Dose escalation study for defining the maximum tolerated dose of continuous oral trofosfamide in pretreated patients with metastatic lung cancer.

BACKGROUND: Trofosfamide is increasingly used in the treatment of patients with several types of malignancies. However, the optimal dose of trofosfamide for patients with advanced cancer has not been systematically investigated yet. The aim of this study was to define the maximum tolerated dose (MTD) of continuous oral trofosfamide. PATIENTS AND METHODS: 16 patients with advanced lung cancer (14 nonsmall cell lung cancer, 2 small cell lung cancer; 10 male, 6 female; median age 64 years (range 46-82); median Karnofsky status 70%; median number of organs involved 3 (range 1-6)) were enrolled. All patients were previously treated with chemotherapy (median 2x, range 1-6) and 8/16 (50%) with radiotherapy. Patients received trofosfamide p.o. administered in 3 doses per day for 3 weeks (1 cycle) using a 3-patient-cohort dose-escalation strategy. Toxicities were graded according to the WHO Criteria. RESULTS: Patients received a median of 2 cycles of trofosfamide (range 1-4) at 3 dose levels (90, 125, and 175 mg/m2). Grade 3 and 4 neutropenia, anemia, and thrombocytopenia were observed in 20, 13.3, and 6.6%, respectively. Dose-limiting toxicities during the first cycle were grade 3 muscle weakness and anorexia observed in 1/6 patients in cohort 1 (trofosfamide 90 mg/m2), grade 3 neutropenia in 1/6, and encephalopathy in 1/6 patients in cohort 3 (trofosfamide 175 mg/m2). Therefore, the dose level of 125 mg/m2 was defined as the MTD. CONCLUSION: Trofosfamide at 125 mg/m2 administered in 3 doses per day was well tolerated. This dose level is recommended for further clinical studies.

Expression of a novel factor, short-type PB-cadherin, in Sertoli cells and spermatogenic stem cells of the neonatal rat testis.

In the rodent testis, contact-mediated interactions between gonocytes, or neonatal stem cells, and Sertoli cells are critical for development. Previously, we showed that the neural cell adhesion molecule (NCAM) serves as a Sertoli cell-gonocyte attachment factor in neonates. Its expression decreases dramatically by 1 week of age and eventually disappears in vivo, and appears to be down-regulated by thyroid hormone (tri-iodothyronine (T(3))). In this study, we used a cDNA microarray to screen for additional adhesion factors which might be important in testes of developing rats and detected expression of a novel factor, short-type PB-cadherin (STPB-C). Next, RT-PCR was used to generate cDNA for STPB-C from total RNA isolated from co-cultures, cDNA was cloned into pPCR-Script Amp SK(+) cloning vector, and plasmid DNA was isolated and sequenced to confirm the fidelity of the STPB-C cDNA portion of the plasmid. In situ hybridization analyses of testicular sections indicated that STPB-C expression in neonates is localized in the cytoplasm of many, but not all, gonocytes and in the cytoplasm of most of the surrounding Sertoli cells. Parallel hybridizations carried out on co-cultures also demonstrated a strong cytoplasmic signal in some gonocytes and in the great majority of the Sertoli cells of the underlying monolayer. With Northern analyses we found that STPB-C is expressed in vivo at high levels between days 1 and 5, with a subsequent large drop by day 10 and thereafter, suggesting that its expression may be associated with Sertoli or germ cell differentiation. Subsequent analyses of co-cultures exposed under a variety of conditions to T(3) suggest that, unlike NCAM, STPB-C is not regulated by this hormone. Next, we studied production of STPB-C protein by using an antiserum recognizing a peptide sequence unique to this factor in Western blotting and in immunolocalization. Signal was detected both intracellularly and at cell surfaces in most Sertoli cells and many gonocytes, although many of the latter cell type were also found to be negative for the protein, suggesting a potential role for STPB-C in survival and further development of some of these germ cells from which all subsequent spermatogenic cells originate.

[Semiquantitative analysis of bone regeneration on porous metallic wire-mesh specimens with or without HA-coating in comparison to pure titanium in animal experiments]. / Semiquantitative Analyse des Knocheneinwachsverhaltens von Kobalt-Chrom-Molybdän-Prüfkörpern mit und ohne HA-Beschichtung im Vergleich zu Reintitan im Tierversuch.

