Seasonal differences in ram seminal plasma revealed by partition in an aqueous two-phase system

Seminal plasma plays an important role in maturation of spermatozoa through hormonal, enzymatic and surface-modifying events. We have previously shown that adsorption of seminal plasma proteins (SPPs) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we investigate the hypothesis that seasonal differences in seminal plasma could affect its ability to recover membrane integrity of cold-shocked sperm. The effect of seminal plasma proteins, obtained in breeding (bsSPPs) and non-breeding (nbsSPPs) season, on cold-shocked ram spermatozoa previously freed from seminal plasma, was analysed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system as well as membrane integrity determination by fluorescence markers. Cold-shock treatment greatly lowered cell viability in both breeding and non-breeding season spermatozoa. The cold-shocked sperm viability obtained was approximately 20%. The loss of heterogeneity and the decrease in viability revealed by CCCD analysis was reversed by the addition of increasing amounts of bsSPP, which induced restoration of the surface characteristics of viable-like spermatozoa, as well as an increase in the number of recovered viable sperm. However, this restoring effect was much lower when nbsSPPs were added, even in a sixfold higher concentration than used with bsSPPs. Incubation of cold-shocked cells with both kinds of proteins performed in both seasonal periods, showed that the recovering effect was related to the season when the plasma sample was obtained rather than to the semen season. The addition of bsSPPs to cold-shocked sperm accounted for a nearly 50% reversion for both studied breeding seasons. However, the reversion percentages obtained with nbsSPPs were significantly lower (P<0.05) than those found with bsSPPs in both studied seasonal periods. This different reversion capacity of bsSPPs and nbsSPPs was related to a different protein composition, as revealed by comparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The bands of 20, 21, 24, 36 and 67 kDa of the bsSP sample profile decreased in winter–spring SP, and were even less intensely stained in summer SP. Densitometric analysis of the stained gel patterns allows automatic comparison among the separated bands, and revealed an important decrease in the content of several bands. The 21.5 kDa band showed the highest decrease, lowering to 14% in June–August plasma with respect to the value obtained in September–December plasma.     

The repeatability and effect of season on seminal characteristics and computer-aided sperm analysis in the stallion

The within-stallion repeatability and effect of season on sperm movement characteristics, determined by computer-aided sperm analysis (CASA), were compared with those of other seminal characteristics. The computer-aided determinations of sperm movement were more repeatable than the seminal characteristics of gel-free volume and sperm cell concentration based on coefficients of variation obtained from the analysis of multiple ejaculates from the same stallions. A significant (P<0.05) seasonal effect on the computer-aided movement characteristic of mean sperm linearity was observed, with a reduction in sperm linearity in the winter months. The percentage of motile and progressively motile cells and mean sperm velocity determined by CASA also tended to be lower in the winter months. These changes in sperm movement characteristics paralleled changes in other seminal characteristics. The use of CASA may have value in the potential fertility evaluation of a stallion in that it can provide relatively precise quantification of sperm movement characteristics from the evaluation of only a few ejaculates. Some CASA derived sperm movement characteristics may be lower during the physiological nonbreeding season than during the breeding season.     

The quantification of lipid and protein oxidation in stallion spermatozoa and seminal plasma: seasonal distinctions and correlations with DNA strand breaks, classical seminal parameters and stallion fertility

