Characterization of maturation-dependent extrinsic proteins of the rat sperm surface.

Mammalian spermatozoa must mature in the epididymis before they can fertilize an egg. It is known that modification of the protein composition of the sperm surface is an important part of the maturation process. In this paper, we present data on two related glycoproteins that can be extracted from mature but not immature spermatozoa. Cell surface radioiodination has shown that these proteins are on the sperm surface, and immunofluorescence microscopy, by use of monospecific antibodies to the proteins, has indicated that their localization is restricted to the periacrosomal region of the sperm head. We have also shown that in vitro, these proteins will bind to the identical region of immature sperm. Immunohistochemical localization of the proteins in the epididymis shows that they are produced and secreted by the cauda region. The significance of the addition of these proteins to the sperm surface in both maturation and fertilization is discussed.

Arrangement of tubulin subunits and microtubule-associated proteins in the central-pair microtubule apparatus of squid (Loligo pealei) sperm flagella.

This study provides a comprehensive, high-resolution structural analysis of the central-pair microtubule apparatus of sperm flagella. It describes the arrangement of several microtubule-associated "sheath" components and suggests, contrary to previous thinking, that microtubules are structurally asymmetric. The two microtubules of the central pair are different in several respects: the C2 tubule bears a single row of 18-nm-long sheath projections with an axial periodicity of 16 nm, whereas the C1 tubule possesses rows of 9-nm globular sheath components with an axial repeat of 32 nm. The lumen of the C2 tubule always appears completely filled with electron-dense material; that of the C1 tubule is frequently hollow. The C2 tubule also possesses a series of beaded chains arranged around the microtubule; the beaded chains are composed of globular subunits 7.5-10 nm in diameter and appear to function in the pairing of the C1 and C2 tubules. These findings indicate: that the beaded chains are not helical, but assume the form of lock washers arranged with a 16-nm axial periodicity on the microtubule; and that the lattice of tubulin dimers in the C2 tubule is not helically symmetric, but that there are seams between certain pairs of protofilaments. Proposed lattice models predict that, because of these seams, central pair and perhaps all singlet microtubules may contain a ribbon of 2-5 protofilaments that are resistant to solubilization; these models are supported by the results of the accompanying paper (R. W. Linck, and G. L. Langevin. 1981. J. Cell Biol. 89: 323-337.

Isolation and molecular characterization of AKAP110, a novel, sperm-specific protein kinase A-anchoring protein.

Agents that increase intracellular cAMP are potent stimulators of sperm motility. Anchoring inhibitor peptides, designed to disrupt the interaction of the cAMP-dependent protein kinase A (PKA) with A kinase-anchoring proteins (AKAPs), are potent inhibitors of sperm motility. These data suggest that PKA anchoring is a key biochemical mechanism controlling motility. We now report the isolation, identification, cloning, and characterization of AKAP110, the predominant AKAP detected in sperm lysates. AKAP110 cDNA was isolated and sequenced from mouse, bovine, and human testis libraries. Using truncated mutants, the RII-binding domain was identified. Alignment of the RII-binding domain on AKAP110 to those from other AKAPs reveals that AKAPs contain eight functionally conserved positions within an amphipathic helix structure that are responsible for RII interaction. Northern analysis of eight different tissues detected AKAP110 only in the testis, and in situ hybridization analysis detected AKAP110 only in round spermatids, suggesting that AKAP110 is a protein found only in male germ cells. Sperm cells contain both RI, located primarily in the acrosomal region of the head, and RII, located exclusively in the tail, regulatory subunits of PKA. Immunocytochemical analysis detected AKAP110 in the acrosomal region of the sperm head and along the entire length of the principal piece. These data suggest that AKAP110 shares compartments with both RI and RII isoforms of PKA and may function as a regulator of both motility- and head-associated functions such as capacitation and the acrosome reaction.

Progesterone and implanting blastocysts regulate Muc1 expression in rabbit uterine epithelium.

