Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.     

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.     

Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year

Overall, significantly more antral follicles greater than or equal to 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 +/- 3 (mean +/- s.e.m.) follicles per ewe, 23 sheep; Day 9-10 of oestrous cycle, 24 +/- 1 follicles per ewe, 22 sheep; P less than 0.01), although the mean numbers of preovulatory-sized follicles (greater than or equal to 5 mm diam.) were similar (anoestrus, 1.3 +/- 0.2 per ewe; oestrous cycle, 1.0 +/- 0.1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9-10 of the oestrous cycle there were significantly more (P less than 0.001) high-amplitude LH peaks (i.e. greater than or equal to 1 ng/ml) in plasma and significantly fewer (P less than 0.001) low amplitude peaks (less than 1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P less than 0.05, prolactin, P less than 0.001). It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.     

Effect of GnRH antagonist-induced prolonged follicular phase on follicular atresia and oocyte developmental competence in vitro in superovulated heifers

A GnRH antagonist (Antarelix) was used to suppress endogenous pulsatile secretion of LH and delay the preovulatory LH surge in superovulated heifers to study the effect of a prolonged follicular phase on both follicle and oocyte quality. Oestrous cycles were synchronized in 12 heifers with progestagen (norgestomet) implants for 10 days. On day 4 (day 0 = day of oestrus), heifers were stimulated with 24 mg pFSH for 4 days and luteolysis was induced at day 6 with PGF2 alpha (2 ml Estrumate). Animals in the control group (n = 4) were killed 24 h after the last FSH injection. At this time, heifers in group A36h (n = 4) and group A60h (n = 4) were treated with 1.6 mg of Antarelix every 12 h for 36 and 60 h, respectively, and then killed. After dissection of ovarian follicles, oocytes were collected for individual in vitro maturation, fertilization and culture; follicular fluid was collected for determination of steroid concentrations, and granulosa cells were smeared, fixed and stained for evaluation of pycnosis rates. Granulosa cell smears showed that 90% of follicles were healthy in the control group. In contrast, 36 and 58% of the follicles in group A36h showed signs of early or advanced atresia, respectively, while 90% of the follicles in group A60h showed signs of late atresia. Intrafollicular concentrations of oestradiol decreased (P < 0.0001) from healthy follicles (799.14 +/- 40.65 ng ml-1) to late atretic follicles (3.96 +/- 0.59 ng ml-1). Progesterone concentrations were higher (P < 0.0001) in healthy follicles compared with atretic follicles, irrespective of degree of atresia. Oestradiol:progesterone ratios decreased (P < 0.0001) from healthy (4.58 +/- 0.25) to late atretic follicles (0.07 +/- 0.009). The intrafollicular concentrations of oestradiol and progesterone were significantly higher (P < 0.0001) in the control than in the treated groups. The oestradiol:progesterone ratio was higher (P < 0.0001) in the control (4.55 +/- 0.25) than in the A36h (0.40 +/- 0.05) and A60h (0.07 +/- 0.009) groups. Unexpectedly, the cleavage rate of fertilized oocytes, blastocyst rate and number of cells per blastocyst were not significantly different among control (85%, 41% and 95 +/- 8), A36h (86%, 56% and 93 +/- 5) and A60h (88%, 58% and 79 +/- 4) groups. In addition, there were no significant differences in the blastocyst rates from oocytes derived from healthy (45%), early atretic (54%), advanced atretic (57%) and late atretic follicles (53%). In conclusion, the maintenance of the preovulatory follicles in superovulated heifers with a GnRH antagonist induced more atresia and a decrease in oestradiol and progesterone concentrations. However, the developmental potential in vitro to day 8 of the oocytes recovered from these atretic follicles was not affected.     

