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.

Regulation of germ line stem cell homeostasis.

Mammalian spermatogenesis is a complex process in which spermatogonial stem cells of the testis (SSCs) develop to ultimately form spermatozoa. In the seminiferous epithelium, SSCs self-renew to maintain the pool of stem cells throughout life, or they differentiate to generate a large number of germ cells. A balance between SSC self-renewal and differentiation is therefore essential to maintain normal spermatogenesis and fertility. Stem cell homeostasis is tightly regulated by signals from the surrounding microenvironment, or SSC niche. By physically supporting the SSCs and providing them with these extrinsic molecules, the Sertoli cell is the main component of the niche. Earlier studies have demonstrated that GDNF and CYP26B1, produced by Sertoli cells, are crucial for self-renewal of the SSC pool and maintenance of the undifferentiated state. Down-regulating the production of these molecules is therefore equally important to allow germ cell differentiation. We propose that NOTCH signaling in Sertoli cells is a crucial regulator of germ cell fate by counteracting these stimulatory factors to maintain stem cell homeostasis. Dysregulation of this essential niche component can lead by itself to sterility or facilitate testicular cancer development.

Human spermatogonial stem cells: a possible origin for spermatocytic seminoma.

In mammals, spermatogenesis is maintained throughout life by a small subpopulation of type A spermatogonia called spermatogonial stem cells (SSCs). In rodents, SSCs, or Asingle spermatogonia, form the self-renewing population. SSCs can also divide into Apaired (Apr) spermatogonia that are predestined to differentiate. Apaired spermatogonia produce chains of Aaligned (Aal) spermatogonia that divide to form A1 to A4, then type B spermatogonia. Type B spermatogonia will divide into primary spermatocytes that undergo meiosis. In human, there are only two different types of A spermatogonia, the Adark and Apale spermatogonia. The Adark spermatogonia are considered reserve stem cells, whereas the Apale spermatogonia are the self-renewing stem cells. There is only one generation of type B spermatogonia before differentiation into spermatocytes, which makes human spermatogenesis less efficient than in rodents. Although the biology of human SSCs is not well known, a panel of phenotypic markers has recently emerged that is remarkably similar to the list of markers expressed in mice. One such marker, the orphan receptor GPR125, is a plasma membrane protein that can be used to isolate human SSCs. Human SSCs proliferate in culture in response to growth factors such as GDNF, which is essential for SSC self-renewal in mice and triggers the same signalling pathways in both species. Therefore, despite differences in the spermatogonial differentiation scheme, both species use the same genes and proteins to maintain the pool of self-renewing SSCs within their niche. Spermatocytic seminomas are mainly found in the testes of older men, and they rarely metastasize. It is believed that these tumours originate from a post-natal germ cell. Because these lesions can express markers specific for meiotic prophase, they might originate from a primary spermatocyte. However, morphological appearance and overall immunohistochemical profile of these tumours indicate that the cell of origin could also be a spermatogonial stem cell.

Micro-bioreactors for fed-batch fermentations with integrated online monitoring and microfluidic devices.

In this study an array of micro-bioreactors based on the format of 48-well microtiter plates (MTP) is presented. The process parameters pH-value and biomass are monitored online by a combination of different sensors, the biolector measurement technology and conductance measurements. A microfluidic device dispenses two fluids individually into each well for controlling the pH-value of fermentations. The micro-bioreactor consists of four wells and two reservoirs. In each well a polyimide foil with platinum electrodes for conductance measurements is integrated. The microfluidic device is fabricated using softlithographic techniques and utilizes pneumatically actuated microvalves. The device is able to dispense volumes below 5nl. Finally, fermentations of Escherichia coli are carried out in the micro-bioreactor system. During the fermentation, the pH-value is measured optically and the biomass development is monitored by the scattered light signal. Meanwhile, the pH-value is controlled by dispensing sodium hydroxide and phosphoric acid. This micro-bioreactor demonstrates the possibility of online monitored and pH-controlled fermentations in micro-scale. The pH-value in the uncontrolled culture varies within the range of 6.46-8.83 whereas the pH-value in the controlled cultures can be kept within 6.85-7.07. This results in an increase in biomass in the pH-controlled culture compared to the nearly completely inhibited pH-uncontrolled culture.

Regulation of the spermatogonial stem cell niche.