Mammalian uteri are unreceptive to blastocyst implantation except during a relatively brief period. The transmembrane, cell surface mucin, Muc1, is present on epithelial cells of nonreceptive uteri in various species and has been demonstrated to have antiadhesive properties. These activities of Muc1 may prevent interaction of the embryonic trophoblast cells with the uterine epithelium. A previous study indicated that Muc1 expression in the rabbit, as in primates, is up-regulated by progesterone. This response would be expected to create a nonadhesive uterine surface during the progesterone-dominated receptive phase. In the current study, Northern blot analysis was used to evaluate Muc1 messenger RNA expression in the endometrium of estrous and progesterone-treated estrous rabbits and in endometrium from different stages of pregnancy or pseudopregnancy. Steady state levels of Muc1 messenger RNA were increased 10-fold when estrous animals were treated with progesterone for 5 days. Muc1 message was elevated 2- to 6-fold over estrous levels in endometrium of pseudopregnant females and 30-fold in preimplantation stage (6.75 days postcoitum) uteri. During implantation (7.25 day postcoitum), the high level of Muc1 expression continued in nonimplantation regions, but was dramatically reduced in endometrium from implantation sites. Using immunofluorescence localization, Muc1 protein was present on the apical surface of epithelial cells of estrous, pseudopregnant (4 and 6.75 days), preimplantation (6.75 days), and implantation (7.25 day) stage uteri. At the latter stage, luminal epithelium apposed to blastocysts had a marked reduction or absence of Muc1 immunostaining. Muc1-immunoreactive cells included luminal and cryptal epithelium in pregnant/pseudopregnant uteri, whereas the glandular cells stained weakly. Short term coculture of uterine epithelial cells with trophoblastic vesicles derived from 6.75-day blastocysts also resulted in a local reduction in apical epithelial Muc1 staining. These findings demonstrate that Muc1 expression is up-regulated by progesterone in the rabbit uterine epithelium and increases incrementally during pre- and periimplantation stages. Removal of Muc1 from the epithelial surface at implantation sites is accomplished locally via signals apparently produced by the blastocyst.

Gene duplication gives rise to a new 17-kilodalton lipocalin that shows epididymal region-specific expression and testicular factor(s) regulation.

Using transgenic mice, we have recently shown that 5 kb of the 5'-flanking region of the mouse epididymal retinoic acid-binding protein (mE-RABP) gene contains all of the information required for spatial and temporal gene expression in the epididymis. To identify the important cis-DNA regulatory element(s) involved in the tissue-, region-, and cell-specific expression of the mE-RABP gene, the 5-kb DNA fragment was sequenced. A computer analysis of the nucleotide sequence showed the presence of a new gene located 1.7 kb upstream from the mE-RABP gene transcription initiation site. The analysis of the open reading frame showed that the new gene encoded a putative 17-kDa lipocalin (named mEP17) related to mE-RABP. A 600-bp complementary DNA encoding mEP17 was cloned by rapid amplification of 3'-cDNA ends from epididymal total RNA. Two mEP17 RNA species (1 and 3.1 kb in size) were detected by Northern blot in the epididymis, but not in other tissues tested. In situ hybridization analyses showed that, unlike mE-RABP messenger RNA (mRNA), which is expressed in the distal caput epididymidis, mEP17 mRNA was detected only in the principal cells of the initial segment. The spatial expression and homology with mE-RABP suggest that mEP17 may act as a retinoid carrier protein within the epididymis. mEP17 mRNA expression disappeared 5 days postcastration. Four days after unilateral castration, mEP17 mRNA had nearly disappeared in the epididymis from the castrated side, but not from the intact side. In addition, testosterone replacement to bilaterally castrated mice failed to restore gene expression. We conclude that mEP17 gene expression is dependent on testicular factors circulating in the luminal fluid. Together our results suggest that mE-RABP and mEP17 genes were generated by duplication and that evolution led to a different region-specific gene expression and regulation in the epididymis.

Specific expression of haptoglobin mRNA in implantation-stage rabbit uterine epithelium.

