Kenneth P. Roberts

Microdeletions in the Y Chromosome of Infertile Men

Background Some infertile men with azoospermia or severe oligospermia have small deletions in regions of the Y chromosome. However, the frequency of such microdeletions among men with infertility in general is unknown. We sought to determine the prevalence of Y-chromosome microdeletions among infertile men and to correlate the clinical presentation of the men with specific deletions. Methods We studied 200 consecutive infertile men. Each man was evaluated comprehensively for known causes of infertility, and Y-chromosome microdeletions were studied with use of the polymerase chain reaction to amplify specific regions of the chromosome. The Y chromosomes of 200 normal men were also analyzed. Results Fourteen infertile men (7 percent) and four normal men (2 percent) had microdeletions of the Y chromosome. Nine of the infertile men had azoospermia or severe oligospermia (sperm concentration, <5 million per milliliter), four had oligospermia (sperm concentration, 5 million to <20 million per milliliter), and one had normospermia (sperm concentration, ≥20 million per milliliter). The size and location of the deletions varied and did not correlate with the severity of spermatogenic failure. The fathers of six infertile men with microdeletions were studied; two had the same deletions as their sons, and four had no deletions. Conclusions A small proportion of men with infertility have Y-chromosome microdeletions, but the size and position of the deletions correlate poorly with the severity of spermatogenic failure, and a deletion does not preclude the presence of viable sperm and possible conception.

Inhibition of Capacitation-Associated Tyrosine Phosphorylation Signaling in Rat Sperm by Epididymal Protein Crisp-1

Abstract Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3−. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3−, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.

Analysis of the Human Sperm Proteome

Abstract: As part of our effort to identify putative protein targets for the development of male contraceptives, we performed an in‐depth proteomic analysis of human sperm by liquid chromatography and tandem mass spectrometry. Motile sperm were collected from a single fertile individual and fractionated into detergent‐soluble and detergent‐insoluble fractions. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis separation of these fractions, followed by manual cutting of the gel, yielded 35 gel sections for each fraction to include proteins across the full range of electrophoretic mobility. Proteomic analysis of these gel sections identified more than 1,760 proteins with high confidence, with 1,350 proteins identified in the soluble fraction, 719 identified in the insoluble fraction, and 309 identified in both fractions. This characterization of the human sperm proteome provides a high‐resolution, physiologically relevant index of the proteins that comprise human sperm.

Cryopreservation of Equine Sperm: Optimal Cooling Rates in the Presence and Absence of Cryoprotective Agents Determined Using Differential Scanning Calorimetry

Abstract Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5°C/min and 20°C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 μm and a radius of 0.66 μm with an osmotically inactive cell volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at ≤0.3°C/min in an Equitainer-I from 37°C to 4°C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined best-fit parameters of water transport (at both 5°C/min and 20°C/min) in Kenney extender (absence of CPAs) are Lpg = 0.02 μm min−1 atm−1 and ELp = 32.7 kcal/mol with a goodness-of-fit parameter R2 = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are Lpg[cpa] = 0.008 μm min−1 atm−1 and ELp[cpa] = 12.1 kcal/mol with R2 = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is ∼29°C/min and is ∼60°C/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the ∼5% of initial osmotically active water volume trapped inside the cells at −30°C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap ∼3.4% and ∼7.1% of the intracellular water when cooled at 20°C/min and 100°C/min, respectively. As an independent test of this prediction, the percentage of viable equine sperm was obtained after freezing at 6 different cooling rates (2°C/min, 20°C/min, 50°C/min, 70°C/min, 130°C/min, and 200°C/min) to −80°C in the CPA medium. Sperm viability was essentially constant between 20°C/min and 130°C/min.

Identification of Rat Cysteine-Rich Secretory Protein 4 (Crisp4) as the Ortholog to Human CRISP1 and Mouse Crisp4

Abstract Cysteine-rich secretory proteins (CRISPs) are present in a diverse population of organisms and are defined by 16 conserved cysteine residues spanning a plant pathogenesis related-1 and a C-terminal cysteine-rich domain. To date, the diversification of mammalian CRISPs is evidenced by the existence of two, three, and four paralogous genes in the rat, human, and mouse, respectively. The current study identifies a third rat Crisp paralog we term Crisp4. The gene for Crisp4 is on rat chromosome 9 within 1 Mb of both the Crisp1 and Crisp2 genes. The full-length transcript for this gene was cloned from rat epididymal RNA and encodes a protein that shares 69% and 91% similarity with human CRISP1 and mouse CRISP4, respectively. Expression of rat Crisp4 is most abundant in the epididymis, with the highest levels of transcription observed in the caput and corpus epididymis. In contrast, rat CRISP4 protein is most abundant in the corpus and cauda regions of the epididymis. Rat CRISP4 protein is also present in caudal sperm extracts, appearing as a detergent-soluble form at the predicted MWR (26 kDa). Our data identify rat Crisp4 as the true ortholog to human CRISP1 and mouse Crisp4, and demonstrate its interaction with spermatozoa in the epididymis.

A Comparative Analysis of Expression and Processing of the Rat Epididymal Fluid and Sperm-Bound Forms of Proteins D and E

Abstract The mammalian epididymis secretes numerous proteins important for sperm maturation. Among these are proteins D and E, which belong to the CRISP family (cysteine-rich secretory proteins) and are the product of the Crisp-1 gene. These proteins have been the focus of a number of studies and have been implicated in sperm/egg fusion. Protein D and protein E have been purified to apparent homogeneity in several laboratories. Polyclonal antibodies raised against each protein typically cross-reacted with both proteins, suggesting that they were immunologically similar, if not identical. Our laboratory has previously reported the generation of a monoclonal antibody (mAb 4E9) that recognizes only protein E. Using mAb 4E9, the localization of protein E was shown to be domain specific on the sperm surface and there is processing of the protein in the fluid, with only the lowest molecular weight form associating with sperm. Subsequent purification and amino acid sequencing of protein D confirmed that proteins D and E are nearly identical and differ only by presence of the 4E9 epitope on protein E. Here we report the generation of antibodies to regions of amino acid sequence identity in proteins D and E. Using these antibodies, we demonstrate that protein D associates with the sperm head and that a portion of this protein may be proteolytically processed. In addition, we demonstrate that the proteolytic processing of protein E occurs in the carboxy terminal region of this protein. The data also suggest that a portion of protein D may also undergo processing, similar to that of protein E. Finally, we use these antibodies to demonstrate that proteins D and E are differentially expressed by the epididymal epithelium. Taken together, these data suggest that proteins D and E may have individual roles in sperm function.

Proteome of Human Calcium Kidney Stones

OBJECTIVES Idiopathic calcium oxalate (CaOx) stones are believed to develop attached to papillary subepithelial deposits called Randalls plaques. Calcium phosphate (CaP) stones, conversely, are thought to arise within the inner medullary collecting ducts, enlarging and damaging surround tubular structures as they expand. If this is true, we theorize that differences will be seen within the organic portion (matrix) of CaOx stones compared with CaP stones using a mass spectroscopy (MS) approach. METHODS From a cohort of 47 powdered stones, 25 calculi (13 CaOx, 12 CaP) were confirmed to contain a dominant mineral content of >80% by powder x-ray diffraction. Matrix proteins were then extracted, purified, and digested. Peptide tandem MS data were acquired, and spectra were searched against a large human protein database to identify protein matches. RESULTS No significant differences were seen between pattern profiles of CaOx and CaP stones. However, variations in protein expression patterns were seen within individual CaOx (monohydrate and dihydrate) and CaP (apatite and brushite) mineral subtypes, suggesting a relationship between crystal-surface binding properties and matrix composition. Both groups contain a large number of inflammatory proteins and a catalog of common proteins is included. CONCLUSIONS Calcium kidney stone matrix contains hundreds of proteins and is predominated by proteins associated with inflammatory response. Many of the same proteins were identified in both CaOx and CaP stones, suggesting inflammation as a unifying origin or a common secondary role in calcium stone pathogenesis.

Second prize: Comprehensive proteomic analysis of human calcium oxalate monohydrate kidney stone matrix

BACKGROUND AND PURPOSE Previous efforts to identify the protein content of stone matrix have been limited by the lack of technology necessary to analyze the highly insoluble protein-crystalline complex. Our study objective is to characterize the matrix of calcium oxalate monohydrate (COM) stones using a comprehensive proteomics approach. MATERIALS AND METHODS Seven pure COM stones were powdered, and proteins were extracted using four different buffer solutions. Detergent cleanup spin columns or concentrators were used to remove detergent and to exchange buffers before trypsin digestion. Tryptic peptides were analyzed with reversed-phase, high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) using a QSTAR Pulsar i quadrapole time of flight mass spectrometer. Tandem mass spectra were searched against National Center for Biotechnology Information human nonredundant database using ProteinPilot 1.0 software (Applied Biosystems, Inc.) for protein hits; peptide MS/MS spectra were manually inspected. RESULTS Of the four buffers, only 2% sodium dodecyl sulfate (SDS) samples had normal HPLC and MS/MS elution patterns. We identified 68 distinct proteins with 95% confidence. More than 50 of the proteins have not been previously identified in stone matrix. Of particular note, a significant number of inflammatory proteins were identified, including immunoglobulins, defensin -3, clusterin, complement C3a, kininogen, and fibrinogen. CONCLUSIONS SDS reducing buffer was efficient at solubilizing proteins from stone matrix for further MS-based proteomic analysis. A variety of cellular, structural, and plasma proteins comprise COM stone matrix. Several of the stone proteins are involved in cell injury pathways, which suggests that inflammation plays a role in human COM stone formation.

Regional Distribution of 5α-Reductase Type 1 and Type 2 mRNA Along the Human Epididymis

OBJECTIVE To determine the regional distribution and relative expression of 5alpha-reductase type 1 and type 2 mRNA within the human testis and regions of the epididymis. DESIGN Prospective observational study. SETTING University academic medical center. PATIENT(S) Two young adult male organ donors. INTERVENTION(S) None MAIN OUTCOME MEASURE(S) The distribution of 5alpha-reductase type 1 and type 2 mRNA in the testis and regions of the epididymis was detected by Northern blot analysis. The relative abundance of each 5alpha-reductase mRNA was evaluated using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in which cyclophilin mRNA, a house-keeping gene product, was coamplified as the reference standard. RESULT(S) Northern blot analysis revealed the 5alpha-reductase type 2 transcript in the midcaput, distal caput, corpus, and proximal cauda of the epididymis, but the transcript was undetectable in the testis, proximal caput, and distal cauda region. No transcript for the type 1 isozyme was detected by Northern blot. The more sensitive RT-PCR showed low levels of type 1 mRNA in the testis and epididymis, with the highest abundance in the proximal caput. Type 2 mRNA of 5alpha-reductase was most abundant in the midcaput, was decreased in the more distal regions, and was more abundant than type 1 mRNA in all epididymal regions except for the proximal caput. CONCLUSION(S) Both 5alpha-reductase type 1 and type 2 mRNAs are present in the human epididymis. The type 2 isozyme mRNA is predominant, being more highly expressed than the low-abundance type 1 mRNA.

Feasibility of Using a Computer Modeling Approach to Study SUI Induced by Landing a Jump

Stress urinary incontinence (SUI) occurs due to anatomic and/or neurologic factors involving connective tissues, muscles and nerves. Although SUI is more common in post-menopausal and multiparous women, studies have also shown a high prevalence of SUI in young, physically fit female athletes. With a goal toward dynamic subject-specific mechanical characterization of the interaction between anatomical structures during physical activities that elicit SUI in females during physical or daily activities, a computer aided design (CAD)-based computer model of the female pelvis has been developed to test the feasibility of the computer modeling approach in understanding the measurable differences between stress-continent and stress-incontinent women. In the present study, a fluid–structure interaction analysis was conducted by using the finite element (FE) analysis technique based on the CAD-based computer model of the female pelvis to investigate the urine leakage in females during jumping. To the best of our knowledge, this is the first application of a fluid–structure interaction FE analysis approach in understanding the mechanisms of SUI in females. Through a series of computer simulations, the effects of varying impact forces determined by jumping height and bladder volume were investigated. The dynamic computer simulation results revealed that jumping heights have a significant influence on the volume of urine leakage caused by the landing impact of jumping. Bladder volume did not have a significant influence on leakage when the jumping heights were smaller than 1 ft, which indicates that normal walking (corresponds to a jumping height smaller than 0.1 ft) is not the primary cause of urine leakage for healthy females. The computer simulation results also showed that the deformation difference between the anterior and posterior portion of the female pelvis causes opening of the urethra and resultant urine leakage. The present study demonstrates the feasibility of using a computer modeling approach to study female SUI during physical and daily activities.