Cryopreservation modifies the distribution of the prostate-derived lectin SL15 on the llama (Lama glama) sperm.

Lectin-like molecules play a key role in mammalian sperm functionality. These multifunctional proteins have been proven to be involved in sperm capacitation, sperm motility, and viability, formation of the oviductal sperm reservoir, and in sperm-oocyte interaction. In a previous study, we reported the presence of a novel seminal plasma lectin, sperm lectin 15 kDa (SL15), adsorbed to the llama sperm. In order to gain knowledge in the understanding of SL15 and its functions, the aims of this study were to (a) elucidate the presence and localization of SL15 in the llama male reproductive tract and sperm, and (b) determine whether the sperm cryopreservation process of cooling and freeze-thawing affects the SL15 levels and distribution on llama sperm. We found that SL15 protein was expressed along the male reproductive system: testis, epididymis, prostate, and bulbourethral glands, being the prostate the main site of SL15 secretion. SL15 was localized on the sperm head, following different localization patterns. In order to understand if sperm cryopreservation induces modifications in the SL15 adsorption pattern, immunocytochemistry and flow cytometry analysis were carried out on fresh, 24 h cooled, and frozen-thawed sperm. Both cooled and frozen sperm showed particular SL15 patterns, that were not observed in the freshly ejaculated, indicating loss of SL15. Flow cytometry analysis also exhibited a decrease of SL15 in the cooled sperm (P < 0.05), whereas a tendency to decrease was found in frozen-thawed sperm (P < 0.1) when compared with freshly ejaculated sperm. This study extends the knowledge about the SL15 in the llama male physiology and provides evidence that cryopreservation-related techniques disrupt SL15 adsorption to the sperm membrane, possibly affecting sperm functionality and fertility.

ß-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells.

The beta-nerve growth factor (ß-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama ß-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with ß-NGF, ß-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of ß-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3ß-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. ß-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that ß-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that ß-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

Effect of Cooling and Freezing on Llama (Lama glama) Sperm Ultrastructure.

Semen cryopreservation in South American camelids has a low efficiency. Post-thaw viability of sperm is low, and poor results are obtained when artificial insemination is performed with cryopreserved semen, impeding advances both in accelerated genetic progress and selection. This study aimed to describe the effect of a conventional method of camelid semen cryopreservation on the llama sperm ultrastructure during cooling and freezing, using transmission and scanning electron microscopy (TEM, SEM). Sperm motility, vigor, viability, and DNA integrity during those steps were also examined. Ejaculates from five fertile adult llama males were obtained by electroejaculation. For cooling, semen samples were washed with Hepes-balanced salt solution (HBSS), diluted in Tris-citric acid-fructose egg yolk extender (TCF-EY), and then cooled until 5°C for 24 h. For freezing, sperm samples were washed with HBSS, diluted in TCF-EY and cooled until 5°C for 2.5 h. Samples were equilibrated with TCF-EY, supplemented with 6% glycerol at 5°C for 20 min, and then stored in liquid nitrogen for a month before thawing. TEM and SEM analyses were carried out on sperm samples prior to cryopreservation, after cooling down until 5°C for 2.5 and 24 h, and after the freeze-thaw process. Ultrastructural injury was noticed during cooling, even though sperm motility, vigor, viability, and DNA integrity were not significantly affected. Analysis revealed plasma membrane and acrosome damage, loss of mitochondria, and axoneme and periaxonemal structure disorganization after 2.5 h of cooling. During freezing, a significant decrease in sperm motility and viability was observed after thawing. TEM and SEM revealed prominent signs of post-thawing damage. The plasma membrane was lost or exhibited various degrees of swelling, undulation, and perforations. Besides, the sperm presented vacuoles in the nucleus and broken acrosomes. Mitochondria in the midpiece showed vacuolization and structural disorganization. In conclusion, SEM and TEM revealed that cryopreservation induced ultrastructural damages in llama sperm that initiated during cooling and intensified during freezing. These details provide valuable data for further studies to minimize cryodamage in camelid sperm.

Effect of mating on mRNA and protein expression of beta nerve growth factor and its receptor, TrKA, in the oviduct of llama (Lama glama).

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of ß-nerve growth factor (ß-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. ß-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both ß-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative ß-NGF abundance was higher in the UTJ of copulated females (p < .05). ß-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the ß-NGF/TrKA system and that an increase in ß-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas.

