Expression and Function of Fibroblast Growth Factor 18 in the Ovarian Follicle in Cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Follicular dynamics in Mangalarga mares.

Ovarian follicular activity was studied by ultrasonography during 17 oestrous cycles in 9 Mangalarga mares during the second half of the ovulatory season. Sixteen oestrous cycles were considered normal and one 3-wave cycle showing a prolonged luteal phase was considered atypical. Daily ultrasonographic examinations were performed and the compiled data on follicular dynamics were studied retrospectively. One major wave of follicular growth was observed in 13 of the 16 normal cycles (81.25%), whereas 2 major waves occurred in 3 cycles (18.75%). The mean (+/- s.d.) days of emergence of the primary wave of follicular development in cycles containing one or 2 waves were Day 6.0 +/- 2.3 and Day 11.0 +/- 1.0, respectively. The secondary wave of follicular development in 2-wave cycles emerged on Day 0.0 +/- 3.6. The day of wave divergence for primary waves of follicular development in cycles which exhibited one or 2 major waves were Day 12.2 +/- 3.5 and Day 17.3 +/- 3.0, respectively. Divergence of secondary waves occurred in only one of the 3 cycles which exhibited 2 major follicular waves (Day 7). The mean (+/- s.d.) maximum diameters of the dominant follicle in the primary wave of oestrous cycles exhibiting one and 2 major waves were 39.0 +/- 3.9 mm and 34.7 +/- 2.5 mm, respectively. The mean (+/- s.d.) maximum diameter of the dominant follicle present in the secondary wave was 34.3 +/- 11.0 mm. The mean (+/- s.d.) lengths of the interovulatory intervals for cycles containing one and 2 major waves were 19.4 +/- 2.2 and 23.3 +/- 2.5 days, respectively. These data indicate that most Mangalarga mares show one major follicular wave during the oestrous cycle but a small percentage of mares show 2 major waves.

Temporal interrelationships among luteolysis, FSH and LH concentrations and follicle deviation in mares

The effect of altered LH concentrations on the deviation in growth rates between the 2 largest follicles was studied in pony mares. The progestational phase was shortened by administration of PGF2α on Day 10 (Day 0=ovulation; n=9) or lengthened by daily administration of 100 mg of progesterone on Days 10 to 30 (n=11; controls, n=10). All follicles ≥5 mm were ablated on Day 10 in all groups to initiate a new follicular wave. The interovulatory interval was not altered by the PGF2α treatment despite a 4-day earlier decrease in progesterone concentrations. Time required for growth of the follicles of the new wave apparently delayed the interval to ovulation after luteolysis. The FSH concentrations of the first post-ablation FSH surge were not different among groups. A second FSH surge with an associated follicular wave began by Day 22 in 7 of 11 mares in the progesterone group and in 0 of 19 mares in the other groups, indicating reduced functional competence of the largest follicle. A prolonged elevation in LH concentrations began on the mean day of wave emergence (Day 11) in the prostaglandin group (19.2 ± 2.2 vs 9.0 ± 0.7 ng/mL in controls; P<0.05), an average of 4 d before an increase in the controls. Concentrations of LH in the progesterone group initially increased until Day 14 and then decreased so that by Day 18 the concentrations were lower (P<0.05) than in the control group (12.9 ± 1.6 vs 20.2 ± 2.6 ng/mL). Neither the early and prolonged increase nor the early decrease in LH concentrations altered the growth profile of the second-largest follicle, suggesting that LH was not involved in the initiation of deviation. However, the early decrease in LH concentrations in the progesterone group was followed by a smaller (P<0.05) diameter of the largest follicle by Day 20 (26.9 ± 1.7 mm) than the controls (30.3 ± 1.7 mm), suggesting that LH was necessary for continued growth of the largest follicle after deviation. (C) 2000 by Elsevier B.V.

Morphology of the ventral lobe of the prostate and seminal vesicles in an ethanol-drinking strain of rats (UChA and UChB)

Chronic alcoholism alters reproduction and therefore may be responsible for alterations of prostate and seminal vesicles, which are the subject of this analysis in UCh ethanol-drinking rats. The prostate and seminal vesicles of 20 animals were submitted to macroscopic, light microscopy, electron microscopy and morphometric analysis. The UCh rats showed atrophy of the epithelium and reduction of the weight of the prostate and seminal vesicle, liver hypertrophy and fat infiltration and alterations of the hypothalamus-pituitary axis. Ethanol induces changes in the weight and in the epithelium of prostate and seminal vesicles and hypothalamus-pituitary axis of UCh rats.

Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B®) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V® 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin®, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean ± S.E.M.) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8 ± 1.8, 6.1 ± 1.3, 51.5), P48 (12.6 ± 1.9, 7.1 ± 1.0, 52.3), P60 (10.5 ± 1.6, 5.7 ± 1.3, 40.0) and D60 (10.3 ± 1.7, 5.0 ± 1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol. © 2002 Elsevier B.V. All rights reserved.

Inhibin B and ovarian function after total abdominal hysterectomy in women of reproductive age

The aim of this prospective study was to assess ovarian function using clinical and endocrine parameters in women of reproductive age who underwent total abdominal hysterectomy. Sixty-one women, aged ≤ 40 years, were allocated into two groups: group 1, consisting of 31 patients who had hysterectomy, and group 2, consisting of 30 normal women. Inclusion criteria were normal ovarian function at baseline, normal body weight, no hormonal diseases and basal follicle stimulating hormone (FSH) level of < 15 mIU/ml. FSH, luteinizing hormone (LH), estradiol and inhibin B levels as well as maturation value (MV) were measured by vaginal cytology on three occasions: baseline, and 6 and 12 months after hysterectomy. Analysis of variance, the Friedman test, Mann-Whitney test and t-test statistics were employed to compare the two groups. At baseline the groups were homogeneous. At months 6 and 12, hysterectomized women showed decreased median values of inhibin B, increased median values of estradiol (p < 0.05), unchanged median values of FSH and LH, and decreased median values of MV (p < 0.05). In the hysterectomy group, 12.9% (4/31) of the patients had FSH levels of > 40 mIU/ml, estradiol of < 20 pg/ml and inhibin B of < 5 ng/ml, compatible with ovarian failure. In the control group, all the parameters studied remained unchanged. These results suggest that total abdominal hysterectomy accelerates the decline in ovarian function in women of reproductive age.

LH Response (in vivo and in vitro) to an LHRH Agonist Administered to Domestic Male Cats

We investigated plasma luteinizing hormone (LH) concentration in domestic male cats challenged with Luteinizing Hormone Releasing Hormone Analog (LHRH-A) [des Gly10, (DTrp6)-LHRH ethylamide] that mediates the function of the hypothalamic-piruitary-gonadal axis (HPG). Plasma LH concentrations in cats treated daily with LHRH (10 μg/ 100 μl/kg/day, subcutaneously - sc) for 19 days (LHRH group) and in controls treated with saline (NaCl - 0.9%, same volume - SAL group) were chronically studied. LHRH administration (sc) for 15 days induced a significant fall (P < 0.05) in plasma LH concentrations during the chronic study. After the 15th day of treatment the groups were divided once more into animals treated with LHRH (10 μg/100 μl/kg) or saline (iv), and a time course study (300 min) was performed (acute study). Next, four groups of cats were compared in an acute study involving the sc/iv administration of SAL/SAL, SAL/LHRH, LHRH/SAL, and LHRH/LHRH. The responses of the SAL animals challenged by acute iv administration of LHRH (group SAL/LHRH) were significantly higher (P < 0.01) than those of animals treated with LHRH (sc) (group LHRH/LHRH). LH release was also significantly increased in the latter group (P < 0.05), although the effect was short lasting, being recorded only at the first observation (45 min). An in vitro study with the pituitaries was also performed on day 20. Mean (±SEM) LH concentrations in the culture medium containing pituitaries with LHRH (10-7 M) or saline were determined. In vitro analysis of these pituitaries demonstrated a significantly reduced response (P < 0.05) by animals treated sc with LHRH for 19 days. This study represents a source of data for the domestic cat going beyond its own physiology. Serving as a model, this animal provide important information for the study of reproductive physiology in other members of its family (Felidae), almost all of them threatened with extinction.

Morphology of the vas deferens in an ethanol-drinking strain of rats (UChA and UChB)

Chronic alcoholism alters reproduction and therefore may be responsible for alterations of vas deferens, which are the subject of this analysis in UCh ethanol-drinking rats. The proximal and distal segments of the vas deferens of 20 animals were submitted to macroscopic, light microscopy, electron microscopy and morphometric analysis. The UCh rats showed atrophy of the epithelium of the vas deferens and alterations of the hypothalamus-pituitary axis. Ethanol induces changes in the epithelium of the vas deferens and hypothalamus-pituitary axis of UCh rats.

