Lipopolysaccharide effects on activation and apoptosis of primordial ovarian follicles in heifers / Efeitos de lipopolissacarídeos na ativação e apoptose de folículos ovarianos primordiais em novilhas

The objective of the present study was to evaluate the effect of lipopolysaccharide (LPS) administration on activation and apoptosis of primordial follicles. There was no difference in the total number of follicles as well as in the different types of follicles. Furthermore, the LPS challenge didn't modulate the expression of genes related with ovarian reserve (HAM), oocyte survival (Survivin), activation rate (Pten, KIT, KITL1, KITL2, AKT1, SIRT1), and follicular abnormalities. Therefore, the LPS exposure with 24h interval had no effect on activation rate and primordial follicles abnormalities, and also had no effect on expression of anti-apoptotic genes and genes related with ovarian reserve, oocyte survival, activation rate, and primordial follicles abnormalities.

O objetivo do presente estudo foi avaliar o efeito da administração de lipopolissacarídeo (LPS) na ativação e a apoptose de folículos primordiais. Dez novilhas saudáveis (Bos taurus taurus), com idade média de 14 meses, alojadas em sistema de confinamento e alimentadas com TMR, foram utilizadas neste experimento. Os animais foram distribuídos aleatoriamente em dois grupos: grupo LPS (LPS; n = 5), que recebeu duas injeções intravenosas de 0,5µg/kg de peso corporal de lipopolissacarídeo (Sigma Aldrich®) diluído em 2mL de solução salina (0,9% de NaCl), com intervalo de 24h; e grupo controle (CTR; n = 5), que recebeu duas injeções intravenosas de 2mL de solução salina (0,9% de NaCl), com intervalo de 24h. A primeira injeção de LPS foi realizada no d 1, e no d 5 os animais foram abatidos, os ovários foram pesados e as amostras dos ovários foram coletadas para avaliação histológica e molecular. Não houve diferença no número total de folículos, bem como nos diferentes tipos de folículos. Além disso, o desafio com LPS não modulou a expressão de genes relacionados à reserva ovariana (HAM), à sobrevivência oocitária (Survivin), à taxa de ativação (Pten, KIT, KITL1, KITL2, AKT1, SIRT1) e às anormalidades foliculares. Portanto, a exposição ao LPS com intervalo de 24h não teve efeito sobre a taxa de ativação e as anormalidades dos folículos primordiais, bem como não teve efeito sobre a expressão de genes antiapoptóticos e de genes relacionados com a reserva ovariana, a sobrevivência oocitária, a taxa de ativação e as anormalidades dos folículos primordiais.

Natriuretic peptide system regulation in granulosa cells during follicle deviation and ovulation in cattle.

Natriuretic peptides (NPs) are known to regulate reproductive events in polyovulatory species, but their function and regulation in monovulatory species remain to be fully characterized. Using a well-established in vivo model, we found that bovine granulosa cells from follicles near the deviation stage express mRNA for the three NP receptors (NPR1, NPR2 and NPR3), but not for NP precursors (NPPA, NPPB and NPPC). The abundance of NPR3 mRNA was higher in dominant compared to subordinate follicles at the expected time of follicular deviation. After deviation, mRNA for all NP receptors was significantly more abundant in the dominant follicle. Intrafollicular inhibition of oestrogen receptors downregulated NPR1 mRNA in dominant follicles. In granulosa cells from preovulatory follicles, NPPC mRNA increased at 3 and 6 h after systemic GnRH treatment, but decreased at 12 and 24 h to similar levels observed in samples collected at 0 h. After GnRH treatment, NPR1 mRNA was upregulated at 24 h, NPR3 mRNA gradually decreased after 3 h, while NPR2 mRNA was not regulated. The mRNA expression of the enzyme FURIN increased at 24 h after GnRH treatment. These findings revealed that the expression of mRNA encoding important components of the NP system is regulated in bovine granulosa cells during follicular deviation and in response to GnRH treatment, which suggests a role of NP system in the modulation of these processes in monovulatory species.

Expression of paraoxonase types 1, 2 and 3 in reproductive tissues and activity of paraoxonase type 1 in the serum and seminal plasma of bulls.

The paraoxonases types 1, 2 and 3 (PON1, PON2 and PON3, respectively) are enzymes that degrade lipid peroxides, preventing oxidative damages relevant for male reproductive function. This study determined the expression of those three paraoxonases in reproductive tissues of bulls and evaluated correlations among the activity of PON1 in the serum and seminal plasma with breeding soundness parameters in bulls. The expression of PON1, PON2 and PON3 was characterised by RT-PCR in samples of testicular parenchyma, vesicular glands and epididymis collected from three slaughtered bulls. All three paraoxonases were expressed in the testicular parenchyma, PON2 and PON3 were both expressed in the epididymis head and PON3 was also expressed in the epididymis tail. The PON1 activity was determined in samples of serum and seminal plasma from 110 bulls submitted to breeding soundness evaluation. There was a strong correlation (r = .90) between the activity of the PON1 in both serum and seminal plasma (p < .0001). The PON1 activity in the seminal plasma was positively correlated with ejaculate's colour, sperm mass activity (p = .04), motility, vigour and viability (all p < .01). Thus, PON1 may be a potential marker for sperm motility and viability in bulls.

mRNA expression profile of the TNF-α system in LH-induced bovine preovulatory follicles and effects of TNF-α on gene expression, ultrastructure and expansion of cumulus-oocyte complexes cultured in vitro.

