Biomedical subjects

K Boekelheide

Publications and source records attributed to K Boekelheide.

At least 37 records · Page 2Linked to original sources

PubMed1996

Fate of germ cells in 2,5-hexanedione-induced testicular injury. II. Atrophy persists due to a reduced stem cell mass and ongoing apoptosis.

The Sertoli cell toxicant 2,5-hexanedione (2,5-HD) causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few spermatogonia remain in the seminiferous epithelium. In this study, the number, type, and fate of the remaining germ cells were determined. Male Sprague-Dawley rats were exposed to 1% 2,5-HD in drinking water for 5 weeks and then sacrificed 12 or 40 weeks after the start of exposure. Cell counts determined that the stem cell population was diminished in size, but made up a significant portion of the remaining germ cells. The remaining germ cells were primarily type A spermatogonia. Modeling of spermatogonial divisions suggested that most spermatogonia undergo degeneration at the level of type A3 spermatogonia after 2,5-HD-induced atrophy. Apoptosis was demonstrated to occur in the remaining germ cells by nuclear morphology and in situ analysis of DNA fragmentation. Quantitation indicated that apoptosis occurred in a majority of stem cell progeny. We conclude that the irreversibility of 2,5-HD-induced testicular injury results from the reduced size of the stem cell population as well as a block in germ cell development at the level of type A spermatogonia.

Animals↗

PubMed1996

Exogenous stem cell factor (SCF) compensates for altered endogenous SCF expression in 2,5-hexanedione-induced testicular atrophy in rats.

2,5-Hexanedione (2,5-HD) is a Sertoli cell toxicant that causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few committed type A spermatogonia remain in the seminiferous epithelium. A majority of the stem cell progeny differentiate into type A spermatogonia, but then, rather than continuing to differentiate, undergo apoptosis. We hypothesized that the cause for germ cell apoptosis was, at least in part, a deficiency in the function of stem cell factor (SCF), a paracrine growth factor normally made by Sertoli cells. To test this hypothesis, rats were exposed to 1% 2,5-HD for 5 wk and killed at various times after toxicant exposure. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine that, unlike what was observed in control testes, the majority of SCF was expressed in the soluble form after 2,5-HD injury. In vitro co-culture experiments were used to establish the appropriate dose of SCF to administer in vivo. A continuous intratesticular delivery system was established and used to expose 2,5-HD-treated rats to SCF for 2 wk. Animals were exposed to bromodeoxycytidine (BrdCyd) for 2 days before being killed in order to assess the effect of SCF on germ cell proliferation. SCF caused a statistically significant increase in the number of germ cells positive for bromodeoxyuridine (BrdUrd), indicating that SCF promoted survival and/or stimulated proliferation of the remaining germ cells. We conclude that SCF expression is disrupted after 2,5-HD-induced testicular atrophy and that exogenous administration of SCF promotes recovery of spermatogenesis.

Animals↗

PubMed1996

Kinesin localizes to the trans-Golgi network regardless of microtubule organization.

Knowledge of the mechanisms of intracellular membrane trafficking is critical for understanding cell function. Here, the microtubule motor kinesin is localized to the Sertoli cell trans-Golgi network with the SUK4 kinesin monoclonal antibody. Using immunoelectron and immunofluorescence microscopy, kinesin was shown to localize to the trans-Golgi network in primary Sertoli cell cultures. Kinesin immunostaining was not observed in brefeldin A-induced trans-Golgi network-derived membrane tubules and remained associated with trans-Golgi network remnants. Despite dramatic differences in microtubule organization between Sertoli cells in vivo and in vitro, kinesin immunostaining was consistently associated with the trans-Golgi network. In cryosections of control testis and testis treated with colchicine to disrupt microtubules, kinesin immunostaining was juxtaposed with immunostaining for a Golgi cisternal protein; however, brefeldin A exposure partitioned the kinesin immunostaining from the Golgi cisternal protein, indicating that kinesin was associated with the trans-Golgi network of Sertoli cells in vivo. These results are discussed in relation to known Golgi-associated microtubule-dependent transport events in other cell types and suggest that kinesin functions locally at the Sertoli cell trans-Golgi network.

