Determining the role of nerve growth factor-beta in seminal plasma on bovine reproduction
Nerve growth factor-β (NGF) is a protein found in the seminal plasma of a variety of mammalian species. While initially known for regulating neuronal survival and differentiation, it was later discovered to interact within the endocrine and reproductive systems as well. Most notable is its function in camelids as an ovulation inducing agent with a dose-dependent luteotrophic effect. While also present in the seminal plasma of spontaneously ovulating species, such as the bull, the role of NGF was initially thought to be limited to regulating sperm physiology. However, upon discovering its effect on ovulation induction in camelids, the potential functions of NGF within the female reproductive system of other species came to light. The luteotrophic effect of NGF is of much interest to the cattle industry where sustainability relies on reproductive success. While many cow studies have investigated factors such as nutrition and estrous cycle manipulation, there appears to still be a bottleneck in improving reproductive efficiency due to early embryonic losses. The majority of conception failure in cattle is thought to occur prior to maternal recognition of pregnancy as a result of luteal insufficiency. When corpus luteum formation and progesterone production are inadequate following fertilization, the conceptus will fail to produce the threshold level of interferon tau needed to stimulate maternal recognition of pregnancy. As a result, luteolysis and embryonic loss will occur. There is, therefore, a need to uncover novel techniques that will minimize these potential losses in cattle. Increasing evidence is available to suggest that seminal plasma proteins play an important role in regulating female fertility, such as the luteotrophic effect of NGF. However, with the rapid expansion of artificial insemination use in the cattle industry, these physiological mechanisms may become lost. Frozen semen used in artificial insemination is extended to achieve the lowest effective breeding dose and maximize the number of straws acquired per collection. However, this practice also dilutes out the seminal plasma proteins to sub-physiological concentrations. Given that many of these proteins, such as NGF, have been associated with bull fertility, more work is needed to understand their specific roles in male and female reproduction. Not only could they improve sperm function, but they may also regulate events throughout ovulation within the female reproductive tract that contribute to successful pregnancies. Therefore, it is necessary to evaluate the effects of NGF within the female reproductive system and as an additive to semen extender to determine its potential usefulness within the cattle industry. Therefore, it was hypothesized that intramuscular administration of purified bovine NGF would improve corpus luteum function and conceptus development in cows. To test this hypothesis, cows underwent estrus synchronization and were randomly allocated to one of two treatment groups: (1) CONT- 12 mL phosphate-buffered saline; or (2) NGF- 296 µg purified NGF in 12 mL phosphate-buffered saline administered intramuscularly at artificial insemination (day 0). Blood collections were performed to measure plasma progesterone concentrations (days 0, 3, 7, 10, 14, 19), plasma pregnancy-specific protein B concentrations (day 24), and expression of interferon-stimulated genes in peripheral blood leukocytes (day 19). Corpus luteum size (days 0, 3, 7, 10, 14, 19), pregnancy status (days 28, 45, 66), and embryonic/fetal crown-rump length (days 28, 45, 66) were assessed using transrectal ultrasonography. It was found that despite no change in corpus luteum size between treatments, NGF cows had increased plasma progesterone levels over CONT cows from days 10 to 19. Pregnancy rates were 75% in NGF cows and 59% in CONT cows. Fold-change expression of interferon-stimulated genes Isg15 and Mx2 at day 19 were greater in pregnant NGF cows than in pregnant CONT cows. Consistently, plasma pregnancy-specific protein B concentrations at day 24 were greater in pregnant NGF than in pregnant CONT cows, and fetal crown rump length tended to be increased in NGF cows at day 66. Herein, it was demonstrated that systemic NGF administration to cows at artificial insemination improves luteal function and enhances conceptus development as determined by upregulation of interferon-stimulated genes and increased pregnancy-specific protein B production. Upon confirming the luteotrophic effect of NGF in cattle, my next interest was uncovering its mechanism of action. Therefore, it was hypothesized that NGF interacts with the pre-ovulatory follicle to increase vascularity and steroidogenesis, which persists throughout ovulation to create a highly functional and vascular corpus luteum. To test this hypothesis, heifers underwent estrus synchronization and were ovariectomized upon formation of a pre-ovulatory follicle. Pieces of follicle wall tissue (theca + granulosa cells) were excised and cultured in media containing luteinizing hormone, follicle stimulating hormone, +/- NGF. Culture media was replaced and frozen at 3, 6, 12, 24, 48, and 72 hours for steroid hormone measurement. Follicle wall tissue pieces were flash-frozen at completion of culture (72 hours) to assess expression of steroidogenic