Biomedical subjects

D G de Rooij

Publications and source records attributed to D G de Rooij.

At least 19 recordsLinked to original sources

PubMed2007

Asthenoteratozoospermia in mice lacking testis expressed gene 18 (Tex18).

Testis expressed gene 18 (Tex18) is a small gene with one exon of 240 bp, which is specifically expressed in male germ cells. The gene encodes for a protein of 80 amino acids with unknown domain. To investigate the function of (Tex18) gene, we generated mice with targeted disruption of the (Tex18) gene by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fertile, while they are subfertile on the 129/Sv background, although mating is normal. We showed that Tex18(-/-) males are subfertile because of abnormal sperm morphology and reduced motility, which is called asthenoteratozoospermia, suggesting that (Tex18) affects sperm characteristics. Maturation of spermatids is unsynchronized and partially impaired in seminiferous tubules of Tex18(-/-) mice. Electron microscopical examination demonstrated abnormal structures of sperm head. In vivo experiments with sperm of Tex18(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility, as determined by the computer-assisted semen analysis system, is significantly affected, compared to wild-type spermatozoa. Generation of transgenic mice containing Tex18-EGFP fusion construct revealed a high transcriptional activity of (Tex18) during spermiogenesis, a process with morphological changes of haploid germ cells and development to mature spermatozoa. These results indicate that (Tex18) is expressed predominantly during spermatid differentiation and subfertility of the male Tex18(-/-) mice on the 129/Sv background is due to the differentiation arrest, abnormal sperm morphology and reduced sperm motility.

Acrosome Reaction↗

PubMed2006

Expression of stress inducible protein 1 (Stip1) in the mouse testis.

Phthalate esters are considered endocrine disruptors that interfere with the endocrine balance and development of the mammalian testis. Mono-2-ethylhexyl phthalate (MEHP), the active metabolite of the ubiquitously used plasticizer di-2-ethylhexyl phthalate (DEHP), acts upon Sertoli cells as initial target. By subtractive cDNA libraries we identified genes deregulated as response to MEHP in primary cultures of mouse Sertoli cells. The expression of mouse stress inducible protein 1 (Stip1) was detected as upregulated as a result of MEHP exposure. Stip1 is a cochaperone protein that is homologous to the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). To assess the presence and localization of Stip1 in mouse testis and its potential role in stress defense, we studied the expression pattern of the Stip1 protein by immunohistochemistry and of the mRNA by in situ hybridization. Both the protein and the mRNA of Stip1 were mainly found in the cytoplasm of all types of spermatogonia and spermatocytes up till zygotene, the expression decreased during late pachytene and was very weak in diplotene spermatocytes and round spermatids. Interestingly, this expression pattern resembled the pattern of stress sensitivity of spermatogenic cells in that the most sensitive cell types show the weakest expression of Stip1. This suggests an important role for Stip1 in the ability of germ cells to survive in stress conditions including high temperatures.

Animals↗

PubMed2004

Kinetics of meiosis in azoospermic males: a joint histological and cytological approach.

We have developed a protocol for the identification of aberrant chromosome behavior during human male meiosis up to metaphase of the secondary spermatocyte. Histological evaluation by the Johnsen score of a testicular biopsy was combined with immunofluorescence of first meiotic prophase spermatocytes, using antibodies against synaptonemal complex protein 3 (SYCP3) and the product of the ataxia telangiectasia and rad3-related gene (ATR). This combination enables accurate meiotic prophase substaging and the identification of pachytene spermatocytes with asynapsis. Furthermore, we also investigated the competence of late pachytene primary spermatocytes to complete the first meiotic division up to metaphase and of secondary spermatocytes to transform into metaphase by an in vitro challenge with okadaic acid (OA). We tested this protocol on five males with normal Johnsen scores that presented with obstructive azoospermia, five males with low Johnsen scores and non-obstructive azoospermia and six vasectomized control males of proven fertility and normal Johnsen scores. In all azoospermics, the profiling of meiotic prophase stages by immunofluorescence increases the resolving power of the Johnsen score. In both obstructive and non-obstructive azoospermic patients, relatively more leptotene meiotic prophase stages were counted compared to the controls. In non-obstructive azoospermics, a marked heterogeneity in spermatogenesis was found, after combining the results of all three approaches, pointing at functional mosaicism of the germinal epithelium. Asynaptic pachytene spermatocytes were rarely encountered. Also, when first meiotic metaphase could be induced by OA, chiasma counts were normal. In none of the non-obstructive azoospermic males did the pattern of spermatogenesis resemble that of knock-out mouse azoospermics. We conclude that this combined histological and cytological approach enables a detailed phenotypic classification of infertile males, at a level comparable to that applied for male-sterile knock-out mice with a meiotic defect. This may facilitate the identification of candidate genes for human male infertility.

