Biomedical subjects

F M van Dissel-Emiliani

Publications and source records attributed to F M van Dissel-Emiliani.

11 recordsLinked to original sources

PubMed2000

Spermatogonial stem cell transplantation.

The development of the spermatogonial transplantation technique has given new impetus to research on spermatogonial stem cells. Possibilities opened by this technique include: (a) New ways to study fundamental aspects of spermatogenesis; (b) Generation of transgenic large domestic animals; (c) Protection of (young) male cancer patients from infertility due to chemotherapy or radiotherapy. Spermatogonial stem cell transplantation for the above purposes encompasses a number of steps. First, the stem cells have to be isolated and possibly purified. Second, it should be possible to cryopreserve the stem cells, for example till the children have reached puberty. Third. it should be possible to culture spermatogonial stem cells for a prolonged period of time which would also allow transfection and subsequent selection of stably transfected cells. Fourth, in case of animal studies. the host testis should be emptied from endogenous stem cells. This is probably best done by local irradiation. Finally, the stem cells will have to be transplanted.

Animals↗

PubMed1997

Immunomagnetic isolation of fetal rat gonocytes.

PROBLEM: An efficient method to obtain highly enriched populations of viable gonocytes from rat embryos at Day 18 and Day 20 postcoitum (pc) is described. METHOD: Single-cell suspensions with high cell yield were obtained by a collagenase/ trypsin digestion of the decapsulated testis. The gonocytes were purified by a direct immunoseparation technique, using magnetizable beads coated with rat anti-mouse immunoglobulin M (IgM) and a monoclonal antibody 4B6.3E10, which specifically reacted with a differentiation antigen on the fetal germ cells. RESULTS: Populations of 8.3 +/- 2.7 (x10(3); 18 days pc) or 1.2 +/- 0.25 (x10(4); 20 days pc) viable gonocytes per testis with purities of 91 +/- 6.5% and 92 +/- 4.3%, respectively, as determined by Nomarski microscopy were obtained. CONCLUSION: The cells were successfully used for culture studies and as starting material for the investigation of gene expression.

Animals↗

PubMed1997

Ontogeny and localization of an oncostatin M-like protein in the rat testis: its possible role at the start of spermatogenesis.

Oncostatin M (OSM), a member of the interleukin 6 family of cytokines, was found to be highly expressed in the late fetal and early neonatal rat testis, as well as in the maturing and adult testis. Two different forms of OSM were observed, one of M(r) 22,000 and the other of M(r) 36,000. In the prepubertal rat testis [19 days post coitum, 8 days post partum (dpp), and 15 dpp], the form with the higher molecular weight prevailed, whereas in the maturing testis (30 dpp, 45 dpp, and 12 weeks post partum), a shift toward the lower molecular weight form was observed, as well as a decrease in its relative amount. By immunohistochemistry on testicular sections, OSM-specific immunostaining was observed in the interstitial tissue at every age studied. In contrast, OSM immunoreaction was localized in the Sertoli cells exclusively around the start of spermatogenesis, being strongest at 3 dpp. In vitro studies revealed that neonatal Sertoli cells produce OSM. The possible role of OSM at the start of spermatogenesis was investigated by using a coculture of Sertoli cells and gonocytes isolated from newborn rats. OSM significantly increased the survival of both Sertoli cells and gonocytes in a dose-dependent manner. The proliferative activity of the Sertoli cells was not affected by OSM, whereas that of gonocytes was increased by almost 60% after 6 days of culture. Comparison of the effect of OSM on these cocultures with other members of the interleukin 6 family of cytokines demonstrated that this factor is more potent than leukemia inhibitory factor or ciliary neurotrophic factor. On the basis of these findings, it can be concluded that OSM is present in the rat testis, and it is likely to play an important role at the start of spermatogenesis.

Animals↗

PubMed1996

Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes.

A method for isolating A spermatogonia from the adult vitamin A-deficient (VAD) rat testis is described. After removal, the testes were decapsulated and tubules were dissected. An enzymatic digestion with collagenase, hyaluronidase, and trypsin was performed first to eliminate most of the interstitial cells. A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high number of A spermatogonia. The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient. By this procedure, purification of the suspension to 70-90% A spermatogonia was obtained. In the seminiferous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present. In our rats, the A spermatogonia are almost all arrested in the G1 phase of the cell cycle before the S phase of A1 spermatogonia, and presumably before their differentiation into A1 spermatogonia. After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia. The isolation of these synchronized A spermatogonia opens ways to investigate the regulation of differentiation and proliferation of A spermatogonia and the biochemical characteristics of the subsequent types of A spermatogonia.

