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E. D. Watson and K. Hinrichs

Summary. Fluid was aspirated from the preovulatory follicle of Group 1 mares (N = 6) when follicles reached 32–34 mm in diameter. Group 2 mares each received an i.v. injection of hCG when the preovulatory follicle reached 35 mm. Aspiration of follicular fluid was performed 28–32 h after treatment. Follicular fluid was aspirated from Group 3 mares 28–32 h after the preovulatory follicle reached 35 mm in diameter.

Concentrations of progesterone were significantly higher in follicular fluid from Group 2 mares than in that from mares in Groups 1 and 3. Testosterone was significantly higher in follicular fluid from Groups 2 and 3 than in Group 1 mares. There were no significant differences among groups in concentrations of oestradiol and prostaglandin F (PGF) in follicular fluid.

Keywords: oestradiol; progesterone; testosterone; PGF; follicle; mare

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E. D. Watson and S. R. M. Thomson

Aromatase P-450 (P-450arom) is a crucial regulatory enzyme that is necessary for conversion of androgens to oestrogens. Corpora lutea and follicles were obtained from the ovaries of cyclic mares and from mares at day 20 and days 40–70 of pregnancy. The presence of P-450arom within specific cell types was investigated by immunostaining to determine potential sites of oestrogen synthesis. Immunoreactivity for P-450arom was confined to the granulosa layer of non-atretic follicles > 5 mm in diameter and to corpora lutea at all stages of the oestrous cycle and during pregnancy. These findings confirm that aromatization of androgens occurs within the granulosa cells of the preovulatory follicle of the mare and that the corpus luteum of the mare has the capacity for oestrogen production if adequate androgen substrate is available. Granulosa cells in ovarian tissue from three mares with granulosa cell tumours showed little staining for P-450arom, which suggests that these tumours have little aromatizing capacity.

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E. D. Watson and H. G. Zanecosky

Summary. Fluid was aspirated from the preovulatory follicles of mares before and 12, 24 and 36 h after intravenous administration of hCG. Follicular fluid significantly (P < 0·001) reduced lymphocyte blastogenesis in vitro and, at a dilution of 1:100, fluid collected at 36 h after administration of hCG was significantly more suppressive (P < 0·01) than fluid collected before 36 h. Suppression of blastogenesis was reduced by extracting the follicular fluid with ether or by charcoal treatment (P < 0·01) or by heating at 56°C for 30 min (P < 0·05). Preincubation of lymphocytes with 2 of 5 follicular fluid samples expressed subsequent blastogenesis. Follicular fluid inhibited blastogenesis of T-cell growth factor (TCGF)-dependent Con A lymphoblasts (P < 0·05) and the degree of inhibition was related to time of addition of the TCGF and time of collection of the follicular fluid. These results indicate that preovulatory follicular fluid in the mare is increasingly suppressive to lymphocytes as time of ovulation approaches and that this immunosuppression is associated with an alteration of the response to lymphokine stimulation.

Keywords: mare; immunosuppression; follicular fluid

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E. D. Watson and P. L. Sertich

Summary. Corpora lutea (CL) were collected from mares during early (Day 4–5), mid- (Day 8–9), and late (Day 12–13) dioestrus. Dispersed cell suspensions were obtained by enzymic digestion of tissue. Two distinct luteal cell populations (large and small) were observed. The proportion of small luteal cells significantly increased as age of CL advanced.

Cells (2 × 106) from CL which were incubated for 24 h secreted prostaglandin (PG) F, PGE-2 and 6-keto-PGF-1α (the stable metabolite of prostacyclin). Higher concentrations of all PGs were produced by cells from CL at early dioestrus than from those at mid- or late dioestrus. The ratio of PGF:PGE-2 increased from 0·33 in CL of early dioestrus to 1·34 in CL of mid-dioestrus, whereas ratios of PGF:6-keto-PGF-1α remained relatively constant (∼0·6). The ratio of PGE-2:6-keto-PGF-1α from CL decreased between early (3·27) and mid-dioestrus (0·43). Addition of LH, dbcAMP, or ionophore to cell cultures did not consistently affect secretion of progesterone or PGs by luteal cells. It is suggested that prostaglandins produced by luteal cells of mares may contribute to control of luteal function and that the changing ratios of prostaglandins may be more important in controlling the lifespan of the CL than absolute concentrations of each.

Keywords: mare; corpus luteum; prostaglandins; progesterone

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E. D. Watson and P. L. Sertich

Summary. Embryos, endometrial biopsies, and uterine lavage fluid were collected from pregnant and non-pregnant mares 14 days after ovulation. Embryos were cultured for 20·5 h with and without endometrial tissue from pregnant mares, and endometrial tissue was cultured alone. Endometrial content of PGF tended to be higher (P = 0·06) in non-pregnant than in pregnant mares, but the amount of PGF released from tissue during culture was similar for pregnant and non-pregnant mares. Lavage fluid from non-pregnant mares also tended (P = 0·08) to contain higher concentrations of PGF. Co-incubation of embryos with endometrium from pregnant mares significantly (P = 0·01) lowered concentrations of PGF in medium. Tissue concentrations and release of PGE-2 and 6-keto-PGF-1α were similar in endometrial samples from pregnant and non-pregnant mares and prostaglandin production was unaffected by the presence of an embryo during incubation. Horse embryos released all three prostaglandins during a 20·5-h incubation.

Keywords: mare; prostaglandins; pregnancy; embryo

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D. F. Lawler, J. Hopkins and E. D. Watson

Recent evidence indicates that the cells of the immune system and their large network of secretory products, or cytokines, play an active role in the ovary throughout the oestrous cycle. In the present study, immune cell populations (T and B lymphocytes, macrophages, granulocytes and eosinophils) and expression of major histocompatibility complex (MHC) class II were investigated in corpora lutea from mares in early (days 2–4), mid- (days 7–10) and late (days 12–14) dioestrus, the post-luteolytic phase (days 16–17) and early pregnancy. The number of T lymphocytes within the corpus luteum increased in the late luteal phase. CD4+ cells did not increase until day 16, whereas the number of CD8+ cells increased before functional luteolysis; an apparently selective luteal infiltration of CD8+ cells was observed. MHC class II expression by non-steroidogenic cells was increased in samples from days 16–17, as was the number of infiltrating macrophages. Flow cytometry revealed very low expression of MHC class II by large luteal cells at all stages of the oestrous cycle. In early pregnancy, the number of CD4+ and CD8+ cells and macrophages decreased, as did MHC class II expression, compared with mid-dioestrous samples. B cells were present in very small numbers in all samples examined. Eosinophils were similarly sparsely distributed and numbers decreased further in pregnancy. After exogenous PGF administration, populations of CD4+ cells and non-specific esterase staining cells were significantly smaller than after natural luteolysis, whereas eosinophil numbers were increased compared with samples from days 16–17. However, the number of CD8+ and CD5+ cells and MHC class II expression were not significantly different from those observed after natural luteolysis. These findings indicate that populations of immune cells in the equine corpus luteum vary during its lifespan. The selective increase in CD8+ cells before functional luteolysis indicates that they have a physiological role in the regression of the corpus luteum.

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E. D. Watson, C. R. Stokes, J. S. E. David and F. J. Bourne