Suppression of plasma FSH concentrations with bovine follicular fluid blocks ovulation in GnRH-treated seasonally anoestrous ewes

The specific requirement for FSH in the final stages of preovulatory follicle development was assessed in seasonally anoestrous ewes given 2-h injections of GnRH (250 ng/injection), with (N = 10) or without (N = 10) concurrent treatment with bovine follicular fluid (bFF: 2 ml given i.v. at 8-h intervals). Treatment with bFF significantly (P less than 0.01) suppressed plasma FSH concentrations, but, at least for the first 30 h of treatment, did not influence the magnitude of GnRH-induced LH episodes (mean max. conc. 3.00 +/- 0.39 and 3.63 +/- 0.51 ng/ml for bFF-treated and control ewes, respectively). Of 10 animals treated with GnRH for 72 h, 5/5 control ewes showed oestrus and ovulated whereas 0/5 bFF-treated ewes showed oestrus or ovulated in response to GnRH treatment. There was, however, a transient (13.2 +/- 1.0 h) increase in plasma LH concentrations in the ewes given bFF (mean max. conc. 4.64 +/- 1.57 ng/ml), which was coincident with the preovulatory LH surge recorded in animals given GnRH alone. In 10 GnRH-treated ewes slaughtered after 32 h of treatment, the mean diameter of the largest antral follicle was significantly (P less than 0.001) greater in control ewes (5.92 +/- 0.17 mm) than in animals that were also given bFF (3.94 +/- 0.14 mm). In addition, the incidence of atresia in the 3 largest antral follicles present at this time was greater in bFF-treated ewes. These results show that, when plasma FSH concentrations are suppressed by administration of bFF, although the magnitude of GnRH-induced LH episodes is unchanged, preovulatory follicular development is impaired and ovulation does not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeability of rat ovarian follicles to LH during development and luteinization

A 'double isotope' technique has been used to describe the temporal relationship between plasma and follicular concentrations of LH after injection of 51Cr and 125I-rat LH into immature rats. Radiolabelled LH was detectable in all follicles 1 min after injection. Concentrations in small antral and large preovulatory follicles were not significantly different at any time and reached a maximum of 34.2 +/- 3.0% of plasma concentrations at 40 min. Concentrations of LH in preovulatory follicles exposed to an ovulatory dose of hCG 4 h previously were significantly greater (P less than 0.05) than those in small antral and preovulatory follicles at all times, and reached a maximum of 46.2 +/- 1.7% of plasma concentrations after 1 h. Polyacrylamide gel electrophoresis and immunoprecipitation with an antibody specific for rat LH indicated that radioactivity in plasma and follicular fluid represented radio-iodinated LH. Steroidogenic activities, light microscopy and measurements of follicular volume of each class of follicle confirmed that small antral, preovulatory follicles and preovulatory follicles exposed to an ovulatory dose of hCG in vivo could be isolated specifically. Based on these findings it is possible to calculate that, during an endogenous pulse of LH secretion, follicular concentrations of LH never exceed 20% of peak plasma concentrations. Pronounced increases in functional activities during antral growth were not correlated with increased follicular permeability. Only after acute exposure to an ovulatory dose of hCG in vivo was permeability significantly increased. We conclude that entry of LH into antral follicles is restricted and that exposure to an ovulatory dose of hCG results in greater amounts of LH entering preovulatory follicles.     

Decreased pulsatile LH secretion in heifers superovulated with eCG or FSH

This study was designed to test the hypothesis that treatment with super-ovulatory drugs suppresses endogenous pulsatile LH secretion. Heifers (n=5/group) were superovulated with eCG (2500 IU) or FSH (equivalent to 400 mg NIH-FSH-P1), starting on Day 10 of the estrous cycle, and were injected with prostaglandin F(2alpha) on Day 12 to induce luteolysis. Control cows were injected only with prostaglandin. Frequent blood samples were taken during luteolysis (6 to 14 h after PG administration) for assay of plasma LH, estradiol, progesterone, testosterone and androstenedione. The LH pulse frequency in eCG-treated cows was significantly lower than that in control cows (2.4 +/- 0.4 & 6.4 +/- 0.4 pulses/8 h, respectively; P<0.05), and plasma progesterone (3.4 +/- 0.4 vs 1.8 +/- 0.1 ng/ml, for treated and control heifers, respectively; P<0.05) and estradiol concentrations (25.9 +/- 4.3 & 4.3 +/- 0.4 pg/ml, for treated and control heifers, respectively; P<0.05) were higher compared with those of the controls. No LH pulses were detected in FSH-treated cows, and mean LH concentrations were significantly lower than those in the controls (0.3 +/- 0.1 & 0.8 +/- 0.1, respectively; P<0.05). This suppression of LH was associated with an increase in estradiol (9.5 +/- 1.4 pg/ml; P<0.05 compared with controls) but not in progesterone concentrations (2.1 +/- 0.2 ng/ml; P>0.05 compared to controls). Both superovulatory protocols increased the ovulation rate (21.6 +/- 3.9 and 23.0 +/- 4.2, for eCG and FSH groups, respectively; P>0.05). These data demonstrate that super-ovulatory treatments decrease LH pulse frequency during the follicular phase of the treatment cycle. This could be explained by increased steroid secretion in the eCG-trated heifers but not in FSH-treated animals.     

