Characterization of galactose-binding proteins in equine testis and spermatozoa

Carbohydrate-binding proteins are thought to be involved in a myriad of sperm functions including sperm-oviductal and sperm-zona interactions. Recent studies in our laboratory have characterized galactose-binding proteins on equine spermatozoa as possible candidate molecules for sperm adhesion to oviduct epithelial cells. In the current study, equine sperm membrane proteins were subjected to galactose-affinity chromatography, and bound proteins were eluted with excess galactose in a calcium-free buffer. The eluted fraction recovered after galactose-affinity chromatography was used for generation of a polyclonal antibody which was immobilized on an affinity column to recover a purified protein from equine sperm extracts. Several protein bands of approximately 70, 25, and 20-18 kDa were detected with a major band at 25k Da on immunoblots which was subjected to N-terminal amino acid sequencing. These galactose binding proteins (GBP) were specific to sperm and testis and were absent in all the somatic tissues tested. Based upon immunocytochemistry, GBP were localized over the sperm head. In noncapacitated sperm, fluorescent labeling was observed over the rostral sperm head as well as the postacrosomal area; whereas in capacitated sperm, the labeling was localized primarily in the equatorial segment. Immunohistochemistry of equine testis demonstrated abundant staining in the adluminal region of the seminiferous tubules corresponding to round spermatids. In summary, this study demonstrates the presence of testis- and sperm-specific galactose binding proteins in the horse. The function of these proteins remains to be determined.     

Myosin-like microfilaments in the human normal and pathological testis.

The immunofluorescent staining of the human normal testis shows that both peritubular and tubular cells bind antimyosin-like antibodies. The same cells are provided with a large amount of microfilaments within the cytoplasm. The thin microfilaments observed by electron microscopy in various cells of the human testis likely correspond to the sites of immunofluorescent staining. In pathological specimens there can be observed a decrease in peritubular smooth muscle cells associated with a decrease in cytoplasmic microfilaments and in the immunofluorescent staining of some cells.     

Glucose metabolism in the testis of the normal and streptozotocin diabetic rat

In the present work, the metabolism of [U-14C]glucose was studied in the testicular tissue of normal and streptozotocin diabetic rats. The results show that diabetes alters 14CO2 production and 14C incorporation into lipids from [U-14C]glucose. No differences were found in the [14C]proteins and [14C]nucleic acids between experimental groups. Results are discussed in relation to an insulin deficiency and/or an alteration in the hypothalamic-hypophyseal-gonadal axis.     

Histology and histochemistry of the testis and ovary of the platyfish,Xiphophorus maculatus,from birth to sexual maturity

Summary The gonads of 3-day- to 7-month-old male and female platyfish (Xiphophorus maculatus) were examined for the presence of 5-3-hydroxysteroid dehydrogenase (3-HSD) and glucose-6-phosphate dehydrogenase (G6PD) by histochemical means. In 3-day-old males a positive response for both enzymes is localized in the Leydig cells. With subsequent testicular development, these cells increase in number and display greater activity at the periphery of the testis and around the efferent ducts. In 3-day-old females 3-HSD and G6PD are localized in the stromal cells of the ovary. These cells increase in number and activity as the animals become sexually mature. Sertoli cells, efferent duct epithelium, and ovarian granulosa cells are negative at all stages of development examined. Our findings suggest that the gonads of neonatal fish possess the potential for steroidogenesis. The role played by sexsteroid hormones in the maturation of the brain-pituitary-gonad axis is discussed.     

Interleukin-6 expression during normal maturation of the mouse testis

In this study, we examined the cellular origin and the expression levels of interleukin-6 (IL-6) during normal maturation of mouse testis. The levels of IL-6 (protein and mRNA) were higher in testicular homogenates of sexually immature than mature mice. Immunohistochemical staining of testicular tissues of sexually immature and adult mice show that testicular germ cells, at different stages of differentiation, Leydig cells/interstitial cells and peritubular cells express IL-6. Our results demonstrate, for the first time, overexpression of IL-6 in testicular tissues of immature mice, as compared to mature mice, as well as the expression of IL-6 in germ cells of testicular tissues of adult and sexually immature mice. Thus, our results may indicate the involvement of the endocrine system (gonadotropins and testosterone) in the regulation of IL-6, which is involved in the regulation of testicular development, functions and spermatogenesis.     

