Significant expression levels of transgenic PPP1CC2 in testis and sperm are required to overcome the male infertility phenotype of Ppp1cc null mice.

PPP1CC2, one of four isoforms of the ser/thr protein phosphatase PP1, is a mammalian-specific splice variant of the Ppp1cc 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 Ppp1cc product, is expressed in many tissues including testis, the only phenotype resulting from deletion of Ppp1cc gene is male infertility. To determine which of the products of Ppp1cc is essential for male fertility, we created two PPP1CC2 transgenes, eTg-G2 and pTg-G2, where Ppp1cc2 expression was driven by the putative endogenous promoter of Ppp1cc or by the testis specific human Pgk2 promoter, respectively. Our results demonstrate that the 2.6-kb genomic region directly upstream of the Ppp1cc structural gene can drive expression of Ppp1cc2, 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 Ppp1cc 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 Ppp1cc +/- male. We conclude that the endogenous Ppp1cc 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.

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit.

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.

Inhibition of Ser/Thr phosphatases induces capacitation-associated signaling in the presence of Src kinase inhibitors.

Signaling events leading to mammalian sperm capacitation rely on activation/deactivation of proteins by phosphorylation. This cascade includes soluble adenylyl cyclase, an atypical bicarbonate-stimulated adenylyl cyclase, and is mediated by protein kinase A and the subsequent stimulation of protein tyrosine phosphorylation. Recently, it has been proposed that the capacitation-associated increase in tyrosine phosphorylation is governed by Src tyrosine kinase activity. This conclusion was based mostly on the observation that Src is present in sperm and that the Src kinase family inhibitor SU6656 blocked the capacitation-associated increase in tyrosine phosphorylation. Results in the present manuscript confirmed these observations and provided evidence that these inhibitors were also able to inhibit protein kinase A phosphorylation, sperm motility, and in vitro fertilization. However, the block of capacitation-associated parameters was overcome when sperm were incubated in the presence of Ser/Thr phosphatase inhibitors such as okadaic acid and calyculin-A at concentrations reported to affect only PP2A. Altogether, these data indicate that Src is not directly involved in the observed increase in tyrosine phosphorylation. More importantly, this work presents strong evidence that capacitation is regulated by two parallel pathways. One of them requiring activation of protein kinase A and the second one involving inactivation of Ser/Thr phosphatases.

Expression of transgenic PPP1CC2 in the testis of Ppp1cc-null mice rescues spermatid viability and spermiation but does not restore normal sperm tail ultrastructure, sperm motility, or fertility.

Two isoforms of phosphoprotein phosphatase 1, PPP1CC1 and PPP1CC2, are translated from alternatively spliced transcripts of a single gene, Ppp1cc, and differ only at their extreme C-termini. While PPP1CC1 expression is almost ubiquitous, PPP1CC2 is largely restricted to testicular germ cells and mature spermatozoa. Targeted deletion of Ppp1cc leads to sterility of -/- males due to a combination of gross structural defects in developing spermatids resulting in apoptosis and faulty spermiation. Because PPP1CC2 is the only PP1 isoform that demonstrates high-level expression in wild-type meiotic and postmeiotic male germ cells, we have tested whether its loss in Ppp1cc-/- males is largely responsible for manifestation of this phenotype by expressing PPP1CC2 transgenically in the testis of Ppp1cc-/- mice (rescue mice). Herein, we demonstrate that PPP1CC2 expression in the Ppp1cc-/- testis is antiapoptotic, thus reestablishing spermatid development and spermiation. However, because aberrant flagellar morphogenesis is incompletely ameliorated, rescue males remain infertile. Because these results suggest that expression of PPP1CC2 in developing germ cells is essential but insufficient for normal spermatogenesis to occur, appropriate spatial and temporal expression of both PPP1CC isoforms in the testis during spermatogenesis appears to be necessary to produce structurally normal fertility-competent spermatozoa.

PP1gamma2 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, PP1gamma1 and PP1gamma2, 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 PP1gamma2, 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, PP1gamma2 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 PP1gamma-null testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1gamma-null testis expressing transgenic PP1gamma2. PPP1R11 also appears to be ubiquitinated in PP1gamma-null testis. The levels of PP1gamma2 and PPP1R11 were increased in phenotypically normal PP1alpha-null testis. However, in PP1alpha-null spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1gamma2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis.

Analysis of Ppp1cc-null mice suggests a role for PP1gamma2 in sperm morphogenesis.