The goal of this work was to correlate oxidative stress caused by reactive oxygen species (ROS) and DNA damage with classic semen parameters in spermatozoa and seminal plasma of fertile and subfertile stallions. Oxidation was measured in both lipids and proteins, using the thiobarbituric acid reactive species (TBARS) assay and the DNPH carbonyl groups assay, respectively. Sperm DNA damage was monitored using the TUNEL assay. These parameters were monitored in samples obtained during the breeding and the non-breeding seasons. In general, fertile stallions showed better classical semen parameters, and those parameters improved from the non-breeding to the breeding season, although an increase in sperm production was accompanied by a decrease in the semen quality from subfertile stallions in the breeding season. In terms of oxidation levels we found that there were clear differences whether lipids or proteins were considered. In the breeding season there seemed to be a tendency towards normalizing lipid oxidation in spermatozoa and seminal plasma, and protein oxidation in the seminal plasma, of both fertile and subfertile animals. Thus, differences monitored in the non-breeding season were no longer visible. Interestingly, a higher level of protein oxidation was found in the sperm of fertile animals in the breeding season. Considering that there were positive correlations between sperm protein oxidation and sperm motility and vitality, these results suggests that the oxidation of semen proteins may be important for sperm function. On the other hand, lipid oxidation in the seminal plasma seemed to be a general indicator for sperm damage. In the non-breeding season positive correlations between lipid and protein oxidation levels in both sperm and seminal plasma and several defects in sperm function were found, but only for subfertile animals, thus suggesting that lipid and protein oxidation may aid in the identification of subfertile stallions during the non-breeding season. Levels of ROS production never seemed to result in compromised sperm DNA integrity, indicating that measurements were within physiological levels and/or that there is an efficient antioxidant activity in stallion sperm cells.     

Seminal plasma proteins prolong the viability of rainbow trout (Oncorynchus mykiss) spermatozoa

Rainbow trout (Oncorhynchus mykiss) spermatozoa were incubated in artificial sperm motility inhibiting saline solution (SMIS), in SMIS containing seminal plasma proteins or in pure seminal plasma. In SMIS containing the total seminal plasma protein fraction or the <50 kDa protein fraction or in pure seminal plasma, significantly higher motility rates and swimming velocities could be activated than in SMIS without seminal plasma proteins and in SMIS containing the >50 kDa protein fraction. These preliminary results indicated that seminal plasma proteins have physiological functions in prolongation and stabilization of sperm viability when using sperm motility as viability index.     

Testicular Expression of NGF,TrkA and p75 During Seasonal Spermatogenesis of the Wild Ground Squirrel (Citellus Dauricus Brandt)

The nerve growth factor (NGF) not only has an essential effect on the nervous system, but also plays an important role in a variety of non-neuronal systems, such as the reproductive system. The aim of this study was to compare the quality and quantity in expression of NGF and its receptors (TrkA and p75) in testes of the wild ground squirrel during the breeding and nonbreeding seasons. Immunolocalization for NGF was detected mainly in Leydig cells and Sertoli cells in testes of the breeding and nonbreeding seasons. The immunoreactivity of TrkA was highest in the elongated spermatids, whereas p75 in spermatogonia and spermatocytes in testes of the breeding season. In the nonbreeding season testes, TrkA showed positive immunostainings in Leydig cells, spermatogonia and primary spermatocytes, while p75 showed positive signals in spermatogonia and primary spermatocytes. Consistent with the immunohistochemical results, the mean mRNA and protein level of NGF and TrkA were higher in the testes of the breeding season than in non-breeding season, and then decreased to a relatively low level in the nonbreeding season. In addition, the concentration of plasma gonadotropins and testosterone were assayed by radioimmunoassay (RIA), and the results showed a significant difference between the breeding and nonbreeding seasons with higher concentrations in breeding season. In conclusion, these results of this study provide the first evidence on the potential involvement of NGF and its receptor, TrkA and p75 in the seasonal spermatogenesis and testicular function change of the wild ground squirrel.Key words: NGF, p75, seasonal spermatogenesis, TrkA, wild ground squirrel     

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = −0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = −0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.     

Effect of season on bovine seminal plasma proteins in Thailand

Although season has been shown to affect bull sperm quality and fertility in some studies, the effect of season on seminal plasma proteins has not been examined. In the present study, seminal plasma proteins were analysed by Fast Protein Liquid Chromatography (FPLC), to separate the phosphorylcholine-binding proteins and heparin-binding proteins from the other proteins. Semen samples were collected from bulls in three seasons: winter, summer and the rainy season. Sperm quality was analysed by flow cytometry and computer assisted sperm analysis, and further aliquots of semen were used to prepare the seminal plasma for FPLC. Meteorological data were available from a location close to the bull station. There were slight differences in sperm kinematics between seasons, but other parameters of sperm quality were not different. Minor differences in the phosphorylcholine-binding proteins were detected according to season, being lower in summer than in winter or in the rainy season, although there were no changes in the heparin-binding proteins. Temperature, humidity and rainfall differed between winter and the rainy season, but no differences were observed between summer and the rainy season except in the temperature humidity index (THI). However, the THI was above the threshold indicative of heat stress in all seasons, which could explain why few seasonal differences in protein composition were detected in this study. Alternatively, the bulls could have been well-adapted to heat stress. In conclusion, there were only slight differences in bull sperm quality and seminal plasma proteins between seasons during this study.     

Characterization of seminal plasma proteins stabilizing the sperm viability in rainbow trout (Oncorhynchus mykiss)

In seminal plasma of the rainbow trout 12 proteins were detected by SDS-PAGE, ranging in their molecular weight from 135 to 16 kDa. Only those proteins with a molecular weight of 65, 54, 47 and 16 kDa occurred in all investigated seminal plasma samples. The 65 and the 54 kDa protein were found in highest quantities (34-45% of the total quantified protein content) followed by the 47 and the 16 kDa protein (6-7% of the total quantified protein content). The 65 and the 48 kDa protein were glycoproteins as they stained positively with Periodic-Acid-Schiff reagent (PAS) specific for carbohydrates as well as with Coomassie Blue. The 90 and 19 kDa protein were found in 82-91% of the investigated samples, all other proteins in lower frequencies of 36-73%. Seminal plasma contained no lipoprotein as staining with Sudan black B was negative. To find out which proteins positively affected the sperm viability (defined as sperm motility which could be activated) spermatozoa were incubated in sperm motility inhibiting saline solution containing different seminal plasma protein fractions. Sperm motility which could be activated after an incubation period of 48 h was highest in those fractions which shared the 54, 47, and the 16 kDa protein. When spermatozoa were incubated in untreated seminal plasma sperm viability was still higher than in the seminal plasma protein fractions indicating that other components of the seminal plasma positively affected sperm viability, too. The possible influence of seminal plasma proteins on sperm physiology is discussed.     

Seasonal variation in pituitary-gonadal function in free-ranging impala (Aepyceros melampus).

Blood, testicular biopsies and electroejaculates were collected from adult male impala, free-ranging in the Kruger National Park (Republic of South Africa), during the breeding (rut; April-May) and nonbreeding (September-October) seasons. Blood samples were collected at 5-min intervals for 120 min from anaesthetized males (n = 7 impala/group) treated intravenously with saline, gonadotrophin-releasing hormone (GnRH: 1 microgram/kg body weight) or human chorionic gonadotrophin (hCG: 10 or 30 iu/kg). Semen was collected from six more animals during the breeding season and 12 animals during the nonbreeding season using a standardized electroejaculation protocol. Ejaculates obtained during the nonbreeding season were of inferior quality to those collected during the breeding season, and were characterized by lower sperm concentrations, poorer sperm motility and more morphologically abnormal sperm forms. Within season, there were no differences in testosterone secretion between the two hCG doses, and these responses were similar to those observed after GnRH, but during the rut, testosterone secretion stimulated by both GnRH and hCG was approximately nine times greater than during the nonbreeding season. This seasonal increase in testosterone production was associated with a doubling in testicular volume and concentrations of luteinizing hormone (LH) receptors. Although concentrations of testicular follicle-stimulating hormone (FSH) receptors were similar between seasons, receptor content increased during rut as a result of increased testicular volume. In contrast to testosterone secretion, basal LH and FSH secretions were unaffected by season and GnRH-induced gonadotrophin secretion was reduced during rut.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protracted reproductive seasonality in the male giant panda (Ailuropoda melanoleuca) reflected by patterns in androgen profiles, ejaculate characteristics, and selected behaviors

