Small RNA shuffling between murine sperm and their cytoplasmic droplets during epididymal maturation

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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.     

Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5‐(octa‐decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity. Mol. Reprod. Dev. 52:207–215, 1999. © 1999 Wiley‐Liss, Inc.     

Boar sperm surface glycoproteins: Isolation,localization, and temporal expression during spermatogenesis

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent-solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins. Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region. Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex-acrosome region during spermatogenesis.     

Testis size,epididymis weight,and sperm competition in rhesus macaques

The intensity of sperm competition is often measured using the gonadosomatic index (testes/body weight). But sperm competition could be mediated more by size of the epididymis than by size of the testicles, and little information is available on the relationship between testicular and epididymal size. We found that both organs were positively correlated in size among male rhesus macaques. Body weight accounted for over 70% of the variance in testicle size and volumetric estimates of testicle size accurately reflected testicle weight. We conclude that methods for ascertaining testicle size are accurate, but the covariation in size between testicles and epididymis will hamper understanding of the physiological mechanisms involved in sperm competition in primates. © 1993 Wiley-Liss, Inc.     

Lectin binding sites on the plasma membrane of epididymal and ejaculated chimpanzee sperm

Lectins have been used to analyze variations in the distribution and density of exposed saccharides of the sperm plasma membrane during physiologic maturation and after ejaculation. Studies have been conducted in a number of nonprimate species but have been conducted to only a limited extent in nonhuman primates. In this study, pure suspensions of chimpanzee sperm from the caput and cauda epididymis and from the ejaculate were labeled with lectins conjugated to fluorescein isothiocyanate in order to visualize changes in the distribution of exposed membrane glycocomponents. The lectins used were Con A, DBA, RCA-I, and WGA. Con A binding showed minimal change during epididymal transit, with an increased binding to the flagellum after ejaculation. DBA binding was relatively constant in all specimens. RCA-I showed distinct changes in binding pattern between epididymal and ejaculated sperm. On ejaculated sperm strong fluorescence was limited to the posterior head and to the midpiece. WGA binding increased during epididymal passage and decreased after ejaculation. There appears to be a wide variety of saccharide groups available for lectin binding on the surface of epididymal and ejaculated chimpanzee sperm. The general similarity in binding patterns of caput and cauda epididymal chimpanzee sperm exposed to Con A and DBA might reflect the fact that sperm morphology does not change during epididymal transit in this species, thus implying a more stable membrane structure than is present in other primates so far studied.     

Partial purification and properties of the cytoplasmic factors modulating adenylate cyclase activity in rat lung plasma membranes

The adult rat lung supernatant contains some factors which markedly enhance adenylate cyclase activity in membranes (Nijjar, M.S. (1979) Biochim. Biophys. Acta 584, 43–50). These factors were separated into two less active components (peaks 1 and 2) by DEAE-cellulose chromatography. However, their recombination restored the full activation of adenylate cyclase. Further purification and characterization of these factors revealed that the activation in peak 1 contained two proteins of low (14 500) and high (65 000) molecular weight whereas the activator in peak 2 contained only one protein of 65 000. The kinetics of adenylate cyclase activation revealed that both the K_m and V values were affected. The data also demonstrate that calmodulin was not involved in the cytoplasmic activation of adenylate cyclase in rat lungs.     

Effects of FSH receptor deletion on epididymal tubules and sperm morphology, numbers, and motility

