a mutant allele, whereas wild-type GH is produced from the other allele. Several studies have demonstrated a dominant negative effect of del32-71 GH on wild-type GH secretion, but the precise molecular mechanisms remain unclear. We hypothesized that unfolded del32-71 GH accumulates in the endoplasmic reticulum (ER) and causes ER stress and apoptosis in somatotrophs, promoting GH deficiency. To evaluate del32-71 GH-mediated ER stress, we established GH4C1 cell lines with doxycycline (dox)-controlled del32-71 GH expression. In 20 of 23 dox-controlled cell lines, the concentration of wild-type GH in the culture medium significantly decreased with del32-71 GH induction, demonstrating the dominant negative effect of this mutant. Cell viability, mRNA abundance of ER stress-response genes, caspase activation, and DNA fragmentation were evaluated in 5 dox-controlled cell lines selected as cellular models. In 4 of the 5 cell lines, del32-71 GH induction decreased cell viability, increased expression of 3 major ER stress response pathways (PRKR-like endoplasmic reticulum kinase [PERK], activating transcription factor-6 [ATF6], and inositol requirement 1 [IRE1]), and induced caspase-3 and caspase-7 activation. In 1 of the 4 cell lines, DNA fragmentation was demonstrated. Finally, overexpression of XBP1(S), a nuclear transcription factor downstream of IRE1, completely reversed the observed caspase activation. These data suggested that del32-71 GH-mediated ER stress and apoptosis contributed to the decrease in wild-type GH secretion observed in GH deficiency due to the GH1 gene slice-site mutations.

The absence of somatotroph proliferation during continuous stress is a result of the lack of extracellular signal-regulated kinase 1/2 activation.

The integrity of homeostasis can be affected by chronic stress, and hyposomatotropism is evident in chronic stress-associated illnesses. In the present study, we demonstrated that a continuous stress (CS) severely affected somatotrophs among hormone-secreting cells in the anterior lobe (AL) of the pituitary by using a rat CS model. Among AL cells, the proliferation of somatotrophs was almost entirely suppressed in rats that had 3-5 days of CS (5dCS), although other hormone-secreting cells continued to proliferate. The cell size of somatotrophs was reduced at 5dCS (P<0.01), the number of secretory granules was increased at 3dCS (P<0.01) and serum growth hormone (GH) was on declining trend during 1 to 5dCS, suggesting that GH release was inhibited. GH-releasing hormone (GHRH) mRNA level in the arcuate nucleus was transiently decreased, whereas its receptor expression in the AL was significantly increased in CS rats. When 5dCS rats were injected with GHRH, transient GH secretion was observed, whereas proliferation of somatotrophs did not occur. The GHRH administration failed to stimulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and the nuclear translocation of ERK in somatotrophs. These results suggest that somatotrophs of 5dCS rats expressed sufficient GHRH receptor, which could transfer a signal for GH release. However, the GHRH-induced proliferation signal was blocked somewhere between the receptor and ERK1/2. Because significant increase of corticosterone in the initial stage (the 1-3dCS) was observed in this model, the corticosterone may affect the signalling. Although the mechanism underlying the blockage of the proliferation signal in somatotrophs under CS remains unclear, these somatotrophic disorder, suggesting that the present animal model may be useful for understanding the molecular mechanisms of chronic stress-associated illnesses.

Ultrastructural characterization of the pars distalis of the Indian female Sheath-tailed bat, Taphozous longimanus (Hardwicke) / Caracterización ultraestructural de la pars distalis del murciélago cola de vaina hembra indio, Taphozous longimanus (Hardwicke)

The present ultrastructural observations demonstrate the presence of six cell types in the pars distalis of non-pregnant and pregnant bats of Taphozous longimanus. In the pars distalis of T. longimanus, STH cells are round to oval with eccentrically placed nucleus, numerous secretory granules and well developed Golgi indicate a cell under vigorous synthetic activity while those filled with secretory granules with reduced Golgi complex suggest reserve or storage state of cells. LTH cell is characterized by the large secretory granules, dilated endoplasmic reticulum and numerous mitochondria in the cytoplasm which indicate that these cells are hypertrophied and synthetically very active during pregnancy. ACTH cells are found either singly or in groups and are elongated or angular with long cytoplasmic processes. The size and peripheral arrangement of secretory granules are characteristic of ACTH cell. TSH cells are distributed mostly towards the periphery of the pars distalis of T. longimanus. They are elongated, polygonal or triangular in shape. The secretory granules are small, electron dense, 150-200 nm in diameter. The rough endoplasmic reticulum is very well developed. In FSH, the secretory granules are small (200 to 400 nm) and less in number and are distributed towards the periphery of the cell. FSH cells show well developed mitochondria, Golgi and rough endoplasmic reticulum indicating active state of FSH during estrus and pregnancy. The hypertrophy of FSH and LH cells during pregnancy is associated with filigreed cytoplasmic pattern giving a bizarre appearance. At late pregnancy, FSH and LH cells are highly active and synthesize large quantities of hormone as indicated by the development of cell organelles.

Las observaciones ultraestructurales actuales demuestran la presencia de seis tipos de células en la pars distalis de murciélagos Taphozous longimanus preñadas y no preñadas. En la pars distalis del T. longimanus, las células STH son redondas u ovaladas con un núcleo excéntrico, numerosos gránulos de secreción y un Golgi bien desarrollado que indican una célula en actividad de síntesis vigorosa, mientras que las llenas de gránulos de secreción con un complejo de Golgi reducido sugieren un estado celular de reserva o almacenamiento. Las células LTH se caracterizan por grandes gránulos de secreción, el retículo endoplásmico dilatado y numerosas mitocondrias en el citoplasma, indicando que estas células están hipertrofiadas y con una actividad sintética muy activa durante el embarazo. Células de ACTH se encuentran de forma individual o en grupos, son alargadas o angulares, con largos procesos citoplásmicos. El tamaño y la disposición periférica de los gránulos de secreción de ACTH son característicos de la célula. Células de TSH se distribuyen principalmente hacia la periferia de la pars distalis del T. longimanus. Ellos son alargadas, poligonales o de forma triangular. Los gránulos de secreción son pequeños, electrodensos, de 150-200 nm de diámetro. El retículo endoplasmático rugoso está muy bien desarrollado. En células FSH, los gránulos de secreción son pequeños (200 a 400 nm), menores en número y se distribuyen hacia la periferia de la célula. Células FSH muestran mitocondrias bien desarrolladas, Golgi y retículo endoplasmático rugoso que indica el estado activo de la FSH durante el estro y la preñez. La hipertrofia de las células de FSH y LH durante la preñez se asocia con un patrón citoplasmático filigrana dando una extraña apariencia. Al final de la preñez, las células de FSH y LH son muy activas y sintetizan grandes cantidades de hormonas, como producto del desarrollo de las organelos celulares.