Acute reduction in serum progesterone concentrations after feed intake in dairy cows

This study tested the hypothesis that high feed consumption will acutely decrease circulating progesterone concentrations. In the first experiment, a Latin Square design was used to test whether feeding pattern would alter circulating progesterone in pregnant lactating Holstein cows (n = 12). Feed was removed for 12 h before the experiment and cows were then either fed 100% of the total mixed ration (TMR), 50% of TMR every 12 h, 25% of TMR every 6 h, or left unfed for an additional 12 h. Blood samples were taken every hour for 24 h. Provision of 100 or 50% of TMR decreased circulating progesterone by 1 h after feeding and progesterone remained depressed until 8-9 h after feeding. Feeding 25% of TMR did not reduce circulating progesterone concentrations. Experiment 2 used a crossover design to measure the effect of acute feeding on circulating progesterone and LH concentrations during delivery of a constant amount of exogenous progesterone (Eazi-Breed CIDRs) in lactating Holstein cows (n = 8) and nonpregnant dry Holstein cows (n = 6). Blood samples were taken every 15 min for 8 h. There was no change in serum progesterone during the 8 h treatment period in unfed cows; however, feeding decreased (P < 0.05) circulating progesterone between 2 and 6 h after feeding. In lactating cows, feeding increased mean LH (P < 0.05). There were more LH pulses (P = 0.01) in lactating than nonlactating cows. Thus, acute feeding reduced circulating progesterone in pregnant lactating cows apparently due to an increase in progesterone metabolism. Interestingly, feeding multiple smaller meals eliminated the acute effect of feeding on circulating progesterone. © 2003 Published by Elsevier B.V.

Uso da suplementação de LH recombinante na estimulação ovariana com protocolos de FSH recombinante e agonistas do GnRH: Uma revisão da literatura

The purpose of this review was to assess the efficacy of recombinant LH (r-LH) supplementation for controlled ovarian stimulation in recombinant FSH (r-FSH) and GnRH-agonist (GnRH-a) protocol for IVF/ICSI cycles. Search strategies included on-line surveys of databases from 1990 to 2006. Four trials fulfilled the inclusion criteria (Lisi et al. 2002, Humaidan et al. 2004, Marrs et al. 2004, Tarlatzis et al. 2006). When the review was carried out advantages were observed for the r-LH supplementation protocol with respect to a fewer days of stimulation, a fewer total amount of r-FSH administered and a higher serum estradiol levels on the day of hCG administration. However, these differences were not observed in number of oocyte retrieved, number of mature oocytes, clinical pregnancy per oocyte retrieval, implantation and miscarriage rates. Nevertheless, more randomized controlled trials are necessary before evidence-based recommendations regarding exogenous r-LH supplementation in ovarian stimulation protocols with r-FSH and GnRH-a for assisted reproduction treatment can be provided.

O emprego de hormônio luteinizante recombinante associado ao hormônio folículo-estimulante recombinante nos protocolos de estimulação ovariana com antagonista do GnRH

The purpose of this investigation was to verify the efficacy of recombinant LH supplementation for controlled ovarian stimulation in GnRH-antagonist protocol for assisted reproductive technologies cycles. Search strategies included on-line surveys of databases from 1990 to 2006. In this review and meta-analysis, the observed advantages for the LH supplementation protocol were a higher serum estradiol levels on the day of hCG administration and a higher number of mature oocytes. However, there were no differences observed in the total amount of r-FSH administered, days of stimulation, number of oocyte retrieved, the clinical pregnancy rate per oocyte retrieval, the implantation rate and miscarriage rate. This result demonstrates that the association of r-LH with r-FSH may prevent any decrease in estradiol after antagonist administration and a significant higher number of mature oocytes was obtained. Nevertheless, additional randomized controlled trials are needed confirm these observations.

Recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-antagonist protocol: A meta-analysis

This study aims to compare the efficacy of recombinant LH (rLH) supplementation for ovarian stimulation in gonadotrophin-releasing hormone-antagonist protocol for IVF/intracytoplasmic sperm injection cycles. Search strategies included online surveys of databases. The fixed effects model was used for odds ratio (OR) and effect size (weighted mean difference, WMD). Five trials fulfilled the inclusion criteria. When the meta-analysis was carried out, advantages were observed for the LH supplementation protocol with respect to higher serum oestradiol concentrations on the day of human chorionic gonadotrophin administration (P < 0.0001; WMD: 514, 95% CI 368, 660) and higher number of mature oocytes (P = 0.0098; WMD: 0.88, 95% CI 0.21, 1.54). However, these differences were not observed in the total amount of recombinant FSH (rFSH) administered, days of stimulation, number of oocytes retrieved, the clinical pregnancy rate per oocyte retrieval, the implantation rate and miscarriage rate. This result demonstrates that the association of rLH with rFSH may prevent any decrease in oestradiol after antagonist administration and that a significantly higher number of mature oocytes was available for laboratory work. Nevertheless, it failed to show any statistically significant difference in clinically significant end-points in IVF (implantation and pregnancy rates). Additional randomized controlled trials are needed to confirm these results further. © 2007 Published by Reproductive Healthcare Ltd.

The effect of type of vaginal insert and dose of pLH on embryo production, following fixed-time AI in a progestin-based superstimulatory protocol in Nelore cattle

The objective was to analyze and report field data focusing on the effect of type of progesterone-releasing vaginal insert and dose of pLH on embryo production, following a superstimulatory protocol involving fixed-time artificial insemination (FTAI) in Nelore cattle (Bos taurus indicus). Donor heifers and cows (n = 68; 136 superstimulations over 2 years) received an intravaginal, progesterone-releasing insert (CIDR® or DIB®, with 1.9 or 1.0 g progesterone, respectively) and 3-4 mg of estradiol benzoate (EB) i.m. at random stages of the estrous cycle. Five days later (designated Day 0), cattle were superstimulated with a total of 120-200 mg of pFSH (Folltropin-V®), given twice daily in decreasing doses from Days 0 to 3. All cattle received two luteolytic doses of PGF2α at 08:00 and 20:00 h on Day 2 and progesterone inserts were removed at 20:00 h on Day 3 (36 h after the first PGF2α injection). Ovulation was induced with pLH (Lutropin-V®, 12.5 or 25 mg, i.m.) at 08:00 h on Day 4 with FTAI 12, 24 and in several cases, 36 h later. Embryos were recovered on Days 11 or 12, graded and transferred to synchronous recipients. Overall, the mean (±S.E.M.) number of total ova/embryos (13.3 ± 0.8) and viable embryos (9.4 ± 0.6) and pregnancy rate (43.5%; 528/1213) did not differ among groups, but embryo viability rate (overall, 70.8%) was higher in donors with a DIB (72.3%) than a CIDR (68.3%, P = 0.007). In conclusion, the administration of pLH 12 h after progesterone removal in a progestin-based superstimulatory protocol facilitated fixed-time AI in Nelore donors, with embryo production, embryo viability and pregnancy rates after embryo transfer, comparable to published results where estrus detection and AI was done. Results suggested a possible alternative, which would eliminate the need for estrus detection in donors. © 2006 Elsevier Inc. All rights reserved.

Tamoxifen inhibits transforming growth factor-α gene expression in human breast carcinoma samples treated with triiodothyronine

Objectives: To examine the effects of triiodothyronine (T3), 17β-estradiol (E2), and tamoxifen (TAM) on transforming growth factor (TGF)-α gene expression in primary breast cancer cell cultures and interactions between the different treatments. Methods and results: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3-mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T3; dish 3: T3+TAM; dish 4: TAM; dish 5: E2; dish 6: E2+TAM. TGF-α mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T3 for 48 h significantly increased TGF-α mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-α mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. Conclusion: We demonstrate that TGF-α mRNA expression is more efficiently upregulated by T3 than E2. Concomitant treatment with TAM had a mitigating effect on the T3 effect, while E2 induced TGF-α upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-α, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER α and β; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E2. ©2008, Editrice Kurtis.

LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n = 12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B ®) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2α (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38 h after the first PGF2α injection, blood sampling and ovarian ultrasonography took place every 4 h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7 ± 4.9 and 72.3 ± 3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin ®, total dose = 200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2α, to minimize stress, only seven blood samples were collected at 4 h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6 ± 1.6 and 59.5 ± 1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0 ± 2.6 ng/ml (mean ± S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12 h before the LH surge observed after an induced estrus without ovarian superstimulation. © 2008 Elsevier B.V. All rights reserved.