Testicular Androgen-binding Protein Research Articles

Vitamin E ameliorates testicular histological features and androgen binding protein levels in testicle of rats induced by allethrin.

Objective:This study aims to determine the ameliorative effect of vitamin E (vit E) on histological features and androgen binding protein (ABP) levels in rats induced by allethrin.Materials and Methods:Thirty sexually mature male Wistar rats weighing between 200 and 300 gm, and aging 3 months were taken for this study and were divided into three groups: negative control (NC), positive control (PC), and treatment (T) groups. The PC and T groups were induced by allethrin 12 h per day for 31 days; however, only the T group was given vit E orally at 1 ml/gm body weight (BW) each day for 14 days. The paraffin block method was used to measure tubules’ diameter, thickness of the seminiferous epithelial layer, and Sertoli cell number. The ABP levels were measured by enzyme-linked immunosorbent assay.Results:The results showed that vit E gave significant effect (p < 0.05) on tubular diameter at NC 123.67 ± 12.77, PC 147.16 ± 10.64, and T 130.08 ± 10.00; tubular epithelial thickness at NC 33.55 ± 3.21, PC 30.02 ± 1.53, and T 32.96 ± 2.81; Sertoli cells number at NC 55.48 ± 5.9, PC 43.84 ± 3.77, and T 53.44 ± 4.26; and ABP levels at NC 72.35 ± 39.06, PC 38, 48 ± 18.78, and T 86.10 ± 35.77, respectively.Conclusion:This study concludes that vit E has an ameliorative effect against the toxic effects of allethrin at testicular histological features and ABP levels.

Testosterone Retention Mechanism in Sertoli Cells: A Biochemical Perspective

Mechanism(s) involved in regulating Intratesticular Testosterone levels (iT) have assumed importance in recent years, from the point of view of hormonal contraception. Contraceptives using Testosterone (T) in combination with Progestins (P), for more effective suppression of pituitary gonadotropins thereby iT, are not 100% effective in suppressing spermatogenesis in human males, likely due to pesrsistence of Intratesticular Dihydrotestosterone (iD) in poor-responders. Several lacunae pertaining to the mechanism of action of principal male hormone T during spermatogenesis remain to be resolved. Notably, the mechanism through which T brings about the stage-specific differentiation of germ cells lacking Androgen Receptors (AR). Testosterone is a highly anabolic steroid with a rapid tissue clearance rate. T is intratesticular substrate for synthesis of Dihydrotestosterone (DHT) and Estradiol (E2) involved in spermtaogenesis. Therefore, it is important to delineate the mechanism(s) for retention of iT, in order to understand regulation of its bioavailability in testis. In depth studies, pertaining to the role of androgen-binding protein(s) in sequestration, retention and bioavailability of T/DHT are required to understand male fertility regulation. The appropriate approach to overcome this lacuna would be development of mice lacking functional testicular Androgen-Binding Protein (ABPKO), but not deficient T/DHT, Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH), in order to understand its physiological functions. Insights gained about androgen retention mechanism(s) from the ABPKO murine model will be of immense help in improving the efficacy of male hormonal contraceptives and infertility management.

Effects of phosphodiesterase-5 inhibitor vardenafil on testicular androgen-binding protein secretion, the maintenance of foci of advanced spermatogenesis and the sperm fertilising capacity in azoospermic men

We evaluated the effects of vardenafil on testicular androgen-binding protein secretion (ABP). Bilaterally obstructed azoospermic (OA)-men (n = 19) (group A) underwent unilateral testicular biopsy. A group of nonobstructed azoospermic (NOA)-men (n = 68) (group B) underwent bilateral testicular biopsy. ABP secretion in vitro by testicular tissue was assessed in each participant of every group. In addition, intracytoplasmic sperm injection (ICSI) cycles were performed in several couples of group A or group B using frozen/thawed spermatozoa from the biopsy material. Ten OA-men (group A1), 14 NOA-men (group B1), and nine different NOA-men (group B2) had been positive for spermatozoa in the biopsy but pregnancies were not achieved in the respective female partners. Men of groups A1, B1 and B2 were treated with vardenafil, vardenafil and L-carnitine respectively. Then, the men of groups A1, B1 and B2 underwent a second testicular (unilateral) biopsy. Within the group A1 and within the group B1, ABP secretion rate was significantly larger after vardenafil treatment than prior to vardenafil treatment. In addition, fertilisation rates in ICSI cycles within groups A1 or B1 were not affected by vardenafil administration. Vardenafil administration in NOA-men increased ABP secretion and did not affect detrimentally the presence of testicular foci of advanced spermatogenesis.

