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Uterine luminal fluid protein content in viable and non-viable conceptuses in sheep.

CONTENIDO DE PROTEINAS DEL FLUIDO LUMINAL UTERINO EN CONCEPTUS OVINOS VIABLES Y NO VIABLES

INTRODUCTION

Embryonic mortality (EM) is considered the most important cause of prenatal losses in sheep (Ovis aries), having an incidence of 36.7% [12]. An important proportion of EM occurs during implantation, when the embryo is particularly labile, since it evolves in a slow and gradual manner. It ensues near the embryo at day 15 of gestation and spreads peripherally thereafter, and it is not complete before day 60 of gestation, when placentomes are well developed [5, 8, 9]. The ovine conceptus remains floating within the uterine lumen before its attachment to the uterine endometrium. Its nutrition depends on the small volume of uterine luminal fluid that probably contains all necessary nutrients for the development in this phase, and even after implantation [13].

The embryo depends for its survival, among other factors, on molecules secreted by the uterus, such as growth factors and proteins. They act as growth promoters and immunomodulators [18]. There is extensive work in the identification of proteins secreted by the endometrium, which are controlled by estrogens and progesterone [1, 6, 17]. However, information is lacking on the possible changes in protein secretion with the advancement of gestation during implantation in sheep. Furthermore, it is feasible that the absence of one or more molecules could be crucial for embryonic survival.

Induction of multiple pregnancies by superovulation in sheep, offers reasonably possibilities to obtain both, viable and non-viable conceptuses for comparative studies [15].

The present trial was undertaken to characterize the electrophoretic profiles of secreted proteins into the uterine lumen of sheep at 20, 28 and 35 days of gestation in both, viable and non-viable embryos.

MATERIALS AND METHODS

The work was carried out in the correlation of laboratory experimentation and Morphophysiology and Histopathology of the Graduate School of Veterinary Medicine and Zootecnia of the Universidad Nacional de Colombia, Bogota DC, approx. 2640 m, with an annual average temperature, relative humidity and rainfall of 12[grados]C, 79% and 938 mm / year, respectively [10].

Twelve crossbred sheep, healthy and multiparous (two to five births) randomly allocated into the following groups: a) days 20 (n= 4), b) 28 (n= 4) and, c) 35 (n= 4) of gestation.

The animals were fed with natural pasture (Pennisetum clandestinum) using a daily supplement of concentrated commercial feed containing 14% protein, mineralized salt and water ad libitum.

Estrous detection was carried out with the assistance of a caudoepididymectomised ram. Daily progesterone levels were assessed by radioimmunoassay (RIA), using commercial kits (1), to verify normal luteal function (average levels of 0.1621 and 4.761 nanograms of [P.sub.4] for days 0 and 12, respectively). This test has a sensitivity of 0.02 ng / mL, and a very low cross-reactivity with other components present in the sample. A treatment samples were measured [P.sub.4] levels by RIA in different tests. The intra-assay coefficient of variation average was 8.001% and the intra-assay variation of 8.31%.

When animals fulfilled the established gestational age, were anesthesized and then slaughtered by exsanguination.

The initiation of estrus was set as day 0 of the estrous cycle. Sheep were synchronized for estrous using intravaginal sponges, containing 60 mg of medroxyprogesterone acetate (2), for 13 days. Forty eight hours prior to sponge withdrawal, animals received 1500 IU (im) of Pregnant Mare Serum Gonadotropin (PMSG) (3). Each female was given 2.5 mL (iv) of PMSG (4) antibodies at day 0, as to neutralize possible secondary effects of PMSG. The animals were allowed to have 2 estrous cycles before they were naturally mated.

The reproductive tract was extracted and uterine luminal fluid (ULF) was obtained at site location of each embryo, using a sterile aqueous solution of NaCl (0.15M) for local washes. Protease inhibitors were added to all samples, which were then centrifuged in sterile vials at 10000 rpm for 10 minutes at 4[grados]C (cooling centrifuge. Eppendorf, 5417R, USA). The supernatant was then conserved at -20[grados]C (Freezer Indufrial, BGL-320, Colombia).

Before the analysis, all samples were centrifuged and the sediment placed in culture media for bacterial and fungal cultures. Afterwards, they were stained with Gram's and observed under the light microscope (Olympus microscope CX21, Tokio, Japan) to verify the absence of bacteria, fungi or blood cells.

