BRK phosphorylates SMAD4 for proteasomal degradation and inhibits tumor suppressor FRK to control SNAIL, SLUG, and metastatic potential.

The tumor-suppressing function of SMAD4 is frequently subverted during mammary tumorigenesis, leading to cancer growth, invasion, and metastasis. A long-standing concept is that SMAD4 is not regulated by phosphorylation but ubiquitination. Our search for signaling pathways regulated by breast tumor kinase (BRK), a nonreceptor protein tyrosine kinase that is up-regulated in ~80% of invasive ductal breast tumors, led us to find that BRK competitively binds and phosphorylates SMAD4 and regulates transforming growth factor-ß/SMAD4 signaling pathway. A constitutively active BRK (BRK-Y447F) phosphorylates SMAD4, resulting in its recognition by the ubiquitin-proteasome system, which accelerates SMAD4 degradation. Activated BRK-mediated degradation of SMAD4 is associated with the repression of tumor suppressor gene FRK and increased expression of mesenchymal markers, SNAIL, and SLUG. Thus, our data suggest that combination therapies targeting activated BRK signaling may have synergized the benefits in the treatment of SMAD4 repressed cancers.

Characteristics of peaks in serum concentrations of follicle-stimulating hormone and estradiol, and follicular wave dynamics during the interovulatory interval in cyclic ewes.

There are three or four ovarian follicular waves in the interovulatory interval of cyclic ewes. Each follicular wave is preceded by a transient peak in serum follicle-stimulating hormone (FSH) concentrations. Serum concentrations of estradiol also increase concurrent with the growth of follicle(s) in each wave. In the current study, we investigated the patterns of follicular wave development and characteristics of FSH and estradiol peaks in all follicular waves of the interovulatory interval and after induction of a supraphysiologic FSH peak in cyclic ewes (Ovis aris). In Experiment 1, 19 ewes underwent daily ovarian ultrasonography and blood sampling for a complete interovulatory interval. In Experiment 2, seven ewes received two administrations of ovine FSH (oFSH), 8h apart (1 microg/kg; sc), at the expected time of the endogenous FSH peak preceding the second follicular wave of the interovulatory interval. In Experiment 1, the amplitude of the FSH peaks decreased (up to 50%), whereas basal serum FSH concentrations increased across the interovulatory interval (P<0.05). Maximum follicular diameter was greater (P<0.05) for Wave 1 and the Ovulatory wave (6.0+/-0.3 and 6.1+/-0.2 mm, respectively) than for Waves 2 and 3 (5.3+/-0.1 and 5.4+/-0.3 mm, respectively). Life span was greater for follicles in Wave 1 compared with other waves (P<0.05). Treatment with oFSH increased the amplitude of an FSH peak by 5- to 6-fold. This treatment increased estradiol production (P<0.05) but had little effect on other characteristics of the subsequent follicular wave. We concluded that changes in the amplitude and duration of the peaks in serum concentrations of FSH that precede follicular waves across the interovulatory interval do not influence the characteristics of the follicular waves that follow.

The effect of parity of the dam on sexual maturation, serum concentrations of metabolic hormones and the response to luteinizing hormone releasing hormone in bull calves.

