D. Scarlet

Expression and immunolocalisation of follicle-stimulating hormone receptors in gonads of newborn and adult female horses

In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animals age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.

Effect of stallion age on the expression of LH and FSH receptors and aromatase P450 in equine male reproductive tissues

The aim of the present study was to characterise receptors for LH and FSH (LHR and FSHR, respectively) and aromatase in epididymal and testicular tissue from stallions of different ages (prepubertal, young, mature and old). Gene and protein expression were assessed by real-time quantitative polymerase chain reaction (real-time qPCR), immunohistochemistry and multiple immunofluorescence labelling. There were no differences in LHR mRNA expression in epididymal and testicular parenchyma in stallions of different age. In contrast, expression of FSHR and CYP19A1 in caput, corpus and cauda epididymis and in testicular parenchyma increased with age (P<0.001). Immunolabelling for LHR, FSHR and aromatase was influenced by puberty. In postpubertal stallions, positive staining for LHR and aromatase was detected in Leydig cells, whereas protein expression of FSHR was present in Sertoli cells and primary spermatocytes. In prepubertal colts, staining for LHR, FSHR and aromatase was detected in seminiferous tubules. In epididymal tissue, aromatase was present in the cauda epididymis only, regardless of age. In conclusion, the results highlight the significance of gonadotropin action and oestrogen production for the maturation of male reproductive tissue in the horse. The presence of FSHR in the seminiferous tubules suggests effects of FSH on spermatogenesis in this species. The importance of oestrogen production for maintenance of testicular function in stallions was confirmed.

The Orthology Clause in the Next Generation Sequencing Era: Novel Reference Genes Identified by RNA-seq in Humans Improve Normalization of Neonatal Equine Ovary RT-qPCR Data

Background Vertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0). Results We hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively. Conclusions The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

120 ANTRAL FOLLICLE COUNT OF 2-YEAR-OLD MARES IS HIGHLY CORRELATED WITH ANTI-MÜLLERIAN HORMONE CONCENTRATIONS AT 24 to 28 WEEKS OF AGE

Success of assisted reproductive techniques, as determined by the response to hormonal treatments and embryo quality, can successfully be predicted by the concentration of anti-Müllerian hormone (AMH) in plasma of several species. Being able to predict ovarian follicular reserve of prepubertal female horses (fillies) would help to select fertile broodmares and reduce costs associated with animal upkeep. The objectives of this work were to (1) assess AMH dynamics in female horses during the first year of life and (2) determine whether AMH concentrations detected in plasma of prepubertal fillies are correlated with AMH concentrations and antral follicle count (AFC) after puberty. Warmblood fillies (n=14) born from February to May of 1 year in the same stud were used. Blood samples for AMH determinations were collected from birth onward every 4 weeks up to the age of 1 year. At 2 years, blood samples were collected and AFC was determined by transrectal ultrasonography. The AMH concentrations were determined by ELISA (AL-115, Ansh Laboratories, Webster, TX, USA). Transrectal ultrasonography was used to determine the AFC, which corresponds to the total number of antral follicles detected with ultrasound. Statistical analysis was done with the SPSS Statistics 24 software (SPSS Inc., Chicago, IL, USA). The AMH was detectable in the plasma of all animals from birth onward. At birth, mean AMH concentration was 4.5±1.2ngmL-1. The AMH concentration increased and peaked between 24 weeks (8.7±4.4ngmL-1) and 28 weeks (6.7±2.1ngmL-1) and subsequently decreased again (52 weeks: 3.9±0.9ngmL-1). Very high variation among individuals during the first year was lost at 2 years of age. The AMH concentration at 2 years was highly correlated with AMH concentration at birth (r=0.62, P<0.05) and with AFC (r=0.78, P<0.001). Also, AMH concentration (r=0.73, P<0.01) and AFC (r=0.6, P<0.05) at 2 years were highly correlated with AMH concentrations at 24 and 28 weeks. Gestational length (337±1 days), parity of the dam (4.6±0.8), and placental weight (6983±352g) did not influence AMH concentrations at any time. Our results demonstrate that AMH is detectable in blood of female horses from birth onward. Despite its high variability between individuals up to 52 weeks, strong correlations were observed during the first 2 years of life. High correlations to AFC at 2 years suggest that determination of AMH in prepubertal female horses helps to predict the ovarian reserve and fertility in postpubertal life.

Applicability of a new cell culture device for cooled-storage of stallion semen.

A new device for storage and shipping of cell cultures--the Petaka G3 cell management device--was tested for its applicability for cooled-storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg/l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15 °C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection with computer-assisted semen analysis. In experiment 1 (extender without antibiotics), total motility, progressive motility and viability of spermatozoa significantly decreased over time (p < 0.05). The decrease was significantly faster at 15 °C than at 5 °C (p < 0.05). In the presence of gentamicin (experiment 2), this difference was no longer present. It can be concluded that cooled-storage of equine semen in sophisticated devices for cell culture is not advantageous to syringes for successful maintenance of semen longevity.