Biomedical subjects

C A Suarez-Quian

Publications and source records attributed to C A Suarez-Quian.

At least 19 recordsLinked to original sources

PubMed1999

Laser capture microdissection of single cells from complex tissues.

Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 microns. A computer-controlled arm precisely positions a 40-micron-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.

Animals↗

PubMed1999

Synthesis and properties of an EGF-like domain (residues 361-406) in the extreme N-terminal region of the mouse EGF precursor.

Various proteins contain EGF-like domains that are not ligands for the EGF receptor. In the present study a cognate polypeptide for residues 361-406 of the mouse EGF precursor was synthesized by the solid-phase method. The product was renatured under oxidative conditions since it probably has an EGF-like array of three cystine disulfide bonds in its native state. HPLC analysis of the renaturation reaction revealed formation of a peak material with no apparent free-SH groups. Accordingly, the HPLC retention time of this product was readily increased by treatment (reduction of disulfides) with dithiothreitol. The renatured 46-mer (PEGF-1) did not displace 125I-EGF bound to rat liver membranes and 125I-PEGF-1 did not exhibit specific binding to membrane preparations from the mouse liver, mammary gland, or kidney, with or without Ca2+ in the binding medium. Although PEGF-1 contains a putative Ca2+ binding motif, specific binding of this cation by the polypeptide could not be demonstrated by electromobility shiff or incubation with 45Ca2+. Immunoassay of PEGF-1 and EGF in fractions obtained following gel filtration of mouse urine revealed multiple peaks of PEGF-1 immunoreactivity with the major peaks eluting at an Mr > 30 kDa. In contrast, virtually all the EGF immunoreactivity eluted at a volume similar to that of 125I-EGF. These data suggest that selective cleavage of the PEGF-1 domain from the precursor does not occur with the proclivity known for that of EGF. Instead, the PEGF-1 probably functions coordinately with other EGF-like domains while tethered to the precursor backbone. Finally, localization of PEGF-1 immunoreactivity occurred only in cell populations of the mouse previously demonstrated as sites for EGF/EGF precursor, which suggests that PEGF-1 is exclusively a domain of the EGF precursor.

Amino Acid Sequence↗

PubMed1994

Two Golgi integral membrane proteins (GIMPS) exhibit region- and cell type-specific distribution in the epididymis of the adult rat.

The epididymis participates in the post-testicular maturation and storage of spermatozoa by secreting proteins into the tubule lumen in a region-specific fashion. The underlying molecular mechanisms leading to biogenesis of these region-specific differences, however, are not known, although components of the Golgi complex membrane container must undoubtedly be intimately involved. Two monoclonal antibodies raised against Golgi integral membrane proteins, recognizing either the cis (GIMPc) or trans Golgi (GIMPt) cisternae, were used as molecular probes of these regions to begin the characterization of the Golgi complex of in vivo and in vitro epididymal cells. Immunolocalization of GIMPs was performed on frozen sections and in cultured cells using biotin-streptavidin-peroxidase immunocytochemistry. In tissue sections, immunostaining of GIMPt was extremely robust in the supranuclear cytoplasm throughout the epididymis. In contrast, no GIMPc immunostaining was detected in the initial segment or in clear cells of the distal caput, corpus, and cauda. Immunodetection of GIMPc and GIMPt in epididymal cells in vitro revealed a reticular, perinuclear pattern, and NH4Cl treatment preferentially disrupted the GIMPt immunolocalization. These results characterizing the molecular components of the Golgi complex will form the basis of additional studies to gain further insight into mechanisms leading to generation of regional differences in epididymal function.

Animals↗

PubMed1994

Relationship between submandibular gland epidermal growth factor and spermatogenesis in C3H mice.

Epidermal growth factor (EGF), a potent mitogen produced primarily in the submandibular gland of adult male mice, has been implicated in modulating processes known to be of vital importance in the regulation of spermatogenesis. In the present investigation we demonstrate that submandibular gland EGF from adult male mice is indeed capable of displacing radiolabeled EGF from testicular membranes. Scatchard analysis of this binding site reveals that it is of high affinity (Kd = 0.77 nM) and low capacity (Bmax = 8.15 fmol/mg protein). Cross-linking of 125I-EGF to the identical membrane preparation resulted in the SDS-PAGE/autoradiography identification of a single band at approximately 170 kDa. Next, we examined the cellular distribution of the EGF receptor in the testis using biotin-streptavidin immunoperoxidase and employing different antisera probes generated to a conserved sequence of the EGF receptor. The Scatchard and cross-linking data described above, along with the immunocytochemistry results, suggest strongly that there is only one functional binding site for EGF in the adult testis and that this receptor is present in Sertoli and Leydig cells.