Animals↗

PubMed1995

Distribution of Sertoli cell microtubules, microtubule-dependent motors, and the Golgi apparatus before and after tight junction formation in developing rat testis.

Sertoli cells are polarized epithelial cells of the seminiferous epithelium which provide structural and physiological support for differentiating germ cells. They establish different basal and adluminal environments for the selective nurturing of pre- and post-meiotic germ cells within the seminiferous epithelium, segregated by the Sertoli-Sertoli cell tight junctional complex, the blood-testis barrier. Tight junction formation between epithelial cells in vitro is a critical polarizing event associated with changes in polarized targeting of membrane-specific proteins and reorganization of microtubules, centrioles, and the Golgi apparatus. To investigate whether tight junction formation is associated with organelle reorganization in Sertoli cells in vivo, we have characterized distribution patterns of Sertoli cell microtubules, the mechanoenzymes kinesin and cytoplasmic dynein, and the Golgi apparatus during tight junction formation in developing rat testis. Immunocytochemistry on samples taken at 5, 10, 15, 20, and 25 days of age was used to examine the distribution of these proteins during the extensive cellular reorganization that culminates in the formation of the blood-testis barrier at 19 days of age. Our data show that the distribution patterns reflect the extensive intercellular repositioning of tubule cells in developing seminiferous tubules, but that changes in intracellular organization are not temporally associated with formation of the blood-testis barrier.

Age Factors↗

PubMed1995

2,5-Hexanedione exposure alters microtubule motor distribution in adult rat testis.

2,5-Hexanedione (2,5-HD) exposure in rats causes a progressive Sertoli cell injury culminating in testicular atrophy. Morphological injury is preceded by alterations in the assembly characteristics of tubulin isolated from exposed rat testes. This is followed by decreased seminiferous tubule fluid (STF) secretion by Sertoli cells and an increase in the number and size of Sertoli cell vacuoles. The possible involvement of microtubules and microtubule motor-dependent transport processes in STF secretion by Sertoli cells prompted us to examine the immunodistribution of the microtubule motors cytoplasmic dynein and kinesin during and after 2,5-HD exposure in rats. Three weeks following the commencement of exposure (1% 2,5-HD in the drinking water), the intensity of apical Sertoli cell cytoplasmic dynein immunofluorescence declined. This staining deficit became statistically significant by 4 weeks of exposure. Accompanying this change, there was progressive disruption of the immunodistribution of cisternal Golgi elements and associated kinesin immunoreactivity. The decrease in apical Sertoli cell cytoplasmic dynein immunofluorescence and disruption of Golgi and kinesin immunoreactivity suggest that 2,5-HD-induced alterations in Sertoli cell-mediated transport and secretory events could involve deficits in microtubule-dependent motor function.

Animals↗

PubMed1995

Stem cell kinetics in rat testis after irreversible injury induced by 2,5-hexanedione.

Stem cells provide a continuous supply of committed progenitor cells for the process of spermatogenesis. In rodents, stem cells have been identified as single, undifferentiated type A spermatogonia. The rate of stem cell division has not been definitively determined because of difficulty in locating stem cells among a normal compliment of germ cells. The testicular toxicant 2,5-hexanedione (2,5-HD) induces irreversible testicular atrophy with only Sertoli cells and spermatogonia remaining after injury. Stem cell kinetics could be assessed in this toxicant model because of the absence of most mature germ cells. It is also not known if 2,5-HD-exposed rats possess an active stem spermatogonia population. Charles River CD rats were exposed to 1% 2,5-HD in drinking water for 5 wk. At 7 or 35 wk following toxicant exposure, rats were exposed to bromodeoxycytidine continuously via Alzet mini-pumps for 1-28 days. Serial cross sections of testis were used to identify single stem spermatogonia and to determine whether the cells were positive or negative for bromodeoxyuridine incorporation. We obtained a continuous labeling index for stem cells from rats 7 and 35 wk after 2,5-HD exposure and found that stem cells had a cell cycle time of approximately 8-14 days at both time points after toxicant exposure. In conclusion, we have developed a method for the assessment of stem cell kinetics and verified the presence of an actively dividing stem cell population in irreversibly injured testes.