Spermatogonial stem cells (SSCs) reside within specialized microenvironments called 'niches', which are essential for their maintenance and self-renewal. In the mammalian testis, the main components of the niche include the Sertoli cell, the growth factors that this nursing cell produces, the basement membrane, and stimuli from the vascular network between the seminiferous tubules. This review focuses on signalling pathways maintaining SSCs self-renewal and differentiation and describes potential mechanisms of regulation of the spermatogonial stem cell niche.

[The value of the Kapandji-Sauvé procedure with considering clinical results and measurement of bone density. A clinical study]. / Die Wertigkeit der Operation nach Kapandji unter Berücksichtigung von klinischer Nachuntersuchung und postoperativer Knochendichtemessung - Eine klinische Studie -

Between 1989 and 1995, 33 patients were treated with a Kapandji-Sauvé procedure for malunited fracture of the distal radius and instabilities of the distal radioulnar joint. Thirty patients were followed up with a mean follow-up time of 91 months. Fourteen patients underwent a measurement of bone density of the distal forearm. Twenty-eight patients showed good ossification of the distal radioulnar arthrodesis. Forearm rotation improved by 17.3 %. Mean grip strength was 72 % of that of the contralateral hand. Evaluation by the Cooney score resulted in 10 % very good, in 65 % good, 22 % fair and in 3 % poor results. The measurement of bone density of the distal radius showed an increase of rotation and flexure firmness. The cortical density remained constant. In the subcortical bone of the distal radius, we found a decrease of the trabecular density in the radial part.

Immunomagnetic isolation and long-term culture of mouse type A spermatogonia.

In the mammalian testis, type A spermatogonia proliferate and differentiate into sperm under the tight control of both endocrine and paracrine factors. In order to study the complex process of spermatogenesis at the molecular level, an in vitro system must be devised in which type A spermatogonia can be cultured for a prolonged period of time. Therefore, cocultures including type A spermatogonia and Sertoli cells, which act as nurse cells to the developing germ cells, are desirable. We have developed a method for the specific isolation of type A spermatogonia using magnetic beads and antibodies that recognize the c-kit receptor or the homophilic adhesion molecule, Ep-CAM. Purified spermatogonia could survive for a period of 25 days when cocultivated on Sertoli cell monolayers. Moreover, we recently established Sertoli cell lines that produce growth factors that are essential for the maintenance of spermatogonia in a proliferative state. Some of these Sertoli cell lines are able to reorganize into tubular structures when cultivated on a layer of Matrigel as extracellular matrix. We show here that type A spermatogonia associate specifically with the Sertoli cell tubules, and are able to replicate their DNA in this environment. Thus, these in vitro culture systems could be used for the long-term culture of primary, nonimmortalized type A spermatogonia.

Transcriptional pathways induced by fluid shear stress in mouse preosteoblast cells.

Recent findings support the idea that fluid movement in the bone is a key factor in bone formation and related cellular responses. However, the precise molecular mechanisms are not known. Our data demonstrates that fluid shear stress is a key factor in the activation of specific transcriptional pathways in murine preosteoblast (MC3T3E1) cells. MC3T3E1 cells have increased expression of the egr-1 and p57kip2 genes after being subjected to 0.3 dynes/cm2 of fluid shear stress. The MC3T3E1 cells are already known to express egr-1, a multipotent transcription factor, after high levels of fluid shear stress, but this is the first demonstration of egr-1 expression after low levels of fluid shear stress. Moreover, this is the first study showing expression of p57kip2 by the MC3T3E1 cells after fluid shear stress. The expression of p57kip2, a cyclin dependent kinase inhibitor, is a strong indicator that the cells are exiting the cell cycle and are beginning to differentiate. Our data shows decreased 3H-thymidine incorporation up to 18 hours post stress, which agrees with the upregulation of p57kip2 as a result of fluid shear stress.

An in vitro tubule assay identifies HGF as a morphogen for the formation of seminiferous tubules in the postnatal mouse testis.