Animals↗

PubMed1994

Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis.

The distribution of the androgen receptor (AR) in the adult rat testis was determined by biotin-streptavidin immunoperoxidase, employing tissue embedded in polyester wax which preserves antigenicity without compromising tissue preservation. The antibody probe used, which has been characterized previously, was an affinity purified, rabbit polyclonal antibody raised to the amino terminus peptide of the rat AR. Within the interstitial compartment, AR immunostaining was detected in some Leydig cells and all smooth muscle cells forming the walls of blood vessels, but endothelial cells of blood vessels were negative. Furthermore, in those Leydig cells that were clearly identified as exhibiting AR immunostaining, the intensity of the reaction varied. In the seminiferous tubules AR immunostaining was observed in all peritubular myoid cell nuclei, but not in the distal layer of lymphatic endothelial cells. In Sertoli cells, nuclear AR immunostaining was stage specific. Moderate AR immunostaining first became evident at late stage IV or early stage V of the cycle, reached a robust peak at stages VII-VIII, and then disappeared completely. Specific AR immunostaining was also discerned in the nuclei of stage XI elongated spermatids, the spermatids in which nuclear elongation is apparent but chromatin condensation has not yet begun. Next, with onset of chromatin condensation, nuclear AR immunostaining in elongated spermatids was not discerned concomitant with its detection in the cytoplasm of the germ cells. These results are interpreted in the following manner: 1) The presence of AR in Leydig cells is consistent with the hypothesis that androgens modify Leydig cell activity in an autocrine fashion. Further, that not all Leydig cells exhibited AR immunostaining at steady state suggests a differential, functional activity of these cells within the population. 2) The intense AR immunostaining of smooth muscle cells present in the interstitium indicates that these cells are targets for androgens. 3) AR immunoreactivity in both Sertoli and peritubular myoid cells suggests their involvement in the androgenic control of spermatogenesis. The stage specific AR immunoreactivity in Sertoli cells, however, may be more indicative of a specific androgen response during these stages, whereas peritubular cells may participate in the tonal maintenance of spermatogenesis. 4) The specific presence of AR in step 11 elongated spermatids may suggest that androgens can act directly on germ cells to regulate spermatogenesis.

Androgens↗

PubMed1994

Modulation of transferrin secretion by epidermal growth factor in immature rat Sertoli cells in vitro.

The modulation of transferrin secretion by FSH and epidermal growth factor (EGF) was studied in highly pure, primary cultures of immature rat Sertoli cells grown on a reconstituted basement membrane (Matrigel) in bicameral chambers. Sertoli cell purity was assessed by (1) morphometry, (2) alkaline phosphatase cytochemistry (a specific marker enzyme for peritubular cells) and (3) immunocytochemistry for the alpha-isoform of smooth muscle actin in contaminating peritubular cells. Results revealed a less than 0.5% peritubular cell contamination. During initial periods of culture with EGF or FSH alone or in combination, both EGF and FSH alone maintained transferrin secretion over basal values and their effects were additive. At subsequent times, EGF alone maintained transferrin secretion, but to less extent than did FSH alone, and inhibited significantly the ability of FSH to maintain transferrin secretion. The ratio of polarized transferrin secretion in response to FSH, EGF, or in combination was also examined. FSH significantly reversed the polarity of transferrin secretion, whereas EGF, although significantly reducing the ratio of apical to basal transferrin secretion, did not lead to a preferential basal secretion of transferrin. The change in the apical:basal transferrin secretion ratio, however, was not due to a reversal of the apically secreted transferrin towards a basal direction, but rather to an increase in the total basally secreted transferrin. The effects of cell density effects on transferrin secretion were then examined. At low cell density, the relative ability of EGF and FSH together to maintain transferrin secretion was greater than at high cell density, but overall transferrin secretion was greater as cell density increased. The inhibition of FSH by EGF on transferrin secretion was also density dependent: EGF significantly inhibited FSH effects at low cell density, but failed to do so at high cell density. These results suggest that regulation of transferrin secretion by Sertoli cells appears to be under dual control, involving both FSH and EGF and may provide an explanation for the mechanism by which EGF exerts a regulatory role in spermatogenesis.