Animals↗

PubMed2004

The mammalian mid-pachytene checkpoint: meiotic arrest in spermatocytes with a mutation in Atm alone or in combination with a Trp53 (p53) or Cdkn1a (p21/cip1) mutation.

ATM, the protein product of the gene mutated in the human autosomal recessive disorder ataxia telangiectasia, is involved in detection of double strand breaks (DSBs) and is a key component of the damage surveillance network of cell cycle proteins. In somatic cells ATM phosphorylates many other proteins including p53, an important regulator of cell cycle control. Mice deficient for Atm are male sterile with arrest and apoptosis occurring at testis epithelial stage IV, which in normal spermatocytes corresponds to mid-pachynema. Unlike the situation in somatic cells, we find no evidence that disruption of the Trp53 (p53) gene, or its down-stream target Cdkn1a (p21/Cip1) results in even a partial rescue of the Atm defect.

Animals↗

PubMed2003

Specific arrests of spermatogenesis in genetically modified and mutant mice.

In naturally occurring mutant mice but also in mice genetically modified for the study of other organs, relatively often a spermatogenic arrest is seen. In a number of cases the arrests appear to be very specific causing apoptosis of germ cells at a particular step in their development, while before this step cells progress normally. These steps include: proliferation/migration of primordial germ cells, the production of differentiating spermatogonia by gonocytes, the regulation of stem cell renewal/differentiation, the differentiation of A(al) into A1 spermatogonia, proliferation of A1-A4 spermatogonia, germ cell density regulation, start of meiosis, epithelial stage IV checkpoint of pachytene spermatocytes, the first meiotic division, the formation of the acrosomic vesicle in spermatids and several other steps in spermatid development. In addition, there are many mice that have not been studied in enough detail for a proper categorization. In this review an overview is given of the various mutations and genetically modified mice showing a direct effect on specific spermatogenic cell types. In addition, the relevance of these models to our understanding of the spermatogenic process is discussed.

Animals↗

PubMed2002

Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation.

With a novel method of eliminating spermatogenesis in host animals, male germ cells isolated from mice with targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) were transplanted to evaluate their ability to reproduce the phenotype previously found in the transgenic animals. Successful depletion of endogenous spermatogenesis was achieved using fractionated ionizing irradiation. A dose of 1.5 Gy followed by a dose of 12 Gy after 24 h reduced the percentage of tubule cross-sections displaying endogenous spermatogenesis to approximately 3% and 10% as evidenced by histologic evaluation of testes at 12 and 21 wk, respectively, after irradiation. At this dose, no apparent harmful side effects were noted in the animals. Upon transplantation, GDNF-overexpressing germ cells were found to be able to repopulate the irradiated testes and to form clusters of spermatogonia-like cells resembling those found in the overexpressing donor mice. The cluster cells in transplanted host testes expressed human GDNF, as had been shown previously for clusters in donor animals, and both were strongly positive for the tyrosine kinase receptor Ret. Thus, we devised an efficient method for depleting the seminiferous epithelium of host mice without appreciable adverse effects. In these host mice, GDNF-overexpressing cells reproduced the aberrant phenotype found in the donor transgenic mice.

Animals↗

PubMed2002

Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture.

The culture of spermatogonial cells under well-defined conditions would be an important method for elucidating the mechanisms involved in spermatogenesis and in establishing tissue regeneration in vivo. In this study, a serum-free culture system was established, with type A spermatogonia isolated from adult vitamin A-deficient mice. At days 1, 3 and 7 of culture, the viability and proliferation of cells were monitored. The viability of the cells decreased by day 7 to 10% of the cells present. Proliferation occurred mainly during day 1, when 1% of the germ cells was proliferating. Co-labelling for a germ cell marker (heat shock protein-90alpha, Hsp90alpha), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed that this proliferation was restricted to germ cells. In an attempt to improve these parameters, medium containing fetal calf serum (FCS) was used. Viability was not influenced by serum, but proliferation was markedly enhanced. However, after day 7 of incubation with FCS, co-immunolocalization for Hsp90alpha and BrdU showed a preferential proliferation of somatic cells. Comparison of cultures of adult cells with cultures of prepubertal germ cells, commonly used in studies of spermatogenesis, showed that prepubertal germ cells are twice as viable. In addition, a different proliferation profile was observed, with a peak at day 3. Here, a distinct proliferation of somatic cells was also noted. The results from the present study indicate that the origin of isolated germ cells partly determines culture outcome and that cultures of prepubertal germ cells may not be representative for adult spermatogenesis. Moreover, adding FCS to the culture medium invokes the risk of profound and undesirable effects on cell composition, also underlining the need for identification of germ cells during culture.

