The role of IGF-I in Leydig cell maturation was studied by evaluation of: 1) steady state levels for nine mRNA species expressed specifically in Leydig cells of 35- and 50-d-old IGF-I-null mice and wild-type controls; 2) protein levels for 17 alpha-hydroxylase/C17-20 lyase, cholesterol side-chain cleavage, and type I 5 alpha-reductase (5 alpha R-1) in Leydig cells by immunocytochemistry; and 3) serum testosterone (T) and testicular interstitial fluid IGF-I levels. Expression levels of all mRNA species associated with T biosynthesis were lower in the absence of IGF-I stimulation. In contrast, androgen-metabolizing enzyme mRNA species had either normal (3 alpha-hydroxysteroid dehydrogenase) or higher expression (5 alpha R-1) levels in IGF-I-null mice (P < 0.05) relative to wild-type controls. None of the mRNA species studied changed developmentally in the mutant, whereas there were increases or decreases between d 35 and 50 in normal controls. Parallel trends were observed for average Leydig cell 5 alpha R-1 immunostaining intensity. T levels in mutants were initially higher during d 14-21, equivalent to normal on d 28, and then failed to increase pubertally, remaining at 30% of control levels (P < 0.01) in 90-d-old adult animals. In normal wild-type mice, interstitial fluid and plasma IGF-I levels were highest (P < 0.05) on d 24, indicating that the action of this growth factor on the testis peaks during pubertal development. These results show that in the absence of IGF-I, there is a failure of adult Leydig cells to mature, and that the reduced capacity for T production is caused by disproportionate expression of T biosynthetic and metabolizing enzymes.

The arrangement of blood vessels serving the testis-epididymis vas investigated microscopically in the mouse, rat, and rabbit. Blood vessels were visualized by infusing liquid silicone rubber into the vessels and subsequently clearing the surrounding tissue. Comprehensive illustrations of the vasculature were prepared from three-dimensional examinations. Arterial and venous vessels serving the testis-epididymis follow similar routes in all three species. However, the arrangements and characteristics of the blood vessels demonstrate dramatic species differences. For example, arteries within the testes have tight coils in the mouse and artery-artery anastomoses in the rat. Veins form vascular pathways that connect the testis and efferent ductules in all three species but also form a connection between the testis and cauda epididymidis in the rabbit only. Testosterone concentrations were determined in blood obtained by micropuncture of selected testis-epididymal veins. The measurements establish that the highest levels of testosterone are found in testicular surface veins. Also, vas deferential veins of the rabbit had significant amounts of testosterone. Studies of the blood-vessel volumes suggested that the volumes of arteries and veins in the testis are similar, whereas venous volumes exceed arterial volumes in all of the other organs examined. The studies provide comprehensive information about the architecture and physiology of blood vessels serving the testis-epididymis in the mouse, rat, and rabbit. Each species exhibits diversity in the vasculature and testosterone content of the veins. Veins connecting the testis to the efferent ductules and cauda epididymidis may provide for the preferential delivery of testicular secretions to androgen-dependent organs before the secretions are metabolized or diluted in the systemic blood.

Genetically defined mouse models of male infertility are described in the present report. The mice were rendered infertile by one of the following gene mutations: Ames dwarf, dwarf, flipper-arm, hightail, hypothyroid, little, pygmy, stubby. The effects of each gene mutation on testicular steroidogenesis and spermatogenesis were elucidated by a comparison of the mutant mice to their normal siblings. Testicular steroidogenesis was assessed directly by determining steroid secretion by testes perfused in vitro. The study provides the first comprehensive assessment of testicular function in the mutant mice. The eight gene mutations can be classified into two groups based on the results. One group of gene mutations (Ames dwarf, dwarf, flipper-arm, pygmy) specifically depress spermatogenesis and testicular steroidogenesis. The infertility of these mutant mice can be linked to the lowered total sperm production. The second group of gene mutations (hightail, hypothyroid, little, stubby) do not specifically depress either spermatogenesis or testosterone secretion. Subsequently, the etiology of the male infertility of the second group of mutant mice is unknown. We propose that these mutant mice provide valuable experimental tools for the study of male infertility and male reproduction.

The hyt/hyt mouse (BALB/cBY-hyt, C.hytRF) provides a useful model for exploring the effect of inherited severe primary hypothyroidism. Studies were undertaken to try to define the basis of the primary hypothyroidism in mice homozygous for the autosomal recessive gene, hyt. These mice had congenital hypothyroidism of fetal onset after 15 days post conception. Through their lifetime, the hyt/hyt mice had reduced serum thyroxine (T₄), triiodothyronine (T₃), reduced thyroid gland intralumenal colloid on electron microscopy and a 100-fold elevation of TSH-like activity compared to hyt/+ littermates. Thyroglobulin made in hyt/hyt animals was similar in size to normal thyroglobulin which was inconsistent with a major structural thyroglobulin gene defect. The thyroglobulin was iodinated. Marked, erratic dilation of rough endoplasmic reticulum (RER) was noted in hyt/hyt mouse follicular cells. Despite these ultrastructural findings, pulse chase and immunoprecipitation studies with isolated hyt/hyt and normal thyroid glands indicated that normal thyroglobulin processing occurred in the RER and Golgi of the hyt/hyt mice. The hyt/hyt thyroid glands were hypoplastic compared to hyt/ + littermates. Histologically, the hyt/hyt thyroid glands demonstrated an increase in smaller follicular cells, and greater variability in follicular size compared to hyt/ + littermates. Histological and ultrastructural abnormalities in the gland were similar to those seen in certain cases of human congenital hypothyroidism with TSH receptor insensitivity of the thyroid gland. These findings along with the significant TSH elevation, the reduction in colloid and in serum T₃ and T₄, the efficacy of the hypothalamo-pituitary-thyroid feedback system, and previous observations of reduced iodine uptake and intrathyroidal T4 [3], suggested that primary hypothyroidism in the hyt/hyt mouse might be due to a defect in TSH responsivity of the thyroid gland.

The studies described here provide information about the genetic and morphological bases for the significant differences in testis size among three closely related C57BL mouse substrains: C57BL/6J, C57BL/6ByJ, and C57BL/10J. C57BL/6J mice have normal-size testes while the other two substrains have small-size testes. Genes controlling testis size are postulated to be among the estimated forty genes that differ between the C57BL/6J and C57BL/6ByJ substrains. The number of genes involved in testis size regulation was examined using recombinant inbred mouse strains. An investigation of the role of Y chromosome genes was performed by completing molecular analyses with a mouse Y chromosome-specific probe. Sertoli and germ cell counts provided insight into the morphological basis for the different testis sizes. The experimental results suggest that there are at least two autosomal testis-size genes and that they control testis size by regulating the number of Sertoli cells.