Restoration of the apoptosis pathways' proteins levels after orchiectomy in testicular tumour patients.

Seminal plasma proteins already demonstrated to reflect the testicular environment function and important regulatory mechanisms. However, it is crucial to understand which of these proteins participate in probable altered pathways in testicular germ cell tumours and after unilateral orchiectomy. In this study, we proposed to verify, by a multiplex approach, the levels of DNA damage and apoptosis pathways' proteins, in seminal plasma of men before and after unilateral orchiectomy, and also in control men. Comparing pre- and post-orchiectomy groups, just the apoptosis pathways' proteins presented different levels, in which Bad was lower and Bcl2, Akt, caspase-9, p53 and caspase-8 were higher after orchiectomy. When comparing pre- and post-orchiectomy groups with control, both presented lower levels of ChK1, Chk2, H2AX, p53 and p21, for DNA damage pathway. Regarding the apoptosis pathway, lower levels of JNK, Bcl2, Akt, caspase-9, p53 and caspase-8 and higher levels of Bad were observed before orchiectomy. The post-orchiectomy group did not differ from controls, demonstrating a probable restoration on its proteins levels. We can conclude that testicular tumours can alter both of the assessed pathways, and its removal is associated with a probable restoration of the apoptosis pathway.

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways link capacitation with apoptosis and seminal plasma proteins protect sperm by interfering with both routes†.

The mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38) signaling cascades are involved in triggering apoptosis in somatic cells. Given that spermatozoa are able to undergo apoptosis, we tested the hypothesis that these pathways might be functional in ram spermatozoa as two signal transduction mechanisms that contribute to the modulation of capacitation and apoptosis. Indirect immunofluorescence and western blot analysis evidenced the presence of JNK and p38 in ram spermatozoa. To verify the involvement of these enzymes in sperm physiology, we determined the effect of specific inhibitors of JNK or p38 on in vitro capacitation induced with either cAMP-elevating agents or epidermal growth factor (EGF). Both inhibitions reduced the EGF-induced capacitation with a decrease in the chlortetracycline capacitated-sperm pattern, protein tyrosine phosphorylation, phosphatidylserine externalization, caspase-3 and -7 activation, and the proportion of DNA-damaged spermatozoa. No significant changes were found in the high-cAMP capacitated samples. The addition of 3.4 mg/ml seminal plasma proteins (SPPs) to the EGF-containing samples, either alone or together with each inhibitor, resulted in a decreased proportion of capacitated sperm pattern, protein tyrosine phosphorylation, loss of plasma membrane integrity, and apoptotic alterations. Furthermore, SPPs significantly reduced the phosphorylation level of JNK and p38 MAPK (active forms). These findings show a relationship between capacitation and apoptosis, and represent a step forward in the knowledge of the SPP protective mechanism in spermatozoa.

Use of biochemical and protein profiles of seminal plasma to prediction of semen quality and fertility in stallions.

The identification of various substances in seminal plasma has opened the way to study their functionality. It was aimed to identify the electrophoretic protein profile (EPP) and biochemical parameters (BP) of seminal plasma (SP) as predictors of semen quality and fertility in stallion. Forty-six ejaculates from 7 fertile stallions, aged between 6-26 years, were collected from May to July and 117 mares were used to obtain fertility data. For each ejaculate, volume, sperm motility, concentration were determined and seminal plasma samples were collected to perform one- -dimensional electrophoresis and biochemical profiling. Following the estrus detection, mares were inseminated with fresh sperm. Pregnancy rates and foal rates were recorded. The concentration of 15-18 kDa molecular weight (MW) proteins has shown a positive correlation with sperm concentration and foal rate. Besides, a strong positive correlation was found between sperm concentration and 23-28 kDa MW proteins (r=0.77). The volume of 19-22 kDa MW proteins was negatively correlated with pregnancy and foal rate. Similarly, the volume of high MW proteins (173-385 kDa) correlated negatively with sperm motility and foal rate. Apart from the protein profile, while Magnesium and Glucose levels were negatively correlated with sperm quality and foal rate, Cholesterol level was a positive indicator of the quality of semen as well as the foaling rate. Moreover, the total protein level was correlated negatively with the sperm concentration whereas triglyceride was correlated positively. In conclusion, EPP and BP of seminal plasma are valuable clinical tools as predictors of fertility and semen quality in the stallion.

