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K Sabeur and B A Ball

Reactive oxygen species (ROS) play an important role in normal sperm function, and spermatozoa possess specific mechanisms for ROS generation via an NAD(P)H-dependent oxidase. The aim of this study was to identify the presence of an NADPH oxidase 5 (NOX5) in equine testis and spermatozoa. The mRNA of NOX5 was expressed in equine testis as detected by northern blot probed with human NOX5 cDNA and by RT-PCR. Immunoblotting with affinity purified α-NOX5 revealed one major protein in equine testis and other tissues. Immunolocalization of NOX5 showed labeling over the rostral sperm head with some labeling in the equatorial and post-acrosomal regions. In the testis, there was abundant staining in the adluminal region of the seminiferous tubules associated with round and elongating spermatids. The RT-PCR and sequence analysis revealed a high homology with human NOX5. This study demonstrates that NOX5 is present in equine spermatozoa and testes and therefore represents a potential mechanism for ROS generation in equine spermatozoa.

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J Almeida, A J Conley, L Mathewson and B A Ball

In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and estrogens. 3β-hydroxysteroid dehydrogenase/Δ54-isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. Androgens produced by P450c17 serve as substrate for estrogen synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3βHSD, and P450arom and to determine changes in expression during development in the prepubertal, postpubertal, and adult equine testis based upon immunohistochemistry (IHC) and real-time quantitative PCR. Based on IHC, 3βHSD immunolabeling was observed within seminiferous tubules of prepubertal testes and decreased after puberty. On the other hand, immunolabeling of 3βHSD was very weak or absent in immature Leydig cells of prepubertal testes and increased after puberty. HSD3B1 (3 β HSD gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal testes (P=0.0041). P450c17 immunolabeling was observed in small clusters of immature Leydig cells in prepubertal testes and increased after puberty. CYP17 (P450c17 gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.030) and postpubertal testes (P=0.0318). A weak P450arom immunolabel was observed in immature Leydig cells of prepubertal testes and increased after puberty. Similarly, CYP19 (P450arom gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal (P=0.0001) testes. In conclusion, Leydig cells are the primary cell type responsible for androgen and estrogen production in the equine testis.

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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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B Chanrachakul, B Matharoo-Ball, A Turner, G Robinson, F Broughton-Pipkin, S Arulkumaran and RN Khan

Large-conductance calcium-activated potassium (BKCa) channels play an important role in the control of myometrial excitability. The aim of the present study was to determine the localization and protein expression of the alpha subunit of BKCa channels in the pregnant and parturient human uterus. An anti-alpha BKCa channel monoclonal antibody (anti-alpha(995-1113)) was used to localize and quantitate immunoreactive BKCa channel protein in myometrium of singleton term pregnant women undergoing either elective (n=26) or emergency Caesarean section following the onset of spontaneous labour (n=25). Data are presented as medians (interquartile range). Differences between groups were analysed using the Mann-Whitney U test. Immunohistochemistry studies localized the alpha subunit of the BKCa channel to the plasma membrane and the cytosol of myometrial cells with similar reaction end product in pregnant women who were or were not undergoing labour. Expression of this subunit, observed as a 125 kDa band in western blots, was significantly higher in pregnant women who were not undergoing labour (30.6% (20.3, 43.9)) than in those who were undergoing labour (15.7% (11.3, 22.4); P<0.01). Reduced BKCa alpha subunit expression in pregnant women during labour may underlie the initiation of uterine contractility during parturition.

