Injection Of Follicular Fluid Research Articles

Abstract TMIM-074: IGF2 IN HUMAN FOLLICULAR FLUID PROMOTES LYMPHOMAGENESIS IN MAMMARY FAT PAD OF TRP53-NULL MICE, DEPENDING ON ESTROGEN IN MICROENVIRONMENT

Abstract Ovulation overdrive is the most important risk factor of ovarian cancer. In previous study we discovered that preovulatory follicular fluid (FF) collected from IVF women, upon weekly injections into the mammary fat pad of Trp53-null mice for 7 weeks, could induce early onset of B cell lymphomas. In subsequent studies (Abstract by Chu TY et al.), we disclosed the IGF-axis signaling conferred by FFs is responsible for the stemness, clonal expansion and transformation of fallopian tube fimbrial epithelial cells where IGF2 in FF bound IGF-1R on secretory cells to activate the downstream PI3K/Akt/mTOR and PI3K/Akt/NANOG signaling pathways for the transformation. In this study, we identified the same IGF2 is also responsible for the FF-induced lymphomagenesis in Trp53-null mice. When IGF2 was depleted from FF, the tumorigenesis rate reduced from 10/18 (56%) to 1/6 (17%). On the other hand, injections with pure IGF2 (100 mg/ml) grew tumors in 33% (2/6) of mice. The same injection to Trp53 wild type did not grow tumor. We also found a microenvironment of adipose tissue seemed to be important for the FF-induced lymphomagenesis. The same injections of FF into the subcutis and other sites did not grow tumor. Additionally, tumor development depends not only on the injection site but also on the female sex hormone and ER function. No tumor growth was observed in castrated female mice and in male mice. Adding fulvastrant to the injections resulted in complete shrinkage of tumor. Meanwhile, extended injection of FF from 7 weeks to 13 weeks promoted tumor progression but did not increase tumor incidence. The findings suggest a transformation activity of human preovulatory FF largely conferred by IGF2. In the context of germline Trp53 loss, this activity likely promotes expansion of the lymphocytic progenitors that have already been transformed. Meanwhile, we found ER is expressed in the tumor adjacent adipocytes but not in the lymphoma cells. Also, ER antagonist, although largely confined tumor growth, did not decrease the tumor incidence. In this tumorigenesis model conferred by Trp53 loss and FF-IGF2, estrogen seems to play an essential role in tumor progression but not in tumor initiation, and this promotion effect is through the adipocyte microenvironment. Citation Format: Hsuan-Shun Huang, Sung-Chao Chu, Tang-Yuan Chu. IGF2 IN HUMAN FOLLICULAR FLUID PROMOTES LYMPHOMAGENESIS IN MAMMARY FAT PAD OF TRP53-NULL MICE, DEPENDING ON ESTROGEN IN MICROENVIRONMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-074.

Direct effects of ovine follicular fluid on ovarian steroid secretion and expression of markers of cellular differentiation in sheep.

The objective of this study was to assess the effect of ovine follicular fluid (FF) treatment (with or without FSH replacement) during the late follicular phase on plasma concentrations of gonadotrophins and the development of the ovulatory follicle. Ovarian steroid secretion and expression of mRNA encoding inhibin alpha and beta A, beta B subunits, P450 aromatase and P450 17 alpha-hydroxylase were used as endpoints. After induction of luteolysis by injection of 100 micrograms cloprostenol on days 10-12, Scottish Blackface ewes were allocated to one of three groups: (1) control (n = 7): no further treatment; (2) FF (n = 9): subcutaneous injections of 3 ml steroid-free ovine follicular fluid at 9 h intervals, 18 and 27 h after cloprostenol injection; (3) FF + FSH (n = 8): injections of follicular fluid as above plus subcutaneous injections of 0.36 iu ovine FSH at 6 h intervals, 18, 24, and 30 h after cloprostenol injection. Jugular venous blood samples were obtained via indwelling cannulae at 6 h intervals from 0 to 36 h after cloprostenol injection, and at 10 min intervals from 12 to 18 h (control phase) and from 30 to 36 h after cloprostenol injection (treatment phase). At laparotomy, 36 h after cloprostenol injection, ovarian venous blood was collected and ovaries were removed and processed for in situ hybridization. Plasma concentrations of FSH, luteinizing hormone (LH) and oestradiol were determined by radioimmunoassay. Follicular fluid treatment resulted in a decrease (P < 0.001) in FSH concentrations associated with an acute decrease in ovarian steroid secretion (P < 0.01) and a specific depression in P450 aromatase, (P < 0.001), inhibin-activin beta B subunit (P < 0.05) and thecal LH receptor (P < 0.001) expression. Follicular fluid treatment had no effect on inhibin-activin alpha and beta A, subunit or P450 17 alpha-hydroxylase expression. FSH co-treatment with follicular fluid restored circulating FSH concentrations to normal values and reversed some of the effects of follicular fluid (androstenedione, testosterone and progesterone secretion, and inhibin beta B and thecal LH receptor expression) but not oestradiol secretion or P450 aromatase expression. It was concluded that the actions of follicular fluid are mediated via both central effects on pituitary FSH secretion and by direct ovarian effects on granulosa cell aromatase activity. The results indicate that follicular fluid contains a factor that inhibits aromatase activity of granulosa cells directly and may play a role in the selection of the dominant follicle.

