ZAG alleviates HFD-induced insulin resistance accompanied with decreased lipid depot in skeletal muscle in mice.

Over the past two decades, intramuscular lipids have been viewed as a cause of insulin resistance due to their ability to suppress insulin-stimulated glucose uptake in skeletal muscle. Zinc-α2-glycoprotein (ZAG) is an adipokine involved in lipolysis of white adipose tissue (WAT). To investigate the action of ZAG on insulin resistance induced by a high-fat diet (HFD), which affects the intramuscular fat, mice were divided into three groups, normal diet, HFD, and ZAG treatment under HFD (HFZ). The results showed that the insulin sensitivity of ZAG-treated mice was significantly improved. The body weight, WAT weight, and intramuscular fat were significantly decreased in the HFZ group compared with the HFD group. The lipolytic enzymes, including phosphorylation of hormone-sensitive lipase and adipose triglyceride lipase, were significantly upregulated in the skeletal muscle of mice that received the ZAG treatment compared with the HFD group. Insulin signaling proteins, such as phosphorylation of insulin receptor substrate 1 and cell membrane glucose transporter type 4, were also significantly increased in the skeletal muscle of the ZAG-treated group. Furthermore, a metabolic rate study showed that ZAG overexpression increases the respiratory exchange ratio and heat production. In vitro, ZAG treatment promotes glucose uptake and decreases intracellular lipids in C2C12 myotubes. Taken together, these data showed that overexpression of ZAG alleviates HFD-induced insulin resistance in mice, along with decreasing the lipid content of skeletal muscle.

Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates.

It has been proposed that seminal plasma proteins (SPP) support survival of ram spermatozoa, exerting a dual effect, both capacitating and decapacitating. In this study, changes in motility patterns of ram spermatozoa capacitated in the presence of epidermal growth factor (EGF) were evaluated. Clustering procedures were used to determine the presence of sperm subpopulations with specific motion characteristics. Four sperm subpopulations (SP) were defined after the application of a principal component analysis procedure. Progressive spermatozoa with high straightness (STR) were found in SP1, reflected in the high linearity (LIN) and STR values and low amplitude of lateral head movement (ALH; rapid, non-hyperactivated spermatozoa). SP2 spermatozoa seemed to be starting to acquire hyperactivated motility, while the SP3 group consisted of rapid, hyperactivated spermatozoa. SP4 showed less-vigorous spermatozoa, with non-linear motility. The addition of SPP before in vitro capacitation with EGF induced a decrease in SP1 and an increase in SP3. However, a reduction in the chlortetracycline-capacitated sperm rate and protein tyrosine phosphorylation was found, which corroborates with the hypothesis that the SPP protective effect on spermatozoa is related to their decapacitating role. These findings allow us to deduce that ram spermatozoa are able to undergo capacitation with no hyperactivation and that SPP are able to induce hyperactivation in spermatozoa but maintain them in a decapacitated state.

Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice.

Mammalian zygotic development is initiated by sperm-mediated intracellular calcium oscillations, followed by activation of metaphase II-arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte-activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm-induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain-binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte-derived WWI domain protein substrate of PAWP for successful fertilization. Sperm-delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in nonmammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.-

Role of ß-adrenergic receptors in the anti-obesity and anti-diabetic effects of zinc-α2-glycoprotien (ZAG).

OBJECTIVES: The goal of the current study is to determine whether the ß-adrenoreceptor (ß-AR) plays a role in the anti-obesity and anti-diabetic effects of zinc-α2-glycoprotein (ZAG). MATERIAL AND METHODS: This has been investigated in CHO-K1 cells transfected with the human ß1-, ß2-, ß3-AR and in ob/ob mice. Cyclic AMP assays were carried out along with binding studies. Ob/ob mice were treated with ZAG and glucose transportation and insulin were examined in the presence or absence of propranolol. RESULTS: ZAG bound to the ß3-AR with higher affinity (Kd 46±1nM) than the ß2-AR (Kd 71±3nM) while there was no binding to the ß1-AR, and this correlated with the increases in cyclic AMP in CHO-K1 cells transfected with the various ß-AR and treated with ZAG. Treatment of ob/ob mice with ZAG increased protein expression of ß3-AR in gastrocnemius muscle, and in white and brown adipose tissues, but had no effect on expression of ß1- and ß2-AR. A reduction of body weight was seen and urinary glucose excretion, increase in body temperature, reduction in maximal plasma glucose and insulin levels in the oral glucose tolerance test, and stimulation of glucose transport into skeletal muscle and adipose tissue, were completely attenuated by the non-specific ß-AR antagonist propranolol. CONCLUSION: The results suggest that the effects of ZAG on body weight and insulin sensitivity in ob/ob mice are manifested through a ß-3AR, or possibly a ß2-AR.

