Fig 1.
(A) XY. The Y short arm (Yp) gene complement of an XY male (represented to scale in the magnified view) comprises nine single copy genes, two duplicated genes and one multi copy gene. The pseudoautosomal region (PAR) located distally on the Y long arm mediates pairing and crossing over with the X PAR during meiosis to generate the XY sex bivalent. Centromeres are represented by a dot on the chromosome. (B–D) The diminishing Yp gene complements for the three XO male mouse models that lack the Y long arm. (B) XSxraO. The Yp-derived Sxra attached distal to the X PAR provides an almost complete Yp gene complement. (C) XEif2s3ySxrbO. The Sxra-derived deletion variant Sxrb has a 1.3 Mb deletion (ΔSxr-b) removing 6 single copy genes and creating a Zfy2/1 fusion gene spanning the deletion breakpoint (†). The deleted gene Eif2s3y is necessary for normal spermatogonial proliferation, so an X-located Eif2s3y transgene has been added. (D) XEif2s3yOSry. This model has only two Yp genes—the testis determinant Sry provided as an autosomally located transgene and the spermatogonial proliferation factor Eif2s3y provided as the X-located transgene. E. Y*X. This mini sex-chromosome is an X chromosome with a deletion from just proximal to Amelx to within the DXHXF34 repeat adjacent to the X centromere († marks the deletion breakpoint). This X chromosome derivative has a complete PAR that can pair and crossover with the PAR of XSxra, XSxrb or X to form a ‘minimal sex bivalent’. Scale bar for magnified views is 150 kb.
Fig 2.
The normal steps of spermiogenesis in relation to testis tubule stages in an XY mouse.
This figure presents a schematic depiction of the 16 steps of mouse spermiogenesis based on the stage of the spermatogenic cycle (stages I–XII). Photos of step 1–12 spermatid nuclei are shown with PAS staining at the foot of the diagram. The transition from round to elongating spermatids takes place during stage VIII; this marks the beginning of sperm morphogenesis. The shaping of the sperm head and formation of the sperm tail are essentially complete by step 12. However, the sperm are not shed until the following stages VIII-IX; consequently two generations of spermatids are present in stages I-VIII.
Fig 3.
Addition of Y*X to XEOSry or XESxrbO models does not improve spermiogenic progression.
Periodic acid Schiff/hematoxylin stained testis sections illustrating the extent of spermiogenic progression. Roman numerals denote estimated tubule stages. (A) In XEOSry, the predominantly diploid spermatids do not elongate and the acrosomes (stained dark pink) remain randomly orientated relative to the basement membrane of the tubule (insets in VIII and XII). The spermatid nuclei show signs of pycnosis by stage XII (inset) and the cells have been eliminated by stages II-IV. The abundant round cells at stages II-IV are the new generation of round spermatids with early stages of acrosome development (dark pink ‘acrosomal granules’ in inset) [for more details see Vernet et al 2012]. In XEY*XSry the block to spermiogenesis remains with elimination of the arrested cells once again evident by stages II-IV (see inset). (B) In XESxrbO, at stage VIII the spermatids have not elongated and they are randomly orientated relative to the tubule basement membrane. However, as previously reported (Vernet et al 2012), spermatid elongation is delayed rather than absent, and is apparent by stage XII. Nuclear condensation is also delayed as it is not evident at stage XII, but many of the elongating spermatids survive to stages II-IV at which point nuclear condensation is now evident. In XEY*XSxrb spermatid elongation and nuclear condensation is similarly delayed, but there appear to be fewer elongating spermatids surviving to stages II-IV [note the now evident haploid (h) as well as diploid (d) spermatids]. Scale bar is 40 μm (insets are x3 magnification).
Fig 4.
Addition of Zfy2 (but not Zfy1) to XEY*XSry enables re-orientation of spermatids, together with clear head and tail morphogenesis; however the Zfy2 transgene is not as effective as Sxra in supporting sperm morphogenesis.
Hematoxylin and eosin (H & E) stained testis tubule sections. (A) By comparison with XEY*XSry, it is clear that the Zfy1 transgene addition has no beneficial effect on spermiogenic progression whereas the Zfy2 addition leads to the formation of correctly orientated elongated spermatids which have condensed sperm heads and some tail formation. (B) By comparison of XE,Z2Y*XSry with XY*XSxra (and XY controls) it is clear that Sxra is more effective than the Zfy2 transgene in supporting the sperm morphogenesis. In agreement with the illustration in Fig 2, in XY males at stage XI sperm head morphogenesis has progressed to the early ‘hooked tip’ stage and nuclear condensation is evident. As previously reported, in XY*XSxra at this stage some of the sperm from the previous cycle have not yet been shed (stained dark blue); the spermatids from the new cycle are retarded with respect to elongation and nuclear condensation is not evident. In XE,Z2Y*XSry spermatid elongation is further retarded. Nevertheless, by stage I-III many spermatids with condensed chromatin are found in XY, XY*XSxra and XE,Z2Y*XSry. (C) Images of testicular sperm found in silver stained testicular cell smears. All three genotypes proved to have some sperm present in testicular cell smears. In XY*XSxra, as previously reported for epididymal sperm and testicular sperm, the sperm heads rarely have a hooked tip. In XE,Z2Y*XSry the developing sperm heads show limited elongation. In all three genotypes the tails were well developed. (D) Hematoxylin/eosin stained epididymal tubule sections. In XY there are abundant sperm present with the heads showing the characteristic hooked tip. In XY*XSxra the sperm heads rarely have a hooked tip. In XE,Z2Y*XSry males no sperm could be identified; instead degenerating round spermatids are found suggesting that there has been some shedding of round spermatid stages despite the addition of Zfy2. Scale bars: A = 30 μm, B = 40 μm, C = 20 μm, D = 100 μm. Insets in D = 3x magnifications.
Fig 5.
Sperm head and tail morphogenesis in XE,Z2Y*XSry males as compared to controls.
(A) Electron micrographs of developing sperm heads from 6 week old XY, XY*XSxra and XE,Z2Y*XSry males. Spermatids at round to elongating transitional stage in XE,Z2Y*XSry present no apparent ultrastructural defects (bottom left picture). However, vacuoles (V) appear in the cytoplasm of elongating and condensing spermatids. Irregular spread of the acrosomal cap (arrows) distorting the spermatid nuclei is observed in XE,Z2Y*XSry and XY*XSxra. It was again evident that the sperm heads in XE,Z2Y*XSry fail to elongate properly (stars). (B) Electron micrographs of sperm tail sections showing a normal 9x2+2 axoneme pattern with a central microtubule pair (p) in addition to the nine outer doublets (d) in all three genotypes. Scale bars: A = 1 μm, B = 0.5 μm (Insets = 3x magnification).
Fig 6.
Levels of Cypt-dependent transcripts for XYSxrb, XY, XE,Z2Y*XSry and XEY*XSxrb.
The RT-PCR bands quantified are those from the RT-PCR assay in S1 Fig. The transcripts expected for each genotype (n = 2 represented by two bars on the chart) are indicated above each genotype label (2/1 denotes Cypt-dependent Zfy2/1 transcripts and 2 denotes Cypt-dependent Zfy2 transcripts). (A) The PCR primers for this assay amplified Zfy2/1 and Zfy2 transcripts; only XYSxrb has both transcripts. It can be seen that the level of Cypt-Zfy2/1 transcripts in XEY*XSxrb is comparable to the level of Cypt-Zfy2 transgene transcripts in XE,Z2Y*XSry. (B) For the left panel the PCR primers are specific for Zfy2/1; the second and third genotypes lack the Zfy2/1 fusion gene so the low level signal represents ‘background’. For the right panel the PCR primers are specific for Zfy2; in this case it is the fourth genotype that lacks Zfy2. The genotypes lacking the Y long arm (Yq-) have substantially higher transcript levels than the genotypes with a complete Y (Yq+).
Fig 7.
Mapping of Prssly and Teyorf1 to Sxra and Sxrb.
Prssly and Teyorf1 map to the Yp-derived Sxra chromosomal fragment (here attached distal to the PAR of one X of an XXSxra male). As expected from the known breakpoints for the ΔSxr-b deletion, Prssly and Teyorf1 are also present in Sxrb, whereas the Tspy pseudogene is absent.