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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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B. A. Ball, T. V. Little, J. A. Weber and G. L. Woods

Summary. The estimated embryonic loss rate between Days 4 and 14 after ovulation for young, normal mares (9%) was significantly lower (P < 0·01) than the estimated embryonic loss rate for aged subfertile mares (62%). Fertilization rates, which were based on the recovery of embryos at Day 4 after ovulation, were 96% and 81% (P < 0·1) for normal and subfertile mares, respectively. Day-4 embryos were collected from the oviducts of normal and subfertile donor mares. These embryos were transferred to the uteri of synchronized, normal recipient mares to test the hypothesis that the high incidence of embryonic loss in subfertile mares was related to embryonic defects. The hypothesis was supported because embryo survival rates were significantly higher (P < 0·05) for Day-4 embryos from normal compared to subfertile mares. These defects may have been intrinsic to the embryo or might have arisen due to the influence of the oviducal environment before Day 4 after ovulation.

Keywords: horse; embryonic loss; embryo transfer; embryonic defects; oviduct

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J. K. Critser, B. W. Arneson, D. V. Aaker, A. R. Huse-Benda and G. D. Ball

Summary. A series of 4 experiments was conducted to examine factors affecting the survival of frozen–thawed 2-cell mouse embryos. Rapid addition of 1·5 m-DMSO (20 min equilibration at 25°C) and immediate, rapid removal using 0·5 m-sucrose did not alter the frequency (mean ± s.e.m.) of blastocyst development in vitro when compared to untreated controls (90·5 ± 2·7% vs 95·3 ± 2·8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (−0·3°C/min) at −40°C with subsequent rapid thawing (∼1500°C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at −80°C with subsequent slow thawing (+ 8°C/min) (36·8 ± 5·6% vs 63·9 ± 5·7%). When slow cooling was terminated between −40 and −60°C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below −60°C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below −60°C and thawed slowly. However, when slow cooling was terminated at −32°C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to −80°C and thawed slowly (52·4 ± 9·5%, 59·5 ± 8·6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages. However, for the 2-cell stage, to achieve high survival rates with rapid thawing the temperature at which slow cooling is terminated is greater (∼ −32°C) than values reported for later stages.

Keywords: mouse; 2-cell embryos; freezing rate; thawing rate; development to blastocysts

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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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Erin L Legacki, C J Corbin, B A Ball, M Wynn, S Loux, S D Stanley and A J Conley

Mammalian pregnancies need progestogenic support and birth requires progestin withdrawal. The absence of progesterone in pregnant mares, and the progestogenic bioactivity of 5α-dihydroprogesterone (DHP), led us to reexamine progestin withdrawal at foaling. Systemic pregnane concentrations (DHP, allopregnanolone, pregnenolone, 5α-pregnane-3β, 20α-diol (3β,20αDHP), 20α-hydroxy-5α-dihydroprogesterone (20αDHP)) and progesterone) were monitored in mares for 10days before foaling (n=7) by liquid chromatography–mass spectrometry. The biopotency of dominant metabolites was assessed using luciferase reporter assays. Stable transfected Chinese hamster ovarian cells expressing the equine progesterone receptor (ePGR) were transfected with an MMTV-luciferase expression plasmid responsive to steroid agonists. Cells were incubated with increasing concentrations (0–100nM) of progesterone, 20αDHP and 3α,20βDHP. The concentrations of circulating pregnanes in periparturient mares were (highest to lowest) 3α,20βDHP and 20αDHP (800–400ng/mL respectively), DHP and allopregnanolone (90 and 30ng/mL respectively), and pregnenolone and progesterone (4–2ng/mL). Concentrations of all measured pregnanes declined on average by 50% from prepartum peaks to the day before foaling. Maximum activation of the ePGR by progesterone occurred at 30nM; 20αDHP and 3α,20βDHP were significantly less biopotent. At prepartum concentrations, both 20αDHP and 3α,20βDHP exhibited significant ePGR activation. Progestogenic support of pregnancy declines from 3 to 5days before foaling. Prepartum peak concentrations indicate that DHP is the major progestin, but other pregnanes like 20αDHP are present in sufficient concentrations to play a physiological role in the absence of DHP. The authors conclude that progestin withdrawal associated with parturition in mares involves cessation of pregnane synthesis by the placenta.

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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.