INTRODUCTION: The aim of this study was to analyse the bone regeneration on porous metallic wire-mesh specimens of pure titanium, uncoated CoCrMb and HA-coated CoCrMb in a comparative animal experiment. MATERIAL AND METHODS: Uncoated and hydroxyapatite-coated CoCrMb (one-sided) specimens were tested in an animal experiment. The statistical interpretation was done macroscopically as well as microscopically. RESULTS: With regard to the histological and quantitative evaluation, it has been shown that the proximal coated CrCoMb implants were superior to the other specimen. The bone regeneration with the most distinctive extent of osseointegration has been seen by the HA-coated CrCoMb specimen. The uncoated distal CoCrMb specimen demonstrated the worst results. DISCUSSION: This study shows that specimens of titanium and uncoated CrCoMb implanted at the proximal metaphysis deliver comparable effects on the bone regeneration and osseointegration. The best results with regard to the bone regeneration and osseointegration were seen with the implanted hydroxyadaptite-coated CrCoMb specimen. In addition, it has been observed that the bone regeneration and osseointegration of the specimen depends on the place of implantation.

Biological activity of a C-terminal fragment of Pasteurella multocida toxin.

The protein toxin of Pasteurella multocida PMT is a potent mitogen and activator of phospholipase Cbeta. In this study different toxin fragments were investigated. A C-terminal fragment encompassing amino acids 581 through 1285 (PMT581C) was constructed, which was inactive toward intact embryonic bovine lung (EBL) cells after addition to culture medium but caused reorganization of the actin cytoskeleton and rounding up of cells when introduced into the cells by electroporation. As the holotoxin, the toxin fragment PMT581C induced an increase in total inositol phosphate levels after introduction into the cell by electroporation. A C-terminal fragment shorter than PMT581C as well as N-terminal fragments were inactive. Exchange of cysteine-1165 for serine in the holotoxin resulted in a complete loss of the ability to increase inositol phosphate levels. Correspondingly, the mutated toxin fragment PMT581C.C1165S was inactive after cell introduction by electroporation, suggesting an essential role of Cys-1165 in the biological activity of the toxin.

Characterization of the X-linked murine centrin Cetn2 gene.

A multi-gene family (Cetn1, Cetn2, and Cetn3) encodes the calcium-binding protein, centrin, in the mouse. This work characterizes the Cetn2 gene. Structurally, Cetn2 consists of five exons and four introns, and contains a classical TATA-less promoter. Cetn2 has two alternate transcription start sites, and a single length 3' untranslated region. Fluorescence in situ hybridization demonstrates that Cetn2 is an X-linked gene whose alleles replicate asynchronously during S-phase. Cetn2 encodes a 172 amino acid protein, with a predicted molecular mass of 19,795 Da (pI=4.71), that contains all of the defining characteristics of centrin. Northern blot analysis indicates that Cetn2 is ubiquitously expressed in the tissues of adult mice. RT-PCR shows that Cetn2 and Cetn3, but not Cetn1, are expressed in NIH 3T3 cells. Immunofluorescence microscopy demonstrates that mouse centrin 2 protein localizes to the region immediately surrounding the centrioles in the centrosome of NIH 3T3 cells.

Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape.

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

Gonocyte-Sertoli cell interactions during development of the neonatal rodent testis.

During neonatal testicular development in the rat, events critical for subsequent germ cell development occur that set the stage for fertility later in life. Some gonocytes resume mitotic activity and/or migrate to the surrounding basal lamina, and use of a carefully defined Sertoli cell-gonocyte coculture system indicates that these crucial events occur without added factors or hormones and are hence likely to depend on interaction with adjacent Sertoli cells. Coupling of the Kit receptor protein on gonocytes to stem cell factor from Sertoli cells is vital for successful migration by gonocytes, as antagonism of the former suppresses and addition of the latter stimulates gonocyte migration. During the neonatal period, intercellular adhesion is modified in a developmental manner such that neural cell adhesion molecule (NCAM) is the main adhesive molecule expressed and functioning at birth, with a progressive decline as development proceeds. This decline in NCAM expression is supported by the addition of exogenous 3,3',5-triiodothyronine in vitro, and because this factor is recognized as supporting Sertoli cell differentiation, it seems likely that changing intercellular adhesion is a function of progressive development of Sertoli cells. Other avenues whereby maturing testicular cells influence each other doubtless exist, including secretion of growth factors and other peptides and developmentally important changes in the makeup of the extracellular matrix, which Sertoli cells and gonocytes contact. Continued investigation in these areas will be very valuable in enlarging our understanding of how neonatal testicular development provides the basis for successful spermatogenesis.