Animals↗

PubMed1994

Murine PGK-1 promoter drives widespread but not uniform expression in transgenic mice.

Pgk-1 is an X-linked gene encoding 3-phosphoglycerate kinase, an enzyme necessary in every cell for glycolysis. The regulatory sequences of the Pgk-1 gene were used to drive the E. coli lacZ reporter gene and 2 strains of transgenic animals created with this Pgk-lacZ transgene carried on autosomes. The levels of expression of Pgk-1 varied from one adult tissue to another and the transgene was similarly regulated. However, in situ staining of the beta-galactosidase encoded by the transgene indicated extensive cell-to-cell variability in its level of expression. A reproducible subset of cells stained darkly for the transgene product. Some of these beta-galactosidase positive cells were rapidly proliferating while others appeared to be metabolically very active, suggesting that the Pgk-1 promoter is regulated so as to be more active in cells requiring high levels of glycolysis. Although Pgk-1 is X-linked and subject to X chromosome inactivation, the transgenes were not inactivated in either female somatic or male germ cells. Thus, the Pgk-1 promoter drives transgene expression in all tissues but the levels of expression are not uniform in each cell.

Aging↗

PubMed1994

Microtubules with altered assembly kinetics have a decreased rate of kinesin-based transport.

Microtubules treated with the gamma-diketone 2,5-hexanedione (2,5-HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5-HD-induced microtubule modification. 2,5-HD-treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin-based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5-HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3-Acetyl-2,5-hexanedione (AcHD), a pyrrole forming, non-crosslinking congener of 2,5-HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5-HD. However, glutaraldehyde, a pyrrole-independent crosslinking agent which alters microtubule assembly in the same way as 2,5-HD, slowed microtubule transport. These results indicate that a 2,5-HD-induced microtubule modification, possibly a crosslink-related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule-motor interaction.

Animals↗

PubMed1994

Seminiferous tubule fluid secretion is a Sertoli cell microtubule-dependent process inhibited by 2,5-hexanedione exposure.

Mammalian Sertoli cells are responsible for the formation and secretion of seminiferous tubule fluid (STF) which provides the nutritional and hormonal microenvironment necessary for spermatogenesis. Exposure of rats to 2,5-hexanedione (2,5-HD) results in testicular injury characterized by a decrease of STF secretion which immediately precedes bulk germ cell necrosis. The earliest biochemical change in 2,5-HD-exposed rats is an alteration in testicular microtubule assembly kinetics. In this study, we investigate the relationship between microtubule-dependent processes and STF secretion in adult Sprague-Dawley CD rats by exposing seminiferous tubules to two types of toxicants: (1) those which alter microtubules (colchicine and 2,5-HD) and (2) an inhibitor of protein secretion (brefeldin A, [BFA]). Secretion of STF is quantitated by monitoring the rate of transport of a microinjected oil droplet in the lumen of isolated seminiferous tubules using time-lapse stereoscopic microscopy. The rate of oil droplet transport in seminiferous tubules isolated from testis pretreated in vivo for 2 hr with colchicine (40 micrograms/testis) was significantly decreased. Exposure of isolated seminiferous tubules to BFA (10 micrograms/ml) for 40 min also significantly decreased the transport of lumenal oil droplets. Exposure of rats to 1%, 2,5-HD in drinking water decreased transport of injected oil droplets in seminiferous tubules beginning at 3 weeks of exposure in the absence of significant alterations in testicular morphology. These data demonstrate that normal STF secretion requires an intact, microtubule-dependent intracellular membrane transport pathway and strengthen the association between 2,5-HD-induced disruption of Sertoli cell STF secretion and 2,5-HD-induced alterations in Sertoli cell microtubules.