Animals↗

PubMed1995

Heterogeneity in the in vitro survival and proliferation of human seminoma cells.

The in vitro culture conditions allowing survival and initial proliferation of murine primordial germ cells from 10.5 days post coitum embryos, which include the use of a murine embryonal fibroblast (STO) feeder, were applied to 21 human seminomas, composed of tumour cells which are considered as the malignant counterparts of human primordial germ cells. Cells from 18 seminomas attached poorly to STO, and only a few survived through day 10. In contrast, three seminomas showed a higher degree of attachment. Two of them showed initial proliferation and enhanced survival: 30 days for tumour SE1 and 25 days for tumour SE3. Tumour SE1 was more extensively studied, using the culture conditions allowing the derivation of pluripotent embryonic stem cells from 8.5 days post coitum murine primordial germ cells, which include the use of STO feeder, stem cell factor, leukaemia inhibitory factor and basic fibroblast growth factor. The presence of stem cell factor was necessary and sufficient for colonies of tumour cells to form during the first 3 days of culture. While the cell number decreased after day 3 in medium without fetal calf serum, it increased until day 9 in medium containing fetal calf serum. No reprogramming of SE1 cells to pluripotent stem cells was observed. Our data indicate that seminomas form a tumour population with a heterogeneous in vitro behaviour not equivalent to that of 8.5-10.5 days post coitum murine primordial germ cells.

Bromodeoxyuridine↗

PubMed1995

Characteristics of A spermatogonia and preleptotene spermatocytes in the vitamin A-deficient rat testis.

The proliferative activity and other characteristics of germ cells in the vitamin A-deficient (VAD) rat testis were investigated. In the VAD testis, A spermatogonia and preleptotene spermatocytes were found. The A spermatogonia in the VAD testis showed a bromodeoxyuridine (BrdU) labeling index of 6.6 +/- 1.1% and a mitotic index of 2.8 +/- 0.5%. After continuous labeling with BrdU for up to four days, the ultimate labeling index of A spermatogonia was 11.6 +/- 2.5%, which is less than expected. It is concluded that in the VAD rat testis, many of the proliferating A spermatogonia degenerate. During the first 18 h after administration of vitamin A, no increase was observed in either the labeling index or the mitotic index of the A spermatogonia. However, after 24 h the first wave of A spermatogonia in S phase was found, and the first wave in mitosis was found after 48 h. Furthermore, in the VAD testis the DNA content of most of the A spermatogonia was similar to that of Sertoli cells, i.e., 2n. It is concluded that in the VAD situation, nearly all A spermatogonia are arrested before the S phase of the A1 spermatogonia. The hypothesis is put forward that in the VAD testis, the remaining A spermatogonia are the undifferentiated spermatogonia that are unable to differentiate into A1 spermatogonia. The preleptotene spermatocytes in the VAD testis showed a BrdU labeling index of 20.3 +/- 3.5%, while the DNA content of most of these cells was between 3n and 4n.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Survival and proliferation of rat gonocytes in vitro.

Quiescent gonocytes were isolated from fetal testes of rat 18-day post coitum and cultured alone or on monolayers of somatic cells from different origins. The gonocytes specifically adhered to Sertoli cells, isolated from 21 to 23-day-old rat testes; this adherence was necessary for their survival in vitro. Addition of follicle-stimulating hormone and testosterone to these cultures did not increase the viability of the gonocytes. Serum was found to be deleterious to the germ cells. Electron-microscopic examination of Sertoli-cell-gonocyte co-cultures revealed the presence of numerous adhesion plaques between these cells, indicating that Sertoli cells and gonocytes are able to communicate in vitro. Gonocytes, in co-culture with Sertoli cells, were viable for at least 9 days. The gonocytes did not spontaneously resume proliferation. The simple culture system described in the present paper should be useful in studying the nature of the factors that are responsible for sending the quiescent gonocytes into the cell cycle and for stimulating the formation of A spermatogonia, a process characterizing the start of spermatogenesis.

Animals↗

PubMed1993

A monoclonal antibody recognizing a differentiation marker on rat gonocytes.

