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Phthalate exposure impairs testis development and function; however, whether phthalates affect nonreproductive functions is not well understood. To investigate this, C57BL/6J mice were fed 1-500 mg di-n-butyl phthalate (DBP) in corn oil, or vehicle only, daily from 4 to 14 days, after which tissues were collected (prepubertal study). Another group was fed 1-500 mg/kg·d DBP from 4 to 21 days and then maintained untreated until 8 weeks for determination of adult consequences of prepubertal exposure. Bones were assessed by microcomputed tomography and dual-energy X-ray absorptiometry and T by RIA. DBP exposure decreased prepubertal femur length, marrow volume, and mean moment of inertia. Adult animals exposed prepubertally to low DBP doses had lower bone mineral content and bone mineral density and less lean tissue mass than vehicle-treated animals. Altered dynamics of the emerging Leydig population were found in 14-day-old animals fed 100-500 mg/kg·d DBP. Adult mice had variable testicular T and serum T and LH concentrations after prepubertal exposure and a dose-dependent reduction in cytochrome p450, family 11, subfamily A, polypeptide 1. Insulin-like 3 was detected in Sertoli cells of adult mice administered the highest dose of 500 mg/kg·d DBP prepubertally, a finding supported by the induction of insulin-like 3 expression in TM4 cells exposed to 50 μM, but not 5 μM, DBP. We propose that low-dose DBP exposure is detrimental to bone but that normal bone mineral density/bone mineral content after high-dose DBP exposure reflects changes in testicular somatic cells that confer protection to bones. These findings will fuel concerns that low-dose DBP exposure impacts health beyond the reproductive axis.
Targeted mutagenesis of Fgf9 in mice causes male-to-female sex reversal. Among the four FGF receptors, FGFR2 showed two highly specific patterns based on antibody staining, suggesting that it might be the receptor-mediating FGF9 signaling in the gonad. FGFR2 was detected at the plasma membrane in proliferating coelomic epithelial cells and in the nucleus in Sertoli progenitor cells. This expression pattern suggested that Fgfr2 might play more than one role in testis development. To test the hypothesis that Fgfr2 is required for male sex determination, we crossed mice carrying a floxed allele of Fgfr2 with two different Cre lines to induce a temporal or cell-specific deletion of this receptor. Results show that deletion of Fgfr2 in embryonic gonads phenocopies deletion of Fgf9 and leads to male-to-female sex reversal. Using these two Cre lines, we provide the first genetic evidence that Fgfr2 plays distinct roles in proliferation and Sertoli cell differentiation during testis development.
We have investigated the developmental pattern of expression and activity of 17alpha-hydroxylase/C-17,20-lyase cytochrome P450 (cytochrome P450c17) in the liver, stomach, duodenum, and testis of rats from day 18 of pregnancy to adulthood. In the male liver, the enzyme became detectable at birth (135 pmol/mg protein x min) at a level comparable to that in the testis (188 pmol/mg protein x min). The activity then increased dramatically, reaching a peak at 8 days (691 pmol/mg protein x min), which was more than 4-fold the testicular levels in rats of the same age or in adults. Thereafter it declined steadily, becoming undetectable from puberty onward. The hepatic peak followed a depression in testicular activity (58 pmol/mg protein x min) on day 6. Northern and immunoblot analyses showed a good temporal correlation between enzyme activity and the occurrence of P450c17 messenger RNA (mRNA) and protein. The same patterns of mRNA and protein occurrence were observed in female rat liver, indicating that the hepatic CYP17 expression is not sexually dimorphic. Sequencing confirmed a complete identity in the coding region between hepatic and gonadal mRNAs. Hepatic P450c17 mRNA, however, was 150-200 bases longer than the gonadal counterparts. No significant expression of mRNAs encoding P450scc and P450arom was observed in liver of either sex at any age. In stomach and duodenum, enzyme activity was much lower (maxima at 25 and 14 pmol/mg protein x min, respectively) than that in liver, but persisted from the time of weaning onward. It is suggested that the hepatic peak in P450c17 activity may serve to convert circulating progestogens into androgens for gonadal aromatization during Sertoli and granulosa cell proliferation.
