Addition of Y*^X to X^<i>E</i>O<i>Sry</i> or X^<i>E</i><i>Sxr</i>^<i>b</i>O models does not improve spermiogenic progression.

<p>Periodic acid Schiff/hematoxylin stained testis sections illustrating the extent of spermiogenic progression. Roman numerals denote estimated tubule stages. <b>(A)</b> In X^<i>E</i>O<i>Sry</i>, 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 X^<i>E</i>Y*^X<i>Sry</i> the block to spermiogenesis remains with elimination of the arrested cells once again evident by stages II-IV (see inset). <b>(B)</b> In X^<i>E</i><i>Sxr</i>^<i>b</i>O, 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 X^<i>E</i>Y*^X<i>Sxr</i>^<i>b</i> 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).</p

The XO and XY*^X mouse models.

<p><b>(A)</b> 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>(B–D)</b> The diminishing Yp gene complements for the three XO male mouse models that lack the Y long arm. <b>(B)</b> X<i>Sxr</i>^<i>a</i>O. The Yp-derived <i>Sxr</i>^<i>a</i> attached distal to the X PAR provides an almost complete Yp gene complement. <b>(C)</b> X^{<i>Eif2s3y</i>}<i>Sxr</i>^<i>b</i>O. The <i>Sxr</i>^<i>a</i>-derived deletion variant <i>Sxr</i>^<i>b</i> has a 1.3 Mb deletion (Δ^<i>Sxr-b</i>) removing 6 single copy genes and creating a <i>Zfy2/1</i> fusion gene spanning the deletion breakpoint (†). The deleted gene <i>Eif2s3y</i> is necessary for normal spermatogonial proliferation, so an X-located <i>Eif2s3y</i> transgene has been added. <b>(D)</b> X^{<i>Eif2s3y</i>}OSry. This model has only two Yp genes—the testis determinant <i>Sry</i> provided as an autosomally located transgene and the spermatogonial proliferation factor <i>Eif2s3y</i> provided as the X-located transgene. E. Y*^X. This mini sex-chromosome is an X chromosome with a deletion from just proximal to <i>Amelx</i> 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 X<i>Sxr</i>^<i>a</i>, X<i>Sxr</i>^<i>b</i> or X to form a ‘minimal sex bivalent’. Scale bar for magnified views is 150 kb.</p

Sperm head and tail morphogenesis in X^{<i>E</i>,<i>Z2</i>}Y*^X<i>Sry</i> males as compared to controls.

<p><b>(A)</b> Electron micrographs of developing sperm heads from 6 week old XY, XY*^X<i>Sxr</i>^<i>a</i> and X^{<i>E</i>,<i>Z2</i>}Y*^X<i>Sry</i> males. Spermatids at round to elongating transitional stage in X^{<i>E</i>,<i>Z2</i>}Y*^X<i>Sry</i> 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 X^{<i>E</i>,<i>Z2</i>}Y*^X<i>Sry</i> and XY*^X<i>Sxr</i>^<i>a</i>. It was again evident that the sperm heads in X^{<i>E</i>,<i>Z2</i>}Y*^X<i>Sry</i> fail to elongate properly (stars). <b>(B)</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).</p

Levels of <i>Cypt</i>-dependent transcripts for XY<i>Sxr</i>^<i>b</i>, XY, X^{<i>E</i>,<i>Z2</i>}Y*^X<i>Sry</i> and X^<i>E</i>Y*^X<i>Sxr</i>^<i>b</i>.

<p>The RT-PCR bands quantified are those from the RT-PCR assay in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145398#pone.0145398.s002" target="_blank">S1 Fig</a>. The transcripts expected for each genotype (n = 2 represented by two bars on the chart) are indicated above each genotype label (2/1 denotes <i>Cypt</i>-dependent <i>Zfy2/1</i> transcripts and 2 denotes <i>Cypt-</i>dependent <i>Zfy2</i> transcripts). <b>(A)</b> The PCR primers for this assay amplified <i>Zfy2/1</i> and <i>Zfy2</i> transcripts; only XY<i>Sxr</i>^<i>b</i> has both transcripts. It can be seen that the level of <i>Cypt-Zfy2/1</i> transcripts in X^<i>E</i>Y*^X<i>Sxr</i>^<i>b</i> is comparable to the level of <i>Cypt-Zfy2</i> transgene transcripts in X^{<i>E</i>,<i>Z2</i>}Y*^X<i>Sry</i>. <b>(B)</b> For the left panel the PCR primers are specific for <i>Zfy2/1</i>; the second and third genotypes lack the <i>Zfy2/1</i> fusion gene so the low level signal represents ‘background’. For the right panel the PCR primers are specific for <i>Zfy2</i>; in this case it is the fourth genotype that lacks <i>Zfy2</i>. The genotypes lacking the Y long arm (Yq-) have substantially higher transcript levels than the genotypes with a complete Y (Yq+).</p

Mapping of <i>Prssly</i> and <i>Teyorf1</i> to <i>Sxr</i>^<i>a</i> and <i>Sxr</i>^<i>b</i>.

<p><i>Prssly</i> and <i>Teyorf1</i> map to the Yp-derived <i>Sxr</i>^<i>a</i> chromosomal fragment (here attached distal to the PAR of one X of an XX<i>Sxr</i>^<i>a</i> male). As expected from the known breakpoints for the Δ^<i>Sxr-b</i> deletion, <i>Prssly</i> and <i>Teyorf1</i> are also present in <i>Sxr</i>^<i>b</i>, whereas the <i>Tspy</i> pseudogene is absent.</p