Fractionated Seminal Plasma of Boar Ejaculates Analyzed by LC-MS/MS: Its Effects on Post-Thaw Semen Quality.

This study aimed to characterize the protein composition of fractionated seminal plasma (SP) by liquid chromatography mass spectrometry (LC-MS/MS) analysis and investigate its effects on survival of frozen-thaw (FT) boar spermatozoa following storage. Seminal plasma (SP) was fractionated by gel filtration chromatography to give two fractions, SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). SP1 and SP2 were subjected to LC-MS/MS and bioinformatics analysis. Following cryopreservation, FT boar semen (n = 7) was thawed in Beltsville Thawing Solution (BTS), BTS + SP1 or BTS + SP2, stored at different periods and subjected to post-thaw (PT) quality assessment. A total of 52 and 22 abundant proteins were detected in SP1 and SP2, respectively. FN1, ANGPTL1, and KIF15 proteins were more abundance in SP1, whereas a high abundance of spermadhesins (PSP-I and PSP-II) was detected in SP2. Proteins of the fractionated SP were involved in various biological processes, such as cell motility and signal transduction. The dominant pathway of SP1 proteins was the apelin signaling pathway (GNA13, MEF2D, SPHK2, and MEF2C), whereas a pathway related to lysosome (CTSH, CTSB, and NPC2) was mainly represented by SP2 proteins. In most of the boars, significantly higher motility characteristics, membrane integrity, and viability were observed in FT spermatozoa exposed to SP1 or SP2 compared with BTS. The results of our study confirm that a combination of several proteins from the fractionated SP exerted beneficial effects on the sperm membrane, resulting in improved quality characteristics following PT storage.

Bull seminal plasma stimulates in vitro production of TGF-ß, IL-6 and IL-8 from bovine endometrial epithelial cells, depending on dose and bull fertility.

Seminal plasma (SP) regulates immune responses in the female reproductive tract through specific cytokines. It is not known whether SP from high fertility bulls (H) differs from SP from low fertility bulls (L). In this study, the cytokine response of bovine endometrial epithelial cells (bEEC) in culture was investigated after challenge with SP from two bulls of below average (L) or three bulls of above average fertility (H). The bEECs were challenged with 1% or 4% SP from l- or H-fertility bulls (L1, L4, H1, H4, respectively) or 1% or 4% PBS as control (C1, C4) for 72 h. The culture media were analysed for concentrations (pg/million cells) of transforming growth factor beta (TGF-ß1, TGF-ß2 and TGF-ß3) by Luminex, and Interleukin 6 and 8 (IL-6, IL-8) by ELISA. Challenge significantly affected production of TGF-ß1, TGF-ß2 and IL-8 compared to controls and was affected by bull fertility (p < 0.0001), SP concentration (p < 0.0001) and their interaction (p < 0.0001). A higher production of TGF-ß1, TGF-ß2 and IL-8 (p < 0.0001), and also IL-6 (p < 0.01), resulted from challenge with high doses of SP, being higher for L than H (p < 0.05). For TGF-ß3, fertility of bull (p < 0.05). For TGF-B3, fertility of bull (p < 0.05) and the interaction between fertility and concentration of SP were significant (p < 0.01). In conclusion, 4% SP from L bulls stimulated more TGF-ß1, TGF-ß2, TGF-ß3, IL-6 and IL-8 production than SP from H bulls, indicating that stimulation of the endometrium is relevant for fertility. Seminal plasma from high fertility bulls seems to affect cytokine production in utero positively in inseminated cows.

Identification of proteoforms of albumin and kallikrein in stallion seminal plasma and their correlations with sperm motility.

