The Sertoli cell: one hundred fifty years of beauty and plasticity.

It has been one and a half centuries since Enrico Sertoli published the seminal discovery of the testicular 'nurse cell', not only a key cell in the testis, but indeed one of the most amazing cells in the vertebrate body. In this review, we begin by examining the three phases of morphological research that have occurred in the study of Sertoli cells, because microscopic anatomy was essentially the only scientific discipline available for about the first 75 years after the discovery. Biochemistry and molecular biology then changed all of biological sciences, including our understanding of the functions of Sertoli cells. Immunology and stem cell biology were not even topics of science in 1865, but they have now become major issues in our appreciation of Sertoli cell's role in spermatogenesis. We end with the universal importance and plasticity of function by comparing Sertoli cells in fish, amphibians, and mammals. In these various classes of vertebrates, Sertoli cells have quite different modes of proliferation and epithelial maintenance, cystic vs. tubular formation, yet accomplish essentially the same function but in strikingly different ways.

Testisimmune privilege - Assumptions versus facts.

The testis has long enjoyed a reputation as an immunologically privileged site based on its ability to protect auto-antigenic germ cells and provide an optimal environment for the extended survival of transplanted allo- or xeno-grafts. Exploration of the role of anatomical, physiological, immunological and cellular components in testis immune privilege revealed that the tolerogenic environment of the testis is a result of the immunomodulatory factors expressed or secreted by testicular cells (mainly Sertoli cells, peritubular myoid cells, Leydig cells, and resident macrophages). The blood-testis barrier/Sertoli cell barrier, is also important to seclude advanced germ cells but its requirement in testis immune privilege needs further investigation. Testicular immune privilege is not permanent, as an effective immune response can be mounted against transplanted tissue, and bacterial/viral infections in the testis can be effectively eliminated. Overall, the cellular components control the fate of the immune response and can shift the response from immunodestructive to immunoprotective, resulting in immune privilege.

Testis immune privilege - Assumptions versus facts

The testis has long enjoyed a reputation as a n immunologically privileged site based on its ability to protect auto - antigenic germ cells and provide an optimal environment for the extended survival of transplanted allo - or xeno - grafts. Exploration of the role of anatomical, physiological, immunologica l and cell ular components in testis immune privilege reveal ed that the tolerogenic environment of the test i s is a result of the immunomodulatory factors expressed or secreted by testicular cells (mainly Sertoli cells, peritubular myoid cells, Leydig cells , and resident macrophages ). The b lood - testis barrier/Sertoli cell barrier, is also important to seclude advanced germ cells but its requirement in testis immune privilege needs further investigation . T esticular immune privilege is not permanent , as an effe ctive immune response can be mounted against transplanted tissue , and bacterial/viral infections in the testis can be effectively eliminated . Overall, the cellular components control the fate of the immune response and can shift the response from immunodestructive to immunoprotective, resulting in immuneprivilege.

Genetically engineered Sertoli cells are able to survive allogeneic transplantation.

The immunoprotective nature of the testis has led to numerous investigations for its ability to protect cellular grafts. Sertoli cells (SCs) are at least partially responsible for this immunoprotective environment and survive allogeneic and xenogeneic transplantation. The ability of SCs to survive transplantation leads to the possibility that they could be engineered to deliver therapeutic proteins. As a model to test this hypothesis, we examined the ability of SCs that produce green fluorescent protein (GFP) to survive transplantation and continue expressing GFP. SCs were isolated from transgenic mice engineered to express GFP and transplanted as aggregates under the kidney capsule of severe combined immunodeficient (SCID) and Balb/c mice. Using this paradigm, it was possible to compare the survival of transgenic SCs directly in both immunodeficient and immunocompetent recipients. Fluorescence microscopy of the kidney capsule and immunohistochemistry of the grafts for GFP and GATA-4 revealed the presence of GFP-expressing SCs under the kidney capsule of SCID and Balb/c mice at both 30 and 60 days post-transplantation. In contrast, islets transplanted to Balb/c mice were rejected. Thus, SCs survive transplantation and continue to express GFP raising the possibility that SCs can be engineered using transgenic technology to produce proteins, such as insulin, factor VIII, or dopamine for the treatment of diabetes, hemophilia or Parkinson's disease, respectively.

In vitro maturation of neonatal porcine islets: a novel model for the study of islet development and xenotransplantation.

