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M K Skinner

Publications and source records attributed to M K Skinner.

At least 91 records · Page 5Linked to original sources
PubMed1988

Rat thecal/interstitial cells produce a mitogenic activity that promotes the growth of granulosa cells.

To test the hypothesis that the cells outside the basal lamina of the follicle secrete paracrine factors that influence the cells on the inside of the follicle, two ovarian cell populations were obtained from diethylstilbestrol-treated rats. Granulosa cells were obtained by extrusion from the follicles and an ovarian cell preparation, termed thecal/interstitial, was derived from the granulosa cell-depleted ovaries. Light microscopy showed that each cell population in culture had distinctive morphologies. Electrophoretic examination of the radiolabeled proteins secreted by the two ovarian cell preparations revealed that each secreted unique protein components into the culture medium. Rat thecal/interstitial cell-conditioned medium promoted [3H]thymidine incorporation into normal rat kidney cell line (NRK) DNA and into bovine granulosa cell DNA. The growth-promoting activity was stable to heating at 70 degrees C for 5 min whereas native fibroblast growth factor (FGF) lost its activity, showing that the factor was not characteristic of FGF. To further characterize the growth-promoting activity thecal/interstitial cell-conditioned medium was concentrated and the proteins separated by size exclusion high performance liquid chromatography. The growth-promoting activity eluted with an apparent molecular weight between 15,000 and 25,000. The finding that thecal/interstitial cells in culture secrete growth-promoting factors suggests that those cells that are in close proximity to the granulosa cells may secrete protein factors that diffuse into the follicular antrum and influence granulosa cell proliferation.

Animals↗
PubMed1988

Developmental and hormonal regulation of bovine granulosa cell function in the preovulatory follicle.

Bovine granulosa cells were isolated from small antral, medium antral, and large Graffian follicles (i.e. small, medium, and large preovulatory follicles). Serum-free cultures of granulosa cells were established and found to be viable for 3-6 days of cell culture. Radiolabeled granulosa cell-secreted proteins were obtained and analyzed electrophoretically. No major changes were detected in the protein profiles of small, medium, and large follicle granulosa cells. FSH and insulin, however, had a dramatic effect on granulosa cell-secreted proteins and increased the apparent production of 200K, 65K, 25K, and 15K proteins. The effects of these hormones on the radiolabeled secreted proteins were similar for small, medium, and large follicle granulosa cells. Aromatase activity was high for the first day of serum-free granulosa cell culture and subsequently declined to low levels. Both FSH and insulin alone stimulated aromatase activity, while a combination of hormones resulted in an additive response similar to the stimulation observed with 10% calf serum. Although the level of aromatase activity increased slightly with the size of the follicle, the effects of hormones were independent of follicle size. Progesterone production was low on days 1 and 2 of serum-free granulosa cell culture and high on days 3 and 6 of cell culture. Interestingly, FSH and insulin suppressed progesterone production on day 1 of cell culture for small and medium follicle granulosa cells, but not for large follicle cells. In contrast, hormones stimulated progesterone production on days 3 and 6 of granulosa cell culture, and the level of progesterone production increased with the size of the follicle. The stimulatory effects of hormones on days 3 and 6 of the culture were similar for medium and large follicle granulosa cells, but were altered for small follicle cells. Results indicate that when aromatase activity is high and stimulated by hormones, progesterone levels are low and generally suppressed by the same regulatory agents. Conversely when progesterone levels are high and hormone responsive, aromatase activity is low. The inverse relationship between aromatase activity and progesterone production implies that bovine granulosa cells alter their differentiated state in culture from an estrogen-producing cell to a progesterone-producing cell. Combined observations indicate that the results obtained on day 1 of culture probably reflect the developmental and hormonal regulation of granulosa cell function in the preovulatory follicle, while data obtained at later times in culture reflect the ability of the cell to synthesize progesterone and develop a luteinization-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Regulation of ovarian cell growth through the local production of transforming growth factor-alpha by theca cells.