The female giant panda (Ailuropoda melanoleuca) experiences a brief (24-72 h) seasonal estrus, occurring once annually in spring (February-May). Our aim was to determine the existence and temporal profile of reproductive seasonality in the male of this species. The study was facilitated by 3 yr of access to eight giant panda males living in a large breeding center in China. Seasonal periods for the male were defined on the basis of female reproductive activity as prebreeding, breeding (early, peak, late), and nonbreeding seasons. Testes size, fecal androgen excretion, ejaculated sperm density, and frequency of reproductive behaviors (i.e., locomotion, scent marking, vocalizations) increased (P < 0.05) from the prebreeding period (October 1-January 31) to the early breeding season (February 1-March 21). Testes volume and sperm concentration were maximal from March 22 through April 15, a period coinciding with maximal female breeding activity. The occurrence of male reproductive behaviors and fecal androgen concentrations began declining during peak breeding and continued from April 16 through May 31 (late breeding period), returning to nadir throughout the nonbreeding interval (June 1-September 30). Reproductive quiescence throughout the latter period was associated with basal testes size/volume and aspermic ejaculates. Our results reveal that testes morphometry, fecal androgen excretion, seminal quality, and certain behaviors integrated together clearly demonstrate reproductive seasonality in the male giant panda. The coordinated increases in testes size, androgen production, sperm density, and sexual behaviors occur over a protracted interval, likely to prepare for and then accommodate a brief, unpredictable female estrus.     

Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.     

Increased daily sperm production in the breeding season of stallions is explained by an elevated population of spermatogonia

Seasonal variation in number of spermatogonia and germ cell degeneration was evaluated to determine which mechanism might explain seasonal differences in daily sperm production per testis (DSP/testis) or per g parenchyma (DSP/g) in stallions. Comparing 28 adult stallions (4 to 20 yr old) in each of the nonbreeding (December-January) and breeding (June-July) seasons, the population of type A spermatogonia was more than two times greater (P less than 0.01) in the breeding season. While the number of type B spermatogonia also was elevated (P less than 0.01) in the breeding season, the number of type B spermatogonia/type A spermatogonium was similar (P greater than 0.05) between seasons. Daily sperm production/testis based on each cell type from type B spermatogonia to spermatids with elongated nuclei was lower (P less than 0.01) in the nonbreeding season. Based on DSP/g, there was significant degeneration during the meiotic divisions in the nonbreeding season. However, this reduction in potential spermatozoan production was not significant (P greater than 0.05) when considering DSP/testis. Significant germ cell degeneration also occurred in the breeding season between type B spermatogonia and primary spermatocytes. However, the type A spermatogonial population was sufficiently elevated to override this degeneration and to explain elevated production of sperm in the breeding season of stallions.     

Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility

Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simple centrifugation and density gradient centrifugation of the sperm were compared. In 2009 and 2010 breeding seasons (experiment 2, n=125 mares), the effect of the addition of 5% seminal plasma to the extender after sperm selection was evaluated. In all mares deep horn uterine insemination was performed with 1 ml containing 50×10(6) morphologically normal progressive motile spermatozoa, 24-30 h after induction of ovulation with hCG. Pregnancy diagnosis by ultrasonography was performed 14 days following ovulation. Results showed a higher per cycle pregnancy rate (P>0.05) when sperm selection through a density gradient was used (62% vs. 42.3%, exp 1), while the addition of 5% seminal plasma did not influence the outcome (45.9% vs. 47.6%, exp 2) (P>0.05). An age-related decrease in the fertility of the stallion was observed when comparing the results from the different breeding seasons (P<0.05). In conclusion, sperm selection through a discontinuous density gradient enabled a normal per cycle pregnancy rate to be achieved from an oligospermic-subfertile stallion in a commercial breeding program, and no differences were observed regarding the addition of seminal plasma.     

