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R. Y. Ball, C. P. E. Naylor and M. J. Mitchinson

Summary. Spermatozoa were found in a para-aortic lymph node 1 year after vasectomy in a man undergoing laparotomy. Such access of spermatozoa to the lymph nodes is one route by which autosensitization against spermatozoa could arise.

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J. E. Ellington, B. A. Ball and X. Yang

The objective of this study was to determine whether coculture of stallion spermatozoa and mare oviductal (uterine tubal) epithelial cells induced sperm cell capacitation in vitro. Capacitation as determined by zona binding and chlortetracycline staining of the sperm cells was compared for stallion spermatozoa: (1) incubated with medium alone (negative control), (2) treated with calcium ionophore A23187 (positive control) or (3) cultured with mare oviductal epithelial cells (OEC) for 4 h. Chlortetracycline staining patterns of sperm cells bound to the zonae were used to group spermatozoa as uncapacitated, capacitated or acrosome reacted. The zonae and attached spermatozoa were stained for evaluation after initial binding (pulse) and after 1 h of co-incubation (chase). More sperm cells in the ionophore and OEC treatments bound to the zonae at both the pulse and chase than in control medium (P < 0.001). More bound sperm cells were capacitated at the pulse, and acrosome reacted at the chase, for the ionophore and co-culture groups than for the controls (P < 0.001). Staining patterns for sperm cells not bound to the zona pellucida in each of the treatments differed (P < 0.05) from the population of sperm cells that bound to the zona pellucida. There was a higher percentage of capacitated spermatozoa and a lower percentage of acrosome-reacted spermatozoa bound to the zonae at the pulse than were represented in the treatment suspensions of sperm cells. The co-culture treatment resulted in a higher (P < 0.05) proportion of sperm cells in suspension with the capacitated staining pattern and a lower (P < 0.05) proportion with the uncapacitated pattern than those in the ionophore treatment.

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S C Loux, K E Scoggin, M H T Troedsson, E L Squires and B A Ball

The cervical mucus plug (CMP) is believed to play an integral role in the maintenance of pregnancy in the mare, primarily by inhibiting microbial entry. Unfortunately, very little is known about its composition or origin. To determine the proteomic composition of the CMP, we collected CMPs from mares (n = 4) at 9 months of gestation, and proteins were subsequently analyzed by nano-LC–MS/MS. Results were searched against EquCab2.0, and proteomic pathways were predicted by Ingenuity Pathway Analysis. Histologic sections of the CMP were stained with H&E and PAS. To identify the origin of highly abundant proteins in the CMP, we performed qPCR on endometrial and cervical mucosal mRNA from mares in estrus, diestrus as well as mares at 4 and 10 m gestation on transcripts for lactotransferrin, uterine serpin 14, uteroglobin, uteroferrin, deleted in malignant brain tumors 1 and mucins 4, 5b and 6. Overall, we demonstrated that the CMP is composed of a complex milieu of proteins during late gestation, many of which play an important role in immune function. Proteins traditionally considered to be endometrial proteins were found to be produced by the cervical mucosa suggesting that the primary source of the CMP is the cervical mucosa itself. In summary, composition of the equine CMP is specifically regulated not only during pregnancy but also throughout the estrous cycle. The structural and compositional changes serve to provide both a structural barrier as well as a physiological barrier during pregnancy to prevent infection of the fetus and fetal membranes.

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S. P. Brinsko, B. A. Ball, P. G. Miller, P. G. A. Thomas and J. E. Ellington

This study was designed to investigate the development of day 2 embryos obtained from young and aged mares, co-cultured with oviductal epithelial cells obtained from mares in each age group in a 2 × 2 crossover design. Young, fertile mares (n = 19; 2–7 years of age) and aged, subfertile, mares (n = 16; 17–24 years of age) were used as embryo and oviductal epithelial cell donors. Embryos (n = 37) were collected from the oviducts 2 days after ovulation and were paired (embryos obtained from young mares with embryos obtained from aged mares) so that eight pairs were co-cultured with young mare oviductal epithelial cells and eight pairs were co-cultured with aged mare oviductal epithelial cells. Five additional embryos obtained from young mares were co-cultured with oviductal epithelial cells from either young mares or aged mares but were not paired. Embryos were co-cultured for 7 days at 38.5°C in 5% CO2 or until morphological degeneration was detected. The proportions of paired embryos that reached the blastocyst stage were similar for embryos obtained from young mares and embryos obtained from aged mares after co-culture with oviductal epithelial cells from young mares (6 of 8 versus 5 of 8) or from aged mares (6 of 8 versus 5 of 8), respectively. Although the overall rate of development of embryos to blastocyst from both young mares and aged mares was similar, blastocysts developing from embryos obtained from aged mares were inferior to blastocysts obtained from young mares in terms of number of cell nuclei, quality score, and diameter at day 7. The results of this experiment indicate that the high rate of early embryonic loss in aged, subfertile mares may be due to inherent developmental defects in their embryos, but does not appear related to the ability of embryos from aged, subfertile mares to reach the blastocyst stage.