Effects of the presence of a dominant follicle and exogenous oestradiol on the duration of the luteal phase of the bovine oestrous cycle.

The presence of a developing dominant follicle may be a factor in the control of the luteolytic cascade mechanism and the number of follicular waves during the bovine oestrous cycle. In this study, ovaries of all animals were examined once a day by transrectal ultrasonography. It was expected that heifers (n = 18) would have two follicular waves if the second wave occurred later than day 10 after oestrus (Expt 1) and that cows (n = 14) would have three waves if the second wave occurred on or before day 10 (Expt 2). The objective of Expt 1 was to determine if absence of a large follicle late in the luteal phase delays luteal regression in heifers that are expected to have two follicular waves. Nine heifers were injected i.v. with 10 ml charcoal-treated bovine follicular fluid three times a day for 4 days, starting on the day after initiation of the second follicular wave, to delay growth of the second wave dominant follicle. Nine heifers were injected with 0.9% NaCl as controls. The duration of the luteal phase (calculated as the number of days that serum progesterone was > 0.5 ng ml-1) was greater (P < 0.01) in the follicular fluid-treated group compared with the controls (18.7 versus 14.1 days). FSH and follicular growth were suppressed during the period of injection of follicular fluid (P < 0.01 and 0.03, respectively). The objective of Expt 2 was to determine the effect of increased oestradiol on the duration of the luteal phase in cows that were expected to have three follicular waves. Seven cows were injected i.m. three times a day for 4 days with 1 ml oestradiol (100 micrograms ml-1 in corn oil) and seven cows were similarly injected three times a day with 1 ml 0.9% NaCl (control) starting the day after cessation of growth of the second wave dominant follicle. Luteal phase duration was shorter in oestradiol-treated animals than in the controls (14.0 versus 19.0 days; P < 0.04). Serum oestradiol concentrations were higher in the oestradiol-treated group during the period of injection (P < 0.01). In summary, luteolysis was delayed when follicular growth was suppressed with follicular fluid (Expt 1). Exogenous oestradiol administration during the development of uterine oestradiol responsiveness initiated luteolysis earlier compared with control animals (Expt 2).

Effects of charcoal-extracted follicular fluid on reproductive function in postpartum cows

In order to determine the role of follicle-stimulating hormone (FSH) on the resumption of ovarian function in cows early postpartum (PP), bovine follicular fluid (FF) was used to selectively suppress concentrations of FSH. Calves were removed from all cows within 24 hr of birth. Follicular fluid that was treated with charcoal to remove steroids (15 ml; n=14) or serum (S) from an ovariectomized cow (15 ml, n=14) was injected i.m. twice daily from days 1 to 10 PP. Blood samples were collected before each injection and frequent samples (every 15 min for 6 hr) taken on days 5 and 10 PP. Eight cows from each group (FF and S) were slaughtered on the morning of day 11 PP and pituitaries and ovaries collected. The remaining cows (n=6) were observed for estrus. Treatment with FF delayed follicular growth (P<0.01), as evidenced by the largest follicle per cow observed at time of slaughter (3.6 ± 0.42 vs 11.5 ± 1.77 mm dia; FF vs S). The intervals from parturition to first estrus (P<0.11) and to first progesterone rise (25.3 ± 1.97 vs 18.0 ± 3.62 d; P<0.06) tended to be delayed by treatment with FF vs S. Many of the cows treated with S ovulated by day 10 PP, they were divided retrospectively into those that had ovulated by (n=9) or after (n=5) day 10 PP for analysis. Cows treated with FF had lower (P<0.05) and less variable (P<0.01) serum FSH concentrations while levels of luteinizing hormone (LH) tended (P<0.08) to be greater on days 5 and 10 PP. Follicular fluid decreased levels of FSH (P<0.001), but not LH (P>0.15), in the samples obtained twice daily compared to S-treated cows that did not ovulate by day 10 PP. Anterior pituitaries were dissociated, and cells from each cow were cultured in order to ascertain whether treatment with FF in vivo would affect gonadotropin secretion in vitro. Estradiol-17β (E) was incubated with pituitary cells to determine the effect of E on gonadotropin secretion from cells of PP cows, and to ascertain whether treatment with FF in vivo and with E in vitro would interact to affect secretion of FSH and LH in culture. After 2 d of incubation, cells were treated with 10 −9 M E or vehicle (1% ethanol). Two days later, cells were treated with gonadotropin-releasing hormone (GnRH) and after 6 hr, medium collected. Injections of FF did not affect GnRH-induced release of FSH or LH from pituitary cells in culture (P>0.20). Incubation with E decreased the release of LH in response to GnRH (P<0.005). There was a greater suppression of GnRH-induced LH release due to E pretreatment in cells from FF-treated cows than in cells from cows treated with S (P<0.03). Thus, treatment with FF decreased FSH concentrations and delayed follicular development and ovulation, even in the presence of elevated or control levels of LH. Therefore, FSH does play a role in folliculogenesis in PP cows. Based on results obtained in vitro, we conclude that E inhibited LH secretion and this phenomenon may be inherent to the PP animal. Even though FF treatment in vivo did not significantly decrease the secretion of FSH or LH in vitro, it did synergize with E resulting in a much greater suppression of LH than with either E or FF alone.