The aim of this study was to identify the proteoforms of albumin and kallikrein in stallion seminal plasma (SP), and to determine their correlations with sperm motility parameters. The experimental material consisted of ejaculates from 8 stallions, which were collected during the breeding and non-breeding seasons (BS and NBS, respectively). SP proteins were identified by 2-D PAGE and mass spectrometry (MALDI TOT/TOF MS). Sperm motility parameters were analyzed using the CASA system. Protein expression (integrated optical density-IOD) of albumin proteoforms 1 (ALB 1) and 2 (ALB 2) and kallikrein proteoforms 1 (KAL 1) and 2 (KAL 2) was correlated (p⟨0.05) with sperm motility parameters (total motility and progressive motility) during the BS. No significant correlations were found between the expression of albumin or kallikrein and sperm motility parameters during the NBS. The presence of correlations between the expression of ALB 1, ALB 2, KAL 1, KAL 2 and selected sperm motility parameters could suggest that the analyzed components of the SP belong to the group of fertility-associated proteins (FAPs).

Expression and secretion of albumin in male turkey (Meleagris gallopavo) reproductive tract in relation to yellow semen syndrome1.

Yellow semen syndrome (YSS) is the most widely recognized problem among male turkeys. Yellow semen is of low quality and, when used for insemination, results in reduction of fertility and hatchability. Elevated level of serum albumin-like protein accession no. XP_003205725 is a characteristic feature of yellow seminal plasma suggesting albumin role in YSS pathology. However, knowledge regarding the expression of albumin in the reproductive tract in relation to YSS is very limited. The aim of this study was to identify albumin secretion and localization sites in the turkey reproductive tract in relation to YSS. Reproductive tract tissues and liver originating from turkeys producing white semen (WS) and YSS were used for analysis of albumin mRNA expression and its localization using immunohistochemistry. Moreover, albumin abundance in tissues, blood and seminal plasma was analyzed using two dimensional electrophoresis and western blot analysis. Albumin mRNA expression was found in all parts of the reproductive tract. Apart from the liver, the highest expression of albumin was found in the ductus deferens in YSS turkeys. The testicular spermatids, Leydig, and myoid cells and the epithelium of the epididymis and ductus deferens were the main secretion sites of albumin in the reproductive tract in turkeys. Higher albumin abundance was found in the reproductive tract and seminal plasma of YSS toms compared to WS toms. Our results demonstrated that germ cells from spermatocytes to spermatids, Leydig cells, and myoid cells synthesized and secreted albumin in turkey testis, and epithelial cells are the main secretion sites in epididymis and ductus deferens. Ductus deferens secretion of albumin seems to be mostly responsible for YSS. Over-secretion by the ductus deferens may be the main origin of albumin abundance in YSS semen. Knowledge regarding disturbances of albumin secretion in relation to YSS may be useful for future work on studies related to better understanding the molecular basis of YSS.

Chromatography analysis of seminal plasma proteins in buffalo semen samples with high and low cryotolerance.

The aim of this study was to analyse and identify specific buffalo seminal plasma proteins (SPPs) responsible for sperm cryotolerance during low temperature storage. Computer Assisted Sperm Analysis (CASA) of the motility and viability of buffalo spermatozoa was performed before freezing and after thawing. Two sample groups were formed - ejaculates with high cryotol- erance (group A) and low cryotolerance (group B). CASA demonstrated that the initial progres- sive motility after thawing of the spermatozoa in group A is significantly higher than in group B (p⟨0.001). Group B showed a significant increase in the percentage of static and non-progressive spermatozoa at 240 min, when compared to group A (p⟨0.05). SPPs, proteins in the cryoprotec- tive medium (PM) and proteins in the mixture of PM and SP were separated by High Perfor- mance Liquid Chromatography (HPLC). Comparative analysis of the chromatographic profiles was performed to identify specific proteins related to sperm cryotolerance. SPPs profiles showed 5 distinct protein peaks in both groups, ranging from 500 kDa to 50 Da. Chromatograms of group A and group B showed quantitative and qualitative differences in protein content. Chromato- grams of proteins in PM showed 11 well-expressed peaks. HPLC analysis of the mixtures of SPPs from the two groups and PM visualized the formation of a new bio-complex structure expressed by a protein peak specific for group A (7.674 min, AU 1.50). This protein peak can be referred as a phenotypic trait for buffalo ejaculates with high sperm cryotolerance.