Morphometric analysis of Leydig cells in the normal rat testis

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis. The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future. Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy. In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15.7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume. Each cubic centimeter (contained approximatelyy 1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 micron3 and its plasma membrane had a surface area of 1,520 micron2. The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of approximately 10,500 micron2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell. The total surface area of Leydig SER per cubic centimeter of testis tissue is approximately 2,300 cm2 or 0.23 m2. There were 3.0 mg of Leydig mitochondria in 1 g of testis tissue. The average Leydig cell contained approximately 622 mitochondria, measuring on the average 0.35 micron in diameter and 2.40 micron in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 micron2/cell, which is 1.9 times that of the plasma membrane. There were 644 cm2 of inner mitochondrial membrane/cm3 of testis tissue. These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes approximately 0.44 pg of testosterone/d or 10,600 molecules of testosterone/s. The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/s: the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s.     

Spermatogenesis, testicular composition and the concentration of testosterone in the equine testis as influenced by season

The influence of season on spermatogenesis, testicular composition and the concentration of testosterone in the equine testis was evaluated using testes from 45 stallions. Testes were obtained through a commercial abbatoir during September, December-January, March and July. The weights of the testes, the tunica albuginea and testicular parenchyma and the proportion of the testicular parenchyma occupied by seminiferous tubules or interstitial tissue were similar during each season. How ever, diameter of the seminiferous tubules was greater in July than during other months of the study. In addition, the concentration of testosterone within the testicular parenchyma was twice as great during July as during the fall and winter, and this period of peak testicular testosterone concentrations was associated with spermatozoal production rates, which were 65% greater than those observed in September.     

Survival of equine embryos transferred to normal and subfertile mares

To test the hypothesis that an abnormal uterine environment was a cause of early embryonic loss in subfertile mares, morphologically normal embryos were transferred to normal mares (n = 20) and subfertile mares (n = 20), and embryo survival rates were compared. Embryos were recovered nonsurgically at Days 7 to 8 postovulation and transferred surgically to normal and subfertile mares that had ovulated on the same day or within 2 d after a donor. Survival of transferred embryos was monitored by ultrasonography of the recipient mare's uterus from Day 9 through Day 28 postovulation. There were no significant differences (P > 0.5) in the embryo survival rates at Day 12 (11 20 vs 9 20 ) or Day 28 (10 20 vs 8 20 ) for normal or subfertile mares, respectively. The uterine environment of subfertile mares was apparently adequate to support the development of transferred embryos from Days 7 or 8 through Day 28 postovulation.     

Identification of a dendritic cell population in normal testis and in chronically inflamed testis of rats with autoimmune orchitis

Experimental autoimmune orchitis (EAO) in the rat is the primary chronic animal model for the investigation of one of the main causes of male infertility, viz., testicular inflammation. Dendritic cells (DC) are potent antigen-presenting cells that play a fundamental role in autoimmune disease. We investigated the number of DC in normal testis and examined whether DC infiltrated the testis during the development of EAO. EAO was induced by active immunization with testis homogenate and adjuvants in two strains of rat (Wistar and Sprague Dawley). The presence of DC in testis was determined, 50 and 80 days after the first immunization, by immunohistochemical staining with specific antibodies (OX-62 and CD11c), and then the total number of DC was measured by stereological analysis. Labeled cells were found only in the interstitial compartment and within granulomas of EAO animals. The number of DC in EAO testes increased compared with control rats in both strains, whereas the number of OX-62+ and CD11c+ cells in adjuvant controls remained unchanged compared with untreated rats. Interspecies variations in the quantity of DC were found, with the total number of DC per testis in untreated and adjuvant control Sprague-Dawley rats being about three times higher than that seen in Wistar rats. Moreover, the increase in DC numbers at 80 days was less prominent in EAO testes of Sprague-Dawley rats than in the Wistar strain in which EAO was more severe and showed a higher number of granulomae. Thus, we have identified the DC population in normal and chronically inflamed testis. The increase in DC observed in EAO suggests that, under inflammatory conditions, the modified action(s) of these cells is a factor in the induction of the autoimmune response in testis.This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Germany (Me 1323/4–1 &4–2) and grants from Universidad de Buenos Aires (UBA) and Consejo Nacional de Investigaciones Científícas y Técnicas (CONICET, Argentina). The authors are Research Members (L.L.) or Fellows (C.R.) of CONICET and the UBA (V.A.G.).