A simple in vivo bioassay for inhibin-like activity using ovariectomized ewes

Long term ovariectomized ewes were used in a bioassay for inhibin-like activity. The concentration of FSH 6 to 7 hr after injection of follicular fluid (a rich source of inhibin), as a percentage of pretreatment, regressed on the log of the dose had a slope of −26.0 ± 7.6 (5 replications, mean ± SD) and an index of precision of .32 ± .04. This system was rapid, relatively easy and specific for in vivo inhibin-like activity. This bioassay was also used to determine the relative potency of an affinity-purified fraction of follicular fluid.

Changes in FSH and the pulsatile secretion of LH during treatment of ewes with bovine follicular fluid throughout the luteal phase of the oestrous cycle.

Treatment of Damline ewes with twice-daily i.v. injections of bovine follicular fluid during the luteal phase for 10 or 2 days before prostaglandin-induced luteolysis resulted in a delay in the onset of oestrous behaviour and a marginal increase in ovulation rate. During the treatment cycle, blood samples were withdrawn at 15-min intervals for 25 h from 08.00 h on days 1, 6 and 10 (day 0 = oestrus). At all three stages of the luteal phase, plasma FSH concentrations were suppressed relative to controls 3 h after the 09.00 h injection of follicular fluid and remained low until 06.00 h on the following day. In the 10-day treatment group LH pulse amplitude was significantly greater than that of controls on days 6 and 10. Pulse frequency remained high throughout treatment and was significantly higher relative to controls on day 10 despite normal progesterone levels. The results suggest that the higher pulsatile LH secretion during the luteal phase is due to reduced negative feedback effects of oestradiol occurring as a result of the follicular fluid-induced reduction in FSH.

The effects of food restriction and hypophysectomy on numbers of primordial follicles and concentrations of hormones in rats.

Three experiments were done to examine the effects of food restriction, beginning at 21 days of age, on loss of primordial follicles and on concentrations of gonadotropins and sex steroids in rats. In Experiment 1, food restriction (FR) from 21 to 51-55 days of age had no effect on number of primordial follicles, but increased the plasma concentration (p less than 0.05) of follicle stimulating hormone (FSH). (p less than 0.05). In Experiment 2, comparisons were made of groups of rats (1) fed ad libitum (AL) (2) hypophysectomized at 21 days of age and fed ad libitum (AL-HY), (3) food restriction from 21 to 52-58 days of age (FR), and (4) food restriction with twice-daily injections of follicular fluid (FR-FF). Hypophysectomy was the only treatment that decreased the loss of primordial follicles (p less than 0.001). Concentrations of FSH were decreased in AL-HY and increased in FR and FR-FF rats (144 +/- 13, 53 +/- 15, 275 +/- 30 and 359 +/- 56 ng/ml in AL, AL-HY, FR and FR-FF rats, respectively). Concentrations of luteinizing hormone (LH) were lower (p less than 0.05) in AL-HY, FR and FR-FF rats than in AL rats. In Experiment 3, AL and FR rats were unilaterally ovariectomized (ULO) at 30 days of age. Blood samples were taken 5 days prior to ULO, at ULO and at 12 h, 5 days, and 22-28 days after ULO.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of changes in FSH induced by bovine follicular fluid and FSH infusion in the preovulatory phase on subsequent ovulation rate and corpus luteum function in the ewe.