A single dose of Di-(2-ethylhexyl) phthalate in neonatal rats alters gonocytes, reduces sertoli cell proliferation, and decreases cyclin D2 expression.

In this study, we explored the impact on both Sertoli cells and gonocytes of a single, relatively low dose of di-(2-ethylhexyl) phthalate (DEHP; 20-500 mg/kg) administered in vivo to 3-day-old rat pups. In parallel, we assessed the potential for two immediate metabolites of DEHP to produce similar testicular changes and began to explore the possible mechanisms involved. Morphological examination revealed the presence of many abnormally large, multi-nucleated germ cells by 24 h posttreatment with DEHP and with its metabolite, mono-ethylhexyl phthalate (MEHP), but not with another metabolite, 2-ethylhexanol (2-EH; all at 1.28 mmol/kg) or with vehicle alone. These cells persisted through 48 h posttreatment, the longest time point examined in our study. We also assessed the rate of Sertoli cell proliferation in pups at intervals after dosage with either chemical or vehicle by administering bromodeoxy uridine (BrdU) 3 h before euthanasia. By 24 h after treatment with DEHP or MEHP, but not 2-EH or vehicle, the number of BrdU-labeled Sertoli cells was obviously diminished in testicular sections. Quantitation of DEHP-treated pups and controls indicates that a dose-response relationship exists between chemical treatment and labeling index (LI) of Sertoli cells, with a LI at the highest DEHP dose tested that was only 20% of that in controls. In addition, when we examined the time course of the effect of an intermediate dose of DEHP, we found that there the LI of Sertoli cells rebounds by 48 h after dosage, when we found the rate of proliferation in treated pups to be significantly higher than in controls. We also explored the potential mechanism involved in the response to DEHP and found serum levels of FSH to be unaffected by the chemical. In addition, study of cell cycle-related proteins including p27kip1 and cyclins D1, D2, and D3 with Western and Northern analysis indicated that cyclin D2 mRNA is specifically down-regulated by DEHP in a dose-dependent manner, and this decrease is manifest as a small, transient but reproducible reduction in the amount of cyclin D2 protein detectable in samples from treated pups compared to controls. Our findings characterize the changes in neonatal Sertoli cells and gonocytes that follow in vivo to low levels of DEHP and its metabolite, MEHP, as well as providing new information on the underlying mechanism and highlighting the extreme sensitivity of the neonatal testis to injury by this toxicant.

Clonal expansion of Melan A-specific cytotoxic T lymphocytes in a melanoma patient responding to continued immunization with melanoma-associated peptides.

Peptides derived from human tumor antigens have been used in a number of clinical trials to induce specific immune responses against autologous tumors in cancer patients. Although favorable clinical results were observed in single patients, immune responses correlating with tumor regression were either not detected or in case of responses, the T-cell specificity was difficult to demonstrate. In this study, we analyzed antigen-specific T-cell responses induced in the skin and in peripheral blood lymphocytes (PBL) in an HLA-A2-positive melanoma patient. The patient showed major regression of metastatic melanoma under continued immunization with peptides derived from the melanocyte differentiation antigens Melan A/MART-1, tyrosinase and gp100/Pmel17. Based on the identification of different T-cell receptor (TCR) families reactive with Melan A/MART-1, we have demonstrated that i.d. immunization with peptides alone leads to oligoclonal expansion of Melan A/MART-1-specific cytotoxic T lymphocytes (CTL), detectable in local delayed-type hypersensitivity (DTH) reactions and PBL. A monoclonal expansion of a Melan A/MART-1-specific TCR VB 16 CTL was reproducibly observed after in vitro stimulation with Melan A/MART-1 peptides. The same TCR VB 16 CTL clone was detected in skin biopsies taken from vitiligo areas. Our findings provide strong evidence for the effective induction of specific T-cell responses to Melan A/MART-1 by i.d. immunization with peptide alone, which accounts for dermal depigmentation, specific cytotoxicity against Melan A/MART-1-expressing melanoma cells and clinical tumor regression.