A glycoprotein, termed GP42, was previously identified in uterine fluid obtained from peri-implantation-stage rabbits. N-terminus amino acid sequencing of purified GP42 demonstrated identity through the first 13 amino acids with the beta subunit of liver haptoglobin. The present study was undertaken to determine if GP42 is indeed identical to haptoglobin and, if so, to determine whether it is expressed in the uterus as opposed to being present as a transudate from plasma. Reverse transcription-PCR amplification of poly(A)+ RNA prepared from implantation-stage rabbit endometrium with GP42- and haptoglobin-specific primers yielded a predicted 667 bp cDNA product. Sequence analysis of the cloned cDNA confirmed the identity of GP42 with beta-haptoglobin. Northern blot analysis demonstrated the specific expression of haptoglobin mRNA in the peri-implantation-stage endometrium and the absence of its expression in the estrous or day 4 pseudopregnant endometrium. Non-isotopic in situ hybridization revealed that the haptoglobin mRNA was restricted to the epithelium lining the luminal surface and mucosal folds of day 6(3/4) pregnant or pseudo-pregnant uteri and that no haptoglobin mRNA was detectable in the epithelium of the deep glands or cells of the stroma or myometrium. Similarly, in situ hybridization revealed no expression of haptoglobin mRNA in any cell types of the estrous uterus. These data establish the identity of GP42 with beta-haptoglobin and demonstrate that it is expressed in a stage-specific manner just prior to implantation, correlating with uterine receptivity to blastocyst implantation. Endometrial GP42 mRNA expression is not dependent on the presence of blastocysts.

Cytoskeletal assemblies of mammalian spermatozoa.

It is becoming increasingly evident that the different domains of the mammalian spermatozoa possess distinct cytoskeletal assemblies. In this paper we have discussed three assemblies, found in the acrosomal, postacrosomal, and midpiece segment, respectively. Each has a distinct substructure and associates with specific sperm-membrane systems. Their protein compositions are currently unidentified, and they may be comprised of sperm-specific polypeptides. Analysis of their formation and fate during sperm-egg interaction should provide valuable insight into their role in the development of cell polarity and in the membrane-mediated steps of fertilization.

Effect of various corticosteroids upon the phagocytic bactericidal activity of neutrophils.

Corticosteroid compounds are used broadly in surgical practice, although mechanisms remain unclarified and efficacy in some situations remains unproved. A recognized adverse effect of steroids in all doses is the potentiation of infection. Specific derivatives of the glucocorticoids appear to have varying degrees of effectiveness in the enhancement of bacterial infection. To evaluate such effects objectively, a series of experiments was undertaken to measure the phagocytic-bactericidal activity of polymorphonuclear leukocytes. This study examines the differential effects of clinical dose equivalents of four glucocorticoid derivatives in depressing in vitro neutrophil phagocytic-bactericidal function. Neutrophils separated from normal human plasma were incubated in vitro in the presence of hydrocortisone sodium phosphate, hydrocortisone sodium succinate, dexamethasone sodium phosphate, and methylprednisolone sodium succinate. Hydrocortisone sodium phosphate, hydrocortisone sodium succinate, and dexamethasone sodium phosphate produce variable, short-term inhibitory effects upon the systems within the neutrophil which are responsible for its bactericidal competency. Methylprednisolone sodium succinate appears to be free of these adverse effects. These in vitro experiments indicate that diminished risk of infection should attend the use of methylprednisolone sodium succinate, although the precise mechanism have not been defined.

A silver method for staining normal axis cylinders of central nervous system structures.

The fink-Heimer stain for degenerating axons has been modified for demonstration of normal axis cylinders. The technique stains tissue prepared from a variety of fixation protocols. Vibratome prepared sections, coupled with an efficient use of laboratory time makes this procedure a simple, quick, and repeatable general technique. This preparation provides valuable insight into the histological organization of the intact central nervous system.

Regional differences in luminal fluid polypeptides of the rat testis and epididymis revealed by two-dimensional gel electrophoresis.