Modifications of extracellular matrix features in the left and right uterine horns during the embryo pre-implantation period in Vicugna pacos.

More than 98% of the pregnancies in South American camelids is carried out in the left uterine horn (LUH). Hence, embryos originated from right-ovary ovulations have to migrate to the contralateral or left uterine horn (LUH) to implant and survive. A reason for this unique pattern of embryo implantation has not been elucidated yet. In general, embryo implantation involves an extensive extracellular matrix (ECM) remodeling within the endometrium, in which collagen and matrix metalloproteinases (MMPs) play an essential role. Deregulation of collagen and MMPs has been related to embryo implantation failure, miscarriage, and infertility. Therefore, we hypothesized that ECM components in camelids could be involved in differential embryo implantation and consequently the high incidence of left horn gestations. The aim of this study was to describe and compare changes in ECM components in the left and right uterine horn of non-pregnant and 15 days pregnant alpacas. To test this hypothesis, the collagen content was evaluated by specific staining with Picrosirius Red and using ImageJ 1.42q software. Subsequently, gene expression of the following components of the MMP pathway was determined: MMP-2, -3, -7, -9, and -14, MMP substrates (COL1A2 and COL3A1), MMP inhibitors (TIMP1 and TIMP2), LGMN, an MMP activator, and EMMPRIN, an extracellular matrix metalloproteinase inducer. Uterine horns of pregnant alpacas exhibited a marked decrease in collagen content. In contrast, transcript expression of COL1A2 and COL3A1 was higher in the LUH of pregnant alpacas. Gene expression of MMP-3, -7, -9, -14, LGMN, and EMMPRIN were also higher in the LUH of pregnant animals, whereas MMP-2 gene expression was higher in the LUH of both pregnant and non-pregnant alpacas. Expression of TIMP1 and TIMP2 increased during pregnancy, with higher values in the LUH. In conclusion, expression of ECM components displayed a specific pattern depending on the uterine side and the physiological status (pregnant vs non-pregnant) of the animal. The increased expression of ECM transcripts in the left uterine horn during early pregnancy in alpacas suggests the involvement of these molecules in a highly regulated process leading to the implantation process.

New Insights Into the Role of ß-NGF/TrKA System in the Endometrium of Alpacas During Early Pregnancy.

One striking reproductive feature in South American camelids is that more than 90% of gestations are established in the left uterine horn (LUH). This phenomenon could be related to a differential vascular irrigation of the LUH. An increase of vascularization in llama endometrium was observed after systemic administration of Beta Nerve Growth Factor (ß-NGF), a neurotrophin present in the uterus and placenta of various mammals that is involved in pregnancy development. We hypothesized that the ß-NGF signaling pathway is related to embryo implantation in the LUH in camelids. The aim of this study was to characterize the spatial expression of ß-NGF and its high-affinity receptor, TrKA, between LUH and right uterine horn (RUH) of non-pregnant (NP) and early pregnant alpacas (15 and 30 days of gestation, 15 and 30P, respectively). In addition, ß-NGF, TrKA, and Vascular Endothelium Growth Factor A (VEGFA) temporal gene expression patterns and counting of blood vessels were evaluated among groups. The ß-NGF and TrKA were localized in the luminal, glandular, and vascular epithelium of the alpaca uterus and in the embryonic membranes of the 30-days-old conceptus. ß-NGF and TrKA immunosignal were stronger in 15P females than that of NP and 30P. In addition, TrKA signal was higher in the LUH luminal epithelium of NP and 15P alpacas than that of NP-RUH and 15P-RUH. ß-NGF mRNA relative abundance was higher in the 30P-RUH than that of NP-RUH; whereas TrKA mRNA abundance only differed between 15P-RUH and NP-LUH. VEGFA mRNA relative abundance was higher in NP females compared to the LUH of 15P and 30P alpacas, and lower to their right counterparts. The number of vessels per field was higher in 15P than that of 30P. A positive correlation was observed between the number of vessels per field and ß-NGF immunosignal in 15P-LUH. In contrast, the area occupied by vessels was higher in 30P alpacas than of NP and 15P females. The changes of ß-NGF/TrKA expression pattern in the peri-implantation endometria between LUH and RUH and their localization in the extraembryonic membranes support the implication of the neurotrophin during implantation and pregnancy development in South American Camelids.