Protein concentration was then determined using the Bradford's method [4], with bovine serum albumin (BSA) as the standard. Equal amounts of proteins were employed for two-dimensional -PAGE, which was carried out following the procedure described by O'Farrell [15], using an electrophoresis chamber MINI PROTEAN III, Bio-Rad, USA,. Briefly, up to 10 [micron]g of total protein was loaded onto each prefocused 7.5 cm x 25 mm (i.d.) tube gel containing 1.6% (w:v) ampholytes (pH 5-7) and 0.4 (w:v) ampholytes (pH 3.6-9.5), and proteins were separated by isoelectric focusing. The gels were extruded from the tubes, equilibrated with 62.5 mM Tris-HCl pH 6.8 containing 10% (w:v) glycerol, 5% (v:v) 2-mercaptoethanol, and 2.3% (w:v) sodio dodecilsulfato (SDS), then attached to 0.075 x 7 x 8-cm 11 and 15% (w:v) acrylamide SDS gels, and subjected to electrophoresis in the second dimension. After electrophoresis, proteins were detected by non-ammonium silver staining, a preparation where band staining depends on the union of silver with more than one chemical compound in the protein. The range of detection was 2-5 ng/protein. For analysis, the gels were registered photographically, using a digital camera SONY Mavica, 1.3 Mega Pixels, model MVC-FD88, Canada.

In order to verify pH gradients and validate the standarization for the first dimension, some isoelectric focus gels were cut in 5mm sections and then placed in individual vials, in which 2 mL of a 9.2 M urea degasified aqueous solution had been added. The obtained pH reading for each sample was within the expected range, following O'Farrell's recommendations [15].

Embryonic viability was estimated by the macroscopical characteristics of the embryonic membranes, degree of vascularization of the allantochorion and embryo development, according to that described by Boshier y Guillomot [3, 8].

Descriptive statistics, and correlative studies, using the Minitab Program 7.2 [14]. Multivariate analysis was used, to determine possible differences in protein electrophoretic profiles among variables.

RESULTS AND DISCUSSION

Sixty embryos were found in all studied sheep, with a mean value of 5 embryos/ewe. Thirty six percent of them were classified as non-viable, based on at least 1 of the following macroscopic characteristics: absence of blood vessels in the allantochorion, yellowish colour of embryonic membranes and absence of an embryo.

This is the first report of protein content of secretory products adjacent to non viable and viable ovine conceptuses.

There were no individual differences in the electrophoretic profile of the embryos in the same condition (viable or nonviable). Hence, for the 2D-PAGE electrophoretic analysis, data were pooled for each condition of the embryos (viable or nonviable) in each age studied. The employed experimental procedures allowed the detection of proteins having 6.5 to 205 kDa, within an isoelectric point ranging from 3.6 to 9.5.

[FIGURA 1 OMITIR]

At the embryonic sites of viable embryos, there was a greater amount of proteins in the ULF adjacent to viable embryos as compared to correspondent sites of non-viable conceptuses: 35, 19.2 and 21% at 20, 28 and 35 days of gestation, respectively. The quantitative differences encountered were represented by 4 complex groups, namely, protein spots 39-44, 47-52, 63-67, and 210-214, with molecular weight (MW) of 66 kDa with Isoelectric Points (Ip) ranging from 7.18-8.3; 60-58 kDa (pI 4.8-5.6); 59-56 kDa (Ip 5.8-6.4); 14.2 kDa (Ip 5.2-6.2), respectively.

At 20 days of gestation, there were several proteins in the ULF adjacent to viable embryos, which were absent in the correspondent sites of non-viable conceptuses. Those molecules were represented by several protein complexes (FIG. 1): 1) MW ~32 kDa with Ip ranging from ~3.7 a 5.15 (protein spots 132 - 144), 2) MW ~30 kDa and Ip ~3.84-5.03 (spots 151 -160), 3) MW ~80 kDa and Ip ~6.8-7.3 (spots 23 -25), those which migrated in a similar position to that of the group of proteins identified by Lee et al. [13], as transferrin in 2D gels of the ovine uterine luminal fluid proteins, and 4) MW ~31 kDa and Ip ~6.6-6.8 (spots 148 - 149.1). The latter protein, did migrated to a similar position to that of a molecule known as aldose reductase, identified by Lee et al. [13], using 2D electrophoresis, this enzyme is responsible for the synthesis of sorbitol and fructose, but its role in implantation is unknown. It is possible that this protein has more unidentified functions [13]. Likewise, non-grouped proteins were found (TABLE I). It is tempting to propose those proteins can be taken as markers for embryonic viability. Some of these proteins corresponding to spots 132-144 and 151-160), might be of maternal origin, since they were also encountered in the uterine luminal fluid of cycling animals (unpublished data). Furthermore, regional differences in protein secretion could be the result of either uterine incompetence to produce certain compounds or, that a viable embryo induces the uterine epithelium to secrete specific proteins. Also, the abovementioned difference could be interpreted as the result of inhibition to uterine secretion, by a non-viable conceptus. Some groups of proteins, at day 20 of gestation, were found in both, viable and non-viable conceptuses. However, there were a higher amount of them in the viable conceptuses, as compared to the non-viable ones (FIG. 1).

