H-2M3 encodes the MHC class I molecule presenting the maternally transmitted antigen of the mouse

Mta, the maternally transmitted antigen of mice, is a hydrophobic, N-formylated mitochondrial peptide, MTF, presented on the cell surface to cytotoxic T lymphocytes by a novel major histocompatibility complex class I molecule, encoded by H-2M3. We have cloned and sequenced two alleles of M3, which differ in their ability to present MTF despite greater than 99% identity in the coding regions. M3 is as divergent from classical, antigen-presenting H-2 molecules as from other class I genes of the Hmt and the Qa/Tla regions. Amino acids critical for folding of class I molecules are conserved in M3. Noncharged amino acids lining the peptide-binding groove and phenylalanine 171 may explain the unique interaction with MTF, and leucine 95 appears critical for immunological activity.     

Protein dynamics in sperm membranes: implications for sperm function during gamete interaction.

A number of mammalian sperm plasma membrane antigens have been implicated as playing a functional role in sperm-egg interaction, by virtue of the fact that antibodies against these antigens interfere with fertilization. Two such mouse sperm plasma membrane antigens are M42, a 200/220 kD glycoprotein doublet, and M5, a 150-160 kD glycoprotein. We show that both of these antigens are concentrated on the posterior region of caudal epididymal and capacitated mouse sperm heads and are relatively diffusible, as determined by fluorescence recovery after photobleaching measurements (D = 3-8 x 10(-9) cm2/s with approximately 23% diffusing). Crosslinking of these antigens with bivalent antibodies causes them to redistribute into the anterior region (acrosomal crescent) of the sperm head. In contrast, we describe a third antigen, P220, which is also localized to the posterior region of the sperm head on caudal epididymal sperm but which exhibits very little diffusion and does not redistribute upon crosslinking. Bivalent anti-M42 blocks the ZP3-induced acrosome reaction. We have found that monovalent Fab fragments of anti-M42 do not block the ZP3-induced acrosome reaction, but that inhibition is restored by addition of a second antibody which crosslinks the Fabs. Thus, crosslinking is required for both inhibition of the acrosome reaction and redistribution. This suggests that redistribution of antigen away from the posterior region of the head may be part of the mechanism of inhibition of the ZP3-induced acrosome reaction.     

Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth

The extent of Sertoli cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output. To gain further knowledge of the factors that regulate Sertoli cell proliferation, the present study used a new approach to analyse changes in morphology and proliferation in the postnatal testis by combining organ culture with morphometric analysis. Fragments of rat testes from days 0 to 10 postpartum were cultured in contact with DMEM for 6 h or 72 h and fixed. The effects of ovine follicle-stimulating hormone (FSH) and activin were studied in an additional 72-h organ culture experiment using day 9 testes. Bromodeoxyuridine (BrdU) was added for the last 6 h of culture to mark proliferating cells. Two-microm sections of the fragments were analysed for morphological changes of the seminiferous cords, and the proportion of BrdU-labelled Sertoli and germ cells was determined. Assessment of 6-h samples revealed growth characteristics consistent with those observed in vivo during days 1-10 of postnatal development. From day 2 onwards, the volume fraction of seminiferous cords began to increase, while significant growth in cross-sectional area of the cords occurred only after day 6. In these culture conditions, germ cell proliferation and testicular architecture was consistent with that expected for the age of the tissue at time of explant. The proportion of dividing Sertoli cells declined from 15-20% at days 0-4 postpartum to below % at day 10 postpartum in the 6-h culture, and it was low or abolished in the 3-day culture at all time points. Activin and FSH together, but not singly, stimulated Sertoli cell proliferation in the 72-h culture. This paper presents a new approach to analysis of in vitro testis development. The combination of fragment culture and stereological analysis permits rigorous and detailed assessment of developmental changes in the postnatal testis.     

Aspects of cancer immunotherapy

Cancer immunotherapy has traditionally undergone a 'revolution' every decade, from the use of Bacille Calmette-Guérin by scarification in the 1970s, to interleukin-2 therapies in the 1980s, and monoclonal antibody treatments in the early 1990s. Usually the early reports on the use of such agents were encouraging, but when more patients were studied in multiple centres, the initial promising results could not be confirmed. Now in a new century, we have more reagents and methods available than ever before - indeed, with such a plethora of reagents it is difficult to envisage them being fully and appropriately tested within the next decade, by which time there will be even more reagents to test. However, there have been three major advances which should lead to substantial progress in cancer immunotherapy: (1) the widespread use of genetic engineering, enabling identification of candidate vaccine proteins and manipulation of their sequences; (2) the production of antigens, antibodies and cytokines in large amounts by recombinant technologies, and (3) an understanding of the mode of presentation of peptides by major histocompatibility complex Class I and Class II molecules and their recognition by T cells. Despite these advances, there are major problems facing cancer immunotherapy, such as the ability of tumours to mutate and evade the immune system and the difficulty of precisely defining the interactions of effector cells in mediating 'rejection' or destruction of a tumour. There are clearly immunological similarities with diseases such as malaria and schistosomiasis, where the invading foreign organisms can use a variety of strategies to resist an elicited immune response. The failure to find a suitable vaccine for these diseases must lead to some pessimism for the development of immunotherapy for an autologous tumour. However, there are promising studies now in progress which should give an indication of the most important directions to follow. This review provides a commentary on aspects of cancer immunotherapy and in particular will deal with: (1) the selection of antigens as vaccine components; (2) the modes of presentation of antigens, particularly by major histocompatibility complex Class I molecules; and (3) new modes of delivery of vaccine immunogens.     

Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH

We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin beta(A) subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin beta(A) subunit, the inhibin alpha subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis.     

Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development

The mammalian urogenital sinus (UGS) develops in a sex specific manner, giving rise to the prostate in the male and the sinus vagina in the embryonic female. Androgens, produced by the embryonic testis, have been shown to be crucial to this process. In this study we show that retinoic acid signaling is required for the initial stages of bud development from the male UGS. Enzymes involved in retinoic acid synthesis are expressed in the UGS mesenchyme in a sex specific manner and addition of ligand to female tissue is able to induce prostate-like bud formation in the absence of androgens, albeit at reduced potency. Functional studies in mouse organ cultures that faithfully reproduce the initiation of prostate development indicate that one of the roles of retinoic acid signaling in the male is to inhibit the expression of Inhba, which encodes the βA subunit of Activin, in the UGS mesenchyme. Through in vivo genetic analysis and culture studies we show that inhibition of Activin signaling in the female UGS leads to a similar phenotype to that of retinoic acid treatment, namely bud formation in the absence of androgens. Our data also reveals that both androgens and retinoic acid have extra independent roles to that of repressing Activin signaling in the development of the prostate during fetal stages. This study identifies a novel role for retinoic acid as a mesenchymal factor that acts together with androgens to determine the position and initiation of bud development in the male UGS epithelia.