Effect of GnRH antagonists treatment on gonadotrophin secretion, follicular development and inhibin A secretion in goats

The aim of this study was to determine, for goats, the effects of daily doses of GnRH antagonist on ovarian endocrine and follicular function. Ten does were given 45 mg FGA intravaginal sponges and then five were treated with daily injections of 0.5mg of the GnRH antagonist Teverelix for 11 days from 2 days after the day of sponge insertion, while five does acted as controls. Pituitary activity was monitored by measuring plasma FSH and LH daily from 2 days before the first GnRH injection to Day 12. Follicular activity was determined by ultrasonographic monitoring and by assessing plasma inhibin A levels during the same period. In treated does, the FSH levels decreased linearly (0.8 +/- 0.1 ng/ml to 0.5 +/- 0.1 ng/ml, P < 0.01) and remained lower than the mean concentration in control goats (0.8 +/- 0.1 ng/ml, P < 0.005). LH levels were also lower during the period of antagonist treatment (0.6 +/- 0.2 ng/ml versus 0.4 +/- 0.1 ng/ml, P < 0.0005). During GnRH antagonist treatment, there was a significant decrease in the number of large follicles (> or = 6 mm) from Day 3 of treatment (1.2 +/- 0.6, P < 0.0001), with no large follicles from Day 9. The number of medium follicles (4-5 mm in size) also decrease during the period of treatment (4.2 +/- 0.7 to 1.0 +/- 0.6, P < 0.0001), leading to a significant decrease in inhibin A levels when compared to the control (143.7 +/- 31.3 pg/ml versus 65.2 +/- 19.1 pg/ml, P < 0.00005). In contrast, the number of small follicles (2-3 mm) increased in treated goats from Day 4 of treatment (9.6 +/- 2.9 to 20.2 +/- 6.3, P < 0.005). Such data indicate that GnRH antagonist reduced plasma levels of FSH and LH with suppression of the growth of large dominant ovarian follicles and a two-fold increase in number of smaller follicles. The results confirm that GnRH antagonist treatment can be used in goats to control gonadotrophin secretion and ovarian follicle growth in superovulatory regimes.     

Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH

Preantral follicles of cyclic hamsters were isolated on proestrus, estrus and diestrus I, incubated for 3 h in 1 ml TC-199 containing 1 microgram ovine luteinizing hormone (LH) (NIH-S22), and the concentrations of progesterone (P), androstenedione (A) and estradiol (E2) determined by radioimmunoassay. At 0900-1000 h on proestrus (pre-LH surge) preantral follicles produced 2.4 +/- 0.3 ng A/follicle per 3 h, less than 100 pg E2/follicle and less than 250 pg P/follicle. At the peak of the LH surge (1500-1600 h) preantral follicles produced 1.8 +/- 0.2 ng P and 1.9 +/- 0.1 A and less than 100 pg E2/follicle. After the LH surge (1900-2000 h proestrus and 0900-1000 h estrus) preantral follicles were unable to produce A and E2 but produced 4.0 +/- 1.0 and 5.0 +/- 1.1 ng P/follicle, respectively. By 1500-1600 h estrus, the follicles produced 8.1 +/- 3.1 ng P/follicle but synthesized A (1.6 +/- 0.2 ng/follicle) and E2 (362 +/- 98 pg/follicle). On diestrus 1 (0900-1000 h), the large preantral-early antral follicles produced 1.9 +/- 0.3 ng A, 2.4 +/- 0.4 ng E2 and 0.7 +/- 0.2 ng P/follicle. Thus, there was a shift in steroidogenesis by preantral follicles from A to P coincident with the LH surge; then, a shift from P to A to E2 after the LH surge. The LH/follicle-stimulating hormone (FSH) surges were blocked by administration of 6.5 mg phenobarbital (PB)/100 g BW at 1300 h proestrus. On Day 1 of delay (0900-1000 h) these follicles produced large quantities of A (2.2 +/- 0.2 ng/follicle) and small amounts of E2 (273 +/- 27 pg/follicle) but not P (less than 250 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of thecal androstenedione production by exogenous progesterone in the cyclic hamster