Animals↗

PubMed1993

Localization of secretory, membrane-associated and cytoskeletal proteins in rat testis using an improved immunocytochemical protocol that employs polyester wax.

Immunocytochemistry is a compromise between maintaining antigenicity and preserving tissue morphology. In the testis, successful immunostaining results at the level of resolution provided by the light microscope have been obtained through use of either frozen or paraffin sections, although both techniques are fraught with limitations. With freezing, tissue preservation is not optimum, whereas with paraffin embedding, antigenicity is often destroyed. These limitations are not trivial and have led to numerous ambiguous results in the literature. In the present study we wish to report the results of immunocytochemical localization of various proteins in testis fixed by perfusion with Bouin's fluid and embedded in polyester wax, a ribboning embedding medium for histology. The advantages of this medium are that it does not require clearing of tissues in xylene solvents before embedding and that unlike paraffin, it liquifies at 38 degrees C. Because of these two properties, the polyester was appears to adequately maintain antigenicity as compared to that observed in frozen sections, yet because it is a ribboning wax, it preserves detailed structure as well as paraffin does. Proteins that were immunolocalized included cytoskeletal proteins (tubulin, actin, vinculin, vimentin) and cell-specific markers: 1) androgen-binding protein (ABP) for Sertoli cells; 2) peripheral type benzodiazepine receptor (PBR) for Leydig cells; and 3) nuclear lamins for germ cells. Biotin-streptavidin peroxidase immunocytochemistry was employed to determine the specific distribution of the various proteins, and both rabbit antisera and mouse monoclonal antibodies were used with equal success. In addition, fluorochrome-labeled second antibodies combined with confocal microscopy were used to examine the disposition of the antigens in the testis. Results revealed maintenance of antigenicity and morphology far superior to that obtained with paraffin and frozen sections, respectively, they also showed that within the seminiferous epithelium, germ cell or Sertoli cell-specific proteins were unambiguously immunolocalized to their respective cells. Specific observations made possible through use of this protocol suggest that neither tubulin or vimentin immunostaining patterns in Sertoli cells are altered during the cycle of the seminiferous epithelium. Similarly, ABP staining appeared constant throughout the cycle. Further, we wish to report that anti-PBR is a specific probe for Leydig cells in vivo and that an anti-nuclear lamin antibody appears to serve as a specific probe for spermatogonia and pachytene spermatocytes, but that the commercially available anti-smooth muscle alpha-actin monoclonal antibody immunostains both the myoid and lymphatic endothelial cells forming the peritubular cells layer of the seminiferous tubule.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen-Binding Protein↗

PubMed1993

Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms.

Previous studies demonstrated that the polypeptide diazepam binding inhibitor (DBI) and its receptor, the peripheral-type benzodiazepine receptor (PBR), are involved in the regulation of steroid biosynthesis and that one site of PBR action resides in mitochondria. In the present investigation, evidence is presented that a functional form of PBR is also present at the cell surface. First, PBR was immunolocalized in the rat testis using biotin-streptavidin peroxidase immunocytochemistry, and results revealed that PBR was present exclusively in the interstitial Leydig cells. Next, the distribution of PBR in MA-10 Leydig cells was further examined using confocal microscopy. MA-10 cells were either fixed and immunostained or fixed/permeabilized and immunostained for PBR, followed by generation of confocal microscope optical sections, three-dimensional reconstructions of these sections, and then generation of vertical confocal sections of the three-dimensional reconstruction. In the fixed/unpermeabilized cells, PBR immunostaining at the cell surface was clearly evident, whereas in the fixed/permeabilized cells, intracellular PBR distribution was more robust. These results suggest that the plasma membrane fraction of the receptor could mediate the action of extracellular PBR ligands on Leydig cell function. Next, we examined whether DBI, the naturally occurring PBR ligand, is secreted by testicular cells and whether it could activate the cell surface PBR. Immunolocalization of DBI demonstrated that it was present in both Leydig and Sertoli cells. Further, using an immunoblot assay, we demonstrated that DBI is present in rat testicular interstitial fluid. Metabolic labeling of cultured immature rat Sertoli cells and MA-10 mouse tumor Leydig cells, followed by immunoprecipitation of the secreted proteins with an anti-DBI antiserum, demonstrated that both Leydig and Sertoli cells secrete DBI and could serve as a cell source for the interstitial fluid DBI. Then, we partially purified the DBI present in conditioned medium and interstitial fluid by reverse phase chromatography and demonstrated it to be bioactive, based on displacement of a radiolabeled benzodiazepine (Ro5-4864)-specific ligand for PBR; pronase treatment of different preparations eliminated all bioactivity. We then examined the effects of DBI on Leydig cell function. DBI added to MA-10 cells affected DNA synthesis and cell growth in a biphasic manner; at low concentrations (1 nM), DBI was mitogenic, increasing [3H]thymidine incorporation and cell numbers by 30-40%, while at high concentrations (1 microM), DBI inhibited cell growth (30-40%). Similar effects on cell growth were obtained using the benzodiazepine Ro5-4864.