Luminal fluid samples were collected by micropuncture of the seminiferous tubule, rete testis, and defined levels of the epididymal tubule. After removal of spermatozoa by centrifugation, the supernatant fluids were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and an ultrasensitive silver staining procedure to define the sequential change in protein composition along the excurrent duct system. Fluid from each segment displayed a characteristic 2-D PAGE map composed of numerous polypeptides. Seminiferous tubule fluid contained a wide array of polypeptides, with most concentrated in the 45 Kd to 90 Kd range, but, in contrast, rete testis fluid lacked most of these polypeptides. The major complex of rete testis fluid comigrated with serum albumin and was present in all distal segments. Other major rete testis components were not noted distally. Fluid from the caput was characterized by new major components of 30 to 37 Kd, 28 to 30 Kd, 24 Kd, and 23 Kd, each of which consisted of multiple spots of apparent isoelectric variants; all except the 30 to 37 Kd complex were present in the fluid from more distal segments. Proceeding distally, there was a temporal appearance of new polypeptides, especially in the molecular weight range below 30 Kd. Two-dimensional PAGE analysis of detergent extracts of washed spermatozoa indicate that a specific subset of these fluid polypeptides are sperm associated.

Structural features of cultured epithelial cells from the adult rat epididymis.

Epithelial cells isolated from the caput epididymidis of adult rats were placed in primary culture and examined daily for ten days for changes in external anatomy, reorganization of cytoskeletal components, maintenance of characteristic cytoplasmic features, and response to media formulated to minimize nonepithelial cell proliferation. Significant cell attachment to the substrate began after the first 24 hours of culture. After attachment, the cells underwent a progressive flattening and became closely applied to the substrate. This was accompanied by a redistribution of microvilli on the cell surface and a reorganization of cytoskeletal elements within the cell. After flattening, the cultured cells displayed an extensive array of 10-nm filaments which were associated with the desmosomes attaching adjacent cells. Immunofluorescence studies demonstrated that these were keratin-containing intermediate filaments and 2-D gel electrophoresis of intact cells and cell cytoskeletons revealed that a family of "keratin-like" polypeptides were major components of the cells. Epithelial cell attachment, morphology, and maintenance in the primary culture were unaffected by D-valine, cytosine arabinoside, or both; however, these agents, either individually or in combination, reduced significantly the number of cells incorporating 3H-thymidine. These data show that isolated epithelial cells retain some differentiated structural features that characterize the intact cell and that enriched cultures of epithelial cells can be maintained under conditions where fibroblast proliferation is inhibited.

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis.

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.

Changes in intramembranous particle distribution in the plasma membrane of Didelphis virginiana spermatozoa during maturation in the epididymis.

Structural specializations in the plasma membrane of opossum spermatozoa obtained from different levels of the epididymis have been analyzed in thin sections and freeze-fracture replicas. The maturation process was accompanied by a redistribution of intramembranous particles in the flagellar midpiece region. Caput epididymal spermatozoa are immotile, and freeze-fracture replicas of the midpiece plasma membrane reveal a random arrangement of intramembranous particles. As spermatozoa transit the corpus epididymis, the intramembranous particles in the midpiece plasma membrane are redistributed from a random arrangement to an organized packing pattern. This redistribution apparently involves the formation of chains of intramembranous particles which gradually increase in length, orient parallel to the flagellar long axis, and ultimately form numerous parallel rows, each three to five particles wide. In cauda epididymal spermatozoa the intramembranous particles within the rows are packed in an organized manner, and few free intramembranous particles are noted between rows. Analysis of thin sections revealed that the reorganization of intramembranous particles is accompanied by the deposition of a mat of amorphous material at the cytoplasmic face of the membrane. No striking changes in intramembranous particle distribution during epididymal maturation were found in other flagellar segments or in the plasma membrane overlying the sperm head.

Substructure of a cytoskeletal complex associated with the hamster sperm acrosome.