Animals↗

PubMed2002

Isolation and purification of type A spermatogonia from the bovine testis.

The aim of this study was to isolate and purify bovine type A spermatogonia. Testes from 5-7-month-old calves were used to isolate germ cells using a two-step enzymatic digestion. During the isolation and purification steps, the viability of cells was determined using live/dead staining. The identity of type A spermatogonia during isolation and purification was determined under a light microscope equipped with a Nomarski lens. Isolated cells were characterized further by using specific markers for type A spermatogonia, including Dolichos biflorus agglutinin (DBA) and c-kit. The cell suspension was transplanted into immunodeficient recipient mouse testes and the colonization was assessed 1-3 months after transplantation, to assess the stem cell population among the isolated cells. After isolation, a cell suspension was obtained containing about 25% type A spermatogonia, which was enriched further by differential plating and separation on a discontinuous Percoll gradient. Finally, fractions containing 65-87% pure type A spermatogonia were obtained. Large and small type A spermatogonia with different numbers and sizes of nucleoli were found. DBA stained both large and small type A spermatogonia and its application in fluorescence-activated cell sorting (FACS) resulted in comparable percentages of type A spermatogonia to those determined by morphological examination under a light microscope equipped with a Nomarski lens. Nearly all of the large type A spermatogonia showed strong c-kit immunoreactivity, indicating that these cells had undergone at least an initial differentiation step. In contrast, approximately half of the small type A spermatogonia were negative for c-kit, indicating the presence of the spermatogonial stem cells in this population. At 3 months after transplantation, groups of bovine type A spermatogonia were found in most tubule cross-sections of the recipient mouse testes, showing the presence of spermatogonial stem cells among the isolated cells.

Animals↗

PubMed2001

Transient disruption of spermatogenesis by deregulated expression of neurturin in testis.

Two related ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), are expressed by Sertoli cells, but their cognate ligand-binding co-receptors, GDNF family receptor alpha1 and alpha2, are displayed by different germ cells suggesting different targets for the ligands. GDNF regulates cell fate decision of undifferentiated spermatogonia 'Science 287 (2000) 1489'. The role of NRTN was now approached by targeted overexpression in mouse testis. Between 3 and 5 weeks of age, transient degeneration of spermatogenic cells was observed in approximately 20% of all five transgenic lines generated. Spermatids and pachytene spermatocytes underwent segmental degeneration, if the rete testis was undilated. When it was dilated, the spermatids and spermatocytes were more generally depleted. After 5 weeks of age, spermatogenic defects were no more observed and the NRTN overexpressing mice were fertile. The data suggest that NRTN might regulate survival and differentiation of spermatocytes and spermatids, but the low penetrance indicates that either the transgene expression has not been high enough or NRTN is not as essential as GDNF for spermatogenesis.

Animals↗

PubMed2001

Role for c-Abl and p73 in the radiation response of male germ cells.

p53 plays a central role in the induction of apoptosis of spermatogonia in response to ionizing radiation. In p53(-/-) testes, however, spermatogonial apoptosis still can be induced by ionizing radiation, so p53 independent apoptotic pathways must exist in spermatogonia. Here we show that the p53 homologues p63 and p73 are present in the testis and that p73, but not p63, is localized in the cytoplasm of spermatogonia. Unlike p53, neither p63 nor p73 protein levels were found to increase after a dose of 4 Gy of X-rays. Although p73 protein levels did not increase, its interaction with the non-receptor tyrosine kinase c-Abl and its phosphorylation on tyrosine residues did. c-Abl and p73 co-localize in the cytoplasm of spermatogonia and spermatocytes and in the residual bodies. Furthermore, c-Abl protein levels increase after irradiation. p63 was not found to co-localize or interact with c-Abl neither before nor after irradiation. In conclusion, in the testis ionizing radiation elevates cytoplasmic c-Abl that in turn interacts with p73. This may represent an additional, cytoplasmic, apoptotic pathway. Although less efficient than the p53 route, this pathway may cause spermatogonial apoptosis as observed in p53 deficient mice.