The objectives of this study were to determine the effect of parity of the dam on age at which a scrotal circumference (SC) ≥ 28 cm was attained and the LH response to Luteinizing Hormone Releasing Hormone (LHRH) in bull calves. We also wanted to confirm, in a large group of bull calves, that the LH response to LHRH could be used to select early maturing bulls. Body weight and SC of the bull calves were measured every other week. At 15, 20 and 25 weeks of age, calves received 4.12 nm/kg body weight of LHRH ischio-rectally and blood samples were taken every 15 min for 4 h. Calves from primiparous and multiparous dams were separated into two sub-groups based on age at which an SC ≥ 28 cm was attained (early or late). An SC ≥ 28 cm was attained earlier in calves born to multiparous as compared with primiparous dams (p < 0.05). At 20 and 25 weeks of age, peak serum LH concentrations (LH-peak) and area under the LH response curve (LH-AUC) in response to LHRH were higher (p < 0.01) in calves born to multiparous as compared with primiparous dams. In calves born to multiparous dams the LH-peak at 15 and 25 weeks of age and the LH-AUC at 15 weeks of age were lower (p < 0.05) in calves that attained an SC ≥ 28 cm early as compared with late. The LHRH-challenge test sensitivity and specificity ranged from 46% to 86%. We concluded that parity of the dam affected age at which SC ≥ 28 cm was attained and the LH response to LHRH in bull calves. Serum LH responses to LHRH at 15 and 25 weeks of age, in calves born to multiparous dams, show some promise for development into a test to select early maturing bull calves.

Postnatal changes in testicular concentrations of transforming growth factors-alpha and-beta 1, 2 and 3 and serum concentrations of insulin like growth factor I in bulls.

Based on work largely in laboratory animals, transforming growth factors (TGF) and insulin like growth factors (IGF) could be regulators of testicular development. The aim of this study was to see if TGF-alpha and -beta 1, 2 and 3 are present in the bovine testis and to monitor concentrations of these factors in the testis and IGF-I in serum during reproductive development. Separate groups of Hereford x Charolais calves (n = 6) were castrated every 4 weeks from 5 to 33 weeks of age and at 56 weeks of age. A week prior to castration, from 5 to 33 weeks of age, blood was collected every 15 min for 10 h. Serum IGF-I concentrations increased from 8 to 12 weeks of age, decreased from 24 to 28 weeks and increased to 32 weeks of age (p < 0.05). Testicular TGF-alpha concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks and from 33 to 56 weeks of age (p < 0.05). Testicular TGF-beta 1 concentrations decreased from 17 to 21 weeks of age, increased to 25 weeks and decreased from 25 to 33 weeks of age (p < 0.05). Testicular TGF-beta 2 concentrations increased from 5 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks and decreased at 29 weeks of age (p < 0.05). Testicular TGF-beta 3 concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks and from 25 to 29 weeks of age (p < 0.05). We concluded that TGF-alpha and TGF-beta 1, 2 and 3 are present in the testis of the bull calf, and changes in concentrations with age suggest a functional role in the development of the testis.

Post-natal changes in testicular concentrations of interleukin-1 alpha and beta and interleukin-6 during sexual maturation in bulls.

Based on observations in laboratory animals interleukins could be regulators of testicular development. The objects of this study were to see if interleukins (IL-1 and IL-6) are present in the developing bull testis and to establish the temporal patterns of concentrations of IL-1 and IL-6 in the bovine testis during development. Separate groups of six bull calves were castrated every 4 weeks from 5 to 33 weeks of age, and at 56 weeks of age. Mean testicular IL-1 alpha concentrations decreased (p < 0.01) from 5 to 9 weeks of age and 13 to 21 weeks of age. Mean testicular IL-1 beta concentrations decreased (p < 0.01) from 13 to 17 weeks of age and from 29 to 33 weeks of age. Mean IL-1 bioactivity increased from 13 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks, decreased to 29 weeks and decreased from 33 to 56 weeks of age (p < 0.05). Mean testicular IL-6 concentrations decreased (p < 0.05) from 9 to 13 weeks of age, increased (p < 0.05) to 21 weeks, decreased (p < 0.05) to 25 weeks, increased (p < 0.05) to 29 weeks and decreased (p < 0.01) to 56 weeks of age. In conclusion, testicular IL-1 alpha, IL-1 beta and IL-6 were found in the bovine testis and concentrations were age dependent. Testicular IL-1 alpha and IL-1 beta concentrations were highest in the early post-natal period; however, IL-1 bioactivity and IL-6 concentrations were greatest in the immediate pre-pubertal period. These findings suggest a functional role for interleukins in testicular development in the bull.