Repression of the Human Sex Hormone-binding Globulin Gene in Sertoli Cells by Upstream Stimulatory Transcription Factors

Expression of the sex hormone-binding globulin gene (SHBG) in the liver produces SHBG, which transports sex steroids in the blood. In rodents, the SHBG gene is also expressed in Sertoli cells giving rise to the testicular androgen-binding protein, which is secreted into the seminiferous tubule where it presumably controls testosterone action. Evidence that the SHBG gene functions in this way in the human testis is lacking, and mice containing a human SHBG transgene (shbg4) under the control of its own promoter sequence are characterized by SHBG gene expression in the liver but not in the testis. A potential cis-element, defined as footprint 4 (FP4) within the human SHBG promoter, is absent in SHBG promoters of mammals that produce the testicular androgen-binding protein, and we have produced mice harboring a shbg4 transgene in which FP4 was deleted to evaluate its functional significance. Remarkably, these mice express the modified human SHBG transgene in the testis as well as the liver. Human SHBG transcripts were found within their Sertoli cells, primary cultures of which secrete human SHBG, and this was increased by treatment with follicle-stimulating hormone, retinoic acid, and estradiol but not testosterone. We have also found that the upstream stimulatory factors (USF-1 and USF-2) bind FP4 in vitro by electromobility shift assay of Sertoli cell nuclear extracts and in vivo by chromatin immunoprecipitation assay and conclude that USF transcription factors repress human SHBG transcription in Sertoli cells through an interaction with FP4 within its proximal promoter.

Effects of sexual intercourse on testicular function

Objective: We evaluated the effects of sexual intercourse on testicular function in fertile and infertile rats. Design: Markers of Leydig and Sertoli cell secretory functions were evaluated prior to and during sexual intercourse in fertile and infertile rats. Materials/Methods: Each one of four-month-old-Wistar male rats of groups A1 (n=10; fertile rats), A2 (n=10; fertile rats), B1 (n=10; bilaterally cryptorchid infertile rats), and B2 (n=10; bilaterally cryptorchid infertile rats) was placed at the same cage with 2 female rats in oestrus. Immediately after the entrance of the male rat into the cage all the males of groups A1 and B1 were killed. Intratesticular testosterone profiles (ITPs), testicular transferrin profiles and androgen binding protein profiles (ABPPs) in testicular cytosols were evaluated. Rats of groups A2 and B2 were allowed to have a sexual intercourse. Immediately after the first ejaculation (first postejaculatory interval) rats of groups A2 and B2 were killed and ITPs, testicular transferrin levels, and ABPPs were evaluated. Prior to sacrificing each rat of groups A1, A2, B1 and B2 blood was collected from the tail vein of this male rat and the serum levels of FSH, LH, testosterone and prolactin were evaluated. Detected spermatozoa in the vaginae of the females confirmed the performance of complete intercourses. Ten and 12 additional four-month-old fertile male Wistar rats (group C and group D, respectively)were allowed to have sexual intercourse with female rats in oestus (one male was placed in the same cage with two females). However, at the time of intercourse the males of group C were disturbed by visual and audial stimuli. ITPs and ABPPs were evaluated immediately after the first ejaculation in all rats of group C. Rats of group D were killed during intromissions prior to ejaculation. ITPs and ABPPs were evaluated in each rat of group D. Results: ITPs were significantly larger (p <0.05; Wilcoxon’s test) in the group A2 (136 ± 13 ng/g testis) than in group A1 (114 ± 10 ng/g testis) and in the group B2 (91 ± 11 ng/g testis) than in group B1 (73 ± 7 ng/g testis). In contrast there were no significant differences in serum FSH, LH, testosterone and prolactin levels and in testicular ABPPs and transferrin concentrations between groups A1 and A2, and between groups B1 and B2. ITPs and testicular ABPPs were significantly smaller in groups C and D than in group A2. The latter parameters were significantly larger in groups C and D than in group A1. Conclusions: Sexual intercourse results in an increase in the ITPs in both fertile and infertile subjects. In contrast sexual intercourse does not affect Sertoli cell secretory function. This increase in ITPs appears not to be due to an enhanced pituitary gonadotropin secretion and is partially inhibited when the sexual intercourse is interrupted or the male is disturbed during sexual intercourse. Whether multiple sexual intercourses in infertile subjects increasing ITPs have a positive influence in spermatogenesis needs to be evaluated. Supported by: Research Grant from MFC Clinic, Yonago, Japan.

