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

Design of transgenic rescue constructs.

<p>(<b>A</b>) Transgene mini cassette construct where transgene expression was driven by the putative endogenous promoter fragment of <i>Ppp1cc</i> gene. A genomic fragment of ∼2.6 kb immediately upstream of the transcription start site was employed as the endogenous promoter to drive the expression of full length mouse <i>Ppp1cc2</i> cDNA (with intact 5′UTR and 3′UTR). (<b>B</b>) Testis specific human <i>Pgk2</i> promoter driven transgene cassette. In this mini gene the mouse <i>Ppp1cc2</i> coding sequence used lacked the 5′ and 3′UTRs. In both constructs the SV40 PolyA signal sequence was added 3′ of the cDNA construct.</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

Morphological abnormalities of spermatozoa as seen in rescue lines expressing low levels of PPP1CC2.

<p>(<b>a, b, c, d</b>) Mature epididymal spermatozoa with aberrant bending between mid-piece and principle piece. The angular variation of the bend ranges from 45° (<b>a</b>) to 90° (<b>c, d</b>) to 180° (<b>b</b>). (<b>e–i</b>) A series of frequently observed morphological defect involving jack-knifing of the head between the capitulum and the proximal mid-piece. (<b>j–m</b>) Other less common deformities, including shortened mitochondrial sheaths (<b>j, m</b>) and malformed heads (<b>k, l, m</b>). All micrographs were photographed under DIC optics with an Olympus 70 light microscope. Abnormal regions of the spermatozoa are denoted by white arrows and its corresponding normal regions are denoted by black arrows. These observations are representative of multiple samples from cauda epididymal preparations of different rescue animals.</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