A.M.M. van Pelt

Transplantation of Germ Cells from Glial Cell Line-Derived Neurotrophic Factor-Overexpressing Mice to Host Testes Depleted of Endogenous Spermatogenesis by Fractionated Irradiation

Abstract 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.

Regulation of spermatogonial proliferation.

In nonprimates the spermatogonial compartment can be subdivided into (morphologically) undifferentiated spermatogonia and differentiating spermatogonia. Each cycle of the seminiferous epithelium the proliferative activity of the undifferentiated spermatogonia is stimulated, probably by factors secreted by Sertoli cells. Subsequently, during a period of active proliferation many Aal spermatogonia are formed. In the normal situation around epithelial stage III, proliferation is inhibited by the differentiating spermatogonia by way of a negative feedback system, probably involving a spermatogonial chalone. Then most of the Aal spermatogonia formed differentiate into Al spermatogonia. For this differentiation vitamin A, or factors secreted by Sertoli cells under the influence of vitamin A, is/are necessary. In the normal situation there is no regulation of the density of the undifferentiated spermatogonia. Different tubular areas can contain widely varying numbers of stem cells and other undifferentiated spermatogonia and consequently can produce widely varying numbers of Al spermatogonia. Only in extreme circumstances, such as after irradiation, the stem cells change the ratio between self-renewing and differentiating divisions in favor of self-renewal. Furthermore, in this situation the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III because of the lack of differentiating spermatogonia. Density regulation does take place during the development of the differentiating spermatogonia. In the chinese hamster it appeared that despite the variation in the numbers of Al spermatogonia produced in different areas, the density of the preleptotene spermatocytes was very much the same. It was found that the even distribution of spermatocytes in the epithelium was achieved by a density-dependent degeneration of differentiating spermatogonia in such a way that many of the latter cells degenerated in high density areas and only few or none in low density areas. In primates the undifferentiated spermatogonia can be subdivided into Ap and Ad spermatogonia. Both Ap and Ad spermatogonia can be seen to be topographically arranged in clones of 1 or 2n cells in situations in which their density is low. The Ad spermatogonia do not proliferate, but after cell loss these cells were found to transform into Ap spermatogonia that start to proliferate. The Ap spermatogonia only divide once every epithelial cycle, renewing themselves and giving rise to B spermatogonia. In the monkey the number of Ap spermatogonia could be increased by FSH treatment. Hence, there may be a correlation between FSH levels and the numbers of Ap spermatogonia. Fu

Isolation of gonadotrops from the pituitary of the African catfish, Clarias lazera

SummaryDispersed pituitary cells from male African catfish, Clarias lazera, were fractionated in a density gradient of Percoll. Five fractions were isolated, consisting of about 6, 19, 39, 95 and 83% gonadotrops, respectively. The gonadotrops were identified by their ultrastructural characteristics, by immunocytochemistry, and by measuring their hormone content. After one day in culture, in each fraction the secretion of gonadotropin could be stimulated by a luteinizing hormone-releasing hormone analogue, indicating that the cells had retained their functional integrity. Since the regulatory mechanisms of different cell types from the pituitary have some similarity, purification of the gonadotrops provides a model to study the regulation of gonadotropin secretion.

Role of retinoids in spermatogonial proliferation and differentiation and the meiotic prophase.

A detailed study was made of the events taking place during the early onset of vitamin A deficiency (VAD). Pregnant Wistar rats (18–20 days p.c.) were fed a VAD diet or a normal balanced diet. Newborn male rats received the same diet. Body growth of the animals on the VAD diet was found to slow down after the rats were 40 days old. Cell counts revealed that at a very early stage, between 44 and 46 days, soon after the slowing down of the body growth, the division of A1- into A2-spermatogonia no longer occurred, indicating an arrest at this stage of the spermatogenic process. All other types of differentiating spermatogonia were found to develop normally into spermatocytes except for the intermediate (In) spermatogonia, some of which were seen to degenerate. As a result of the inhibition of the formation of the A2-spermatogonia, a maturation depletion was found in which cell numbers decreased to those normally present in stage VIII. This depletion could be calculated to lead to a complete depletion at about day 58.

A rapid immunogold-silver staining for detection of bromodeoxyuridine in large numbers of plastic sections, using microwave irradiation.

SummaryA rapid and convenient method for the large scale, immunogold-silver staining (IGSS) of bromodeoxyuridine (BrdU) incorporated by S phase cells, by means of a monoclonal antibody (anti-BrdU) is described. Nineteen slides at a time can be incubated with the antibodies and the protein A-gold (PAG) in staining jars. The antibody and protein A-gold solutions could be used at least five times to incubate new batches of slides. The incubation times with these solutions were shortened by means of microwave irradiation. In this way 200 slides carrying at least 800 sections could be easily processed under the same conditions in one day, using 1.25ml neat antibody solutions of anti-BrdU and rabbit anti-mouse.For light microscopy bothpplastic embedding systems: methylmethacrylate (MMA) and glycolmethacrylate (GMA) can be stained with this technique. The MMA sections, of which the plastic has to be removed before the IGSS, has the advantage of a stronger labelling intensity. The GMA plastic, which contains a cross-linking, agent cannot be removed and consequently for GMA sections it is necessary to incubate the sections with a proteolytic enzyme (trypsin) before the IGSS, to reexpose the antigenic binding sides. However, the GMA sections can be allowed to air dry during the IGSS without negative effects on the morphology. This makes it possible to perform the antibody and the PAG-incubating steps on one day and to finish the IGSS the next day. In this way twice as many GMA slides can be incubated with the same antibody and PAG solutions than with MMA slides.In both plastic embedding systems the intensity of the BrdU labelling was found to be stronger in Carnoys than in Bouins fixed sections.