Sexual maturation in the bull.

In this review, we describe the process of sexual maturation in the bull calf. The testes of the bull grow relatively slowly until approximately 25 weeks of age and then a rapid phase of growth occurs until puberty, at 37-50 weeks of age. During the early postnatal phase of slower growth of the testis pre-spermatogonia and some spermatogonia are established, adult Leydig cells appear and undifferentiated Sertoli cells are produced. The rapid testicular growth, after 25 weeks of age, consists of marked increases in the diameter and length of the seminiferous tubules, dramatic proliferation and differentiation of germ cells, with mature spermatozoa occurring between 32 and 40 weeks of age. The adult Leydig cell population is largely in place by 30 weeks of age and that of Sertoli cells by 30-40 weeks of age. Serum concentrations of LH increase from 4 to 5 weeks of age, to an early postnatal peak at 12-16 weeks of age, followed by a decline to 25 weeks of age. Serum FSH concentrations are high postnatally, declining to approximately 25 weeks of age. Serum testosterone concentrations increase during the phase of rapid testicular growth. Hypothalamic opioidergic inhibition may abate transiently to allow the early postnatal increase in LH secretion, while testicular androgenic negative feedback probably contributes to the decline in gonadotropin secretion to 25 weeks of age. Several lines of study have led us to suggest that early postnatal gonadotropin secretion is pivotal in initiating the process of sexual maturation in the bull calf.

Effects of treatment with GnRH from 4 to 8 weeks of age on the attainment of sexual maturity in bull calves.

In bull calves an early transient increase in circulating concentrations of LH occurs between 6 and 20 weeks of age. This has been shown to influence reproductive development and performance later in life. In an attempt to hasten the onset of sexual maturity, bull calves (Hereford x Charolais) were treated (im) with 120 ng/kg of GnRH (n=6) twice every day from 4 to 8 weeks of age; control calves received saline (n=6). Injection of GnRH resulted in an LH pulse in all animals. GnRH treated bulls displayed more rapid testicular growth rates between 22 and 44 weeks of age. Sexual maturity (SC>or=28 cm) was achieved earlier in GnRH treated bulls compared to saline treated bulls (41.7+/-2.22 and 47.0+/-0.45 weeks of age, respectively) and this was confirmed by age of sexual maturity based on ejaculate characteristics (>50 million spermatozoa, >10% motility; 45.0+/-0.86 and 49.0+/-1.13 weeks of age for GnRH and control treated bull calves, respectively; P<0.05). We concluded that treatment with GnRH, twice daily, from 4 to 8 weeks of age, prior to the endogenous early increase in plasma LH concentrations, could increase in plasma LH concentrations, advance testicular development and reduce age at puberty in beef bull calves. This may provide the basis for a simple regimen to hasten sexual development in the bull calf.

Postnatal changes in testicular gonadotropin receptors, serum gonadotropin, and testosterone concentrations and functional development of the testes in bulls.

The primary objectives of this study were to follow the temporal patterns of testicular LH and FSH receptor (LH-R and FSH-R) concentrations and affinity (Ka) during sexual maturation in bulls and to see if such patterns could help explain the control of rapid testicular growth that occurs after 25 weeks of age, when serum gonadotropin concentrations are low. Separate groups of Hereford x Charolais calves (n = 6) were castrated every 4 weeks from 5 to 33 weeks of age and at 56 weeks of age. A week prior to castrations, from 5 to 33 weeks of age, blood was collected every 15 min for 10 h. The transition from indifferent supporting cells to Sertoli cells in seminiferous tubules was rapid between 13 and 25 weeks and rapid testis growth occurred after 25 weeks of age. Serum LH and FSH concentrations were transiently elevated at 12 weeks of age (P < 0.05). LH-R concentrations decreased from 13 to 25 weeks of age and increased to 56 weeks of age (P < 0.05). LH-RKa decreased from 9 to 17 weeks of age, increased to 29 weeks of age and declined to 33 weeks of age (P < 0.05). FSH-R concentrations declined from 17 to 25 weeks of age then increased to 56 weeks of age (P < 0.05). FSH-RKa increased from 17 to 25 weeks of age (P < 0.05). High concentrations of gonadotropins and their receptors may be critical to initiate testis growth postnatally and support it after 25 weeks of age in the face of low serum gonadotropin concentrations.

