Mechanisms governing protective pregnancy-induced adaptations of the pelvic floor muscles in the rat preclinical model.

BACKGROUND: The intrinsic properties of pelvic soft tissues in women who do and do not sustain birth injuries are likely divergent. However, little is known about this. Rat pelvic floor muscles undergo protective pregnancy-induced structural adaptations-sarcomerogenesis and increase in intramuscular collagen content-that protect against birth injury. OBJECTIVE: We aimed to test the following hypotheses: (1) the increased mechanical load of a gravid uterus drives antepartum adaptations; (2) load-induced changes are sufficient to protect pelvic muscles from birth injury. STUDY DESIGN: The independent effects of load uncoupled from the hormonal milieu of pregnancy were tested in 3- to 4-month-old Sprague-Dawley rats randomly divided into the following 4 groups, with N of 5 to 14 per group: (1) load-/pregnancy hormones- (controls), (2) load+/pregnancy hormones-, (3) reduced load/pregnancy hormones+, and (4) load+/pregnancy hormones+. Mechanical load of a gravid uterus was simulated by weighing uterine horns with beads similar to fetal rat size and weight. A reduced load was achieved by unilateral pregnancy after unilateral uterine horn ligation. To assess the acute and chronic phases required for sarcomerogenesis, the rats were sacrificed at 4 hours or 21 days after bead loading. The coccygeus, iliocaudalis, pubocaudalis, and nonpelvic tibialis anterior musles were harvested for myofiber and sarcomere length measurements. The intramuscular collagen content was assessed using a hydroxyproline assay. An additional 20 load+/pregnancy hormones- rats underwent vaginal distention to determine whether the load-induced changes are sufficient to protect from mechanical muscle injury in response to parturition-associated strains of various magnitude. The data, compared using 2-way repeated measures analysis of variance followed by pairwise comparisons, are presented as mean±standard error of mean. RESULTS: An acute increase in load resulted in significant pelvic floor muscle stretch, accompanied by an acute increase in sarcomere length compared with nonloaded control muscles (coccygeus: 2.69±0.03 vs 2.30±0.06 µm, respectively, P<.001; pubocaudalis: 2.71±0.04 vs 2.25±0.03 µm, respectively, P<.0001; and iliocaudalis: 2.80±0.06 vs 2.35±0.04 µm, respectively, P<.0001). After 21 days of sustained load, the sarcomeres returned to operational length in all pelvic muscles (P>.05). However, the myofibers remained significantly longer in the load+/pregnancy hormones- than the load-/pregnancy hormones- in coccygeus (13.33±0.94 vs 9.97±0.26 mm, respectively, P<.0001) and pubocaudalis (21.20±0.52 vs 19.52±0.34 mm, respectively, P<.04) and not different from load+/pregnancy hormones+ (12.82±0.30 and 22.53±0.32 mm, respectively, P>.1), indicating that sustained load-induced sarcomerogenesis in these muscles. The intramuscular collagen content in the load+/pregnancy hormones- group was significantly greater relative to the controls in coccygeus (6.55±0.85 vs 3.11±0.47 µg/mg, respectively, P<.001) and pubocaudalis (5.93±0.79 vs 3.46±0.52 µg/mg, respectively, P<.05) and not different from load+/pregnancy hormones+ (7.45±0.65 and 6.05±0.62 µg/mg, respectively, P>.5). The iliocaudalis required both mechanical and endocrine cues for sarcomerogenesis. The tibialis anterior was not affected by mechanical or endocrine alterations. Despite an equivalent extent of adaptations, load-induced changes were only partially protective against sarcomere hyperelongation. CONCLUSION: Load induces plasticity of the intrinsic pelvic floor muscle components, which renders protection against mechanical birth injury. The protective effect, which varies between the individual muscles and strain magnitudes, is further augmented by the presence of pregnancy hormones. Maximizing the impact of mechanical load on the pelvic floor muscles during pregnancy, such as with specialized pelvic floor muscle stretching regimens, is a potentially actionable target for augmenting pregnancy-induced adaptations to decrease birth injury in women who may otherwise have incomplete antepartum muscle adaptations.

Recurrent urinary tract infection: Association of clinical profiles with urobiome composition in women.

AIMS: Clinical profiles of women with recurrent urinary tract infection (RUTI) are correlated with their urinary microbes. METHODS: This IRB-approved, cross-sectional study enrolled adult women with RUTI. Urine samples (catheterized and voided) underwent culture by expanded quantitative urine culture (EQUC) and standard urine culture (SUC) methods. A validated symptom questionnaire, relevant clinical variables, and EQUC were used to identify symptom clusters and detect associations with specific urinary microbes. RESULTS: Most (36/43) participants were postmenopausal; the average age was 67 years. 51% reported vaginal estrogen use; 51% reported sexual activity. Although single symptoms were not associated with specific urinary microbes, EQUC results were correlated with five distinct clinical profile clusters: Group A: odor, cloudiness, and current vaginal estrogen use (no culture result association). Group B: frequency, low back pain, incomplete emptying, and vaginal estrogen (significantly increased proportion of Lactobacillus-positive cultures). Group C: pain/burning, odor, cloudiness, and urgency (high proportions of UTI-associated microbe-positive cultures). Group D: frequency, urgency, pain/burning, and current vaginal estrogen use (increased number of no growth cultures). Group E: frequency, urgency, pain/burning, odor, overactive bladder, and sexually active (significantly increased proportion of Klebsiella-positive cultures). CONCLUSIONS: Distinct clinical profiles are associated with specific urinary microbes in women with RUTI. Refined assessments of clinical profiles may provide useful insights that could inform diagnostic and therapeutic considerations.

Age-associated changes in the mechanical properties of human cadaveric pelvic floor muscles.

Proper function of the female pelvic floor requires intact pelvic floor muscles (PFMs). The prevalence of pelvic floor disorders (PFDs) increases substantially with age, in part due to clinically identified deterioration of PFM function with age. However, the etiology of this decline remains largely unknown. We previously demonstrated that PFMs undergo age-related fibrotic changes. This study sought to determine whether aging also impacts PFMs' passive mechanical properties that are largely determined by the intramuscular extracellular matrix. Biopsies from younger (≤52y) and older (>52y) female cadaveric donors were procured from PFMs, specifically coccygeus (C) and two portions of the levator ani - iliococcygeus (IC) and pubovisceralis (PV), and the appendicular muscles - obturator internus (OI) and vastus lateralis (VL). Muscle bundles were subjected to a passive loading protocol, and stress-sarcomere length (Ls) relationships calculated. Muscle stiffness was compared between groups using 2-way ANOVA and Sidak pairwise comparisons, α < 0.05. The mean age was 43.4 ±â€¯11.6y and 74.9 ±â€¯11.9y in younger (N = 5) and older (N = 10) donors, respectively. In all PFMs, the quadratic coefficient of parabolic regression of the stress-Ls curve, a measure of stiffness, was lower in the younger versus older group: C: 33.7 ±â€¯13.9 vs 87.2 ±â€¯10.7, P = 0.02; IC: 38.3 ±â€¯12.7 vs 84.5 ±â€¯13.9, P = 0.04; PV: 24.7 ±â€¯8.8 vs 74.6 ±â€¯9.6, P = 0.04. In contrast, non-PFM stiffness was not affected by aging: OI: 14.5 ±â€¯4.7 vs 32.9 ±â€¯6.2, P = 0.8 and VL: 13.6 ±â€¯5.7 vs 30.1 ±â€¯5.3, P = 0.9. Age-associated increase in PFM stiffness is predicted to negatively impact PFM function by diminishing muscle load-bearing, excursional, contractile, and regenerative capacity, thus predisposing older women to PFDs.

Uncovering changes in proteomic signature of rat pelvic floor muscles in pregnancy.

BACKGROUND: Structural and functional changes of the rat pelvic floor muscles during pregnancy, specifically, sarcomerogenesis, increase in extracellular matrix content, and higher passive tension at larger strains protect the integral muscle components against birth injury. The mechanisms underlying these antepartum alterations are unknown. Quantitative proteomics is an unbiased method of identifying protein expression changes in differentially conditioned samples. Therefore, proteomics analysis provides an opportunity to identify molecular mechanisms underlying antepartum muscle plasticity. OBJECTIVE: To elucidate putative mechanisms accountable for pregnancy-induced adaptations of the pelvic floor muscles, and to identify other novel antepartum alterations of the pelvic floor muscles. MATERIALS AND METHODS: Pelvic floor muscles, comprised of coccygeus, iliocaudalis, and pubocaudalis, and nonpelvic limb muscle, tibialis anterior, were harvested from 3-month-old nonpregnant and late-pregnant Sprague-Dawley rats. After tissue homogenization, trypsin-digested peptides were analyzed by ultra-high-performance liquid chromatography coupled with tandem mass spectroscopy using nano-spray ionization. Peptide identification and label free relative quantification analysis were carried out using Peaks Studio 8.5 software (Bioinformatics Solutions Inc., Waterloo, ON, Canada). Proteomics data were visualized using the Qlucore Omics Explorer (New York, NY). Differentially expressed peptides were identified using the multi-group differential expression function, with q-value cutoff set at <0.05. Proteomic signatures of the pelvic floor muscles were compared to nonpelvic limb muscle and between nonpregnant and pregnant states. RESULTS: Unsupervised clustering of the data showed clear separation between samples from nonpregnant and pregnant animals along principal component 1 and between pelvic and nonpelvic muscles along principal component 2. Four major gene clusters were identified segregating proteomic signatures of muscles examined in nonpregnant vs pregnant states: (1) proteins increased in the pelvic floor muscles only; (2) proteins increased in the pelvic floor muscles and tibialis anterior; (3) proteins decreased in the pelvic floor muscles and tibialis anterior; and (4) proteins decreased in the pelvic floor muscles alone. Cluster 1 included proteins involved in cell cycle progression and differentiation. Cluster 2 contained proteins that participate in mitochondrial metabolism. Cluster 3 included proteins involved in transcription, signal transduction, and phosphorylation. Cluster 4 comprised proteins involved in calcium-mediated regulation of muscle contraction via the troponin tropomyosin complex. CONCLUSION: Pelvic floor muscles gain a distinct proteomic signature in pregnancy, which provides a mechanistic foundation for the antepartum physiological alterations acquired by these muscles. Variability in genes encoding these proteins may alter plasticity of the pelvic floor muscles and therefore the extent of the protective pregnancy-induced adaptations. Furthermore, pelvic floor muscles' proteome is divergent from that of the nonpelvic skeletal muscles.

Exosomes mediate embryo and maternal interactions at implantation and during pregnancy.

Shedding of exosomes and microvesicles is now a well-recognized, important method of cell-cell communication in a number of different cell types. However, their importance in the female reproductive tract and in mediating embryo-maternal interactions during pregnancy has only recently been recognized. Here we review the current literature as to release of extracellular vesicles by uterine cells, the embryo,, and placental trophoblast cells; how release is regulated; and the different types of signaling molecules and genetic information contained within such vesicles. We also discuss the role of these exosomes and microvesicles in regulating critical processes during implantation and pregnancy such as angiogenesis, matrix remodeling, alterations in immune function and pathological effects in gestational diseases. A better understanding of the role of exosomes and microvesicles in reproduction may lead to the development of new therapeutic approaches for treatment of infertility and pregnancy complications.

Characterization of store-operated Ca2+ channels in pancreatic duct epithelia.

Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. Previously we demonstrated that Ca2+ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca2+ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca2+ influx as Ca2+ was released from intracellular stores. Direct measurement of [Ca2+] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La3+ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca2+ such as salt and mucin secretion from the exocrine pancreatic ducts.

Stimulation of GPR30 increases release of EMMPRIN-containing microvesicles in human uterine epithelial cells.

CONTEXT: Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. OBJECTIVE: The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). DESIGN: We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. RESULTS: We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. CONCLUSIONS: These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology.

Allurin, an amphibian sperm chemoattractant having implications for mammalian sperm physiology.

Eggs of many species are surrounded by extracellular coats that emit ligands to which conspecific sperm respond by undergoing chemotaxis and changes in metabolism, motility, and acrosomal status in preparation for fertilization. Here we review methods used to measure sperm chemotaxis and focus on recent studies of allurin, a 21-kDa protein belonging to the Cysteine-RIch Secretory Protein (CRISP) family that has chemoattraction activity for both amphibian and mammalian sperm. Allurin is unique in being the first extensively characterized Crisp protein found in the female reproductive tract and is the product of a newly discovered amphibian gene within a gene cluster that has been largely conserved in mammals. Study of its expression, function, and tertiary structure could lead to new insights in the role of Crisp proteins in sperm physiology.

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family.

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm-egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.

Egg jelly proteins stimulate directed motility in Xenopus laevis sperm.

Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose-dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488-conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0-1 µg/ml range and correlated well with previously published dose-dependent sperm attraction data. Binding was rapid with a half-time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm-orienting behavior.

Two types of assays for detecting frog sperm chemoattraction.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm. Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg. Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 µm diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer. The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Testicular expression of Adora3i2 in Adora3 knockout mice reveals a role of mouse A3Ri2 and human A3Ri3 adenosine receptors in sperm.

Adenosine is a candidate modulator of sperm motility in the female reproductive tract that increases sperm flagellar beat frequency in vitro. Past work suggested that this acceleration may involve equilibrative (ENT) and concentrative (CNT) nucleoside transporters. Here we show that Slc29a1 (ENT-1) is the predominant nucleoside transporter expressed in the mouse testis. Unexpectedly, the beat of Slc29a1-null sperm still accelerates in response to 2-chloro-2'-deoxyadenosine (Cl-dAdo). Moreover, in wild-type sperm neither blockade of CNTs by removal of external Na(+), nor inhibition of ENTs with nitrobenzylthioionosine, prevents acceleration of the sperm beat by Cl-dAdo. In contrast, pertussis toxin produces strong blockade, indicating involvement of a Gα(i/o)-coupled adenosine receptor. Although agonists selective for adenosine receptors A1R, A2aR, and A2bR are ineffective, A3R-selective agonists Cl-IB-MECA and IB-MECA do accelerate the beat. Consistent with this pharmacological profile, the predominant Adora transcripts in the testis are products of the nested Adora3i1 and Adora3i2 genes. Surprisingly, Cl-IB-MECA and Cl-dAdo still accelerate the beat of Adora3i1-null sperm indicating that the remaining Adora3i2 transcript produces an A3R that functions in sperm. When cloned Adora3i2 is heterologously expressed in tsA-201 cells, Cl-dAdo decreases forskolin-evoked accumulation of cAMP, indicating that Adora3i2 specifies a functional A3Ri2 adenosine receptor that couples through Gα(i). Database mining reveals that mouse Adora3i2 is expressed primarily in testis, almost exclusively in spermatids. Expression of the orthologous ADORA3i3 transcript also is most prominent in human testis; presumably producing an A3Ri3 receptor that is functional in sperm and that may be a target for development of male-directed contraceptives.

A semi-automated analysis method of small sensory nerve fibers in human skin-biopsies.

Computerized detection method (CDM) software programs have been extensively developed in the field of astronomy to process and analyze images from nearby bright stars to tiny galaxies at the edge of the Universe. These object-recognition algorithms have potentially broader applications, including the detection and quantification of cutaneous small sensory nerve fibers (SSNFs) found in the dermal and epidermal layers, and in the intervening basement membrane of a skin punch biopsy. Here, we report the use of astronomical software adapted as a semi-automated method to perform density measurements of SSNFs in skin-biopsies imaged by Laser Scanning Confocal Microscopy (LSCM). In the first half of the paper, we present a detailed description of how the CDM is applied to analyze the images of skin punch biopsies. We compare the CDM results to the visual classification results in the second half of the paper. Abbreviations used in the paper, description of each astronomical tools, and their basic settings and how-tos are described in the appendices. Comparison between the normalized CDM and the visual classification results on identical images demonstrates that the two density measurements are comparable. The CDM therefore can be used - at a relatively low cost - as a quick (a few hours for entire processing of a single biopsy with 8-10 scans) and reliable (high-repeatability with minimum user-dependence) method to determine the densities of SSNFs.

Pharmacological targeting of native CatSper channels reveals a required role in maintenance of sperm hyperactivation.

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca(2+) entry evoked by alkaline depolarization. In the absence of external Ca(2+), Na(+) carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na(+)](i)-reporting probe SBFI in populations of intact sperm. Removal of external Ca(2+) increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na(+)](i) is greater for sperm alkalinized with NH(4)Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na(+)](i) rise is slowed by candidate CatSper blocker HC-056456 (IC(50) approximately 3 microM). HC-056456 similarly slows the rise of [Ca(2+)](i) that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO(3) (-) but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca(2+) entry through the CatSper channel is terminated by removal of external Ca(2+). Thus, open CatSper channels and entry of external Ca(2+) through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.

Crisp proteins and sperm chemotaxis: discovery in amphibians and explorations in mammals.

Crisp proteins appear to play multiple roles in the life history of sperm. One of these roles is to act as a sperm chemoattractant. Allurin, a 21 kDa Crisp protein rapidly released from the egg jelly of at least two frogs, X. laevis and X. tropicalis, elicits directed motility in both homospecific and heterospecific sperm. In X. tropicalis, allurin is coded for by the newly documented Crisp A gene. Recently, the observation that allurin can also elicit chemotaxis in mouse sperm raises the question of whether allurin-like proteins might act as sperm chemoattractants in mammals. Although an allurin gene has yet to be documented in mammals, Crisp proteins truncated post-translationally appear to exist in both the male and female reproductive tract of mammals.

Xenopus tropicalis allurin: expression, purification, and characterization of a sperm chemoattractant that exhibits cross-species activity.

Previously we reported the identification of the first vertebrate sperm chemoattractant, allurin, in the frog Xenopus laevis (Xl) and demonstrated that it was a member of the CRISP family of proteins. Here we report identification, purification, and characterization of Xenopus tropicalis (Xt) allurin, a homologous protein in X. tropicalis. "Egg water" as well as purified allurin from both species exhibit efficient cross-species sperm chemoattractant activity. Western blots show that Xt egg water contains a single anti-allurin cross-reactive protein whose molecular weight (20,497 Da by MALDI MS) agrees well with the molecular weight of the hypothetical gene product for a newly recognized "Crisp A" gene in the X. tropicalis genome. A recombinant form of the protein, expressed in 3T3 cells, exhibits chemoattraction for both Xt and Xl sperm and cross reacts with anti-allurin antibodies. Examination of Crisp protein expression in the Xt oviduct using RT-PCR showed that of five documented Xt Crisp genes (Crisps 2, 3, LD1, LD2 and A) only Crisp A was expressed. In contrast, Crisp 2, Crisp 3, Crisp LD1, and Crisp LD2, but not Crisp A, were all found to be expressed in the Xt testes while subsets of Crisp proteins where expressed in the Xt ovary. These data suggest that Crisp proteins in amphibians may play multiple roles in sperm production, maturation and guidance just as they are thought to in mammals indicating that Crisp protein involvement in reproduction may not be limited to mammals.