The rapid proliferation of a tissue often requires the local production of a specific growth factor. The ovarian follicle is a rapidly growing tissue which contains two primary somatic cell types, granulosa cells and theca cells. Theca cells and granulosa cells were isolated from bovine ovaries and cultured to assess the possible local production of a growth factor within the ovarian follicle. Serum-free conditioned medium from theca cells, but not from granulosa cells, was found to contain a component that specifically bound to the epidermal growth factor (EGF) receptor. Therefore, theca cells appear to produce an EGF-like substance as a potential regulator of follicle cell growth. This result provides physiological significance to the previous observation that granulosa cells contain EGF receptors and respond to EGF to increase cell proliferation. Transforming growth factor-alpha (TGF alpha) is a protein that is structurally and functionally related to EGF and binds to the EGF receptor. Using a molecular probe to TGF alpha, theca cells were found to express the TGF alpha gene, which is consistent with the presence of an EGF-like substance in conditioned medium, but granulosa cells had no detectable TGF alpha gene expression. Similar analysis with a molecular probe to EGF demonstrated the apparent lack of EGF gene expression in theca cells or granulosa cells. As previously demonstrated with granulosa cells, the data presented indicate that theca cells also contain high affinity EGF receptors. TGF alpha was found to stimulate the growth of both granulosa and theca cells. These observations imply that within the ovarian follicle TGF alpha is produced by theca cells, which can subsequently have both a paracrine and an autocrine role in regulating follicle cell proliferation. Results presented demonstrate production of TGF alpha by a normal adult mesenchymal tissue and provide an example of a growth factor-mediated mesenchymal-epithelial cell interaction between theca cells and granulosa cells.

Animals↗
PubMed1987

Rete testis fluid (RTF) proteins: purification and characterization of RTF albumin.

A major 68-kDa protein in ram rete testis fluid (RTF) is shown to be chemically and immunologically indistinguishable from albumin in ovine serum. Data obtained with two-dimensional gel electrophoresis of RTF demonstrate the presence of additional proteins with a molecular mass of 68 kDa that do not react with antisera against sheep serum albumin. Biochemical characteristics of albumin preparations isolated by immunoaffinity chromatography from ovine serum and from RTF were compared. Albumin from both sources had the same apparent molecular mass of 68 kDa, the same isoelectric point of approximately 4.2, and neither bound specifically to Concanavalin A. Analysis of tryptic peptide maps, obtained with reverse-phase high-pressure liquid chromatography, indicated no significant differences between digests of the two purified albumin preparations. Results indicate that RTF albumin and serum albumin are the same protein, which implies that RTF albumin may originate from serum. Albumin levels in RTF, collected from different rams and measured by radioimmunoassay, varied between 46 and 164 micrograms/ml, constituting between 11 and 17% of total RTF protein, while albumin levels in sheep plasma were 40,000 micrograms/ml. The protein composition of RTF is discussed in relation to the relative amounts of various components contributed by testis cells and the amounts derived from serum.

Albumins↗
PubMed1987

Cell-cell interactions in the testis.

In conclusion, the information available indicates that the interactions between different cell types in the testis play an important role in the control and maintenance of testicular functions. Further characterization of these interactions will clearly provide insight into the cell biology of the testis and into the regulation of cellular differentiation, function, and growth. It is apparent that no testicular cell type is autonomous, but that there is communication and cooperation between all cell types in the testis.

Animals↗
PubMed1987

Ovarian thecal cells produce transforming growth factor-beta which can regulate granulosa cell growth.

Ovarian thecal cells in culture were found to synthesize and secrete transforming growth factor-beta (TGF beta). A component in thecal cell-conditioned medium was immunologically similar to TGF beta, as assessed with a RIA, and inhibited specific binding of TGF beta to its cell surface receptors. Thecal cell-secreted proteins also contained TGF beta biological activity, which was determined by stimulation of soft agar colony formation by AKR-2B indicator cells. Specific TGF beta antibodies precipitated a 25 K protein from radiolabeled thecal cell-secreted protein that comigrated with purified platelet-derived TGF beta. Both bovine thecal cell and rat thecal/interstitial cell preparations produced TGF beta, which required acid treatment to obtain fully active samples. The physiological significance of TGF beta production by thecal cells was addressed through an analysis of the effects of TGF beta on bovine granulosa cell growth. TGF beta inhibited epidermal growth factor stimulation of granulosa cell growth, but alone it had no apparent influence. Observations indicate that ovarian thecal cells produce TGF beta, which can regulate granulosa cell growth and differentiation. Discussion of thecal cell-granulosa cell interactions and the possible functions of TGF beta in the ovary is presented.

Animals↗
PubMed1987

Ovarian thecal/interstitial cells produce an epidermal growth factor-like substance.