Follicle stimulating hormone (FSH) interacts with its cognate receptor (R) on Sertoli cells within the testis and plays an important role in the maintenance of spermatogenesis. Male FSH-R knockout (FORKO) mice show fewer Sertoli cells and many that are structurally abnormal and as a consequence fewer germ cells. Lower levels of serum testosterone (T) and androgen binding protein (ABP) also occur, along with reduced fertility. To assess the effects of FSH-R depletion as an outcome of testicular abnormalities, sperm from the cauda epididymidis were counted and examined ultrastructurally. As reduced fertility may also reflect changes to the epididymis, the secondary responses of the epididymis to lower T and ABP levels were also examined by comparing differences in sizes of epididymal tubules in various regions of FORKO and wild type (WT) mice. Sperm motility was evaluated in FORKO mice and compared to that of WT mice by computer assisted sperm analysis (CASA). Quantitatively, the data revealed that epithelial areas of the caput and corpus epididymidis were significantly smaller in FORKO mice compared to WT mice. Cauda epididymal sperm counts in FORKO mice were also much lower than in WT mice. This resulted in changes to 9 out of 14 sperm motility parameters, related mostly to velocity measures, which were significantly lower in the FORKO mice. The greatest change was observed relative to the percent static sperm, which was elevated by 20% in FORKO mice compared to controls. EM analyses revealed major changes to the structure of the heads and tails of cauda luminal sperm in FORKO mice. Taken together these data suggest a key role for the FSH receptor in maintaining Sertoli cells to sustain normal sperm numbers and proper shapes of their heads and tails. In addition, the shrinkage in epididymal epithelial areas observed in FORKO mice likely reflect direct and/or indirect changes in the functions of these cells and their role in promoting sperm motility, which is noticeably altered in FORKO mice.     

CASK is in the mammalian sperm head and is processed during epididymal maturation

Upon adhesion to the zona pellucida or egg extracellular matrix, sperm undergo regulated exocytosis of the acrosomal vesicle. CASK is an adaptor protein that has been implicated in coupling neuronal cell adhesion to regulated exocytosis. In neurons, this scaffolding molecule is associated with several types of transmembrane receptor complexes and connects cell adhesion molecules with ion channels, the actin cytoskeleton, and the cell's exocytotic machinery. We hypothesized CASK might also be an important link between zona pellucida binding and the sperm acrosome reaction. RT-PCR experiments indicated CASK is transcribed in mouse testis. The full size (120 kDa) CASK protein was present in testis from mouse and pig. Immunoblots of mature porcine and murine sperm revealed that the 120 kDa molecule was much less abundant than in testis but the antibody also recognized a group of smaller proteins migrating at 55-65 kDa. Immunofluorescence experiments indicated both the full length and smaller CASK immunoreactive products were found only in the acrosomal region of spermatids and mature sperm and not in other testicular cell types. CASK immunofluorescence was lost following the acrosome reaction. During epididymal maturation, the abundance of the full size CASK decreased and the CASK fragments increased. These results suggest that CASK may be proteolytically processed during epididymal maturation. Because sperm acquire the ability to bind the zona pellucida, acrosome react, and fertilize eggs during epididymal maturation, CASK processing may play a role in the acquisition of these functions.     

Possible involvement of a sialylated component of the sperm plasma membrane in sperm-zona interaction in the mouse

We have studied the molecular mechanisms of gamete interaction in vitro in the laboratory mouse, Mus musculus. In particular, we were interested in whether this interaction is similar to a lectin-hapten-mediated process. Inhibition of sperm-zona binding was examined using various concentrations (.25 mM to 50 mM) of different sugars (sialic acid α-methylmannose, glucose, fucose, galactose, and N-acetyl-glucosamine). Sperm-zona binding was significantly decreased when eggs were pretreated with 10 mM of sialic acid or α-methylmannose but not by other sugars tested. Furthermore, treatment of capacitated sperm with neuraminidase destroyed their ability to bind and fertilize eggs. We have also used a specific lectin for sialic acid from the horseshoe crab (Limulus polyphemus) to agglutinate mouse sperm. The lectin (.120 ng/ml) mediated agglutination of mouse sperm (10⁵ sperm/ml) was routinely observed to increase from a 10% agglutination immediately following their isolation from the epididymis to 100% agglutination 90 minutes later. Collectively, these results suggest the appearance of specific sugar moieties on the surface of the sperm plasma membrane which, in this particular species of mouse, are sialylated glycoproteins acting as ligands for specific receptors on the surface of the egg. These are the first data to indicate that sperm-egg recognition and attachment is a lectin-hapten-mediated process in the mouse.     

