Androgen-binding Protein Research Articles

The Pattern of Inhibin/Activin - and B-Subunit Messenger Ribonucleic Acid Expression in Rat Testis after Selective Leydig Cell Destruction by Ethylene Dimethane Sulfonate

To further investigate the regulatory mechanisms responsible for the control of testicular inhibin/activin subunit gene expression, inhibin-α, -βA, and -βB messenger RNA (mRNA) levels were assessed after ethylene dimethane sulfonate (EDS)-induced destruction of Leydig cells (LC) in different animal models: the intact rat, the rat treated with high doses of testosterone, and the unilaterally cryptorchid rat. In intact rats, EDS selectively eliminates the mature adult-type LCs, activating the proliferation and differentiation of preexisting LC precursors into a new population of functionally active LCs. In this model, a single dose of EDS (75 mg/kg BW, ip) induced a significant increase in testicular inhibin-α and -βB mRNA levels 5 days after treatment (5.0- and 5.5-fold increases, respectively), whereas inhibin-βA mRNA remained undetectable upon Northern hybridization in control and EDS-treated testes. Moreover, in situ hybridization analysis demonstrated that the increased expression of inhibin-α and -βB mRNAs observed 5 days after EDS takes place mainly in Sertoli cells. Along with LC repopulation, the expression level of inhibin-α and -βB messages declined, and inhibin-α mRNA returned to control values on day 40 after EDS. This treatment, however, failed to alter the pattern of testicular expression of FSH receptor and androgen-binding protein mRNAs, thus suggesting selectivity for the above effects. In EDS-treated rats supplemented with high doses of testosterone, the preexisting mature LCs are destroyed, but, due to elevated testosterone concentrations, disruption of spermatogenesis is attenuated, and the post-EDS rise in serum gonadotropins is blocked; the latter prevents LC regeneration. In this model, a 5.0-fold increase in inhibin-α mRNA levels, similar to that found in intact animals, was detected 5 days after EDS administration, but the rise in inhibin-βB levels was partially delayed. In addition, the blockade of LC repopulation resulted in permanent elevation of inhibin-α and -βB messages throughout the study period. In unilaterally cryptorchid rats, the abdominal testis shows disrupted spermatogenesis and altered paracrine environment that expedites LC repopulation after EDS treatment. In this model, the abdominal testes showed a significant 2.5-fold increase in inhibin-α mRNA levels 5 days after EDS, but no effect was found in those of inhibin-βB. Further, the faster rate of LC repopulation resulted in precocious decline of inhibin-α mRNA levels. Finally, the expression of inhibin/activin subunit mRNAs was monitored during postnatal testicular development, specifically at the time of regression of fetal-type LCs and appearance of those of the adult type. High levels of expression of inhibin-α and -βB mRNAs were detected in neonatal and infantile testes. A sharp decline in both messages took place between days 15–20, i.e. at the time when fetal-type Leydig cells are replaced by adult-type cells. From this time point onward, inhibin-α and -βB mRNA levels remained low, ranging between 15–30% of the maximum. In conclusion, our results suggest that the adult-type LCs differentially modulate the expression of inhibin/activin subunit genes and point to a major inhibitory role in this cell type on expression of the inhibin-α gene.

The pattern of inhibin/activin alpha- and betaB-subunit messenger ribonucleic acid expression in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