An ovarian thecal/interstitial cell preparation was obtained by enzymatic digestion of immature rat ovaries depleted of granulosa cells. A component in thecal/interstitial cell-conditioned medium inhibited specific binding of [125I]iodo-epidermal growth factor ([125I]EGF) to its cell surface receptor, as determined with an EGF radioreceptor assay. Thecal/interstitial cell secreted proteins also stimulated growth of an EGF-dependent cell line. Granulosa cells isolated from the same ovaries did not produce detectable levels of an EGF-like substance, as determined by both EGF RRA and EGF growth assay. Fractionation of thecal/interstitial cell secreted proteins by size exclusion HPLC resulted in a 35K component being detected by both the EGF RRA and the growth assay. An EGF-like substance was also detected when thecal/interstitial cell secreted proteins were fractionated by reverse phase HPLC. Several biochemical properties of the EGF-like substance examined were different from those of authentic mouse EGF. However, the biological activities of the EGF-like substance and mouse EGF were similar in their ability to promote the growth of an EGF-dependent cell line. In addition to the production of an EGF-like substance, the thecal/interstitial cell preparation also produced an EGF inhibitory activity. This thecal/interstitial cell secreted product was fractionated from the EGF-like substance and inhibited the ability of EGF to stimulate cell growth. EGF receptors are present on granulosa cells, and EGF is known to influence both the growth and differentiation of these cells. Observations indicate that ovarian thecal/interstitial cells produce an EGF-like substance that may act as a paracrine factor to regulate granulosa cell growth and differentiation. In addition, an EGF inhibitory activity is produced that also may have a role in regulating follicular cell growth.

Animals↗
PubMed1986

Identification of a non-mitogenic paracrine factor involved in mesenchymal-epithelial cell interactions between testicular peritubular cells and Sertoli cells.

Seminiferous peritubular cells have previously been shown to secrete a protein termed P-Mod-S which modulates the functions of Sertoli cells. The present study provides an initial characterization of P-Mod-S and examines the actions of P-Mod-S on Sertoli cells. Gel filtration chromatography demonstrates that P-Mod-S has an apparent molecular weight of 70 000 that could not be dissociated to a lower molecular weight form. A 40- to 90-fold purification of P-Mod-S was obtained with a predicted half maximal effective concentration for Sertoli cells of less than 10(-9) M. Through an analysis of the actions of P-Mod-S on Sertoli cells it is demonstrated that P-Mod-S stimulates the Sertoli cell to a greater extent than any single hormone or vitamin known to influence the cell. P-Mod-S maximally stimulates testicular transferrin and androgen-binding protein production by Sertoli cells, but does not stimulate levels of plasminogen activator activity. P-Mod-S also appears to induce the synthesis of several proteins that are not detected in control non-treated Sertoli cell cultures. One such protein whose synthesis was stimulated by P-Mod-S treatment of Sertoli cells was a component having a molecular mass of 20 kDa. This 20 kDa Sertoli cell-secreted protein was specifically immunoprecipitated with an antibody against an epididymal lactalbumin-like protein. This implies that P-Mod-S can induce Sertoli cells to synthesize and secrete a lactalbumin-like protein. P-Mod-S was found not to contain mitogenic activity. Data presented indicate that testicular peritubular cells synthesize and secrete a 70 kDa non mitogenic paracrine factor termed P-Mod-S which has a dramatic influence on Sertoli cell functions. Results are discussed with respect to modulation of epithelial (Sertoli) cell functions by components produced by mesenchymal (peritubular) cells.

Androgen-Binding Protein↗
PubMed1986

Cytodifferentiation of granulosa cells induced by gonadotropin-releasing hormone promotes fibronectin secretion.

An analysis of the hormonal regulation of fibronectin production by granulosa cells showed that GnRH stimulated fibronectin secretion. GnRH increased fibronectin production 2.5- to 5-fold over that of control untreated cultures and greater than 20-fold over that of FSH-treated cultures. The GnRH concentration required for a minimal response was 10(-10) M, and that required for a maximal response was 10(-8) M. In contrast to the effects of GnRH, FSH suppressed fibronectin production to low or undetectable levels. In addition, FSH abolished the actions of GnRH on fibronectin production. Treatment of cultured granulosa cells with either (Bu)2cAMP or methylisobutylxanthine to influence cellular cAMP levels mimicked the actions of FSH on fibronectin secretion. Similar to FSH, both (Bu)2cAMP and methylisobutylxanthine abolished the actions of GnRH on fibronectin production. These results indicated that an increase in cAMP levels resulted in suppression of fibronectin secretion by granulosa cells and inhibition of the actions of GnRH. Insulin treatment of granulosa cells also suppressed fibronectin secretion, but to a lesser extent than FSH. In addition, insulin inhibited the actions of GnRH on fibronectin production by approximately 40-50% of stimulated levels. GnRH promoted a state of cytodifferentiation of the granulosa cell which had a high level of fibronectin production and, as shown previously, a low level of steroidogenesis. In contrast, FSH promoted a state of cytodifferentiation which had a low level of fibronectin production and a high level of steroidogenesis Both fibronectin and steroidogenic enzymes (e.g. aromatase) provide useful markers for an analysis of the cytodifferentiation of granulosa cells between these two distinct differentiated states. Results are discussed in relation to the possible mechanisms controlling granulosa cell cytodifferentiation and the possible functions of fibronectin in the ovary.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells.

