Higher systemic progesterone in the immediate post-conception period is associated with an increa... more Higher systemic progesterone in the immediate post-conception period is associated with an increase in embryonic growth rate, interferon-tau production and pregnancy rate in cattle. The objective of this study was to examine the effect of increasing progesterone concentration on Day 3 on subsequent embryo survival and development. Oestrus (Day 0) was synchronised in beef-cross heifers (n = 210) and approximately two-thirds of the heifers were inseminated with semen from a proven sire, while the remainder were not inseminated. In order to produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the oestrous cycle, which was left in situ until slaughter. The four treatment groups were: (i) pregnant, high progesterone; (ii) pregnant, normal progesterone; (iii) non-pregnant, high progesterone; and (iv) non-pregnant, normal progesterone. Animals were blood-sampled twice daily from Days 0 to 8 and once daily thereafter until slaughter on Days 5, 7, 13 or 16, corresponding to the 16-cell stage, the blastocyst stage, the beginning of elongation and the day of maternal recognition of pregnancy, respectively. Embryos were recovered by flushing the tract with phosphate-buffered saline and characterised by stage of development and, in the case of Days 13 and 16, measured. Data were analysed by mixed models ANOVA, Chi-square analysis and Student's t-test where appropriate. Insertion of a PRID on Day 3 increased (P < 0.05) progesterone concentrations from Day 3.5 onwards. There was no difference between treatments in the proportion of embryos at the expected stage of development on Days 5 or 7 (P > 0.05). While not significantly different, the proportion of viable embryos recovered was numerically greater in the high progesterone group on both Day 13 (58 v. 43%) and Day 16 (90 v. 50%). Elevation of progesterone significantly increased embryonic length on Day 13 (2.24 ± 0.51 mm v. 1.15 ± 0.16 mm, P = 0.034) and Day 16 (14.06 ± 1.18 cm v. 5.97 ± 1.18 cm, P = 0.012). In conclusion, insertion of a PRID on Day 3 of the oestrous cycle increased serum progesterone concentrations on subsequent days, which, while having no phenotypic effect on embryonic development on Days 5 or 7, was associated with an increase in embryonic size on Days 13 and 16.
Forde N. Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, ... more Forde N. Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, and -3 binding protein in the luteal phase and early pregnancy in cattle. induced blocking factor (PIBF) and galectins modulate the maternal immune response during pregnancy. We hypothesized that the relative transcript abundance of the above genes would be different during the luteal phase/early pregnancy and would be affected by progesterone supplementation. To further test this, hypothesis protein expression analyses were carried out to evaluate the abundance and localization of LGALS9 and PIBF. Following estrus synchronization, heifers were inseminated (n ϭ 140) or not (n ϭ 70). Half the heifers in each status (cyclic or potentially pregnant) were randomly assigned to receive a progesterone-releasing intravaginal device (PRID) on day 3 after estrus, which elevated progesterone concentrations from day 3.5 to 8 (P Ͻ 0.05), resulting in four treatment groups: cyclic and pregnant heifers, each with normal and high progesterone. After confirmation of pregnancy status in inseminated animals, uterine tissue was collected on days 5, 7, 13, or 16 of the luteal phase of the cycle/ pregnancy. Gene and protein expression was determined using Q-RT-PCR and IHC, respectively, on 5 heifers per treatment per time point (i.e., 80 in total). Progesterone concentrations did not affect expression of any of the genes (P Ͼ 0.05).
The cellular form of the prion protein (PrP C ) has been detected in many tissues including repro... more The cellular form of the prion protein (PrP C ) has been detected in many tissues including reproductive tissues. While its function is unclear, it has been suggested to act as a receptor for an unidentified ligand and/or as an antioxidant agent. We tested the hypothesis that PrP C is differentially expressed in dominant, growing, compared to subordinate bovine ovarian follicles. Using both microarray analysis and quantitative real-time PCR, the level of prion protein mRNA (Prnp) in both theca and granulosa cells was measured. We found that levels of Prnp were significantly higher in the theca cells of dominant compared to subordinate follicles but similar among granulosa cells from different follicles. This difference was apparent immediately after selection of the dominant follicle and continued to the dominance stage of the follicle wave. Levels of the protein for PrP C were also higher (P < 0.05) in theca cells of dominant compared to subordinate follicles. In conclusion, elevated PrP C was associated with ovarian follicle growth and development and we suggest that it may play a role in the success of follicle development. Mol.
