Different expression of Defensin-B gene in the endometrium of mares of different age during the breeding season.

BACKGROUND: Despite being one of the major causes of infertility in mares, the mechanisms responsible for equine endometrosis are still unclear and controversial. In the last few years, many investigations focused on local immune response modulation. Since it is generally accepted that endometrial fibrosis increases with age, we hypothesize that older mares could show altered local immune modulation, initiating a pro-inflammatory and tissue remodeling cascade of events that could lead to endometrosis. The aim of this study, indeed, is to evaluate and describe the local gene expression of genes involved in acute inflammatory response and fibrosis (COL1A1, COL3A1, TNFA, MMP9, IL6, TGFB1 and TGFBR1), together with others associated to immune modulation (DEFB4B, IDO1 and FOXP3), in uterine specimens from mares of different age. RESULTS: Twenty-five Standardbred mares were involved in the study with age ranging from 7 to 19 years (mean 10.40 ± 4.42). They were divided by age into two groups: G1 (n = 15, less than 10 years old) and G2 (N = 10, greater than 11 years old). Specimens from the uterus' right horn-body junction were collected and processed for histology evaluation and RT-qPCR assay.Gene expression of DEFB4B, MMP9 and TNFA was higher in younger mares, suggesting a balance in immune modulation and tissue remodeling. Interleukin-6 and COL3A1 gene expressions were greater in older animals, probably indicating inflammatory pathways activation and fibrosis increase. Although no differences in fibrosis and inflammation distribution could be found with histological examination among G1 and G2, our results suggest a possible involvement of DEF4BB in regulating the local immune response in younger mare's uterus (G1); age may contribute to the dis-regulation of DEFB4B transcription and, indirectly, influence the extracellular matrix homeostasis. Transcription of IDO1 and FOXP3 genes, instead, does not seem to be age related, or to be involved in local immune-response and tissue remodeling functions. CONCLUSIONS: Further investigations are needed in order to clarify the interactions between the expression of DEFB4B, IL6, TNFA, COL3A1 and MMP9 and other local signals of immune-modulation and tissue remodeling, in mares in a prospective study design.

Transcriptome analysis of bovine granulosa cells of preovulatory follicles harvested 30, 60, 90, and 120 days postpartum.

The nutritional requirements of high-producing dairy cows increase rapidly in the weeks postpartum due to elevated milk production, and most cows enter a state of negative energy balance (NEB). Cows in this condition may preferentially divert nutrients away from reproduction, thereby experiencing a period of anovulatory anestrus, delayed ovulation of large follicles, and impaired fertility. To better understand the changes occurring in these large follicles as a function of time postpartum, granulosa cells from preovulatory follicles were collected at different times after calving: 30 days, 60 days, 90 days, and 120 days from a minimum of six animals at each time point. Three subjects for each period were selected for the analysis of the transcriptome, which was performed using a global bovine oligoarray microarray to identify differences in gene expression and cellular functions that occur in the follicular microenvironment during the progressive recovery from NEB in dairy cows. The time point 120 days was chosen as a reference period, as cows are then fully recovered from NEB. Data clustering indicated that the most different period from 120 days was 60 days with several 100 genes deregulated at that time. The principal cellular functions affected were cell proliferation, which was reduced, and deregulation of steroidogenesis, indicating immaturity of the follicles from the 60-day period relative to 120 days. One of the most interesting downregulated pathways was p38 mitogen-activated protein kinase, which is sensitive to the amount of retinoic acid or vitamin A, suggesting a possible treatment to improve postpartum fertility.

Ultrasonographic features of the mule embryo, fetus and fetal-placental unit.