The mechanisms involved in islet neogenesis have remained largely unexplored due to lack of an appropriate model. Furthermore, with the recent advances in islet transplantation, the need for alternative islet tissue sources is greater than ever. Therefore, the authors have refined a neonatal porcine islet (NPI) maturation model that offers an ideal tool to gain insight into islet growth as well as an alternative source of transplantable tissue. Recent knowledge in islet growth has resulted in endocrine tissue being derived from human pancreatic precursor tissue in vitro. The potential for large scale production of endocrine tissue in vitro has been indicated, however, more investigation must be done on the various signals and pathways involved in pancreatic development to optimize this technique. The authors believe that their NPI in vitro maturation model provides an ideal tool to study islet growth and maturation. Transduction of the NPI to overexpress genes of interest (i.e., PDX-1) or exposure of the NPI to various culture conditions will allow us to determine the effects on islet maturation. An understanding of NPI development gained will not only allow us to mature this unlimited tissue source for optimal xenotransplantation, but also elude to how human pancreatic endocrine precursor cells may be used to solve the current islet tissue supply problem.

Cellular and subcellular localization of six retinoid receptors in rat testis during postnatal development: identification of potential heterodimeric receptors.

Vitamin A is required in the testis for germ cell development. It acts through two families of retinoid receptors, retinoic acid receptors (RAR) and retinoid X receptors (RXR), each with three subtypes alpha, beta, and gamma. These receptors are postulated to dimerize and regulate the transcription of retinoid-responsive genes that are crucial for germ cell development. In this study, we determined the cellular and subcellular localization of six retinoid receptors in the developing rat testis to identify the specific cellular sites and times of receptor expression. Immunohistochemical results revealed the expression of RARalpha, RARbeta, RXRalpha, and RXRgamma proteins in somatic and germ cells throughout postnatal development. In contrast, the expression of RARgamma and RXRbeta did not increase until 30-35 days of age in somatic cells from the testis. Interestingly, RARalpha and RXRalpha had a similar subcellular localization pattern in Sertoli cells throughout postnatal testis development, while RARalpha and RXRgamma were both present in the nucleus of spermatocytes and elongating spermatids. These results suggest that RARalpha may potentially dimerize with RXRalpha in Sertoli cells and with RXRgamma in germ cells. In addition, we demonstrate that the only RAR in the nucleus of early meiotic germ cells is RARalpha.

Action of retinoids on embryonic and early postnatal testis development.

The current study investigates the hypothesis that retinoids have a role in embryonic testis development. The action of retinoids on testis development and the expression of retinoic acid receptors (RAR alpha, RAR beta, RAR gamma) were examined. In embryonic day 13 (E13; plug date = E0) testis organ cultures an RAR-selective agonist and all-trans retinoic acid completely inhibited seminiferous cord formation. In contrast, an RAR alpha-selective antagonist had no effect. RT-PCR demonstrated that RAR alpha messenger RNA (mRNA) was expressed at all developmental time points evaluated, which included embryonic day 14 (E14) through postnatal day 30 (P30). Expression of RAR beta mRNA was present at E15 through P2, whereas RAR gamma mRNA was expressed at E18 through P2. Cellular localization of receptors by immunohistochemistry indicated that RAR alpha was localized to the interstitium at E18 and to the seminiferous cords by P0. RAR beta and RAR gamma were detected in both interstitium and cords at E16 and by E18 were mainly expressed in the cords. At P0 RAR beta and RAR gamma were localized to the germ cell populations. To examine retinoid actions, the growth of P0 testis cultures were investigated. Interestingly, retinol and retinoic acid did not inhibit growth of P0 testis cultures but did inhibit the action of growth stimulators. Retinoic acid inhibited FSH, EGF, and 10% calf serum stimulated growth in P0 testis cultures. The hypothesis tested was that the inhibitory effects of retinoids on P0 testis growth may be mediated through the growth inhibitor, transforming growth factor-beta (TGF beta). The action of retinoids on TGF beta mRNA expression was examined in P0 testis cultures. Retinoic acid stimulated TGFbeta3 mRNA expression within 24 h and increased expression of TGFbeta1 and TGFbeta2 after 72 h. Retinol increased expression of TGFbeta1 and TGFbeta2 but not TGFbeta3 after 72 h of treatment. These observations indicate that retinoic acid can influence seminiferous cord formation and testis growth. The inhibitory actions of retinoids may in part be mediated through increased expression of TGFbeta isoforms.

Ligand-dependent regulation of retinoic acid receptor alpha in rat testis: in vivo response to depletion and repletion of vitamin A.

