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.

Expression of urokinase type plasminogen activator receptor (uPAR) in the bovine oviduct: Relationship with uPA effect on oviductal epithelial cells.

Urokinase type plasminogen activator (uPA) is an oviductal fluid component whose activity is regulated by binding to urokinase type plasminogen activator receptor (uPAR). In this study uPAR and uPA gene expression in bovine oviduct were evaluated and similar expression patterns for both uPAR and uPA mRNAs were observed during the estrous cycle. Immunolocalization of uPAR at the apical zone of epithelial cells suggests that uPA action would be focalized in the oviductal lumen, triggering intracellular signaling pathways. As uPAR expression was also observed in in vitro cultures of oviductal epithelial cells, the effect of uPA was explored using this culture model. Real-time RT-PCR demonstrated that c-fos expression in oviductal cell cultures increases under uPA stimulation. These results suggest that uPA/uPAR binding would be involved in signaling pathways that activate transcription factors and would regulate the synthesis of molecules concerned with the arrangement of a particular oviductal microenvironment.

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.

Expression of DNA methyltransferase genes in four-cell bovine embryos cultured in the presence of oviductal fluid.

The possible epigenomic effect of oviductal fluid on expression of DNA methyltransferase (DNMT) genes was examined in early bovine embryos (4-cell stage). Real-time quantitative PCR was performed to determine the relative expression of DNMT1, DNMT3a and DNMT3b transcripts in embryos cultured in vitro in the presence or absence of oviductal fluid. Expression of DNMT1 significantly increased when cultured with oviductal fluid, whereas DNMT3a and DNMT3b transcripts were unaffected by the addition of oviductal fluid. These results may help reveal the role of oviductal factors in the regulation of DNMT expression.

In vivo and in vitro sperm interaction with oviductal epithelial cells of llama.

Sperm reservoirs in South American Camelids would be crucial for successful fertilization. Since ovulation occurs approximately 36 h after mating, the maintenance of the sperm viability in the oviduct waiting for the ovum is a critical reproductive event. Our study aimed at determining whether the isthmus or the utero tubal junction (UTJ) could function as a sperm reservoir in llama by means of in vivo and in vitro experiments. For the in vivo experiments, the oviducts of adult females with a dominant follicle bigger than 7 mm were examined for the presence of sperm at 6, 18, 24, 28 and 35 h after mating. The results using scanning and transmission electron microscopy showed ultrastructural differences between isthmus and UTJ with respect to (1) predominance of secretory cells in the UTJ and ciliated cells in the isthmus epithelium and (2) cytoplasmic bulbous projection of the secretory cells in the UTJ. Sperm adhered by a mucus-like substance were seen only in the UTJ at 6, 18, 24 and 28 h postmating. Lack of sperm adhered to oviductal mucosa was observed around ovulation (35 h). In vitro experiments demonstrated higher ability of UTJ epithelial cell explants with respect to isthmus explants to bind sperm in a co-cultured system. The anatomical features and the presence of a sperm bonding agent in the UTJ together with the in vitro differential binding of sperm to UTJ explants strongly suggest that both may be feasible mechanisms that facilitate sperm storage in this oviductal region in llama.

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.

Potential involvement of C(3) complement factor in amphibian fertilization.

We have assessed the potential involvement of C(3), the third complement factor, and its receptor in Bufo arenarum fertilization. We show that a polyclonal antibody against a B. arenarum C(3)-like factor (C(3)Ba) reacts specifically with components of the extracellular matrix (ECM) of coelomic eggs and the cell membrane of uterine eggs. Interestingly, we have identified a 163 kD protein immunoreactive with a monoclonal antibody against the CD11b alpha chain of the human C(3) receptor on the cell membrane of the animal pole of uterine eggs, the site of entry of the sperm, but not in coelomic eggs (CR3Ba). Treatment of coelomic eggs with a pars recta oviductal-like protease, trypsin, induced the translocation of C(3)Ba from the ECM to the cell membrane. Furthermore, inhibition of CR3Ba by trypan blue, as well as inhibition of C(3)Ba by anti-C(3)Ba on uterine eggs impaired fertilization, whereas identical treatment on sperm cells did not alter percentage fertilization. Our results suggest, (A) that changes in the localization of C(3)Ba from the ECM to the cell membrane may be triggered by trypsin-like proteases during passage of eggs through the oviduct; and (B) that C(3)Ba/CR3Ba may be involved in B. arenarum fertilization.