The aim of this study was to establish baseline ultrasound data concerning the mule conceptus during gestation. Ten multiparous Trotter mares were artificially inseminated with chilled semen from an Amiatino jack donkey. Daily transrectal ultrasonography was carried out from the day of ovulation until Day 50 of gestation to determine the following: first detection of the embryonic vesicle (EV), mobility phase, EV diameter, day of EV fixation, changes in EV shape, date of yolk sac regression and embryo crown-rump length. Monthly ultrasonic assessments from Day 50 of gestation to term were carried out. These assessments included an evaluation of fetal well-being and the growth of the mule conceptus, which were monitored using the following variables: cardiac activity, fetal activity and presentation, fetal fluid echogenicity, combined thickness of the utero-placenta unit and fetal orbital and aortic diameter. Mule EV first detection was observed earlier (37% at Day 8) than that observed in the equine pregnancy. EV diameter at first detection was 4.6 ± 1.1 mm. At Day 10, 75% of EVs were detected. EV fixation occurred on Day 17.1 ± 1.1, with a mean EV diameter of 2.5 ± 0.2 cm. EV growth rate was 4.04 mm/day from Days 11 to 16, 0.4 mm/day from Days 16 to 28 and 1.78 mm/day from Days 28 to 45 of pregnancy. The embryo proper was first detected on Day 19.9 ± 1.9 (average length 2.4 ± 1.4 mm), and the embryonic heartbeat was first detected on Day 24 ± 2.4. The fetal carotid pulse was observed at six months of gestation and provided a good means by which to estimate fetal cardiac activity in advanced gestation. The fetal heart rate was recorded from Month 2 of gestation to term. The mean ± SD of the combined uteroplacental thickness was assessed at the cervical-placental junction and at the ventral abdomen in mares between Months 2 and 5 until term, respectively. An abnormal fetal-placental unit and fetal inactivity was observed in association with abortion. Mule-conceptus biometric measurements correlated significantly with the gestational age, and these data were used to predict an unusually large mule fetus, which might result in dystocia. In conclusion, we can assume that early diagnosis of pregnancy failure and assessment of fetal biophysical profile and growth charts could improve the chances of gestation completion in mule-pregnant mares. The early detection of mares at risk for an abnormal pregnancy or delivery may increase the success of prompt treatments, therefore preventing costly emergency procedures and allowing proper obstetrical and neonatal assistance.

Improving calving management to further enhance reproductive performance in dairy cattle.

The aim of this study was to develop a system for the monitoring of calving to both reduce perinatal mortality and improve dairy cow fertility by preventing the majority of post-partum reproductive pathologies. Eighty dairy cows were assigned to the protocol of calving monitoring using GSM (Global System for Mobile Communications) technology. The application of GSM technology and the proper management of calving facilities comprise reliable approaches for calving assistance and improvements in reproductive efficiency and neonatal viability. Based on the results of this study, we advocate the use of GSM technology on large farms for intensive milk production.

Phospholipid hydroperoxide glutathione peroxidase in bull spermatozoa provides a unique marker in the quest for semen quality analysis.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoperoxidase accounting for most of the selenium content in mammalian testis, which has been found to be linked to fertility in humans. In this study, we addressed the issue whether PHGPx content in spermatozoa could be a predictive index of fertilization capacity for sire selection in bulls. Measurement of PHGPx in spermatozoa of 92 yearling bulls of three different Italian breeds (Chianina, Romagnola, and Marchigiana) revealed the presence of two quite well separated populations. A PHGPx activity of 130 mU/mg separated the high-PHGPx group (H-PHGPx, n=73) from the low-PHGPx group (L-PHGPx, n=19). Forward motility was markedly higher in the H-PHGPx group, which also contained a lower percentage of detached heads, abnormal midpiece, and proximal droplets. On the other hand, differently from the human studies, no correlation was observed between PHGPx activity and number of spermatozoa in the ejaculate. Apart from sperm count, which typically differed among breeds, and number of detached heads in the L-PHGPx group, which correlated with higher sperm count, no other significant difference in seminal parameters among breeds was apparent. The assay for sperm PHGPx activity therefore emerges as a unique tool to evaluate semen quality for sire selection.

Grape polyphenols affect mRNA expression of PGHS-2, TIS11b and FOXO3 in endometrium of heifers under ACTH-induced stress.

Stress activates the hypothalamo-pituitary-adrenal axis leading to enhanced glucocorticoid secretion and concurrently disrupts ovarian cycle. Plant polyphenols are known to posses antioxidant and anti-inflammatory proprieties. This could be of interest for ovarian cycle when stressing conditions lead to progesterone enhancement and hamper normal reproduction activity. The present study examined whether ovarian follicular development and progesterone secretory pattern are affected by exogenous ACTH administration in heifers. Moreover, the effect of grape polyphenols in endometrium of heifers, under adrenocorticotropic hormone challenge, is evaluated in terms of transcriptional patterns of genes related to inflammation, oxidative stress and endometrial functions. At day 14 of synchronized estrous cycle, Holstein Friesian heifers received injections of either saline (CTR group) or adrenocorticotropic hormone (ACT group) agonist every 12 h for 7 days. Another group (POL group) of animals received the same treatment plus an oral supplementation of 15 g/day of grape skin extract. Cortisol and progesterone were analysed in the blood samples collected at days 0, 3, 6, 9, 12, 14, 17, 21, 24 of the estrous cycle. Endometrial biopsies were collected at diestrus (day 18) and at estrus and a panel of gene expressions were quantified by real-time PCR. ACTH administration increased both cortisol (P<0.001) and progesterone concentrations (P<0.01) compared to CTR group. PGHS-2 was significantly (P<0.01) up-regulated in the POL group compared to ACT and CTR groups at diestrus and at estrus. FOXO3 and TIS11b were down-regulated in the CTR group compared to ACT and POL groups. The PGHS-2, SOD2 (P<0.05), FOXO3 and TIS11b (P<0.10) genes were down-regulated at estrus in all groups compared to diestrus. An interesting role of polyphenols in modulating the expression levels of PGHS-2 in endometrial tissue and on the activation of TIS11b and SOD2 through c-AMP-dependent signalling was suggested.