Dynamics of testicular germ cell proliferation in normal mice and transgenic mice overexpressing rat androgen-binding protein: a flow cytometric evaluation.

Transgenic mice carrying rat androgen-binding protein (ABP) genomic DNA express high amounts of testicular ABP and develop a progressive impairment of spermatogenesis. To understand the mechanism of these changes, we have studied the pattern of testicular germ cell proliferation from 7 to 360 days of age in wild-type (WT) control and transgenic homozygous (ABP-TG) mice by flow cytometry after labeling DNA in isolated germ cells with propidium iodide. At all ages studied, the body weight of the ABP-TG mice was lower than that of age-matched WT controls. Significantly reduced testicular weight and total germ cell number in the ABP-TG mice were evident from Day 30 and Day 60, respectively. Flow cytometric analysis of isolated germ cells revealed that the number of germ cells undergoing proliferation (S-phase cells) was identical in WT control and ABP-TG mice up to Day 14. Subsequently, the number of germ cells in S-phase was consistently higher in ABP-TG than in WT mice. The number of primary spermatocytes was significantly increased starting from Day 60, and the numbers of round and elongated spermatids were significantly reduced in the ABP-TG animals from Day 21 and Day 60 onwards, respectively. Immunocytometry for intracellular ABP at 90 days of age revealed that the percentage of ABP-containing germ cells was greater in ABP-TG than in WT mice. The continuous presence of ABP in mouse seminiferous tubules at greater than physiological concentrations facilitates the formation of primary spermatocytes but impairs subsequent transformation to round and elongated spermatids. Based on our observations and the analysis of the available literature, the most likely mechanism for production of these effects is sustained reduction in the bioavailability of androgens.

Regulatory influence of germ cells on sertoli cell function in the pre-pubertal rat after acute irradiation of the testis.

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.

Impact of Neonatal Onset Hypothyroidism on Sertoli Cell Number, Plasma and Testicular Interstitial Fluid Androgen Binding Protein Concentration

The impact of neonatal onset hypothyroidism from day 1 postpartum through different postnatal developmental events on rat testis was studied in vivo. Hypothyroidism was induced in neonates by feeding the lactating mother or directly with 0.05% methimazole (MMI) through drinking water from the day of birth and were killed at day 10, 15, 30, 40 and 60 postpartum. Hypothyroidism was confirmed by radioimmunoassay of thyroid hormones and TSH. Sertoli cell number, plasma and testicular interstitial fluid (TIF) androgen binding protein (ABP) concentration was quantified. Sertoli cell number was consistently decreased in all hypothyroid rats. Plasma ABP was also decreased irrespective of the duration of hypothyroidism. Unlike plasma ABP, TIF ABP concentration in hypothyroid rats increased at day 10, and 15 postpartum and decreased in other age groups. Plasma FSH level was increased significantly in all hypothyroid groups. The present investigation points out that suppression of T3 during the critical period of Sertoli cell proliferation affects their number and functional activity.

