Characterization of proteases in the seminal plasma and spermatozoa of llama.

Camelids' semen has peculiar characteristics that differentiate it from other species, including the highly viscous aspect of seminal plasma that greatly difficult sperm manipulation and the development of techniques such as cryopreservation, artificial insemination, and/or in vitro fertilization. The presence of proteases in the seminal plasma is responsible for semen liquefaction, and sperm functionality to achieve fertilization. The enzymatic and molecular composition of the semen of llama remains unknown. Therefore, the goal of the study was to characterize the protease activity and composition of the seminal plasma and sperm of llama semen. The proteolytic activity was performed using gelatine zymography and the composition by mass-spectrometry. Metallo-proteases were the major source of gelatinolytic activity in seminal plasma, while serine-peptidases were the main enzymes of sperm cells. Matrix Metalloproteinase 2 (MMP2) was the most prominent metallo-protease of llama seminal plasma characterized under the exposure of different inhibitors (EDTA and benzamidine) and by a specific immunodetection. Moreover, the prostate and epididymis were identified as potential sites of its synthesis and secretion. Outstandingly, this metalloproteinase was undetectable in llama sperm. Regarding, the molecular composition of semen by mass-spectrometry, 4 metallo-, 9 serine-, 8 threonine-, and 1 aspartic-peptidases were identified alongside 15 regulators in the sperm cell; where 24 were directly or indirectly interacting. Whereas 6 metallo-, 12 serine-, 3 cysteine-, and 1 aspartic-peptidases were identified, besides 7 inhibitors and 5 regulators in llama seminal plasma where 30 of them were directly or indirectly interconnected. This is the first study describing a partial degradome of llama seminal plasma and spermatozoa suggesting significant differences especially the absence of MMP2 in spermatozoa in contrast to data observed in other species. The characterization of proteases in llama semen will provide a better understanding of the molecular mechanisms involved in the in vivo or in vitro fertilization process in this species.

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.

Optimization of the surface properties of nanostructured Ni-W alloys on steel by a mixed silane layer.

Ni-W nanostructured coatings electrodeposited on steel by galvanostatic pulses were functionalized by tetraethoxysilane (TEOS) and octadecyltrichlorosilane (OTS) in a two-step procedure. A silica-rich layer is formed by the reaction of TEOS with the metal coating surface oxides, which allows a further reaction with OTS forming a hydrocarbon-silica outer network. This mixed silane layer provides hydrophobicity and improves the corrosion behavior of the Ni-W surface coatings without modifying their excellent mechanical properties.

Biochemical composition and protein profile of alpaca (Vicugna pacos) oviductal fluid.

Knowledge and assessment of the constituents of the oviductal fluid (OF) in camelids is necessary for a correct formulation of specific culture media for the development of reproductive biotechnology. This study is the first describing the biochemical composition and SDS-PAGE protein profile of alpaca oviductal fluid in non-pregnant animals and animals that have completed the first month and second month of gestation. Samples were also classified into oviducts that were ipsilateral or contralateral to the ovary with corpus luteum. No differences were found between both oviducts, whereas pregnant and non-pregnant females displayed significant differences in the biochemical composition and protein profile of the oviductal fluid. Relative albumin content was higher in non-pregnant females. Relative creatinine content in OF from females that have completed the second month of gestation was lower than non-pregnant females and females that have completed the first month of gestation. Ion Na(+) concentration was higher in OF from non-pregnant females when compared with pregnant ones. The protein profile of non-pregnant females showed five protein bands of 70, 42, 25, 24 and 19kDa that were significantly more intense compared with pregnant animals. Bands were identified as moesin, actin cytoplasmic 2, hydroxypyruvate isomerase, ferritin light chain and peroxiredoxin-6 with MALDI/MS. Our results encourage more thorough future studies, in order to unravel the complex reproductive processes of the South American camelid oviduct.

Biochemical and protein profile of alpaca (Vicugna pacos) uterine horn fluid during early pregnancy.

South American camelids show high embryo loss rate, during the first 60 days of pregnancy. One of the factors which may be related to this situation is that over 98% of the embryos implant in the left uterine horn (LUH) even though both ovaries contribute similarly to ovulation. There is scarce information about the uterine environment of female camelids at any physiological state that could explain the capability of the LUH to attract the embryo and maintain pregnancy. We describe, for the first time, the biochemical and protein profile of uterine fluid (UF), addressing the right and LUH environment in non-pregnant and pregnant alpacas. Different substrates, electrolytes and metabolites were assayed in both uterine horn fluids. Small changes were observed in glucose and total protein levels, which were more noticeable during pregnancy. In addition, 10 specific proteins were found in the left horn fluid in 5-week-pregnant alpacas, and two protein bands were identified in non-pregnant alpaca right horn fluid. These results would provide basic information for identification of possible markers for pregnancy diagnosis, reproductive diseases and hormone-treated animals evaluation and hence contributing to improve the pregnancy rate.

Detection of the matrix metalloproteinases MMP-2 and MMP-9 and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 in llama (Lama glama) oviduct.

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in several reproductive events like oocyte-spermatozoa interaction and semen liquefaction. In order to study their role in the llama oviductal reproductive process, MMP activity in oviductal fluid (OF) was assayed. Considering that llama genome sequences are partially known, a strategy to procure cDNA sequences of MMP-2, MMP-9, TIMP-1 and TIMP-2 was designed. Afterwards, their expression patterns in the different llama oviductal segments were assayed. Gelatine zymograms detected 62 and 94 kDa protease activities that matched MMP-2 and pro-MMP-9, respectively. Expression pattern analysis showed that MMP and TIMP mRNAs were present in ampulla, isthmus, utero-tubal junction (UTJ) and papilla. Altogether, these findings support the argument that MMPs/TIMPs are produced in the oviduct and secreted into the oviductal lumen. Our results encourage further studies to elucidate the role of these proteins in reproductive oviductal events.

Llama oviductal sperm reservoirs: involvement of bulbourethral glands.

The aim of this study was to elucidate the role of llama seminal plasma in the formation of oviductal sperm reservoirs. Female llamas with follicles in the mature phase were mated with a bulbourethral glands-removed male. Females mated with nonbulbourethral glands-removed males were used as control. Oviducts were obtained by surgery 24 h after mating. The uterotubal junction and isthmus were examined by scanning electron microscopy, and mucopolysaccharides were identified by Alcian blue staining. To know the proteins probably involved in sperm reservoir formation, SDS-PAGE of seminal plasma (8% and 18% resolving gel) was made. Spermatozoa only adhered to the oviductal mucosa surface of uterotubal junction of females mated with nonbulbourethral glands-removed males confirming that seminal plasma and, in particular, bulbourethral secretions are related with the oviductal sperm reservoir formation. Histological sections showed sperm in the lumen, immersed in substance, positive for acid mucopolysaccharides. Alcian blue staining of seminal plasma proteins SDS-PAGE showed a band of high molecular weight containing mucopolysaccharides, only present in nonbulbourethral glands-removed males. Bulbourethral glands would secrete at least eight different proteins that most likely participate in the process of sperm storage in the oviduct.

Progesterone receptors: their localization, binding activity and expression in the pig oviduct during follicular and luteal phases.

Estrogens (E) and progesterone (P) are known to require their respective steroid receptors in order to exert structural and functional effects on the oviduct. Cyclic changes in progesterone receptor (PR) localization in the oviductal tissue of female pigs were determined using an immunohistochemical technique with mouse monoclonal antibody mPRI against PR. The variations observed during the estrous cycle in the progesterone receptor (PR) intensity and proportion between ampulla and isthmus probably reflect different response of these regions to progesterone. Immediately before ovulation, during follicular phase, no staining was observed in either the ampulla or the isthmus stroma. However, a low expression of PR in the epithelium of the ampulla was observed. After ovulation, during luteal phase, PR immunostaining was more intense in the whole oviduct. According to immunohistochemical assays, the binding assays for nuclear and cytosolic PR (PRn and PRc, respectively), by using [3H] R5020 at 4 degrees C for 15 h, also showed a higher specific binding during luteal phase. However, the PR mRNA in the oviduct, analyzed by RT-PCR, showed similar levels at both stages of the estrous cycle. Although this methods could not be quantitative, indicate the possibility that a post-transcriptional control could differentially regulate the PR in the pig oviduct.

Paracoccidioidomycosis and larynx carcinoma.

Report of a case of paracoccidioidomycosis associated with a carcinoma: both located in the larynx in a patient whose therapeutic response to antifungal treatment produced a recovery of physical conditions. This case shows the importance of taking into account the diagnosis of paracoccidioidomycosis in all patients with problems in the larynx, especially those who inhabit or inhabited endemic areas of Paracoccidioides brasiliensis.