Decrease in glutathione (GSH) content in bovine sperm after cryopreservation: comparison between two extenders.

Growing evidence suggests that among the causes which deteriorate qualitative and functional characteristics of semen after freezing and thawing, there are those linked to oxidative stress and decrease detoxification of antioxidants present in the cells and seminal plasma. Overall, cryopreservation reduces the spermatozoal GSH content. In the present study we assessed the motility, capacitation status, ability to undergo acrosome reaction and levels of GSH in bovine spermatozoa after freezing and thawing, and compared the effects of the egg yolk tris-citrate and a commercial extenders (Bioxcell). Six semen samples were collected from each of five bulls and frozen and thawed in the two extenders. The level of total GSH of bovine spermatozoa detected in the raw semen ranged from 246.5 to 776.2 pmoles/mg of protein. The Bioxcell extender was superior in preserving GSH content (647.2+/-300.1 versus 223.6+/-154.0 pmoles/mL; P<0.05), motility levels (44.3+/-4.8 versus 41.8+/-4.0%; P<0.05), non-capacitated state (68.3+/-13.6 versus 61.3+/-17%; P<0.05) and a lower proportion of acrosome reacted spermatozoa post thaw (3.8+/-2.1 versus 6.3+/-3.1%; P<0.001) compared to egg yolk tris-citrate extender. These findings could be partially ascribed to the high GSH level contained in the commercial extender which seem able to alleviate oxidative damages to spermatozoa surviving freezing thawing procedures. Further researches are required in order to validate in vivo these results.

Breeding soundness examination of Chianina, Marchigiana, and Romagnola yearling bulls in performance tests over a 10-year period.

The objectives of the present study were (i) to establish the mean value of scrotal circumference (SC), sperm motility, concentration and morphology at 13+/-1 months of age for Chianina, Marchigiana, and Romagnola breeds and (ii) to assign Italian beef bulls at the end of a growth performance test to a potential breeder category by applying the guidelines of the Society for Theriogenology in 1993 (SFT93). Of 1,315 bulls, 869 were not given the breeding soundness examination for the following reasons: not passing the growth performance test (n=445), no training for semen collection (n=404), and presence of genital abnormalities (n=20). Testicular length and diameter and SC exhibited a logarithmic trend over time, with an R(2) value of 0.963, 0.979, and 0.978 (P<0.001), respectively. The SC of Romagnola (33.82+/-2.47 cm) was higher than those of Chianina (33.28+/-2.65 cm, P<0.001) and Marchigiana (33.05+/-2.20 cm, P<0.001). Sperm concentration in Romagnola (875.89+/-416.13x10(6)cells/mL) was higher than those in Chianina (751.63+/-444.45 x 10(6)cells/mL, P<0.05) and Marchigiana (862.57+/-421.87 x 10(6) cells/mL). Progressive sperm motility was 61.30+/-11.24%, 62.18+/-11.17%, and 58.48+/-14.40% in Romagnola, Marchigiana, and Chianina, respectively. Total spermatozoal abnormalities were higher in Chianina (23.35+/-15.41%). Sperm concentration was positively related to testicular length (P<0.01), diameter (P<0.001), and SC (P<0.001). Satisfactory breeders presented high sperm motility compared with deferred and unsatisfactory ones, whereas unsatisfactory breeders had a higher number of abnormal spermatozoa. By applying the SFT93 guidelines, we showed that 74.72%, 78.01%, and 80.16% of Chianina, Marchigiana, and Romagnola bulls, respectively, have been classified as satisfactory potential breeders.

Mammary cell turnover in lactating ewes is modulated by changes of energy fuels.

