Saskia K.M. van Daalen

Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection

Many human Y-chromosomal deletions are thought to severely impair reproductive fitness, which precludes their transmission to the next generation and thus ensures their rarity in the population. Here we report a 1.6-Mb deletion that persists over generations and is sufficiently common to be considered a polymorphism. We hypothesized that this deletion might affect spermatogenesis because it removes almost half of the Y chromosomes AZFc region, a gene-rich segment that is critical for sperm production. An association study established that this deletion, called gr/gr, is a significant risk factor for spermatogenic failure. The gr/gr deletion has far lower penetrance with respect to spermatogenic failure than previously characterized Y-chromosomal deletions; it is often transmitted from father to son. By studying the distribution of gr/gr-deleted chromosomes across the branches of the Y chromosomes genealogical tree, we determined that this deletion arose independently at least 14 times in human history. We suggest that the existence of this deletion as a polymorphism reflects a balance between haploid selection, which culls gr/gr-deleted Y chromosomes from the population, and homologous recombination, which continues to generate new gr/gr deletions.

Chimpanzee and human Y chromosomes are remarkably divergent in structure and gene content

The human Y chromosome began to evolve from an autosome hundreds of millions of years ago, acquiring a sex-determining function and undergoing a series of inversions that suppressed crossing over with the X chromosome. Little is known about the recent evolution of the Y chromosome because only the human Y chromosome has been fully sequenced. Prevailing theories hold that Y chromosomes evolve by gene loss, the pace of which slows over time, eventually leading to a paucity of genes, and stasis. These theories have been buttressed by partial sequence data from newly emergent plant and animal Y chromosomes, but they have not been tested in older, highly evolved Y chromosomes such as that of humans. Here we finished sequencing of the male-specific region of the Y chromosome (MSY) in our closest living relative, the chimpanzee, achieving levels of accuracy and completion previously reached for the human MSY. By comparing the MSYs of the two species we show that they differ radically in sequence structure and gene content, indicating rapid evolution during the past 6 million years. The chimpanzee MSY contains twice as many massive palindromes as the human MSY, yet it has lost large fractions of the MSY protein-coding genes and gene families present in the last common ancestor. We suggest that the extraordinary divergence of the chimpanzee and human MSYs was driven by four synergistic factors: the prominent role of the MSY in sperm production, ‘genetic hitchhiking’ effects in the absence of meiotic crossing over, frequent ectopic recombination within the MSY, and species differences in mating behaviour. Although genetic decay may be the principal dynamic in the evolution of newly emergent Y chromosomes, wholesale renovation is the paramount theme in the continuing evolution of chimpanzee, human and perhaps other older MSYs.

Propagation of human spermatogonial stem cells in vitro.

CONTEXT Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility. OBJECTIVE To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation. DESIGN, SETTING, AND PARTICIPANTS Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation. MAIN OUTCOME MEASURES Propagation of spermatogonial stem cells over time. RESULTS Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and increased 18,450-fold within 64 days in the germline stem cell subculture. CONCLUSION Long-term culture and propagation of human spermatogonial stem cells in vitro is achievable.

High mutation rates have driven extensive structural polymorphism among human Y chromosomes

Although much structural polymorphism in the human genome has been catalogued, the kinetics of underlying change remain largely unexplored. Because human Y chromosomes are clonally inherited, it has been possible to capture their detailed relationships in a robust, worldwide genealogical tree. Examination of structural variation across this tree opens avenues for investigating rates of underlying mutations. We selected one Y chromosome from each of 47 branches of this tree and searched for large-scale variation. Four chromosomal regions showed extensive variation resulting from numerous large-scale mutations. Within the tree encompassed by the studied chromosomes, the distal-Yq heterochromatin changed length ≥12 times, the TSPY gene array changed length ≥23 times, the 3.6-Mb IR3/IR3 region changed orientation ≥12 times and the AZFc region was rearranged ≥20 times. After determining the total time spanned by all branches of this tree (∼1.3 million years or 52,000 generations), we converted these mutation counts to lower bounds on rates: ≥2.3 × 10−4, ≥4.4 × 10−4, ≥2.3 × 10−4 and ≥3.8 × 10−4 large-scale mutations per father-to-son Y transmission, respectively. Thus, high mutation rates have driven extensive structural polymorphism among human Y chromosomes. At the same time, we found limited variation in the copy number of Y-linked genes, which raises the possibility of selective constraints.

