The mouse t complex distorter/sterility candidate, Dnahc8, expresses a γ-type axonemal dynein heavy chain isoform confined to the principal piece of the sperm tail

AbstractHeterozygosity for a t haplotype (t) in male mice results in distorted transmission (TRD) of the t-bearing chromosome 17 homolog to their offspring. However, homozygosity for t causes male sterility, thus limiting the spread of t through the population at large. The Ca2+-dependent sperm tail curvature phenotypes, “fishhook”, where abnormally high levels of sperm exhibit sharp bends in the midpiece, and “curlicue”, where motile sperm exhibit a chronic negative curving of the entire tail, have been tightly linked to t-associated male TRD and sterility traits, respectively. Genetic studies have indicated that homozygosity for the t allele of Dnahc8, an axonemal γ-type dynein heavy chain (γDHC) gene, is partially responsible for expression of “curlicue”; however, its involvement in “fishhook”/TRD, if any, is unknown. Here we report that the major isoform of DNAHC8 is copiously expressed, carries an extended N-terminus and full-length C-terminus, and is stable and equally abundant in both testis and sperm from +/+ and t/t animals. By in silico analysis we also demonstrate that at least three of the seventeen DNAHC8t mutations at highly conserved positions in wild-type DHCs may be capable of substantially altering normal DNAHC8 function. Interestingly, DNAHC8 is confined to the principal piece of the sperm tail. The combined results of this study suggest possible mechanisms of DNAHC8t dysfunction and involvement in “curlicue”, and support the hypothesis that “curlicue” is a multigenic phenomenon. They also demonstrate that the accelerated “fishhook” phenotype of sperm from +/t males is not directly linked to DNAHC8t dysfunction

PP1γ2 and PPP1R11 Are Parts of a Multimeric Complex in Developing Testicular Germ Cells in which their Steady State Levels Are Reciprocally Related

Mice lacking the protein phosphatase 1 gamma isoforms, PP1γ1 and PP1γ2, are male-sterile due to defective germ cell morphogenesis and apoptosis. However, this deficiency causes no obvious abnormality in other tissues. A biochemical approach was employed to learn how expression versus deficiency of PP1γ2, the predominant PP1 isoform in male germ cells, affects spermatogenesis. Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry. We report for the first time that in wild-type testis, PP1γ2 forms an inactive complex with actin, protein phosphatase 1 regulatory subunit 7 (PPP1R7), and protein phosphatase 1 regulatory subunit 11 (PPP1R11), the latter, a potent PP1 inhibitor. Interestingly, PPP1R11 protein, but not its mRNA level, falls significantly in PP1γ-null testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1γ-null testis expressing transgenic PP1γ2. PPP1R11 also appears to be ubiquitinated in PP1γ-null testis. The levels of PP1γ2 and PPP1R11 were increased in phenotypically normal PP1α-null testis. However, in PP1α-null spleen, where PP1γ2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1γ2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis

Significant Expression Levels of Transgenic PPP1CC2 in Testis and Sperm Are Required to Overcome the Male Infertility Phenotype of <em>Ppp1cc</em> Null Mice

<div><p>PPP1CC2, one of four isoforms of the ser/thr protein phosphatase PP1, is a mammalian-specific splice variant of the <em>Ppp1cc</em> gene, and the only isoform whose expression is confined almost completely to spermatogenic cells. Additionally, PPP1CC2 is the sole isoform found in mammalian spermatozoa. Although PPP1CC1, the other <em>Ppp1cc</em> product, is expressed in many tissues including testis, the only phenotype resulting from deletion of <em>Ppp1cc</em> gene is male infertility. To determine which of the products of <em>Ppp1cc</em> is essential for male fertility, we created two PPP1CC2 transgenes, eTg-G2 and pTg-G2, where <em>Ppp1cc2</em> expression was driven by the putative endogenous promoter of <em>Ppp1cc</em> or by the testis specific human <em>Pgk2</em> promoter, respectively. Our results demonstrate that the 2.6-kb genomic region directly upstream of the <em>Ppp1cc</em> structural gene can drive expression of <em>Ppp1cc2,</em> and recapitulate the wild-type tissue specificity of PPP1CC2 in transgenic mice. More importantly, we show that expression of PPP1CC2 alone, via either promoter, is able not only to restore normal spermatogenesis, but the fertility of <em>Ppp1cc</em> null mice as well, provided that transgenic PPP1CC2 expression in testis reaches at least a lower threshold level equivalent to approximately 50% of its expression by a <em>Ppp1cc +/−</em> male. We conclude that the endogenous <em>Ppp1cc</em> promoter normally functions in the testis to maintain a sufficient level of PPP1CC2 expression for normal spermatogenesis to occur, and that production of spermatozoa capable of fertilization in vivo can take place in the complete absence of PPP1CC1 expression.</p> </div