The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy, at arou... more The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy, at around Day 16 postconception. The origin of the embryo can have a significant impact on the dynamics of embryo mortality. The aim of this study was to examine the temporal changes in transcriptional profile as the embryo develops from a spherical blastocyst on Day 7 to an ovoid conceptus at the initiation of elongation on Day 13 and to highlight differences in these temporal gene expression dynamics between in vivo-and in vitro-derived blastocysts that may be associated with embryonic survival/mortality using the bovine Affymetrix microarray. All embryos were produced either in vitro by in vitro fertilization or in vivo by superovulation. A proportion of Day 7 blastocysts were snap frozen, and the remainder were transferred (n ¼ 10 per recipient) to synchronized heifers, recovered on Day 13, and snap frozen individually. Three pools of Day 7 blastocysts (n ¼ 25 per pool) and Day 13 conceptuses (n ¼ 5 per pool) were used for microarray analysis. In Day 7 blastocysts, 50 genes were found to be differentially expressed (P , 0.05), of which 19 were upregulated and 31 down-regulated in the in vivo compared to in vitro embryos. In Day 13 conceptuses, 288 genes were found to be differentially expressed (P , 0.05), of which 133 were upregulated and 155 down-regulated in the in vivo compared to in vitro embryos. The comparison between Day 7 and Day 13 embryos revealed significant temporal changes in transcript profile with 1806 and 909 transcripts differentially expressed in the in vitro-and in vivo-derived embryos, respectively. Across the three array comparisons between Day 7 and Day 13 embryos, 444 genes were consistently exclusively present in the in vivo embryos, whereas 1341 were exclusively present in the in vitro embryos. Regardless of the origin of the embryo, 465 differentially expressed genes between Day 7 and 13 were common to both in vivo-and in vitro-derived embryos; these genes are likely critical for the transition between the blastocyst (Day 7) and ovoid conceptus (Day 13) stages of embryo development. In order to validate the microarray findings, differences in the expression of six genes (CYP51A1, FADS1, TDGF1, HABP2, APOA2, and SLC12A2) were confirmed by quantitative real-time PCR on in vivo-and in vitro-derived embryos on Day 7 and Day 13 using independent samples from those used for the microarray. Subsequent mapping of these differentially expressed genes into relevant functional groups and pathways identified important pathways involved in conceptus elongation in cattle. In conclusion, this analysis has identified genes and pathways crucial for the transition from a spherical blastocyst to an ovoid conceptus as well as those uniquely associated with a greater likelihood of embryonic survival (those unique to in vivo embryos) or loss (those unique to in vitro embryos).
Successful growth and development of the posthatching blastocyst and pregnancy establishment are ... more Successful growth and development of the posthatching blastocyst and pregnancy establishment are a result of the interaction between a competent embryo and a receptive uterine environment. We examined the global transcriptome profiles of the Day 16 bovine conceptus and pregnant endometrium tissues using RNA-Seq to identify genes that contribute to the dialogue during the period of pregnancy recognition. Using stringent filtering criterion, a total of 16 018 and 16 262 transcripts of conceptus and pregnant endometrium origin, respectively, were identified with distinct tissue-specific expression profiles. Of these, 2261 and 2505 transcripts were conceptus and endometrium specific. Using Cytoscape software, a total of 133 conceptus ligands that interact with corresponding receptors on the endometrium and 121 endometrium ligands that interact with corresponding receptors on the conceptus were identified. While 87 ligands were commonly detected, 46 were conceptus specific and 34 endometrium specific. This study is one of the first to provide a comprehensive list of potentially secreted molecules in the conceptus that interact with receptors on the endometrium and vice versa during the critical window of maternal recognition of pregnancy. The identified tissue-specific genes may serve as candidates to study pregnancy recognition and they or downstream products may represent potential early markers of pregnancy.
Dietary restriction in growing cattle and severe negative energy balance in lactating cows have b... more Dietary restriction in growing cattle and severe negative energy balance in lactating cows have been associated with altered gonadotropin secretion, reduced follicle diameter, reduced circulating oestradiol concentrations and anovulation. Therefore, we hypothesised that acute dietary restriction would influence the fate and function of the dominant follicle by altering the expression for genes regulating gonadotrophin and IGF response in ovarian follicles. Newly selected dominant follicles were collected 7-8 days after prostaglandin F 2␣ (PGF) administration from heifers (n = 25) that were individually fed a diet supplying 1.2 maintenance (M; control, n = 8) or 0.4 M (restricted, n = 17) for a total duration of 18-19 days. Heifers within 0.4 M were ovulatory (n = 11) or anovulatory (n = 6) depending on whether the dominant follicle present at PGF ovulated or became atretic following luteolysis. Control animals were all ovulatory. Acute dietary restriction decreased IGF-I (P < 0.001) and insulin (P < 0.05) in circulation; oestradiol (P < 0.01) and IGF-I (P < 0.01) in follicular fluid; and mRNA for FSHR (P < 0.01) in granulosa cells but increased mRNA for IGFBP2 (P < 0.05) in theca cells of the newly selected dominant follicle. However, this only led to anovulation when dietary restriction also decreased mRNA for CYP19A1 (P < 0.05), IGF2 (P < 0.01) and IGF1R (P < 0.05) in granulosa cells and LHCGR (P < 0.05) in theca cells of follicles collected from heifers fed 0.4 M. These results suggest that the catabolic environment induced by dietary restriction may ultimately cause anovulation by reducing oestradiol synthesis, FSH-responsiveness and IGF signaling in granulosa, and LH-responsiveness in theca cells of dominant follicles.
Higher systemic progesterone in the immediate post-conception period is associated with an increa... more Higher systemic progesterone in the immediate post-conception period is associated with an increase in embryonic growth rate, interferon-tau production and pregnancy rate in cattle. The objective of this study was to examine the effect of increasing progesterone concentration on Day 3 on subsequent embryo survival and development. Oestrus (Day 0) was synchronised in beef-cross heifers (n = 210) and approximately two-thirds of the heifers were inseminated with semen from a proven sire, while the remainder were not inseminated. In order to produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the oestrous cycle, which was left in situ until slaughter. The four treatment groups were: (i) pregnant, high progesterone; (ii) pregnant, normal progesterone; (iii) non-pregnant, high progesterone; and (iv) non-pregnant, normal progesterone. Animals were blood-sampled twice daily from Days 0 to 8 and once daily thereafter until slaughter on Days 5, 7, 13 or 16, corresponding to the 16-cell stage, the blastocyst stage, the beginning of elongation and the day of maternal recognition of pregnancy, respectively. Embryos were recovered by flushing the tract with phosphate-buffered saline and characterised by stage of development and, in the case of Days 13 and 16, measured. Data were analysed by mixed models ANOVA, Chi-square analysis and Student's t-test where appropriate. Insertion of a PRID on Day 3 increased (P < 0.05) progesterone concentrations from Day 3.5 onwards. There was no difference between treatments in the proportion of embryos at the expected stage of development on Days 5 or 7 (P > 0.05). While not significantly different, the proportion of viable embryos recovered was numerically greater in the high progesterone group on both Day 13 (58 v. 43%) and Day 16 (90 v. 50%). Elevation of progesterone significantly increased embryonic length on Day 13 (2.24 ± 0.51 mm v. 1.15 ± 0.16 mm, P = 0.034) and Day 16 (14.06 ± 1.18 cm v. 5.97 ± 1.18 cm, P = 0.012). In conclusion, insertion of a PRID on Day 3 of the oestrous cycle increased serum progesterone concentrations on subsequent days, which, while having no phenotypic effect on embryonic development on Days 5 or 7, was associated with an increase in embryonic size on Days 13 and 16.
Forde N. Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, ... more Forde N. Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, and -3 binding protein in the luteal phase and early pregnancy in cattle. induced blocking factor (PIBF) and galectins modulate the maternal immune response during pregnancy. We hypothesized that the relative transcript abundance of the above genes would be different during the luteal phase/early pregnancy and would be affected by progesterone supplementation. To further test this, hypothesis protein expression analyses were carried out to evaluate the abundance and localization of LGALS9 and PIBF. Following estrus synchronization, heifers were inseminated (n ϭ 140) or not (n ϭ 70). Half the heifers in each status (cyclic or potentially pregnant) were randomly assigned to receive a progesterone-releasing intravaginal device (PRID) on day 3 after estrus, which elevated progesterone concentrations from day 3.5 to 8 (P Ͻ 0.05), resulting in four treatment groups: cyclic and pregnant heifers, each with normal and high progesterone. After confirmation of pregnancy status in inseminated animals, uterine tissue was collected on days 5, 7, 13, or 16 of the luteal phase of the cycle/ pregnancy. Gene and protein expression was determined using Q-RT-PCR and IHC, respectively, on 5 heifers per treatment per time point (i.e., 80 in total). Progesterone concentrations did not affect expression of any of the genes (P Ͼ 0.05).
The cellular form of the prion protein (PrP C ) has been detected in many tissues including repro... more The cellular form of the prion protein (PrP C ) has been detected in many tissues including reproductive tissues. While its function is unclear, it has been suggested to act as a receptor for an unidentified ligand and/or as an antioxidant agent. We tested the hypothesis that PrP C is differentially expressed in dominant, growing, compared to subordinate bovine ovarian follicles. Using both microarray analysis and quantitative real-time PCR, the level of prion protein mRNA (Prnp) in both theca and granulosa cells was measured. We found that levels of Prnp were significantly higher in the theca cells of dominant compared to subordinate follicles but similar among granulosa cells from different follicles. This difference was apparent immediately after selection of the dominant follicle and continued to the dominance stage of the follicle wave. Levels of the protein for PrP C were also higher (P < 0.05) in theca cells of dominant compared to subordinate follicles. In conclusion, elevated PrP C was associated with ovarian follicle growth and development and we suggest that it may play a role in the success of follicle development. Mol.
The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy, at arou... more The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy, at around Day 16 postconception. The origin of the embryo can have a significant impact on the dynamics of embryo mortality. The aim of this study was to examine the temporal changes in transcriptional profile as the embryo develops from a spherical blastocyst on Day 7 to an ovoid conceptus at the initiation of elongation on Day 13 and to highlight differences in these temporal gene expression dynamics between in vivo-and in vitro-derived blastocysts that may be associated with embryonic survival/mortality using the bovine Affymetrix microarray. All embryos were produced either in vitro by in vitro fertilization or in vivo by superovulation. A proportion of Day 7 blastocysts were snap frozen, and the remainder were transferred (n ¼ 10 per recipient) to synchronized heifers, recovered on Day 13, and snap frozen individually. Three pools of Day 7 blastocysts (n ¼ 25 per pool) and Day 13 conceptuses (n ¼ 5 per pool) were used for microarray analysis. In Day 7 blastocysts, 50 genes were found to be differentially expressed (P , 0.05), of which 19 were upregulated and 31 down-regulated in the in vivo compared to in vitro embryos. In Day 13 conceptuses, 288 genes were found to be differentially expressed (P , 0.05), of which 133 were upregulated and 155 down-regulated in the in vivo compared to in vitro embryos. The comparison between Day 7 and Day 13 embryos revealed significant temporal changes in transcript profile with 1806 and 909 transcripts differentially expressed in the in vitro-and in vivo-derived embryos, respectively. Across the three array comparisons between Day 7 and Day 13 embryos, 444 genes were consistently exclusively present in the in vivo embryos, whereas 1341 were exclusively present in the in vitro embryos. Regardless of the origin of the embryo, 465 differentially expressed genes between Day 7 and 13 were common to both in vivo-and in vitro-derived embryos; these genes are likely critical for the transition between the blastocyst (Day 7) and ovoid conceptus (Day 13) stages of embryo development. In order to validate the microarray findings, differences in the expression of six genes (CYP51A1, FADS1, TDGF1, HABP2, APOA2, and SLC12A2) were confirmed by quantitative real-time PCR on in vivo-and in vitro-derived embryos on Day 7 and Day 13 using independent samples from those used for the microarray. Subsequent mapping of these differentially expressed genes into relevant functional groups and pathways identified important pathways involved in conceptus elongation in cattle. In conclusion, this analysis has identified genes and pathways crucial for the transition from a spherical blastocyst to an ovoid conceptus as well as those uniquely associated with a greater likelihood of embryonic survival (those unique to in vivo embryos) or loss (those unique to in vitro embryos).
Successful growth and development of the posthatching blastocyst and pregnancy establishment are ... more Successful growth and development of the posthatching blastocyst and pregnancy establishment are a result of the interaction between a competent embryo and a receptive uterine environment. We examined the global transcriptome profiles of the Day 16 bovine conceptus and pregnant endometrium tissues using RNA-Seq to identify genes that contribute to the dialogue during the period of pregnancy recognition. Using stringent filtering criterion, a total of 16 018 and 16 262 transcripts of conceptus and pregnant endometrium origin, respectively, were identified with distinct tissue-specific expression profiles. Of these, 2261 and 2505 transcripts were conceptus and endometrium specific. Using Cytoscape software, a total of 133 conceptus ligands that interact with corresponding receptors on the endometrium and 121 endometrium ligands that interact with corresponding receptors on the conceptus were identified. While 87 ligands were commonly detected, 46 were conceptus specific and 34 endometrium specific. This study is one of the first to provide a comprehensive list of potentially secreted molecules in the conceptus that interact with receptors on the endometrium and vice versa during the critical window of maternal recognition of pregnancy. The identified tissue-specific genes may serve as candidates to study pregnancy recognition and they or downstream products may represent potential early markers of pregnancy.
Dietary restriction in growing cattle and severe negative energy balance in lactating cows have b... more Dietary restriction in growing cattle and severe negative energy balance in lactating cows have been associated with altered gonadotropin secretion, reduced follicle diameter, reduced circulating oestradiol concentrations and anovulation. Therefore, we hypothesised that acute dietary restriction would influence the fate and function of the dominant follicle by altering the expression for genes regulating gonadotrophin and IGF response in ovarian follicles. Newly selected dominant follicles were collected 7-8 days after prostaglandin F 2␣ (PGF) administration from heifers (n = 25) that were individually fed a diet supplying 1.2 maintenance (M; control, n = 8) or 0.4 M (restricted, n = 17) for a total duration of 18-19 days. Heifers within 0.4 M were ovulatory (n = 11) or anovulatory (n = 6) depending on whether the dominant follicle present at PGF ovulated or became atretic following luteolysis. Control animals were all ovulatory. Acute dietary restriction decreased IGF-I (P < 0.001) and insulin (P < 0.05) in circulation; oestradiol (P < 0.01) and IGF-I (P < 0.01) in follicular fluid; and mRNA for FSHR (P < 0.01) in granulosa cells but increased mRNA for IGFBP2 (P < 0.05) in theca cells of the newly selected dominant follicle. However, this only led to anovulation when dietary restriction also decreased mRNA for CYP19A1 (P < 0.05), IGF2 (P < 0.01) and IGF1R (P < 0.05) in granulosa cells and LHCGR (P < 0.05) in theca cells of follicles collected from heifers fed 0.4 M. These results suggest that the catabolic environment induced by dietary restriction may ultimately cause anovulation by reducing oestradiol synthesis, FSH-responsiveness and IGF signaling in granulosa, and LH-responsiveness in theca cells of dominant follicles.
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