To further investigate the regulatory mechanisms responsible for the control of testicular inhibin/activin subunit gene expression, inhibin-alpha, -betaA, and -betaB messenger RNA (mRNA) levels were assessed after ethylene dimethane sulfonate (EDS)-induced destruction of Leydig cells (LC) in different animal models: the intact rat, the rat treated with high doses of testosterone, and the unilaterally cryptorchid rat. In intact rats, EDS selectively eliminates the mature adult-type LCs, activating the proliferation and differentiation of preexisting LC precursors into a new population of functionally active LCs. In this model, a single dose of EDS (75 mg/kg BW, ip) induced a significant increase in testicular inhibin-alpha and -betaB mRNA levels 5 days after treatment (5.0- and 5.5-fold increases, respectively), whereas inhibin-betaA mRNA remained undetectable upon Northern hybridization in control and EDS-treated testes. Moreover, in situ hybridization analysis demonstrated that the increased expression of inhibin-alpha and -betaB mRNAs observed 5 days after EDS takes place mainly in Sertoli cells. Along with LC repopulation, the expression level of inhibin-alpha and -betaB messages declined, and inhibin-alpha mRNA returned to control values on day 40 after EDS. This treatment, however, failed to alter the pattern of testicular expression of FSH receptor and androgen-binding protein mRNAs, thus suggesting selectivity for the above effects. In EDS-treated rats supplemented with high doses of testosterone, the preexisting mature LCs are destroyed, but, due to elevated testosterone concentrations, disruption of spermatogenesis is attenuated, and the post-EDS rise in serum gonadotropins is blocked; the latter prevents LC regeneration. In this model, a 5.0-fold increase in inhibin-alpha mRNA levels, similar to that found in intact animals, was detected 5 days after EDS administration, but the rise in inhibin-betaB levels was partially delayed. In addition, the blockade of LC repopulation resulted in permanent elevation of inhibin-alpha and -betaB messages throughout the study period. In unilaterally cryptorchid rats, the abdominal testis shows disrupted spermatogenesis and altered paracrine environment that expedites LC repopulation after EDS treatment. In this model, the abdominal testes showed a significant 2.5-fold increase in inhibin-alpha mRNA levels 5 days after EDS, but no effect was found in those of inhibin-betaB. Further, the faster rate of LC repopulation resulted in precocious decline of inhibin-alpha mRNA levels. Finally, the expression of inhibin/activin subunit mRNAs was monitored during postnatal testicular development, specifically at the time of regression of fetal-type LCs and appearance of those of the adult type. High levels of expression of inhibin-alpha and -betaB mRNAs were detected in neonatal and infantile testes. A sharp decline in both messages took place between days 15-20, i.e. at the time when fetal-type Leydig cells are replaced by adult-type cells. From this time point onward, inhibin-alpha and -betaB mRNA levels remained low, ranging between 15-30% of the maximum. In conclusion, our results suggest that the adult-type LCs differentially modulate the expression of inhibin/activin subunit genes and point to a major inhibitory role in this cell type on expression of the inhibin-alpha gene.

Sex-hormone binding globulin from sheep serum: purification and effects of pregnancy and treatment with exogenous estradiol

Sex-hormone binding globulin (SHBG) is a protein that binds sex steroids in the serum of many species. SHBG binds androgens and estrogens in humans and primates with high affinity, but behaves as an androgen binding protein in other species. Here we purified SHBG from ewe and ram sera to homogeneity, by a simple and rapid method. The K D of the purified protein was found to be 3.63 nM for testosterone and around 600 nM for estradiol. We also studied the effect of pregnancy on SHBG levels in ewes and the effect of exogenous estradiol administration either orally or parenterally on SHBG levels in rams. Basal levels of SHBG in sheep are not affected by pregnancy or exposure to exogenous estradiol. It is concluded that SHBG regulation of expression in ewes and rams differs from that in humans in that it is not affected by estrogen and possibly is species specific.

Effect of surgically induced varicocele on testicular blood flow and Sertoli cell function

Objectives. To evaluate the effect of varicocele on testicular blood flow and expression by Sertoli cells of transferrin and androgen-binding protein (ABP), to determine whether varicoceles impair Sertoli cell function. Methods. Experimental varicocele was established in male Sprague-Dawley rats by partial ligation of the left renal vein. The control group received a sham operation. At 30 minutes after surgery, rats underwent a xenon-133 washout study, and at 30 days after surgery, transferrin, ABP, and testicular blood flow were evaluated. Expression of transferrin and ABP were evaluated using immunohistochemical techniques. Testicular blood flow was measured using xenon-133 clearance techniques. Statistical analyses were done with an independent t test. Results. The testicular blood flow was 16.7 ± 1.25 mL/100 g/min in varicocele-bearing rats and 21.01 ± 0.46 mL/100 g/min in sham-operated rats 30 minutes after surgery. Testicular blood flow remained decreased at 30 days in varicocele-bearing rats (15.12 ± 1.08 mL/100 g/min) and remained stable in the control group (19.45 ± 0.55 mL/100 g/min). The expression of transferrin and ABP was significantly reduced in varicocele-bearing rats compared with the control group. Conclusions. Our study suggests that a decrease in testicular blood flow may lead to impaired Sertoli cell function in varicocele-bearing rats.

Effects of hypercholesterolaemia on Leydig and Sertoli cell secretory function and the overall sperm fertilizing capacity in the rabbit.