This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor9 (GDF9) gene are associated to sterility or,paradoxically, increased ovulation rate in ewes. Despiteits importance, the exact function of GDF9 in ovarianphysiology is still poorly understood. This study aimedto investigate GDF9 function during dominant folliclegrowth and its regulation in follicular fluid. Theregulation of GDF9 receptors in GnRH/LH-stimulatedgranulosa cells was also investigated. In a firstexperiment, a new follicular wave was induced and theintrafollicular GDF9 treatment into the largest growingfollicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml(n = 4) had no effect on follicular growth, estrusmanifestation and ovulation compared to control (PBSinjected)follicles (n = 3). In a second experiment,follicles were obtained just after follicular deviation(day 4 after follicular emergence) and the abundance ofGDF9 in follicular fluid did not differ between healthydominant (n = 4) and atretic subordinate follicles (n =4), as assessed by western blot analysis. Finally, mRNAexpression of BMPR2 and TGFBR1 receptors wasevaluated in granulosa cells obtained from preovulatoryfollicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 hafter i.m. GnRH administration (n = 4-5follicles/moment). Both receptors were significantly upregulated 12 h after GnRH treatment. Present results donot confirm the hypothesis that GDF9 inhibits dominantfollicle growth and suggests a minor role in determiningfollicle fate. In the other hand, GDF9 receptorsregulation in GnRH/LH-stimulated granulosa cellsprovides the first in vivo evidence of its involvement inthe complex cascade of events that culminates inovulation and luteinization in cattle.(AU)

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor9 (GDF9) gene are associated to sterility or,paradoxically, increased ovulation rate in ewes. Despiteits importance, the exact function of GDF9 in ovarianphysiology is still poorly understood. This study aimedto investigate GDF9 function during dominant folliclegrowth and its regulation in follicular fluid. Theregulation of GDF9 receptors in GnRH/LH-stimulatedgranulosa cells was also investigated. In a firstexperiment, a new follicular wave was induced and theintrafollicular GDF9 treatment into the largest growingfollicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml(n = 4) had no effect on follicular growth, estrusmanifestation and ovulation compared to control (PBSinjected)follicles (n = 3). In a second experiment,follicles were obtained just after follicular deviation(day 4 after follicular emergence) and the abundance ofGDF9 in follicular fluid did not differ between healthydominant (n = 4) and atretic subordinate follicles (n =4), as assessed by western blot analysis. Finally, mRNAexpression of BMPR2 and TGFBR1 receptors wasevaluated in granulosa cells obtained from preovulatoryfollicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 hafter i.m. GnRH administration (n = 4-5follicles/moment). Both receptors were significantly upregulated 12 h after GnRH treatment. Present results donot confirm the hypothesis that GDF9 inhibits dominantfollicle growth and suggests a minor role in determiningfollicle fate. In the other hand, GDF9 receptorsregulation in GnRH/LH-stimulated granulosa cellsprovides the first in vivo evidence of its involvement inthe complex cascade of events that culminates inovulation and luteinization in cattle.

Regulation of Anti-Müllerian Hormone and Its Receptor Expression around Follicle Deviation in Cattle.

The anti-Müllerian hormone (AMH) is an important marker of ovarian reserve and for predicting the response to superovulatory treatments in several species. The objective of this study was to investigate whether AMH and its receptor (AMHR2) are regulated in bovine granulosa cells during follicular development. In the first experiment, granulosa cells were retrieved from the two largest follicles on days 2 (before), 3 (at the expected time) or 4 (after deviation) of follicular wave. In the second experiment, four doses of FSH (30, 30, 20 and 20 mg) or saline were administered twice a day starting on Day 2 of the first follicular wave of the cycle. Granulosa cells and follicular fluid were collected from the two largest follicles 12 h after the last injection of FSH or saline. AMH mRNA abundance was similar in granulosa cells of the two largest follicles (F1 and F2) before deviation (Day 2), but greater in dominant (DF) than subordinate follicles (SF) at the expected time (Day 3) and after (Day 4) deviation (p < 0.05). In experiment 1, AMH mRNA levels declined in both DF and SF near the expected time and after deviation when compared to before deviation. There was no difference in AMHR2 mRNA levels before and during follicular deviation (p > 0.05), but they tended to be greater in DFs than SFs (p < 0.1) after deviation. Experiment 2 showed that AMH and AMHR2 mRNA in granulosa cells and AMH protein abundance in follicular fluid were similar (p > 0.05) between both co-dominant follicles collected from the FSH-treated cows. These findings indicate the followings: AMH mRNA levels decrease in both DFs and SFs during follicular deviation; granulosa cells from heathy follicles express more AMH mRNA compared to subordinate follicles undergoing atresia and FSH stimulates AMH and AMHR2 mRNA expression in granulosa cells of co-dominant follicles.