Effects of NGF Addition on Llama (Lama glama) Sperm Traits After Cooling.

To provide new insights into the mechanisms through which seminal plasma proteins can protect sperm from damage caused during refrigeration, we evaluate the possibility that ß-NGF can contribute to the improvement of sperm quality after cooling. First, ß-NGF was detected in refrigerated sperm and compared with unrefrigerated sperm by western blotting of the proteins adsorbed by sperm, showing that native ß-NGF is still present even 24 h after cooling only as an active form. Then, the effect of exogenous ß-NGF on sperm quality after cooling was evaluated. A total of 12 ejaculates from male llamas (three ejaculates per male), were obtained by electro-ejaculation, diluted 4:1 with buffer Hepes-balanced salt solution and centrifuged at 800 × g for 8 min to remove the seminal plasma. Sperm were suspended in Tris-citrate-fructose-egg yolk diluent for a final concentration of 30 ×106/ml and cooled at 5°C for 24 h. After refrigeration, the extended sperm were equilibrated for 5 min at 37°C and divided into the following subgroups: sperm samples without treatment (control) and sperm samples supplemented with exogenous human ß-NGF (10, 100, and 500 ng/ml). At 5, 30, and 60 min of incubation sperm were evaluated for sperm viability (using eosin/nigrosin stain), sperm motility and vigor (observed under light microscopy), and mitochondrial activity (using the JC-1 fluorescent marker). Vigor data were analyzed with the nonparametric Kruskal-Wallis test. The rest of the variables were analyzed with a mixed models approach. Mean comparisons were performed using Fisher's LSD test with a confidence level of 95%. A principal components analysis was performed to analyze the relationships between variables. Treatment of 24 h cooled sperm with 10 or 100 ng/ml of human ß-NGF increased the percentage of total motility and vigor (p < 0.05). Besides, an incubation time of 60 min would be adequate to improve sperm quality, since all variables are positively related. The significant improvement observed in the motility and vigor of post-refrigerated sperm suggests that supplementation with exogenous ß-NGF may be profitable for the improvement of cooled llama sperm.

Embryo presence regulates NODAL/LEFTY2 system in the rat oviduct in vivo.

To gain further insight in the mechanisms of the embryo-maternal dialog in the oviduct, expression of members of the transforming growth factor-ß superfamily, NODAL, its inhibitor, LEFTY2, and their coreceptor, CFC1, were studied in the oviduct of 3-day post copula (3 dpc) females with and without embryos (E and NE), pseudopregnant rats (SP3), and in 3-day embryos. Nodal transcripts in SP3 oviducts showed a steady-state relative abundance when compared with proestrus stage and the 3 dpc. In contrast, Lefty2 and Cfc1 relative abundance levels in proestrus and 3 dpc were higher. When comparing E with NE oviducts, Nodal and Lefty2 expression levels decreased, while Cfc1 expression increased in the presence of embryos. Nodal messenger RNA (mRNA) was observed in the embryo, but Lefty2 and Cfc1 transcripts were not found. In addition, an increase in Lefty2 expression coincided with increased levels of matrix metalloproteinases 9 mRNA and protein in the oviduct and in the oviductal fluid, respectively. These observations have shed new light on the relevance of the NODAL/LEFTY2 pathway in the oviduct during early embryo development and the role of the embryo in modulating this pathway.

Expression of ß-NGF and high-affinity NGF receptor (TrKA) in llama (Lama glama) male reproductive tract and spermatozoa.

ß-Nerve growth factor (ß-NGF) is a seminal plasma element, responsible for inducing ovulation in camelids. The main organ of ß-NGF production remains nondescript. The aims of this study were to (a) characterize gene expression and protein localization of ß-NGF and its main receptor tyrosine kinase receptor A (TrKA) in the llama male reproductive tract, and (b) determine whether the seminal ß-NGF interacts with ejaculated sperm by localizing ß-NGF and TrKA in epididymal, ejaculated, and acrosome-reacted (AR) sperms and, additionally, by identifying ß-NGF presence in sperm-adsorbed proteins (SAP). Both ß-NGF and TrkA transcripts are widely expressed along the male reproductive tract, with a higher expression level of ß-NGF at prostate (p < 0.05). ß-NGF immunolabeling was only positive for prostate, whereas TrKA label was present in epithelial and muscular cells of testis, prostate, bulbourethral glands, and epididymis. Using an immunofluorescent technique, ß-NGF was colocalized with TrKA in the middle piece of ejaculated and AR sperm. However, only TrKA was observed in epididymal sperm indicating that ß-NGF could have a seminal origin. This was also confirmed by the identification of four ß-NGF isoforms in SAP. This study extends the knowledge about the participation of ß-NGF/TrkA in llama reproduction, providing evidence that may have roles in the regulation of sperm physiology.