Changes in serum luteinizing hormone (LH) concentrations in response to luteinizing hormone releasing hormone (LHRH) in bull calves that attained puberty early or late.

The objectives of this study were to determine if the response to luteinizing hormone releasing hormone (LHRH) could be used to select bull calves capable of early sexual maturation and to establish the optimum route and dose of LHRH. In Trial 1, at 4, 10 and 20 week of age, 20 calves were treated iv with 2 microg/kg body weight of LHRH 1 and 5h after commencing a 9-h period of blood sampling. Bulls were separated into early and late maturing (n=10), based on age at puberty (scrotal circumference (SC) of >or=28 cm). At 4 and 20 week of age, peak serum LH concentrations and area under the LH response curve in response to LHRH were lower (P<0.05) in early- versus late-maturing bulls. In Trial 2, calves at 20 week of age were given LHRH as follows: 2 microg/kg body weight iv (n=6), im (n=6) or sc (n=6); 5 microg/kg im (n=6), or ischio-rectally (ir, n=6) or sc (n=6); and 10 microg/kg im (n=6) or sc (n=6). Serum LH concentrations were at a plateau from 30 to 165 min after treatment with 5 microg/kg of LHRH (im or ir; P>0.05). We concluded that the LH responses to LHRH in calves at 4 and 20 week of age could facilitate the development of a simple test (one blood sample prior to treatment with LHRH and a second during the period of sustained response to LHRH) to select early-maturing bulls.

Patterns of expression of steroidogenic enzymes during the first wave of the ovine estrous cycle as compared to the preovulatory follicle.

The expression patterns of steroidogenic enzymes in ovarian antral follicles at various stages of growth in a follicular wave have not been reported for sheep. Ovaries were collected from ewes (n=4-5 per group) when the largest follicle(s) of the first wave of the cycle, as determined by ultrasonography, reached (i) 3 mm, (ii) 4 mm, (iii) > or =5 mm in diameter or when there was a single (iv) preovulatory follicle in the last wave of the cycle, 12h after estrus detection. The expression pattern of steroidogenic enzymes was quantified using immunohistochemistry and grey-scale densitometry. The expression of CYP19 in the granulosa and 3beta-HSD and CYP17 in the theca increased (P<0.01) progressively from 3 to > or =5 mm follicles in the first wave of the cycle and was lower (P<0.01) in the preovulatory follicle compared to > or =5 mm follicles. However, the expression of 3beta-HSD in the granulosa increased (P<0.05) from 3 to > or =5 mm follicles and was maintained (P<0.05) at a high level in the preovulatory follicles. The amount of CYP19 in the granulosa of the growing follicles correlated positively (r=0.5; P<0.03) with the concurrent serum estradiol concentrations. We concluded that the expression pattern of steroidogenic enzymes in theca and granulosa of follicles growing in each wave in the ewe, paralleled with serum estradiol concentrations, with the exception that concentrations of 3beta-HSD in granulosa increased continuously from follicles 3mm in diameter to the preovulatory follicle.

Does injection of prostaglandin F(2alpha) (PGF2alpha) cause ovulation in anestrous Western White Face ewes?