3T3 Cells↗

PubMed1992

Characterization of Sertoli cell perinuclear filaments.

Sertoli cell nuclei are characterized by deep invaginations and, in addition, the orientation of the nuclei with respect to the wall of the seminiferous tubules varies during the cycle of the seminiferous epithelium. These events may be the result of cytoplasmic filaments acting at the level of the nuclear capsule and may represent significant changes in Sertoli cell activity. Thus, a study was performed to characterize the nature of the perinuclear filaments of Sertoli cells in vivo and in vitro. In Sertoli cells in vivo, microtubules and microfilaments were often detected in the perinuclear cytoplasm, and these cytoskeletal components were observed to course either parallel to, or abut at, the nuclear capsule. In Sertoli cells in vitro, the nuclear infoldings are retained and the perinuclear cytoskeleton was shown to contain microtubules, f-actin, and intermediate filaments. A fixation-permeabilization protocol employing tannic acid-saponin was used and it significantly enhanced the preservation of cytoskeletal components. The presence of f-actin was demonstrated by using the S1 fragment of muscle myosin to decorate the microfilaments. Treatment of the cultured cells with either microtubule or f-actin depolymerizing agents had no effect on nuclear shape. Thus, at present, the function of the prominent perinuclear cytoskeletal components remains unknown.

Actins↗

PubMed1992

Lysosomal integral membrane proteins exhibit region and cell type specific distribution in the epididymis of the adult rat.

The epididymis, a post-testicular site required for maturation and storage of spermatozoa, is actively involved in exocytic and endocytic events, two phenomena likely to depend on the integrity of the lysosomal system. To study the lysosomal system of the epididymis, five monoclonal antibodies, previously characterized as recognizing five distinct lysosomal integral membrane proteins (LIMPs 1-5), were used as molecular probes of lysosome distribution in cells lining the epithelium. Immunocytochemical localization of LIMPs, using biotin-streptavidin immunoperoxidase methodology, was performed on frozen sections of adult rat epididymides and in cell cultures prepared from either the caput or cauda epididymis. In frozen sections, a heterogeneous distribution of the different LIMPs along the length of the epididymis was observed. For example, the distribution of LIMP 1 (35-50 K) was detected in all cells of the caput and quite dramatically in clear cells of the distal caput, corpus, and cauda epididymis, but specifically not in the principal cells of the distal caput, corpus, and cauda. In contrast, LIMP 2 (64-71 K) was present in all cells of the epididymis, except clear cells. LIMPs 4 and 5 (93 K and 93 K) were detected in all epididymal cells, including the clear cells. Finally, whereas the regional and cell type distribution of LIMP 3 (74 K) in the epididymis was identical to that of LIMPs 4 and 5, the nature of the vesicles immunostained was distinct. In cultured cells, the general immunostaining patterns observed in vivo were maintained during the duration of the primary cultures for all five LIMPs. Our results begin to address the molecular heterogeneity of the lysosomal system along the length of the epididymis, and may suggest in part a basis for underlying structural and functional characteristics of the epididymis leading to the sequential maturation of sperm.

Animals↗

PubMed1992

Partitioning of the Golgi apparatus in rat primary and secondary spermatocytes during meiosis.

We examined the disassembly and reformation of the Golgi apparatus as a function of the cycle of the seminiferous epithelium in adult rats during stages XIII and XIV, i.e., just prior to and during meiosis I and II. Serial section analysis of primary spermatocytes at metaphase I demonstrated the presence of two Golgi complexes. At the ultrastructural level, these Golgi complexes were shown to be composed of stacks of cisternae and vesicles, with each stack having a varying number of saccules. Although Golgi complex intermediates resulting from the process of organelle disassembly were not clearly identified in diplotene spermatocytes immediately prior to nuclear envelope vesiculation, we did observe clusters of vesicles resembling the "nuage," with each cluster varying in size and number of vesicles. Meiosis I results in the formation of secondary spermatocytes that exhibit a well-formed spherical Golgi complex approximately half the size of the diplotene spermatocyte Golgi. Next, secondary spermatocytes enter meiosis II. In contrast to metaphase I, during metaphase II reformation of the Golgi apparatus into stacks was not observed and only small clusters of vesicles at two poles of dividing cells were detected. In addition, "nuage"-like structures were not identified during meiosis II. Our results begin to characterize the process by which Golgi apparatus partitioning is accomplished during meiosis, presumably resulting in the delivery of equal complements of this organelle to four round spermatids. We suggest that partitioning of the Golgi apparatus takes place prior to metaphase I and that the two steps of meiosis may exhibit subtle differences with respect to Golgi partitioning.

Animals↗

PubMed1992

Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex.

Previous studies demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) regulates steroid biosynthesis. In this study we investigate further PBR action by examining its subcellular localization in mouse adrenal gland using anti-peptide PBR antiserum and employing biotin-streptavidin peroxidase immunocytochemistry. Results demonstrated PBR immunostaining exclusively in the cortex. Within this region, however, PBR staining was homogeneously distributed in cells of the zona glomerulosa, whereas in cells of the zona fasciculata both cytoplasmic and prominent plasma membrane immunostaining was evident. Next, PBR distribution was examined using confocal microscopy. Confocal optical sections were obtained, 3-D reconstructions of these sections generated, and vertical, z-sections of the 3-D reconstruction recreated. The immunostaining pattern observed was consistent with a cell surface distribution of PBR. The demonstration of a subset of PBR at the plasma membrane may account for actions of PBR ligands not related to mitochondrial function.

Adrenal Cortex↗

PubMed1992

Characterization of epidermal growth factor in mouse testis.

Considerable evidence exists to suggest that epidermal growth factor (EGF) influences spermatogenesis directly. The tissue source of this EGF, however, is not yet clear. In this study we examine whether the testis itself can serve as a source of EGF. Gel filtration fractions of acid extracted testes exhibited the ability to displace 125I-EGF from testis membranes. The testicular fractions containing the 125I-EGF displacement activity coeluted within the same range as those of submandibular gland (SG) fractions containing mature EGF and prepared in an identical fashion. Next, we employed specific antisera probes to investigate first, whether the testis synthesizes this EGF displacement activity and second, to determine the cell distribution of the testicular EGF. Two types of antisera probes were employed: 1) commercially available antisera to mature EGF (EGFm), i.e. the 6,000 M(r) peptide, and 2) polypeptide specific antisera to the C-terminus of the EGF precursor (EGFp), i.e. the 140,000 M(r) integral membrane molecule which exhibits seven EGF-like repeats in addition to the EGFm. Metabolic labeling of testis with 35S-methionine was performed, followed by immunoprecipitation with the anti-EGFm antisera. Parallel studies using kidney and SG were used as positive controls. Fluorograms exhibited a prominent band at M(r) 140,000 for testis and kidney, corresponding to the EGFp. There was, in addition, a M(r) 50,000 band present for the testis. In SG, a band at M(r) 6,000, corresponding to EGFm, in addition to bands at M(r) 21,000 and 46,000 were observed also. Immunoblotting of testis, kidney, and SG membrane preparations with the specific antisera to either the EGFm or EGFp also resulted in identifying the EGFp at M(r) 140,000, as well as other lower mol wt bands. Preadsorption of anti-EGFm antisera with excess EGFm eliminated all of the specific bands that were immunoblotted. Peroxidase immunocytochemistry of testis, kidney, and SG was also performed using the specific antisera to either EGFm or EGFp. EGFp and EGFm staining in SG and kidney was identical to previously published results in which the distribution of EGFm in these tissues was established. In testis, EGFm immunostaining showed positive results in Sertoli cells, pachytene spermatocytes and round spermatids. In contrast, EGFp immunostaining was limited to pachytene spermatocytes and round spermatids. These results suggest that the testis must now be included in the list of tissues capable of synthesizing EGFp. Specifically, EGFp synthesis appears limited to the post meiotic germ cells.