Thyroid hormone down-regulates neural cell adhesion molecule expression and affects attachment of gonocytes in Sertoli cell-gonocyte cocultures.

Contact-mediated interactions between Sertoli cells and gonocytes are important for testicular development. Specifically, down-regulation of neural cell adhesion molecule (NCAM)-based intercellular adhesion during postnatal maturation is likely to be important for appropriate differentiation of testicular cells. Besides NCAM, P-cadherin is also present in neonatal testicular cords, at least in mice, and seems to disappear from the seminiferous epithelium after the first postnatal week. Another factor known to be important in regulating development of the neonatal testis is thyroid hormone (T3). T3 is involved in control of Sertoli cell proliferation and differentiation. Therefore, we examined the effect(s) of T3 on adhesive factors found within the testis using Sertoli cells and gonocytes isolated from neonates and maintained in coculture. T3 (100 nM) down-regulated NCAM expression in vitro, as assessed by Western blotting and immunofluorescent staining. This contrasted with the continued expression of NCAM in cultures without added T3 but mimicked the disappearance of NCAM from the neonatal rat testis in vivo. In addition, Western analysis confirmed that P-cadherin is highly expressed in the developing rat testes, as it is in those of mice. We found that P-cadherin is strongly expressed in gonocytes and weakly expressed in Sertoli cells. Moreover, unlike NCAM, P-cadherin expression diminishes with time in vitro in the absence of added hormones. In parallel with our observations for NCAM, expression of P-cadherin was also apparently decreased by T3 (100 nM). Subsequent quantitative analyses of cultures exposed to a range of T3 levels (0.1-100 nM) indicated that T3 causes detachment of many gonocytes in a dose- and time-dependent manner (approximately 80% detached at 100 nM). In addition, Western blotting indicated that lower concentrations of T3 down-regulate NCAM but not P-cadherin. From this we conclude that the apparent decrease in P-cadherin induced by 100 nM T3 and detected on Western blots reflects loss of gonocytes. In contrast, even low levels of T3 appear to down-regulate NCAM production before any significant detachment of gonocytes. Finally, low levels of T3 that did not affect numbers of adherent Sertoli cells nevertheless caused detachment of gonocytes. Thus, our observations identify T3 as a regulator of NCAM expression in neonatal testicular cells and as a modifier of gonocyte/Sertoli cell adhesion in vitro.

Testis-specific murine centrin, Cetn1: genomic characterization and evidence for retroposition of a gene encoding a centrosome protein.

Centrin is a centrosome component in species from yeast to humans. Here, the mouse centrin 1 gene (Cetn1) is analyzed with respect to its genomic structure, chromosome localization, tissue-specific expression, and phylogenetic relationship to the other mouse centrin genes and their human orthologs. Cetn1 is an intronless gene located on chromosome 18A2 that encodes a 172-amino-acid protein with a predicted molecular mass of 19,696 Da (pI 4.61) and all of the structural features common to centrin. Cetn1 possesses the sequence features of an expressed retroposon: the gene lacks introns, the open reading frame is not interrupted by stop codons, and the coding region is flanked by a pair of direct repeats. Reverse transcriptase-polymerase chain reaction and Northern blot analysis demonstrate that Cetn1 expression is limited exclusively to the testis in adult male mice. Cetn1 expression is first seen in the neonatal testis at 14 days postpartum, reaching adult levels by day 17. These observations provide new insight into the regulation, function, and evolutionary history of centrin in higher eukaryotes.

Human bone marrow cell culture: a sensitive method for determination of the biocompatibility of implant materials.

The objective of this study was to develop a test method for determining the cytotoxicity and biocompatibility of various biomaterials that are used in orthopaedic surgery. This method is based on the use of a human bone marrow cell culture and was developed as an alternative to animal experiments. Human bone marrow cell culture has certain advantages over other cell culture models, as its results show a greater conformity with animal experimental results and clinical studies. Primary cell adherence, cell number, cell proliferation, production of extracellular matrix, cell viability and cell differentiation were used as indicative parameters of biocompatibility. After 2 weeks in culture, differences could be observed between the biomaterials with respect to these parameters. Cell numbers were greatest on the hydroxyapatite ceramic specimens, but were decreased on the titanium alloy specimens. Extracellular matrix hydroxyapatite production was high for ceramics, but reduced for titanium specimens. The polymers allowed only a few cells to adhere, and there were no signs of extracellular matrix production. The influence of biomaterials on differentiation of large numbers of cells was analysed by using flow cytophotometry. There were similar populations of T cells and monocytes on all specimens. However, extended B cell and granulocyte populations were observed with titanium and polyethylene.