Analysis of Variance↗

PubMed1994

Microtubules are oriented with their minus-ends directed apically before tight junction formation in rat Sertoli cells.

We have examined the polarity of microtubules in Sertoli cells of the seminiferous epithelium during testicular development to test the hypothesis that microtubules change their polarity during tight junction formation. Microtubules in a number of polarized epithelial cells, including Sertoli cells, are oriented with their minus-ends directed toward the apical surface of the cell. Indirect evidence from cultured epithelial cell models suggests that this orientation may be achieved during the relocation of centrioles, reorganization of microtubules, and repositioning of the Golgi that occurs during tight junction formation. Using the microtubule hook decoration technique, we have determined the polarity of Sertoli cell microtubules at 5 and 15 days postnatally, prior to the establishment of the tight junctional complex of the blood testis barrier at 19 days. Our results indicate that, although centrioles and the Golgi apparatus migrate from an infranuclear location at 5 days to a supranuclear location by 15 days, the minus-ends of microtubules are already directed toward the apical surface of the cell by 5 days of age. These data indicate that the establishment of the apically directed minus-end orientation of microtubules of mature Sertoli cells precedes rearrangement of the centrioles and Golgi and is not temporally related to the formation of the tight junctional complex of the blood testis barrier.

Animals↗

PubMed1993

Colchicine disrupts the cytoskeleton of rat testis seminiferous epithelium in a stage-dependent manner.

Sertoli cell microtubules play an important role in the process of spermatogenesis. We investigated the effects of colchicine, a microtubule-disrupting agent, on the seminiferous epithelium. Rats were injected intratesticularly with 0.004-40 micrograms colchicine/testis. Colchicine had a dose-related effect on seminiferous tubule fluid secretion and completely blocked secretion at a dose of 40 micrograms colchicine/testis. Colchicine also resulted in a dose-related decrease in testes weight 2 and 8 wk after injection. When 40 micrograms colchicine/testis was used, testis morphology showed a time-dependent increase in the incidence of sloughing over a time course of 1, 3, 6, and 16 h. Quantitative analysis demonstrated that stage IX-XIV seminiferous tubules were most sensitive to sloughing. Changes in the distribution of tubulin immunostaining within Sertoli cells occurred preferentially in stage VII-VIII seminiferous tubules, which were most resistant to sloughing. In addition, colchicine resulted in disruption of vimentin filaments in stage IX-XIV seminiferous tubules, which correlated with the stage-dependent sensitivity of sloughing. We propose that the stage dependence of colchicine-induced effects reflects the dynamic and stage-dependent role of microtubules in spermatogenesis. Furthermore, cellular structures other than microtubules, such as vimentin filaments, may be important for maintaining the structural integrity of the seminiferous epithelium.

Animals↗

PubMed1993

Visualization of Golgi complexes and spermatogonial cohorts of viable, intact seminiferous tubules.

Golgi complexes of cells within the intact, viable seminiferous tubule were examined by light microscopy using the vital Golgi stain C6-NBD-ceramide. This staining technique provided a quick and simple method to visualize all Golgi complexes of cells within the seminiferous tubule that are directly accessible to the basal compartment. Thus, peritubular, spermatogonial, and Sertoli cell Golgi complexes were visualized. Peritubular cells contained simple Golgi complexes which did not change with the stage of the seminiferous epithelium. Both solitary spermatogonial Golgi complexes which varied in size and number with the stage of the seminiferous epithelium and cohorts of spermatogonia connected by intercellular bridges were easily visualized. The Golgi complexes of Sertoli cells were located in the basal, perinuclear cytoplasm except in Stages VII-VIII, when they extended towards the lumen. Exposure of isolated seminiferous tubules to the fungal metabolite brefeldin A caused the Sertoli cell Golgi complex staining pattern to become diffuse or to co-localize with heads of elongate spermatids. The Golgi complexes of the peritubular cells and spermatogonia were resistant to brefeldin A. The C6-NBD-ceramide vital staining method should be useful for studying stage-dependent Sertoli cell Golgi complex movement and spermatogonial maturation.

