Friederike L. Jayes

Phosphoproteome Analysis of Capacitated Human Sperm EVIDENCE OF TYROSINE PHOSPHORYLATION OF A KINASE-ANCHORING PROTEIN 3 AND VALOSIN-CONTAINING PROTEIN/p97 DURING CAPACITATION

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.

Opposing LSD1 complexes function in developmental gene activation and repression programmes

Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1—a component of the CoREST-CtBP co-repressor complex—is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.

Differential extraction and enrichment of human sperm surface proteins in a proteome: Identification of immunocontraceptive candidates

The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two‐dimensional (2‐D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim‐up sperm were either solubilized directly or solubilized after surface labeling with sulfo‐succinimidyl‐6‐(biotinamido)hexanoate (sulfo‐NHS‐LC‐biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA‐630; (ii) 1% Triton X (TX)‐100; (iii) 1.7% TX‐114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high‐resolution 2‐D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA‐1), the acrosome (SP‐10), and the cytoskeleton (α‐tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20‐s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX‐100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX‐100, TX‐114, and 1 M NaCl. Extraction with TX‐114 followed by phase‐partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

Biological and biochemical consequences of global deletion of exon 3 from the ERα gene

To address issues resulting from α estrogen receptor-knockout (αERKO) residual N-terminal truncated estrogen receptor α, and to allow tissue-selective deletion of ERα, we generated loxP-flanked exon 3 mice. Initial characterization of global sox2 cre-derived exon 3-deleted Ex3αERKO mice indicated no ERα protein in uterine tissue and recapitulation of previously described female phenotypes, confirming successful ablation of ERα. Body weights of Ex3αERKO female mice were 1.4-fold higher than wild-tupe (WT) females and comparable to WT males. Microarray indicated the Ex3αERKO uterus is free of residual estrogen responses. RT-PCR showed Nr4a1 is increased 41-fold by estrogen in WT and 7.4-fold in αERKO, and not increased in Ex3αERKO. Nr4a1, Cdkn1a, and c-fos transcripts were evaluated in WT and Ex3αERKO mice following estrogen, IGF1, or EGF injections. All 3 were increased by all treatments in WT. None were increased by estrogen in Ex3αERKO. Nr4a1 increased 24.5- and 14.7-fold, Cdkn1a increased 14.2- and 12.3-fold, and c-fos increased 20.9-fold and 16.2-fold after IGF1 and EGF treatments, respectively, in the Ex3αERKO mice, confirming that growth factor regulation is independent of ERα. Our Ex3α ERα model will be useful in studies of complete or selective ablation of ERα in target tissues.

SLLP1, A Unique, Intra-acrosomal, Non-bacteriolytic, c Lysozyme-Like Protein of Human Spermatozoa

Abstract We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by β-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P ≤ 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, ∼1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.

Transdermal iontophoretic delivery of luteinizing hormone releasing hormone (LHRH) : effect of repeated administration

The transdermal iontophoretic delivery of the reproductive peptide hormone, luteinizing hormone releasing hormone (LHRH) is investigated in the isolated perfused porcine skin flap model (IPPSF). LHRH is delivered twice in a single flap experiment in efforts to identify factors inherent to iontophoretic delivery that might effect the drug flux of a subsequent iontophoretic episode. Initial iontophoretic delivery of LHRH is quite reproducible; however, subsequent iontophoretic episodes result in widely divergent fluxes thought to be caused by iontophoretic influences on the skin. Iontophoretic application of a drug on a previous active site, enhances the flux during the second application. A mass balance study is performed to explain these findings. By iontophoretically delivering I125 labelled LHRH in the isolated perfused porcine skin flap model, the entire iontophoretic dose is identified and quantified. A drug depot is identified in the skin underlying the electrode which is approximately two times as large as the entire mass of drug delivered systemically.

Outer Dense Fiber Proteins Are Dominant Postobstruction Autoantigens in Adult Lewis Rats

Abstract Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.