[Acoustic emission analysis of human bones within the scope of clinical diagnosis]. / Schallemissionsanalyse an menschlichen Knochen im Rahmen der klinischen Diagnostik.

Fractures occurring in human bones produce an acoustic signal, analysis of which permits an evaluation of its source. In the industrial setting acoustic emission analysis (AEA) is used to non-invasively monitor the function of stressed technical systems or parts of systems. During servicing and monitoring of technical systems, acoustic signals emitted by cracks or material deformation are located with the aid of a few acoustic sensors and evaluated for risk-identification purposes. With appropriate technology, therefore, both cortical and trabecular bone can be monitored by acoustic emission analysis. A search is currently ongoing for suitable acoustic technology capable of assessing the extent and location of bone defects and predicting associated risks of fractures occurring. In the present study a system for the measurement and analysis of acoustic emission is described which permits the measurement and analysis of acoustic signals obtained from processed and fresh human and porcine femora. In slightly modified form this system was then used to assess the type and extent of acoustic emission obtained from explanted human femora exposed to cyclical torsional loading until fracture occurred.

Expression of 140-kDa neural cell adhesion molecule in developing testes in vivo and in long-term Sertoli cell-gonocyte cocultures.

The basis for cell-cell adhesion in the seminiferous epithelium of the developing testis is doubtless critical in supporting events that are essential for the onset and maintenance of normal spermatogenesis. In this study, we applied immunoblotting and immunolocalization approaches for the following reasons: 1) to ask whether neural cell adhesion molecule (NCAM) underlies cell-cell interactions in vivo, as we previously showed for cells in vitro, 2) to characterize the isoform or isoforms of NCAM expressed during testicular development, and 3) to study NCAM expression in long-term Sertoli cell-gonocyte cocultures and to compare and contrast this pattern of expression with that in vivo. Our findings indicate that NCAM is found ubiquitously at cell-cell interfaces within the seminiferous cord from birth through day 10 and thereafter is restricted to interstitial cells. Moreover, only polysialic acid-negative 140-kDa NCAM is expressed in the testis or in coculture, an isoform whose properties are compatible with the concept of NCAM as both a direct modifier of cell function and an indirect influence on cell responses mediated by other external factors. In addition, we found that germ cells, potentially gonocytes or Type A spermatogonia, persist in long-term cocultures maintained for 15 days after isolation from 5-day-old rat pups and that NCAM continues to be expressed at high levels in these cultures. This observation is in marked contrast to our observation that NCAM gradually decreases and eventually disappears in vivo by postnatal day 15. Thus, our findings indicate that 140-kDa NCAM is prominent in neonatal testes but is down-regulated by as yet unidentified mechanisms thereafter. Our findings also indicate that down-regulation of NCAM fails to occur in hormone- and serum-free Sertoli cell-germ cell cocultures.

Biocompatibility analysis of different biomaterials in human bone marrow cell cultures.

A cell culture system for biocompatibility testing of hip implant materials is described. Human bone marrow cells have been chosen because these cells are in direct contact with the biomaterial after implantation in situ. The sensitivity of this method is evaluated for materials which are already being used as implants in humans and animal, e.g., hydroxyapatite (HA) ceramic, pure titanium, and ultra-high-molecular-weight polyethylene (UHMWPE). As indicative parameters of biocompatibility primary cell adherence, cell number, cell proliferation, production of extracellular matrix, cell vitality, and cell differentiation are described. After 2 weeks in culture, obvious differences between the biomaterials with respect to the indicative parameters could be observed. Cell numbers were greatest on the HA specimens. In the case of titanium alloys, we observed a decreased number of cells. The production of extracellular matrix was high for the HA ceramics but reduced for titanium specimens. The polymers allowed only a few adherent cells and showed no signs of extracellular matrix production. The results can be correlated astonishingly well to animal experiments and clinical experiences. Therefore, we suggest that this cell culture system seems to be a useful tool for biocompatibility testing of bone implantation materials. It also helps reduce animal experiments. With the help of flow cytophotometry, we analyzed the influence of biomaterials on large numbers of cells with respect to differentiation. There were similar populations of T cells and monocytes on all specimens tested. Extended B-cell and granulocyte populations, however, were observed with titanium and UHMWPE. Most osteocalcin-containing cells adhered to the HA ceramics.