Gene copy number reduction in the azoospermia factor c (AZFc) region and its effect on total motile sperm count

The azoospermia factor c (AZFc) region harbors multi-copy genes that are expressed in the testis. Deletions of the AZFc region lead to reduced copy numbers of these genes. Four (partial) AZFc deletions have been described of which the b2/b4 and gr/gr deletions affect semen quality. In most studies, (partial) AZFc deletions are identified and characterized using plus/minus sequence site tag (STS) polymerase chain reaction (PCR). However, secondary duplications increase the gene copy number without re-introducing the STS boundary marker. Consequently, the actual copy number of AZFc genes cannot be determined via STS PCR. In the current study, we first set out to determine by quantitative real-time PCR the actual copy number of all AZFc genes in men with (partial) AZFc deletions based on STS PCR. We then analyzed whether reduced gene copy numbers of each AZFc gene family were associated with reduced total motile sperm count (TMC), regardless of the type of deletion. We screened 840 men and identified 31 unrelated men with (partial) deletions of AZFc based on STS PCR. Of these 31 men, 6 men (19%) had one or more secondary duplications. For all AZFc genes, we found an association between a reduction in the copy number of each individual AZFc gene and reduced TMC. In gr/gr-deleted men, restoration of reduced gene copy numbers restored their TMC to normal values. Our findings suggest that the gene content of the AZFc region has been preserved throughout evolution through a dosage effect of the AZFc genes on TMC safeguarding male fertility.

Enrichment of spermatogonial stem cells from long-term cultured human testicular cells

OBJECTIVE To evaluate the degree of enrichment of spermatogonial stem cells (SSCs) from human testicular cell cultures by ITGA6+, HLA-/ITGA6+, GPR125+, and HLA-/GPR125+ magnetic-assisted cell sorting (MACS). DESIGN Experimental basic science study. SETTING Reproductive biology laboratory. PATIENT(S) Multiple samples of cryopreserved human testicular cells from two prostate cancer patients with normal spermatogenesis. INTERVENTION(S) Cultured human testicular cells subjected to four sorting strategies based on MACS and xenotransplanted to the testes of mice to determine the enrichment for SSCs. MAIN OUTCOME MEASURE(S) Enrichment for human spermatogonia and SSCs tested by expression analysis of spermatogonial markers ITGA6, GPR125, ZBTB16, UCHL1, and ID4 using quantitative real-time polymerase chain reaction (qPCR) and by xenotransplantation into the testes of mice, respectively. RESULT(S) Compared with the nonsorted cultured testicular cells, only the ITGA6+ and HLA-/GPR125+ sorted cells showed enrichment for ID4. No difference in expression of ZBTB16 and UCHL1 was observed. Xenotransplantation of the sorted cell fractions showed a 7.1-fold enrichment of SSCs with ITGA6+. CONCLUSION(S) Magnetic-assisted cell sorting of cultured human testicular cells using ITGA6 allows for enrichment of SSCs, which aids in further molecular characterization of cultured human SSCs and enhances testicular colonization upon transplantation in future clinical settings.

Clinical relevance of partial AZFc deletions

To determine the number of DAZ gene clusters in the Y-bearing spermatozoa of patients who underwent intracytoplasmic sperm injection (ICSI) and to compare the outcome with the number of clusters found in the spermatozoa of normospermic men. Prospective study. Academic hospital.Forty-seven patients with impaired spermatogenesis who were attending our clinic for ICSI and 56 semen donors. Peripheral blood was drawn to obtain somatic DNA for polymerase chain reaction (PCR) analysis and leukocytes for karyotyping and FISH analysis. Three-color FISH was performed on the spermatozoa remaining after ICSI and on the spermatozoa of semen donors to determine the presence of the X and Y chromosome as well as the number of DAZ gene clusters. Number of DAZ gene clusters in Y-bearing spermatozoa. Five patients had only one DAZ gene cluster, one patient had a complete AZFc deletion, and one patient had three clusters on average. One of the semen donors also showed three DAZ gene clusters in his Y-bearing spermatozoa. None of the semen donors had only one DAZ gene cluster. Besides complete AZFc deletions, partial deletions are also associated with impaired spermatogenesis. As a result, these partial deletions that are not recognized by routine PCR are reintroduced into the population by the ICSI technique.