4-Chloro-7-nitrobenzofurazan↗

PubMed1993

A tumorigenic murine Sertoli cell line that is temperature-sensitive for differentiation.

The Sertoli cell is the epithelial cell within the seminiferous tubule responsible for supporting germ cells. Most current in vitro studies of Sertoli cell function use primary cultures because of the limited number of available Sertoli cell lines. In addition, few in vivo models of Sertoli cell malignancy have been described. In this study, a tumorigenic Sertoli cell line was developed by infection of isolated murine Sertoli cells by simian virus 40 tsA255; the ts mutation causes the inactivation of the large T antigen at elevated temperatures. A cloned Sertoli cell line, called S14-1, demonstrated temperature-dependent growth in soft agar and formed tumors in nude mice. Electron microscopy of the S14-1-derived tumor revealed extensive basal intercellular junctions and tubulobulbarlike processes supporting its Sertoli cell origin. Cytogenetic analysis showed that S14-1 cells were aneuploid with an average of 70 chromosomes per cell. At the nonpermissive (40 C) temperature, S14-1 cells in vitro demonstrated a reduced growth rate, enhanced secretion of transferrin, and increased expression of sulfated glycoprotein-2 messenger RNA, indicating the cells manifested increased differentiation following large T antigen inactivation. The murine S14-1 Sertoli cell line should be useful for both in vitro studies of Sertoli cell function and in vivo studies of Sertoli cell malignancy.

Animals↗

PubMed1992

cis-diamminedichloroplatinum (II) (cisplatin) alters microtubule assembly dynamics.

Cisplatin is an highly effective chemotherapeutic agent which produces a cumulative dose-limiting peripheral neuropathy. In this study, a possible role for microtubule abnormalities in cisplatin-induced toxicity was explored. CD rats (300 g) were injected daily ip with five doses of cisplatin (2 mg/kg) or every other day ip with two doses of cisplatin (10 mg/kg). The day after the last dose, the rats were killed and tubulin was purified from their testes. The maximal rate of cold-induced microtubule disassembly was consistently slower for testis tubulin purified from cisplatin-treated rats compared with control rats. Overnight in vitro coincubation of polymerized bovine brain tubulin with cisplatin followed by a purifying cycle of assembly and disassembly yielded tubulin capable of forming morphologically normal but short microtubules (average length: cisplatin-treated, 2.5 microns; control, 3.7 microns). Cisplatin coincubation markedly reduced the rate of cold-induced microtubule disassembly, producing a half-maximal effect at approximately 0.3 mM. The cold stability of cisplatin-treated microtubules could be partially reversed by diethyldithiocarbamate. Carboplatin, a cisplatin analog which does not cause clinical peripheral neuropathy, was less capable of producing microtubule disassembly abnormalities. These findings demonstrate the ability of cisplatin to alter microtubule disassembly by direct tubulin modification, an abnormality which may contribute to cisplatin-induced peripheral neuropathy.

Animals↗

PubMed1992

Sertoli cells isolated from adult 2,5-hexanedione-exposed rats exhibit atypical morphology and actin distribution.

Sertoli cells were isolated from 2,5-hexanedione (2,5-HD)-exposed, cryptorchid and 21-day-old rats in order to examine alterations in in vitro Sertoli cell transferrin secretion, germ cell adhesion, in vitro morphology, and cytoskeletal organization which might be involved in the irreversibility of 2,5-HD-induced testicular injury. Sertoli cells isolated from 21-day-old, cryptorchid and 2,5-HD-exposed rats exhibited similar transferrin secretion as measured using an enzyme-linked immunosorbent assay. Germ-cell adhesion was measured using [3H]leucine-labeled immature rat germ cells and revealed similar levels of germ-cell binding in Sertoli cell cultures isolated from the three groups of rats. Differential interference contrast microscopy demonstrated that Sertoli cells isolated from 2,5-HD-exposed rats possessed an atypical spindle shape and long cytoplasmic processes. The immunofluorescent distribution of tubulin and vimentin corresponded with the morphological appearance of the cells with well-defined microtubule and intermediate filament networks which, in the cells isolated from 2,5-HD-exposed rats, extended into the cytoplasmic processes. Rhodamine-conjugated phalloidin-labeled actin stress fibers were decreased in density within the 2,5-HD-exposed rat Sertoli cells. The altered morphology and distribution of actin filaments within Sertoli cells isolated from adult 2,5-HD-exposed rats may reflect an underlying insult which is involved in the irreversible nature of 2,5-HD intoxication.

Actins↗

PubMed1992

Tau, the neuronal heat-stable microtubule-associated protein, is also present in the cross-linked microtubule network of the testicular spermatid manchette.

The seminiferous tubule of the testis contains a rich variety of microtubule networks and of microtubule-associated proteins (MAPs). Tau is a heat-stable MAP previously believed to be limited in its expression in mammals to the nervous system. We have identified tau in rat and bovine testis, a unique non-neuronal location, using biochemical, molecular, and immunologic approaches. SDS-PAGE of ammonium sulfate-fractionated, testis heat-stable MAPs resulted in an enrichment of bands that comigrated with rat brain tau. Only the 35-45% precipitated ammonium sulfate fraction induced microtubule assembly. Immunoblotting with monoclonal anti-tau antibodies demonstrated tau immunoreactivity in these testis MAP preparations. Northern analysis of total rat testis RNA demonstrated a 1.7-kb band that hybridized with a 51-nucleotide oligomer complementary to a conserved portion of the tau transcript. This 51-mer identified a similar 1.7-kb minor band and an additional 6-kb major band in Northern analysis of total rat brain RNA. Finally, in the bull testis, immunohistochemistry localized tau to the spermatid manchette, a transient, cross-linked microtubule network of unknown function. As spermatid elongation begins, the manchette forms a sheath around the posterior aspect of the nucleus, but, by the completion of nuclear condensation, the manchette is largely disassembled. Tau most likely plays a structural role in the manchette; however, tau immunoreactivity also was observed in late stage I spermatids prior to manchette formation, suggesting that tau may serve a function in manchette assembly.

Animals↗

PubMed1992

Distribution of the microtubule-dependent motors cytoplasmic dynein and kinesin in rat testis.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.

Animals↗

PubMed1992

Binding between mammalian spermatid-ectoplasmic specialization complexes and microtubules.

Ectoplasmic specializations (ESs) are submembrane specializations that consist of Sertoli cell plasma membrane linked by an ordered array of actin filaments to a cisterna of endoplasmic recticulum (ESER). They are thought to function in the spermatid-Sertoli cell adhesion junction. Microtubules occur adjacent to the cytoplasmic face of the ESER and are oriented parallel to the long axis of the Sertoli cell, the direction of spermatid translocation during spermatogenesis. Our hypothesis that spermatid orientation and translocation in the seminiferous epithelium is microtubule dependent predicts that microtubules bind to ESs. To test for binding between microtubules and ESs, we have developed an in vitro assay in which spermatid-ES complexes were isolated from the seminiferous epithelium and incubated with bovine brain microtubules that were labeled with [3H]GTP and stabilized with taxol. Binding was determined by scintillation counts from gradient fractions enriched for spermatid-ES complexes and depleted of unbound microtubules by differential centrifugation. Our data indicate that microtubules bind to spermatid-ES complexes in a substrate concentration-dependent manner and can be released with 5 mM GTP or 10 mM MgATP. Binding is competitively reduced with excess unlabeled microtubules and is inhibited by 100 microM vanadate and 2 mM N-ethylmaleimide (NEM). The amount of binding is unchanged by 10 microM vanadate, 2 mM erythro-(2-hydroxy-3-nonyl)adenine (EHNA) or 1 mM 5'-adenylylimidodiphosphate (AMP-PNP). Immunofluorescence and autoradiographic data confirm that labeled microtubules bind to ES locations on spermatid-ES complexes. These data are consistent with the hypothesis that spermatid translocation is a microtubule-based transport event.

Actins↗