Effects of relatively low levels of mono-(2-ethylhexyl) phthalate on cocultured Sertoli cells and gonocytes from neonatal rats.

Di-(2-ethylhexyl) phthalate (DEHP), one of the abundant man-made environmental chemicals, induces testicular damage in both developing and adult animals. However, the nature and mechanism underlying the action of phthalates on testicular development remain largely unexplored. In the present study, we used cocultures of neonatal Sertoli cells and gonocytes (precursors of spermatogonia) to characterize in detail the effects of mono-(2-ethylhexyl) phthalate (MEHP; the active metabolite of DEHP) on these cells and to explore the underlying mechanism(s). Sertoli cells and gonocytes were isolated from rat pups on the 2nd day after birth, cocultured, and exposed to MEHP at concentrations of 0.01, 0.1, or 1.0 microM, or to 0.5% DMSO (vehicle control), or 10 microM DEHP (negative control) for a total of 48 h. We found that exposure to MEHP induced gonocyte detachment from the Sertoli cell monolayers in a time- and dose-dependent manner. When exposed to 1.0 microM MEHP, many gonocytes started to detach after 12 h of exposure and most gonocytes were lost during the media change at 24 h. Gonocyte detachment was also observed in cocultures treated with 0.1 microM MEHP for 24 h of exposure, but not in cultures treated with 0.01 microM MEHP for 48 h. Detached gonocytes were viable as indicated by their ability to exclude trypan blue. Furthermore, when proliferation of cultured Sertoli cells was detected by BrdU labeling and subsequently quantified, we found that exposure to 0.1 or 1.0 microM MEHP for 48 h resulted in a decrease in labeling indices of 33.6 and 83.6%, respectively, compared to the vehicle control (p < 0.01), while the labeling index was unchanged by treatment with 0.01 microM MEHP. In addition, we also tested the potential effect of MEHP on FSH-stimulated Sertoli cell proliferation by simultaneously treating cultures with 200 ng/ml human FSH and different concentrations of MEHP for 48 h. Exposure to 0.1 or 1.0 microM MEHP resulted in decreases of 24.2 and 74.2%, respectively, in FSH-stimulated Sertoli cell proliferation (p < 0. 01). Furthermore, MEHP also inhibited dibutyl cAMP-stimulated Sertoli cell proliferation, regardless of whether dibutyl cAMP was added to the cultures before or at the same time as MEHP. Finally, addition of FSH or dibutyl cAMP had no effect on MEHP-induced gonocyte detachment, and none of the observed effects on either Sertoli cells or gonocytes were detected in control cultures treated with 0.5% DMSO only or with 10 microM DEHP. Therefore, short exposure to low levels of MEHP disrupted adhesion of gonocytes to Sertoli cells and inhibited both basal and FSH-stimulated Sertoli cell proliferation in a dose-dependent manner. The lowest effective dose of MEHP in vitro was 0.1 microM, which is about 10- to 1, 000-fold lower than the dose shown to affect Sertoli cells from prepubertal animals. Moreover, our data indicate that MEHP impairs division of neonatal Sertoli cells by acting at a post-cAMP site in the FSH-response pathway or via a mechanism independent of FSH. These data provide direct new evidence that relatively low levels of MEHP disrupt Sertoli cell-gonocyte physical interactions and suppress Sertoli cell proliferation in neonates via mechanisms specific to neonatal testis where the foundations of adult fertility are established. The results also highlight the neonatal period of testicular development as one particularly sensitive to environmental chemicals.

Use of in vitro systems to study male germ cell development in neonatal rats.

The aim of this review is to summarize ways in which in vitro approaches have allowed us to investigate several aspects of gametogenesis in the male. In our laboratory, we have established both organ culture and cell co-culture methodologies and applied them to questions focused on cellular and molecular events important for development of primitive spermatogonia, or gonocytes, in testes of neonatal rats. We have described their postnatal reinitiation of mitosis and their migration to the basal lamina in anticipation of basal compartment formation and, through use of these in vitro systems, we have identified several mechanisms regulating these processes. These include matrix influence on mitosis and migration, adhesive mechanisms active between gonocytes and Sertoli cells, and involvement of the Kit receptor on germ cells and its ligand from Sertoli cells in supporting gonocyte migration, as described below.