Duration-Dependent Effect of Transient Neonatal Hypothyroidism on Sertoli and Germ Cell Number, and Plasma and Testicular Interstitial Fluid Androgen Binding Protein Concentration

The impact of transient neonatal hypothyroidism on growth and function of puberal testis during different milestones of postnatal testicular development was studied in Wistar rats. Rat pups were made hypothyroid for 10, 15, 30, 40 and 60 days of postnatal age from birth by providing 0.05% (W/V) methimazole (MMI) in the drinking water of the mother, from day 1 postpartum till weaning (25 days postpartmn) and thereafter in the drinking water. Control rats were raised without MMI treatment. Sertoli cell number and its function was assessed on day 60 postpartum. Sertoli cell number increased consistently in 10, 15, 30 and 40 days transient hypothyroid rats but decreased in rats subjected to continuous hypothyroidism from birth to 60 days postpartum. Rats subjected to continuous hypothyroidism from birth showed spermatogenic arrest at puberty and had only a single layer of spermatogonia. Transient neonatal hypothyroidism for 10 (or) 15 days from birth increased spermatocytes (pachytene and zygotene), spermatids (elongated and round) whereas, that of 30 and 40 days decreases the number of germ cells. Plasma androgen binding protein (ABP) concentration decreased in puberal rats belonging to all groups, whereas the testicular interstitial fluid (TIF) concentration of ABP increased significantly in 10 and 15 days hypothyroid rats while it decreased in all other groups. These findings indicate that the mitogenic activity of Sertoli cell is increased irrespective of the duration of transient neonatal hypothyroidism. However, the functional activity of Sertoli cells (ABP production) in these puberal rats varies depending upon the postnatal penod at which the animals were in hypothyroid state.

Establishment of a Mouse Sertoli Cell Line Producing Rat Androgen-binding Protein (ABP)

The ultimate goal of this study was to compare the fate of rat testicular germ cells cocultured with mouse Sertoli cells that either do or do not produce rat androgen-binding protein (ABP). As a first step, we stably transfected a rat ABP expression construct into an immortalized mouse Sertoli cell line (TM4), which does not produce ABP when growing on plastic without hormones. The transfection of the pRc/CMV- rat ABP cDNA expression vector containing a neomycin resistance gene was made by either the liposome method (Dotap) or by polyethyleneimine transfection (PEI) into TM4 cell cultures. Neomycin-resistant clones were selected by adding Geneticin to the culture medium for 3 weeks. Analysis of over 25 clones revealed the presence of recombinant rat ABP when cell extracts and culture media were probed with a rabbit polyclonal antibody raised against rat testicular ABP, indicating the translation and secretion of a protein similar to rat testicular ABP. Transfected TM4 cells maintain the secretion of rat ABP for more than 40 days, with immunopositive rat ABP localized within cytoplasmic granules in the Golgi region and along cytoplasmic processes in TM4 transfected with either vector. Electron microscopic study revealed a higher development of cytoplasmic organelles involved in protein secretion.

New and traditional approaches for the assessment of testicular toxicity

In this study, the suitability of several methods for the assessment of testicular damage, including histopathology, flow cytometry (FCM), testicular sperm head counts, and secretion of androgen binding protein (ABP), has been evaluated. Testicular toxicity after acute exposure of adult rats to different doses of the known toxicant 1,3-dinitrobenzene (DNB) was analyzed. The effects showed dose dependence, in spite of the large variability within each dose group. Histopathology and FCM showed germ cell depletion, particularly of round spermatids; testicular sperm head counts were reduced and ABP production was increased. All evaluated methods showed similar sensitivities. The increased testicular ABP levels support the theory that the Sertoli cell is the likely target of DNB induced testicular toxicity, producing subsequent germ cell depletion. The presented results show the suitability of FCM for the analysis of testicular damage and also support the usefulness of including a metabolic marker for Sertoli cell function.

Characterization and developmental expression patterns of testicular androgen-binding protein in the Djungarian hamster (Phodopus sungorus).

Studies were conducted to characterize Djungarian hamster androgen-binding protein and examine its expression during development. The cDNA encoding the full length testicular androgen-binding protein was cloned and, except for two suspected polymorphisms, shared a common primary sequence with hepatic sex hormone-binding globulin. A single androgen-binding protein/sex hormone-binding globulin gene was identified and a 1.7 kb mRNA encoding androgen-binding protein/sex hormone-binding globulin was present in both the testis and liver. Testicular homogenates contained specific 5 alpha-dihydrotestosterone-binding activity that was identified as androgen-binding protein. In the prepubertal testis, immunoreactive androgen-binding protein/sex hormone-binding globulin subunits ranged from 46 kDa to 60 kDa, with the majority of isoforms > 50 kDa. These subunits were distinct from the major 55 kDa and 51 kDa isoforms of sex hormone-binding globulin present in the serum of prepubertal hamsters. Deglycosylation studies demonstrated that size heterogeneities were the result of developmentally specific glycosylation patterns. Expression of androgen-binding protein by the testis was upregulated during puberty and coincided with a decline in serum sex hormone-binding globulin activity. The tissue- and age-dependent expression of specific androgen-binding protein and sex hormone-binding globulin variants suggests that these proteins play different roles in steroid-mediated sexual development.

Expression and distribution of androgen-binding protein/sex hormone-binding globulin in the female rodent reproductive system.

Androgen-binding protein (ABP)/sex hormone-binding globulin gene expression has been described in the rat testicular Sertoli cell and brain. The extracellular protein is thought to regulate the bioavailability of sex steroids, but may have a more complex function as a hormone or growth factor. Transgenic mice were developed with a 5.5-kilobase (kb) rat DNA fragment containing the ABP gene with all 8 exon sequences and 1.5 kb upstream of the transcription start site. Expression of the gene was observed in the testis and brain, but not in other examined tissues of the transgenic mice. In this paper we describe ABP gene expression in ovaries of transgenic mice that contain the rat gene; a lower level of ABP mRNA was also detected in the transgenic uterus. Northern blot analysis also detected ABP mRNA in rat ovary. The hybridizing species in the rat and transgenic mouse ovaries and uteri were the size of testicular ABP mRNA (1.7 kb). Except in the transgenic mouse brain, there was no detectable hybridizing RNA in the other transgenic tissues examined. The plasma, ovary, and uterus of the transgenic mice all contained elevated ABP (dihydrotestosterone [DHT]-binding) activities as compared to those of wild-type littermates; other wild-type and transgenic tissues were negative for DHT binding. Immunohistochemistry revealed increased immunoreactivity in the transgenic oviduct and uterus, but not the ovary. In the oviduct, the intense immunoreactivity was associated with the epithelium, whereas in the uterus it was primarily associated with the luminal epithelium and glands. Phenotypic abnormalities of the homozygous transgenic mice included reduced fecundity resulting in small litters. We conclude that ABP may function in the female reproductive system to increase the local concentrations of sex steroids or to sequester them in key target organs. Studies in the female will aid in elucidating the functions of ABP in male and female reproduction.

Sex hormone-binding globulin: gene organization and structure/function analyses.

Human plasma sex hormone-binding globulin (SHBG) and testicular androgen-binding protein (ABP) are homodimeric glycoproteins with a single steroid-binding site. They share the same primary structure and differ only with respect to the types of oligosaccharides attached to them. Both are products of a single gene (Shbg), the expression of which has been detected in several tissues including liver, testis, placenta, brain and endometrium. The transcript encoding SHBG in hepatocytes and ABP in Sertoli cells is identical. Several other transcripts result from differential exon utilization in sex steroid-responsive tissues, but their biological significance remains obscure. Wild-type and mutant forms of human SHBG have been produced in mammalian cells and Escherichia coli, and have provided insight into the structural and functional properties of SHBG and its related gene products.

Effects of photoperiod on testicular inhibin-α and androgen binding protein mRNA expression during postnatal development in siberian hamsters, [formula omitted

The effects of short photoperiod (SPP) on testicular inhibin-α and androgen binding protein (ABP) mRNA expression were investigated in Siberian hamsters during postnatal development. Hamsters were raised in either long photoperiod (LPP; 16L:8D) or in a SPP (6L:18D). Hamsters were sacrificed at the following developmental ages: preweaning (18–20 days), immature (28–30 days), pubertal (38–40 days), postpubertal (48–50 days) and adult (60–62 days; n = 7–12 animals per group). Body and testicular weights were determined and plasma FSH levels were measured by validated RIA. As expected, testicular weight and circulating FSH levels were significantly lower in hamsters raised in SPP than in LPP animals. Photoperiod had no significant effect on body weight except during postpubertal age. The northern analyses of inhibin-α and ABP expression in the testes revealed that the corresponding relative mRNA levels were higher in animals exposed to SPP than in animals from LPP. These findings suggest that the testes of hamsters exposed to a SPP can synthesize inhibin-α and ABP and we speculate that the release of these proteins may be regulated by photoperiod and the role of FSH in the control of inhibin-α and ABP gene expression in male Siberian hamsters may be minimal.