The study of cell turnover of mammary gland helps to gain understanding of the subtle modulation that largely determines milk production. To evaluate the effect of diet composition on cell turnover of mammary gland, pluriparous sheep were allotted to three experimental groups and fed from day 33 after lambing a control diet (BD), a high starch (HS) diet or a high fat (HF) diet. Biopsies of mammary gland tissue were collected at 50 days after lambing for in situ detection of cell death and RT-PCR analysis of bax, bcl-2, caspase-3 and GST expressions. Both apoptotic and proliferation indexes were significantly higher (P<0.05) in the BD group compared to HS and HF groups. The relative expressions of GST were significantly greater (P<0.05) in HF group compared to the BD and HS groups. There was a significant increase in the ratio of bcl-2 to bax mRNA in the HS and HF groups. Availability of energy substrates for mammary gland can interfere with the cell fate, modulating genes involved in the control of oxidative stress which, in turn, can indirectly regulate cell apoptosis and proliferation.

The effect of Mycoplasma mycoides ssp. mycoides LC of bovine origin on in vitro fertilizing ability of bull spermatozoa and embryo development.

Several Mycoplasma species may adversely affect bovine spermatozoa viability and embryo development. Mycoplasma mycoides ssp. mycoides large-colony (LC) has been isolated from naturally aborted bovine fetuses and from bull semen. The objective of this study was to evaluate whether M. mycoides ssp. mycoides LC contaminated bovine ejaculates could (i) impair in vitro fertilizing ability of bull spermatozoa, (ii) impair embryo development, and (iii) evaluate potential spread by reproductive technologies. In the present study, spermatozoa of 10 fertile bulls were contaminated with M. mycoides ssp. mycoides LC, at a final concentration of 1.5 million CFU/ml and incubated for 60 min before evaluating spermatozoa motility and acrosome reaction inducibility with calcium ionophore. In addition, in vitro contaminated semen of a bull previously shown to have a good in vitro fertilizing ability, was used in an IVF procedure. Embryo development stage on Day-7 of culture was evaluated. Spermatozoa and embryos at morula and blastocyst stages were routinely processed for transmission electron microscopy observation. Both mean total and progressive motility decreased (P < 0.01 ) upon spermatozoa incubation with Mycoplasma. One-hour incubation with calcium ionophore increased the percentage of acrosome-reacted spermatozoa, although Mycoplasma contamination reduced calcium ionophore treatment efficacy (P < 0.05). Ultrastructurally, Mycoplasma microorganisms appeared as moderately electron-dense sphere-shaped particles, adhering to cell membranes. Sperm mid-piece sections showed numeric aberrations of the central singlets such as nine + zero or nine + one of the axonemal complex. Further morphological abnormalities included partial or total absence of dinein arms and radial fibers, with lack of the bridge and the central ring in 35.00 +/- 4.20% of contaminated cells, whereas these abnormalities were not observed in uninfected ones. The IVF trials showed that two-four cell blocks were higher (P < 0.05) in the infected group. Ultrastructure of Day-7 contaminated embryos showed Mycoplasma particles adhering and infiltrating the outer layer of the zona pellucida. Our investigations suggest that M. mycoides ssp. mycoides LC contaminating the bovine ejaculate induced adverse effects on in vitro spermatozoa-fertilizing ability and embryonic development. Some satisfactory quality transferable embryos could be produced in contaminated IVF systems. This could imply a potential transmission of this microorganism through reproductive technologies.

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions.

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.

Localization of the lectin reactive sites in adult and prepubertal horse testes.

The testes of prepubertal and adult horses were investigated using 10 horseradish peroxidase conjugated lectins combined with sialidase digestion and potassium hydroxide treatment, to localise the oligosaccharide sequences of glycoconjugates during spermatid maturation. In adult animals, the lectins showed a variable affinity for spermatids and Sertoli cell apical extensions. Soybean agglutinin (SBA), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA-I) and wheat germ agglutinin (WGA) bound to the acrosomal structures of spermatids, whereas Griffonia simplicifolia agglutinin (GSA-II) labelled these structures only during Golgi and cap phases. These results suggested that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides and that these carbohydrate chains undergo modifications during spermiogenesis. Sialic acid residues were not detected throughout the acrosomal development. The lectin binding pattern of Sertoli cells was very similar to that of acrosome of spermatids during the maturation phase. In sexually immature horses, only the degenerated germinal cells and the Leydig cells showed reactivity towards lectins. The first cells reacted with SBA and Dolichos biflorus agglutinin (DBA), the latter with SBA, PNA, WGA, GSA-II, Canavalia ensiformis agglutinin (ConA), Lens culinaris agglutinin (LCA) and also with DBA after sialidase digestion.