Expression of the c-kit gene is critical for migration of neonatal rat gonocytes in vitro.

Rat gonocytes migrate to the basement membrane during the first postnatal week, a change in position crucial for their survival. These cells express the c-kit gene from the day of birth through Day 5 in vivo and develop the ability to migrate in Sertoli cell-gonocyte cocultures. In this study, we asked whether c-kit expression and synthesis of Kit protein are required for pseudopod production by gonocytes in vitro. To determine whether gonocyte migration in vitro is invariably accompanied by c-kit expression, we quantified percentages of gonocytes expressing c-kit with increasing time in vitro and correlated these data with pseudopod development by individual cells. We also determined the effect of exposure to Kit antibodies on gonocyte migration in vitro, and, conversely, asked whether addition of exogenous stem cell factor (SCF), the Kit ligand, stimulates pseudopod development. We found that 1) increasing numbers of gonocytes express c-kit with increasing time in vitro; 2) once these cells begin migrating in vitro, the appearance of a pseudopod on a gonocyte is absolutely correlated with kit expression by that cell; 3) incubating cocultures with Kit antibodies significantly reduces the number of cells with pseudopods, without any detectable decrease in numbers of gonocytes; and 4) addition of exogenous SCF to cocultures prepared on Day 5 results in a transient but significant increase in the percentage of gonocytes with pseudopods even though we found that Sertoli cells in the cultures produce endogenous SCF. Thus, our findings provide evidence to support a role for c-kit expression by neonatal gonocytes and, presumably, SCF expression by neonatal Sertoli cells in stimulating migration of these germ cells in vitro.

Arrest of spermatogenesis and defective breast development in mice lacking A-myb.

The Myb gene family currently consists of three members, named A-, B- and c-myb. These genes encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In adult male mice, A-myb is expressed predominantly in male germ cells. In female mice, A-myb is expressed in breast ductal epithelium, mainly during pregnancy-induced ductal branching and alveolar development. We report here that mice homozygous for a germline mutation in A-myb develop to term but show defects in growth after birth and male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb-/- males revealed that the germ cells enter meiotic prophase and arrest at pachytene. In adult homozygous null A-myb female mice, the breast epithelial compartment showed underdevelopment of breast tissue following pregnancy and the female mice were unable to nurse their newborn pups. These results demonstrate that A-myb plays a critical role in spermatogenesis and mammary gland development.

Hst7: a male sterility mutation perturbing sperm motility, flagellar assembly, and mitochondrial sheath differentiation.

Hst7, a mouse hybrid sterility locus, has been mapped in close linkage to four other hybrid sterility loci, on proximal chromosome 17 within the t complex. When an allele (s) of Hst7 from the species Mus spretus is crossed into the Mus musculus domesticus (laboratory mouse) background, all male offspring are sterile. This occurs regardless of whether the Hst7 allele on the other chromosome 17 homolog is wild-type (+) or an allele (t) derived from the structurally variant homolog known as a t haplotype. Males of the Hst7 genotype s/+ produce sperm that, after release from the cauda epididymis, display moderate asthenospermia (straight line velocity = 49 +/- 4 microm/second, significantly lower than 102 +/- 7 microm/second for congenic wild-type controls) and normal morphology. However, males of the Hst7 genotype s/t produce sperm whose forward movement is below the detectable limit of the sperm motion analysis system. In addition, these sperm exhibit a variety of flagellar abnormalities, with about one third having normal heads attached to sacklike caudal regions. These sacks consist of membrane-delimited cytoplasm containing disorganized and/or misshapen axonemal elements. The remainder of the sperm from s/t mice have flagella with seemingly normal axonemes, although many exhibit enlarged areas of cytoplasm in their midpieces with extra layers of misaligned mitochondria. The s/t sperm mitochondria also display diffuse and vacuolated matrices reminiscent of meiotic germ cell and spermatid mitochondria. Observations of developing spermatids in the s/t testis reveal an unusual phenotype in which gaps of varying length occur in the mitochondrial wrapping of the midpiece. These data suggest that both the s and t alleles of Hst7 are defective alleles that contribute differentially to the severe asthenospermia phenotype and interact genetically to perturb flagellar development.