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K. P. McNatty, N. Hudson, M. Gibb, K. Ball, K. M. Henderson, D. A. Heath, S. Lun and L. E. Kieboom

Summary. Injection of steroid-free bovine follicular fluid (bFF; 2 × 5 ml s.c. 12h apart) into anoestrous ewes lowered plasma FSH concentrations by 70% and after 24 h had significantly (P < 0·01) reduced the number of non-atretic follicles (≥ 1 mm diam.) without influencing the total number of follicles (≥ 1 mm diam.) compared to untreated controls. Hourly injections of FSH (10 μg i.v. NIH-FSH-S12) for 24 h did not influence the number of non-atretic follicles but did negate the inhibitory effects of bFF on follicular viability. Hourly injections of FSH (50 μg i.v., NIH-FSH-S12) + bFF treatment for 24 h significantly increased the total number of non-atretic follicles, and particularly the number of medium to large non-atretic follicles (≥ 3 mm diam.) compared to the untreated controls (both P < 0·01). The 10μg FSH regimen (without bFF) significantly increased aromatase activity in granulosa cells from large ( ≥ 5 mm diam.; P < 0·01) but not medium (3–4·5 mm diam.) or small (1–2·5 mm diam.) follicles compared to controls. The 10 μg FSH + bFF regimen had no effect on granulosa-cell aromatase activity compared to the controls. However, the 50 μg FSH plus bFF regimen increased the aromatase activity of granulosa cells from large, medium and small non-atretic follicles 2·6-, 8·3- and ≥ 11-fold respectively compared to that in the control cells.

Ewes (N = 11) that ovulated 2 follicles had significantly higher plasma FSH concentrations from 48 to 24 h and 24 to 0 h before the onset of a cloprostenol-induced follicular phase (both P < 0·01) than in the ewes (N = 12) that subsequently ovulated one follicle. Hourly FSH treatment (1·6 μg i.v., NIAMDD-FSH-S15) for 24 h but not for any 6 h intervals between 48 and 24 h or 24 and 0 h before a cloprostenol-induced luteolysis also resulted in significant increases (P < 0·05) in the number of ewes with 2 ovulations.

We conclude that (1) the number of non-atretic antral follicles in sheep ovaries is influenced by plasma FSH concentrations; (2) the level of follicular oestradiol biosynthesis can be enhanced by FSH treatment; and (3) sustained elevations of plasma FSH concentrations for 24 h but not 6 h within 48 h of the onset of luteolysis significantly enhances the ovulation rate in Romney ewes.

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Erin L Legacki, C Jo Corbin, Barry A Ball, Kirsten E Scoggin, Scott D Stanley and Alan J Conley

Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) crucial for progesterone and androgen synthesis, respectively, as well as aromatase cytochrome P450 (P450arom) that converts Δ4-androgens to estrogens. 5α-reductase is another important enzyme in equine placentas because 5α-dihydroprogesterone (DHP) sustains pregnancy in the absence of progesterone in the second half of equine pregnancy. DHP and its metabolites decline dramatically days before foaling, but few studies have investigated placental enzyme activity before or at parturition in mares. Thus, key enzyme activities and transcript abundance were investigated in equine placentas at 300 days of gestation (GD300) and post-partum (term). Equine testis was used as a positive control for P450c17 activity. Substrates were incubated with microsomal preparations, together with enzyme inhibitors, and products were measured by liquid chromatography tandem mass spectrometry or radiometric methods (aromatase). Equine placenta expressed high levels of 3βHSD, 5α-reductase and aromatase, and minimal P450c17 activity at GD300 compared with testis (600-fold higher). At foaling, 3βHSD and aromatase activities and transcript abundance were unchanged but 5α-reductase (and P450c17) was no longer detectable (P < 0.05) and transcript was decreased. Trilostane inhibited 3βHSD significantly more in testis than placenta, suggesting possible existence of different 3βHSD isoforms. Equine placentas have significant capacity for steroid metabolism by 5α-reductase, 3βHSD and aromatase but little for androgen synthesis lacking P450c17. Declining pre-partum 5α-reduced pregnane concentrations coincide with selective loss of placental 5α-reductase activity and expression at parturition in horses.

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K. M. Henderson, M. D. Prisk, N. Hudson, K. Ball, K. P. McNatty, S. Lun, D. Heath, L. E. Kieboom and J. McDiarmid

Summary. Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0·1, 0·5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1·0 ml bFF twice daily (1·9 ± 0·2 ovulations/ewe, mean ± s.e.m. for N = 34) or 5·0 ml once daily (2·0 ± 0·2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1·4 ± 0·1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (≥ 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17β in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17β and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1–2·5 mm diam.) or medium (3–4·5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.