Serine/threonine protein phosphatase 1 (PP1) consists of four ubiquitously expressed major isoforms, two of which, PP1gamma1 and PP1gamma2, are derived by alternative splicing of a single gene, Ppp1cc. PP1gamma2 is the most abundant isoform in the testis, and is a key regulator of sperm motility. Targeted disruption of the Ppp1cc gene causes male infertility in mice due to impaired spermiogenesis. This study was undertaken to determine the expression patterns of specific PP1 isoforms in testes of wild-type mice and to establish how the defects produced in Ppp1cc-null developing sperm are related to the loss of PP1gamma isoform expression. We observed that PP1gamma2 was prominently expressed in the cytoplasm of secondary spermatocytes and round spermatids as well as in elongating spermatids and testicular and epididymal spermatozoa, whereas its expression was weak or absent in spermatogonia, pachytene spermatocytes, and interstitial cells. In contrast, a high level of PP1gamma1 expression was observed in interstitial cells, whereas much weaker expression was observed in all stages of spermatogenesis. Another PP1 isoform, PP1alpha, was predominant in spermatogonia, pachytene spermatocytes, and interstitial cells. Examining the temporal expression of PP1 enzymes in testes revealed a striking postnatal increase in PP1gamma2 levels compared with other isoforms. Testicular sperm tails from Ppp1cc-null mice showed malformed mitochondrial sheaths and extra outer dense fibers in both the middle and principal pieces. These data suggest that in addition to its previously documented role in motility, PP1gamma2 is involved in sperm tail morphogenesis.

The mouse T complex gene Tsga2, encoding polypeptides located in the sperm tail and anterior acrosome, maps to a locus associated with sperm motility and sperm-egg interaction abnormalities.

Previous studies of sperm from mice heterozygous for a t haplotype (t) and heterospecific combinations of the t complex identified two tightly linked genetic factors responsible for t/t male sterility related to expression of the flagellar waveform aberration, curlicue. Dnahc8, an axonemal dynein heavy chain gene, is a strong candidate for the proximal factor, Ccua, but the identity of the distal factor, Ccub, is unknown. In the present study, we employ motility assays of sperm from males heterozygous for t and novel heterospecific combinations of the t complex to demonstrate that Ccub is a composite of at least two synergic elements, Ccub1, positioned within a genomic interval spanning approximately 0.6 Mb immediately distal to Dnahc8, and Ccub2, situated in a region approximately 4-7 Mb distal to Ccub1. We also show that Tsga2, a testis-restricted gene, fulfills many of the prerequisites required to make it a strong candidate for Ccub1. These include: 1) its location within the aforementioned genomic interval; 2) a highly reduced level of testis expression by its heterospecific allele relative to the level of expression of its t allele; 3) determination that TSGA2(t) carries numerous nonsynonymous mutations in residues otherwise highly conserved in all known orthologous proteins; 4) the detection of major TSGA2 polypeptides in sperm protein extracts; and 5) the apparent distribution of these polypeptides in major sperm tail structures. Surprisingly, these TSGA2 isoforms appear to localize in the vicinity of the anterior acrosome, as well, suggesting that Tsga2 may also play a role in sperm-egg interaction. Finally, our results indicate that a TSGA2 polypeptide with apparent similarities to the smaller of the two sperm isoforms is expressed by epididymal cells.

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

Heterozygosity 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 Ca(2+)-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 gamma-type dynein heavy chain (gammaDHC) 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 DNAHC8(t) 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 DNAHC8(t) 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 DNAHC8(t) dysfunction.

The T complex distorter 2 candidate gene, Dnahc8, encodes at least two testis-specific axonemal dynein heavy chains that differ extensively at their amino and carboxyl termini.

Homozygosity for the t haplotype allele of the testis-specifically expressed axonemal dynein heavy chain (axDHC) gene, Dnahc8, has been linked to male sterility resulting from aberrant sperm motility. However, the near absence of Dnahc8 expression has been associated with male sterility resulting from an early breakdown in sperm flagellar development. Although axDHCs are integral participants in flagellar motility, a role in flagellar morphogenesis has never been attributed to a member of this highly conserved gene family. To gain a better understanding of this presumed novel role for Dnahc8, we have studied the organization and expression of full-length Dnahc8(+) and Dnahc8(t) transcripts. Our results demonstrate the existence of at least two alternatively spliced, testis-specific Dnahc8 mRNAs transcribed from both the + and t alleles. A highly expressed isoform encodes a protein with significant homology nearly throughout to the gamma heavy chain of the Chlamydomonas axonemal outer arm dynein, while a more poorly expressed isoform codes for a protein whose sequence diverges significantly from that of other axDHCs at both its N and C termini. While in situ hybridization studies demonstrate that both mRNA species accumulate exclusively in mid to late spermatocytes, each isoform shows spatial independence. Additional experiments demonstrate the existence of a testis-expressed mRNA with no significant open reading frame, a portion of which is antisense to the 5'-untranslated region of the highly divergent Dnahc8 isoform. The cumulative data imply that Dnahc8 may have acquired functional plasticity in the testis through the tightly controlled expression of both typical and unusual isoforms.