Seasonal Expression of Androgen Receptor,Aromatase, and Estrogen Receptor Alpha and Beta in the Testis of the Wild Ground Squirrel (Citellus Dauricus Brandt)

The aim of this study was to investigate the seasonal expression of androgen receptor (AR), estrogen receptors α and β (ERα and ERβ) and aromatase cytochrome P450 (P450arom) mRNA and protein by real-time PCR and immunohistochemistry in the wild ground squirrel (WGS) testes. Histologically, all types of spermatogenic cells including mature spermatozoa were identified in the breeding season (April), while spermatogonia and primary spermatocytes were observed in the nonbreeding season (June), and spermatogonia, primary spermatocytes and secondary spermatocytes were found in pre-hibernation (September). AR was present in Leydig cells, peritubular myoid cells and Sertoli cells in the breeding season and pre-hibernation with more intense staining in the breeding season, whereas AR was only found in Leydig cells in the nonbreeding season; P450arom was expressed in Leydig cells, Sertoli cells and germ cells during the breeding season, whereas P450arom was found in Leydig cells and Sertoli cells during pre-hibernation, but P450arom was not present in the nonbreeding season; Stronger immunohistochemical signal for ERα was present in Sertoli cells and Leydig cells during the breeding season; ERβ was only expressed in Leydig cells of the breeding season. Consistent with the immunohistochemical results, the mean mRNA level of AR, P450arom, ERα and ERβ were higher in the testes of the breeding season when compared to pre-hibernation and the nonbreeding season. These results suggested that the seasonal changes in spermatogenesis and testicular recrudescence and regression process in WGSs might be correlated with expression levels of AR, P450arom and ERs, and that estrogen and androgen may play an important autocrine/paracrine role to regulate seasonal testicular function.Key words: Wild ground squirrels, testes, seasonal expression, androgen and estrogen receptors, aromatase cytochrome P450, Citellus dauricus Brandt     

Isolation and characterization of the major proteins of ram seminal plasma

Mammalian seminal plasma contains among others, two major families of proteins, namely spermadhesins and those proteins that contain fibronectin type II domains. Spermadhesins are the major proteins of boar and stallion seminal plasma and homologous proteins have been identified in the bull. These proteins appear to be involved in capacitation and sperm-egg interaction. In bovine seminal plasma, proteins containing fibronectin type II domains are the major proteins and are designated BSP proteins. These proteins play a role in sperm capacitation. In this study, we present the isolation and characterization of the major proteins of ram seminal plasma. Precipitated proteins from Suffolk ram seminal plasma were loaded onto a gelatin-Agarose column. The unadsorbed (fraction A) and retarded proteins (fraction B) were removed by washing the column with phosphate buffered-saline and the adsorbed proteins (fraction C) were eluted with 5 M urea. SDS-PAGE of fraction B indicated the presence of a 15.5 kDa protein, which is the major protein of ram seminal plasma (approximately 45% of total protein by weight) and was identified as a spermadhesin by N-terminal sequencing. SDS-PAGE analysis of fraction C revealed the presence of four proteins, which represented approximately 20% of total ram seminal plasma proteins by weight, and were identified as proteins of the BSP family and named RSP proteins. These RSP proteins were designated RSP-15 kDa, RSP-16 kDa, RSP-22 kDa, and RSP-24 kDa. Only RSP-15 kDa and -16 kDa proteins cross-reacted with antibodies against BSP proteins. Ram spermadhesin and RSP proteins interact with heparin but only RSP proteins bind to hen's egg yolk low-density lipoprotein. In conclusion, spermadhesin is the major protein of ram seminal plasma and other major proteins belong to the BSP protein family.     

Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins

Bovine seminal plasma (BSP) contains a family of major proteins designated BSP-A1/A2, BSP-A3, and BSP-30kDa (collectively called BSP proteins) that bind to sperm at ejaculation and potentiate sperm capacitation. Homologous proteins have been identified in stallion, boar, goat, and ram seminal plasma. We report here the isolation and characterization of homologous proteins from bison seminal vesicle secretions. Seminal vesicle secretory proteins were precipitated by adding cold ethanol and recovered by centrifugation. The precipitates were resuspended in ammonium bicarbonate, dialyzed, and lyophilized. Lyophilized proteins were dissolved in 0.05 M phosphate buffer (PB) and loaded onto a gelatin-agarose column. The unadsorbed proteins and adsorbed proteins were eluted with PB and 5 M urea in PB, respectively. The gelatin-adsorbed fraction was analyzed by SDS-PAGE and revealed the presence of four major proteins designated BiSV-16kDa, BiSV-17kDa, BiSV-18kDa, and BiSV-28kDa (BiSV: bison seminal vesicle proteins). Heparin-Sepharose chromatography allowed the separation of BiSV-16kDa, which did not bind heparin from other BiSV proteins, which bound heparin. Immunoblotting revealed that BiSV-16kDa cross-reacted with BSP-A3 antibodies, BiSV-17kDa and BiSV-18kDa cross-reacted with BSP-A1/-A2 antibodies, and BiSV-28kDa cross-reacted with BSP-30kDa antibodies. Radioimmunoassays indicated that approximately 25% of bison seminal vesicle total proteins are related to BSP proteins. The amino-terminal sequencing indicated that BiSV proteins share almost 100% sequence identity with BSP proteins. In addition, BiSV proteins bind to low-density lipoproteins isolated from hen's egg yolk. These results confirm that BSP protein homologs are present in mammalian seminal plasma and they may share the same biological role.     

Components in seminal plasma regulating sperm transport and elimination

Seminal plasma has been suggested to be involved in sperm transport, and as a modulator of sperm-induced inflammation, which is thought to be an important part of sperm elimination from the female reproductive tract. This article reports on recent experiments on the importance of seminal plasma components in sperm transport and elimination. In Experiment 1, hysteroscopic insemination in the presence (n = 3) or absence (n = 3) of 2 ng/mL PGE showed an increased portion of spermatozoa crossing the utero-tubal junction in the presence of PGE in two mares, while no difference was observed between treatments in a third mare. In Experiment 2, whole seminal plasma, heat-treated seminal plasma (90 degrees C for 45 min), and charcoal-treated seminal plasma were added to: (1) sperm samples during opsonization prior to polymorphonuclear neutrophil(s) (PMN)-phagocytosis assays (n = 5); or to (2) phagocytosis assays (n = 5). Opsonization of spermatozoa was suppressed in the presence of whole seminal plasma, compared with samples without seminal plasma (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma on opsonization, but heat treatment of seminal plasma reduced its suppressive properties (p < 0.05). The addition of whole seminal plasma to opsonized spermatozoa almost completely blocked phagocytosis (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma. However, heat-treated fractions of seminal plasma removed the suppressive effect of seminal plasma on phagocytosis (p < 0.05). In Experiment 3, viable and non-viable (snap-frozen/thawed) spermatozoa were subjected to in vitro assays for PMN binding and phagocytosis with the following treatments (n = 3): (1) seminal plasma (SP), (2) extender; (3) ammonium sulfate precipitated seminal plasma proteins with protease inhibitor (SPP+); or (4) ammonium sulfate precipitated seminal plasma proteins without protease inhibitor (SPP-). Treatment was observed to impact binding and phagocytosis of viable and non-viable spermatozoa (p < 0.05). SP and SPP+ suppressed PMN-binding and phagocytosis of viable sperm. This effect was also seen, but to a lesser degree, in SPP- treated samples. Non-viable spermatozoa showed less PMN-binding and phagocytosis than live sperm in the absence of SP. The addition of SP promoted PMN-binding and phagocytosis of non-viable spermatozoa. SPP- treated samples also restored PMN-binding of non-viable spermatozoa. The addition of protease inhibitors removed this effect. In Experiment 4, seminal plasma proteins were fractionated based on MW by Sephacryl S200 HR columns (range 5000-250,000 kDa). Fractionated proteins were submitted to sperm-PMN binding assays. A protein fraction <35 kDa suppressed PMN-binding to live and snap-frozen spermatozoa. A greater MW protein fraction appeared to promote binding between PMNs and snap-frozen spermatozoa. While the addition of protease inhibitors was necessary to maintain the protective effect of seminal plasma proteins on viable spermatozoa, the promotive effect of seminal plasma on non-viable spermatozoa appeared to require some protease activity. It was concluded from these experiments that components of seminal plasma play active roles in transportation and survival of viable spermatozoa in the female reproductive tract and in the elimination of non-viable spermatozoa from the uterus.     

Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent

Heparin binds to bovine sperm and stimulates capacitation in vitro. Seminal plasma alters the ability of epididymal sperm to bind heparin, and several heparin-binding proteins (HBPs) have been identified in bull seminal plasma. This study had three objectives: 1) to identify production sites of seminal plasma HBPs, 2) to determine which HBPs bound to cauda epididymal sperm, and 3) to determine whether presence of HBPs was testosterone dependent. Proteins from bull or rat seminal vesicles, prostates, and bulbourethral glands were separated by heparin affinity high-performance liquid chromatography. HBPs were found in all accessory glands of rats and bulls, but the major source of bovine seminal plasma HBPs appeared to be seminal vesicles. Between 25% and 50% of the protein from each gland bound to the heparin column, and NaCl concentrations required to elute proteins ranged from 0.15 to 1.4 M. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that major HBPs were relatively small, with molecular weights between 13 and 31 kDa, but some HBPs also exhibited higher molecular weights, between 40 and 100 kDa. Radioiodinated HBPs from each bovine gland were incubated with epididymal sperm. Labeled HBPs binding to sperm exhibited molecular weights of 14, 16, 24, and 30 kDa as determined by SDS-PAGE and autoradiography. The HBP content of the accessory sex glands decreased significantly in castrated rats and was restored to levels of sham-operated controls by testosterone replacement. Heparin-binding proteins may play a role in fertilization by attaching to sperm surfaces, enabling heparin-like glycosaminoglycans in the female reproductive tract to induce capacitation.     

Seasonal androgen cycles in adult male American alligators (Alligator mississippiensis) from a barrier island population

The seasonal patterns of two primary plasma androgens, testosterone (T) and dehydroepiandrosterone (DHEA), were assessed in adult male alligators from the Merritt Island National Wildlife Refuge, a unique barrier island environment and home to the Kennedy Space Center in Florida. Samples were collected monthly from 2008 to 2009, with additional samples collected at more random intervals in 2007 and 2010. Plasma T concentrations peaked in April, coincident with breeding and courtship, and declined rapidly throughout the summer. Seasonal plasma T patterns in smaller though reproductively active adult males differed from those in their larger counterparts during the breeding season. Both size classes showed significant increases in plasma T concentration from February to March, at the beginning of the breeding season. However, smaller adults did not experience the peak in plasma T concentrations in April that were observed in larger adults, and their concentrations were significantly lower than those of larger males for the remainder of the breeding season. Plasma DHEA concentrations peaked in May and were significantly reduced by June. This is the first study to demonstrate the presence of DHEA in a crocodilian, and the high plasma DHEA concentrations that paralleled the animals' reproductive activity suggest a reproductive and/or behavioral role in adult male alligators. Similar to androgen variations in some birds, plasma DHEA concentrations in the alligators were considerably higher than T concentrations during the nonbreeding season, suggesting a potential role in maintaining nonbreeding seasonal aggression.