Spermatogonial Stem Cell-Based Therapies: Taking Preclinical Research to the Next Level.

Fertility preservation via biobanking of testicular tissue retrieved from testicular biopsies is now generally recommended for boys who need to undergo gonadotoxic treatment prior to the onset of puberty, as a source of spermatogonial stem cells (SSCs). SSCs have the potential of forming spermatids and may be used for therapeutic fertility approaches later in life. Although in the past 30 years many milestones have been reached to work towards SSC-based fertility restoration therapies, including transplantation of SSCs, grafting of testicular tissue and various in vitro and ex vivo spermatogenesis approaches, unfortunately, all these fertility therapies are still in a preclinical phase and not yet available for patients who have become infertile because of their treatment during childhood. Therefore, it is now time to take the preclinical research towards SSC-based therapy to the next level to resolve major issues that impede clinical implementation. This review gives an outline of the state of the art of the effectiveness and safety of fertility preservation and SSC-based therapies and addresses the hurdles that need to be taken for optimal progression towards actual clinical implementation of safe and effective SSC-based fertility treatments in the near future.

pH stability of human preimplantation embryo culture media: effects of culture and batches.

RESEARCH QUESTION: How stable is the pH of human preimplantation embryo culture media during IVF culture and is there variation in pH between batches of culture media? DESIGN: To evaluate pH stability, three batches of three culture media were incubated in triplicate without embryos (sham culture) at CO2 levels recommended by the manufacturers (5% or 6%) for 4 days. To evaluate differences in pH between batches, the pH of three batches of five culture media was measured in triplicate during 1 day of sham culture. Linear mixed models were used for the analysis. RESULTS: An increase in pH during 4 days of culture was found in all three culture media, but the observed increased values were within the generally accepted range for clinical practice (pH 7.2-7.4). One medium was pH 7.1 in the first 2 days, but this was within the range provided by the manufacturer for that medium. Three out of five analysed media showed batch variation in pH that exceeded the generally accepted range for clinical practice. CONCLUSIONS: A relevant difference in pH was found between batches of human preimplantation embryo culture media. This suggests that the CO2 level of incubators may need to be adjusted for new batches of culture medium based on measured pH, to anticipate batch variability and safely accommodate limited pH increase over time. This study was unable to identify the cause of the differences in pH between batches, and further investigation on a larger number of batches and other media seems warranted.

[Oocyte vitrification: for whom?]. / Eicelvitrificatie: voor wie eigenlijk? Een dwarsdoorsnede-onderzoek.

OBJECTIVE: To study the indications of oocyte vitrification in women who undergo this intervention. DESIGN: Cross-sectional study. METHOD: We collected the indications of oocyte vitrification in women who underwent, or started ovarian stimulation for, this intervention between May 2006 and December 31st 2013. Indications were subcategorized into six groups: no sperm available during IVF or ICSI treatment, planned gonadotoxic therapy, ovarian surgery, risk on premature ovarian insufficiency , previous gonadotoxic therapy, and anticipated gamete exhaustion. RESULTS: During the study period 298 women vitrified oocytes or started with ovarian stimulation for oocyte vitrification. The majority of the women (33%) vitrified oocytes because of anticipated gamete exhaustion. Planned gonadotoxic treatment was for 81 women (27%) the reason for oocyte vitrification. CONCLUSION: With oocyte vitrification women are able to extend the time at which they can conceive. The future will tell whether the benefits of oocyte vitrification outweigh the risks and costs.

Fertility preservation: a challenge for IVF-clinics.

OBJECTIVE: Acute fertility preservation for women is an interdisciplinary treatment that requires adequate information provision and early referral. This quality management project aimed to improve fertility preservation care by using a practical tool: Strengths, Weaknesses, Opportunities and Threats (SWOT) analysis. STUDY DESIGN: Quality management project was executed between May 2011 and July 2013. This project has been executed in a university affiliated IVF-clinic in cooperation with two oncological sites and used a four-step strategy: (1) monitoring baseline referral process, (2) exploring baseline fertility preservation program by Strengths, Weaknesses, Opportunities and Threats' (SWOT)-analysis, (3) setting up a new fertility preservation program and (4) evaluating the new fertility preservation program by means of SWOT-analysis. RESULTS: During the three-months monitoring period, fertility preservation was requested for a total of 126 women. The mean age of the women was 33.8 years old (range 1-42 years old). Most requests came from women who wanted to cryopreserve oocytes because of age-related decline of fertility (n=90; 71%). Most requests for acute fertility preservation concerned women with breast cancer (n=16; 57%). Information leaflets and pre-consultation questionnaires for women improved the quality of first fertility preservation consultation as evaluated by final SWOT-analysis. Collaboration with oncological centres and information about fertility preservation improved the referral process. CONCLUSIONS: SWOT-analysis proved useful for setting up a new fertility preservation-program and can be recommended as a tool to improve the management and organisation of new types of reproductive care.

[Reproduction with the use of vitrified oocytes]. / Voortplanting met behulp van gevitrificeerde oöcyten.