Animals↗

PubMed1992

Characterization of epidermal growth factor receptor in testis, epididymis and vas deferens of non-human primates.

The presence of the epidermal growth factor receptor (EGFR) in testis, epididymis and vas deferens of monkeys was demonstrated using a polyclonal antibody (RK2) raised against a peptide-specific sequence of the intracellular domain of the human EGFR. Immunoblotting of membrane preparations revealed a specific band at approximately 170 kDa corresponding to those of controls, A431 and monkey liver cells. Cryostat sections were stained by biotin-streptavidin peroxidase immunocytochemistry. The liver showed positive staining along the basolateral membranes of the hepatocytes lining the sinusoids. The testis showed positive staining indicating the presence of EGFR in Leydig cells, Sertoli cells and peritubular cells. In the epididymis, immunostaining of the EGFR was observed on both the basolateral and the luminal borders of the epididymal epithelium. Immunofluorescence studies revealed a similar pattern of EGFR distribution in the epididymis and indicated that the luminal immunostaining was vesicular. In the vas deferens, positive immunostaining was detected in a pattern very similar to that observed in the epididymis. There was no positive staining in the interstitium of the epididymis or in the smooth muscle cell layers of the vas deferens. The sections of all tissues treated with pre-immune serum were negative. These results suggest that EGF in the primate testis may act at the level of somatic cells. In addition, the basolateral and luminal EGFR staining in the epididymis and vas deferens suggest that these cells respond to an EGF, or EGF-like, source both at the basal, luminal or at both sides of the cells, or that these tissues serve as sites of EGF transcytosis across the epithelium.

Animals↗

PubMed1991

The Golgi apparatus of rat pachytene spermatocytes during spermatogenesis.

A morphological and immunocytochemical study of the Golgi apparatus in pachytene spermatocytes was performed in an effort to correlate the structure and function of this organelle during meiotic prophase. In stages I-III of the cycle, the Golgi complex of pachytene spermatocytes is a flattened discoid, 0.5-1 microns in diameter, composed of vesicles interspersed with classically described Golgi cisternae. During subsequent maturation of pachytene spermatocytes (stages IV-XIII), the size of the Golgi complex increases significantly, attaining a size of 2-3 microns. However, unlike pachytene spermatocytes of stages I-III, the majority of the Golgi complex of more mature spermatocytes is characterized by an abundance of distinct stacks of cisternae interspersed with numerous vesicles and tubules. The composition of the Golgi complex was also studied by using two monoclonal antibodies that recognize either the cis or the trans Golgi cisternae, respectively, and employing biotin-streptavidin-peroxidase immunocytochemistry in 5 micron frozen sections of testes. Immunodetection of the distinct cisternae revealed that the increase in size of the Golgi complex during maturation of pachytene spermatocytes was due predominantly to an accumulation of trans Golgi; the amount of cis Golgi remained unchanged. The morphological data presented in this study are consistent with an heightened secretory activity of pachytene spermatocytes during their maturation. In addition, the increase in size of the Golgi apparatus during the extensive prophase of pachytene spermatocytes may suggest that the mechanism employed by germ cells to partition the Golgi complex during the first division of meiosis varies significantly from that of somatic cells undergoing mitosis.

Animals↗

PubMed1989

Epidermal growth factor receptor localization in the rat and monkey testes.

Epidermal growth factors receptor (EGFR) was localized immunocytochemically in the testes of mature and immature rats and immature monkeys. One polyclonal antibody, recognizing the intracellular domain (RK2) of the receptor, was used to carry out the EGFR immunodetection. The RK2 antibody revealed the presence of the EGFR predominantly in Sertoli cells of mature and immature rats and of immature monkeys, although limited interstitial localization of the EGFR was also discerned in the mature rat. In cultured Sertoli cells of immature rats, grown in the absence of epidermal growth factor (EGF), the EGFR was randomly distributed at the cell surface, whereas after the addition of EGF the receptor became aggregated into distinct focal regions. In addition, EGFR of cultured Sertoli cells exhibited autophosphorylation activity upon stimulation with EGF, but failed to transcytose iodinated EGF across a permeability barrier formed by the cultured cells. Instead, all of the added iodinated EGF was internalized and degraded.