Whole mount and thin section preparations of intact and selectively disrupted hamster spermatozoa revealed an organized array of cytoplasmic filaments associated with specific regions of the acrosome. The filaments were localized along the ventral surface of the spermatozoon and extended from its tip, distally to the anterior margin of the equatorial segment. Individual filaments were 11-13 nm in diameter and they were aligned parallel to one another to form a two-dimensional sheet oriented in the long axis of the spermatozoon. The filament complex adhered preferentially to the cytoplasmic surface of the outer acrosomal membrane rather than the plasma membrane. Examination of disrupted spermatozoa revealed that the distribution of this cytoskeletal assembly correlated with the distribution of a specific acrosomal matrix component. The possible role of this complex in the acrosome reaction or in the organization of acrosomal matrix domains is discussed.

Structure of membrane domains and matrix components of the bovine acrosome.

The acrosomal membrane system of bovine spermatozoa was examined by thin-section, freeze-fracture, surface-replica, and negative staining techniques in order to identify structural differentiations of specific acrosomal membrane domains. The outer acrosomal membrane of the apical and principal segments is characterized by a prominent electron-dense complex associated with its luminal face and a random intramembranous particle distribution. In the equatorial segment, the two-dimensional organization of bridging elements extending between the outer and inner acrosomal membrane was determined and correlated to freeze-fracture images. The inner acrosomal membrane lacked the electron-dense assembly noted on the outer acrosomal membrane and in freeze-fracture it appears crystalline. Further studies identified the distribution of the electron-dense subacrosomal material in the space between the inner acrosomal membrane and outer nuclear membrane. Finally, new observations on the structural organization of the acrosomal matrix are presented.

Membrane domains in guinea pig sperm and their role in the membrane fusion events of the acrosome reaction.

In this study, we have examined the structure of domains of the periacrosomal plasma membrane (PM) and outer acrosomal membrane (OAM) of guinea pig sperm and defined their fate during the membrane fusion events of the acrosome reaction. Cauda epididymal sperm were arranged in rouleaux, joined by periacrosomal PM "junctional" zones; in these zones, the PMs were linked by cross bridges formed from a paracrystalline glycocalyx. Bridging elements linked the PM to the OAM on the ventral (concave) but not dorsal (convex) aspect of the apical segment. Parallel filaments were associated with the luminal face of the OAM overlying the dorsal surface of the apical segment. Sperm were induced to undergo a "synchronous" acrosome reaction after preincubation in Ca2+-free medium containing lysolecithin, by the addition of Ca2+. Fusion between the OAM and PM occurred at the boundaries but not within the PM "junctional" zones over the apical segment. In nonjunctional regions on the dorsal surface of the apical segment, sheets of unfenestrated hybrid membranes and parallel arrays of hybrid membrane tubules formed, while branching arrays of hybrid membrane tubules and vesicles were observed on the ventral surface. In the principal segment, networks of branching hybrid membrane tubules initially formed but later transformed into vesicles. Hence, the lysolecithin-mediated guinea pig sperm acrosome reaction involves a complex sequence of membrane fusions, which differs in domains of the periacrosomal PM and OAM. Stable nonfusigenic domains are present in both the PM and OAM of the apical segment; membrane-associated assemblies may maintain these domains and may also provide direction to some of the membrane fusion events of the acrosome reaction.

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.

Structural organization of surface domains of sperm mitochondria.

Sperm mitochondria are assembled into an organized sheath surrounding the outer dense fibers and axoneme of the flagellar midpiece. Each mitochondrion is arranged so that its surface faces three different cellular organelles including the plasma membrane, neighboring mitochondria and the outer dense fiber-axoneme complex. In this manuscript we present data on structural differentiations of these three different surface domains of the outer mitochondrial membrane. We demonstrate that the apposed surfaces of adjacent mitochondria are joined by a two dimensional network of studs unique to this domain. By contrast, the surface domain facing the outer dense fiber-axoneme complex exhibits a different, but highly ordered structural organization, evident as a parcrystalline network of parallel stripes; this domain is further distinguished by its exclusive association with a midpiece-specific cytoskeletal complex. These differentiations are not seen on the surface domain of mitochondria which faces the plasma membrane. The implications of the mosaic composition of the outer mitochondrial membrane in the assembly and function of the mitochondrial sheath are discussed.