Changes in mucins and matrix metalloproteases in the endometrium of early pregnant alpacas (Vicugna pacos).

South American Camelids (SAC) have unique reproductive features, one of which is that 98% of the pregnancies develop in the left uterine horn. Furthermore, early pregnancy is an uncharacterized process in these species, especially in regard to the ultrastructural, biochemical and genetic changes that the uterine epithelial surface undergoes to allow embryo implantation. The present study describes the uterine horn luminal surface and the characteristics of the mucinous glycocalyx in non-pregnant and early pregnant (15 days) female alpacas. In addition, the relative abundance of Mucin 1 and 16 genes (MUC1 and MUC16) was determined, as well as the relative mRNA abundance of matrix metalloproteinases (MMPs) that could be involved in MUC shedding during early pregnancy. Noticeable changes were detected in the uterine luminal epithelium and glycocalyx of pregnant alpacas in comparison to non-pregnant ones, as well as presence of MUCs and MMPs in the endometrial environment. The decrease in glycocalyx staining and in the relative abundance of MUC 1 and MUC 16 transcripts in pregnant females would allow embryo attachment to the luminal epithelium and its subsequent implantation, as has been described in other mammals. These results suggest a crucial role of MUC1 and MUC16 and a possible role of MMPs in successful embryo implantation and survival in alpacas.

Mating induces production of MMP2 in the llama oviduct: Analysis of MMP2 effect on semen.

Ovulation of South American Camelids is induced by mating. After copulation, sperm are stored into the oviduct to be released near ovulation time. To study whether copulation induces matrix metalloproteinase-2 (MMP2) secretion in the oviduct, the occurrence of MMP2 in oviductal tissue and oviductal fluid (OF) from 24 h post-mated was compared with non-mated llama females. There was an incremental increase of MMP2 in the oviductal epithelial cells, and MMP2 activity in OF after copulation. Additionally, MMP2 activator (MMP14), inducer (EMMPRIN) and inhibitor (TIMP2) were present in the oviductal epithelial cells of both non-mated and post-mated females. A post-mating segment-specific regulation occurred because relative abundance of TIMP2 mRNA was greater in the utero tubal-junction which was accompanied with a reduced amount of MMP14 in the ampulla in comparison with the non-mated females. To examine the effect of MMP2 on semen liquefaction and sperm physiology, the effects of addition of recombinant human MMP2 was evaluated. The MMP2 had no effect on semen thread formation and seminal plasma protein profile. Sperm viability and plasma membrane function were not influenced by the enzyme treatment either. In summary, in llamas the oviductal microenvironment changes in response to stimuli induced by copulation, increasing the production and secretion of MMP2.

Segment­ specific expression of MMP/TIMP in the oviduct of llama (Lama glama) and gelatinolytic activity in the oviductal fluid / Expresión segmento específica de MMP/TIMP en el oviducto de llama (Lama glama) y actividad gelatinolítica en el fluido oviductal