Alteration of the human sperm surface during in vitro capacitation as assessed by lectin-induced agglutination

Human sperm incubated in vitro in BWW medium containing 35 mg/ml human serum albumin acquire the capacity to penetrate the human zona pellucida and to fuse with the zona-free hamster oocyte. We have studied changes in lectin-induced agglutination of human sperm during incubation in this medium to detect alterations in the sperm surface which may be correlated with the acquisition of these functions. Sperm incubated for 1, 6, or 24 hr were combined with two-fold dilutions of lectins for 30 min at 37°C, in 5% CO₂, balance air. When pooled data from five donors were analyzed, the average sperm agglutination titer of wheat germ agglutinin (WGA), phytohemagglutinin-E (PHA), Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), and Pisum sativum agglutinin (PSA) was found to increase significantly (P ≤ 0.06) with incubation in vitro, although there was considerable variation between ejaculates. Ulex europaeus and Dolichos biflorus agglutinins did not agglutinate human sperm (≤250 μg/ml). Results of this screening demonstrate the alteration of sperm surface components during in vitro incubation and suggest that WGA, PHA, LCA, and PSA may prove useful in efforts to correlate changes in the sperm surface with the ability of the sperm to fertilize the egg.     

Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: Implications on oocyte maturation, fertilization and developmental competence after ICSI

Lipid droplets (LDs) and mitochondria in the ooplasm are essential for energy production required for maturation, fertilization and embryo development. This study investigates the correlations between cytoplasmic LDs polar aggregation and: (1) nuclear maturation (Experiment 1); (2) mitochondrial (mt) distribution pattern and localization (Experiment 2); (3) fertilization and embryonic development after intracytoplasmic sperm injection (ICSI; Experiment 3) in equine oocytes recovered from slaughtered mares and matured in vitro. Morphologically normal oocytes were selected after culture and categorized as having polar (P) aggregation or uniform (U) distribution of LDs. In Experiment 1, the maturation rate was significantly higher in P compared with U oocytes (69%, 40/58 vs. 32%, 13/41; P < 0.001). In Experiment 2, it was observed that P and U oocytes showed heterogeneous mt distribution at comparable rates (68%, 25/37 vs. 50%, 2/4 for P and U respectively; NS). Moreover, only in 8/25 (32%) of P oocytes, LDs overlapped with mt aggregates in the area containing meiotic spindle. In Experiment 3, normal fertilization (51%, 19/37 vs. 60%, 6/10, for P and U) and cleavage rates (83%, 20/24 vs. 67%, 4/6, for P and U) did not differ between groups, also in oocytes with LDs located nearby the polar body. Overall, P aggregation of LDs was related to cumulus expansion at collection. In conclusion, in equine matured oocytes, P aggregation of LDs is related with cumulus expansion and nuclear maturation. However, it is not related with heterogeneous mt distribution and cannot be considered a predictive indicator for normal fertilization and embryo development.     

Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation,properties, and immunocytochemical localization at the inner side of the cytoplasmic membrane

The fhu operon of Escherichia coli K-12 comprises four genes, termed fhuA,C,D,B, which are involved in the uptake of iron-hydroxamate compounds. The fhuA gene encodes the outer membrane receptor protein. Cells that contained three copies of the fhuACD fragment on the thermoamplifiable plasmid pHK232 accumulated at 37° C large amounts of the proFhuA protein. Most of the overproduced proFhuA protein was not translocated into the outer membrane but instead precipitated at the cytoplasmic side of the inner membrane, presumably at the sites of synthesis. Despite inhibition of export proFhuA synthesis continued.The precipitate formed was sedimented by centrifugation at 8,000xg. The proFhuA protein could be solubilized in 1% sodium dodecyl sulfate. Replacement of sodium dodecyl sulfate by Triton X-100 resulted in a proFhuA protein which exhibited 10% of the phage T5 binding activity of renatured mature FhuA protein. Binding of phage T5 was inhibited by the FhuA-specific ligands ferrichrome, albomycin and colicin M. Limited proteolysis of the isolated pro- and mature form of the FhuA protein with trypsin yielded similar oligopeptide patterns. Addition of ferrichrome affected trypsin cleavage of both proteins in the same way. The common proteolytic intermediates together with phage inactivation indicate a similar conformation of the pro- and mature form.Dedicated to Prof. G. Braunitzer on the occasion of his 60th birthday     

Phagocytosis of sperm cytoplasmic droplets by a specialized region in the epididymis of the brushtailed possum, Trichosurus vulpecula

During sperm maturation in the brushtailed possum, Trichosurus vulpecula, cytoplasmic droplets are shed from maturing spermatozoa in the distal regions of the head of the epididymis. Examination of luminal contents from various regions of the epididymis showed that the proportion of detached droplets in the luminal contents was reduced from about 45% in the proximal corpus epididymidis to less than 10% in the distal corpus and cauda epididymides. In contrast, the proportion of droplet-free spermatozoa increased from about 45% to more than 90% in the luminal contents. Disappearance of detached cytoplasmic droplets from the lumen was found to be associated with a region of specialized principal cells lining Regions 6 and 7 of the epididymis which selectively sequester and phagocytose free droplets from the luminal milieu. The luminal surfaces of these cells are characterized by a complex system of interdigitating processes which appear as waves of microfolds . These processes contrast with the stereocilia which cover the luminal surfaces of principal cells in adjacent, nonphagocytic regions of the duct. Cytoplasmic droplets are phagocytosed with their limiting membrane intact and gradually become condensed as they are transported deeper into the cell. Membrane lamellae are gradually compacted, transformed into concentrically arranged membrane stacks and then condensed into small electron-dense vesicles, which are probably degraded by the epithelial cells. The presence of a specific recognition factor on cytoplasmic droplets is suggested by the observation that phagocytic principal cells are able to selectively remove detached cytoplasmic droplets from the lumen in the presence of sperm-associated droplets and spermatozoa.     

Calcium-binding proteins of boar spermatozoan plasma membranes: Identification and partial characterization

Calcium-binding proteins (CBPs) of boar spermatozoa and boar seminal plasma were identified by using a ⁴⁵Ca overlay technique to detect these proteins on transblots of PAGE-separated proteins. A single CBP (Mr ～ 300 kDa) was detected in seminal plasma. This protein binds specifically to the plasma membrane overlying the principal segment and is removed from sperm during capacitation. The protein was purified for further charac terization by anion exchange chromatography and gel filtration. In addition, six major proteins (30, 35, 38, 42, 52, and 66 kDa) which do not originate from accessory gland secretions were found to be strongly associated with the plasma membrane. Most of these proteins are not integral to the membrane and appear to develop an association with the plasma membrane during cpididymai maturation. Similarly, calmodulin-binding proteins appear to develop strong associations with the plasma membrane during epididymal transit.     

Quantitative changes in sperm head morphology during passage through the male excurrent duct system of the rabbit

A fine adjustment of sperm head size and shape occurs during maturation and storage within the male excurrent duct of the rabbit. This remodelling, as judged by morphometric values of area, perimeter, length, width, and shape factors, takes place mostly in passage from the seminiferous tubules of the testis to the distal caput of the epididymis. The dimensions of sperm heads from the distal corpus of the epididymis break the general tendency toward a reduction in size and more elliptical shapes. A period of transport and storage within the epididymal cauda and vas deferens follows in which there are no further changes in sperm head morphometry. It can be concluded that the period immediately following sperm release from the testis is crucial to the final morphological maturation of spermatozoa. Moreover, the fact that changes are detected in the appearance of sperm heads at successive stages of sperm maturation suggests that the dimensions of a particular epididymal spermatozoon may be taken as an approximate indication of its relative maturity. Mol. Reprod. Dev. 51:203–209, 1998. © 1998 Wiley-Liss, Inc.     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.