Summary. The bactericidal and phagocytic activities of blood neutrophils suspended in uterine washings and the mobilization of neutrophils into the uterine lumen were studied in ovariectomized mares receiving oestradiol benzoate (N = 4), progesterone (N = 4) or oily vehicle (N = 4). Uterine lavage was performed sequentially up to 144 h after induction of endometritis by intrauterine infusion of glycogen (1%). There was no significant difference between the 3 groups in speed of mobilization of neutrophils into the uterus in the first 6 h after infusion but there were significantly more uterine luminal neutrophils in progesterone-treated than in oestradiol-treated mares by 24 h after infusion (P < 0·01). Uterine washings collected from progesterone-treated mares at 0, 24 and 144 h were significantly worse at promoting bactericidal activity by neutrophils than washings from oestradiol-treated and control mares (P < 0·001). In oestrogen-treated and control mares bactericidal activity had increased by 144 h but in progesterone-treated mares bactericidal activity remained low. Neither treatment nor time affected the ability of washings to opsonize yeast blastospores. Elevated concentrations of progesterone in plasma were therefore associated with decreased bactericidal activity of neutrophils suspended in uterine washings but the generation of C3b in washings did not appear to be affected by hormone treatment.

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E. D. Watson, E. S. Aubrey, H. G. Zanecosky and P. L. Sertich

Summary. Glandular epithelial and stromal cells were isolated from the endometrium of mares by collagenase digestion and were incubated on plastic for 7–9 days until the cells formed confluent monolayers. The cells differed in morphology: epithelial cells appeared polyhedral and stromal cells were spindle like. The monolayers were incubated in the presence and absence of oxytocin. Medium was removed from wells after 2, 8 and 24 h of incubation. Concentrations of prostaglandin F (PGF) in the medium increased significantly during this time. Glandular epithelial cells produced significantly more PGF than did stromal cells. Both types of cell responded significantly to oxytocin stimulation by increased secretion of PGF; the response of glandular epithelial cells tended to be greater than that of stromal cells. Secretion of PGF by cultured cells was not affected by cycle stage or pregnancy.

Keywords: endometrium; cell culture; prostaglandins; horse

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S N Schauer, S D Sontakke, E D Watson, C L Esteves and F X Donadeu

Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle anovulatory wave (n=6 mares). Follicular fluid levels of progesterone and estradiol were lower (P<0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P<0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P≤0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24 h after (L) administration of an ovulatory dose of hCG (n=5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (P=0.05) and lower granulosa cell levels of CYP19A1 and LHCGR (P<0.005). Levels of miR-21, miR-132, miR-212, and miR-224 were increased (P<0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets, PTEN, RASA1, and SMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132, and miR-212 in the regulation of cell survival, steroidogenesis, and differentiation during follicle selection and ovulation in the monovular ovary.

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L. A. Penny, D. Armstrong, T. A. Bramley, R. Webb, R. A. Collins and E. D. Watson

Immune cells and their cytokine products have powerful local effects within body tissues. There has been great interest in the potential role of these cells, not only during destruction of the corpus luteum but also during its functional lifespan. In this study, lymphocytes, macrophages and major histocompatibility complex class II molecules were quantified using immunohistochemistry and the reverse transcription–polymerase chain reaction was used to detect mRNA for tumour necrosis factor α and interferon γ within corpora lutea from three groups of cows: (1) corpora lutea collected at an abattoir and assessed visually into four stages (stage I (days 1–5), stage II (days 6–12), stage III (days 13–18) and stage IV (days 19–21) of the oestrous cycle); (2) corpora lutea collected around natural luteolysis (days 14–20); and (3) corpora lutea collected 6, 12 and 24 h after prostaglandin F-induced luteolysis. The numbers of T lymphocytes (CD5+ and CD8+) were significantly higher (P < 0.05) at stage IV and from day 16 onwards, before functional luteolysis. There were significantly higher numbers (P < 0.01) of macrophages at stages I, III and IV compared with stage II in visually staged tissue. Major histocompatibility complex class II molecules were increased (P < 0.05) at stages I and IV compared to stage II and at all times after induced luteolysis. Using reverse transcription–polymerase chain reaction, mRNA encoding tumour necrosis factor α and interferon γ was detected in all luteal tissue collected around natural luteolysis and after induced luteolysis. These findings, particularly the increase in T lymphocytes before functional luteolysis, provide further evidence of a significant role for the immune system in affecting reproductive function in cows.