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

Background Many genetically multi‐modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno‐organs may impair their long‐term function after orthotopic transplantation in non‐human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR‐KO) to reduce the size of donor pigs in one step. Methods Heart weight and proteome profile of myocardium were investigated in GHR‐KO and control pigs. GHR‐KO mutations were introduced using CRISPR/Cas9 in an α1,3‐galactosyltransferase (GGTA1)‐deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple‐modified (4GM) pigs. Results At age 6 months, GHR‐KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin‐like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi‐modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. Conclusion GHR‐KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno‐organ overgrowth in preclinical studies.

show abstract

“…IGF1 concentrations were measured using the iSYS automated chemiluminescent IGF1 assay (Immunodiagnostic Systems) as described previously 21,22 …”

Section: Methodsmentioning

confidence: 99%

Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies

Hinrichs¹,

Riedel²,

Klymiuk³

et al. 2020

Xenotransplantation

View full text Add to dashboard Cite

show abstract

“…V V(GH-GRAN/SOM) was determined, using the point counting method like described above and previously. 13 Per case, 2211.3…”

Section: Determination Of the Volume Of Growth Hormone Granules In So...mentioning

confidence: 99%

“…Sections of approximately 4 μm thickness were stained with hematoxylin and eosin (HE), and subjected to immunohistological detection of growth hormone (GH) using a specific polyclonal goat anti-rat GH antibody (#AF1566 R&D Systems, USA; 1:3000) and biotinylated rabbit anti-goat IgG solution (DAako, USA; 1:100), diaminobenzidine as chromogen and hemalum as nuclear counterstain, as previously described in detail. 13 2.2 | Quantitative morphological analyses 2.2.1 | Determination of the volume of the adenohypophysis (AH) and the total volume of somatotrophs (SOM) in the AH Volume densities (V V(X/Y) ) were estimated based on area densities using the principle of Delesse. 18,19 According to this principle, the volume density of a target structure V (X) in a reference compartment V (Y) is equal to the quotient of their respective cumulative section areas ( P A (X) / P A (Y) ).…”

Section: Generation Of Pituitary Gland Tissue Samplesmentioning

confidence: 99%

See 1 more Smart Citation

Structural and proteomic repercussions of growth hormone receptor deficiency on the pituitary gland: Lessons from a translational pig model

Shashikadze

Franzmeier

Hofmann

et al. 2023

J Neuroendocrinology

View full text Add to dashboard Cite

Growth hormone receptor deficiency (GHRD) results in low serum insulin‐like growth factor 1 (IGF1) and high, but non‐functional serum growth hormone (GH) levels in human Laron syndrome (LS) patients and animal models. This study investigated the quantitative histomorphological and molecular alterations associated with GHRD. Pituitary glands from 6 months old growth hormone receptor deficient (GHR‐KO) and control pigs were analyzed using a quantitative histomorphological approach in paraffin (9 GHR‐KO [5 males, 4 females] vs. 11 controls [5 males, 6 females]), ultrathin sections tissue sections (3 male GHR‐KO vs. 3 male controls) and label‐free proteomics (4 GHR‐KO vs. 4 control pigs [2 per sex]). GHR‐KO pigs displayed reduced body weights (60% reduction in comparison to controls; p < .0001) and decreased pituitary volumes (54% reduction in comparison to controls; p < .0001). The volume proportion of the adenohypophysis did not differ in GHR‐KO and control pituitaries (65% vs. 71%; p = .0506) and GHR‐KO adenohypophyses displayed a reduced absolute volume but an unaltered volume density of somatotrophs in comparison to controls (21% vs. 18%; p = .3164). In GHR‐KO pigs, somatotroph cells displayed a significantly reduced volume density of granules (23.5%) as compared to controls (67.7%; p < .0001). Holistic proteome analysis of adenohypophysis samples identified 4660 proteins, of which 592 were differentially abundant between the GHR‐KO and control groups. In GHR‐KO samples, the abundance of somatotropin precursor was decreased, whereas increased abundances of proteins involved in protein production, transport and endoplasmic reticulum (ER) stress were revealed. Increased protein production and secretion as well as significantly reduced proportion of GH‐storing granules in somatotroph cells of the adenohypophysis without an increase in volume density of somatotroph cells in the adenohypophysis could explain elevated serum GH levels in GHR‐KO pigs.

show abstract

“…The reference-and look-up sections are usually taken from a series of consecutive sections, sectioned with a defined nominal section thickness (d). For determination of N V(EC/SL) , it is recommendable to prepare a series of at least 7 consecutive semithin sections (per sample) [108,109] with a nominal section thickness of 0.5 μm. The mean section profile area of the reference compartment (SL) that is present in the reference-and the look-up section and the disector height define the 3-D reference compartment volume in which the particles of interest (SL-EC) are counted.…”

Section: Estimation Of the Total Number The Total Volume And The Meamentioning

confidence: 99%

A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies

et al. 2020

View full text Add to dashboard Cite

Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300–2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.

show abstract

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

Cited by 6 publications

References 69 publications

Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies

Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies

Structural and proteomic repercussions of growth hormone receptor deficiency on the pituitary gland: Lessons from a translational pig model

A practical guide to unbiased quantitative morphological analyses of the gills of rainbow trout (Oncorhynchus mykiss) in ecotoxicological studies

Contact Info

Product

Resources

About