Insufficient Luteinizing Hormone-Induced Intracellular Signaling Disrupts Ovulation in Preovulatory Follicles Lacking Estrogen Receptor-β

Gonadotropin-stimulated estrogen receptor-beta (ERbeta)-null preovulatory follicles exhibit submaximal estradiol production, insufficient acquisition of LH receptor, and attenuated expression of essential ovulatory genes. These observations lead to low ovulatory rates compared with wild-type (WT) follicles. We hypothesize that insufficient LH receptor results in reduced cAMP production after an ovulatory stimulus. Individual preantral follicles were cultured with FSH for 4 d and then induced to ovulate with a single dose of human chorionic gonadotropin (hCG). cAMP levels 1 h after hCG were 50% lower in ERbeta-null than WT follicles. To determine whether the lack of LH receptor, and resulting lack of cAMP, could be bypassed by direct activation of adenylyl cyclase, WT and ERbeta-null follicles were induced to ovulate with forskolin. Ten micromolar forskolin doubled the ovulatory rate of ERbeta-null follicles compared with treatment with hCG ( approximately 50 vs. 25%, respectively). In WT follicles, 10 microm forskolin reduced the ovulation rate compared with hCG (14 vs. 83%, respectively), indicating that high doses of forskolin inhibited WT ovulation. A 10 microm concentration of forskolin induced cAMP levels in ERbeta-null follicles that were comparable to levels produced in WT follicles after hCG and either partially or completely rescued the attenuated expression of LH-responsive genes. These data indicate that direct activation of adenylyl cyclase, resulting in increased production of cAMP, partially rescues the ovulatory response of ERbeta-null follicles, suggesting that insufficient LH receptor and low cAMP levels contribute to their poor ovulatory rates. We also determined that ERbeta-null ovaries exhibit an alteration in the activation of ERK1/2. Our evaluation of the ERbeta-null ovarian phenotype indicates that ERbeta plays a role in facilitating folliculogenesis. We show that expression of ERbeta in preovulatory follicles is required for adequate cAMP production and propose that an optimal level of cAMP is required for hCG-stimulated ovulation.

The extracellular matrix contributes to mechanotransduction in uterine fibroids.

The role of the extracellular matrix (ECM) and mechanotransduction as an important signaling factor in the human uterus is just beginning to be appreciated. The ECM is not only the substance that surrounds cells, but ECM stiffness will either compress cells or stretch them resulting in signals converted into chemical changes within the cell, depending on the amount of collagen, cross-linking, and hydration, as well as other ECM components. In this review we present evidence that the stiffness of fibroid tissue has a direct effect on the growth of the tumor through the induction of fibrosis. Fibrosis has two characteristics: (1) resistance to apoptosis leading to the persistence of cells and (2) secretion of collagen and other components of the ECM such a proteoglycans by those cells leading to abundant disposition of highly cross-linked, disoriented, and often widely dispersed collagen fibrils. Fibrosis affects cell growth by mechanotransduction, the dynamic signaling system whereby mechanical forces initiate chemical signaling in cells. Data indicate that the structurally disordered and abnormally formed ECM of uterine fibroids contributes to fibroid formation and growth. An appreciation of the critical role of ECM stiffness to fibroid growth may lead to new strategies for treatment of this common disease.

Injectable Clostridium Histolyticum Collagenase as a Potential Treatment for Uterine Fibroids

Purified Clostridium histolyticum collagenase (CHC), an Food and Drug Administration-approved drug that does not affect nerves or blood vessels, was assessed as a potential treatment for fibroids in this proof-of-principle study. Fibroids (1-4 cm, capsules intact) and myometrial specimens from 5 patients were injected posthysterectomy with CHC or vehicle containing methylene blue and incubated for 24 hours. Percentage of collagen-stained area was estimated using Masson-Trichrome-stained slides. Collagen fibers were observed with picrosirius staining. Tissue stiffness was objectively measured by rheometry (complex shear modulus [Pa]). Injected materials spread within and beyond fibroids as visualized by methylene blue. Of the 8 treated fibroids, 7 were softened and some contained liquefied centers. Relative percentage of collagen-stained area (mean ± standard deviation) in treated fibroids (38 ± 12%; n = 7) was less than that in control fibroids (66 ± 17%; n = 5). Treated myometrium (40 ± 30% collagen; n = 3) was similar to control myometrium (53 ± 8%; n = 2). Picrosirius staining demonstrated loss of collagen fibers in treated fibroids. Treated fibroids were less stiff (3630 ± 2410 Pa; n = 4) than controls (5930 ± 830 Pa; n = 4). Treated and control myometrium had similar stiffness (2149 ± 927 Pa; n = 3 and 3314 ± 494 Pa; n = 2, respectively) and were never liquefied. In conclusion, injections of CHC into encapsulated fibroids are feasible and effective. Heterogeneity of collagen types and quantities within individual fibroids may contribute to varied responses and need additional investigation. Further study of collateral effects on myometrium is indicated. Injected CHC has potential for treatment of fibroids.