Monoclonal antibodies (MAb) were raised against a testicular membrane fraction from 18-day post coitum (p.c.) rat testes. One antibody, designated 4B6.3E10 (mu, kappa), was obtained which specifically reacted with gonocytes in the fetal testis. No significant cross-reactivity with other tissues from the 18-day p.c. embryo was found. MAb 4B6.3E10 was reactive with rat gonocytes from 17-day p.c. until the day of birth. Germ cells at later stages of testis development did not show any labelling. The epitope recognized by 4B6.3E10 is a carbohydrate as periodate treatment leads to a loss of reactivity of the antibody. By SDS-PAGE and Western blotting of proteins extracted from a testicular membrane fraction from 18-day p.c. testes, MAb 4B6.3E10 was found to recognize at least 3 protein moieties with apparent molecular weights in the ranges of 80-100, 120, 160-180 kDa (either under reducing- or non-reducing conditions). The results suggest that MAb 4B6.3E10 recognizes a specific differentiation marker for fetal rat gonocytes.

Animals↗

PubMed1991

Significant improvement of the survival of seminoma cells in vitro by use of a rat Sertoli cell feeder layer and serum-free medium.

Seminoma cell lines, essential to the study of the biology of seminoma, do not exist. Tissue culture conditions for establishing such cell lines have to be developed. Under conventional culture conditions, seminoma cells usually die within the first 3 days after plating. The enhanced survival of rat gonocytes when cocultured with rat Sertoli cells in serum-free medium suggests that seminoma cells, the neoplastic counterparts of gonocytes, might benefit from the same conditions. Indeed, when cocultured with rat Sertoli cells in a serum-free medium, viable seminoma cells could be demonstrated on the 11th day of culture. This result is a significant improvement over the results with conventional methods.

Adult↗

PubMed1989

Inhibin reduces spermatogonial numbers in testes of adult mice and Chinese hamsters.

Bovine follicular fluid (bFF) injected ip in mice during 2 days (65,000 U inhibin/day, 1 U inhibin the activity in 1 micrograms bFF protein) caused a significant decrease in the numbers of A4, intermediate (In), and B spermatogonia to 91%, 74%, and 67% of the control values, respectively. The numbers of undifferentiated spermatogonia remained unchanged. These injections suppressed peripheral FSH levels to 6% of the control values, suggesting that FSH might be the modulator of the effects on spermatogenesis. However, in the Chinese hamster, intratesticular injections of bFF during 4 days (6500 U inhibin/day into one testis) also caused a significant decrease in the numbers of A3. In, B1, and B2 spermatogonia to 86%, 61%, 55%, and 94% of the control values, respectively. Similarly, treatment with a partially purified inhibin preparation from rat Sertoli cell-conditioned medium (rSCCM) during 4 days (Mono Q fraction; 1512 U inhibin/day; 37.8 micrograms protein) caused a significant decrease in the numbers of A3, In, B1, and B2 spermatogonia to 90%, 87%, 66%, and 93% of the control values, respectively. Treatment with a highly purified inhibin preparation from rSCCM during 4 days (30K inhibin; 750 U inhibin/day; 100 ng protein) significantly decreased the numbers of In and B1 spermatogonia to, respectively, 87% and 91% of the control values. These effects were limited to the testis into which the material was injected; the contralateral testis or testes injected with control fluid always showed normal numbers of spermatogonia. This implies that the effects on the seminiferous epithelium are not FSH mediated. Intratesticular injections of bFF or pure inhibin did not affect the number of undifferentiated spermatogonia. However, the Mono Q fraction caused a significant increase in the numbers of undifferentiated spermatogonia in stages IV-VII of the cycle, suggesting the presence of a mitogenic factor for undifferentiated spermatogonia in rSCCM which is not present or is counteracted in bFF. The results suggest that inhibin may have a role in the regulation of spermatogonial development in the adult animal.

Animals↗

PubMed1989

Isolation of rat gonocytes by velocity sedimentation at unit gravity.

A method for obtaining enriched populations of gonocytes from rat embryos at 18 days p.c. has been developed. Single cell suspensions with high cell yield and good viability of the cells were obtained by a collagenase/trypsin digestion of the testes. Cells were separated on the basis of size by the Staput technique of velocity sedimentation at unit gravity. Populations of 600,000 gonocytes (70-75% purity), sedimenting at about 12 mm/h, could be obtained from 30-35 fetal rats within 8 h after killing. Purities were determined by Nomarski microscopy and verified in fixed preparations and by Coulter volume measurements.

Animals↗