Bone morphogenetic protein 8B (BMP8B) is a member of the TGFbeta superfamily of growth factors. In the mouse, Bmp8b is expressed in male germ cells of the testis and trophoblast cells of the placenta, suggesting that it has a role in spermatogenesis and reproduction. To investigate these possibilities, we have generated mice with a targeted mutation in Bmp8b. Here, we show that homozygous Bmp8b(tm1blh) mutant males exhibit variable degrees of germ-cell deficiency and infertility. Detailed analysis reveals two separable defects in the homozygous mutant testes. First, during early puberty (2 weeks old or younger) the germ cells of all homozygous mutants either fail to proliferate or show a marked reduction in proliferation and a delayed differentiation. Second, in adults, there is a significant increase in programmed cell death (apoptosis) of spermatocytes, leading to germ-cell depletion and sterility. Sertoli cells and Leydig cells appear relatively unaffected in mutants. This study therefore provides the first genetic evidence that a murine germ cell-produced factor, BMP8B, is required for the resumption of male germ-cell proliferation in early puberty, and for germ-cell survival and fertility in the adult.
Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.
The potential role of transforming growth factor beta (TGF beta) as a mediator of cell-cell interactions within the seminiferous tubule was investigated through an examination of the local production and action of TGF beta. Sertoli cells and peritubular (myoid) cells were isolated and cultured under serum-free conditions. Secreted proteins from Sertoli cells and peritubular cells were found to contain a component that bound to TGF beta receptors in RRA. Reverse-phase chromatography of Sertoli cell and peritubular cell secreted proteins fractionated a protein with similar biochemical properties as TGF beta 1. This fractionated protein also contained TGF beta bioactivity in its ability to inhibit growth of an epidermal growth factor-dependent cell line. Both peritubular cells and Sertoli cells contained a 2.4 kilobase mRNA species that hybridized in a Northern blot analysis with a TGF beta 1 cDNA probe. TGF beta 1 gene expression was not detected in freshly isolated germ cells. TGF beta 1 alone was not found to influence Sertoli cell nor peritubular cell proliferation with cells isolated from a midpubertal stage of development. The effects of hormones and TGF beta on Sertoli cell differentiation and function were assessed through an examination of transferrin production by Sertoli cells. TGF beta 1 had no effect on transferrin production nor the ability of hormones to influence transferrin production. The presence of peritubular cells in a coculture with Sertoli cells also did not affect the inability of TGF beta 1 to act on Sertoli cells. Although Sertoli cell function did not appear to be influenced by TGF beta 1, peritubular cells responded to TGF beta 1 through an increase in the production of a number of radiolabeled secreted proteins. TGF beta 1 also had relatively rapid effects on peritubular cell migration and the promotion of colony formation in culture. Cocultures of Sertoli cells and peritubular cells responded to TGF beta 1 by the formation of large cell clusters with ball-like structures. Data indicate that TGF beta may have an important role in influencing the differentiation and migration of peritubular cells. Observations demonstrate the local production of TGF beta within the seminiferous tubule by Sertoli cells and peritubular cells and suggest that TGF beta may have a role as a paracrine-autocrine factor involved in the maintenance of testicular function.