Animals↗

PubMed1986

Receptor-mediated endocytosis of GnRH analogs: differential processing of gold-labeled agonist and antagonist derivatives.

The hypothalamic decapeptide, GnRH, stimulates LH and FSH release from pituitary gonadotrophs. Many synthetic peptide analogs, both agonist (GnRH-A) and antagonist (GnRH-AT), have been developed which bind specifically to the GnRH receptor. We have utilized highly potent GnRH-A and GnRH-AT analogs labeled with 18 nm colloidal gold to analyze ultrastructurally the events of binding and interiorization of these specific ligands by gonadotrophs in vitro. To examine internalization of GnRH-A-gold, gonadotrophs were cooled to 4 degrees C and equilibrated with the ligand for 1 h. Next, the cells were either fixed immediately or warmed to 37 degrees C for various times (5, 15 and 30 min) and prepared for electron microscopy. For GnRH-AT-gold, which binds slowly at 4 degrees C, the ligand was incubated with gonadotrophs at 37 degrees C for 15, 30 and 60 min, and the cells were processed for electron microscopy at each time point. In both cases, control gonadotrophs were also incubated in an excess of GnRH-A and GnRH-AT, respectively, in the presence of the gold-conjugated ligands. The results indicated that GnRH-A-gold was bound and rapidly internalized via a receptor-mediated endocytic pathway. GnRH-AT-gold was also bound but showed only limited entry into gonadotrophs; the percentage of intracellular GnRH-AT-gold in gonadotrophs was the same as in other pituitary cells contaminating the gonadotroph fraction and did not increase with time. In the gonadotroph, binding of the specific antagonist ligand to GnRH receptors does not stimulate its interiorization, in contrast to the rapid endocytosis and processing of the agonist ligand. These data suggest that specific ligand internalization requires prior receptor activation, and that GnRH-AT which does not activate the receptor remains bound at the cell surface for a prolonged period.

Animals↗

PubMed1986

Pituitary binding and internalization of radioiodinated gonadotropin-releasing hormone agonist and antagonist ligands in vitro and in vivo.

In rat pituitary gonadotrophs, the rates of binding and endocytosis of two GnRH superagonist analogs, [D-Ala6,Pro9-NEt]GnRH and [D-Lys6,Pro9-NEt]GnRH, were compared with those of the potent antagonist analog [N-acetyl-D-pCl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH by quantitative electron microscopic autoradiography. In dispersed pituitary cells, the two agonist analogs showed similar binding kinetics and comparable degrees of sequestration, as measured by their resistance to dissociation by low pH buffer. However, quantification of silver grain localization suggested that cellular internalization of the [D-Ala6]GnRH agonist increased more rapidly than that of the [D-Lys6]GnRH analog. These discrepancies, and the finding that a larger amount of the specifically bound 125I-[D-Ala6]GnRH agonist was removed during glutaraldehyde fixation, indicated that the proportional internalization of this analog was over estimated by quantitative autoradiography owing to loss of cell surface-bound radioligand. We, therefore, employed radioiodinated D-Lys6-substituted analogs to analyze the receptor binding and cellular uptake of GnRH agonist and antagonist derivatives in vivo. After iv injection, a high proportion of the 125I-[D-Lys6]GnRH agonist was translocated into pituitary gonadotrophs within 60 min, whereas the D-Lys6 antagonist was predominantly associated with the plasma membrane during that time. Four hours after injection of the antagonist, an appreciable proportion of silver grains was associated with intracellular organelles, and this trend increased progressively at later time points. The relatively prolonged cellular processing of the GnRH antagonist is consistent with in vivo binding kinetics, and its slower internalization may reflect the basal rate of GnRH receptor turnover in the cell membrane. In addition, the marked difference between the rates of internalization of the bound [D-Lys6]GnRH agonist and antagonist ligands supports the proposal that receptor activation is responsible for the rapid endocytosis of agonist ligands by the GnRH-stimulated pituitary gonadotroph.

Animals↗