Effects of Smoking on Testicular Function, Semen Quality and Sperm Fertilizing Capacity

Purpose The effects of smoking on testicular function and sperm physiology were studied. Materials and Methods Left testicular biopsy was performed in 49 smokers and 28 nonsmokers. Seminal specimens from these men were analyzed. Results Testosterone levels in the left testicular vein, left testicular androgen-binding protein secretion rate (in vitro), sperm motility, percentage of morphologically normal spermatozoa, sperm morphometric parameters and outcome of sperm function tests were significantly lower (p less than 0.05) in smokers than in nonsmokers. Conclusions Morphological sperm abnormalities due to secretory dysfunction of the Leydig and Sertoli cells may be the cause of impaired sperm fertilizing capacity in smokers.

The influence of vasectomy and vasovasostomy on testicular ATPases, cAMP, ABP and androgen receptor in rabbits

The present study was performed to further clarify the influences of vasectomy on functions of testis and to disclose the possible mechanisms of infertility after vasovasostomy (VV). Thirty-one rabbits were divided into sham-operated control group (C), vasectomy control group (V), VV fertility group (VaF) and VV infertility group (Val). Serum testosterone (ST) level, testicular cAMP, androgen binding protein (ABP), nuclear androgen receptor (NAR) concentrations, testis cell membrane Na +−, K +− ATPase, Mg 2+− ATPase activities, sperm density and testis weight were measured. Vasectomy resulted in significantly reduced cAMP, Na +−,K +−ATPase, Mg 2+− ATPase, testis weight and increased ABP; VV completely restore testis weight in VaF and Val, Na +−, K +− ATPase, Mg 2+− ATPase in VaF, partly cAMP in VaF and Val, Na +−, K +− ATPase, Mg 2+− ATPase in Val, but did not restore ABP. The NAR content in Val was significantly lower than those in C, VaF and V. No statistical differences among 4 groups were seen in Kd values for [ 3H]-T. ST levels in VaF, Val and V were insignificantly different compared with C, but the value in VaF was higher than that in Val (p < 0.05). Sperm density after VV reached 122 ± 62 × 10 6/ml in VaF and 10 ± 24 × 10 6/ml in Val, both in VaF and Val were, significantly low compared with C (p < 0.001), and the value in Val was remarkedly lower than that in VaF (p < 0.001). It was shown that sperm density was positively correlated with cAMP content, Na +−, K +−, ATPase, Mg 2+− ATPase activities, but negatively with ABP. These results suggest that vasectomy gives rise to damage to the testis, and vasovasostomy does not appear completely effective in reversing testicular changes.

Demonstration of Androgen-Binding Protein Gene Expression in Primary Neuronal and Astrocyte Cultures

Testicular androgen-binding protein (ABP) and liver (plasma) sex hormone-binding globulin (SHBG) are extracellular carrier proteins. They are encoded by the same gene and have the identical primary amino acid sequence. The ABP/SHBG gene is also expressed in rat brain; immunoreactive neurons are located throughout the male and female brain and immunoreactive fibers are present in the ventral hypothalamus. ABP/SHBG RNA transcripts are synthesized from two promoters, P 1 and P A. P 1 regulates synthesis of the mRNA that encodes secreted ABP, whereas, a P A mRNA encodes a nonsecreted ABP-like protein without steroid binding properties. P A and P 1 transcripts are expressed in the brain. In this study, we demonstrated by Northern hybridization analysis that primary neuronal and astrocyte cultures express ABP mRNA. PCR analysis of ABP cDNA revealed that astroglia express transcripts originating from promoters P 1 and P A, whereas, neuronal cDNAs contain primarily PA transcripts. These ABP cDNA-hybridizing products represent mRNAs that encode secreted ABP and the nonsecreted ABP-like protein. Furthermore, it was shown by immunohistochemistry that cultured astrocytes contain immunoreactive ABP. Western blots of astrocyte proteins yielded immunoreactive ABP migrating as 38,000-, 34,000-, and 24,060- M r proteins. The sizes of these proteins do not correspond to the protein-encoding properties of P 1 or P A ABP mRNA or any known alternatively processed ABP mRNAs, suggesting that they are derived by proteolytic processing of ABP or the ABP-like protein (46,000 Da). The function of the ABP gene products in brain is not obvious. We speculate that ABP may act as a carrier protein for an unknown ligand in axonal transport.