and angiogenic genes. Treatment with NGF increased testosterone production from the follicular tissue throughout the entire culture period compared to untreated controls. There were no differences in progesterone or estradiol production between treatments. Follicle tissue treated with NGF had upregulated steroidogenic enzyme 17-beta-hydroxysteroid dehydrogenase (17β-hsd) gene expression and downregulated fibroblast growth factor 2 (Fgf2) gene expression. Overall, this study demonstrated that NGF interacts directly with the bovine pre-ovulatory follicle to increase thecal cell testosterone production and alter expression of enzymes involved with follicular to luteal reconstruction. Next, heifers underwent estrus synchronization and were randomly allocated to one of two treatment groups: (1) Control- 12 mL phosphate-buffered saline; or (2) NGF- 250 µg purified NGF in 12 mL phosphate-buffered saline administered intramuscularly at day 0 (presence of pre-ovulatory follicle). Transrectal ultrasonography was performed every 4 hours to measure follicle size and vascularity and determine the time to ovulation. Blood was collected every 4 hours up to 32 hours to measure serum estradiol concentrations. After ovulation, transrectal ultrasonography was performed daily to evaluate corpus luteum size and vascularity, and blood was collected every 2 days to measure serum progesterone concentrations. Corpus luteum biopsies were performed on days 9 and 14 to assess gene expression of steroidogenic and angiogenic enzymes, oxytocin, and luteinizing hormone receptor, and to measure the percentage of small luteal cells over large luteal cells. Follicle diameter at treatment was ~13 mm in both groups. There was a main effect of NGF treatment on the pre-ovulatory follicle diameter; however, vascular area and estradiol production were unaffected. The average time to ovulation was 13.8 hours in NGF heifers versus 17.5 hours in CONT heifers. There tended to be an effect of NGF treatment on corpus luteum diameter, but no differences in its vascular area. Serum progesterone concentrations were higher in NGF heifers from days 10 to 12 compared to control heifers. Gene expression of steroidogenic enzymes, steroidogenic acute regulatory protein (Star) and 3-beta-hydroxysteroid dehydrogenase (3β-hsd), were both upregulated in the corpus luteum of NGF heifers, whereas angiogenic enzymes, prostaglandin E2 synthase (Pges) and its receptor (Pger), were downregulated. There was a tendency for increased luteinizing hormone receptor (Lhcgr) gene expression in the corpus luteum of NGF heifers, consistent with an increased percentage of small luteal cells. In conclusion, NGF treatment affected follicle size, which may be attributed to increased follicular edema. Contrary to our hypothesis, NGF did not affect vascularity of the follicle or corpus luteum. Rather, the luteotrophic effect of NGF appears to be mediated through increased small luteal cell number and enhanced steroidogenic function in the corpus luteum. This change is likely secondary to the interactions between NGF and the pre-ovulatory follicle that alter the mechanisms of luteal reconstruction following ovulation. Furthermore, it was hypothesized that NGF is produced predominantly in the bull vesicular gland, is positively associated with sire conception rate scores, and improves sperm cryotolerance. To test this hypothesis, accessory sex glands (ampulla, bulbourethral, prostate, vesicular glands) were harvested post-mortem from mature bulls and either frozen or formalin-fixed for Ngf gene expression and protein localization. Seminal plasma NGF concentrations were measured in the pre-ejaculate and sperm-rich fraction from bulls collected by electroejaculation. Additionally, seminal plasma NGF concentrations were measured in semen collected by artificial vagina at a commercial bull-stud. These bulls were allocated to two categories based on calculated sire conception rate scores: (1) negative/0 deviations; or (2) positive deviations. Lastly, sperm acquired by both electroejaculation and epididymal harvest were incubated with 0 ng/mL (CONT), 0.5 ng/mL (LOW), 5 ng/mL (MED), or 50 ng/mL (HIGH) purified NGF prior cryopreservation to assess its effect on sperm cryotolerance. Computer-aided sperm analysis was used to assess post-thaw motility, whereas flow cytometry was used to evaluate post-thaw sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). Gene expression of Ngf was highest in the vesicular gland, intermediate in the ampulla, and lowest in the prostate and bulbourethral glands. Consistently, highest NGF staining intensity was detected in the ampulla and vesicular glands. Seminal plasma NGF concentrations were higher in the sperm-rich fraction than in the pre-ejaculate and associated with positive sire conception rate scores. Despite these findings, supplementing freezing extender with purified NGF did not improve post-thaw sperm motility, viability, acrosome integrity, or chromatin stability in ejaculated or epididymal derived sperm. Collectively, these results demonstrate that NGF is positively associated with bull fertility, which may be attributed to its beneficial effects within the female reproductive tract rather than on spermatozoal functions alone.
seminal plasma, Bovine, luteotrophic, Nerve growth factor-beta, reproduction
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign