Immunolocalization of the sea urchin sperm receptor in eggs and maturing ovaries.

The sperm receptor from Strongylocentrotus purpuratus eggs is a high molecular weight proteoglycan-like molecule that inhibits fertilization species-specifically in a competition bioassay. A preparation of highly active sperm receptor that contained one major N-terminal sequence was used to generate polyclonal antibody in rabbits. This antibody species-specifically inhibited fertilization at low concentrations without interfering with fertilization envelope elevation. On immunofluorescence microscopy, the antibody recognized determinants on the mature egg cell surface and in the cortical granules just beneath the surface. Preabsorption of the antibody with the calcium-soluble fraction of the exudate from cortical granules rendered the antibody specific for the cell surface in mature eggs and still able to inhibit fertilization at the same concentrations as before treatment with cortical granule exudate. With antibody preabsorbed with cortical granule and by counting antibody-gold particles viewed by electron microscopy, sperm receptor was almost undetectable on the cell surface of immature oocytes in preseason ovaries, present on the cell surface and intracellularly in immature oocytes of ovaries collected at the beginning or at the height of the spawning season, and present only on the cell surface of mature oocytes in the lumen of the ovaries. Our results indicate that receptor is synthesized early in oogenesis and is rapidly moved to the egg cell surface.

Tissue and cell specificity of immobilin biosynthesis.

The mechanisms for the initiation of sperm motility have been poorly understood until recently. Immobilin is a novel mucin glycoprotein of high molecular weight found in the cauda epididymis of the rat that, at concentrations equivalent to those found in native cauda epididymal fluid, reversibly inhibits sperm motility. In this study, immobilin was purified from rat cauda epididymal fluid to apparent homogeneity and used to generate polyclonal antibody in rabbits. The antibody was characterized by immunoblotting, and immunofluorescence was used to localize immobilin in paraffin sections of components of the reproductive system of adult male rats. Immobilin was not detectable in the efferent duct and was first detectable in the apical portion of some epithelial cells of the initial segment of the caput epididymis. Immobilin was detectable intracellularly only in cells of the caput epididymis. In the corpus and cauda epididymis immobilin was detectable only in the lumen of the tubules. Immunoprecipitation of immobilin radiolabeled in vitro confirmed that immobilin biosynthesis in the adult rat is restricted to the caput epididymis. Principal cells in the caput epididymis synthesize immobilin and secrete it into the lumen of the tubules to travel with the sperm into the cauda.

Isolation and characterization of proteolytic fragments of the sea urchin sperm receptor that retain species specificity.

The sea urchin sperm receptor isolated from the eggs of Strongylocentrotus purpuratus is a high molecular weight proteoglycan-like molecule. Previous studies in our laboratory suggested that the sperm receptor has two functional components, glycosaminoglycan chains that are responsible for sperm binding and polypeptide chains that control species specificity in the binding process. We have investigated this idea further by generating fragments of the receptor by limited proteolytic digestion of the egg cell surface. The results of experiments with these receptor preparations support the hypothesis that the species specificity of inhibition of fertilization observed in a competitive bioassay is conferred by the polypeptide portion of the receptor molecule. Studies with various receptor preparations reveal that the presence of at least 30% of the polypeptide by weight is required to inhibit fertilization species specifically. Receptor preparations containing less than 10% protein lack species specificity and inhibit fertilization in both S. purpuratus and Arbacia punctulata.

Identification and partial characterization of sperm receptor associated with the newly formed fertilization envelope from sea urchin eggs.

Prior studies from this laboratory have identified a proteoglycan-like component of high molecular weight from the surface of the egg of the sea urchin Strongylocentrotus purpuratus that serves as a receptor for sperm. In the present study, a glycoconjugate has been isolated from uncrosslinked fertilization envelopes prepared from eggs activated by treatment with ionophore. Based on its high molecular weight (greater than 5 X 10(6)) and its ability to inhibit fertilization by acrosome-reacted sperm, this glycoconjugate has the properties of the previously described sperm receptor. Components of the fertilization envelope of lower molecular weight (less than 10(6)) showed little or no ability to inhibit fertilization.

Characterization of the Strongylocentrotus purpuratus egg cell surface receptor for sperm.

In earlier studies from our laboratory, the intact sperm receptor was partially purified from Strongylocentrotus purpuratus crude egg membranes, but due to its insolubility, it was not possible to purify it to homogeneity. Nonetheless, this receptor preparation bound with species specificity to acrosome-reacted sperm, thereby inhibiting fertilization. Antibodies against the partially pure receptor inhibited fertilization in S. purpuratus (but not Arbacia punctulata) by coating the egg surface, indicating the presence of binding sites that can be species-specifically recognized by both sperm and antibody molecules. Recently we were able to further purify and characterize the receptor from S. purpuratus eggs. Chaotropic agent solubilization of the receptor prepared from crude egg membranes yielded a very high molecular weight glycoconjugate that had many of the properties of a proteoglycan. The receptor interacted with binding in an in vitro assay and bound with species specificity to acrosome-reacted sperm to inhibit fertilization. Unfortunately, this receptor preparation was soluble only in certain chaotropic agents. Exhaustive Pronase digestion of the intact receptor yielded a soluble high-molecular-weight (greater than 10(6)) polysaccharide that was virtually devoid of protein. This glycosaminoglycan-like fragment was highly sulfated, and contained fucose, galactosamine, and iduronic acid. The fragment inhibited fertilization, but did not do so with species specificity. Recently, soluble molecules with receptor activity were generated by treating intact dejellied eggs with trypsin. These proteolytically derived molecules contained (on a weight basis) approximately equal amounts of protein and carbohydrate. Importantly, they inhibited fertilization with species specificity. The results suggested that the binding activity was conferred by the polysaccharide component of the receptor and that the intact receptor and the tryptic fragments contained structural elements in the polypeptide chain necessary for species recognition.

Multihormonal control of tyrosine aminotransferase activity in developing rat liver.

Tyrosine aminotransferase (TAT) enzymatic activity was undetectable in fetal rat liver until 1 day before birth (21 days). In utero injection of (Bu)2cAMP induced both catalytic and mRNA activity. By contrast, in utero injection of hydrocortisone acetate did not elicit the appearance of TAT enzyme or its functional mRNA. Injection of both the steroid hormone and the cyclic nucleotide elicited the appearance of nearly adult levels of the enzyme and its mRNA. By 24 h after injection of (Bu)2cAMP alone or in combination with hydrocortisone acetate, enzymatic activity had returned to basal levels. Functional TAT mRNA levels, however, remained elevated. The role of insulin as a potential repressor of TAT activity in utero was examined. Reducing circulating fetal insulin levels by injection of streptozotocin was not sufficient to induce TAT enzyme activity. In vitro, either (Bu)2cAMP or hydrocortisone acetate alone induced TAT enzymatic activity in liver explants from fetuses as early as the 16th day of gestation. Explants from fetuses in the 20th day of gestation were able to maintain induced levels of TAT enzymatic activity 48 h after removal from medium containing hydrocortisone.

Induction of tyrosine aminotransferase mRNA by glucocorticoids and cAMP in fetal rat liver.

Tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) enzyme and mRNA activity were not detectable in day 20 fetal rat liver. Precocious induction of catalytic activity by in utero injection of dibutyryl cAMP was a direct consequence of the de novo appearance of translatable tyrosine aminotransferase mRNA. In contrast, in utero injection of hydrocortisone acetate failed to elicit fetal liver enzyme activity. This failure was due to the inability of the steroid hormone to induce the appearance of tyrosine aminotransferase mRNA activity. In fetal rat liver explants, either compound was capable of stimulating the synthesis of adult levels of enzyme and mRNA. However, catalytic and mRNA activity comparable with that seen in vivo 24 hr after birth required the concerted action of both inducers.

Testosterone secretion by rat, rabbit, guinea pig, dog, and hamster testes perfused in vitro: correlation with Leydig cell mass.

Testes from guinea pigs, rabbits, dogs, rats, and hamsters perfused in vitro with maximally stimulating concentrations of ovine LH released 9.76 +/- 2.05, 12.80 +/- 3.15, 28.94 +/- 3.01, 3.18 +/- 0.41, and 0.70 +/- 0.12 microgram testosterone (T)/h, respectively. Adjusting for differences in testicular weight did not eliminate significant (P less than 0.01) species variation in testicular capacity for T secretion in response to ovine LH. Similarly, correction for Leydig cell mass, as determined by morphometric analysis, still left significant (P less than 0.01) differences in the testosterone secretion rates in response to ovine LH for guinea pigs (262.5 +/- 38.6 micrograms T/g Leydig cell), rabbits (205.5 +/- 50.7 micrograms T/g Leydig cell), dogs (116.4 +/- 14.8 micrograms T/g Leydig cell), rats (83.55 +/- 21.80 micrograms T/g Leydig cell), and hamsters (18.24 +/- 3.55 micrograms T/g Leydig cell). The data suggest that significant between-species variation of T production in response to ovine LH is not due to quantitative differences in the mass of Leydig cells.