Effect of alpha-tocopherol deprivation on the involution of mammary gland in sheep.

The objective of this study was to investigate the effect of alpha-tocopherol deprivation on mammary gland involution and apoptosis in sheep. Two groups of four single lamb ewes were used. The control group received 100 mg/d of RRR-alpha-tocopherol supplementation and the experimental group received no vitamin E supplementation. After 3 mo of suckling, ewes were dried off, and blood samples from the jugular vein and tissue biopsies from the mammary gland were collected at d 1, 3, 5, and 8 after dry-off. The experimental group had lower plasma concentrations of alpha-tocopherol (1.8 vs. 4.2 micromol/L), lower glutathione peroxidase activity in erythrocytes, and higher concentration of malondialdehyde in plasma than the control group. Immunohistochemical analysis of tissue samples resulted in marked differences of bcl-2 and bax protein expressions during involution and between groups. The bax expression was decreased by alpha-tocopherol deprivation at 1, 3, and 5 d, but not at 8 d, while the bcl-2 score was higher only at 8 d (1.5 vs. 0.0 for experimental and control groups, respectively). As a result, the bcl-2 to bax ratios were increased for the experimental group at 1 and 8 d. During involution, apoptotic counts increased (from 0.12 to 4.06%), but no effects were detected in relation to bcl-2 to bax ratio and alpha-tocopherol. These results indicate that alpha-tocopherol can control bcl-2 expression, but not apoptosis in cells of the mammary gland during involution.

Mycoplasma mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis-affected bulls.

In this study, by using the polymerase chain reaction (PCR) diagnosis for the detection and identification of Mycoplasma, we investigated mycoplasmas contaminating the semen of yearling bulls affected by seminal vesiculitis. The bulls presented neither subclinical nor clinical contagious bovine pleuropneumonia signs and the complement fixation test for specific antibodies was negative. Furthermore, we have investigated mycoplasmas isolated from semen of healthy breeding bulls of several breeds and origins, which routinely underwent breeding soundness examinations and presented no clinical signs of seminal vesiculitis. We were able to demonstrate mycoplasma infection in all tested samples by i) growth on mycoplasma-specific media and ii) a PCR-based method using a mycoplasma-specific MGSO/GPO1 primer set to amplify the 16S fragment rDNA. In addition, the identification of Mycoplasma species was made by PCR using the MSC1/MSC2 primer set that specifically amplifies M. mycoides subsp. mycoides SC or the MM450/MM451 primer set followed by AsnI digestion analysis in order to identify M. mycoides subsp. mycoides LC. The data presented herein clearly show that M. mycoides subsp. mycoides SC infection was associated with seminal vesiculitis while M. mycoides subsp. mycoides LC was only found in bull semen from healthy control animals. Our findings confirm that the M. mycoides subsp. mycoides SC is shed in the sperm making the ejaculate a valuable biological sample for the isolation of these bacteria from serologically negative animals. Although the pathogenic role of M. bovigenitalium in bull seminal vesiculitis has been established, our clinical findings, semen characteristics, microbiological and bacterial genomic analysis strongly suggest that M. mycoides subsp. mycoides SC may contribute to induce vesicular adenitis in the bull.

Glycoconjugates in small antral ovarian follicles of the river buffalo (Bubalus bubalis L.).

Types and distribution patterns of glycoconjugates in antral ovarian follicles were investigated in the buffalo, using periodic-acid Schiff (PAS), high iron diamine (HID), low ion diamine (LID) and lectin histochemical staining methods. HID and LID staining procedures were preceded in some cases by digestion with testicular hyaluronidase, Streptomyces hyaluronidase, chondroitinase ABC and heparitinase (heparinase III). Lectin staining was performed with the use of 12 horseradish peroxidase (HRP) lectin conjugates. Some lectin staining procedures were preceded by neuraminidase digestion and saponification. Large amounts of isomeric chondroitin sulphates and a minor quantity of heparan sulphate and hyaluronic acid and/or chondroitin were found in follicular fluid. Lectin staining of buffalo follicular fluid revealed glycoconjugates with different glucidic determinants such as beta-N-acetylgalactosamine, beta-galactose-(1-3)-N-acetylgalactosamine, beta-galactose-(1-4)-N-acetylglucosamine, N-acetylglucosamine, alpha-fucose and alpha-glucose/alpha-mannose, and sialic acid residues. Glycosaminoglycans were absent in the zona pellucida of oocytes in small antral follicles. Acidic glycoconjugates in the zona pellucida were caused by sulphated groups and sialic acid residues. Our data show few internal glucidic residues, such as N-acetylglucosamine in the buffalo zona pellucida but many subterminal beta-N-acetylgalactosamine, alpha- and beta-galactose determinants masked by sialic acids. These findings demonstrate that buffalo follicular fluid has a very heterogeneous composition that is similar to that found in small and large bovine follicles. No differences in composition of the follicular fluid were observed in the follicles examined.