Male animals are sterile due to testicular degeneration in the absence of retinoic acid (RA) or functional retinoic acid receptor-alpha (RAR alpha). This degeneration can be reversed by injecting retinol, a precursor of RA, into vitamin A-deficient (VAD) rats. To determine the relationship between this ligand-dependent testicular degeneration and regeneration and the expression levels of RAR alpha messenger RNA and protein, testes were depleted and then replenished with retinol in vivo. Results showed that RAR alpha messenger RNA and protein levels declined to VAD amounts after 7 weeks on a VAD diet. This decline was due to decreased RAR alpha levels in early meiotic spermatocytes and the loss of advanced germ cells. Interestingly, the advanced germ cells still contained RAR alpha, but the protein was primarily cytoplasmic instead of nuclear, indicating inactivity as a transcription factor. In VAD testis, RAR alpha levels were low and then increased primarily in Sertoli cells after retinol replenishment. TUNEL analyses showed that most germ cells at the basal aspect of seminiferous tubules were undergoing apoptosis during degeneration. These results indicate that RAR alpha is either down-regulated or inactivated in RA-deficient testis and coincident with that, testes degenerate by apoptosis or selective loss of germ cells.

Retinoic acid receptor alpha gene expression in the rat testis: potential role during the prophase of meiosis and in the transition from round to elongating spermatids.

Mutational studies have identified retinoic acid receptor alpha (RAR alpha) as having an essential role in spermatogenesis. The objective of this study was to determine which cells express RAR alpha within the normal rat testis by conducting in situ analyses for mRNA and protein. Characterization of RAR alpha expression revealed the time and location of the vitamin A requirement during spermatogenesis. In situ hybridization analysis of testis from adult rats showed the highest level of transcripts occurring in round spermatids at stage VIII of the spermatogenic cycle. Analysis in the developing testes revealed that the mRNA level was high from 10 to 15 days of age, both in Sertoli cells and in germ cells, and then declined in 20-day-old rats. Consistent with this, immunohistochemical studies on adult testis demonstrated that the protein was present in the nucleus of the elongating spermatids (stages IX-XI) but not in elongated spermatids. The protein was also expressed in germ cells in the prophase of meiosis and at very low levels in Sertoli cells. These results suggest a role for RAR alpha during meiosis, at the transition from round to elongating spermatids, and in Sertoli cells of developing testis.

Region-specific localization of retinoic acid receptor-alpha expression in the rat epididymis.

Vitamin A has been shown to be involved in spermatozoal maturation in the epididymis. The action of vitamin A in male reproduction is postulated to be mediated at least partly by retinoic acid receptor-alpha (RAR alpha). The objective of this study was to determine whether the RAR alpha gene exhibits regional specificity in its pattern of expression along the length of the epididymis. The results would indicate where in the epididymis vitamin A may be required during maturation of spermatozoa. Northern blot analyses of RNA from the epididymis revealed two major transcripts, 3.4 kb and 2.7 kb, similar to the two major transcripts found in testis. In situ hybridization analyses demonstrated the expression of transcripts in the luminal epithelia to be highest in the proximal caput, low in the corpus, and high again in the distal cauda. This regional specificity in expression of the RAR alpha transcripts along the epididymis was virtually identical to that for the proteins, as visualized by reactions with anti-RAR alpha antibody and immunohistochemical stains. These results suggest roles for RAR alpha in the regions of epididymal epithelium that are postulated to participate in spermatozoal maturation and storage.

Novel compounds inhibit estrogen formation and action.

Estrogens are well known to play a predominant role in human breast cancer. The current endocrine therapy of breast cancer consists in administering an antiestrogen which blocks the action of estrogens at the receptor level. However, the currently available antiestrogens possess mixed estrogenic and antiestrogenic activity, thus limiting their potential therapeutic efficacy. The present data show that a series of new estrogen derivatives demonstrate not only pure antiestrogenic activity in the sensitive in vivo mouse uterus assay, but simultaneously exert potent inhibitory effects on 17 beta-hydroxysteroid dehydrogenase activity, the enzyme responsible for the formation of 17 beta-estradiol from estrone, the last step in estrogen formation. Such compounds having a dual site of inhibitory action, namely on estrogen formation and on the estrogen receptor, could well lead to an improved endocrine therapy of breast and other estrogen-sensitive cancers as well as other nonmalignant estrogen-sensitive diseases.

Synthesis and biological activity of new halo-steroidal antiestrogens.