Animals↗

PubMed2001

Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis.

We show with transgenic mice that targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors, which express germ-cell markers. The tumors are invasive and contain aneuploid cells, but no distant metastases have been found. By several histological, molecular, and histochemical characteristics, the GDNF-induced tumors mimic classic seminomas in men, representing a useful experimental model for testicular germ-cell tumors. The data also show that a deregulated stimulation of a normal proto-oncogene by its ligand can be an initiative event in carcinogenesis.

Aneuploidy↗

PubMed2001

Sox9 induces testis development in XX transgenic mice.

Mutations in SOX9 are associated with male-to-female sex reversal in humans. To analyze Sox9 function during sex determination, we ectopically expressed this gene in XX gonads. Here, we show that Sox9 is sufficient to induce testis formation in mice, indicating that it can substitute for the sex-determining gene Sry.

Animals↗

PubMed2001

Nature of the spermatogenic arrest in Dazl -/- mice.

Dazl encodes an RNA-binding protein essential for spermatogenesis. Mice that are deficient for Dazl are infertile, lacking any formation of spermatozoa, and the only germ cells present are spermatogonia and a few spermatocytes. To gain more insight regarding the timing of the spermatogenic arrest in Dazl -/- mice, we studied the spermatogonial cell types present in testis sections and in seminiferous tubular whole mounts. Most of the seminiferous tubular cross-sections contained A spermatogonia as the most advanced cell type, with only very few containing cells up to pachytene spermatocytes. Both 5-bromodeoxy-uridine incorporation and mitotic index indicated that the remaining A spermatogonia were actively proliferating. C-kit immunohistochemical studies showed that most of the A spermatogonia were positively stained for the c-Kit protein ( approximately 80%). The clonal composition of the A spermatogonia in tubular whole mounts indicated these cells to be A(single) (A(s)), A(paired) (A(pr)), and A(aligned) (A(al)) spermatogonia. It is concluded that the prime spermatogenic defect in the Dazl -/- mice is a failure of the great majority of the A(al) spermatogonia to differentiate into A(1) spermatogonia. As a result, most seminiferous tubules of Dazl -/- mice only contain actively proliferating A(s), A(pr), and A(al) spermatogonia, with cell production being equaled by apoptosis of these cells.

Animals↗

PubMed2001

Localization of two mammalian cyclin dependent kinases during mammalian meiosis.

Mammalian meiotic progression, like mitotic cell cycle progression, is regulated by cyclins and cyclin dependent kinases (CDKs). However, the unique requirements of meiosis (homologous synapsis, reciprocal recombination and the dual divisions that segregate first homologues, then sister chromatids) have led to different patterns of CDK expression. Here we show that Cdk4 colocalizes with replication protein A (RPA) on the synaptonemal complexes (SCs) of newly synapsed axes of homologously pairing bivalents, but disappears from these axes by mid-pachynema. The switch from the mitotic pattern of expression occurs during the last two spermatogonial divisions. Cdk2 colocalizes with MLH1, a mismatch repair protein at sites of reciprocal recombination in mid-late pachynema. In addition Cdk2 localizes to the telomeres of chromosomal bivalents throughout meiotic prophase. The mitotic pattern of expression of Cdk2 remains unchanged throughout the spermatogonial divisions, but is altered in meiosis of the spermatocytes.

Animals↗

PubMed2001

Use of antibodies against LH receptor, 3beta-hydroxysteroid dehydrogenase and vimentin to characterize different types of testicular tumour in dogs.

Testicular tumours in dogs are of Sertoli cell, Leydig cell or germinal origin and mixed tumours are also frequently observed. The cellular components of mixed tumours are usually identified by histological examination but sometimes this is difficult. In this study, a panel of specific antibodies was used to identify the different cell types in testicular tumours by immunohistochemistry. Leydig cells were identified using an antibody against the LH receptor and an antibody against the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD), both of which are characteristic of Leydig cells in testes. Sertoli cells were identified using an antibody against the intermediate filament vimentin. Seminoma cells did not stain with any of these antibodies. Vimentin was used only in histologically complex cases. Eighty-six tumours, diagnosed histologically as 29 Sertoli cell tumours, 25 Leydig cell tumours, 19 seminomas and 13 mixed tumours, were studied. Feminization was observed in 17 dogs. Leydig cell tumours stained positively with the antibodies against the LH receptor and 3beta-HSD, whereas seminomas and Sertoli cell tumours were negative (unstained). The antibody against vimentin stained both Sertoli and Leydig cells, and tumours arising from these cells, but not seminomas. Immunohistochemistry revealed that three tumours identified histologically as Sertoli cell tumours were actually Leydig cell tumours. In 14 dogs the histological diagnosis appeared to be incomplete, as mixed tumours instead of pure types of tumours were identified in 11 dogs, and in three dogs mixed tumours appeared to be pure types. Hence, the histological diagnosis was insufficient in approximately 20% of dogs. Furthermore, immunohistochemical analysis of testis tumours revealed that feminization occurred in dogs with Sertoli cell tumours or Leydig cell tumours and their combinations, but not in dogs with a seminoma. In conclusion, incubation with antibodies against LH receptor and 3beta-HSD proved to be a consistently reliable method for identification of Leydig cell tumours in dogs. Vimentin can be used to discriminate between Sertoli cell tumours and seminomas. Overall, this panel of antibodies can be very useful for determination of the identity of testicular tumours in which histological characterization is complicated and the pathogenesis of feminization is not clear.

3-Hydroxysteroid Dehydrogenases↗

PubMed2001

Proliferation and differentiation of spermatogonial stem cells.

Spermatogonial stem cells (A(s) spermatogonia) are single cells that either renew themselves or produce A(pr) (paired) spermatogonia predestined to differentiate. In turn, the A(pr) divide into chains of A(al) (aligned) spermatogonia that also divide. The ratio between self-renewal and differentiation of the stem cells is regulated by glial cell line-derived neurotrophic factor produced by Sertoli cells, while the receptors are expressed in stem cells. A(s), A(pr) and A(al) spermatogonia proliferate during part of the epithelial cycle forming many A(al) spermatogonia. During epithelial stage VIII, almost all A(al) spermatogonia, few A(pr) and very few A(s) spermatogonia differentiate into A1 spermatogonia. A number of molecules are involved in this differentiation step including the stem cell factor-c-kit system, the Dazl RNA binding protein, cyclin D(2) and retinoic acid. There is no fine regulation of the density of spermatogonial stem cells and consequently, in some areas, many A1 and, in other areas, few A1 spermatogonia are formed. An equal density of spermatocytes is then obtained by the apoptosis of A2, A3 or A4 spermatogonia to remove the surplus cells. The Bcl-2 family members Bax and Bcl-x(L) are involved in this density regulation. Several mechanisms are available to cope with major or minor shortages in germ cell production. After severe cell loss, stem cell renewal is preferred above differentiation and the period of proliferation of A(s), A(pr) and A(al) spermatogonia is extended. Minor shortages are dealt with, at least in part, by less apoptosis among A2-A4 spermatogonia.

Animals↗

PubMed2001

Spermatogenesis and testicular tumours in ageing dogs.

The aims of this investigation were to quantify the changes in canine spermatogenesis that occur during ageing and to study the prevalence of testicular tumours and their effects on spermatogenesis in dogs. Testes from 74 dogs of various breeds without clinically detected testicular disease and from 28 dogs with clinically palpable tumours were examined. Testicular tumours were classified histologically according to the criteria of Nielsen and Kennedy (1990). Spermatogenesis was evaluated using a modified Johnsen score adapted for use in dogs. The diameter of the seminiferous tubules was measured in dogs without testicular disease to examine the possible effects of ageing. The different lifespans of small and large breeds were compensated for by expression as a percentage of the age at which dogs with various body weights are considered to be geriatric. Of the dogs without clinically detected disease, 21 of 74 had small testicular tumours. As in the 28 dogs with clinically detected tumours, multiple types of tumour and bilateral occurrence of tumours were common findings. The prevalence of tumours increased during ageing. Eighty-six per cent of the clinically detected tumours and 57% of the non-clinically detected tumours were found in geriatric dogs. The diameter of the seminiferous tubules did not change with age. Impairment of spermatogenesis was found only in dogs with bilateral tumours and in the affected testis of dogs with clinically detected tumours. In conclusion, it appears that spermatogenesis per se does not decrease during ageing in dogs. However, the occurrence of testicular tumours increases with age and this may affect spermatogenesis significantly.

Aging↗

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↗