New insights into the mechanisms of ram sperm protection by seminal plasma proteins.

To provide new insights into the mechanisms through which seminal plasma proteins (SPP) are able to protect spermatozoa, we tested the hypothesis that apoptosis can contribute to the negative effect of refrigeration on ram spermatozoa, and that SPP prevent this damage. Having proved the presence of key constituents of apoptosis-related pathways in ram sperm protein extracts, we carried out a comparative analysis of the effects of the addition of SPP before refrigeration (15 °C, 30 min) and induced-apoptosis with betulinic acid or fibroblast-associated receptor ligand, assessing sperm quality parameters and apoptotic markers. The protective effect of SPP on plasma membrane integrity and potential, motility and mitochondrial inner membrane potential, and surface (cardiolipin content) was evidenced in refrigerated and induced-apoptosis samples. The addition of SPP resulted in lower values of phosphatidylserine externalization, DNA damage, and caspase activity. Therefore, apoptosis in fresh or refrigerated ram spermatozoa can occur due to activation of both the extrinsic and the intrinsic mediated pathway, and SPP might interfere with both pathways. The addition of SPP also resulted in higher proportions of viable, noncapacitated sperm and fertilizing ability (ZBA rate). This report demonstrates that SPP support survival of ram spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, and proposes the possibility that SPP might interfere with the extrinsic and the intrinsic apoptotic pathways. This opens new, interesting perspectives for the study of cellular regulatory mechanisms in spermatozoa that could be crucial for the improvement of ram semen preservation protocols.

Studies on the anti-obesity activity of zinc-α2-glycoprotein in the rat.

OBJECTIVE: To investigate the anti-obesity effect of the adipokine zinc-α(2)-glycoprotein (ZAG) in rats and the mechanism of this effect. SUBJECTS: Mature male Wistar rats (540 ± 83 g) were administered human recombinant ZAG (50 µg per 100 g body weight given intravenously daily) for 10 days, while control animals received an equal volume of phosphate-buffered saline (PBS). RESULTS: Animals treated with ZAG showed a progressive decrease in body weight, without a decrease in food and water intake, but with a 0.4 °C rise in body temperature. Body composition analysis showed loss of adipose tissue, but an increase in lean body mass. The loss of fat was due to an increase in lipolysis as shown by a 50% elevation of plasma glycerol, accompanied by increased utilization of non-esterified fatty acids, as evidenced by the 55% decrease in plasma levels. Plasma levels of glucose and triglycerides were also reduced by 36-37% and there was increased expression of the glucose transporter 4 in both skeletal muscle and adipose tissue. Expression of the lipolytic enzymes adipose triglyceride lipase and hormone-sensitive lipase in the white adipose tissue (WAT) were increased twofold after ZAG administration. There was almost a twofold increased expression of uncoupling proteins 1 and 3 in brown adipose tissue and WAT, which would contribute to increased substrate utilization. Administration of ZAG increased ZAG expression twofold in the gastrocnemius muscle, BAT and WAT, which was probably necessary for its biological effect. CONCLUSION: These results show that ZAG produces increased lipid mobilization and utilization in the rat.

Studies on the antiobesity effect of zinc-α2-glycoprotein in the ob/ob mouse.