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K. P. McNatty, K. M. Henderson, S. Lun, D. A. Heath, K. Ball, N. L. Hudson, J. Fannin, M. Gibb, L. E. Kieboom and P. Smith

Summary. A marked difference in both the function and composition of individual ovarian follicles was noted in Booroola × Romney ewes (6–7 years of age) which had previously been segregated on at least one ovulation rate record of 3–4 (F+ ewes, N = 21) or <3 (++ ewes, N = 21).

Follicles in F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In F+ ewes (N = 3), the presumptive preovulatory follicles were 4·4 ± 0·5 (s.e.m.) mm in diameter and contained 2·1 ± 0·3 × 106 (s.e.m.) granulosa cells, whereas in ++ ewes (N = 3), such follicles were 7·3 ± 0·3 mm in diameter and contained 6·5 ± 0·8 × 106 cells. During a prostaglandin (PG)-induced follicular phase, the secretion rate of oestradiol from ovaries containing 3 presumptive preovulatory follicles in F + ewes was similar to that from ovaries with only one such follicle in ++ ewes.

We suggest that the putative 'gene effect' in F+ ewes is manifested during early follicular development and that it may be mediated via an enhanced sensitivity of granulosa cells to pituitary hormones. As a consequence, the development of 3 preovulatory follicles in F+ ewes may be necessary to provide a cell mass capable of producing the same quantity of oestradiol as that from one preovulatory follicle in ++ ewes.

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Erin L Legacki, Barry A Ball, C Jo Corbin, Shavahn C Loux, Kirsten E Scoggin, Scott D Stanley and Alan J Conley

Equine fetuses have substantial circulating pregnenolone concentrations and thus have been postulated to provide significant substrate for placental 5α-reduced pregnane production, but the fetal site of pregnenolone synthesis remains unclear. The current studies investigated steroid concentrations in blood, adrenal glands, gonads and placenta from fetuses (4, 6, 9 and 10 months of gestational age (GA)), as well as tissue steroidogenic enzyme transcript levels. Pregnenolone and dehydroepiandrosterone (DHEA) were the most abundant steroids in fetal blood, pregnenolone was consistently higher but decreased progressively with GA. Tissue steroid concentrations generally paralleled those in serum with time. Adrenal and gonadal tissue pregnenolone concentrations were similar and 100-fold higher than those in allantochorion. DHEA was far higher in gonads than adrenals and progesterone was higher in adrenals than gonads. Androstenedione decreased with GA in adrenals but not in gonads. Transcript analysis generally supported these data. CYP17A1 was higher in fetal gonads than adrenals or allantochorion, and HSD3B1 was higher in fetal adrenals and allantochorion than gonads. CYP11A1 transcript was also significantly higher in adrenals and gonads than allantochorion and CYP19 and SRD5A1 transcripts were higher in allantochorion than either fetal adrenals or gonads. Given these data, and their much greater size, the fetal gonads are the source of DHEA and likely contribute more than fetal adrenal glands to circulating fetal pregnenolone concentrations. Low CYP11A1 but high HSD3B1 and SRD5A1 transcript abundance in allantochorion, and low tissue pregnenolone, suggests that endogenous placental pregnenolone synthesis is low and likely contributes little to equine placental 5α-reduced pregnane secretion.

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S C Loux, A J Conley, K E Scoggin, H El-Sheikh Ali, P Dini and B A Ball

Steroid production varies widely among species, with these differences becoming more pronounced during pregnancy. As a result, each species has its own distinct pattern of steroids, steroidogenic enzymes, receptors, and transporters to support its individual physiological requirements. Although the circulating steroid profile is well characterized during equine pregnancy, there is much yet to be explored regarding the factors that support steroidogenesis and steroid signaling. To obtain a holistic view of steroid-related transcripts, we sequenced chorioallantois (45 days, 4 months, 6 months, 10 months, 11 months, and post-partum) and endometrium (4 months, 6 months, 10 months, 11 months, and diestrus) throughout gestation, then looked in-depth at transcripts related to steroid synthesis, conjugation, transportation, and signaling. Key findings include: 1) differential expression of HSD17B isoforms among tissues (HSD17B1 high in the chorioallantois, while HSD17B2 is the dominant form in the endometrium) 2) a novel isoform with homology to SULT1A1 is the predominant sulfotransferase transcript in the chorioallantois; and 3) nuclear estrogen (ESR1, ESR2) and progesterone (PGR) expression is minimal to nonexistant in the chorioallantois and pregnant endometrium. Additionally, several hypotheses have been formed, including the possibility that the 45-day chorioallantois is able to synthesize steroids de novo from acetate and that horses utilize glucuronidation to clear estrogens from the endometrium during estrous, but not during pregnancy. In summary, these findings represent an in-depth look at equine steroid-related transcripts through gestation, providing novel hypotheses and future directions for equine endocrine research.