Oviductal molecules have the potential to improve the reproductive biotechnologies. In camelids, knowledge and assessment of the oviductal environment are necessary to successfully develop species-specific reproductive technologies, especially because of the camelids reproductive particularities. Among the oviductal factors, the matrix metalloproteinases/tissue inhibitor of matrix metalloproteinases system (MMPs/TIMPs) should be investigated more thoroughly due to their participation in reproductive processes. Consequently, the current study assayed gene and protein expression of MMPs throughout the llama oviduct. MMPs zymogen and active forms in the oviductal fluid were also characterized. MMP2 and MMP9 transcripts were detected in ampulla, isthmus, utero-tubal junction and papilla, being MMP2 and MMP9 2.15 and 1.10 folds higher in papilla than in ampulla, respectively. In addition, differences in immunolocalization of MMP2 and MMP9 between the epithelial mucosa layers of the oviductal segments were observed. The presence of MMPs in the epithelium suggests their secretion into the oviductal lumen. Coincidently, bands of 62 and 94 kDa, corresponding to MMP2 and MMP9 were detected by zymography in the oviductal fluid. Treatment with an exogenous activator (APMA) suggests that they are present as proMMPs. TIMP2 and TIMP1, the specific inhibitors of MMP2 and MMP9, respectively, were expressed in each oviductal segment, indicating a well-regulated control of MMP proteolytic activity in the oviduct. These findings prove that the llama oviduct produces and secretes MMPs into the oviductal lumen, suggesting that these enzymes may have an unknown role in the preparation of the oviductal environment for gametes, fertilization and early embryo development in camelids.

Las moléculas oviductales tienen el potencial para mejorar las biotecnologías reproductivas. En los camélidos, debido a sus peculiares características reproductivas, el conocimiento del ambiente oviductal constituye una herramienta útil para el desarrollo de tecnologías reproductivas específicas para estas especies. Entre los factores oviductales de interés se encuentran las metaloproteasas de matriz (MMPs) y sus inhibidores específicos (TIMPs), los cuales han sido involucrados en diferentes procesos reproductivos. Por estas razones, en este trabajo se caracterizó la expresión génica y proteica de MMP2 y MMP9 en el oviducto de llama. Además, se analizó la presencia de las formas activas e inactivas (zimógenos) de estas enzimas en el fluido oviductal. Se observó que todos los segmentos oviductales, ámpula, istmo, unión útero-tubal y papila, expresan MMP2 y MMP9, siendo los niveles de expresión de MMP2 y MMP9 más elevados en papila respecto a ámpula; 2,15 y 1,10 veces respectivamente. Asimismo, se observaron diferencias en la distribución de las MMPs a nivel de la mucosa entre los segmentos oviductales. Consecuentemente, bandas con actividad gelatinolítica de 62 y 94 kDa, se detectaron en el fluido oviductal, las cuales corresponderían a las formas inactivas de la MMP2 y la MMP9, respectivamente. Los inhibidores específicos de MMP2 y MMP9; TIMP2 y TIMP1, también se detectaron en los segmentos oviductales, indicando su probable participación en la regulación de la actividad proteolítica de las MMPs en el oviducto de llama. En conjunto, los datos de este trabajo demuestran que el oviducto de la llama produce y secreta MMPs al lumen oviductal; sugiriendo que estas enzimas pueden participar en la preparación del ambiente oviductal para la recepción de los gametos, la fecundación y el desarrollo embrionario temprano en camélidos.

SL15: A seminal plasma-derived lectin from the sperm of llama (Lama glama).

The oviductal sperm reservoir of South American camelids is formed when sperm bind to N-acetylgalactosamine (GalNAc) on the surface of oviductal epithelium. The aim of this study was to characterize the GalNAc-binding proteins on llama sperm, and to establish their origin. Sperm-adsorbed proteins were extracted with 0.5 M KCl in Hepes-balanced salts. Sperm-adsorbed and seminal plasma proteins were then subjected to ligand blotting for their GalNAc affinity, and the labeled bands were identified by mass spectrometry. Three proteins were identified in seminal plasma versus only one in the sperm-adsorbed population; SL15, a seminal lectin, was common to both. SL15 is a homologue of Zymogen granule protein 16, homolog B-like, which belongs to the Jacalin-related lectin family. This lectin is likely presented to sperm via seminal plasma since epididymal sperm are not capable of binding GalNAc, whereas ejaculated sperm does, and its transcript was enriched predominantly in the prostate and bulbourethral glands. This is the first report of a seminal lectin in South American camelids that originates in the male reproductive tract, and is probably involved in sperm reservoir formation.

Morphometric Study of the Mare Oviductal Mucosa at Different Reproductive Stages.