We have been working with a recently immortalized Sertoli cell line, SF7, that appears to produce sleeves, or hollow tubules, when cultivated on a layer of growth factor-reduced Matrigel (GFR-Matrigel) in medium supplemented with serum. We tried to determine which components of GFR-Matrigel and serum provide the environment needed for tubule formation. While laminin and collagen IV were essential for the formation of flat cords, none of the basement membrane constituents, when taken alone or in combination, would support the formation of tubules in minimal culture medium. Moreover, none of the growth factors present in GFR-Matrigel could induce tubulogenesis. Recently, much attention has been focused upon the role of hepatocyte growth factor (HGF) and its receptor c-met in the induction of tubulogenesis by epithelial cells. Therefore, we investigated the expression of HGF/c-met in the mouse testis at different postnatal stages and in the adult and evaluated the contribution of HGF/c-met in the production of Sertoli cell tubules by SF7 and primary Sertoli cells in vitro. Our results confirm that laminin and collagen IV are essential for the formation of testicular cords and reveal that HGF/c-met are necessary for the further remodeling of these cords into tubules.

Immunomagnetic isolation of osteoprogenitors from human bone marrow stroma.

We report here the efficient and specific isolation of human bone marrow osteoprogenitors using magnetic beads coated with a mouse mAb (STRO-1) that recognizes a stromal cell surface protein. According to their pattern of differentiation, osteoprogenitors accounted for 100% of the STRO-1-positive cell population. Upon long term culture, osteoprogenitors differentiated down the osteoblastic lineage as evidenced by their in vitro morphology, their increased expression of alkaline phosphatase, their production of osteocalcin, and the deposition of a mineralized matrix. Upon differentiation, the cells first expressed alkaline phosphatase on their surface before they began proliferating as phenotypically recognizable osteoblasts. This observation conflicts with previous studies that have characterized human osteoprogenitors as highly proliferative cells that form colonies before differentiating into osteoblasts. The ability to isolate and cultivate pure populations of primary human osteoprogenitors will substantially advance investigations on osteogenesis and bone remodeling.

Establishment of a shear stress protocol to study the mechanosensitivity of human primary osteogenic cells in vitro.

We report here the establishment of a protocol to study the influence of mechanical stress on the behavior of primary human osteogenic cells. We first developed a method for the specific isolation of human bone marrow osteoprogenitors and studied their potential of differentiation into osteoblasts in vitro. Then, using NIH-3T3 cells as a model for cells of mesenchymal origin, we employed a parallel plate flow chamber apparatus to apply shear stress to cells in culture. This technique will be useful to determine the influence of shear stress on the rate of proliferation and differentiation of human osteoprogenitors and osteoblasts. Ultimately, this line of research will extend our knowledge on osteogenesis and bone remodeling.

[Vascular pedicled transposition of the pisiform bone for treatment of lunate malacia]. / Die gefässgestielte Transposition des Os pisiforme zur Behandlung der Lunatummalazie.

In a prospective study we investigated the results of 18 patients with Kienböck's disease stage II as defined by Decoulx, treated with transposition of the pedicled pisiform. In eight cases of minus variance of the ulna, a radius shortening osteotomy was performed. There was an average follow-up of 30 months, X-ray investigations were done every six months after operation. 17 patients had less pain, 14 patients showed an improved range of motion of 30 degrees. Magnetic resonance imaging proved vitalizing of the pisiforme in 16 cases.

[Restoration of grip function in both hands after frostbite of all fingers and toes. A case report]. / Wiederherstellung einer Greiffunktion beider Hände nach Erfrierung sämtlicher Finger und Zehen. Ein Fallbericht.

Evaluation, therapeutical possibilities, and surgical planning for the restoration of bilateral grip function are described, based upon a 34-year-old engineer presenting after severe bilateral frostbite of the hands and feet with loss of all fingers and toes. Finally, the given level of amputation and remaining components of the hand together with distraction-lengthening of both thumbs permitted restoring a basic grip on both sides in combination with bone and soft tissue procedures.

Refinement of the differentiated phenotype of the spermatogenic cell line GC-2spd(ts)

A transformed spermatogenic cell line GC-2spd(ts), recently reported to express a protein marker of spermiogenesis, was tested for the presence of several mRNAs encoded by genes transcribed specifically in the testis and at precise stages of spermatogenesis. Northern blotting and reverse-transcriptase polymerase chain reaction techniques showed that mRNAs for the stage-specific marker proteins LDH-C4 (preleptotene), acrosin (premeiotic), protamine-2 (postmeiotic), and SP-10 (postmeiotic round spermatid stage) were not detected in GC-2spd(ts) cells. Flow cytometric analysis of GC-2spd(ts) failed to detect a peak indicative of the presence of haploid chromosomes. Furthermore, the HS-63 monoclonal antibody, employed in an earlier report to demonstrate putative proacrosomal granules, failed to recognize the SP-10 protein in extracts of human or mouse sperm or in GC-2spd(ts) cells and instead recognized proteins of different masses. In view of interest in this line as a model for analyzing molecular events of spermatogenesis, this refinement of the GC-2spd(ts) phenotype may aid others considering these cells for studies of terminal stages of sperm differentiation.

