Oviductal transcriptional profiling of a bovine fertility model by next-generation sequencing

In cattle, the oviduct plays a fundamental role in the reproductive process. Oviductal functions are controlled by the ovarian sex steroids: estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex steroid milieus differentially impacts the oviductal transcriptional profile. We manipulated growth of the pre-ovulatory follicle to obtain cows that ovulated a larger (LF group) or a smaller (SF group) follicle. The LF group presented greater proestrus/estrus concentrations of estradiol and metaestrus concentrations of progesterone (Gonella-Diaza et al. 2015 [1], Mesquita et al. 2014 [2]). Also, the LF group was associated with greater fertility in timed-artificial insemination programs (Pugliesi et al. 2016 [3]). Cows were slaughtered on day 4 of the estrous cycle and total RNA was extracted from ampulla and isthmus fragments and analyzed by RNAseq. The resulting reads were mapped to the bovine genome (Bos taurus UMD 3.1, NCBI). The differential expression analyses revealed that 325 and 367 genes in ampulla and 274 and 316 genes in the isthmus were up-regulated and down-regulated in LF samples, respectively. To validate the RNAseq results, transcript abundance of 23 genes was assessed by qPCR and expression patterns were consistent between the two techniques. A functional enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) software. Processes enriched in the LF group included tissue morphology changes (extracellular matrix remodeling), cellular changes (proliferation), and secretion changes (growth factors, ions and metal transporters). An overview of the gene expression data was deposited in the NCBI's Gene Expression Omnibus (GEO) and is accessible through the accession number GSE65681. In conclusion, differences in the peri-ovulatory sex steroid milieu modify the oviductal gene expression profiles. Such differences may be associated with the greater fertility of the LF cows. This dataset is useful for further investigations of the oviductal biology and the impact of sex-steroid on the female reproductive tract

Efeito do ambiente endócrino peri-ovulatorio na fisiologia do oviduto de vacas de corte: regulação do transcriptoma, morfologia do tecido, proliferação celular, remodelamento da matriz extracelular, perfil de abundância de microRNAs e composição do fluido ovidutal

In cattle, the oviduct plays an important role in the reproductive process. Oviductal secretions characterize the environment where storage and sperm capacitation, fertilization, and early embryo development take place. Because molecular control of bovine oviduct receptivity is poorly understood, this Thesis proposed a model of receptivity based on the manipulation of pre-ovulatory follicle growth (POF) used to study the effects of periovulatory endocrine profile on oviductal physiology. Growth of POF in Nelore cows (Bos indicus) was manipulated to produce two groups: cows with large POF and large corpus luteum (LF-LCL; higher fertility) and cows with small POF and small CL (SF-SCL; Lower fertility). Ampulla and isthmus samples were collected on day 4 after induction of ovulation with GnRH. In the first study, the transcriptome of the ipsilateral to CL ampulla and isthmus was determined by RNAseq, the regional expression of genes was studied by qPCR, and the distribution of the PGR and ER proteins was assessed by immunohistochemistry. Greater abundance of PGR and ER was found in the oviduct of the LF-LCL animals indicating that there is a greater availability of receptors and, possibly, of signaling-mechanisms stimulated by steroids in both oviductal regions. The transcripts profile showed enriched oviductal functional characteristics that could affect its embryo receptivity. These characteristics include changes in morphology i.e. branching morphogenesis, and changes in cell functioning i.e. cell secretion, that were enriched in the LF-LCL group. In the second study, after morphological analyses, it was concluded that the ampulla of the LF-LCL animals presented more primary folds, a larger perimeter of the luminal epithelium, and a higher proportion of secretory and proliferating cells, when compared to SF-SCL group. There was no difference in isthmus morphology between groups. In the third study, the extracellular matrix remodeling was reserched. It was concluded that in the isthmus region of the LF-LCL animals, there is less type 1 collagen fibers and greater abundance of proteins involved in extracellular matrix remodeling. In the fourth study, it was determined that the periovulatory endocrine milieu affects the expression of components of the microRNAs biosynthesis pathway and the microRNAs profile, both different between groups. Finally, in the fifth study, 205 metabolites were quantified in the oviductal fluid and 37 were found to be in different concentrations when both groups were compared. It was concluded that oviduct of cows of higher fertility presents a profile of transcripts, proteins, and metabolites that is associated with morphological and functional characteristics favorable to the survival and development of the embryo.Em fêmeas bovinas, o oviduto apresenta um importante papel no processo reprodutivo. As secreções ovidutais representam o ambiente onde ocorrem o armazenamento e a capacitação espermática, a fecundação e o desenvolvimento embrionário inicial. O controle molecular da receptividade do oviduto em bovinos é pouco conhecido. Na presente tese, empregou-se um modelo de receptividade baseado na manipulação do crescimento do folículo pré-ovulatório (FPO) para o estudo dos efeitos do perfil endócrino periovulatório na fisiologia do oviduto. O crescimento do FPO de vacas Nelore (Bos indicus) foi manipulado com o objetivo de produzir dois grupos: vacas com FPO e corpo lúteo (CL) grandes (FG-CLG; maior fertilidade) e vacas com FPO e CL pequenos (FP-CLP; menor fertilidade). Amostras da ampola e istmo foram coletadas no dia 4 após da indução da ovulação com GnRH. No primeiro estudo, o transcriptoma da ampola e istmo do lado ipsolateral ao CL foi determinado por RNAseq, à expressão gênica regional e a distribuição das proteínas PGR e ER foram analisadas por qPCR e imunohistoquímica, respectivamente. Houve maior abundância de PGR e ER no oviduto dos animais do grupo FG-CLG, o que indica uma maior disponibilidade de receptores e possivelmente, de mecanismos intracelulares de sinalização estimulados pelos esteroides em ambas as regiões. O perfil global de transcritos mostrou enriquecimento de características funcionais do oviduto que poderiam afetar sua receptividade ao embrião. Tais características incluem mudanças morfológicas, como a ramificação morfogênica, e celulares, como a secreção, que foram aumentadas no grupo FG-CLG. No segundo estudo, após analisarem-se características morfológicas dos tecidos, concluiu-se que a ampola dos animais FG-CLG apresentou maior número de pregas primárias, maior perímetro do epitélio luminal, e maior proporção de células secretoras e de células em proliferação quando comparado aos animais do grupo FP-CLP. Não houve diferença na morfologia do istmo entre os grupos. No terceiro estudo, foi analisado o processo de remodelamento de matriz extracelular. Concluiu-se que no istmo dos animais do grupo FG-CLG existe menor quantidade de fibras de colágeno tipo 1 e maior abundância de proteínas envolvidas no remodelamento de matriz. No quarto estudo, determinou-se que o perfil endócrino periovulatório afeta a expressão de componentes da via de biossíntese e o perfil de microRNAs, que são diferentes entre os grupos. Finalmente, no quinto estudo, foram quantificados 205 metabólitos no fluido ovidutal dos animais. Destes, 37 encontram-se em concentrações diferentes entre os grupos. Concluiu-se que o oviduto de vacas de maior fertilidade apresenta um perfil de transcritos, proteínas e metabólitos que está associado a características morfológicas e funcionais favoráveis à sobrevivência e desenvolvimento do embrião

Sex Steroid-Mediated Control of Oviductal Function in Cattle

In cattle, the oviduct is a tubular organ that connects the ovary and the uterus. The oviduct lumen stages a dynamic set of cellular and molecular interactions to fulfill the noble role of generating a new individual. Specific anatomical niches along the oviduct lumen provide the appropriate microenvironment for final sperm capacitation, oocyte capture and fertilization, and early embryo development and transport. To accomplish such complex tasks, the oviduct undergoes spatially and temporally-regulated morphological, biochemical, and physiological changes that are associated with endocrine events of the estrous cycle. Specifically, elevated periovulatory concentrations of estradiol (E2) and progesterone (P4) influence gene expression and morphological changes that have been associated positively to fertility in beef cattle. In this review, we explore how E2 and P4 influence oviductal function in the beginning of the estrous cycle, and prepare the oviductal lumen for interactions with gametes and embryos

A simulation model to evaluate the economic consequences of insemination programs in dairy herds: timed artificial insemination and sex-sorted semen

The objective of this study was to develop a simulation model to analyse the technical, economic, and financial performance of using different reproductive strategies in dairy herds. Strategies simulated were: artificial insemination (AI) using conventional semen after oestrus detection (AIC), AI using sex-sorted semen after oestrus detection (AIS), timed artificial insemination (TAI) using conventional semen (TAIC), and TAI using sex-sorted semen (TAIS). The total time horizon analysed corresponded to 25 years, divided into 425 periods of 21 days. The model simulates the biological cycle that takes place within the bovine herd, and uses input information (productive parameters, investments, and reproductive program) to calculate output information (animal inventory variance, incomes, costs, and cash flow analysis). Based on the obtained cash flow, the payback period, net present value, and internal rate of return were calculated. The payback for AIC, AIS, TAIC, and TAIS occurred in 26, 27, 23, and 25 periods. The net present value and the internal rate of return per year of the investment for AIC, AIS, TAIC, and TAIS were US$557773 and 59.44$ 520469 and 54.76%; US$741800 and 70.22$ 662891 and 63.52%, respectively. The mean culling rate over 25 years for AIC, AIS, TAIC, and TAIS was 43.30%, 64.89%, 21.12%, and 36.40%, respectively. The simulation clearly demonstrated the economic and technical benefits of using TAI in dairy herds. These benefits are greater when TAI is used with conventional semen, despite the large investment in technology that is required. Using this mathematical model, future studies could be conducted when the assessment of the technical and economic viability of new scenarios is require