Implants of progesterone on the day of dioestrus II in the hamster induced on the following day an increase in circulating levels of progesterone (6.0 +/- 0.7 ng/ml, N = 8; sesame oil controls, less than 0.5 ng/ml, N = 6) and a decline in serum levels of LH (5.3 +/- 0.4 ng/ml; controls 12 +/- 2 ng/ml) and oestradiol (10 +/- 2 pg/ml; controls 69 +/- 5 pg/ml). The production of androstenedione and oestradiol by antral follicles in vitro was reduced in progesterone-treated hamsters when compared with controls, but progesterone production was not affected. Aromatizing activities of antral follicles were the same in progesterone-treated and sesame oil-treated hamsters. Androstenedione production by theca was significantly less in progesterone-treated hamsters than in controls. On dioestrus II, LH replacement therapy (200 micrograms ovine LH by osmotic minipump inserted s.c.) prevented the decline in follicular androstenedione and oestradiol production induced by progesterone alone, and also prevented the decline in thecal androstenedione production in vitro. The results indicate that exogenous progesterone on dioestrus II lowers circulating levels of LH by the following day, inhibits thecal androstenedione production and thus reduces follicular oestradiol production without alteration in aromatizing ability.     

Follicular atresia and LH concentrations during the follicular phase of the estrous cycle in the goat (Capra hircus)

The objective of the study was to identify the effects of LH on the final follicle maturation process as well as the incidence of atresia during the follicular phase of the goat's estrous cycle. In Experiment 1, concentrations of the LH were measured during the follicular phase of a synchronized cycle in 8 Canary goats. In Experiment 2, the same animals were synchronized again. On each day of a 4-day experimental period (day 0=day of sponges withdrawal), 2 of the goats were bilaterally ovariectomized. Follicles with a diameter >1 mm were dissected out to obtain qualitative histological data in normal, early atretic I, early atretic II, advanced atretic I and advanced atretic II follicles. The total interval from sponge withdrawal to LH peak was 77.5±9.8 h. LH peak concentration averaged 44±5.3 ng/ml and the mean length of the preovulatory surge (amounts over 10 ng/ml) was 8.9±0.9 h. During the total follicular phase, there were more atretic follicles than normal follicles (58 vs. 30, P<0.05). The number of early and advanced atretic follicles was similar. There were more early atresia I than early atresia II follicles (23 vs. 6, P<0.05). On day 2, the number of advanced atretic follicles was greater than early atretic follicles (10 vs. 4, P<0.05). There was an increase in the number of early atretic follicles from day 2 to day 4 (4 vs. 9, P<0.05), which was consistent with the effects of the preovulatory LH surge.     

Experimental assumption of dominance by a smaller follicle and associated hormonal changes in mares.