Can insights into urodele limb regeneration be achieved with cell cultures and retroviruses?

Recent advances in the field of amphibian limb regeneration have provided insights into its cellular and molecular events. This review summarizes the development of cell lines from limb tissues and their application to the study of transdifferentiation and limb regeneration. In addition, the availability of suitable retroviral vectors for salamanders is discussed for it has opened new avenues for experimentation at the molecular level.

A haploid and a diploid cell coexist in an in vitro immortalized spermatogenic cell line.

We have recently established a conditionally immortalized germ cell line [GC-2spd(ts)] that, at the permissive temperatures of 37 degrees C and 32 degrees C, is able to undergo meiosis in vitro and form round spermatids [Hofmann et al., (1994): Proc Natl Acad Sci USA 91:5533-5537]. In this report, we provide data that indicate that the GC-2spd(ts) cell line consists of two cell populations undergoing a haploid (n/2n) cell cycle and a diploid (2n/4n) cell cycle, respectively. The cells containing 2n DNA, when sorted by fluorescence-activated cell sorting, are able to reconstitute the full population of n/2n/4n DNA cells, indicating that they are able to commit to the reductive meiotic division and form haploid spermatids or to continue self-replication through a diploid cell cycle. This GC-2spd(ts) cell line provides a valuable tool to study the molecular mechanisms involved in the cellular decision between self-renewal by mitosis and commitment to meiosis.

Immortalized germ cells undergo meiosis in vitro.

Establishing mammalian germ-cell lines capable of differentiation in vitro would greatly facilitate the study of gametogenesis and the meiotic process that is so fundamental for reproduction and the maintenance of genetic diversity of the species. We have established two germ-cell lines [GC-2spd(ts) and GC-3spc(ts)] by cotransfecting primary mouse testicular germ cells with the simian virus 40 large tumor antigen gene and the gene coding for a temperature-sensitive mutant of p53. Both cell lines express the germ cell-specific lactate dehydrogenase C4 isozyme and cytochrome ct isoform. At the permissive temperature of 37 degrees C, the GC-2spd(ts) line generates cells with a haploid DNA content and morphologic and biochemical features of round spermatids, including the appearance of an acrosomic granule. The identification of a flagellar axoneme when these cells are cultured at 32 degrees C further indicates that these cells correspond to the early spermatid stages of spermiogenesis.

Genomic structure and promoter activity of the human testis lactate dehydrogenase gene.

The structure of the gene encoding the human testis-specific isozyme of lactate dehydrogenase (LDH) has been characterized and a regulatory region identified by promoter activity. The single-copy ldh-c gene has two alternative 5' noncoding exons and seven coding exons comprising an approximately 40-kb locus. The gene does not contain the canonical TATA or CAAT promoter sequences, and ribonuclease protection experiments suggest multiple transcription start sites. In the present study an immortalized murine germ cell line was used to detect promoter activity driven by 5' sequence of human ldh-c with lacZ as the reporter gene. Reporter gene activity was nondetectable when promoter constructs were transfected into nongerminal cells.

Developmental expression of alkaline phosphatase genes; reexpression in germ cell tumours and in vitro immortalized germ cells.

Human germ cell alkaline phosphatase (GCAP) is developmentally expressed in primordial germ cells and in trace amounts in the testis and thymus. The equivalent mouse isozyme, embryonic alkaline phosphatase (EAP), is similarly expressed in the testis and thymus but also from the 2-cell to blastocyst stage of preimplantation development. EAP has been found to be transiently expressed in M-phase spermatogenic cells in the mouse testis. These alkaline phosphatase isozymes serve as markers of germ cell differentiation and in the management of germ cell tumors. GCAP is expressed in carcinoma-in-situ and seminoma where serum GCAP levels are often elevated and may provide clinically useful information. However, GCAP is a polymorphic enzyme, and monoclonal antibodies to be used in the clinical evaluation of tumor tissues or fluids should be carefully evaluated for their ability to detect all allelic variants of GCAP.