A single dose of FSH or hCG during a split-time AI program did not enhance follicular growth or pregnancy per artificial insemination in beef heifers

ABSTRACTThe objective of this study was to evaluate the effect of a low dose of hCG or FSH on follicle growth and pregnancy per artificial insemination (AI) in crossbreed beef heifers subjected to a split-time AI program. All heifers (n = 386) were subjected to a 7-d Co-synch protocol and an intravaginal controlled internal drug release (CIDR) device (GnRH and CIDR insertion; 7 days later PGF2α, CIDR removal, and application of a tail head estrus detection patch; 36 hours later AI of heifers detected in estrus, 24 hours later timed AI of remaining heifers). At CIDR removal, heifers were randomly assigned to receive either 150 IU of hCG, 20 mg of FSH, or remained as untreated controls. The diameter of the preovulatory follicle and the pregnancy per AI did not differ among treatments. A smaller (P = 0.035) proportion of heifers in the hCG group were detected in estrus compared with control. Additionally, the incidence of premature ovulation was greater (P < 0.01) among heifers treated with hCG compared with control. In conclusion, using a low dosage of hCG or FSH at the time of device removal did not affect pre-ovulatory follicle growth or the proportion of pregnant heifers

Impact of hormonal modulation at proestrus on ovarian responses and uterine gene expression of suckled anestrous beef cows

Abstract Background This study evaluated the impact of hormonal modulation at the onset of proestrus on ovarian response and uterine gene expression of beef cows. Methods A total of 172 anestrous beef cows were assigned to one of four groups according to the treatment with estradiol cypionate (ECP) and/or equine chorionic gonadotropin (eCG) [CON (n = 43), ECP (n = 43), eCG (n = 44) and ECP + eCG (n = 42)]. Results ECP-treated cows (ECP and ECP + eCG groups) presented greater occurrence of estrus (44.6% vs. 65.4%; P = 0.01) and pregnancy per AI [47.1% vs. 33.3%; P = 0.07], but similar progesterone (P4) concentration at subsequent diestrus than cows not treated with ECP (CON and eCG groups). Nonetheless, eCG-treated cows (eCG and ECP + eCG groups) presented larger follicle at timed AI (12.6 ± 0.3 vs. 13.5 ± 0.3 mm; P = 0.03), greater ovulation rate (96.5% vs. 82.6%; P = 0.008) and greater P4 concentration at d 6 (3.9 ± 0.2 vs. 4.8 ± 0.2 ng/mL; P = 0.001) than cows not treated with eCG (CON and ECP groups). Next, cows with a new corpus luteum 6 d after TAI were submitted to uterine biopsy procedure. Uterine fragments [CON (n = 6), ECP (n = 6)] were analyzed by RNA-Seq and a total of 135 transcripts were differentially expressed between groups (73 genes up-regulated by ECP treatment). Subsequently, uterine samples were analyzed by qPCR (genes associated with cell proliferation). ECP treatment induced greater abundance of PTCH2 (P = 0.07) and COL4A1 (P = 0.02), whereas suppressed EGFR (P = 0.09) expression. Conversely, eCG treatment increased abundance of HB-EGF (P = 0.06), ESR2 (P = 0.09), and ITGB3 (P = 0.05), whereas it reduced transcription of ESR1 (P = 0.05). Collectively, supplementation with ECP or eCG at the onset of proestrous of anestrous beef cows influenced ovarian responses, global and specific endometrial gene expression. Conclusion Proestrus estradiol regulate the endometrial transcriptome, particularly stimulating proliferative activity in the endometrium

Search for diphoton events with large missing transverse momentum in 1 fb(-1) of 7 TeV proton-proton collision data with the ATLAS detector ATLAS Collaboration

A search for diphoton events with large missing transverse momentum has been performed using 1.07 fb(-1) of proton-proton collision data at root s = 7 TeV recorded with the ATLAS detector. No excess of events was observed above the Standard Model prediction and 95% Confidence Level (CL) upper limits are set on the production cross section for new physics. The limits depend on each model parameter space and vary as follows: sigma < (22-129) fb in the context of a generalised model of gauge-mediated supersymmetry breaking (GGM) with a bino-like lightest neutralino, sigma < (27-91) fb in the context of a minimal model of gauge-mediated supersymmetry breaking (SPS8), and sigma < (15-27) fb in the context of a specific model with one universal extra dimension (UED). A 95% CL lower limit of 805 GeV, for bino masses above 50 GeV, is set on the GGM gluino mass. Lower limits of 145 TeV and 1.23 TeV are set on the SPS8 breaking scale Lambda and on the UED compactification scale 1/R, respectively. These limits provide the most stringent tests of these models to date. (C) 2012 CERN. Published by Elsevier B.V. All rights reserved