A two-follicle model was used to study the nature of selection of the dominant follicle in mares by ablating neither or one of the two follicles on the day the larger follicle reached >/= 20 mm (Day 0). The larger follicle became the dominant follicle in all mares in which both follicles (n = 8) or only the larger follicle (n = 10) was retained. When only the smaller follicle (n = 9) was retained, it became dominant and ovulated in six mares and became atretic in three mares; the difference in diameter between the two follicles on Day 0 was less (p < 0.01) in mares in which the retained smaller follicle grew and ovulated (2.2 +/- 0.6 mm) than in the mares in which the follicle became atretic (5.9 +/- 1.2 mm). A decline (p < 0. 0001) in FSH concentrations occurred over Days -4 (8.4 +/- 0.7 ng/ml) to 0 (5.9 +/- 0.3 ng/ml), averaged over all groups, and the decline continued for several more days in the groups with both follicles or with only the larger follicle retained. In the group with only the smaller follicle retained, compared to the group with both follicles retained, FSH concentrations and diameter of the smaller follicle increased between Days 0 and 1 (significant interaction for each end point). After Day 1, FSH concentrations continued to increase when the smaller retained follicle became atretic; concentrations decreased when the smaller retained follicle became dominant. An increase (p < 0.0001) in LH concentrations occurred over Days -4 (12.2 +/- 1.1 pg/ml) to 0 (21.1 +/- 2.0 pg/ml), averaged over the three groups. In 23 of 27 mares, a transient peak in LH concentrations occurred within 2 days of Day 0. In the groups with both follicles or with only the larger follicle retained, an increase (p < 0.0001) in systemic estradiol concentrations occurred between Day 0 (5.3 +/- 0.6 pg/ml) and Day 2 (7.5 +/- 0.4 pg/ml). When only the smaller follicle was retained, estradiol did not begin to increase until Day 2, and it increased only when the retained follicle grew and became dominant. The beginning of an increase in estradiol and continued decrease in FSH at the expected beginning of deviation were attributable to the future dominant follicle; there was no indication that the smaller follicle was involved.     

Superovulation of immature hypothyroid rdw rats by thyroxine therapy and the development of eggs after in vitro fertilization.

The aim of this study was to examine the effect of thyroxine on ovulation in immature rdw rats and the fertilization and development of the eggs. Serum thyroxine concentrations at 30 days of age were significantly lower in rdw rats than in normal rats (P < 0.001), and greatly increased after thyroxine replacement therapy (P < 0.001). Although few eggs (1-5 +/- 1-2) were obtained from immature rdw rats treated with gonadotrophins alone, females treated with gonadotrophins and thyroxine ovulated significantly more eggs (85 +/- 5). As a control, normal littermates ovulated 21-45 eggs when treated with gonadotrophins alone, and 68 eggs when administered with gonadotrophins and thyroxine. Of the eggs collected from rdw rats treated with gonadotrophins and thyroxine, and inseminated with spermatozoa from mature F1 males, 98% were penetrated and in almost all (99%) of these eggs, male and female pronuclei formed. Forty-seven per cent of the pronuclear eggs developed to the blastocyst stage in vitro. After transfer to recipients, 21% (14/66) of one-cell and 22% (8/37) of two-cell embryos developed to offspring, and 62% (8/13) of pups were of rdw/rdw genotype. The average body weight (6.9 versus 7.8 g) of offspring derived from one-cell embryos was lower than that for two-cell embryos. The morulae and blastocysts did not develop to term, although 41% implanted in the uterine horns of recipients. In conclusion, in immature rdw rats, superovulation was induced by gonadotrophins combined with thyroxine therapy and the superovulated oocytes were fertilized and developed in vitro and developed to term after embryo transfer.     

Effects of the Booroola Fec gene on ovarian follicular populations in superovulated Romanov ewes pretreated with a GnRH antagonist

