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Corpus luteum growth and endocrine function are closely dependent on the formation of new capillaries. The objectives of this study were to evaluate (i) tissue growth and microvascular development in the equine cyclic luteal structures; (ii) in vitro angiogenic activity of luteal tissues in response to luteotrophic (LH, PGE 2 ) and luteolytic (PGF 2␣ ) hormones and (iii) to relate data to luteal endocrinological function. Our results show that microvascular density was increased in the early and mid luteal phase, followed by a fall in the late luteal phase and a further decrease in the corpus albicans. Hyperplasia of luteal tissue increased until the mid luteal phase and it was followed by tissue regression. Luteal explants were cultured with no hormone added, or with PGF 2␣ , LH, PGE 2 , LH + PGE 2 or LH + PGF 2␣ . Media conditioned by equine luteal tissue from different stages of the luteal phase were able to stimulate mitogenesis of bovine aortic endothelial cells (BAEC), suggesting the presence of angiogenic activity. No difference was observed among luteal structures on their mitogenic capacity, for any treatment used. Nevertheless, Late-CL conditioned-media with PGF 2␣ showed a significant decrease in BAEC proliferation (p < 0.05) and LH + PGF 2␣ a tendency to reduce mitogenesis. Thus, prostaglandin F 2␣ may play a role on vascular regression of the CL during the late luteal phase in the * Corresponding author. Tel.: +351 213652859; fax: +351 213652889. -mail address: gmlfdias@fmv.utl.pt (G. Ferreira-Dias Ferreira-Dias et al. / Domestic Animal Endocrinology 30 (2006) [247][248][249][250][251][252][253][254][255][256][257][258][259] mare. These data suggest that luteal angiogenesis and vascular regression in the mare are coordinated with the development of non-vascular tissue and might be regulated by many different factors. E

Contents We have shown that bacteria induce neutrophil extracellular traps (NETs) in mare endometrium. Besides killing pathogens, NETs may contribute for endometrosis (chronic endometrium fibrosis). Since elastase (ELA) is a NETs component that regulates fibrosis and prostaglandin (PG) output, the aim was to evaluate if inhibition of ELA would affect collagen 1 (COL1) transcription and PGs secretion by endometrium explants, in different estrous cycle phases. Follicular‐FP (n = 8) and mid luteal–MLP (n = 7) phases explants were cultured for 24–48 hr with medium alone (Control), ELA (0.5 μg/ml,1 μg/ml), sivelestat ‐ ELA inhibitor (INH,10 μg/ml), or ELA (0.5 μg/ml,1 μg/ml) + INH (10 μg/ml). COL1 gene transcription was done by qRT‐PCR and PGE2 and PGF2α determination in culture medium by EIA. In FP, at 24 hr, ELA0.5 increased COL1 transcription (p < 0.001) but its inhibition (ELA0.5 + INH10) decreased COL1 transcription (p < 0.01) and PGF2α production (p < 0.05). Also, ELA0.5 + INH10 or ELA1 + INH10 raised PGE2 production (p < 0.01). At 48 hr, ELA1 increased COL1 transcription (p < 0.01) and PGF2α production (p < 0.001), but its inhibition (ELA1 + INH10) decreased these actions (p < 0.01; p < 0.05, respectively). Besides, ELA1 + INH10 incubation increased PGE2 (p < 0.05). PGF2α also augmented with ELA0.5 (p < 0.001), but lowered with ELA0.5 + INH10 (p < 0.01). In MLP, ELA0.5 up‐regulated COL1 transcription (24 hr, p < 0.01; 48 hr, p < 0.001), but ELA0.5 + INH10 decreased it (24 hr, p < 0.05; 48 hr, p < 0.001). At 48 hr, incubation with ELA1 also increased COL1 transcription and PGF2α production (p < 0.05), but PGF2α production decreased with ELA1 + INH10 incubation (p < 0.05). PGE2 production was higher in ELA1 + INH10 incubation (p < 0.05). Therefore, ELA inhibition may reduce the establishment of mare endometrial fibrosis by stimulating the production of anti‐fibrotic PGE2 and inhibiting pro‐fibrotic PGF2α.

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa.

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Neutrophils can originate neutrophil extracellular traps (NETs). Myeloperoxidase (MPO) is a peroxidase found in NETs associated to equine endometrosis and can be inhibited by 4-aminobenzoic acid hydrazide (ABAH). Metallopeptidases (MMPs) participate in extracellular matrix stability and fibrosis development. The objectives of this in vitro work were to investigate, in explants of mare’s endometrium, (i) the ABAH capacity to inhibit MPO-induced collagen type I (COL1) expression; and (ii) the action of MPO and ABAH on the expression and gelatinolytic activity of MMP-2/-9. Explants retrieved from the endometrium of mares in follicular or mid-luteal phases were treated with MPO, ABAH, or their combination, for 24 or 48 h. The qPCR analysis measured the transcription of COL1A2, MMP2, and MMP9. Western blot and zymography were performed to evaluate COL1 protein relative abundance and gelatinolytic activity of MMP-2/-9, respectively. Myeloperoxidase elevated COL1 relative protein abundance at both treatment times in follicular phase (p < 0.05). The capacity of ABAH to inhibit MPO-induced COL1 was detected in follicular phase at 48 h (p < 0.05). The gelatinolytic activity of activated MMP-2 augmented in mid-luteal phase at 24 h after MPO treatment, but it was reduced with MPO+ABAH treatment. The activity of MMP-9 active form augmented in MPO-treated explants. However, this effect was inhibited by ABAH in the follicular phase at 48 h (p < 0.05). By inhibiting the pro-fibrotic effects of MPO, it might be possible to reduce the development of endometrosis. Metallopeptidase-2 might be involved in an acute response to MPO in the mid-luteal phase, while MMP-9 might be implicated in a prolonged exposition to MPO in the follicular phase.

Endometrosis is a chronic degenerative disease of the mare's endometrium characterized by the presence of collagen fibers. Besides several causes, such as recurrent endometritis, endometrosis has been highly related to mare aging. Biopsy of mare endometrium has been used either as a diagnostic tool to assess inflammatory and fibrotic changes, or as a prognosis means of reproductive performance. We have evaluated if the presence of mare endometrial fibrosis was related to fibrosis in the oviduct. Collagen type I (COL1) and collagen type III (COL3) fibers were present in mare endometrium and in all portions of the oviduct (infundibulum, ampulla, isthmus), as detected by specific collagen protein staining with picrosirius red (PSR), and by real-time polymerase chain reaction (qPCR). When the oviduct was considered as a whole, a decrease in COL3 mRNA levels was noted in the oldest mares (>17), when compared to the youngest ones. A correlation between oviduct and endometrium fibrosis was examined. The protein COL3 was mostly found in the youngest mares' endometria and oviducts, in contrast to COL1 detected in older mares with endometrosis. In conclusion, since collagen increased in equine endometrium and oviduct with mares aging, the existence of a possible correlation between fibrosis in the endometrium and oviduct is suggested. To the best of our knowledge this is the first study that relates endometrosis to the presence of fibrosis in the mare oviduct. Thus, the histopathological evaluation of mare endometrium could give additional information on oviduct fibrosis, where a biopsy for diagnostic purposes cannot be performed.