A simple procedure for isolation of Bufo arenarum C3 complement fraction and preparation of antiserum.

Because of the need for antibodies in our studies involving the third component of complement in Bufo arenarum, we performed a simple procedure to purify C3 from B. arenarum serum to use as antigen in the preparation of the antiserum. The strategy was based on the well-known ability of C3 to bind to zymosan (Zy), a yeast cell wall extract comprised of polysaccharides. The Zy-bound fraction showed cross reactivity with a commercial antibody to human C3 as well as a similar electrophoretic profile (SDS-PAGE) to C3 from other species. The Zy-C3 complex resulting from binding Zy to B. arenarum serum was injected into rabbits and the antiserum against this C3-like fraction was purified by protein A-Sepharose chromatography. The purified C3 antibody was found to be suitable for immunochemical studies.

Phosphorylation of endogenous and TEST-yolk buffer proteins by intact human sperm.

Protein kinase activity of intact, motile sperm was assessed by measuring the transfer of the terminal phosphate from [32P]ATP to tricholoroacetic acid (TCA)-precipitable casein. The action of TEST (TES and Tris) yolk buffer (TYB) treatment on phosphorylation of sperm and TYB proteins was studied by detecting labelled phosphoproteins by autoradiography of polyacrylamide gel electrophoresis (PAGE). Results demonstrate that intact, forward-motile sperm have cell surface protein kinase activities. Although the difference between the kinase activity of freshly ejaculated sperm incubated in TYB was not significant, the protein phosphorylation during incubation in TYB showed that: (i) specific sperm surface proteins were phosphorylated to different degrees during the course of treatment; (ii) TYB proteins were phosphorylated by sperm during incubation; (iii) solubilised [32P]-labelled surface proteins were similar in molecular weight to TYB-labelled proteins. Taking into account that specific proteins on the human sperm surface undergo phosphorylation during incubation in TYB and that the sperm enzyme also acts specifically on some TYB proteins that become attached to the surface of the sperm, working hypotheses are proposed that suggest some correlation between the preservation of semen in TYB and the phosphorylation of proteins by intact human sperm.

Involvement of blood serum in amphibian fertilization

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization

Involvement of blood serum in amphibian fertilization

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization

Involvement of blood serum in amphibian fertilization.

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization.

Characterization and properties of steroid binding protein in Bufo arenarum serum.

Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol-17 beta (E2) with high affinity (10(-7) M-1 - 10(8) M-1) and fair capacity (10(-6) M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control.

In vivo and in vitro protein synthesis of oviducal pars recta by estradiol effect.

1. Proteins secreted by toad oviductal pars recta are involved in fertilization and their biological activity is regulated by estrogens. 2. Effect of 17 beta-estradiol on protein synthesis was examined on castrated animals by different in vivo and in vitro experimental approaches. 3. Different routes of L-[3H]leucine administration were assessed. An intralymphatic route was the most efficient for incorporating radioactivity per mg of trichloroacetic acid insoluble proteins. 4. Ligature of pars recta induces protein synthesis at a similar level to exogenous estradiol. 5. Electrophoretic pattern of radioactive proteins did not show synthesis of a specific protein related to the zone with biological activity. 6. Pars recta releases newly synthesized proteins in vivo into its fluid secretion as much as in vitro into culture medium.

Oviducal pars recta as factor in fertilization properties and hormonal regulation in the toad Bufo arenarum.