Again, at 28 and 35 days of gestation, there were proteins in the ULF adjacent to viable embryos, which were lacking in the correspondent sites of non-viable conceptuses. They corresponded to compounds of MW ~32 y 30 kDa (Ip ~3.7-5.15 and ~3.84-5.03, respectively). However, there were differences in the amount of these molecules between the abovementioned gestation-ages (FIG. 2). As observed for 20, 28 and 35 days of gestation, protein spots 215 (~16 kDa, Ip ~7.3), 219 (~14.9 kDa and Ip ~6.2), and 23 - 25 (~80 kDa and Ip ~6.8-7.3) denominated transferrin and protein spots 148-149.1 (aldose reductase), were non-detectable in non-viable embryos. The following compounds were found in viable embryos, and absent in the non viable ones at 28 days: protein spots 36-38, 148, 149, 149.1, 200, 203, 205 y 223), with corresponding MW and pI were: ~80 kDa (Ip ~6.8 - 7.3); ~70 kDa (Ip ~6.9-7.1); ~31 kDa (Ip ~6.2); ~31 kDa (Ip ~6.8); ~30.5 kDa (Ip ~6.6); ~18.5 kDa (Ip ~5.7); ~17.03 kDa (Ip ~4.0); ~18.5 kDa (Ip ~4.1); and <6.5 kDa (Ip ~5.01), respectively (TABLE I; FIG. 2). At day 35, additional qualitative differences were represented by the absence in non-viable embryos of two protein complexes: one, with MW ~27-24 kDa (Ip ~7.0-7.9, spots 184190). Non-grouped proteins were also found (TABLE I; FIG. 3). Therefore, it could also be thought that differences in protein expression after 20 days might be taken as indicative of embryonic decay.

At all gestational ages studied, spots 215 and 219 (MW 16kDa (Ip 7.3) and 14.9 kDa (Ip 8.2), respectively were absent in non-viable conceptuses, but present in viable ones. Nevertheless, there are no reports about proteins in ULF with a similar pattern of migration in two dimension gels, as presently found.

An important difference in the three studied gestation ages was the presence, only in viable embryos, of a protein which migration in 2D gels coincides with the physical characteristics of transferrin (protein spots 23-25), shown to derive from both the maternal and the conceptus side; and which is believed to be important for proliferation, differentiation and cellular function [13]. Given the role of this protein in iron transportation and binding, its absence in the ULF near non viable embryos could be interpreted as a lack of production of transferrin by the conceptus. Transferrin appears to be involved in the formation of blood vessels [13], which then could account for the absence of those structures in the membranes of non viable conceptuses.

At all gestational ages studied, proteins identified at spots 47-53 and 63-67, as well as protein at spot 210-213 coincide with reports by Lee et al. [13]. The latter, is important for the establishment of pregnancy according to Lee et al. [13].

Proteins encountered during the follicular and luteal phases of the estrous cycles (Rodriguez and Hernandez, unpublished data) were also found in viable embryos at 28 and 35 day-old embryos. At these stages of gestation the protein profile in the ULF showed quantitative differences, since there were grater amounts of protein near viable embryos than in the correspondent sites of non viable ones (FIG. 2 and 3).

The electrophoretic profile became more complex with the advancement of gestation, from MW ~148 proteins at 20 days, up to ~198 at day 35 of gestation. Also, with the advancement of gestation, the complexity of the protein profile in the uterine luminal fluid increases, coincidently, trophoblastic growth becomes evident, spreading from embryonic sites, towards the extremities of the conceptus [5, 16]. It is also clear, that glandular growth is going on simultaneously. Godkin et al. [5], also found an increment in protein secretion in vitro, concomitant with placental development. As pregnancy progresses, the pattern of protein secretion to the uterine lumen, becomes more uniform.

[FIGURA 2 OMITIR]

[FIGURA 3 OMITIR]

Quantitative differences among viable and non-viable embryos, at different gestational ages, are shown in FIG. 1, 2, and 3.