Endocrine control of follicular growth was studied in mature Romanov ewes carrying (RF+) or not carrying (R+2) the Booroola Fec gene during an oestrous cycle after gonadotrophin-dependent follicles were suppressed by treatment with an antagonist of GnRH (Antarelix, 0.5 mg per day) and superovulatory treatment was administered. The left ovary was removed after 10 days of treatment (saline or Antarelix) and the right ovary was removed at the end of the superovulatory treatment. Ewes of both genotypes treated with Antarelix had lower plasma LH concentrations than did controls from day 0 to day 10. The inhibitory effect of Antarelix on LH concentration increased with day of treatment. The variability in FSH concentrations during the initial 10 days was reduced by Antarelix treatment in both genotypes. Plasma FSH concentrations were higher in RF+ ewes than in R+2 ewes. In both genotypes, FSH concentrations varied significantly with day of treatment, with the lowest concentrations at day 8 and the highest concentrations at day 5. RF+ ewes had a greater total and atretic number of antral follicles 0.62-1.12, 1.12-2.00 and 2.00-3.00 mm in diameter (classes 2, 3 and 4) than did R+2 ewes before and after superovulatory treatment. After superovulatory treatment, the total number of atretic and non-atretic follicles > 3.00 mm in diameter (class 5) increased in both genotypes. Superovulatory treatment also increased the number of total and atretic class 4 follicles in RF+ only. Conversely, superovulatory treatment decreased the mean number of class 3 follicles in both genotypes, while the number of atretic follicles was decreased only in R+2 ewes. Antarelix treatment significantly reduced the percentage of follicles > 2.00 mm in diameter in RF+ but not in R+2 ewes. Antarelix treatment before superovulatory treatment increased the total number of class 4 follicles in both genotypes but the increase was more significant in RF+ than in R+2 ewes. These results indicate that Antarelix pretreatment favours a greater superovulatory response in Romanov ewes carrying the Fec gene because ovulatory follicles are recruited from a wider range of follicular size classes.     

Estradiol-induced blockade of ovulation in the cow: effects on luteinizing hormone release and follicular fluid steroids

Administration of 10 mg estradiol valerate (EV) to nonlactating Holstein cows on Days 16 of the estrous cycle prevented ovulation in 7 of 8 cows for 14 days post-injection. In these 7 cows, the timing of luteolysis and the luteinizing hormone (LH) surge was variable but within the normal range. At 14 days post-treatment, each of these cows had a large (greater than 10 mm) follicle, with 558 +/- 98 ng/ml estradiol-17 beta, 120 +/- 31 ng/ml testosterone, and 31 +/- 2 ng/ml progesterone in follicular fluid (means +/- SE). A second group of animals was then either treated with EV as before (n = 22), or not injected (control, n = 17) and ovariectomized on either Day 17, Day 18.5, Day 20, or Day 21.5 (24, 60, 96, or 132 h post-EV). Treatment with EV did not influence the timing of luteolysis, but surges of LH occurred earlier (59 +/- 8 h post-EV vs. 100 +/- 11 h in controls). The interval from luteolysis to LH peak was reduced from 44 +/- 6 h (controls) to 6.9 +/- 1.5 h (treated). Histologically, the largest follicle in controls tended to be atretic before luteolysis, but nonatretic afterwards, whereas the largest follicle in treated animals always tended to be atretic. Nonatretic follicles contained high concentrations of estradiol (408 +/- 59 ng/ml) and moderate amounts of testosterone (107 +/- 33 ng/ml) and progesterone (101 +/- 21 ng/ml), whereas atretic follicles contained low concentrations of estradiol (8 +/- 4 ng/ml) and testosterone (12 +/- 4 ng/ml), and either low (56 +/- 24 ng/ml) or very high (602 +/- 344 ng/ml) concentrations of progesterone. This study suggests that EV prevents ovulation by inducing atresia of the potential preovulatory follicle, which is replaced by a healthy large follicle by 14 days post-treatment.     

Restoration of endocrine and ovarian function after stopping GnRH antagonist treatment in goats