In this communication I have attempted to present an overview of some contributions to the understanding of the oviduct-egg interaction in amphibians. According to data from other authors, the vitelline envelope of the newly ovulated egg constitutes a barrier for the passage of spermatozoa. Our results demonstrated that only after they have been affected by substances released by the first 1-3 cm of the oviduct (pars recta), is the envelope sensitive to spermlysins and the oocytes fertilizable. This functional change is matched by biological, physicochemical and morphological differences in the vitelline envelope. The fact that the pars recta activity is affected by the sexual cycle and that in ovariectomized females - devoid of active pars recta - the biological activity can be restored by steroid hormones, strongly suggests that the molecules involved in fertilization are synthesized and secreted during specific steps of the reproductive cycle. The pars recta-oocyte interaction probably involves more than one type of molecules, considering the observations made on the carbohydrate metabolism of coelomic eggs, which could be altered by the oviducal secretions. Several explanations for the pars recta mechanism of action have been suggested. One is a direct action on the sperm; pars recta molecules - engulfed in the vitelline envelope - would trigger the acrosome reaction. We propose the unmasking of specific vitelline envelope sites for sperm interaction. Material on the outer surface of the VE can be removed or altered by the enzymatic activity - similar to plasmin and trypsin - detected in the pars recta secretions.

Properties of an oviducal protein involved in amphibian oocyte fertilization.

The secretion produced at the most cephalic portion of the oviduct of the toad (pars recta in Bufo arenarum) is involved in fertilization. Although the present study indicates that a prerequisite for the fertilization of coelomic eggs is either their passage through the pars recta (PR) or their treatment with PR secretion fluid prior to insemination, the exact role of this secretion in the fertilization process is still not clearly understood. A protein acting upon the vitelline envelope (VE) of Bufo arenarum coelomic eggs has been purified from secretion fluid (pars recta protein, PRP). Some properties of both this protein and the secretion fluid are examined in an effort to understand their mechanism of action. It was shown that PRP partially dissolves the VE of coelomic oocytes in a way that resembles the action of proteolytic enzymes. PRP has also proved to be active on synthetic substrates of proteolytic enzymes such as p-Tosyl-L-arginine methyl ester-HCl (TAME), alpha-N-benzoyl-L-arginine ethyl ester-HCl (BAEE), and alpha-N-benzoyl-DL-arginine-p-nitroanilide HCl (BAPNA). PR enzyme is activated by calcium ions, shows a broad peak of maximum activity at pH 7.8, is stable at alkaline pH, and is inhibited by soybean trypsin inhibitor (SBTI) and N-alpha-p-tosyl-L-lysine chloromethyl ketone HCl (TLCK) but not by lima bean trypsin inhibitor (LBTI) or L-1-tosyl-amide-2-phenylethylchloro-methyl-ketone (TPCK).

A trypsin-like oviducal proteinase involved in Bufo arenarum fertilization.

When the jelly-less eggs removed from the most cephalic region of the oviduct (pars recta) of the toad Bufo arenarum were inseminated at a high sperm concentration, high frequencies of fertilization were obtained. On the other hand, control eggs removed from the pleuroperitoneal cavity (coelomic eggs)) were neither fertilized upon insemination under identical conditions, nor with the water extract of the jelly. Under these inseminating conditions, however, a high frequency of fertilization was obtained when coelomic eggs were preincubated in the presence of the fluid secreted by the epithelium of the pars recta or of an extract prepared from pars recta homogenate. Experimental evidence is presented showing that the component responsible for this effect acts on the vitelline envelope of the egg, increasing its susceptibility to sperm lysin. It is probable, therefore, that it induces successful fertilization of coelomic eggs by making the vitelline envelope more easily penetrable by sperm. The active factor was partially purified by Sephadex chromatography. The product obtained was of high activity and, as judged by its inhibition with soybean trypsin inhibitor and lima-bean trypsin inhibitor, it is likely to be a trypsin-like enzyme. The molecular weight of the factor was estimated to be 47000 by Sephadex chromatography. Secretion of the pars recta factor is hypophysis-dependent and its activity is not influenced by pH within the range testes (6.0--8.4).

An oviducal enzyme isolated by affinity chromatography which acts upon the vitelline envelope of Bufo arenarum coelomic oocytes.

A trypsin-like oviducal proteinase acting upon the vitelline envelope of Bufo arenarum coelomic oocytes has been purified to apparent homogeneity by gel filtration on Sephadex G-200 and by affinity chromatography on a column of Sepharose 4-B containing covalently bound concanavlin A (Con A). The biologically active molecule migrated as a single band of protein upon SDS polyacrylamide gel electrophoresis.