Antiestrogen therapy is the most widely used endocrine manipulation for the treatment of breast cancer, especially in postmenopausal women. Unfortunately, the compounds presently available possess mixed agonistic/antagonistic activity, thus potentially limiting their therapeutic efficacy. Following the observations that an aliphatic chain at the 7 alpha-position of 17 beta-estradiol does not prevent binding to the estrogen receptor while halogenation of estradiol can increase the affinity of its binding (expressed as RBA) to the estrogen receptor, we have synthesized a series of new steroidal antiestrogens (6-10) which possess both an 7 alpha-undecanamide group and an halogen atom (Cl, Br, or I) at the 16 alpha-position. The stereochemistry of these compounds was unambiguously established by high-field (400-MHz) nuclear magnetic resonance. Some of the compounds obtained possess potent in vivo antiestrogenic activity. At the low twice daily 3-micrograms dose, 16 alpha-chloro 3,17 beta-diol amide, 16 alpha-iodo 3,17 beta-diol amide, 16 alpha-bromo 3,17 beta-diol amide, 16 alpha-chloro 3,17 alpha-diol amide, and 16 alpha-bromo 3,17 alpha-diol amide inhibit by 74, 63, 52, 35, and 60%, respectively, the estradiol-induced stimulation of uterine weight in ovariectomized Balb/c mice while 78-99% blockade of estradiol action is achieved at the 20-micrograms dose. These new antiestrogens show no estrogenic activity on uterine weight at the doses used while tamoxifen (2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N- dimethylethanamine) shows full estrogenic activity and is only a weak partial antiestrogen in the same assay.

Antiestrogenic properties of keoxifene, trans-4-hydroxytamoxifen, and ICI 164384, a new steroidal antiestrogen, in ZR-75-1 human breast cancer cells.

The agonistic/antagonistic properties of two non-steroidal antiestrogens, namely trans-4-monohydroxytamoxifen (OH-TAM) and keoxifene (LY156758), and the new steroidal antiestrogen ICI164384, a 7 alpha-alkylamide derivative of estradiol (E2), were assessed by measuring their effect on the proliferation of ZR-75-1 cells, an estrogen-responsive human breast cancer cell line. While subnanomolar concentrations of both OH-TAM and LY156758 had significant estrogenic stimulatory activity on cell growth in the absence of estrogens and higher concentrations were inhibitory, ICI164384 behaved exclusively as a growth inhibitor and more potently so than the two other compounds. The three antiestrogens had similar potency to inhibit the mitogenic effect of E2 and at 300 nM, all antiproliferative effects were completely reversible by the estrogen. ICI164384 was a weaker competitor of 3H-labeled E2 or R2858 (moxestrol) uptake in intact ZR-75-1 cells in a 1-hour assay, partly because of a slower intracellular access to estrogen specific binding sites. Moreover, ICI164384 interacted in a rapidly (approximately 6 h) reversible manner with estrogen-specific binding sites, while the non-steroidal antiestrogens induced a longer-acting (greater than 24 h) down-regulation of specific [3H]R2858 uptake. The present data indicate that, among the antiestrogens studied, ICI164384 is the only compound acting as a pure antiestrogen in ZR-75-1 breast cancer cells, while LY156758 and OH-TAM behave as antiestrogens endowed with partial agonistic activity in this system.

Progestin inhibition of estrogen-dependent proliferation in ZR-75-1 human breast cancer cells: antagonism by insulin.

The effect of R5020 [17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione], a synthetic progestin, was studied in the hormone-responsive ZR-75-1 human breast cancer cell line. Following a 12-day incubation with increasing concentrations of R5020, the mitogenic effect of 17 beta-estradiol (E2, 1nM) was partially (60-80%) antagonized by the progestin, with a half-maximal effective concentration measured at about 30 pM. This effect of R5020 was completely reversed by the addition of physiological concentrations of bovine insulin, as well as by the potent antiprogestin RU486 [17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propynyl)-4, 9-estradien-3-one], but not by the antiandrogen hydroxyflutamide (alpha, alpha, alpha-trifluoro-2-methyl-4'-nitro-m-lactotoluidide). Moreover, the effect of R5020 required the presence of estrogens, thus further indicating a progesterone receptor (PgR)-mediated effect. Low (greater than 100 nM) concentrations of R5020 increased the specific binding of [125I]-insulin up to 2- to 2.5-fold in intact ZR-75-1 cells, an effect which was reversed by RU486. The effect was rapid, being nearly maximal after 24h of incubation with R5020. The PgR-mediated effect of R5020 on cell proliferation was abolished by the addition of a pure steroidal antiestrogen. The present results suggest a physiological role for progestins in increasing the responsiveness to insulin, which could, in turn, reverse the antiproliferative effect of progestins on estrogen action and thus decrease the efficacy of progestins in the treatment of breast cancer.