Lectin-staining pattern in extratesticular rete testis and ductuli efferentes of prepubertal and adult horses.

This study was undertaken to determine the lectin affinity of the extratesticular rete testis and ductuli efferentes epithelial cells in adult and prepubertal horses, using ten different lectin horseradish peroxidase conjugates: Con-A, LCA, WGA, GSA-II, SBA, PNA, RCA-I, DBA, UEA-I, and LTA. In some cases, treatments with sialidase and KOH preceded the lectin staining. In sexually mature and immature horses the results showed the presence of different kinds of sialoglycoconjugates with the terminal sialic acid linked to D-GalNAc and beta-D-Gal residues in the rete testis. In the apical surface and cytoplasm of epithelial cells lining the ductuli efferentes of the adult horse, glycoconjugates with alpha-D-Man and/or alpha-D-Glc, GlcNAc, D-GalNac and beta-D-Gal residues were evidenced, whereas in the prepubertal horse only the apical surface of the ductuli efferentes epithelial cells resulted reactive toward some lectins. The differences observed in the presence of glycoconjugates between adult and prepubertal horse ductuli efferentes, suggest a hormonal control of the function of these tracts of the post-testicular ducts.

Detection of glycoconjugates in the ductus epididymis of the prepubertal and adult horse by lectin histochemistry.

This paper describes an approach for studying the structure of glycoconjugates found in the principal cells lining the epididymal duct in adult and prepubertal horses, using ten different lectin horseradish conjugates: Con-A, LCA, WGA, GSA-II, SBA, PNA, RCA-I, DBA, UEA-I, and LTA. Saponification and sialidase procedures, followed by lectin binding, were employed to visualize the distribution and to reveal the sequence of sialoglycoconjugates in ductus epididymis. In the adult horse the results demonstrated variations in the content and distribution of glycosidic residues of glycoconjugates in different epididymal regions (caput, corpus, cauda) and vas deferens, suggesting that each epididymal segment has a specific function. In particular, staining of the Golgi-zone in the principal cells lining corpus epididymis was interpreted as evidence for synthesis and secretion of glycoconjugates and sialoglycoconjugates. In the prepubertal horse, only the glycocalyx of the epithelial cells lining the epididymal duct showed reactivity toward the different lectins used, suggesting hormonal regulation of the epididymis activity. Additional, the heterogeneity of the lectin staining pattern of the adult horse epididymis reported in this investigation also suggests the existence of different functional segments along the epididymal duct.

Characterization of the complex carbohydrates in the zona pellucida of mammalian oocytes using lectin histochemistry.

The objective of this study was to characterize the glycoconjugates present in the zona pellucida of the follicular oocytes in sheep, goats and pigs. The zona pellucida was stained with periodic acid-Schiff, low iron diamine, high iron diamine, and nine different lectin horseradish conjugates: Con-A, SBA, DBA, PNA, RCA-I, GSA-II, WGA, LTA and UEA-I. Staining with DBA, PNA, SBA and RCA-I was performed with and without saponification with KOH and sialidase digestion. The results showed the presence of neutral and acidic glycoconjugates with different terminal sugars and also sialic acid radicals in the zona pellucida of all the animal studied. In particular, the positive staining with WGA, SBA, PNA and RCA-I suggests the presence of oligosaccharides with N-acetyl-D-glucosamine and sialic acid linked to the penultimate beta-N-acetyl-D-galactosamine and to the disaccharide galactosyl-(beta 1-3)-N-acetyl-D-galactosamine. The terminal trisaccharide sialic acid galactosyl-(beta 1-4)-N-acetyl-D-glucosamine was identified only in the zona pellucida of ovine and porcine oocytes. Thus, the zona pellucida exhibited species-specific variations in the content and distribution of lectin-binding patterns that may reflect the species specificity of gamete interaction.