Technique to simultaneously evaluate ram sperm morphology, acrosome and membrane integrity

Several laboratory methods to evaluate ram sperm quality have been developed. The combination of different fluorescent probes is suitable to simultaneously evaluate different sperm characteristics but the need fora fluorescent microscope restricts its use. The Aim of the present study was to evaluate the efficacy of Hypoosmotic Trypan Blue Giemsa (HTBG) staining to simultaneously detect sperm morphological abnormalities, plasma and acrosomal membrane integrity using phase contrast and fluorescence microscopy as the gold standards. Samples from twelve fresh ejaculates from three rams (4 ejaculates/ram) were used in the study. Sperm cells were evaluated using HTBG, phase contrast (PC) and fluorescent (FLUO) techniques. HTBG was more effective in detecting sperm defects when compared to PC (P < 0.05). No significant differences (P > 0.05) were observed between HTBG and FLUO in the assessment of plasmatic membrane and acrosome integrity. High correlation between HTBG and FLUO techniques was observed when assessing plasma membrane and acrosome (R = 0.97 and 0.96, respectively). In conclusion, the HTBG staining is suitable to assess ram sperm morphology, plasma and acrossomal membrane integrity simultaneously.(AU)

Technique to simultaneously evaluate ram sperm morphology, acrosome and membrane integrity

Several laboratory methods to evaluate ram sperm quality have been developed. The combination of different fluorescent probes is suitable to simultaneously evaluate different sperm characteristics but the need fora fluorescent microscope restricts its use. The Aim of the present study was to evaluate the efficacy of Hypoosmotic Trypan Blue Giemsa (HTBG) staining to simultaneously detect sperm morphological abnormalities, plasma and acrosomal membrane integrity using phase contrast and fluorescence microscopy as the gold standards. Samples from twelve fresh ejaculates from three rams (4 ejaculates/ram) were used in the study. Sperm cells were evaluated using HTBG, phase contrast (PC) and fluorescent (FLUO) techniques. HTBG was more effective in detecting sperm defects when compared to PC (P 0.05) were observed between HTBG and FLUO in the assessment of plasmatic membrane and acrosome integrity. High correlation between HTBG and FLUO techniques was observed when assessing plasma membrane and acrosome (R = 0.97 and 0.96, respectively). In conclusion, the HTBG staining is suitable to assess ram sperm morphology, plasma and acrossomal membrane integrity simultaneously.

Responsivity to PGE2 labor induction involves concomitant differential prostaglandin E receptor gene expression in cervix and myometrium.

Prostaglandin E2 (dinoprostone) is largely used for labor induction. However, one-third of patients do not respond to treatment. One cause of this poor response may be associated with changes in regulation of prostaglandin E receptors (EP1-4). In this study, we investigated EP mRNA expression in the uterine cervix and lower uterine segment myometrium for term births. Biopsies were obtained from women with successful (responders) and failed (non-responders) dinoprostone labor induction, while women that underwent spontaneous labor were included as controls. EP1 mRNA was upregulated in the cervical tissue of women who did not respond to dinoprostone induction. In addition, in the myometrium, significantly higher levels of EP3 mRNA were observed in women treated with dinoprostone, independent of their responsiveness. Dinoprostone-responders presented 3.6-fold higher levels of EP3 mRNA expression than the spontaneous labor group. Significantly higher levels of EP3 mRNA in the myometrium of the dinoprostone-treated group indicated that dinoprostone may regulate the EP3 gene on the transcriptional level. These results highlight the relationship between EP gene expression and delivery and indicate that understanding the regulation of prostaglandin E receptors may lead to improved labor induction.

Effects of estradiol and progestins on follicular regression before, during, and after follicular deviation in postpartum beef cows.

The objective was to evaluate the effect of estradiol benzoate (EB), in association with three progestin protocols, on ovarian follicular regression of suckled beef cows treated at three stages of follicular development (pre-deviation, deviation, or post-deviation). Thirty-six suckled beef cows (60-90 d postpartum, given 125 microg cloprostenol on two occassions, 12h apart). Forty-eight hours after the first cloprostenol treatment, all follicles >5mm were ablated and transrectal ultrasound scanning (8 MHz) was performed every 24h until Day 7 (Day 0=treatment). When the largest follicle reached a designated diameter of 5-7, 8-10 or >10mm, cows were randomly allocated to receive 2mg of EB im in association with an intravaginal device containing 250 mg of medroxyprogesterone acetate (MPA) with or without 100mg of progesterone (P(4)) given im, or an intravaginal device containing P(4) (3 x 3 factorial design). Treatments induced follicular regression in all cows, independent of follicular stage or treatment. There was no interaction between progestin treatment and follicular stage, nor was there any difference in the time of follicular regression or new wave emergence among follicular stages. Treatment with MPA plus P(4) delayed follicular regression. In conclusion, EB in association with various progestins induced regression of growing follicles and emergence of a new follicular wave in postpartum beef cows, regardless of the stage of follicular development.