Cryopreservation of spermatozoa and embryos has been a standard procedure in Dutch fertility laboratories for several decades. Until recently, oocyte cryopreservation was rarely performed because of the low chance of survival. 'Vitrification' is a promising new technique for the freezing of oocytes. This ultra-quick freezing method can be used when no spermatozoa, or no viable spermatozoa, could be obtained during in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI), and for patients with cancer who have to undergo potentially damaging oncology treatment. The Academic Medical Center in Amsterdam has performed oocyte vitrification for 20 patients in the period March 2006 - October 2009. Ten of these patients have undergone ICSI treatment; this resulted in three pregnancies and the birth of two healthy babies from two of these pregnancies.

Ubiquitin ligase activity of c-Cbl guides the epidermal growth factor receptor into clathrin-coated pits by two distinct modes of Eps15 recruitment.

We have demonstrated previously that c-Cbl requires the presence of a functional ubiquitin interacting motif (UIM) in Eps15 to mediate epidermal growth factor receptor (EGFR) endocytosis. Both the ubiquitin ligase activity of c-Cbl and the UIM of Eps15 were necessary for plasma membrane recruitment of Eps15 and entry of ligand-bound EGFR into coated pits and vesicles containing Eps15. This is consistent with a scenario in which ubiquitin moieties appended to activated EGFR complexes act as docking sites for Eps15 and thereby recruit receptors into clathrin coated pits. Here, we have investigated which additional structural features of c-Cbl are required for this process. We find that c-Cbl can guide ligand-bound EGFR into the Eps15 internalization route by two distinct mechanisms. These are either dependent on the phosphotyrosine binding domain of c-Cbl that directly binds to the EGFR or on the region C-terminal of the Ring finger, which allows for indirect binding to an alternative site on the receptor. No strict requirement exists for either ubiquitin modified EGFR or the Cbl binding ubiquitination substrate CIN85 as docking site for the UIM of Eps15. Only in the phosphotyrosine binding-dependent pathway, the EGFR is ubiquitinated and may serve as a site of recruitment for Eps15. Only in this pathway, Eps15 is tyrosine-phosphorylated, but this appears unrelated to its capacity to participate in EGFR internalization.

c-Cbl directs EGF receptors into an endocytic pathway that involves the ubiquitin-interacting motif of Eps15.

c-Cbl associates with the activated EGF receptor before endocytosis. We here reveal that the capacity of c-Cbl to promote receptor internalization depends on its ubiquitin ligase activity, which functionally connects the EGF receptor to Eps15, a mediator of clathrin-coated pit formation. EGF-induced phosphorylation of Eps15, as well as recruitment of Eps15 to the plasma membrane and its co-localization with the EGF receptor in endosomes required the ubiquitin ligase activity of c-Cbl. This suggested that ubiquitin provides a direct or indirect link between the receptor and Eps15. Indeed, EGF-induced redistribution of Eps15 to the plasma membrane and endosomes depended on its ubiquitin-interacting motif. Upon over-expression, the ubiquitin-interacting motif abrogated the capacity of c-Cbl to promote EGF receptor endocytosis and only allowed receptor internalization via a route that lacked Eps15. Our findings disclose a novel function for the c-Cbl ubiquitin ligase and identify ubiquitin as a module that directs the EGF receptor into an endocytic pathway involving Eps15.

Cellular localization and signaling activity of beta-catenin in migrating neural crest cells.

In the vertebrate embryo, development of the neural crest is accompanied by sequential changes in cellular adhesiveness, allowing cells to delaminate from the neural epithelium, to undergo migration through extracellular matrix material, and to coalesce into ganglia of the peripheral nervous system. Because of its dual role in cell adhesion, as a link between cadherins and the actin cytoskeleton, and in cell signaling, as a key mediator of the Wnt-signaling pathway, beta-catenin is a good candidate to play a central role in the control of neural crest cell development. In the present study, we analyzed, by using an in vitro culture system, whether the cellular localization and the signaling activity of beta-catenin are regulated in conjunction with cell migration during ontogeny of trunk neural crest cells in the avian embryo. beta-Catenin molecules were found primarily in association with N-cadherin in the regions of intercellular contacts in most migrating neural crest cells, and only early-migrating cells situated in proximity with the dorsal side of the neural tube showed detectable beta-catenin in their nuclei. This finding indicates that beta-catenin may be recruited for signaling in neural crest cells only transiently at the onset of migration and that sustained beta-catenin signals are not necessary for the progression of migration. The nuclear distribution of beta-catenin within crest cells was not affected upon modification of the N-cadherin-mediated cell-cell contacts, revealing that recruitment of beta-catenin for signaling is not driven by changes in intercellular cohesion during migration. Overstimulation of beta-catenin signals in neural crest cells at the time of their migration, using LiCl treatment or coculture with Wnt-1-producing cells, induced nuclear translocation of beta-catenin and Lef-1 up-regulation in neural crest cells and provoked a marked inhibition of cell delamination and migration. The effect of LiCl and exogenous Wnt-1 on neural crest cells could be essentially attributed to a dramatic decrease in integrin-mediated cell-matrix adhesion as well as a massive reduction of cell proliferation. In addition, although it apparently did not affect expression of neural crest markers, Wnt-1 exposure dramatically affected signaling events involving Notch-Delta, presumably also accounting for the strong reduction in cell delamination. In conclusion, our data indicate that beta-catenin functions primarily in cell adhesion events during migration and may be recruited transiently for signaling during delamination possibly to regulate the balance between cell proliferation and cell differentiation.