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 postcoïtum (pc) is described.

Expression of the Scaffolding Subunit A of Protein Phosphatase 2A During Rat Testicular Development

Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A (PP2A-A) is the major subunit in the formation of a functional PP2A holoenzyme. In this study, we investigated the expression of PP2A-A during testicular development in more detail using in situ hybridization, immunohistochemistry, and Western blot with testes of rats of various ages from 16 days postcoitum (pc) to adulthood. The expression of PP2A-A was detected in fetal proliferative gonocytes at 16 days pc, declining thereafter during the quiescent period of the gonocytes. From the day of birth to the start of spermatogenesis (Day 4 postpartum [pp]), the number of PP2A-A-immunopositive gonocytes increased. At Day 4 pp, the first A1 spermatogonia appeared along the basement membrane; all were PP2A-A positive. In the adult, PP2A-A was upregulated during the differentiation of the As, Apr, and Aal spermatogonia to the A1 spermatogonia and expressed thereafter by all other spermatogonia. Spermatocytes from the pachytene stage onward and all spermatids in the adult testis also showed clear expression of PP2A-A. In Sertoli cells, PP2A-A was detected during their proliferative period at 19 days pc to 15 days pp. The presence of a functional enzyme was confirmed by the additional detection of the catalytic subunit C of PP2A using Western blot analyses at various ages during testicular development. This apparent pattern of expression of PP2A-A during testicular development suggests that PP2A may play an important role in the proliferation of distinct populations of testicular cells and during meiosis and sperm maturation.

Immunocytochemical labelling of isolated catfish pituitary gonadotrops with the protein A-gold method on epon sections

The adenohypophysis of catfish and other teleosts consists of morphologically and functionally different cell types. Hormone synthesis and release in some of these cell types are controlled by similar hypothalamic peptidergic and aminergic systems. In vivo also blood-born steroids may directly and/or indirectly influence the activity of for example corticotropic and gonadotropic pituitary cells. Moreover, different adenohypophyseal cell types tend to influence each other via their respective hormones, both under in vivo and in vitro conditions. Consequently, to study the effect of each single control mechanism on hormone synthesis and release by a single cell type, it is necessary to use isolated cells. Confirmation of the identity of the isolated cell type is an important step in the procedure. The present study describes an ultrastructural method to confirm the isolation of gonadotropic cells from the pituitary of the African catfish, Clarias lasera.

Development of the testis in pre-pubertal boys with cancer after biopsy for fertility preservation

STUDY QUESTION Is testicular growth affected by a testicular biopsy intended for fertility preservation in pre-pubertal boys with cancer? SUMMARY ANSWER Testicular growth of the biopsied testis is not impeded in comparison to the non-biopsied contralateral testis up until 1 year after surgery. WHAT IS KNOWN ALREADY Fertility preservation in pre-pubertal boys by means of testicular biopsy has been conducted for more than 15 years. Although immediate adverse effects of testicular biopsy are rare (1%), no data exist on the effect of biopsy on testicular growth. STUDY DESIGN, SIZE, DURATION In this prospective cohort study, between March 2011 and February 2017, 93 parents of pre-pubertal boys were offered cryopreservation of testicular tissue of their son, of whom 78 consented. Sixty-four boys were included in this follow-up study. PARTICIPANTS/MATERIALS, SETTING, METHODS All boys with cancer at the paediatric oncology department of the Academic Medical Center (AMC) who needed gonadotoxic therapy and were unable to ejaculate were offered cryopreservation of testicular tissue prior to treatment. By testicular ultrasound before and after biopsy (1, 6 and 12 months after biopsy), volume and parenchymal abnormalities were assessed. Data were analysed using mixed-effects modelling. MAIN RESULTS AND THE ROLE OF CHANCE Of the 64 included boys all were followed up at 1 month, 58 at 6 months and 55 at 12 months. Mean testicular volumes after 1, 6 and 12 months after biopsy were 1.7 ± 2.1, 1.7 ± 2.2 and 1.9 ± 2.4 for the biopsied testis and 1.8 ± 2.2, 1.8 ± 2.3 and 2.0 ± 2.2 for the non-biopsied testis, respectively. Biopsy of the testis did not have a significant impact on testicular growth. Immediate adverse effects of the biopsy, i.e. wound infections, were seen in 3/78 boys (3.8%). LIMITATIONS, REASONS FOR CAUTION Although it is the largest cohort available to date, the number of patients included in our follow-up is still relatively small. A larger cohort would be able to evaluate growth more precisely. Follow-up was discontinued in a significant portion of boys, 12/76 (15.8%), mainly because of death due to primary illness but also because they could not be reached or declined further follow-up. WIDER IMPLICATIONS OF THE FINDINGS These reassuring data may be used in counselling future boys who are eligible for fertility preservation and their parents. STUDY FUNDING/COMPETING INTEREST(S) Study funded by KIKA Foundation (Kika 86), Grant from the Netherlands Organisation for Health Research and Development (ZonMW TAS-116003002). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER CCMO-register: NL27690.000.09.

Correction: Spermatogonial stem cell sensitivity to capsaicin: An in vitro study (vol 6, pg 52, 2008)

Since publication of our article Spermatogonial stem cell sensitivity to capsaicin: an in vitro study. Reprod Biol Endocrinol 2008, 6:52., we have realised that the legend of Figure 1 needs to be corrected and should read as follows: