Evaluation of the effect of mating, intrauterine deposition of raw seminal plasma or seminal plasma purified β-NGF on endometrial vascularization in llamas

Urra, Felipe; Ratto, M.H.; Silva, Mauricio

doi:10.1016/j.theriogenology.2018.10.007

Cited by 10 publications

(5 citation statements)

References 47 publications

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“…The ovaries of the llamas were examined daily by transrectal ultrasonography using a 7.5 MHz linear-array transducer (Aloka, SSD-500, International Clinics, Santiago, Chile). When a growing follicle ≥8 mm in diameter was detected [28], llamas were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF ( n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas ( n = 16) were mated once with a fertile male.…”

Section: Methodsmentioning

confidence: 99%

The effect of seminal plasma β-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

Valderrama

Vargas

Silva

et al. 2019

Reprod Biol Endocrinol

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Background Nerve growth factor (β-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified β-Nerve Growth Factor (β-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas. Methods Experiment I: Female llamas ( n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF ( n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas ( n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas ( n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h ( n = 4 replicates) and progesterone secretion at 48 h ( n = 4 replicates) after LH or β-NGF treatment. Results Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified β-NGF with respect to the control group. All treatments except β-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or β-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified β-NGF (10 and 20 h). Experiment II: Only β-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro ...

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Section: Methodsmentioning

confidence: 99%

The effect of seminal plasma β-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

Valderrama

Vargas

Silva

et al. 2019

Reprod Biol Endocrinol

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“…The llamas were examined daily by transrectal ultrasonography using a 7.5-MHz linear-array transducer (Aloka SSD-500; Aloka Co., Ltd., Tokyo, Japan) to detect the emergence of a new preovulatory follicle. When a growing follicle ≥ 8 mm in diameter was detected (17,18,20), llamas were submitted to transvaginal ultrasound-guided follicle aspiration using a 5.0-MHz convexarray ultrasound transducer coupled to a 19-gauge needle as described previously (19,21,22) to collect granulosa cells by flushing the preovulatory follicle.…”

Section: Granulosa Cell Collectionmentioning

confidence: 99%

β-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells

et al. 2020

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P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3β-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. β-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that β-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that β-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

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“…The measurement of MEVA has been reported to be a reliable and sensitive tool to evaluate uterine blood flow during early gestation in mares and heifers (11,16,17). Also, using this ultrasonographic method our research group has demonstrated in previous studies (23,24) that significant changes in uterine blood flow and vascularization area occur in llamas during the follicular growth phase or after mating. However, in the present study MEVA was similar for pregnant and non-pregnant females and between right and left uterine horns during the evaluation period.…”

Section: Discussionmentioning

confidence: 91%

Laterality of Ovulation and Presence of the Embryo Do Not Affect Uterine Horn Blood Flow During the First Month of Gestation in Llamas

2020

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We determined if laterality of ovulation and intrauterine embryo location differentially induces changes in the mesometrial/endometrial vascularization area (MEVA) between uterine horns, during and after embryo migration, elongation and implantation in llamas. Adult, non-pregnant and non-lactating llamas (n = 30) were subjected to daily B-mode ultrasound scanning of their ovaries. Llamas with a growing follicle ≥8 mm in diameter in the left (n = 15) or right (n = 15) ovary were assigned to a single mating with an adult fertile or vasectomized male. Power-doppler ultrasonography was used to determine the MEVA in a cross section of the middle segment of both uterine horns. MEVA was determined by off-line measurements using the ImageJ software. MEVA measurements were performed before mating (day 0) and on days 5, 10, 15, 20, 25, and 30 after mating in pregnant [llamas with left- (n = 6) or right-sided (n = 6) ovulations] and non-pregnant [llamas with left- (n = 6) or right-sided (n = 6) ovulations] females. Ovulation was confirmed by the disappearance of a follicle (≥8 mm) detected previously. Pregnancy was confirmed by the presence of the embryo proper. MEVA was analyzed by one-way ANOVA for repeated measures using the MIXED Procedure in SAS. If significant (P ≤ 0.05) main effects or interactions were detected, Tukey's post-hoc test for multiple comparisons was used. Ovulation rate did not differ (P = 0.4) between females mated to an intact or vasectomized male and between right- or left-sided ovulations. Three females mated to the intact and 3 to the vasectomized male did not ovulate and were excluded of the study. First observation of fluid inside the gestational sac and of embryo proper, were made exclusively in the left uterine horn, on day 15.8 ± 3.8 and 22 ± 2.7, and 16.7± 2.6 and 27.5 ± 2.8 for pregnant llamas ovulating in the right and left ovary, respectively. Although the MEVA of both uterine horns was affected by time (P < 0.05), it was not affected by physiological status (pregnant vs. non-pregnant; P = 0.9) or laterality of ovulation (P = 0.4). Contrary to expectations, regardless of the laterality of ovulation, in pregnant llamas the left horn did not display a greater MEVA before or after embryo arrival, a trend that was observed during the first 30 days of gestation.

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Evaluation of the effect of mating, intrauterine deposition of raw seminal plasma or seminal plasma purified β-NGF on endometrial vascularization in llamas

Cited by 10 publications

References 47 publications

The effect of seminal plasma β-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

The effect of seminal plasma β-NGF on follicular fluid hormone concentration and gene expression of steroidogenic enzymes in llama granulosa cells

β-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells

Laterality of Ovulation and Presence of the Embryo Do Not Affect Uterine Horn Blood Flow During the First Month of Gestation in Llamas

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