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.

Animals↗
PubMed1985

Androgen stimulation of Sertoli cell function is enhanced by peritubular cells.

Rates of production of androgen binding protein (ABP) declined during culture of purified Sertoli cell-enriched aggregates in serum-free minimal essential medium (MEM). This decline was partially prevented by the addition of testosterone to the medium, but not by 17 beta-estradiol addition. The androgen effect was greater in Sertoli cell aggregates in co-culture with peritubular cells than in Sertoli cell preparations depleted of peritubular cells. Initial rates of ABP production by purified Sertoli cell-enriched aggregates were also sustained when preparations were cultured in MEM supplemented with conditioned medium in which peritubular cells had been previously maintained. We interpret data presented to indicate that the stimulatory effects of peritubular cells on functions of Sertoli cells in co-culture, specifically sustained rates of ABP production, are mediated at least partially by component(s) synthesized by peritubular cells and secreted into the medium. We offer the hypothesis that androgens may influence Sertoli cells at two levels: (1) a direct action on Sertoli cells; (2) and indirect influence on Sertoli cells mediated by a direct action on peritubular cells, resulting in the formation of product(s) which modulate the rates of ABP synthesis.

Androgen-Binding Protein↗
PubMed1985

Testicular peritubular cells secrete a protein under androgen control that modulates Sertoli cell functions.

Peritubular cells of the seminiferous tubule synthesize component(s) that stimulate Sertoli cells in culture to increase the production of androgen-binding protein and testicular transferrin. The active peritubular cell component(s) are trypsin-sensitive, heat-sensitive, acid-stable molecule(s) having a molecular weight between 50,000 and 100,000. These specific factors(s) are referred to as P Mod-S to designate protein(s), produced by peritubular cells (P), that modulate the functions of Sertoli cells (S). The degree of stimulation by P Mod-S is comparable to that obtained by maximal hormonal stimulation of the synthesis of ABP and transferrin by Sertoli cells. Levels of P Mod-S secreted into the medium by primary cultures of peritubular cells are increased in the presence of testosterone. Comparable concentrations of 17 beta-estradiol do not stimulate peritubular cells to synthesize P Mod-S. Data are interpreted to indicate that androgens act on testicular peritubular cells to increase the formation of P Mod-S and that P Mod-S may modulate the properties of adjacent Sertoli cells. Findings are discussed in relation to the nature of mesenchymal-epithelial cell interactions in the seminiferous tubule and to the possible role of P Mod-S as a mediator of androgen actions of Sertoli cells.

Androgen-Binding Protein↗
PubMed1985

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components.

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.

Animals↗
PubMed1985

Fibronectin as a marker of granulosa cell cytodifferentiation.

The hormonal regulation of fibronectin secretion by rat granulosa cells in culture was investigated: fibronectin was measured by a competitive enzyme-linked immunoadsorbant assay. Granulosa cells isolated from 25-day-old diethylstilbestrol-primed rats and cultured under defined conditions in the absence of hormones secreted low levels of fibronectin during the first 24 h of culture, after which there was a rapid increase in secretion until 72 h. In contrast, cultures treated with a combination of NIH-FSH-15 (200 ng/ml) and insulin (5 micrograms/ml) secreted low levels of fibronectin throughout the culture period. Subsequently, it was found that both FSH and insulin could independently suppress the increase in fibronectin secretion found in control cultures. Combined treatment with FSH and insulin resulted in a level of fibronectin which was the same as either FSH or insulin alone. The actions of FSH and insulin were dose dependent; 10 ng FSH/ml and 2.5 micrograms insulin/ml were required to produce a maximum suppression. The ability of (Bu)2cAMP (1.0 mM) to suppress fibronectin secretion suggested that the action of FSH on this parameter was mediated via the production of cAMP. Testosterone and estrogen alone did not influence secretion and did not modulate the actions of FSH and insulin. At the time at which FSH induces the cytodifferentiation of granulosa cells in culture, assessed by the increase in aromatase activity, fibronectin secretion is suppressed. The inverse relationship between fibronectin secretion and the induction of those granulosa cell functions essential for the development of the preovulatory follicle indicates that fibronectin may provide a useful marker for the stage of cytodifferentiation and follicular maturation.

Animals↗
PubMed1984

Purification and characterization of testicular transferrin secreted by rat Sertoli cells.

Sertoli cells synthesize and secrete a transferrin-like protein (testicular transferrin) [Skinner & Griswold (1980) J. Biol. Chem. 255, 1923-1925]. The purpose of the present study was to purify and characterize testicular transferrin and to compare it with serum transferrin. Testicular transferrin was obtained from the medium of cultured rat Sertoli cells, whereas serum transferrin was obtained from rat serum. Both proteins were purified with the use of phenyl-Sepharose hydrophobic chromatography and transferrin immunoaffinity chromatography. The purified proteins were shown to have similar molecular masses (75 000 Da) and amino acid compositions. The pattern of tryptic peptides from testicular and serum transferrin were found to be essentially the same when analysed by reverse-phase high-pressure liquid chromatography. The carbohydrate composition of both transferrins was determined by several colorimetric assays and g.l.c. Testicular transferrin, isolated from cell culture medium, had increased amounts of glucose, galactose and glucosamine. Serum transferrin that was incubated with cell culture medium also had a large amount of associated glucose. The results show that testicular transferrin and serum transferrin are structurally very similar and are possibly products of the same gene expressed in two different tissues, the testis and liver. However, the amount of carbohydrate associated with these two proteins is different.

Amino Acids↗
PubMed1984

Fibronectin synthesis is a marker for peritubular cell contaminants in Sertoli cell-enriched cultures.

With indirect immunofluorescent microscopic techniques, we have shown that fibronectin is distributed primarily in or along the basal lamina of the seminiferous tubule boundary tissue in sections of testes from 20-day-old rats. Purified rat Sertoli cell-enriched aggregates, maintained in culture in the presence or absence of serum, exhibit no detectable immunofluorescence with fibronectin antibody, whereas purified peritubular cells in culture do have a positive reaction to fibronectin antibody. Peritubular cells in culture incorporate [35S] methionine into fibronectin which can be immunoprecipitated with a fibronectin antiserum, but Sertoli cells do not. We have used various criteria to estimate the degree of purity of Sertoli cell-enriched preparations. The presence of peritubular myoid cells in conventional Sertoli cell-enriched aggregates, cultured in the presence or absence of serum, can be detected with transmission electron microscopic examination, by the Feulgen staining procedure, and by the immunocytochemical identification of fibronectin. We describe a technique to purify Sertoli cells in conventional Sertoli cell-enriched preparations by treatment with hyaluronidase, resulting in a lesser number of peritubular cells by the above criteria, even in preparations cultured in the presence of serum. Data presented suggest that some of the products previously attributed exclusively to Sertoli cells in Sertoli cell-enriched preparations, particularly those cultured in the presence of serum, may have been contributed by peritubular cells.

Animals↗
PubMed1984

A sulfated glycoprotein synthesized by Sertoli cells and by epididymal cells is a component of the sperm membrane.

We report here the purification, partial characterization and immunofluorescent localization of a dimeric acidic glycoprotein (DAG-protein) secreted by cultures of Sertoli cells of rat testes. Partially purified protein was obtained after chromatography over Sepharose 4B under conditions which favored a soluble, nonaggregated form of the protein. Rechromatography over the same column under reducing conditions yielded very pure monomers of 41,000 daltons and 29,000 daltons. Antibodies were prepared against the mixed monomers and used to immunoprecipitate proteins in spent medium from cultures incubated with [35S] methionine, 35SO4 = or tunicamycin. DAG-protein and another protein (Band 4, 70,000 daltons) were coprecipitated by the antiserum and all contained 35SO4 = in their structures. It was shown by Western blotting that the antiserum cross-reacted very weakly with Band 4 protein. The DAG-protein polypeptides secreted in the presence of tunicamycin were assumed to lack N-glycosylation and exhibited apparent molecular weights of 27,000 and 21,000 daltons. Immunoprecipitations of media from organ cultures of testis and epididymis yielded DAG-protein of slightly lower molecular weight than the protein secreted in Sertoli cell cultures. Indirect immunofluorescence of DAG-protein in paraffin sections of testis and epididymis revealed that the protein was concentrated in the cytoplasm of Sertoli cells, on the stereocilia of epididymal principal cells, in the cytoplasm of epididymal halo cells, and was associated with late spermatids and mature sperm. Sperm were specifically labeled on the acrosome, at the neck, and on the endpiece of the tail. No enzymatic or structural function has been ascribed to DAG-protein as yet, but the protein must play a pivotal role in spermatogenesis because it is secreted by both the testis and epididymis and becomes an integral component of sperm.

Animals↗