The effects of hypercholesterolaemia on testicular endocrine and exocrine function were evaluated. The influence of hypercholesterolaemia on sperm quality, quantity, and fertilizing potential was also determined. Ten mature rabbits (group A) were fed chow containing 3% cholesterol for 12 weeks. Ten control rabbits (group B) were fed normal chow for the same period. At the end of the experimental period testosterone profiles and sperm parameters were evaluated and the sperm reproductive potential was assessed by in vitro fertilization (IVF) techniques. Peripheral serum testosterone responses to testicular stimulation with human chorionic gonadotrophin, androgen-binding protein activity in testicular cytosols, sperm concentration, sperm motility, length of sperm midpiece, and IVF outcome were all significantly lower in group A than in group B. In contrast, serum cholesterol concentrations were significantly higher in group A. There were no significant differences in either testicular versus intra-abdominal temperature differences or cholesterol concentrations in seminal plasma or testicular tissue between groups A and B. The results suggest that hypercholesterolaemia has a detrimental effect on Leydig and Sertoli cell secretory function, spermatogenesis, epididymal sperm maturation process, and the overall sperm fertilizing capacity.

Heregulins or Neu Differentiation Factors and the Interactions between Peritubular Myoid Cells and Sertoli Cells

Interactions between (mesenchymal) peritubular myoid cells and (epithelial) Sertoli cells play an important role in the control of Sertoli cell function and spermatogenesis. The factors involved, however, have only partially been identified. Heregulins or neu differentiation factors (NDFs) mediate mesenchymal-epithelial interactions in a variety of tissues, but their role in the testis has not been investigated. Here we demonstrate that recombinant human heregulin-α (Her-α) and Her-β stimulate transferrin and androgen-binding protein production by cultured rat Sertoli cells up to 2.5-fold. These effects are more pronounced than those of previously identified growth factors active in this assay, such as insulin-like growth factor I and basic fibroblast growth factor. Combination with these factors results in additive effects and in marked morphological changes in the Sertoli cell cultures, including formation of tubule-like structures. Stimulation of androgen-binding protein secretion is paralleled by increased levels of the corresponding messenger RNA. This parallelism was less consistent for transferrin. Semiquantitative RT-PCR indicates that the expression of NDF-α and NDF-β is more pronounced in peritubular cells than in Leydig or Sertoli cells. Conversely, the main receptors for heregulins/NDFs, HER-3 and HER-4, are predominantly expressed in Sertoli cells. A displacement assay confirms the presence of high-affinity binding sites for [125I]Her-β on intact Sertoli cells and reveals parallel displacement curves for Her-β, Her-α, and concentrated peritubular cell-conditioned medium (PTCM; estimated ED50 values: 1 ng/ml, 50 ng/ml, and 130 μg protein/ml, respectively), indicating that peritubular cells secrete one or more factors able to compete for heregulin receptors. It is concluded that heregulins/NDFs may play a role in mesenchymal-epithelial interactions in the testis. Estimates of the concentrations of heregulins in PTCM, however, make it unlikely that they contribute significantly to the effects observed with low concentrations of PTCM and ascribed to the putative mediator PModS (peritubular factor that modulates Sertoli cell function). Further investigations will be required to define the exact role of heregulins in the testis.

Heregulins or neu differentiation factors and the interactions between peritubular myoid cells and Sertoli cells.

Interactions between (mesenchymal) peritubular myoid cells and (epithelial) Sertoli cells play an important role in the control of Sertoli cell function and spermatogenesis. The factors involved, however, have only partially been identified. Heregulins or neu differentiation factors (NDFs) mediate mesenchymal-epithelial interactions in a variety of tissues, but their role in the testis has not been investigated. Here we demonstrate that recombinant human heregulin-alpha (Her-alpha) and Her-beta stimulate transferrin and androgen-binding protein production by cultured rat Sertoli cells up to 2.5-fold. These effects are more pronounced than those of previously identified growth factors active in this assay, such as insulin-like growth factor I and basic fibroblast growth factor. Combination with these factors results in additive effects and in marked morphological changes in the Sertoli cell cultures, including formation of tubule-like structures. Stimulation of androgen-binding protein secretion is paralleled by increased levels of the corresponding messenger RNA. This parallelism was less consistent for transferrin. Semiquantitative RT-PCR indicates that the expression of NDF-alpha and NDF-beta is more pronounced in peritubular cells than in Leydig or Sertoli cells. Conversely, the main receptors for heregulins/NDFs, HER-3 and HER-4, are predominantly expressed in Sertoli cells. A displacement assay confirms the presence of high-affinity binding sites for [125I]Her-beta on intact Sertoli cells and reveals parallel displacement curves for Her-beta, Her-alpha, and concentrated peritubular cell-conditioned medium (PTCM; estimated ED50 values: 1 ng/ml, 50 ng/ml, and 130 microg protein/ml, respectively), indicating that peritubular cells secrete one or more factors able to compete for heregulin receptors. It is concluded that heregulins/NDFs may play a role in mesenchymal-epithelial interactions in the testis. Estimates of the concentrations of heregulins in PTCM, however, make it unlikely that they contribute significantly to the effects observed with low concentrations of PTCM and ascribed to the putative mediator PModS (peritubular factor that modulates Sertoli cell function). Further investigations will be required to define the exact role of heregulins in the testis.