A comprehensive gene mutation screen in men with asthenozoospermia

OBJECTIVE To find novel genetic causes of asthenozoospermia by comprehensively screening known candidate genes derived from mouse models. DESIGN Case-control study. SETTING A fertility center based in an academic hospital. PATIENT(S) Thirty men with isolated asthenozoospermia. INTERVENTION(S) Screening nine candidate genes for mutations: ADCY10, AKAP4, CATSPER1, CATSPER2, CATSPER3, CATSPER4, GAPDHS, PLA2G6, and SLC9A10. To account for a possible effect of heterozygous mutations, assessing imprinting of all candidate genes by studying the expression pattern of heterozygous SNPs in testis biopsies of five unrelated men. MAIN OUTCOME MEASURE(S) Mutations found in patients only. RESULT(S) We identified 10 heterozygous asthenozoospermia-specific mutations in ADYC10 (n = 2), AKAP4 (n =1), CATSPER1 (n = 1), CATSPER2 (n = 1), CATSPER3 (n = 1), CATSPER4 (n = 3), and PLA2G6 (n = 1). These mutations were distributed over six patients. In silico analysis showed that 8 of the 10 mutations either had a negative BLOSUM score, were located in conserved residues, and/or were located in a functional domain. Expression analysis demonstrated that CATSPER1 and CATSPER4 are imprinted. CONCLUSION(S) Given their putative effect on protein structure, their location in conserved sequences or functional domains, and their absence in controls, the identified mutations may be a cause of asthenozoospermia in humans.

Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells

Abstract STUDY QUESTION Is testicular transplantation of in vitro propagated spermatogonial stem cells associated with increased cancer incidence and decreased survival rates in recipient mice? SUMMARY ANSWER Cancer incidence was not increased and long-term survival rate was not altered after transplantation of in vitro propagated murine spermatogonial stem cells (SSCs) in busulfan-treated recipients as compared to non-transplanted busulfan-treated controls. WHAT IS KNOWN ALREADY Spermatogonial stem cell autotransplantation (SSCT) is a promising experimental reproductive technique currently under development to restore fertility in male childhood cancer survivors. Most preclinical studies have focused on the proof-of-principle of the functionality and efficiency of this technique. The long-term health of recipients of SSCT has not been studied systematically. STUDY DESIGN, SIZE, DURATION This study was designed as a murine equivalent of a clinical prospective study design. Long-term follow-up was performed for mice who received a busulfan treatment followed by either an intratesticular transplantation of in vitro propagated enhanced green fluorescent protein (eGFP) positive SSCs (cases, n = 34) or no transplantation (control, n = 37). Using a power calculation, we estimated that 36 animals per group would be sufficient to provide an 80% power and with a 5% level of significance to demonstrate a 25% increase in cancer incidence in the transplanted group. The survival rate and cancer incidence was investigated until the age of 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS Neonatal male B6D2F1 actin-eGFP transgenic mouse testis were used to initiate eGFP positive germline stem (GS) cell culture, which harbor SSCs. Six-week old male C57BL/6 J mice received a single dose busulfan treatment to deplete the testis from endogenous spermatogenesis. Half of these mice received a testicular transplantation of cultured eGFP positive GS cells, while the remainder of mice served as a control group. Mice were followed up until the age of 18 months (497–517 days post-busulfan) or sacrificed earlier due to severe discomfort or illness. Survival data were collected. To evaluate cancer incidence a necropsy was performed and tissues were collected. eGFP signal in transplanted testis and in benign and malignant lesions was assessed by standard PCR. MAIN RESULTS AND THE ROLE OF CHANCE We found 9% (95% CI: 2–25%) malignancies in the transplanted busulfan-treated animals compared to 26% (95% CI: 14–45%) in the busulfan-treated control group, indicating no statistically significant difference in incidence of malignant lesions in transplanted and control mice (OR: 0.3, 95% CI: 0.1–1.1). Furthermore, none of the malignancies that arose in the transplanted animals contained eGFP signal, suggesting that they are not derived from the in vitro propagated transplanted SSCs. Mean survival time after busulfan treatment was found to be equal, with a mean survival time for transplanted animals of 478 days and 437 days for control animals (P = 0.076). LARGE SCALE DATA NA. LIMITATIONS, REASONS FOR CAUTION Although we attempted to mimic the future clinical application of SSCT in humans as close as possible, the mouse model that we used might not reflect all aspects of the future clinical setting. WIDER IMPLICATIONS OF THE FINDINGS The absence of an increase in cancer incidence and a decrease in survival of mice that received a testicular transplantation of in vitro propagated SSCs is reassuring in light of the future clinical application of SSCT in humans. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by KiKa (Kika86) and ZonMw (TAS 116003002). The authors report no financial or other conflict of interest relevant to the subject of this article.

Distinct prophase arrest mechanisms in human male meiosis

ABSTRACT To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX. Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development. Summary: Histological examination and transcriptomic analysis of human meiosis-arrested spermatocytes reveals two prophase arrest mechanisms, each associated with distinct gene expression profiles implicating the involvement of different biological processes.