Comparison of PPP1CC2 levels, testis weight, sperm number and morphology between transgenic rescue lines and control animals.

<p>The Kruskal-Wallis non-parametric one-way ANOVA by rank was performed and results were analyzed <i>post-hoc</i> by Dunn’s procedure for performing two-tailed multiple pair wise comparisons. Differences were considered significant if <i>p</i><0.05 at a confidence interval of 95%.</p><p><b>a, b, c…</b> denote significantly different groups.</p><p><b>SEM</b> denotes Standard Error of the Mean.</p><p><b>n</b> denotes number of samples/group.</p

Comparison of steady state levels of transgenically expressed PPP1CC2 in testis and spermatozoa from all transgenic rescue lines.

<p>(<b>A</b>) Western blot showing the levels of PPP1CC2 expressed in the testis of rescue lines compared with littermate controls. In the left panel protein estimated testis extracts from rescue animals were serially diluted to 20 µg, 10 µg, 5.0 µg and 2.5 µg and 20 µg, 10 µg, 7.5 µg, 5.0 µg and 2.5 µg from positive controls. (<b>*</b>) denotes the lane in which the band corresponding to rescue mouse PPP1CC2 expression is comparable in intensity to a band in a control mouse lane. In the right panels, (*) lanes are shown adjacent to each other for visual comparison. β-Actin was used as loading control. Each blot was repeated with preparations from different animals of the same line to confirm the original data. (<b>B</b>) Western blot of whole sperm protein extract demonstrate PPP1CC2 levels in spermatozoa. Protein extract from 2×10⁶ sperm was loaded in each lane for each rescue line and its littermate control. Upper panel represents PPP1CC2 levels in endogenous promoter driven rescue lines (eTg) while the lower panel represents PPP1CC2 levels in the <i>hPgk2</i> promoter driven rescue lines (pTg). Note that the PPP1CC2 levels in spermatozoa roughly conform to their levels in the testis.</p

Fertility Data.

<p>The Kruskal-Wallis non-parametric one-way ANOVA by rank was employed and results were analyzed <i>post-hoc</i> by Dunn’s procedure for performing two-tailed multiple pairwise comparisons. Differences were considered significant if <i>p</i><0.05.</p><p><b>a, b…</b> denote significantly different groups.</p><p><b>SEM</b> denotes Standard Error of the Mean.</p

Histology of testis section from rescue animals compared to that of control heterozygous mice.

<p>Paraffin embedded adult testis sections from transgenic lines eTg-F1 (<b>a, b</b>), eTg-F10 (<b>c, d</b>), pTg-M26 (<b>e, f</b>) and positive controls (<b>g, h</b>) were stained with haematoxylin for comparison of gross tissue architecture. The stained sections were viewed under 10× and 20× magnification using an Olympus 1×70 microscope. Note that both the size and overall architecture of seminiferous tubules of rescue mouse testes were restored in comparison to <i>Ppp1cc −/−</i> testis, and were comparable to those of positive control mice. Also notice that the seminiferous tubular lumens are replete with mature testicular spermatozoa, clearly seen in all rescue lines (white arrow) and in positive controls (black arrow). These observations are representative of multiple sections prepared from testes of several animals of each rescue and positive control line.</p