The use of rats and mice as animal models in ex vivo bone growth and development studies.

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine.Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610-618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2.

An ultrastructural study of the Sertoli cell in the water buffalo (Bubalus bubalis).

The ultrastructure of Sertoli cell in the water buffalo (Bubalus bubalis) was observed in a transmission electron microscope. The nucleus had homogeneous nucleoplasm, scarce heterochromatin and multivesicular nuclear body (MNB). The MNB was composed of numerous vesicles and ribosome-like dense structures. The vesicles varied in size and number and contained a sparse and flocculent substance. In the indentation of the nucleus, aggregates of ribosomes were frequently observed. In the apical and middle region of the cell, long mitochondria and microtubules were distributed parallel to the long axis of the cell. Non-laminated smooth ER and some ribosomes were also recognizable throughout this region. In the basal region, widely-distributed laminated smooth ER was characteristic. Microfilament bundles at ectoplasmic specialization were irregularly arranged. Frequently-emerged nodular processes occasionally separated from basal lamina and formed round structures within Sertoli cytoplasm. Although these characteristics of buffalo Sertoli cell were very similar to those of the bovine studied, the aggregate of ribosomes was more developed in the buffalo.

The Sertoli cell of the water buffalo--an electron microscopic study.

The ultrastructure of Sertoli cells in the seminiferous tubules of water buffaloes before and during sexual maturity was studied by transmission electron microscopy, with emphasis on the intranucleolar vesicular elements. Sertoli cells of animals under 12 months of age were distinguished from the germ cells by the presence of electron dense membrane bound bodies within their cytoplasm. These cells, referred to as basal indifferent supporting cells, were probably involved in the phagocytosis and elimination of degenerating spermatocytes, which failed to differentiate into spermatids and spermatozoa in animals under one year of age. In 12 month old animals, a few Sertoli cells exhibiting the vesicular elements appeared in the nucleolar region while in animals over 15 months of age Sertoli cells could be positively identified by the characteristic cytoplasm containing microtubules, elongated and electron dense mitochondria, extensive granular endoplasmic reticulum and the presence of spermatids in various stages of spermiogenesis. The vesicular elements in the nucleolar region of the Sertoli cells were most prominent at this stage. Ultrastructural features of the Sertoli cells revealed an abundance of ribosome-like particles surrounding the vesicles of varying size. Some of these vesicular elements contained amorphous material suggesting that they represent the products sequestered in the nuclear region for transport to the cytoplasm and that the process of spermiogenesis may be dependent on the ability of Sertoli cells to generate these products at sexual maturity.

Identification of crossbred buffalo genotypes and their chromosome segregation patterns.

Chromosome analysis on different breed types of water buffaloes (Bubalus bubalis) was undertaken to identify their karyotypes and to determine the pattern of chromosome segregation in crossbred water buffaloes. Altogether, 75 purebred and 198 crossbred buffaloes including 118 from Malaysia and 80 from the Philippines, were analyzed in this study. The diploid chromosome number of the swamp buffalo from both countries was 48 and that of the river buffalo was 50, while all F1 hybrids exhibited 49 chromosomes. The F2 hybrids consisted of three different karyotype categories (2n = 48, 2n = 49, and 2n = 50), whereas the backcrosses included two different karyotype categories each, with 2n = 48 and 2n = 49 in the three quarters swamp types and 2n = 49 and 2n = 50 in the three quarters river types. Chi-square tests on pooled data from Malaysia and the Philippines indicated that the distribution of different karyotype categories of F2 animals did not deviate significantly from the 1:2:1 ratio expected if only balanced gametes with 24 and 25 chromosomes were produced by the F1 hybrids. In the three quarters swamp and three quarters river types, the respective karyotypic categories were in ratios approximating 1:1. The distribution of chromosome categories among the F2 hybrids and backcrosses suggests that only genetically balanced gametes of the F1 hybrids are capable of producing viable F2 and backcross generations.

Morphometry of the functional and regressing corpus luteum of the guinea pig.

A morphometric study of functional and regressing corpora lutea (CL) of guinea pigs (n = 5 per day) was performed on days 9, 12, and 16 of the estrous cycle. On day 9 the functional CL contained congruent to 750,000 cells, which included 565,200 +/- 56,700 (S.D.) endothelial cells or pericytes and 137,300 +/- 7,700 luteal cells. Between days 9 and 12 the only significant change suggesting the onset of regression was a reduction in vascular luminal surface area. During this time the number of luteal cells per CL increased to 204,400 +/- 34,800 (P less than .05), with an accompanying reduction in luteal cell volume from 19.8 +/- 1.8 to 14.4 +/- 2.4 pl/cell (P less than .01). The increase in cell numbers was explicable by cell division, with mitotic indices of 0.83% and 0.97% on days 9 and 12, respectively. Luteal volume was unaltered. Between days 12 and 16, the mean volume of a single CL fell from 3.98 +/- 0.2 to 1.42 +/- 0.3 mm3 (P less than .01) and luteal cell volume was reduced to 5.3 +/- 1.1 pl/cell (P less than .01). Between these 2 days the number of endothelial cells per CL fell from 539,900 +/- 75,500 to 144,400 +/- 63,300 (P less than .01), with an accompanying reduction in vascular luminal surface area and in the volume occupied by vascular lumina. The total number of luteal cells per CL was not reduced significantly. It was concluded that luteal cell numbers in the guinea pig increase up to the time of onset of luteal regression, and that during regression up to day 16, shrinkage of luteal cells is the major cause of loss of luteal volume. During regression, endothelial cell loss occurs much more rapidly than loss of luteal cells.

Mechanism of deletion of endothelial cells during regression of the corpus luteum.

Endothelial cells are deleted rapidly, and in large numbers, during cyclical regression of the corpus luteum of the guinea pig. This paper reports a study of the mechanisms, structural and causal, by which this deletion occurs. Corpora lutea from guinea pigs were examined by transmission electron microscopy on day 9 (functional stage) and day 16 (regressing) of the estrous cycle. Corpora lutea were also studied at 1, 3, 12, 24, and 48 hours after administration of a synthetic luteolytic substance (cloprostenol), and after temporary occlusion of the ovarian blood vessels for 15, 30, 60, or 120 minutes. Early signs of endothelial cell degeneration included protrusion of some individual cells into capillary lumina and the formation of adherens junctions across the lumen. Intermediate stages of degeneration included nuclear and cytoplasmic condensation and cellular and nuclear lobation or fragmentation in cells either protruding into, or lying within, the lumen. Terminal changes included loss of plasma membrane integrity and cytoplasmic density, together with disruption of cell organelles. Some degenerate endothelial cells were engulfed by viable endothelial cells. Macrophages were not seen to be involved in removal of dead endothelial cells, and integrity of the walls of capillaries was maintained while individual endothelial cells were deleted. Experimental findings were consistent with the hypothesis that cessation or reduction of flow of blood along capillaries plays an etiologic rule in endothelial cell deletion.

Effects of a synthetic prostaglandin analogue, cloprostenol, on the corpus luteum of the guinea pig.

The effects of a synthetic prostaglandin analogue, cloprostenol, on luteal function in the guinea pig were studied. At a dose of 250 micrograms, cloprostenol administered I-P on day 9 of the cestrous cycle caused a reduction in the length of the oestrous cycle from 17.4 +/- s.d.0.9 to 14.5 +/- 1.1 days (p less than 0.01). Lower doses were ineffective, and post-treatment cycles were not different in length from pre-treatment cycles. Cloprostenol also caused a dose-dependent reduction in luteal weight, which fell from 3.52 +/- 0.82 to 1.82 +/- 0.41mg (p less than 0.01) 48 h after administration of a 250 micrograms dose on day 9. Plasma progesterone, measured by radioimmunoassay, was reduced from 4.67 +/- 0.59 to 2.69 +/- 0.66 ng ml-1 (p less than 0.01) 48 h after administration of 250 micrograms cloprostenol on day 9. 250 micrograms cloprostenol also reduced blood flow per corpus luteum, measured by 85Sr-labelled 15 microns microspheres, both at 3 h (20.20 +/- 10.36 to 9.40 +/- 4.20 microliters min-1; p less than 0.05) and at 48 h (18.47 +/- 8.27 to 5.23 +/- 1.90 microliters min-1; p less than 0.01) after administration on day 9. No adverse side-effects were observed at any dose level of cloprostenol used. It was concluded that cloprostenol is a useful experimental luteolysin in the guinea pig.