Effects of spinal cord injury on spermatogenesis and the expression of messenger ribonucleic acid for Sertoli cell proteins in rat Sertoli cell-enriched testes.

The study was an examination of the effects of spinal cord injury (SCI) on spermatogenesis and Sertoli cell functions in adult rats with Sertoli cell-enriched (SCE) testes. The effects of SCI on the seminiferous epithelium were characterized by abnormalities in the remaining spermatogenic cells during the first month after SCI. Three days after SCI, serum testosterone levels were 80% lower, while serum FSH and LH levels were 25% and 50% higher, respectively, than those of sham control SCE rats. At this time, the levels of mRNA for androgen receptor (AR), FSH receptor (FSH-R), and androgen-binding protein (ABP) were normal whereas those for transferrin (Trf) had decreased by 40%. Thereafter, serum testosterone levels increased, but they remained lower than those of the sham control rats 28 days after SCI; and serum FSH and LH levels returned to normal. The levels of mRNA for AR, ABP, and Trf exhibited a biphasic increase 7 days after SCI and remained elevated 28 days after SCI. FSH-R mRNA levels were also elevated 90 days after SCI. Unexpectedly, active spermatogenesis, including qualitatively complete spermatogenesis, persisted in > 40% of the tubules 90 days after SCI. These results suggest that the stem cells and/or undifferentiated spermatogonia in SCE testes are less susceptible to the deleterious effects of SCI than the normal testes and that they were able to proliferate and differentiate after SCI. The presence of elevated levels of mRNA for Sertoli cell FSH-R and AR, as well as of that for the Sertoli cell proteins, in the SCE testes during the chronic stage of SCI suggests a modification of Sertoli cell physiology. Such changes in Sertoli cell functions may provide a beneficial environment for the proliferation of the stem cells and differentiation of postmeiotic cells, thus resulting in the persistence of spermatogenesis in these testes.

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.

Aldehyde dehydrogenase gene superfamily. The 1998 update.

Aldehydehydrogenases (ALDHs) represent a group of NAD(P)+-dependent enzymes, which e similar primary structures and oxidize a wide spectrum of endogenous and exnous aldehydes (Lindahl, 1992; Vasiliou et al., 1995). Several ALDHs display broad strate specificities, oxidizing a variety of both aliphatic and aromatic aldehydes, whereather forms possess narrower substrate preferences. Based on substrate specificities, ALDH emes are broadly categorized as:a. semialdehyde dehydrogenases, including hydroxymuconic semialdehyde dehydrogenase (E.C. 1.2.1.32), Escherichia coli (EC 1.2.1.16) and mammalian (EC 1.2.1.24) succinate-semialdehyde dehydrogenase, glutamate semi aldehyde dehydrogenase (E.C. 1.2.1.41), aspartate semialdehyde dehydrogenase (E.C. 1.2.1.11), 2-amino-adipate-6-semialdehyde dehydrogenase (E.C. 1.2.1.31) and metylmalonate- semialdehyde dehydrogenase (E.C. 1.2.1.27), b. nonspecific ALDHs (E.C.I.2.1.3), c. other ALDHs including betaine dehydrogenase (E.C. 1.2.1.8), non phosphorylating glyceraldehyde 3-phosphatedehydrogenase (E.C. 1.2.1.9), phenylacetaldehyde dehydrogenase (EC 1.2.1.39), lactaldehyde dehydrogenase (EC 1.2.1.22), and d. ALDike proteins which represent certain other protein sequences, containing either complete or almost complete ALDH sequences. These include the 10-formyltetrahydrofolate dehydrogenase (E.C. 1.5.1.6), Δ1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12), antiquitin, a human 56-kDa androgen-binding protein, and the crystallins..KeywordsBacillus SubtilisAromatic AldehydeClostridium AcetobutylicumGene SuperfamilyDeinococcus RadioduransThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The effects of environmental hormones on reproduction.