The objectives of this work were to describe some morphometric characteristics and to establish quantitative parameters of different regions of the equine oviductal mucosa from the isthmus, ampullary-isthmic junction (AIJ), and ampulla. Twenty-one mixed-bred mares were used for this study. Mares were selected in the following reproductive phases: anestrus, estrus, and diestrus. The left oviducts were examined with light microscopy, and rights ones were studied through the intraoviductal molds. The isthmus showed the smallest luminal area, mucosal area, epithelial perimeter, and luminal diameter. On the molds surfaces, some grooves extended as longitudinal canals, reducing their depth as they approached to the AIJ. Several small height projections, some obliquely positioned towards utero-tubal junction, were observed in all reproductive phases. These formations may represent pockets or cul-de-sacs in the basal areas of the epithelial folds. The AIJ mucosa gradually changed from the smooth isthmic region toward highly folded ampulla. The number and complexity of epithelial folds showed moderate increase in the same way that many of the morphometric parameters. Multiple curves were observed on the molds of the AIJ, creating a zigzag path in the oviductal lumen. In the ampulla, the high branched epithelial folds occupied most of the lumen, leaving a small luminal area free. A linear relationship between epithelial perimeter and mucosal area was found. The presence of glandular-like structures was observed in all the reproductive stages studied. The equine endosalpinx reveals a highly complex tridimensional arrangement where each region shows very particular and specific designs.

Expression and localization of nodal in bovine oviduct and uterus during different functional stages of oestrus cycle and pregnancy.

Members of TGF-ß superfamily play a major role in the endometrial changes involved in the establishment and maintenance of pregnancy. Their deregulated expression and action could lead to absolute or partial failure of embryo implantation. Nonetheless, the precise function and mechanism of many of these cytokines remain unclear. Nodal, a transforming growth factor beta (TGF-ß) superfamily member, was characterized in the human and rodent uterus and implicated in the tissue remodeling events during menstruation and embryo implantation. In order to study its possible role in the cattle reproductive process, we have analyzed Nodal expression pattern and localization in the oviduct and uterine horn during the oestrus cycle and early pregnancy (day 20). Nodal was detected both in oviduct and uterus during either the oestrus cycle or pregnancy; however, it shows a differential expression profile in the uterine horn at dioestrus and pregnancy, decreasing 1.5 and 1.4 folds in comparison with oestrus. Nodal immunostaining intensity was observed in stromal and in epithelial cells of the surface and the glandular epithelium. The staining pattern correlates with the RT-qPCR expression profile. This work is the first to evidence the presence of Nodal in the bovine reproductive tract; our data suggest that Nodal is a novel cytokine that would be involved in the remodelling occurring in the endometrium of cattle during the oestrus cycle and in the embryo implantation. The identification of new molecules that participate in endometrium cycling and/or pregnancy may be useful for predicting the ability of the uterine tissue to establish and maintain pregnancy or for detecting the infertility processes. These results highlight Nodal as a possible novel marker of the fertility process, nevertheless further studies should be done to determine its role in the reproductive system.

LEFTY2 expression and localization in rat oviduct during early pregnancy.

In mammals, fertilization and preimplantation embryo development occurs in the oviduct. Cross-talk between the developing embryos and the maternal reproductive tract has been described in such a way as to show that the embryos modulate the physiology and gene expression of the oviduct. Different studies have indicated that transforming growth factor beta (TGF-ß) can modulate the oviductal microenvironment and act as an autocrine/paracrine factor on embryo development. LEFTY2, a novel member of the TGF-ß superfamily is involved in the negative regulation of other cytokines in this family such as nodal, activin, BMPs, TGF-ß1 and Vg1. In previous studies, we have reported that LEFTY2 is differentially expressed in the rat oviduct during pregnancy. In this study, we describe the temporal pattern of LEFTY2 in pregnant and non-pregnant rat oviduct by western blotting, which showed higher levels of LEFTY2 on day 4 of pregnancy, a time at which the embryos are ending their journey along the oviduct. The cellular location of LEFTY2 was assessed by immunohistochemistry, which showed immunolabelling in the cytoplasm and at the apical surface of the oviductal epithelial cells. The oviductal fluid also presented a 26 kDa band, which corresponds to the biologically active form of this protein, at the preimplantation period of pregnancy, indicating LEFTY2 secretion to the lumen. As LEFTY2 is expressed at a high level just before the embryos pass to the uterus, its biological effect might be relevant and significant for the preimplantation stage of embryo development in the oviduct. The fact that embryos do not express LEFTY2 at this stage of development supports this hypothesis.

Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.