Cindy M. Korver

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.

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.

Embryonic stem cell-like cells derived from adult human testis

BACKGROUND Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the derivation of ES-like cells from adult human testis. METHODS Testis material was donated for research by four men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were cultured using StemPro medium. Colonies that appeared sharp edged and compact were collected and subcultured under hES-specific conditions. Molecular characterization of these colonies was performed using RT-PCR and immunohistochemistry. (Epi)genetic stability was tested using bisulphite sequencing and karyotype analysis. Directed differentiation protocols in vitro were performed to investigate the potency of these cells and the cells were injected into immunocompromised mice to investigate their tumorigenicity. RESULTS In testicular cell cultures from all four men, sharp-edged and compact colonies appeared between 3 and 8 weeks. Subcultured cells from these colonies showed alkaline phosphatase activity and expressed hES cell-specific genes (Pou5f1, Sox2, Cripto1, Dnmt3b), proteins and carbohydrate antigens (POU5F1, NANOG, SOX2 and TRA-1-60, TRA-1-81, SSEA4). These ES-like cells were able to differentiate in vitro into derivatives of all three germ layers including neural, epithelial, osteogenic, myogenic, adipocyte and pancreatic lineages. The pancreatic beta cells were able to produce insulin in response to glucose and osteogenic-differentiated cells showed deposition of phosphate and calcium, demonstrating their functional capacity. Although we observed small areas with differentiated cell types of human origin, we never observed extensive teratomas upon injection of testis-derived ES-like cells into immunocompromised mice. CONCLUSIONS Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis

The population of early A spermatogonia includes stem cells that possess spermatogonial stem cell properties. Recent reports suggest that these cells have the ability to regain pluripotent properties. Here, we show that expression of the pluripotency marker undifferentiated embryonic cell transcription factor 1 (UTF1) is restricted to distinct germ cells within the testis. In embryonic and neonatal testes, all gonocytes were found to strongly express UTF1. During further testicular development, expression of UTF1 was restricted to a subset of A spermatogonia and with the increase in age the number of cells expressing UTF1 decreased even more. Ultimately, in the adult rat testis, only a small subset of the A spermatogonia expressed UTF1. Remarkably, even in testes of vitamin A-deficient rats, in which the early A spermatogonia (A(s), A(pr), and A(al)) are the only type of spermatogonia, only a subset of the spermatogonia expressed UTF1. In the adult rat testis, expression of UTF1 is restricted to a subpopulation of the ZBTB16 (PLZF)-positive early A spermatogonia. Furthermore, the observed distribution pattern of UTF1-expressing cells over the different stages of the cycle of the seminiferous epithelium suggests that the expression of UTF1 is restricted to those A(s), A(pr), and short chains of A(al) spermatogonia that are in the undifferentiated state and therefore maintain the ability to differentiate into A1 spermatogonia in the next round of the epithelial cycle or possibly even in other directions when they are taken out of their testicular niche.

Y chromosome gr/gr deletions are a risk factor for low semen quality

BACKGROUND Subfertility affects one in eight couples. In up to 50% of cases, the male partner has low semen quality. Four Y chromosome deletions, i.e. Azoospermia factor a (AZFa), P5/proximal-P1 (AZFb), P5/distal-P1 and AZFc deletions, are established causes of low semen quality. Whether a recently identified partial AZFc deletion, the gr/gr deletion, also causes low semen quality is at present unclear. METHODS We used a dual approach to review the effect of the gr/gr deletion on semen quality. First, we conducted a systematic review and meta-analysis of previous association studies, to compare the prevalence of gr/gr deletions between azoo-/oligozoospermic men and normozoospermic men. Secondly, we studied a cohort of 1041 male partners of subfertile couples unselected for semen quality. We employed a cross-sectional design by screening all men for the gr/gr deletion and comparing the semen quality of men with and without the gr/gr deletion. RESULTS Seven studies were included in the meta-analysis. The gr/gr deletion was significantly more prevalent among azoo-/oligozoospermic men than among normozoospermic men (OR 2.4, 95% CI 1.75-3.30). In our cohort, 25 men carried a gr/gr deletion. Men with this genotype had a lower sperm concentration (median 34 x 10(6)/ml versus 53 x 10(6)/ml, P = 0.017), total sperm count (median 108 x 10(6) versus 152 x 10(6), P = 0.006) and total motile sperm count (median 20 x 10(6) versus 50 x 10(6), P = 0.010) than men without the gr/gr deletion. CONCLUSION Y chromosome gr/gr deletions significantly reduce sperm counts and are thus associated with low semen quality.

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.

BMP4-Induced Differentiation of a Rat Spermatogonial Stem Cell Line Causes Changes in Its Cell Adhesion Properties

Spermatogonial stem cells (SSCs) are at the basis of the spermatogenic process and are essential for the continuous lifelong production of spermatozoa. Although several factors that govern SSC self-renewal and differentiation have been investigated, the direct effect of such factors on SSCs has not yet been studied, mainly because of the absence of markers to identify SSCs and the lack of effective methods to obtain and culture a pure population of SSCs. We now have used a previously established rat SSC cell line (GC-6spg) to elucidate the role of BMP4 in SSC differentiation. We found that GC-6spg cells cultured in the presence of BMP4 upregulate KIT expression, which is an early marker for differentiating spermatogonia. GC-6spg cells were found to express three BMP4 receptors and the downstream SMAD1/5/8 proteins were phosphorylated during BMP4-induced differentiation. A time-course DNA micro-array analysis revealed a total of 529 differentially regulated transcripts (≥2-fold), including several known downstream targets of BMP4 such as Id2 and Gata2. Pathway analysis revealed that the most affected pathways were those involved in adherens junctions, focal junctions, gap junctions, cell adhesion molecules, and regulation of actin cytoskeleton. Interestingly, among the genes belonging to the most strongly affected adhesion pathways was Cdh1 (known as E-cadherin), an adhesion molecule known to be expressed by a subpopulation of spermatogonia including SSCs. Overall, our results suggest that BMP4 induces early differentiation of SSCs in a direct manner by affecting cell adhesion pathways.

Are Sequence Family Variants Useful for Identifying Deletions in the Human Y Chromosome

To the Editor: We read with interest the report of a novel deletion of part of the azoospermia factor c (AZFc [MIM 415000]) region of the human Y chromosome (Fernandes et al. 2004). This article reported that the deletion is found only in branch N of the Y-chromosome genealogical tree, occurs through one mutational pathway, is ∼2.2 Mb in size, and has no effect on spermatogenesis. We, too, recently reported this deletion, which Fernandes et al. termed the “g1/g3” deletion and which we termed the “b2/b3” deletion (Repping et al. 2004). Our findings, however, differed from those of Fernandes et al. in several important particulars: (1) our screening of 1,563 men demonstrated that this deletion is not confined to branch N and that it has at least four independent origins; (2) our analysis revealed two mutational pathways, rather than one, that can generate the deletion, and we confirmed the existence of the inverted AZFc organizations that are the intermediate steps in these pathways; (3) on the basis of the reference sequence of the Y chromosome, we concluded that the size of the deletion is 1.8 Mb, rather than ∼2.2 Mb; (4) using interphase FISH, we confirmed the amplicon organization that was postulated in the deletion and also identified three instances of duplication subsequent to the deletion; and (5) because of the possibility of a compensatory factor on Y chromosomes in branch N and because of the limited number of deletions outside this branch, we concluded that a possible effect of this deletion on risk of spermatogenic failure cannot be excluded (Repping et al. 2004). Beyond these differences, however, the characterizations of this and other partial deletions of AZFc (Repping et al. 2003) highlight a more important question. At issue is the relative utility of sequence family variants (Saxena et al. 2000), compared with that of plus/minus STSs, for identification and differentiation of deletions involving AZFc. AZFc is composed entirely of amplicons—repeat units 115–678 kb in length that only differ by ∼1 nt per 3,000 bp. These rare differences are called “sequence family variants” (SFVs). We previously relied on SFVs to map and sequence the AZFc region of one man’s Y chromosome (Kuroda-Kawaguchi et al. 2001). The report by Fernandes et al. (2004) emphasized the use of SFVs in identification of the novel deletion, whereas our analysis relied on plus/minus STSs for identification of the deletion, followed, in most instances, by confirmation with FISH. Two observations led us to ask whether SFVs, as opposed to plus/minus STSs, offer the simpler and more robust means of detecting and distinguishing deletions in AZFc. First, figures 1 and 4 in the report by Fernandes et al. (2004) indicated that negative results at the plus/minus STS sY1192 or 50f2/C combined with positive results at flanking STSs are sufficient to detect the deletion (table 1). Moreover, the b2/b3 deletion and other types of deletions involving AZFc can be distinguished by their plus/minus signatures, without the use of SFVs (table 1). Table 1 Plus/Minus STS Results Distinguishing Different Types of Deletions Involving AZFc[Note] Second, table 2 in the report by Fernandes et al. (2004) showed that the SFV patterns of undeleted chromosomes vary considerably among different branches of the Y-chromosome genealogy and that the patterns also vary among individuals within branches. These observations suggested that the link between SFV patterns and particular types of deletions would likely not be consistent across the worldwide diversity of Y chromosomes. The diversity of SFV patterns in undeleted chromosomes is not surprising, since AZFc is subject to large inversions, deletions, and duplications caused by ectopic homologous recombination between amplicons (Kuroda-Kawaguchi et al. 2001; Repping et al. 2003, 2004). Such events would rearrange the locations of particular variants and would blur the association between SFV patterns and particular types of deletions. The association would likely be further blurred by gene conversion, which frequently erases small sequence differences (i.e., SFVs) between amplicon copies on the Y chromosome (Rozen et al. 2003). We experimentally investigated the consistency of SFV patterns in different types of deletions involving AZFc. First, using the SFVs employed by Fernandes et al. (2004), we typed 20 men reported elsewhere to have the b2/b3 deletion (Repping et al. 2004) (see GenBank Web site for SFV assays). These men represented branch N and three other branches of the Y-chromosome genealogy (fig. 1). Second, using the same SFVs, we typed 40 men reported elsewhere to have the gr/gr deletion, the other common partial AZFc deletion (Repping et al. 2003). These men represented 14 branches of the Y-chromosome genealogy (fig. 1). Figure 1 Genealogical analysis of SFV patterns associated with b2/b3 and gr/gr deletions. In the SFV patterns, “C” indicates the cut variant described by Fernandes et al. (2004), “U” indicates the uncut variant, “B” ... The b2/b3 deletions outside branch N showed diverse SFV patterns, and the gr/gr deletions showed even greater diversity (fig. 1). This greater diversity was likely due to the larger number of independent gr/gr deletions studied. Two branches, F*(xHK) and R1*x, contained numerous deletions and a high diversity of SFV patterns (fig. 1). In these branches, multiple independent deletion events probably account for the high diversity. By contrast, two other branches, D2b and N, contained numerous deletions but uniform SFV patterns. This uniformity is explained by the fact that all chromosomes in these branches descended from deleted founders (Repping et al. 2003, 2004; Fernandes et al. 2004). Thus, the chromosomes in each of these branches represent a single deletion event. Our data also showed that the SFV patterns of b2/b3 and gr/gr deletions are not distinct from each other. For example, the b2/b3 pattern UUUCUU−CUUU (branch F*[xHK]) is more similar to the gr/gr pattern UUCCUU+CBUB (branch F*[xHK], four differences [underlined]) than to the b2/b3 pattern UBBBCU−CCUC (branch N, six differences). In another example, the gr/gr pattern UBBBCU−UBUB (branch R1*x) is more similar to the b2/b3 pattern UBBBCU−CUUC (branch I, three differences) than to the gr/gr pattern BCCCUB+CBCC (branch R1*x, 10 differences). In conclusion, the SFV patterns of b2/b3 and gr/gr deletions vary widely and are not clearly distinct. SFVs can offer insight only if one knows the common SFV organizations in the genealogical branches represented by the Y chromosomes being tested. However, SFV organizations across the Y-chromosome genealogical tree are largely unknown, and SFV patterns vary even among individuals in the same branch. Just as important is that a large number of two-step assays are needed for SFV typing and for determining the Y-chromosome branch. By contrast, six simple plus/minus STSs distinguish between the deletions involving AZFc (table 1). Thus, plus/minus STSs provide a straightforward means of identifying and distinguishing the deletions of part of AZFc, whereas, in most situations, SFVs do not.

A novel partial deletion of the Y chromosome azoospermia factor c region is caused by non-homologous recombination between palindromes and may be associated with increased sperm counts

BACKGROUND The male-specific region of the human Y chromosome (MSY) contains multiple testis-specific genes. Most deletions in the MSY lead to inadequate or absent sperm production. Nearly all deletions occur via homologous recombination between amplicons. Previously, we identified two P5/distal-P1 deletions that did not arise via homologous recombination but most probably via non-homologous recombination (NHR) between palindromes. In the current study, we set out to identify deletions in the azoospermia factor c (AZFc) region caused by NHR between palindromes. METHODS We screened 1237 men using plus/minus and quantitative real-time polymerase chain reaction, fluorescence in situ hybridization and Southern blot analyses for deletions caused by NHR. These 1237 men originated from two series: one series of 237 men with azoospermia or severe oligozoospermia and 148 with normozoospermia and one series of 852 consecutively included men of subfertile couples unselected for sperm count. RESULTS We identified eight unrelated men with deletions caused by NHR. These deletions could be categorized into four classes termed P3a, P3b, P3c and P3d. The P3a and P3b deletions were found in single instances whereas the P3c and P3d deletions were found in three men. Men with a P3c deletion had a higher total sperm count than those without a deletion (median 378.8 × 10(6) versus 153.9 × 10(6), P = 0.040). We did not find an association of the other P3 deletions with altered sperm counts. CONCLUSIONS We have found a novel subclass of partial AZFc deletions that results from NHR. One deletion, the P3c deletion, might be associated with increased sperm count.