Use of heterospermic inseminations and paternity testing to evaluate the relative contributions of common sperm traits and seminal plasma proteins in boar fertility.

The objective of this study was to evaluate relationships between common semen quality estimates including sperm motility, sperm morphology, spontaneous capacitation status and seminal plasma proteins and boar fertility using heterospermic inseminations and subsequent paternity testing. All boars (n=12) used in the study had excellent semen quality (≥70% normal sperm) that resulted in average farrowing rates and litter sizes of 88.9±0.7% and 11.7±0.1 pigs, respectively. Their ejaculates were combined to make heterospermic insemination doses in such a way that each boar was tested against all of his contemporaries. The proportion of piglets sired by each individual was used to separate boars into three fertility groups: High (71.6±4.8%; n=3); Medium (51.6±3.8%; n=6); and Low (25.2%±5.3%; n=3). Ejaculates from High fertility boars had more motile sperm with normal acrosomes that moved faster in a straight-line and were more likely to undergo an acrosome reaction (p≤0.05) compared with their counterparts in the Low fertility group. Ejaculates from High fertility boars contained the greatest concentrations of three seminal plasma proteins (25.9kD/5.9pI; 55.1kD/4.8pI; and 70.1kD/5.2pI; p≤0.05), whereas concentrations of a 19.1kD/6.8pI were highest in semen from Low fertility boars (p≤0.05). Multiple regression analyses indicated that concentrations of the 25.9kD/5.9pI seminal plasma protein explained 66% of the variation observed in the proportion of pigs sired within a litter among boars (p≤0.00001). These results demonstrate that heterospermic inseminations and subsequent paternity testing is an effective technique for defining relationships between common semen quality tests and fertility, especially in situations where reproductive performance of all the boars is high. Motility, normal acrosome morphology, average linear velocity of motile sperm, and the proportion of sperm capable of an acrosome reaction were all positively associated with boar fertility. However, concentrations of a 25.9kD/5.9pI seminal plasma protein were the best single semen characteristic for ranking boars in terms of their fertility.

Topografia de ligação de proteínas do plasma seminal à membrana de espermatozoides bovinos epididimários e ejaculados / Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane

The present study was designed to investigate the topographical distribution of seminal plasma (SP) proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS) and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.

Investigou-se a distribuição topográfica da ligação de proteínas seminais à membrana de espermatozoides bovinos epididimários e ejaculados. Utilizando imunocitoquímica e microscopia confocal, avaliaram-se a topografia de ligação das proteínas BSP-A3, albumina, transferrina, prostaglandina D sintetase (PGDS) e nucleobindina 2 (NUC2) à membrana espermática. Os experimentos foram realizados utilizando espermatozoides de cinco touros. Os resultados mostraram que, para espermatozoides epididimários, somente detectou-se a PGDS na crista do acrossomo. Nos espermatozoides ejaculados, a PGDS ligou-se de forma mais intensa à crista acrossômica, enquanto a NUC2 apresentou sinal bastante fraco na peça intermediária e região equatorial. A BSP-A3 ligou-se a todas as regiões estudadas, de forma mais intensa na peça intermediária e acrossomo. Nenhum sinal foi detectado para albumina ou transferrina, seja em espermatozoides epididimários ou ejaculados. Concluiu-se que PGDS, BSP-A3 e NUC2 interagem diretamente com espermatozoides bovinos, e mostrou distribuição topográfica específica. Estes achados permitem melhor compreensão sobre o papel desempenhado por essas proteínas na regulação da função espermática e da fertilidade.