Treatment of Damline ewes with i.v. injections of various doses (2, 5 or 10 ml) of bovine follicular fluid for 72 h after prostaglandin-induced luteal regression resulted in a significant decrease in plasma concentrations of FSH after a 1.5-2 h delay but did not affect LH. The half life of this decrease in plasma FSH levels (156 min) after injection of follicular fluid was similar to that for clearance (159 min) of ovine FSH after infusion. A significant rebound increase in plasma FSH levels occurred by 13 h after all follicular fluid injections, and the magnitude of this rebound was inversely related to the dose of follicular fluid injected. A significant delay in the onset of oestrus occurred only with 5 and 10 ml bovine follicular fluid. There was no significant effect on ovulation rate or subsequent corpus luteum function as measured by plasma concentrations of progesterone. Infusion of ovine FSH (50 micrograms/h for 48 h) during the period of follicular fluid treatment prevented the delay in onset of oestrus and resulted in a substantial (2-10-fold) increase in ovulation rate. Corpus luteum function in terms of progesterone secretion was also enhanced. These results show that (1) intermittent suppression of FSH during the preovulatory period in the ewe does not affect subsequent ovulation rate or corpus luteum function and (2) the delay in the onset of oestrus induced by bovine follicular fluid can be prevented by exogenous FSH.

Increase in ovulation rate after treatment of ewes with bovine follicular fluid in the luteal phase of the oestrous cycle.

Administration of charcoal-treated bovine follicular fluid to Damline ewes twice daily (i.v.) from Days 1 to 11 of the luteal phase (Day 0 = oestrus) resulted in a delay in the onset of oestrous behaviour and a significant increase in ovulation rate following cloprostenol-induced luteolysis on Day 12. During follicular fluid treatment plasma levels of FSH in samples withdrawn just before injection of follicular fluid at 09:00 h (i.e. 16 h after previous injection of follicular fluid) were initially suppressed, but by Day 8 of treatment had returned to those of controls. However, the injection of follicular fluid at 09:00 h on Day 8 still caused a significant suppression of FSH as measured during a 6-h sampling period. Basal LH levels were higher throughout treatment due to a significant increase in amplitude and frequency of pulsatile secretion. After cloprostenol-induced luteal regression at the end of treatment on Day 12, plasma levels of FSH increased 4-fold over those of controls and remained higher until the preovulatory LH surge. While LH concentrations were initially higher relative to those of controls, there was no significant difference in the amount of LH released immediately before or during the preovulatory surge. These results suggest that the increase in ovulation rate observed during treatment with bovine follicular fluid is associated with the change in the pattern of gonadotrophin secretion in the luteal and follicular phases of the cycle.

Effects of Testicular and Ovarian Inhibin‐Like Activity, Using In Vitro and In Vivo Systems

Inhibin‐like activities in charcoal‐treated bovine follicular fluid (FF) and medium from cultured Sertoli cells (SCCM) were assayed in an in vitro bioassay system, using cultured pituitary cells. Addition of both fluids resulted in parallel dose‐dependent decreases of the concentration of follicle‐stimulating hormone (FSH) in the medium, both in the presence or absence of luteinizing hormone‐releasing hormone (LH‐RH).A single injection of FF into immature and adult male and female rats resulted in decreased peripheral levels of FSH, but not of LH, after 4 or 8 h. This decrease was larger and occurred faster in adult female rats than in prepubertal female animals. Injection of FF into adult female rats, immediately after unilateral ovariectomy (ULO) prevented the specific increase of FSH levels, occurring in control animals. This suppression could not be obtained after treatment with steroids.Daily treatment of adult female or immature male rats for periods longer than 5 days did not result in prolonged suppression of circulating FSH concentrations; LH levels were significantly increased. The female animals showed cyclic vaginal smear changes and normal ovulation; in the male rats testis weight and numbers of spermatogenic cells were reduced.It is concluded that testicular and ovarian inhibin‐like activities have similar properties. Injections of FF into male and female rats cause similar effects on FSH and LH. The effect of FF in ULO‐animals suggests that inhibin could play a role in the short‐term regulation of the number of developing follicles in the ovary. Injection of FF into male rats causes a probably transient impairment of spermatogenesis through an initial suppression of FSH.

Mucification of the Vaginal Epithelium of Immature Mice Following Injections of Follicular Fluid

Mucification of the Vaginal Epithelium of Immature Mice Following Injections of Follicular Fluid