Stephen H. Pilder

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.

A candidate gene family for the mouse t complex responder (Tcr) locus responsible for haploid effects on sperm function

The mouse t complex responder (Tcr) locus plays a central haploid-specific role in the transmission ratio distortion phenotype expressed during germ cell differentiation in t-carrying males. The accumulated data map Tcr to a region of less than 500 kb. Over 400 kb of this region has been cloned and consists entirely of sequences associated with a clustered family of large cross-hybridizing elements of 30 kb to 70 kb in size. We have characterized a gene family within this region that is expressed uniquely in male germ cells with a complex pattern of RNA processing. Antibodies produced against a product of the putative open reading frame recognize a testes-specific polypeptide. Genetic data support the hypothesis that this polypeptide(s) functions to effect the Tcr phenotype.

Analysis of Ppp1cc-Null Mice Suggests a Role for PP1gamma2 in Sperm Morphogenesis

Abstract 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.

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.

A mouse chromosome 17 gene encodes a testes-specific transcript with unusual properties.

We have characterized a novel mouse gene (D17Si11) on chromosome 17 that expresses a major transcript observed uniquely in the testes. TheD17Si11 locus has been mapped to the central region of chromosome 17 betweenH-2 andC3. Sequence analysis demonstrates several unusual features of this locus and its transcript: first is the presence of complementary sets of alternating purine and pyrmidine residues within the 3′ region of the transcript that could form double-stranded, hairpin-like secondary structures with properties similar to that of Z-DNA; second is the existence of a hypothetical, long open reading frame in the nucleotide strand that is complementary to the testes transcripts. This complementary strand open reading frame is three times the size of the longest potential open reading frame present in the transcript itself. Although a function forD17Si11 has yet to be determined, the gene is relatively non-polymorphic in mice and appears conserved in mammals.

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.

Identification and linkage mapping of Hst7, a new M. spretus/M. m. domesticus Chromosome 17 hybrid sterility locus

Species: Mouse Locus name: Hybrid sterility 7 Locus symbol: Hst7 Map position: Because M. spretus Chromosome (Chr) 17 contains an inversion polymorphism (In2) of 4 cM (3.7-7.7 cM from the centromere; see Fig. 1) relative to M. m. domesticus wild-type (+) Chr 17 [1], the approximate genetic size of the interval to which Hst7 s maps on M. spretus Chr 17 has been extrapolated from the addition of two genetic intervals on the M. m. dornesticus map: D17Tul-dp(17)1p (2.50-3.53 cM = 1.03 cM) + T-inv(17)2d (4.007.62 cM = 3.62 cM) = 4.65 cM. The exact position of the Hst7 t allele on the t haplotype map may be different within the same size interval, since a t haplotype carries an additional inversion polymorphism (Inl) [2] relative to both the M. m. domesticus + and the M. spretus Chr 17 homologs (Fig. 1). Thus, the Hst7 + allele must map to either of the sub-intervals used to extrapolate the size of the interval on M. spretus Chr 17 (Fig. 1). Method of mapping: Linkage analysis of the Hst7 phenotype was carried out with C57BL/6 mice each congenic for one of two recombinant M. spretus-M, m. domesticus Chr 17 homologs (Fig. 1). Males (n = 30) carrying the first of the two recombinant chromosomes (referred to as SD 1) and any homolog were sterile, while males (n = 25) carrying the second recombinant chromosome (referred to as SD 2) and any homolog were fertile. SD 2 is a derivative of SD 1 and differs from SD 1 at its proximal end (Fig. 1). Database deposit information: Accession no. is MGD-CREX688. Molecular reagents used f o r mapping: MIT primer pairs for D17Mit156, D17Mit164, D17Mit l71 [3], pr imer pairs for D17TulCA, and D17Pri3 [4]; and clones used for RFLP analysis: Tu48 [5], T u l l 9 [6], Sod2 [7], Tctexl [8], Hba4 [9], and Nkx2-5 [10]. Method of allele detection: Alleles were detected either by Southern blot-RFLP analysis of TaqI-digested DNAs at Tctexl (3.00 cM), D17Leh48 (3.50 cM), D17Lehll91 and II (3.57 and 3.80 cM, respectively), Sod2 (7.60 cM), and Hba-ps4 (11.78 cM); and BglIIdigested DNAs at Nkx2-5 (13.00 cM); by amplification under standard PCR conditions and evaluation of size polymorphisms on 2.5% agarose gels at D17Tul (2.50 cM), D17Mit164 (4.10 cM), D17Pri3 (4.20 cM), D17Mitl71 (5.00 cM), and D17Mit156 (7.10 cM); or by phenotypic analysis of tail length at T (4.00 cM) [12]. Discussion: Four of five hybrid sterility loci previously defined in the mouse (the assignment of a sixth, Hst2, to Chr 9 has been retracted) [13] have been mapped to the t haplotype region (also called the t complex; Fig. 1), an expansive portion of proximal Chr 17, famous for the expression of a male-specific meiotic drive phenotype and its attendant male sterility phenotype [14,15]. Hstl is a M. m. musculus/M, m. domesticus hybrid sterility locus that maps to In3 of the t complex (Fig. 1; [13]); Hst4, 5, and 6 are all M. spretus/M, m. domesticus hybrid sterility loci found tightly linked in In4 (Fig. 1) [11,16]. Hst7 is the fifth hybrid sterility locus found to map to the t haplotype region. M. m. domesticus males carrying Hst7 s and any allele are sterile. Furthermore, the portion of M. spretus chromatin present in recombinant Chr 17 SD 1, subsequently replaced by a recombination event with both + and t chromatin in recombinant Chr 17 SD 2 (Fig. 1), defines the region to which Hst7 maps. Interestingly, the Hst7 s dominant sterility allele is not part of

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

2 (the t haplotype male sterility factor in In4) [15], but instead maps proximal to the t complex responder locus (Fig. 1) in either In2, Inl , between these two inversions, or just proximal to Inl. These results are surprising, since earlier mapping studies had suggested: (A) males carrying M. spretus Chr 17 and its M. m. domesticus + homolog in an essentially M. m. domesticus

Sperm from mice carrying two t haplotypes do not possess a tyrosine phosphorylated form of hexokinase

Abstract 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.

Localization of the Mas proto-oncogene to a densely marked region of mouse chromosome 17 associated with genomic imprinting

Mouse sperm contain a tyrosine phosphorylated form of hexokinase type 1 (HK1; Kalab et al., 1994: J Biol Chem 269:3810–3817) that has properties consistent with an integral plasma membrane protein. Furthermore, this tyrosine phosphorylated form of HK1 has an extracellular domain and HK1 is localized to both the head and flagellum of nonpermeabilized cells (Visconti et al., 1995c). We have characterized HK1 in mature sperm from sterile tw32/tw5 mice (mutant sperm) that have defects in motility and sperm‐egg interaction (Johnson et al., 1995: Dev Biol 168:138–149). Immunoprecipitation of mouse sperm extracts with an antiserum made against purified rat brain HK1 demonstrates the presence of HK1 in mutant sperm. Various biochemical and immunofluorescence assays indicate that at least a portion of the HK1 present in these cells is an integral membrane protein with an extracellular domain located on the sperm head and flagellum. However, immunoblot analysis with anti‐phoshotyrosine antibodies demonstrates that HK1 in mutant sperm is not tyrosine phosphorylated. Northern blot and RT‐PCR analysis does not indicate any obvious abnormalities in the transcription of somatic or germ cell‐specific HK1 isoforms in mutant testes, and RFLP analysis of recombinant mice indicates that no genes specifying HK1 isoforms are located on chromosome 17. We have mapped the locus responsible for the lack of tyrosine phosphorylation of HK1 mutant sperm to the most proximal (to the centromere) of the four inversions within the t haplotype. A male sterility factor is located in this same inversion (Lyon, 1986: Cell 44:357–363). Since the mutant sperm are unable to complete fertilization, there could be a relationship between sterility and the lack of tyrosine phosphorylation of HK1 in these mutant sperm.