Fig 1.
Mouse X and Y chromosomes, variant sex chromosomes, and mouse genotypes relevant to this study.
(A) The mouse Y chromosome contains ~90 Mb of male specific DNA and ~0.7 Mb constituting the pseudoautosomal region (PAR) situated at the end of the long arm. The PAR is the region of homology with the X that mediates pairing and recombination between the X and Y in normal males. The remaining non-pairing male specific part of Y (NPY) contains several genes and gene families. On the short arm (NPYp), there are single-copy genes: Prssly, Teyorf1, Uba1y, Smcy/Kdm5d, Eif2s3y, Uty, Dby/Ddx3y, Usp9y, Sry, duplicated gene Zfy (Zfy1 and 2), duplicated gene H2al2y, and a multi-copy gene Rbmy. The non-pairing region of the long arm (NPYq), representing ~90% of all NPY, contains mostly repetitive sequences, and encodes multiple copies of 5 distinct genes that are expressed in spermatids: Ssty1 and Ssty2, Sly, Srsy, Rbm31y [6]. Y*X is an X chromosome derivative encoding PAR, X centromere and near centromeric region. Sxra is a sex reversal variant Tp(Y)1CtSxr-a encoding almost intact NPYp complement but with Rbmy gene family reduced. Sxrb is a Sxra derivative with a 1.3 Mb deletion that has removed the majority of the NPYp gene complement and created a Zfy2/1 fusion gene. (B) The mice used in this study and their Y chromosome contribution. The X chromosome located Eif2s3y and autosomally located Sry transgenes, are shown in light blue frames. The Zfy2 transgene, shown in brown frame, is located on the X chromosome in the Hrpt locus in close proximity to the Eif2s3y transgene. The genotype designations without the Zfy2 transgene are shown above the diagrammatic representation of sex chromosomes and with the Zfy2 transgene below them (brown font). Sxra and Sxrb gene content is shown in A. n/a = mice with transgenic Zfy2 addition were either not produced or not examined in this study.
Table 1.
Zygotic chromosome analysis after ICSI with sperm from XESxrbO and XESxrbY*X males.
Fig 2.
(A) Exemplary tubules of stage VII-VIII testis sections. XEY*XSry males have meiotic and post-meiotic arrest that only occasionally allow formation of round spermatids that do not develop beyond step 7 of spermatid development. In XESxrbY*X spermatid elongation is observed but usually ceases at step 11–12, with few occurrences of more advanced stages. In XE,Z2Y*XSry males spermatogenesis is progressing with good spermatid elongation and many spermatids developing to step 15–16; these elongated spermatids are morphologically abnormal, which is expected from males lacking NPYq genes [19]. Tubule stages are shown in Roman numerals and steps of spermatid development (St) in Arabic numerals. Bar = 50 μm; insets = x3 magnification. See also S3 Fig emphasizing spermatid at step 7–8. (B) Quantitative analysis of spermatogenesis progression. For each male 10 tubules were examined per stage and the numbers of round spermatid (steps 1–8), elongating/ed spermatid (steps 9–16), and Sertoli cells were counted. The data are expressed as spermatid/Sertoli cell ratios. In wild-type males no round spermatids are present in stages IX-XI so those observed in males with limited Y gene complement represent 'delayed spermatids'. Statistical significance (t-test): a different than XEY*XSry; b different than XESxrbY*X; c different than all other. Three males per genotypes were included in the analysis. For explanation of male genotypes see Fig 1, S1 Table, and text.
Fig 3.
(A) The distribution of specific headshape defect categories among testicular sperm from XE,Z2Y*XSry (n = 41 sperm from 3 males), XESxrbY*X (n = 19 sperm from 2 males), and XY*XSxra (44 sperm from 3 males) males. (B) The categories of headshape defects. Normal: represents a normal shape of testicular sperm head. A ("dolphin"): sperm head is elongated and has some curvature reminiscent of crescent shape typical for mouse sperm, small hooked tip can be differentiated. B ("mushroom"): sperm head is elongated but the curvature is not present, hint of a hooked tip can sometimes be observed. C ("cupcake"): sperm head is no longer elongated, the caudal side is wider than in A and B categories, and opens up to a wide dorsal side; hint of a hooked tip can be sometimes be seen. D ("egg"): sperm head has an oval shape with no mark of a hooked tip. The head is less elongated than in category B; E ("ball"): sperm head has a round shape with no hint of a hooked tip, is smaller than in all other categories, and is strongly stained indicative of high DNA condensation; F ("drumstick"): sperm head is elongated, has no traces of a hooked tip, is longer and thinner than in category A&B but shorter than in G. G ("club"): sperm head is clearly elongated with no hint of a hooked tip, and very poorly condensed. Scale = 5 μm.
Table 2.
The results of round spermatid injection (ROSI) and intracytoplasmic sperm injection (ICSI) with germ cells from males with limited Y gene complement.
Fig 4.
Frequency of the offspring genotypes obtained after assisted reproduction with XE,Z2Y*XSry males. Four predominant genotypes (grey bars) and one rare genotype derived from untypical sex chromosome segregation (black bar on the right side of the dashed line) were observed. These genotypes are expected from XE,Z2Y*XSry males. Number of genotyped progeny was 49 for ROSI and 18 for ICSI. See also S2 Table.
Fig 5.
(A) Zfy1 transcript levels in genotypes of interest (n = 3 per genotype) obtained by real-time PCR. The loading controls were two ubiquitously expressed genes (actin and Sdha) and two spermatid-specific genes (Act and Acrv), and normalization was achieved by geometric averaging of these genes. (B) Zfy1 and Zfy2 (global Zfy) transcript levels were examined as in A. Values are mean ± SEM. Statistical significance: a different than all others (except zero to zero values comparison); * P < 0.05. For explanation of genotypes see Fig 1, S1 Table, and text. Primer sequences are shown in S3 Table. The data normalized to individual reference genes are shown in S5 Fig.