Under the conditions of the present trial, at all gestational stages studied, proteins similar to IFN-t, and other proteins which are dependent for their secretion on IFN-t release, like ubiquitin cross-reactive protein (UCRP) [7, 10, 11, 19] were absent, both in viable and non-viable conceptuses, which could be explained because interpheron-t secretion ends by the 21st day of gestation [1]. In the present study, IFN-t was not found at day 20. Also, in the present model of induced multiple pregnancy, some unidentified changes could imply modifications of gene expressions.

Proteins with MW and Ip compatible with ovine Placental lactogen (oPL) and pregnancy specific protein B were not detected. This could be explained accepting the proposal that they are presumed to be secreted to the endometrium, rather than to the uterine lumen [2, 20].

CONCLUSIONS

There are some proteins in viable embryos, which are absent in non-viable ones, at 20, 28, and 35 days of gestation. They could represent key molecules to determine the viability of embryos, although some of them have not been identified yet. It is to be expected that with cellular death and/or absence of development in the trophoblast, several secretory products would disappear, which could as well be inductors to the secretion of proteins by the uterus.

It is also possible that embryonic death is the result of lack of some proteins secreted by the conceptus or the uterus. In the latter event, this might involve a localized event, to explain present findings, since embryonic decay was not generalized.

ACKNOWLEDEGMENTS

This work was supported by Universidad Nacional de Colombia, Bogota, Colciencias, and Universidad del Zulia, Maracaibo, Venezuela.

Recibido: 06 / 06 / 2011. Aceptado: 07 / 10 / 2011.

BIBLIOGRAPHICS REFERENCES

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[2] BECKERS, J.F.; ZARROUK, A.; BATHALA, E.S.; GARBAYO, J.M.; MESTER, L.; SZENCI, O. Endocrinology of pregnancy: chorionic somatomamotropins and pregnancy-associated glycoproteins: review. Acta Vet. Hung. 46:175-189. 1998.

[3] BOSHIER, D. P. Histological examination of serosal membranes in studies of early embryonic mortality in the ewe. J. Reprod. Fertil. 15: 81-86. 1968.

[4] BRADFORD, M.M. A rapid sensitive method for the quantitative analysis of microgram quantities of protein utilising the principle of protein-dye binding. Annals of Biochemi. 72:248-254. 1976.

[5] GAVIRIA, M.; HERNANDEZ, A. Morphometry of implantation in the sheep. Trophoblast attachment, modification of the uterine lining, conceptus size and embryo location. Theriogenol. 41:1139-1149. 1994.

[6] GODKIN, J.D.; BAZER, F.W.; MOFFATT, J.; SESSIONS, F.; ROBERTS, R.M. Purification and properties of a major, low molecular weight protein released by the trophoblast of sheep blastocysts at day 13-21. J. Reprod. Fertil. 65(1): 141-150. 1982.

[7] GONELLA, A.; GRAJALES, H.; HERNANDEZ, A. Ambiente receptivo uterino: control materno, control embrionario, muerte embrionaria. REVISION. Rev. MVZ. (Cordoba). Colombia. 15(1):1976-1984, 2010.

[8] GUILLOMOT, M. Cellular interactions during implantation in domestic ruminants. J. Reprod. Fertil. 49: 39-51. 1995.

[9] HERNANDEZ, A. The development of the extremities of the placenta of the domestic sheep. University of Bristol. Grade Dissertation. 82pp. 1971.

[10] JOHNSON, G.A.; SPENCER, T.E.; HANSEN, T.R.; AUSTIN, H.J.; BURGHARDT, R.C.; BAZER, F.W. Expression of the interferon tau inducible ubiquitin crossreactive protein in the ovine uterus. Biol. Reprod. 61: 312-318. 1999.

[11] JOHNSON, G.A.; SPENCER, T.E.; BURGHARDT, R.C.; JOYCE, M.M.; BAZER, F.W. Interferon-tau and progesterone regulate ubiquitin cross-reactive protein expression in the ovine uterus. Biol. Reprod. 62: 622-627. 2000.

[12] KAULFUSS, K. H.; MAY, J.; SUSS, R.; MOOG, U. In vivo diagnosis of embryo mortality in sheep by real-time ultrasound. Small Rum. Res. 24 (2): 141-145. 1997.

[13] LEE, R.S.F.; WHEELER, T.T.; PETERSON, A.J. Large format, two-dimensional polyacrylamide gel electrophoresis of ovine periimplantation uterine luminal fluid proteins: Identification of aldose reductase, Cytoplasmic Actin, and Transferrin as conceptus-synthesized proteins. Biol. Reprod. 59: 743-752. 1998.

[14] MINITAB STATISTICAL SOFTWARE. 7.2 V. Minitab, Inc. United States. 1989.

[15] O'FARRELL, P.H. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250 (10): 40074021. 1975.

[16] RODRIGUEZ, J.; JIMENEZ, C.; HERNANDEZ, A. A microscopical study of uterine lining modification, binucleate cell numbers and trophoblastic development, at 14, 20 and 24 of day gestation in single and multiple pregnancies in sheep. Small Rum. Res. 35(2): 163-168. 2000.

[17] SKOPETS, B.; HANSEN, P.J. Identification of predominant proteins in uterine fluids of unilaterally pregnant ewes that inhibit lymphocyte proliferation. Biol. Reprod. 49: 997-1007. 1993.

[18] SCHAFER-SOMI, S. Cytokines during early pregnancy of mammals: a review. Anim. Reprod. Sci. 75 (1-2):73 94. 2003.

[19] SPENCER, T.E.; STAGG, A.G.; OTT, T.L.; JOHONSON, G.A.; RAMSEY, W.S.; BAZER, F.W. Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on the endometrium of cycle ewes. Biol. Reprod. 61: 464-470. 1999.

[20] WOODING, F.B.P. Role of binucleate cells in fetomaternal cell fusion at implantation in the sheep. Amer. J. Anat. 170: 233-250. 1984.

(1) Diagnostic Products, USA.

(2) Depo-Provera, Upjohn, USA.

(3) Folligon, Interver, Holland.

(4) Neutra PMSG, Intervet, Holland.

Jose Rodriguez-Marquez (1) *, Aureliano Hernandez (2), Victor Vera (2) y Roneisa Morales (3)

(1) Unidad de Investigaciones en Ciencias Morfologicas (UNICIM). Facultad de Ciencias Veterinarias, Universidad del Zulia, Apartado 15252. Maracaibo 4005-A, estado Zulia, Venezuela. E-mail: jose.rodriguez@fcv.luz.edu.ve.

(2) Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogota, Colombia.

(3) Medico Veterinario. Profesor Facultad de Ciencias Veterinarias, Universidad del Zulia.
TABLE I

DIFFERENCES IN PROTEIN CONTENTS IN ULF ADJACENT TO VIABLE
AND NON-VIABLE EMBRYOS IN SHEEP DURING IMPLANTATION

                                           Day of Gestation

                                        20                  28

 Prot. #       MW        Ip        E.V      E.N.V      E.V      E.N.V
  (Spot)     (kDa)

23-25          80     6.8-7.3    [marca]   -         [marca]   -
36-38          70     6.9-7.1    [marca]   [marca]   [marca]   -
75            56.8      5.44     -         -         [marca]   [marca]
76             56       5.14     -         -         [marca]   [marca]
96             55       4.7      -         -         -         -
97             55       4.9      -         -         -         -
132-144        32     3.7-5.1    [marca]   -         [marca]   [marca]
148-149.1      31     6.6-6.8    [marca]   -         [marca]   -
150            30       8.5      [marca]   [marca]   [marca]   [marca]
151-160        30     3.8-5.0    [marca]   -         [marca]   [marca]
184-190      24-27    7.0-7.9    -         -         [marca]   [marca]
203          17.03      4.0      -         -         [marca]   -
205           16.5      4.1      -         -         [marca]   -
208           17.5      5.4      [marca]   -         [marca]   [marca]
215            16       7.3      [marca]   -         [marca]   -
219           14.9      8.2      [marca]   -         [marca]   -
223           <6.5      5.0      -         -         [marca]   -

             Day of Gestation

                    35

 Prot. #       E.V      E.N.V
  (Spot)

23-25        [marca]   -
36-38        [marca]   -
75           [marca]   -
76           [marca]   -
96           [marca]   -
97           [marca]   -
132-144      [marca]   [marca]
148-149.1    [marca]   -
150          [marca]   -
151-160      [marca]   [marca]
184-190      [marca]   -
203          -         -
205          -         -
208          [marca]   [marca]
215          [marca]   -
219          [marca]   -
223          -         -

MW = Molecular Weigth, Ip = Isoelectric point, EV = Viable embryo,
NV = Non viable embryo, [marca]: Present; -: absent.
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Author:Rodriguez-Marquez, Jose; Hernandez, Aureliano; Vera, Victor; Morales, Roneisa
Publication:Revista Cientifica de la Facultad de Ciencias Veterinarias
Date:Nov 1, 2011
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