Numerous studies in recent years have elucidated fundamental properties of axoplasmic structure, biochemistry, and function. The structural role of the cytoskeletal elements, the orientation of MTs within the axon, the phenomenon of MT-dependent transport, and the identity and direction of movement of two MT motors--kinesin and MAP-1C--have been revealed. For many years to come, researchers investigating the structure and function of the Sertoli cell cytoskeleton will be able to adapt techniques gleaned from work on the axonal cytoskeleton. Innovative thinking will be required to apply these techniques to the special circumstances of the male reproductive system; however, the underlying questions are similar. For example, knowledge of several fundamental properties of transport processes in the Sertoli cell would facilitate the toxicologic evaluation of this system. What is the orientation of MTs within the Sertoli cell cytoplasm? Are the fast-growing (+) ends of all MTs in the Sertoli cell cytoplasm directed toward the lumen? This is an important question because the direction of MT-dependent transport involving known MT motors is dependent upon the MT orientation. Which of the Sertoli cell transport pathways are MT-dependent pathways? What are the MT motors involved in these pathways? Ultrastructural examination following exposure to specific cytoskeleton-disrupting agents has highlighted the importance of AFs, IFs, and MTs in the Sertoli cell. Future research will focus on the nature of those molecules which integrate these cytoskeletal components into a dynamic whole, the regulatory systems which control this integration, and the role of an integrated cytoskeleton in Sertoli cell function and testicular homeostasis. Toxicology will be an active participant in this process of scientific discovery. The selective nervous system and testicular toxicants may be useful tools in revealing similarities in the cytoskeletal organization of these apparently disparate organ systems. By searching for common targets in the testis and nervous system, the mechanisms of action of these agents may be more easily, and more confidently, determined.
The local production and action of an epidermal growth factor (EGF)-like substance within the seminiferous tubule was investigated as a potential mediator of cell-cell interactions. Peritubular (myoid) and Sertoli cells were isolated and cultured under serum-free conditions. Proteins secreted by Sertoli and peritubular cells were found to contain a component that bound to the EGF receptor in a RRA. Separation of secreted proteins by reverse phase chromatography fractionated a protein that contained EGF bioactivity in its activity to stimulate growth of an EGF-dependent cell line. Biochemical properties examined for both Sertoli and peritubular cell EGF activities were similar with each other, but distinct from murine EGF. Northern blot analysis with an EGF cDNA probe did not detect EGF gene expression in peritubular, Sertoli, or germ cells. The possible production of an EGF-like substance such as transforming growth factor-alpha (TGF alpha) was investigated with a molecular probe to human TGF alpha. Both peritubular and Sertoli cells contained a 4.5-kilobase mRNA species that hybridized in a Northern blot analysis with a human TGF alpha cRNA probe. An immunoblot with a TGF alpha antisera confirmed the production of TGF alpha by the detection of a protein in both Sertoli and peritubular cell secreted proteins. TGF alpha gene expression was not detected in freshly isolated germ cells. Scatchard analysis revealed the presence of high affinity EGF receptors on peritubular cells and the absence of such receptors on Sertoli or germ cells. TGF alpha was found to stimulate peritubular cell proliferation, but had no effect on Sertoli cell growth. The effects of hormones and TGF alpha on Sertoli cell function and differentiation were assayed through an examination of transferrin production by Sertoli cells. TGF alpha had no direct effect on transferrin production or the ability of hormones to influence Sertoli cells. However, the presence of peritubular cells in coculture with Sertoli cells allowed TGF alpha to stimulate transferrin production. TGF alpha was also found to have relatively rapid effects on peritubular cell migration and the promotion of colony formation in culture. Cocultures of peritubular and Sertoli cells also responded to TGF alpha by the formation of large clusters of cells. Observations demonstrate the local production of TGF alpha by Sertoli and peritubular cells, and action of TGF alpha on peritubular cells and, potentially, Sertoli cells. The local production and action of TGF alpha may have a critical role as a paracrine/autocrine factor involved in the maintenance of testicular function.
The ultrastructure and biochemical characteristics of HMW-2, the Sertoli cell cytoplasmic dynein isolated from rat testes, were analyzed. Electron microscopic studies revealed a two-headed two-stem structure with dimensions very similar to other dyneins. We found that, like other cytoplasmic dyneins, both heads have an approximately spherical shape with a central cavity. Heavy chain analysis suggested the presence of only one type of heavy chain, a finding that was supported by the simple Michaelis-Menten kinetics displayed by the HMW-2-associated ATPase activity. In addition, dissociation of the HMW-2 complex resulted in a single type of dynein subunit sedimenting at 11.8 S. This fraction contained all the polypeptides present in the undissociated HMW-2. Ultrastructurally the HMW-2 subunits were composed of one globular domain with a tail. The simplest interpretation is that HMW-2 is a dimer of nearly identical subunits, each containing one heavy chain, one 90-kDa intermediate chain, and two light chains.