We have tested if the high number of unfertilized ova and degenerated embryos found in superovulated goats previously treated with GnRH antagonist can be related to a prolongation of gonadotrophin down-regulation and/or alterations in follicular function during the period of administration of the superovulatory treatment, around 4 days after the end of the antagonist treatment. A total of 15 does were treated with intravaginal progestagen sponges and daily injections of 0.5mg of the GnRH antagonist Antarelix for 6 days, while 5 does acted as controls receiving saline. During the antagonist treatment, the mean plasma LH concentration was lower in treated than control goats (0.5 +/- 0.2 versus 0.7 +/- 0.5 ng/ml, P < 0.0005 ); however, the FSH levels remained unaffected (0.8 +/- 0.4 versus 0.8 +/- 0.5 ng/ml). In this period, treated does also showed an increase in the number of small follicles with 2-3 mm in size ( 10.7 +/- 0.7 versus 8.4 +/- 0.6, P < 0.05), and a decrease in both the number of follicles > or =4 mm in size ( 5.0 +/- 0.3 versus 6.8 +/- 0.5, P < 0.005) and the secretion of inhibin A (120.9 +/- 10.7 versus 151.6 +/- 12.6 pg/ml, P < 0.05). After cessation of the antagonist treatment, there was an increase in LH levels in treated goats from the day after the last Antarelix injection (Day 1), so that LH levels were the same as controls on Day 3 (0.6 +/- 0.1 versus 0.6 +/- 0.2 ng/ml). However, there were even greater numbers of small follicles than during the period of antagonist injections (15.4 +/- 0.6 in treated versus 8.9 +/- 0.7 in control, P < 0.0005 ). Moreover, the number of > or =4 mm follicles and the secretion of inhibin A remained lower in treated goats (3.9 +/- 0.3 follicles and 84.4 +/- 7.0 pg/ml versus 5.4 +/- 0.5 follicles, P < 0.05 and 128.9 +/- 14.2 pg/ml, P < 0.05 ). These results indicate that pituitary secretion of gonadotrophins is restored shortly after the end of antagonist treatment, but activity of ovarian follicles is affected.     

Endocrine, luteal and follicular responses after the use of the short-term protocol to synchronize ovulation in goats

The effect of the so-called Short-Term Protocol (5-day progesterone treatment+PGF(2)alpha) on ovarian activity and LH surge was studied in goats. The goats received 250IU eCG at the time of device withdrawal (eCG group; n=7), or 200microg of EB (estradiol benzoate) 24h after device withdrawal (EB group; n=8), or received neither eCG nor EB (control group; n=8). The Short-Term Protocol induced greater (4.1+/-1.1ng/ml) progesterone serum concentrations at 24h after start of the treatment, that declined to 0.2+/-0.1ng/ml at 12h after device withdrawal. In all of the groups, the maximum concentration of estradiol-17beta was reached at about 36h after device withdrawal. Maximum concentration was greater in the EB group (76.9+/-24.6pmol/l) than in the control group (41.8+/-9.0pmol/l; P<0.01), with the eCG group showing intermediate concentration (70.3+/-32.5pmol/l; P=NS). The LH peak occurred earlier in the eCG group (38.4+/-2.0h after device withdrawal) and in the EB group (41.0+/-4.1h), than in the control group (46.3+/-5.1h; P<0.05). Ovulation occurred earlier in the eCG group (5/7) and in the EB group (8/8) (58.8+/-2.7h and 63.0+/-5.6h, respectively), than in the control group (7/8) (70.2+/-8.3h; P<0.05). In summary, the Short-Term Protocol induced similar concentrations of progesterone among treated goats. In addition, eCG or EB resulted in a similar increase in estradiol-17beta and a similar LH surge, which induced ovulation in most females (86.7%) in a consistent interval (about 60h) after the end of progesterone exposure.     

Role of ovarian failure in reproductive senescence in aged red deer (Cervus elaphus) hinds

Physiological and endocrine factors associated with reproductive senescence were assessed in a group of 19 ageing red deer hinds. Reproductive success, defined as the percentage of hinds weaning a calf successfully, decreased gradually from 89% at 6-7 years of age to 50% at 17 years, and subsequently decreased markedly; only one hind reared a calf at 19-20 years of age. When the 12 surviving hinds were approaching 21 years of age, they were compared with ten mature 7-year-old females over the onset of the breeding season. All hinds were subsequently killed, the reproductive tracts were recovered and antral (>/= 2 mm in diameter) and preantral follicle populations were determined by dissection (n = 7 hinds per age group) or stereological analysis (n = 2 ovaries per age group), respectively. Cyclical ovarian activity (plasma progesterone) was evident in fewer aged hinds compared with mature hinds (3/12 versus 10/10, P < 0.001) and mean plasma LH concentrations were higher in aged animals than in mature animals (0.57 +/- 0.05 and 0.20 +/- 0.05 ng ml(-1), P < 0.001). Mean uterine (44.2 +/- 4.5 and 75.4 +/- 4.2 g; P < 0.001) and ovarian masses (0.88 +/- 0.11 and 1.52 +/- 0.12 g; P < 0.001) were lower in the aged hinds, which also had fewer antral follicles than did mature hinds (0.89 +/- 0.35 and 23.5 +/- 4.5 follicles per hind, respectively; P < 0.001). Only one primordial follicle was observed in one of the ovaries of the aged hinds, compared with 7000-21 000 in the ovaries of mature hinds. The high gonadotrophin concentrations, paucity of primordial and antral follicles and failure of ovulation indicate collectively that waning reproductive performance after 17 years of age is primarily due to ovarian failure.     

Do high progesterone concentrations decrease pregnancy rates in embryo recipients synchronized with PGF2alpha and eCG?

The objective of this study was to evaluate the effects of equine chorionic gonadotropin (eCG) treatment on the number of induced accessory corpora lutea (CL), plasma progesterone concentrations and pregnancy rate in cross-bred heifers after transfer of frozen-thawed (1.5M ethylene glycol) embryos. All recipients received 500 microg PGF2alpha (dl-cloprostenol, i.m.) at random stages of the estrous cycle (Day 0) and were observed for estrus for 7 days. On Day 14, heifers detected in estrus between 2 and 7 days after PGF2alpha treatment were randomly allocated to four groups ( n=83 per group) and given 0 (control), 200, 400, or 600 IU of eCG. Two days later (Day 16), these recipients were given PGF2alpha and observed for estrus. Six to eight days after detection of estrus, plasma samples were collected to determine progesterone concentration and ultrasonography was performed to observe ovarian structures. Heifers with multiple CL or a single CL >15 mm in diameter received an embryo by direct transfer. Embryos of excellent and good quality were thawed and transferred to the recipients by the same veterinarian. Pregnancy was diagnosed by ultrasonography and confirmed by transrectal palpation 21 and 83 days after embryo transfer (ET), respectively. Plasma progesterone concentrations on the day of transfer (Day 7 of the estrous cycle) were 3.9+/-0.7, 4.2+/-0.4,6.0+/-0.4 and 7.8+/-0.6 ng/ml for groups Control, 200, 400, and 600, respectively (Control versus treated groups P=0.009; 200 versus 400 and 600 groups P=0.0001; and 400 versus 600 P=0.012 ). Conception rates 83 days after ET were 41.9, 50.0, 25.0, and 20.9% for groups Control, 200, 400, and 600, respectively (200 versus 400 and 600 groups P=0.0036 ). In conclusion, an increase in progesterone concentration, induced by eCG treatment, did not improve pregnancy rates in ET recipients. Conversely, there was a decline in conception rates in the animals with the highest plasma progesterone concentrations.     

Compensatory ovulatory mechanisms operative after first ovulation in rats unilaterally ovariectomized prepubertally

Ovarian steroid contents and serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured during the days after first ovulation in rats unilaterally ovariectomized in late prepuberty. In addition, follicle counts were made at second estrus and second metestrus. During the cycle following first ovulation, ovarian estradiol contents in unilaterally ovariectomized (ULO) rats were significantly increased as compared to intact rats on the day of metestrus, on diestrus 1 and on second estrus. Ovarian progesterone was significantly increased on the days of metestrus, on diestrus 1, second proestrus and second estrus, but no differences were seen in ovarian androgen contents. After ULO there was an indication of an augmented FSH surge at the first and the second ovulation. Follicle counts revealed that the total number of healthy as well as of atretic antral follicles on the day of second estrus was significantly increased after ULO, due to increased numbers of the smallest antral follicles. At second metestrus the number of larger antral follicles (350-500 micron 3) and the total number of healthy antral follicles was higher after ULO. It is concluded that the compensatory process after ULO involved increased recruitment of small antral follicles. Activities in the remaining ovary were not simply doubled but a new hormonal balance was established.