Mutagenesis of essential functional residues of rat androgen-binding protein/sex hormone-binding globulin.

Testicular androgen-binding protein (ABP) and liver (plasma) sex hormone-binding globulin (SHBG) are extracellular carrier proteins that bind androgens with high affinity. Both proteins are encoded by the same gene and have the same primary amino acid sequence. Previous affinity labeling experiments to identify the steroid-binding site of ABP/SHBG led to ambiguous results, implicating various residues from 134 to near the C-terminus. To aid in elucidation of the essential functional residues of ABP/SHBG, we created mutant rat proteins by deletion and site-directed mutagenesis. The mutants were expressed in COS 7 green monkey kidney cells and analyzed for immunoreactive cellular and medium ABP and dihydrotestosterone (DHT) binding properties. Analysis of truncated ABP proteins revealed that removal of 26 or more residues from the C-terminus eliminates secretion and DHT-binding activity. Alteration of amino acid residues by site-directed mutagenesis from residue 54 to residue 333 resulted in elimination of DHT binding for 9 of 10 mutants and reduced DHT affinity for one altered protein (ABPGly54-57). Only one of the 10 mutant ABP proteins was secreted by the COS cells. This secreted mutant ABP (ABPArg139) exhibited no detectable DHT-binding activity. Thus, our data demonstrate that modifications of the ABP primary sequence throughout the molecule have a detrimental effect on steroid binding and secretion. These data, taken together with previous affinity labeling experiments, mutagenesis studies, and the conserved residues between rat and human ABP/SHBG, indicate that at least part of active site is located in residues 139-150, but most of the protein is required to maintain the conformation of the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of asparagine-linked oligosaccharides in the subunit structure, steroid binding, and secretion of androgen-binding protein.

Testicular androgen-binding protein (ABP) and liver sex hormone-binding globulin are encoded by the same gene. These proteins have the same primary amino acid sequences, but they differ in attached oligosaccharides; the differences are presumably due to cell-specific glycosylation mechanisms. To investigate the role of oligosaccharides in ABP/sex hormone-binding globulin subunit structure, secretion, and steroid binding, mutant rat ABP proteins were constructed that eliminated one or both of the two potential sites of asparagine (Asn)-linked glycosylation. Immunoblot analysis of wild type recombinant ABP yielded the typical heterogeneous banding pattern. Secreted ABP was composed of two protomers of M(r) 46,000 and M(r) 43,000, while cellular ABP yielded three mol wt species (M(r) 43,000, 41,000, and 39,000). Substitution of the Asn residue in either consensus sequence for Asn-linked glycosylation with an Ile residue resulted in increased mobility of the immunoreactive ABP species. These changes are consistent with the loss of an Asn-linked oligosaccharide. Substitution of both Asn residues yielded a single immunoreactive species in the medium and cell extracts that migrated as a M(r) 39,000 protein. These results demonstrate that the mol wt heterogeneity of ABP is due to differential Asn-linked glycosylation of both potential sites. All three mutant forms of ABP were secreted by the COS cells. However, the amount of immunoreactive ABP and [3H]5 alpha-dihydrotestosterone binding in the medium was lower than wild type (100%) in one of the single mutants (65%) and in the double mutant (29%). Unlike the glycosylation mutants, alteration of other residues, not involved in glycosylation, yielded cellular ABP and no detectable medium ABP.(ABSTRACT TRUNCATED AT 250 WORDS)