In a previous study in our laboratory, treatment of non-prolific Western White Face (WWF) ewes with PGF(2 alpha) and intravaginal sponges containing medroxyprogesterone acetate (MAP) on approximately Day 8 of a cycle (Day 0 = first ovulation of the interovulatory interval) resulted in ovulations during the subsequent 6 days when MAP sponges were in place. Two experiments were performed on WWF ewes during anestrus to allow us to independently examine if such ovulations were due to the direct effects of PGF(2 alpha) on the ovary or to the effects of a rapid decrease in serum concentrations of progesterone at PGF(2 alpha)-induced luteolysis. Experiment 1: ewes fitted with MAP sponges for 6 days (n = 12) were injected with PGF(2 alpha) (n = 6; 15 mg im), or saline (n = 6) on the day of sponge insertion. Experiment 2: ewes received progesterone-releasing subcutaneous implants (n = 6) or empty implants (n = 5) for 5 days. Six hours prior to implant removal, all ewes received a MAP sponge, which remained in place for 6 days. Ewes from both experiments underwent ovarian ultrasonography and blood sampling once daily for 6 days before and twice daily for 6 days after sponge insertion. Additional blood samples were collected every 4 h during sponge treatment. Experiment 1: 4-6 (67%) PGF(2 alpha)-treated ewes ovulated approximately 1.5 days after PGF(2 alpha) injection; these ovulations were not preceded by estrus or a preovulatory surge release of LH, and resulted in transient corpora hemorrhagica (CH). The growth phase was longer (P < 0.05) and the growth rate slower (P < 0.05) in ovulating versus non-ovulating follicles in PGF(2 alpha)-treated ewes. Experiment 2: in ewes given progesterone implants, serum progesterone concentrations reached a peak (1.7 2 ng/mL; P < 0.001) on the day of implant removal and decreased to basal concentrations (<0.17 ng/mL; P < 0.001) within 24 h of implant removal. No ovulations occurred in either the treated or the control ewes. We concluded that ovulations occurring after PGF(2 alpha) injection, in the presence of a MAP sponge, could be due to a direct effect of PGF(2 alpha) at the ovarian level, rather than a sudden decline in circulating progesterone concentrations.

Effects of treatment with LH or FSH from 4 to 8 weeks of age on the attainment of puberty in bull calves.

A transient increase in gonadotropin secretion between 6 and 20 weeks of age is critical for the onset of puberty in bull calves. To try and hasten the onset of puberty, bull calves were treated (s.c.) with 3 mg of bLH (n = 6) or 4 mg of bFSH (n = 6) once every 2 days, from 4 to 8 weeks after birth; control calves received saline (n = 6). At 4 and 8 weeks of age, mean LH concentrations were higher (P < 0.05) in bLH-treated (2.3 +/- 0.04 ng/ml and 1.20 +/- 0.04 ng/ml) as compared to control calves (0.50 +/- 0.1 ng/ml and 0.70 +/- 0.10 ng/ml). Mean serum FSH concentrations at 4 and 8 weeks of age, were higher (P < 0.05) in bFSH-treated (1.60 +/- 0.20 ng/ml and 1.10 +/- 0.2 ng/ml) as compared to control calves (0.38 +/- 0.07 ng/ml and 0.35 +/- 0.07 ng/ml). The age at which scrotal circumference (SC) first reached > or = 28 cm, occurred earlier (P < 0.05) in bFSH-treated calves as compared to saline-treated calves (39.3 +/- 1.3 and 44.8 +/- 1.3 weeks of age, respectively). Based on testicular histology at 56 weeks of age, treatment with bFSH resulted in greater (P < 0.05) numbers of Sertoli cells (5 +/- 0.2, 6 +/- 0.3 and 5 +/- 0.3 in bLH-, bFSH- and saline-treated calves, respectively); elongated spermatids (42 +/- 2, 57 +/- 8 and 38 +/- 5 in bLH-, bFSH- and saline-treated calves, respectively) and spermatocytes (31 +/- 3, 38 +/- 3 and 29 +/- 2 in bLH-, bFSH- and saline-treated calves, respectively) per seminiferous tubule. We concluded that treatment of bull calves with bFSH from 4 to 8 weeks of age increased testicular growth (SC); hastened onset of puberty (SC > or = 28 cm); and enhanced spermatogenesis.