Considerable attention has been given in the past few years to the possibility that man-made chemicals (xenobiotics) in the environment may pose a hazard to human reproductive health. The endocrine-disrupting effects of many xenobiotics can be interpreted as interference with the normal regulation of reproductive processes by steroid hormones. Evidence reviewed here indicates that xenobiotics bind to androgen and oestrogen receptors in target tissues, and to androgen-binding protein and to sex hormone-binding globulin. Although environmental chemicals have weak hormonal activity, their ability to interact with more than one steroid-sensitive pathway provides a mechanism by which their hazardous nature can be augmented. A given toxicant may be present in low concentration in the environment and, therefore, harmless. However, we are not exposed to one toxicant at a time, but, rather, to all of the xenobiotics present in the environment. Therefore, numerous potential agonists/antagonists working together through several steroid-dependent signalling pathways could prove to be hazardous to human reproductive health.

Androgen binding protein secretion and endocytosis by principal cells in the adult rat epididymis and during postnatal development.

Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.

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.

Androgen regulation of the Pem homeodomain gene in mice and rat Sertoli and epididymal cells.

Although the role of homeodomain transcription factors during embryogenesis is well known, their developmental function in postnatal animals is only beginning to be understood. We examined the regulation and expression pattern of Pem, a homeodomain protein that may regulate androgen-dependent events in the testis and epididymis. Immunohistochemical analysis showed that Pem protein is expressed selectively in the nuclei of Sertoli cells during the androgen-dependent stage of the seminiferous epithelium cycle in vivo. RNase protection analysis revealed that a proximal promoter was responsible for androgen-dependent mouse Pem expression in testis and epididymis in vivo, whereas a distal promoter was used in placenta. The mouse Pem gene was expressed at approximately 10-fold higher levels in the testis than in the epididymis; conversely, the rat Pem gene was expressed at >10-fold higher levels in the epididymis than in the testis. Because androgen-binding protein has been proposed to transport androgens from the testis to the epididymis, we tested whether the > or = 20-fold higher levels of androgen-binding protein expression in the rat, compared to that of mouse, are responsible for the differential expression of Pem in these two rodent species. Studies with androgen-binding protein transgenic mice demonstrated that the species-specific difference in androgen-binding protein expression is unlikely to be responsible for the species-specific difference in Pem expression. We found that androgen is necessary but not sufficient for Pem expression, since purified Sertoli cells rapidly down-regulated Pem transcripts in culture, regardless of the presence of testosterone. We conclude that Pem gene expression in Sertoli cells requires other cell types or cellular factors in addition to androgen.

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.

Steroid binding by mouse salivary proteins.

The goals of this study were to determine the steroid-binding specificity of the mouse salivary androgen-binding protein (ABP) family and to ascertain whether there might be other proteins in mouse saliva capable of binding steroids. The optimal conditions for testosterone binding by mouse salivary proteins were determined using a small-scale chromatography system to separate bound from unbound steroid. Testosterone binding appeared to be biphasic but was directly proportional to saliva concentration, with an optimum temperature of 37 degrees C in the second phase. These results were used to develop a steroid-binding protocol to study the steroid specificity of salivary proteins separated by electrophoresis. The ABP family bound testosterone and progesterone well and HO-progesterone and DHT to a lesser extent but did not bind either cholesterol or estradiol. Steroid structural comparisons suggest that binding by ABP is governed by the A ring of the steroid. Another protein that is not a member of the ABP family bound cholesterol specifically but no protein that specifically bound estradiol was observed.

Effects of rapamycin and FK 506 on rat Sertoli cells in vitro.

Sertoli cells play an important role in the physical and metabolic support of germ cells developing in the seminiferous tubules within the testis. They are also closely involved in the initiation and control of spermatogenesis (Gray and Beamand, 1984). Many functions of Sertoli cells have been maintained in vitro as the production of androgen binding protein (ABP) and the binding to follicle stimulating hormone (FSH). One role of the Sertoli cells is the production of lactate and pyruvate as substrates for the germ cells. The energy requirements of isolated germ cells are not fully supported by glucose but they are able to utilize the lactate and pyruvate produced by the Sertoli cells. Different compounds have been show to produce testicular damage in experimental animals. Some of these drugs have specific target cells within the testis, including the Sertoli cells.