OBJECTIVE: To investigate the mechanism of the lipid depletion by zinc-α(2)-glycoprotein (ZAG). DESIGN: Studies were conducted in the ob/ob mouse, or on isolated adipocytes from these animals or their lean counterparts. RESULTS: Treatment of these animals for 15 days with ZAG (100 µg, intravenously, daily) resulted in a reduction of body weight of 6.55 g compared with phosphate-buffered saline-treated controls, without a change in food or water intake, but with a 0.4 °C rise in rectal temperature. ZAG-treated mice had a 30% reduction in carcass fat mass and a twofold increase in weight of brown adipose tissue. Epididymal adipocytes from ZAG-treated mice showed an increased expression of ZAG and hormone-sensitive lipase (HSL), and this was maintained for a further 3 days in the absence of ZAG. There was an increased lipolytic response to isoproterenol, which was retained for 3 days in vitro in the absence of ZAG. Expression of HSL was also increased in subcutaneous and visceral adipose tissue, as was also adipose triglyceride lipase (ATGL). There was a rapid loss of labelled lipid from epididymal adipose tissue of ZAG-treated mice, but not from the other depots, reflecting the difference in sensitivity to lipolytic stimuli. The increased expression of HSL and ATGL may involve the extracellular signal-regulated kinase (ERK) pathway, as the active (phospho) form was upregulated in all adipose depots after ZAG administration, whereas in vitro studies showed induction of HSL and ATGL by ZAG to be attenuated by PD98059, an inhibitor of the ERK pathway. CONCLUSION: These results suggest that ZAG not only induces direct lipolysis, but also sensitizes adipose tissue to other lipolytic stimuli.

Cetrorelix suppresses the preovulatory LH surge and ovulation induced by ovulation-inducing factor (OIF) present in llama seminal plasma.

BACKGROUND: The purpose of the study was to determine if the effect of llama OIF on LH secretion is mediated by stimulation of the hypothalamus or pituitary gland. METHODS: Using a 2-by-2 factorial design to examine the effects of OIF vs GnRH with or without a GnRH antagonist, llamas with a growing ovarian follicle greater than or equal to 8 mm were assigned randomly to four groups (n = 7 per group) and a) pre-treated with 1.5 mg of GnRH antagonist (cetrorelix acetate) followed by 1 mg of purified llama OIF, b) pre-treated with 1.5 mg of cetrorelix followed by 50 micrograms of GnRH, c) pre-treated with a placebo (2 ml of saline) followed by 1 mg of purified llama OIF or d) pre-treated with a placebo (2 ml of saline) followed by 50 micrograms of GnRH. Pre-treatment with cetrorelix or saline was given as a single slow intravenous dose 2 hours before intramuscular administration of either GnRH or OIF. Blood samples for LH measurement were taken every 15 minutes from 1.5 hours before to 8 hours after treatment. The ovaries were examined by ultrasonography to detect ovulation and CL formation. Blood samples for progesterone measurement were taken every-other-day from Day 0 (day of treatment) to Day 16. RESULTS: Ovulation rate was not different (P = 0.89) between placebo+GnRH (86%) and placebo+OIF groups (100%); however, no ovulations were detected in llamas pre-treated with cetrorelix. Plasma LH concentrations surged (P < 0.01) after treatment in both placebo+OIF and placebo+GnRH groups, but not in the cetrorelix groups. Maximum plasma LH concentrations and CL diameter profiles did not differ between the placebo-treated groups, but plasma progesterone concentrations were higher (P < 0.05), on days 6, 8 and 12 after treatment, in the OIF- vs GnRH-treated group. CONCLUSION: Cetrorelix (GnRH antagonist) inhibited the preovulatory LH surge induced by OIF in llamas suggesting that LH secretion is modulated by a direct or indirect effect of OIF on GnRH neurons in the hypothalamus.

Ovulation-inducing factor: a protein component of llama seminal plasma.

BACKGROUND: Previously, we documented the presence of ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas. The purpose of the study was to define the biochemical characteristics of the molecule(s) in seminal plasma responsible for inducing ovulation. METHODS: In Experiment 1, llama seminal plasma was centrifuged using filtration devices with nominal molecular mass cut-offs of 30, 10 and 5 kDa. Female llamas (n = 9 per group) were treated i.m. with whole seminal plasma (positive control), phosphate-buffered saline (negative control), or the fraction of seminal plasma equal or higher than 30 kDa, 10 to 30 kDa, 5 to 10 kDa, or < 5 kDa. In Experiment 2, female llamas (n = 7 per group) were given an i.m. dose of seminal plasma treated previously by: 1) enzymatic digestion with proteinase-K, 2) incubation with charcoal-dextran, 3) heating to 65 degrees C, or 4) untreated (control). In Experiment 3, female llamas (n = 10 per group) were given an i.m. dose of pronase-treated or non-treated (control) seminal plasma. In all experiments, llamas were examined by transrectal ultrasonography to detect ovulation and CL formation. Ovulation rate was compared among groups by Fisher's exact test and follicle and CL diameters were compared among groups by analyses of variance or student's t-tests. RESULTS: In Experiment 1, all llamas in the equal or higher than 30 kDa and positive control groups ovulated (9/9 in each), but none ovulated in the other groups (P < 0.001). In Experiment 2, ovulations were detected in all llamas in each treatment group; i.e., respective treatments of seminal plasma failed to inactivate the ovulation-inducing factor. In Experiment 3, ovulations were detected in 0/10 llamas given pronase-treated seminal plasma and in 9/10 controls (P < 0.01). CONCLUSIONS: We conclude that ovulation-inducing factor (OIF) in llama seminal plasma is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K, and has a molecular mass of approximately equal or higher than 30 kDa.

Flow-sorted ram spermatozoa are highly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase.

To determine whether flow sorting increased the susceptibility of spermatozoa to reactive oxygen species (ROS), ram semen was either diluted with Tris medium (100 x 10(6) spermatozoa mL(-1); D) or highly diluted (10(6) spermatozoa mL(-1)) before being centrifuged (DC) at 750g for 7.5 min at 21 degrees C or flow-sorted (S) before cryopreservation. Thawed spermatozoa were resuspended in graded concentrations of hydrogen peroxide to induce oxidative stress. In Experiment 1, following exposure to 30 or 45 muM hydrogen peroxide (H(2)O(2)), the total motility (%) of DC (41.0 +/- 7.3 or 25.7 +/- 6.7, respectively) and S spermatozoa (33.8 +/- 6.3 or 20.1 +/- 6.3, respectively) was lower (P < 0.001) than that of D spermatozoa (58.7 +/- 5.6 or 44.5 +/- 6.7, respectively). In Experiment 2, supplementation of samples containing H(2)O(2) with catalase (150 IU mL(-1)) or seminal plasma proteins (4 mg protein per 10(8) spermatozoa) negated oxidative stress, resulting in comparable values to samples receiving no H(2)O(2)in terms of the proportion of spermatozoa with stable plasmalemma (as determined using merocyanine-540 and Yo-Pro-1) in the D and S groups, the proportion of viable, acrosome-intact spermatozoa (as determined by fluorescein isothiocyanate and propidium iodide staining) in the D group and the motility of control (undiluted) and S spermatozoa. Neither H(2)O(2) nor sperm type (i.e. D, DC or S) had any effect on intracellular concentrations of ROS. These results show that flow sorting increases the susceptibility of spermatozoa to ROS, but the inclusion of anti-oxidants or seminal plasma as part of the sorting protocol improves resistance to oxidative stress.

Seminal plasma proteins inhibit in vitro- and cooling-induced capacitation in boar spermatozoa.

Dilute boar seminal plasma (SP) has been shown to inhibit in vitro capacitation and cooling-induced capacitation-like changes in boar spermatozoa, as assessed by the ability of the spermatozoa to undergo an ionophore-induced acrosome reaction. We hypothesised that the protein component of SP is responsible for this effect. To test this hypothesis, varying concentrations of total SP protein or SP proteins fractionated by heparin binding were assayed for their ability to inhibit in vitro capacitation, as well as cooling- and cryopreservation-induced capacitation-like changes. In vitro capacitation and cooling-induced capacitation-like changes were prevented by 10% whole SP, as well as by total proteins extracted from SP at concentrations greater than 500 microg mL(-1). No amount of SP protein was able to prevent cryopreservation-induced capacitation-like changes. Total SP proteins were fractionated based on their heparin-binding properties and the heparin-binding fraction was shown to possess capacitation inhibitory activity at concentrations as low as 250 microg mL(-1). The proteins in the heparin-binding fraction were subjected to mass spectrometry and identified. The predominant proteins were three members of the spermadhesin families, namely AQN-3, AQN-1 and AWN, and SP protein pB1. We conclude that one or more of these heparin-binding SP proteins is able to inhibit in vitro capacitation and cooling-induced capacitation-like changes, but not cryopreservation-induced capacitation-like changes, in boar spermatozoa.

Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze-thaw damage.

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n=four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37 degrees C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze-thawing.

[Recombinant human testis sperm binding protein increases sperm motility parameters].

OBJECTIVE: To investigate the effects of recombinant human testis sperm binding protein (TSBP) on human sperm motility parameters in vitro. METHODS: Sperm specimens obtained from 22 healthy fertile men were prepared by the Percoll gradient-centrifugation technique. The sperm suspension was incubated with recombinant His6-TSBP at the concentration of 0.01 mg/ml or 0.1 mg/ml at 37 degrees C for 1 or 3 hours in vitro. The combination of the recombinant protein and sperm membrane was determined by Western blot, and the sperm motility parameters were analyzed by computer-aided sperm analysis (CASA). The same procedure was performed for 12 asthenospermia patients. RESULTS: In the 22 healthy volunteers, the percentage of forward motile sperm was increased after incubated with 0.1 mg/ml recombinant protein for 1 h (P < 0.05), both forward motile sperm percentage and motility were increased after incubated with recombinant protein at the same concentration for 3 h (P < 0.05), but no effect was observed after incubation with 0.01 mg/ml recombinant protein. In the 12 asthenospermia patients, the forward motile sperm percentage was increased after incubated with 0.1 mg/ml recombinant protein for 3 h (P < 0.05), but no statistically significant difference was observed in sperm motility. CONCLUSION: Recombinant His6-TSBP at the concentration of 0.1 mg/ml can increase sperm motility in healthy fertile men and the forward motile sperm percentage in both healthy fertile men and asthenospermia patients in vitro.

Recombinant peptide reverses cryo-capacitation in ram sperm and improves in vitro fertilization.

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 µM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ±â€¯2.95; 40.13 ±â€¯11.82 for 0.3 µM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.

Sperm binding glycoprotein (SBG) produces calcium and bicarbonate dependent alteration of acrosome morphology and protein tyrosine phosphorylation on boar sperm.

The oviduct is a dynamic organ which modulates gamete physiology. Two subpopulations of sperm have been described in the oviduct of sows, a majority with normal appearance in the deep furrows and a minority, centrally located, and showing damaged membranes. Sperm-oviduct interaction provides the formation of a sperm storage and allows the selection of sperm with certain qualities. Pig (Sus scrofa) oviductal sperm binding glycoprotein (SBG) binds to sperm and exposes Gal beta1-3GalNAc. This disaccharide may be recognized by boar spermadhesin AQN1, which seems to be involved in sperm interaction with the oviduct. SBG is present at the apical surface of the epithelial cells that surround the lumen of the oviduct rather than at the bottom of the crypts. These characteristics imply it could be involved in sperm interaction with this organ. In this study, we evaluate the effect of SBG over boar sperm. We show that the presence of SBG produces alterations of the acrosome morphology of sperm only when they are incubated in capacitating conditions. SBG binds to the periacrosomal region of sperm undergoing capacitation. Its presence induces an increase on the tyrosine-phosphorylation of a polypeptide of apparent molecular mass 97 kDa, as occurs with a 95 kDa protein in other mammalian sperm upon acrosomic reaction. Altogether, these results suggest that SBG might be involved in sperm selection by alteration of the acrosome of sperm that have already begun the capacitation process when they arrive to the oviduct.

An ectopic expression screen reveals the protective and toxic effects of Drosophila seminal fluid proteins.

In Drosophila melanogaster, seminal fluid regulates the reproductive and immune responses of mated females. Some seminal fluid proteins may provide protective functions to mated females, such as antimicrobial activity and/or stimulation of antimicrobial gene expression levels, while others appear to have negative effects, contributing to a "cost of mating." To identify seminal proteins that could participate in these phenomena, we used a systemic ectopic expression screen to test the effects on unmated females of proteins normally produced by the male accessory gland (Acps). Of the 21 ectopically expressed Acps that we tested for ability to assist in the clearance of a bacterial infection with Serratia marcescens, 3 Acps significantly reduced the bacterial counts of infected females, suggesting a protective role. Of the 23 Acps that we tested for toxicity, 3 were toxic, including one that has been implicated in the cost of mating in another study. We also tested ectopic expression females for other Acp-induced effects, but found no additional Acps that affected egg laying or receptivity upon ectopic expression.

Cloning and identification of a novel sperm binding protein, HEL-75, with antibacterial activity and expressed in the human epididymis.

BACKGROUND: The HEL-75 protein is a beta-defensin that was identified by analyzing a human epididymis cDNA library. Studying its function may not only elucidate the mechanisms of host defense, but may also provide new alternatives for novel therapeutic drugs for reproductive tract infections. METHODS: The HEL-75 gene was amplified by PCR, and its structure and function were predicted and analyzed with bioinformatics tools. Polyclonal serum was raised against recombinant HEL (rHEL)-75 protein. The gene expression pattern was analyzed with RT-PCR and immunofluorescent staining. Finally, the antimicrobial activity and function during fertilization of HEL-75 were analyzed using a colony-forming unit assay and IVF, respectively. RESULTS: The human HEL-75 gene is located on chromosome 20p13 and encodes a 95 amino acid protein with a predicted N-terminal signal peptide of 22 amino acids. The protein has six conserved cysteine residues, characteristic of members of the beta-defensin superfamily, as well as several potential post-translational modification sites. At the transcriptional level, HEL-75 was expressed in the epididymis and lung, but only in the epididymis at the translational level. Immunofluorescent staining showed that HEL-75 protein bound spermatozoa in the epididymis. RHEL-75 protein could kill Escherichia coli in vitro in a dose- and time-dependent fashion. However, no effect was observed on sperm motility nor fertilization when spermatozoa were blocked with anti-rHEL-75 polyclonal serum. CONCLUSION: HEL-75 is a new beta-defensin expressed in the epididymis and on sperm; it may play an important role in host defense.

Postnatal expression of zinc-alpha2-glycoprotein in rat white and brown adipose tissue.

Zinc-alpha(2)-glycoprotein (ZAG), a lipid-mobilizing factor, is a novel adipokine which may act locally to influence adipose tissue metabolism. This study examined the ontogeny of ZAG expression in adipose tissue during postnatal development. White (subcutaneous, gonadal, and perirenal) and brown (interscapular) fat was collected from rats aged 1-32 days. ZAG mRNA was detected from day 1 in subcutaneous fat, which appears the key site of synthesis postnatally. ZAG was detected in perirenal (day 5) and in gonadal (day 7) fat when the tissue was sufficient for analysis. ZAG mRNA and protein levels fell significantly at weaning (day 21) and thereafter. ZAG was also detected in brown fat at day 1 but it fell significantly afterwards. The downregulated ZAG expression in white fat depots at weaning, when fat mass is expanded substantially, suggests that ZAG might be involved in the postnatal development of adipose tissue mass.

Synthetic seminal plasma peptide inhibits testosterone production in frog testis in vitro.

The role of synthetic seminal plasma peptide, designed using biochemical and mass spectroscopy analyses of native peptides extracted from seminal plasma, was studied in amphibian (Rana esculenta) testicular steroidogenesis. Production of testosterone and prostaglandin F(2alpha) was determined by incubating frog testes with synthetic peptide in vitro. Analysis of the data showed a dose-dependent inhibition of testosterone production (43% at 10(-5) M concentration) without prostaglandin F(2alpha) synthesis being affected. Determination of the peptide activity during the annual R. esculenta reproductive cycle showed inhibition of testosterone production in post-reproductive and recovery periods, suggesting a possible involvement of peptide in gonad steroidogenesis.

Effect of egg yolk and seminal plasma heparin binding protein interaction on the freezability of buffalo cauda epididymal spermatozoa.

Egg yolk is routinely used in most of the extenders for cryopreservation of semen, but mechanisms of protection of spermatozoa by egg yolk are not very clear. Investigations with buffalo cauda epididymal sperm have shown that seminal plasma heparin binding proteins have detrimental effects during semen cryopreservation. The present study was conducted to investigate the effect of egg yolk on the detrimental effects of heparin binding proteins during cryopreservation of buffalo cauda epididymal spermatozoa. The results indicated that egg yolk was able to reduce the heparin binding proteins mediated cryoinjury in spermatozoa. One of the mechanisms of protection of spermatozoa from cryoinjury by egg yolk may be due to the inhibition of deleterious actions of heparin binding proteins on the spermatozoa.