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Several germ cell tumors that occur in adult men likely originate from gonocytes that are impaired in their development. To select candidate genes that are involved in the normal development of the gonocytes, we constructed a complementary DNA (cDNA) library from rat gonocytes as well as from single, paired, and aligned A spermatogonia (As, Apr, and Aa1 spermatogonia, respectively), their direct descendants. Five hundred gonocyte clones were differentially screened using both libraries. Successive verification by dot blot assays yielded 7 clones that were consistently highly abundant only in the gonocyte cDNA library. Also, in situ hybridization of these clones confirmed their differential expression. They encoded for, respectively, succinate dehydrogenase, ribosomal protein S15a, and the 65-kilodalton scaffolding subunit (α isoform) of protein phosphatase 2A. No clues regarding the nature of the remaining four clones could be found. Hence, using differential screening on both constructed cDNA libraries, we were able to select several genes that are interesting candidates for studying the molecular mechanisms of normal gonocyte development.

In vitro spermatogenesis has been investigated using embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) or spermatogonial stem cells (SSCs) by many laboratories worldwide. However, for human fertility preservation, patient-specific ESCs are not available and the safety of using these cells to generate gametes that transfer their genome to the next generation has still not been thoroughly investigated. Therefore, in this thesis, we aim to recapitulate spermatogenesis in vitro using SSCs that are available in most of the patients with impaired spermatogenesis and can already be cryopreserved and propagated in vitro. We developed a three-step in vitro culture system to achieve complete meiosis using mouse SSCs cultured on immortalized Sertoli cells. Interestingly, in this culture system, although the in vitro-formed pachytene spermatocytes often displayed unsynapsed chromosomes and did not form sufficient meiotic crossovers, meiosis still progressed to the meiotic metaphase stages, and even sporadically formed spermatid-like cells. This indicated that the meiotic checkpoints that normally eliminate such aberrant pachytene spermatocytes were not completely functional in vitro. Considering that meiotic checkpoints prevent genomic instabilities from being transmitted to the offspring, we proposed that meiotic checkpoints should be thoroughly investigated before considering gametogenesis in vitro for clinical use. Finally, we reviewed the current progress in the field of spermatogenesis in vitro, with specific focus on meiosis and meiotic checkpoint mechanisms and systematically screened current research articles. Also clinical prospects of in vitro-derived gametes and the criteria that are applied to assess in vitro meiosis are discussed.

Hypogonadism, a clinical condition due to low testosterone levels is associated with increased visceral fat, decreased muscle mass, erectile dysfunction, reduced libido, depression and changes in mood. Hypogonadism might occur after testicular sperm extraction, although its exact effect on testosterone levels and consequently the risk on hypogonadism is still unclear. Other causes of hypogonadism can be orchitis, disorders of sexual development, mutations in the luteinizing hormone receptor, medication or graft-versus-host disease. To treat hypogonadism, testosterone replacement therapy is currently a common intervention to increase testosterone levels and with that to alleviate symptoms. A completely different avenue to address hypogonadism is by physiologically restoring testosterone levels by cell therapy. Cell therapy to treat hypogonadism is however currently unavailable, largely because the adequate cell type to use for cell therapy is yet to be identified. An interesting candidate cell type in this respect is the stem Leydig cell, which has the capacity to differentiate into testosterone producing adult Leydig cells. How to identify and properly isolate these cells from the human testis is at present unknown. In this thesis, we aimed at clarifying the risk of hypogonadism after testicular sperm extraction in men with azoospermia. We did this by performing a systemic literature review and a cohort study. In addition, we investigated the expression of multiple proteins in human testicular interstitial cells, and their potential to be used as markers for the isolation of human stem Leydig cells as a first prerequisite step for future cell therapy.

The vast majority of cases of spermatogenic failure, defined as no sperm in semen or low semen quality, are idiopathic, although several genetic factors have been identified. In this thesis, we focused on the effect of Y-chromosome variation including TSPY copy number and AZFc deletions on semen quality and studied the effectiveness and safety of in vitro spermatogonial stem cell propagation for (SSC) transplantation as a potential treatment for spermatogenic failure by increasing the number of SSCs. We found that the variation in TSPY copy number is not associated with total motile sperm count and therefore has no functional consequences for semen quality. AZFc deletions are known to be common genetic causes of spermatogenic failure. We showed that in our previously established culture system, the AZFc-deleted spermatogonia behave similar as spermatogonia from non-deleted men. To further characterize the human SSCs in our culture, we investigated which marker is efficient in enriching for SSCs. We found that ITGA6 (using magnetic assisted cell sorting or MACS) is an efficient method for enrichment of SSCs. We then investigated the genetic and epigenetic stability of human SSCs after long-term culture and showed that genetic stability of SSCs is preserved during culture. However, the methylation status of some of the imprinted genes is changed, which requires further investigation. We concluded that in vitro propagation of human SSCs followed by transplantation could be a good potential treatment option in patients with a low number of functional SSCs due to genetically caused spermatogenic failure.

Dit proefschrift beschrijft onderzoek naar genetische oorzaken van een falende spermatogenese. Wij onderzochten of mutaties in een aantal kandidaatgenen dan wel variatie in genkopie aantallen verband houden met falende spermatogenese. In de Westerse wereld heeft een op de acht paren vruchtbaarheidsproblemen. In ongeveer de helft van de gevallen heeft de man een in meer of mindere mate falende spermatogenese. Helaas is er op dit moment geen behandeling om de semenkwaliteit te verbeteren. Mannen met ernstige stoornissen in de spermatogenese kunnen met behulp van intracytoplasmatische spermacel injectie ofwel ICSI toch biologisch eigen nageslacht verkrijgen. Deze procedure is evenwel belastend voor de niet-zieke vrouwelijke partner. Door onderzoek te doen naar genetische oorzaken van falende spermatogenese hoopten wij aangrijpingspunten voor een toekomstige behandeling te vinden. In een aantal kandidaatgenen werden mutaties gevonden die mogelijk een oorzaak zijn van falende spermatogenese. Ook maakte ons onderzoek duidelijk dat een bepaalde deletie van het Y-chromosoom, de zogenaamde gr/gr deletie, een risicofactor voor falende spermatogenese is. Tenslotte vonden we 24 nieuwe genetische regio's met variabel DNA kopie aantal die mogelijk een extreme vorm van spermatogeen falen, het Sertoli Cell Only Syndroom, veroorzaken. Niettegenstaande deze bevindingen werd ook duidelijk dat genetisch onderzoek op korte termijn geen aanknopingspunten voor oorzakelijke behandelingen van falende spermatogenese zal opleveren. Recente ontwikkelingen op het gebied spermatogoniale stamcellen, in combinatie met genetische studies, zijn echter veelbelovend.

Spermatogoniale stamcellen staan aan de basis van de productie van mannelijke zaadcellen. Het is essentieel dat deze stamcellen een goede balans vinden tussen zelfvernieuwing en differentiatie; een verstoring hierin kan leiden tot onvruchtbaarheid of testiskanker. Gianfranco Carlomagno onderzocht welke groei- en omgevingsfactoren die balans beïnvloeden. Hij ontdekte dat de groeifactoren BMP4 en Activine A spermatogoniale stamcellen tot differentiatie aanzetten, net als verhoging van de kweektemperatuur.