Human Sex Hormone-Binding Globulin Gene Expression in Transgenic Mice

The sex hormone-binding globulin gene (shbg) is expressed in the liver and testis as well as in several other tissues that play important roles in reproduction. Expression of shbg in the human liver results in the production of plasma sex hormone-binding globulin (SHBG), which regulates the bioavailability of sex steroids in the blood. Although shbg is not expressed in rodent livers postnatally, it gives rise to the androgen-binding protein in their testes upon sexual maturation. Human shbg is also expressed in the testis, but its products and their function are less well characterized. To study the expression of human shbg in different tissues and the consequences of overexpressing this gene in vivo, we have produced several lines of mice containing approximately 11-kilobase (kb; shbg11) or 4.3-kb (shbg4) human shbg genomic fragments that comprise all eight exons encoding SHBG as well as approximately 6 kb or approximately 0.9 kb of 5'-flanking DNA, respectively. Northern blots indicated that human shbg transcripts were most abundant in liver, kidney, and testis of the shbg11 mice. The 4.3-kb shbg transgenes were expressed at similar levels in liver and kidney, but the abundance of human shbg transcripts in their testes was much lower than that in shbg11 mice. Primer extension analysis indicated that transcription starts 60 bp from the translation initiation codon for SHBG in liver and kidney of shbg11 mice, and that the shbg transcripts in their testis are derived from a separate promoter flanking an alternative exon that replaces the exon containing the translation initiation codon for SHBG or androgen-binding protein. At the cellular level, the human shbg transgenes are expressed in clusters of hepatocytes located mainly within the periportal region of hepatic lobules and in the epithelial cells lining the proximal convoluted tubules of the kidney. This results in high levels of human SHBG in serum (1.45-1.72 nmol/ml) and urine (6-16 pmol/ml) of mature male shbg mice. The abundance and distribution of human shbg transcripts in the Sertoli cells of shbg11 mice vary throughout the spermatogenic cycle, with levels increasing in the Sertoli cell cytoplasm until stage VII of spermatogenesis and declining after stage IX. At stages X-XII of spermatogenesis, these transcripts concentrate at the adluminal compartment of the Sertoli cells, and this suggests that they have a role in the elongation phase of spermiogenesis. The presence of human SHBG in the blood of shbg transgenic mice may result in serum levels of testosterone that are 10-100 times higher than those in wild-type littermates. Despite this, their reproductive performance is normal, and there is no obvious phenotypic abnormalities even in animals homozygous for the transgenes.

Regulation of gamma-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat epididymis by testicular factors: role of basic fibroblast growth factor.

The objective of this study was to test the hypothesis that gamma-glutamyl transpeptidase (GGT) catalytic activity and protein level in the initial segment are regulated by testicular factors. In the rat epididymis, levels of GGT catalytic activity were initial segment > caput > corpus = cauda. GGT catalytic activity and protein level in the initial segment decreased after efferent duct ligation (EDL) for 3 days, but were recovered when initial segment pieces were incubated with ovine or rat rete testis fluid (oRTF or rRTF, respectively). Factors responsible for the recovery were shown to be greater than 10 kDa and protein-like, but these factors were not androgen-binding protein or testosterone in oRTF. Further experiments were designed to test the hypothesis that growth factors within oRTF and rRTF regulate GGT catalytic activity and GGT protein level. Basic fibroblast growth factor (bFGF) but not epidermal growth factor recovered GGT catalytic activity and protein level in the initial segment following 3-day EDL. Western blot analyses also revealed that bFGF-like proteins were present in rRTF, epididymal luminal fluid, and rat initial segment homogenate, and that the level of bFGF-like proteins declined in the initial segment following 3-day EDL. Using a bFGF monoclonal antibody, a small amount of bFGF-like proteins was found to be also present in oRTF. Our studies suggest that bFGF is one of the testicular factors involved in the regulation of epididymal GGT catalytic activity and protein level. Since decreased GGT catalytic activity caused by 3-day EDL in the rat initial segment was also recovered by a tumor promoter, phorbol 12-O-tetradecanoylphorbol 13-acetate, it is possible that a signal transduction pathway is involved in the regulation of GGT catalytic activity and GGT protein level by testicular factors. Western blot analyses also indicated that the 43-kDa bFGF-like protein in the lumen of the rat epididymis originates from the testis, is concentrated in the initial segment, and is reabsorbed by the epididymal epithelia from proximal to distal epididymal regions.