Effect of FSH infusion on follicle development in GnRH agonist-treated gilts

The aim was to investigate the effect of infusion of purified FSH alone on follicle development in hypogonadotrophic GnRH agonist-treated gilts. Large-White hybrid gilts (n = 12) were treated during the mid-luteal phase and again after 28 days (day 0) with a potent slow releasing GnRH agonist. On day 3, seven gilts were infused for 168 h with 1.5 S1 units oFSH h-1 (equivalent to 1.5 units of bioactivity of NIH-FSH-S1 standard) and blood samples were collected. Ovaries were then recovered and all follicles > or = 1 mm in diameter were dissected and incubated for 2 h in 1 ml Eagle's minimum essential medium. The ovaries were recovered from the remaining five GnRH agonist-treated gilts on day 10 and also from five cyclic gilts during the late follicular phase (controls). Plasma FSH concentrations in GnRH agonist-treated gilts were lower (P < 0.01) than in follicular phase controls, increased (P < 0.001) after 1 h of FSH infusion and reached a plateau similar (P > 0.1) to that of controls after 8 h. Basal LH concentrations were similar (P > 0.1) between GnRH agonist-treated and control gilts and remained unchanged (P > 0.1) throughout the infusion period. GnRH agonist treatment reduced (P < 0.01) basal oestradiol concentrations compared with control gilts. Infusion with FSH alone increased (P < 0.001) plasma oestradiol concentrations after 96 h compared with those before infusion; when the animals were killed oestradiol concentrations were higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH than in controls. This was also apparent by vulval swelling and behavioural oestrus. There were more follicles > or 1 mm in diameter in the GnRH agonist-treated groups than in the controls (184, 153 and 86 per animal; P < 0.01). Infusion with FSH increased the maximum follicle diameter (GnRH agonist: < 4 mm; FSH infused: < 12 mm; controls: < 10 mm) and tended to increase (P < 0.07) the mean number of follicles > or = 6 mm diameter per animal (FSH infused: 53; controls: 21). Total oestradiol production in vitro by follicles > or = 1 mm was higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH and in follicular phase controls than in animals treated with GnRH agonist alone. However, oestradiol and testosterone secretion in vitro per follicle > or = 6 mm in diameter was lower (P < 0.05) in FSH-infused animals than in controls. In summary, although infusion of FSH alone stimulated the growth of multiple follicles of preovulatory size in GnRH agonist-treated gilts, steroidogenic output by individual follicles was impaired.     

Differences in follicular morphology, steroidogenesis and oocyte maturation in naturally cyclic and PMSG/hCG-treated prepubertal gilts

Ovaries were obtained from naturally cyclic pigs on Days 16-17, 18, 19, 20 and 21 of the oestrous cycle and on the basis of observed follicular characteristics were assigned as representative of the early (Group 1), mid- (Groups 2 and 3) or late (after LH; Group 4) follicular phase. Follicular development in cyclic gilts was compared with that in ovaries obtained from late prepubertal gilts 36 (Group 5) or 72 (Group 6) h after treatment with 750 i.u. PMSG alone, or with a combination of 500 i.u. hCG 72 h after PMSG and slaughter 30-40 h later (Group 7). After dissection of all follicles greater than 2 mm diameter, follicular diameter, follicular fluid volume, follicular fluid concentrations of progesterone, oestradiol and testosterone, and the stage of oocyte maturation were determined. Combined PMSG/hCG treatment of immature gilts resulted in a pattern of follicular development different from that in naturally cyclic gilts during the follicular phase. Overall exogenous gonadotrophin treatment also increased (P less than 0.001) the variability in follicular diameter and fluid volume. Comparisons between appropriate groups also established differences in the variability of both morphological (diameter and volume, Group 1 vs Group 5; P less than 0.05) and biochemical development (follicular fluid oestradiol, Group 3 vs Group 6 and Group 4 vs Group 7; both P less than 0.05). Such differences in both morphological and biochemical characteristics between cyclic and PMSG/hCG-treated gilts were particularly evident in the population of larger (greater than 6 mm) follicles. These results indicate that the pattern of follicular development in naturally cyclic and in PMSG/hCG-treated gilts is dissimilar and suggests that the ovaries of gonadotrophin-treated prepubertal gilts are functionally different from the ovaries of mature females.     

Kinetics of follicle growth in the prepubertal gilt.

Follicular growth rates were determined by histological examination of ovaries of five prepubertal gilts following treatment with the stathmokinetic agent colchicine. One ovary from each of five gilts was removed surgically and then colchicine (n = 3) or saline (n = 2) was infused i.v. Precisely 2 h after treatment with colchicine, the remaining ovary was removed. Ovaries were processed for histological analyses and sectioned at 10 microns; every twentieth section was stained with hematoxylin and periodic acid-Schiffe's. Sections were viewed with a projection microscope and individual follicles were measured. Eight classes of follicles were established such that the number of granulosa cells per cross section doubled in each class. Diameters of follicles for each class were as follows: 1) less than 106 microns, 2) 106-148 microns, 3) 148-206 microns, 4) 206-287 microns, 5) 287-400 microns, 6) 400-657 microns, 7) 657-1480 microns, and 8) 1480-3130 microns. A layer of thecal cells was first seen in class 2 follicles, and 76% of class 3 follicles had a thecal layer. Oocyte diameter increased through the first four classes and reached a maximum diameter of approximately 110 microns. Almost all follicles greater than 400 microns had an antrum. Preantral follicles had a lower mitotic index and a higher mitotic time and class time than antral follicles. Growth rate increased with increasing size of follicles. Preantral follicles grew at a rate of 5.2 microns/day whereas antral follicles grew at 313 microns/day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early folliculogenesis in primate ovaries: testing the role of estrogen.

The purpose of this study was to examine the effect of an exogenous estrogen, diethylstilbestrol (DES), on follicle development in the ovary of a juvenile primate. The immature cynomolgus monkey (12-22 mo) was used as a model since ovaries at this age lack endogenous gonadotropin support but are capable of responding to exogenous hormonal stimulation. In addition, the pituitary gland receives virtually no GnRH stimulation and under these conditions lacks responsiveness to estrogen feedback. Two groups of three monkeys each received DES for 14 days. Members of the second group also were given GnRH antagonist to assure no GnRH action upon the gonadotropes. The left ovary of each monkey was removed just prior to Day 1 of DES treatment and served as the control. The right ovary was removed on Day 14 of treatment. Both ovaries from each monkey were prepared for evaluation by light microscopy. Results indicated that both the number of preantral follicles and the mean number of medium-sized (0.5-1 mm in diameter) developing antral follicles decreased significantly (p less than 0.05) in the DES-treated ovaries with no increase in early-atretic antral follicles. These data suggest that DES, at the amount administered, inhibits the growth of both preantral and medium-sized antral follicles in the primate. Whether these effects are manifest directly at the follicle level or are mediated by other mechanisms remains to be determined.     

The effect of a GnRH agonist on follicular dynamics and response to FSH stimulation in prepubertal calves

Endocrine control of follicular growth was determined by observing the left ovary of prepubertal calves previously treated with a potent GnRH agonist for 13 days. The ovarian response to hormonal stimulation was determined using the right ovaries of the same animals. Three-month-old crossbred calves were assigned to one of the two following treatment groups: 1) saline control for 13 days, with purified porcine FSH for the last 3 days (n = 5); and 2) GnRHa for 13 days, with purified porcine FSH for the final 3 days (n = 5). The left ovaries were removed from all calves after 10 days, and the right ovaries were removed at the end of treatment. Plasma concentrations of FSH, LH and oestradiol-17 beta were followed up during the GnRHa and pFSH treatments. The maximum macroscopic diameter of the F1 follicle, as determined by daily ultrasonography, did not differ between GnRHa-treated calves (from 6.6 to 10.4 mm) and the saline control calves (from 6.7 to 10.3 mm). Histological analysis of the ovaries showed that the number of follicles > 0.40 mm in diameter varied greatly for calves of the two groups (from 11 to 220 at 10 days). GnRHa significantly increased the mean number of follicles (total and nonatretic) of size class > 5.4 mm as compared to saline control calves (P < 0.05). The FSH treatment significantly increased the mean number of follicles 3.00-5.4 and > 5.4 mm in diameter (P < 0.05), with no change in the number of follicles smaller than 3.00 mm. The rate of atresia of large follicles (3.01-5.40 mm) was significantly reduced by purified porcine FSH treatment in both groups (P < 0.05). In no case did the GnRHa induce ovulation or luteinization of follicles. The LH and FSH concentrations increased transiently after GnRHa treatment on the first day, but afterwards, both hormones increased to only one sixth of what was observed after the initial GnRHa injection treatment. This increase in LH and FSH was observed 1 h after GnRHa treatment on each consecutive day of the experiment and were significantly different in the control group (0 h versus 1 h versus 2 h x saline control versus GnRH agonists groups; P < 0.01). During the superovulatory treatment, FSH concentrations peaked at around 0.70 ng.mL-1 in both saline- and GnRHa-treated groups on the first day but on the last day of surovulatory treatment, FSH concentrations were higher in GnRHa agonist-treated calves than in the control calves (day 11 versus day 12 versus day 13 x saline control versus GnRH agonist treatment groups; P < 0.01). LH profiles were unchanged by surovulatory treatment. Concentrations of oestradiol-17 beta increased significantly over the three days (P < 0.001) of the superovulatory treatments in both groups (P < 0.01). These results indicate that GnRH agonist treatment allows recruited antral follicles to pursue their growth during the early selection process via sustained FSH and LH secretion allowing more than a single large follicle to maintain their growth without going to atresia.     

Effect of unilateral ovariectomy on compensatory ovarian hypertrophy, peripheral concentrations of follicle-stimulating hormone and luteinizing hormone, and ovarian venous concentrations of estradiol-17 beta in prepuberal gilts

A study was designed to characterize the compensatory ovarian response to unilateral ovariectomy (ULO) in prepuberal gilts and to investigate further the mechanisms involved in compensatory ovarian hypertrophy (COH). Forty-eight crossbred gilts were sham ovariectomized (Sham) or unilaterally ovariectomized at 130 days of age (Day 0). Remaining ovaries in ULO gilts were removed and Sham gilts were bilaterally ovariectomized 2, 4 or 8 days later. A peripheral blood sample was taken before surgery and ovarian venous blood samples were taken before removal of each ovary. Serum estradiol-17 beta (E2) concentrations were determined. Mean wet and dry ovarian weights per ovary on Day 2 for ULO and Sham gilts were 3.4 versus 2.8 and 0.26 versus 0.24 g, respectively. Those weights on Days 4 and 8 were greater (P less than 0.01) for ULO than Sham gilts. Follicular fluid weight per ovary was greater (P less than 0.05) for ULO than Sham gilts on Days 2, 4 and 8. Ovarian venous E2 concentrations were greater (P less than 0.01) for ULO than for Sham gilts on Days 2 and 4 but were similar on Day 8. In a second experiment, 42 prepuberal gilts 130 days of-age were subjected to Sham (n = 18), ULO (n = 18) or bilateral ovariectomy (BLO; n = 6) to evaluate follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion immediately after surgical treatment. Release of FSH within the first 24 h was greater for BLO than ULO and for ULO than Sham gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of follicular fluid treatment on follicle-stimulating hormone, luteinizing hormone and compensatory ovarian hypertrophy in prepuberal gilts

Prepuberal 130-day-old gilts were treated with 10 ml of charcoal-stripped porcine serum (PS), whole porcine follicular fluid (WpFF) or charcoal-stripped pFF (CpFF) twice daily beginning the day before and continuing 8 days after unilateral ovariectomy (ULO). Follicle-stimulating hormone (FSH) declined for the first 14 h after ULO in WpFF and CpFF gilts and then by 24 h returned to values observed at or before ULO, whereas FSH was increased nearly twofold at 14 h in PS gilts. At 8 days after ULO the remaining ovaries from PS-treated gilts were heavier than ovaries from follicular fluid-treated gilts. In a second experiment, ovariectomized 130-day-old gilts were assigned to either a group infused with PS, a group infused with 5 ml CpFF, or a group infused with 10 ml Cpff at 18 and 2 h before a gonadotropin-releasing hormone (GnRH) challenge. Porcine follicular fluid had no effect on luteinizing hormone (LH) response to GnRH, depressed the FSH response to a 10-micrograms challenge of GnRH, but had no effect on FSH response to a 50-micrograms challenge of GnRH. In a third study, gilts were subjected to sham ovariectomy (Sham) or ULO at 130 days of age. GnRH (10 micrograms) was given on Days 1, 2 or 8 after surgery. The response to GnRH in ULO versus Sham gilts did not differ for FSH or LH on any day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of ovarian activity in the gilt and reversal by exogenous gonadotrophin administration

The aim of the current experiment was to study the regulation of follicle development in the pig using a potent GnRH agonist (GnRH-A) to initially suppress follicle development. Large-White hybrid gilts (n = 8) were treated during the luteal phase with GnRH-A. Four of these GnRH-A treated gilts and four control gilts were given a GnRH bolus on days 14 and 28 after GnRH-A administration or during the luteal phase in control gilts. Blood samples were collected for 10 h for FSH and LH, after which 1500 IU PMSG were administered and the ovaries and uteri recovered 72 h later. A further four GnRH-A treated gilts and four control gilts were slaughtered either 28 days after GnRH-A administration or during the luteal phase respectively, and all follicles > or = 1 mm diameter were dissected. The mean basal plasma FSH level was lower (P < 0.01) in GnRH-A treated than control gilts and showed no response to the GnRH challenge although levels increased (P < 0.01) in control gilts. The mean basal plasma LH levels were similar (P > 0.1) in GnRH-A treated and control gilts. Whilst in GnRH-A treated gilts plasma LH levels showed no response to the GnRH challenge, plasma LH levels were increased (P < 0.01) in control gilts. Pulsatile LH secretion was abolished in GnRH-A treated but not in control gilts. Plasma oestradiol levels were lower (P < 0.001) in GnRH-A treated gilts than in control gilts, but nevertheless both GnRH-A treated and control gilts responded to PMSG with increased plasma oestradiol levels. Treatment with GnRH-A reduced both the mean (2.1 vs. 2.7 mm; P < 0.01) and the maximal follicle diameter (4 vs. 6 mm) and reduced (P < 0.01) the total number of follicles > or = 2 mm diameter compared with control gilts. Administration of PMSG increased both mean follicle diameter (5.1 vs. 4.4 mm; P < 0.01) and maximal follicle diameter (7 vs. 9 mm) and caused a reduction (P < 0.001) in the total number of follicles > or = 2 mm diameter in both GnRH-A treated and control gilts. In summary, this study has demonstrated, for the first time in the pig, that the inhibition of follicle development as a result of pituitary down regulation/desensitisation can be reversed by exogenous gonadotrophin treatment. This model will be a powerful tool with which to investigate the precise regulation of follicle development in the pig.     

Decreased follicular steroids and insulin-like growth factor-I and increased atresia in diabetic gilts during follicular growth stimulated with PMSG

Four streptozotocin-diabetic gilts (maintained on exogenous insulin for 3 months) and 4 normoglycaemic gilts were treated with 600 i.u. PMSG. Diabetic gilts had insulin therapy removed at the time of PMSG administration. Plasma glucose averaged 463 +/- 5 mg/100 ml for diabetic gilts and 82 +/- 4 mg/100 ml for control gilts over the 72-h sampling period. Serum insulin was lower in diabetic than in normoglycaemic gilts (glycaemic state by time interaction; P less than 0.0001). At ovary removal 75 h after PMSG, numbers and percentages of large (greater than or equal to 7 mm) and medium (3-6 mm) non-atretic follicles were similar for diabetic and control gilts (31 vs 68%; s.e.m. = 7; P less than 0.05). Diabetic gilts had a greater percentage of atretic follicles over all size classes (50 vs 21%; s.e.m. = 7; P less than 0.03). After PMSG, LH was suppressed within 12 h in control gilts and remained similar to values in diabetic gilts until 72 h, when LH was elevated in 2 diabetic gilts (glycaemic state by time interaction; P less than 0.001). Pulsatile LH patterns during 52-55 h after PMSG were not affected by glycaemic state. Serum concentrations of IGF-I tended (P less than 0.1) to be lower in diabetic gilts. Concentrations of oestradiol and FSH in serum were similar in diabetic and control gilts. Follicular fluid concentrations of oestradiol in follicles greater than or equal to 7 mm were lower in diabetic than normoglycaemic gilts (341 vs 873 ng/ml; s.e.m. = 86; P less than 0.05). Testosterone was higher in follicles 3-6 mm in diameter in diabetic than in normoglycaemic gilts (142 vs 80 ng/ml; s.e.m. = 26; P less than 0.05). Progesterone concentrations in follicular fluid were not affected by glycaemic state. Concentrations of IGF-I in follicles greater than or equal to 7 mm were lower in diabetic than control gilts (150 vs 200 ng/ml; s.e.m. = 13; P less than 0.05). We conclude that follicles of diabetic gilts respond to external gonadotrophic stimulation with decreased hormone production and increased ovarian follicular atresia, despite an absence of effects on circulating gonadotrophin and oestradiol concentrations.     

Passive immunization of the pig against gonadotropin releasing hormone during the follicular phase of the estrous cycle

Three experiments were conducted to determine the effects of passively immunizing pigs against gonadotropin releasing hormone (GnRH) during the follicular phase of the estrous cycle. In Experiment 1, sows were given GnRH antibodies at weaning and they lacked estrogen secretion during the five days immediately after weaning and had delayed returns to estrus. In Experiment 2, gilts passively immunized against GnRH on Day 16 or 17 of the estrous cycle (Day 0 = first day of estrus) had lower (P<0.03) concentrations of estradiol-17beta than control gilts, and they did not exhibited estrus at the expected time (Days 18 to 22). When observed three weeks after passive immunization, control gilts had corpora lutea present on their ovaries, whereas GnRH-immunized gilts had follicles and no corpora lutea. The amount of GnRH antiserum given did not alter (P<0.05) serum concentrations of LH or pulsatile release of LH in sows and gilts. In Experiment 3, prepuberal gilts were given 1,000 IU PMSG at 0 h and GnRH antiserum at 72 and 120 h. This treatment lowered the preovulatory surge of LH and FSH, but it did not alter serum estradiol-17beta concentrations, the proportion of pigs exhibiting estrus, or the ovulation rate. These results indicate that passive immunization of pigs against GnRH before initiation of or during the early part of the follicular phase of the estrous cycle retards follicular development, whereas administration of GnRH antibodies during the latter stages of follicular development does not have an affect. Since the concentration of antibodies was not high enough to alter basal or pulsatile LH secretion, the mechanism of action of the GnRH antiserum may involve a direct ovarian action.     

Mechanism controlling ovulation rate in ewes in relation to seasonal anoestrus.

Three experiments were carried out during seasonal anoestrus in Finnish Landrace and Scottish Blackface ewes, to establish whether the differences between the breeds in ovulation rate are functional during the non-breeding season and are therefore independent of the mechanism controlling ovulation. In Expt 1, follicles greater than or equal to 2 mm in diameter were dissected from the ovaries of both breeds and incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. In both breeds, follicles producing greater than or equal to 500 pg oestrogen/ml/h (oestrogen-active) were readily identifiable from a population producing less (oestrogen-inactive). The number of oestrogen-active follicles in each breed was similar to the number of ovulations near the end of the breeding season. Oestrogen-active follicles also had more luteinizing hormone (LH) receptors and larger diameters than oestrogen-inactive follicles. There were, however, no significant differences between the two follicle types in follicular fluid or in-vitro testosterone concentrations. In Expt 2, seasonally anoestrous Scottish Blackface ewes were unilaterally ovariectomized; the second ovary was removed 7 days later. Follicles from both ovaries were processed as described for Expt 1; oestrogen-active follicles were categorized according to their ability to produce greater than 500 pg/ml/h. There were twice as many oestrogen-active follicles in the second ovary as in the first ovary; the number of oestrogen-active follicles in the second ovary was also similar to the total number of oestrogen-active follicles in both ovaries of the Scottish Blackface ewes in Expt 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between histological signs of atresia, steroids in follicular fluid, and gonadotropin binding in individual bovine antral follicles during postpartum anovulation

Two experiments were conducted to determine the relationship between histological signs of atresia, gonadotropin binding, and steroids in fluid of medium-sized bovine follicles during postpartum anestrus. In Experiment I, ovaries of 21 cows were removed on Days 7, 14, 28, 42, or 56 after parturition. In Experiment II, ovaries of 29 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH; 500 ng/5 ml saline) injections given every 2 h via jugular cannulas. Two to 10 follicles, 4.0-7.9 mm in diameter, were removed per pair of ovaries. Follicles were classified as normal, intermediate, atretic, or luteinized-atretic, depending on their micromorphology. In both Experiments I and II, follicles classified as normal had 50-80% lower (p less than 0.05) concentrations of progesterone and 2- to 7-fold greater (p less than 0.05) concentrations of estradiol than atretic follicles. However, concentrations of androstenedione and gonadotropin-binding sites were similar in normal and atretic follicles. Atretic follicles had degenerative granulosa with several pyknotic nuclei, thick theca, and little distinction between theca and granulosa. Intermediate follicles showed slight signs of degeneration and had 2- to 3-fold greater (p less than 0.05) concentrations of progesterone than normal follicles. Concentrations of estradiol did not differ (p greater than 0.10) between normal and intermediate follicles. Equal proportions of normal and atretic medium-sized follicles were located on the ovaries bearing the corpus albicans from pregnancy (CAP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification at the onset of luteolysis of follicles capable of ovulation in the ewe

Follicles of various sizes at the surface of the ovary were ablated by electrocautery at the time of cloprostenol-induced luteolysis in ewes and the interval from cloprostenol treatment to the onset of the LH surge determined as an index of the time from luteolysis to ovulation. When follicles 2-4 mm or greater than 4 mm diameter remained in the ovaries, the interval from cloprostenol treatment to the onset of the LH surge was similar to that in sham-operated (control) ewes (55-60 h), whereas when the only follicles remaining were less than 2 mm, the interval was extended by 24 h (P less than 0.05). This study demonstrates that follicles capable of ovulating can be selected from those greater than or equal to 2 mm diameter at luteolysis, emphasizing the flexibility of the sheep ovary in its final selection of the ovulatory follicle.     

Ovarian function in nutritionally induced anoestrous cows: effect of exogenous gonadotrophin-releasing hormone in vivo and effect of insulin and insulin-like growth factor I in vitro

Ovarian function of nutritionally induced anoestrus cows was evaluated in vivo (Expt 1) and in vitro (Expt 2). In Expt 1, 32 nutritionally induced anoestrous beef cows were divided into four treatment groups receiving: (1) saline infusions at one pulse every 4 h for 13 days (control); (2) 2 micrograms GnRH at one pulse every 4 h (2 micrograms infused in 1.8 ml saline over 5 min) for 13 days (GnRH-4); (3) 2 micrograms GnRH at one pulse every 1 h for 13 days (GnRH-1); and (4) continuous infusion of 2 micrograms GnRH (a total of 2 micrograms in 34 ml h-1) for 13 days (GnRH-C). On the last day of treatment, cows were killed, ovaries were removed and follicular fluid samples (n = 149) were collected. The percentage of cows with luteal activity on day 13 was significantly different (P < 0.01) among treatments (0, 25, 75 and 25% for control, GnRH-4, GnRH-1 and GnRH-C cows, respectively). Owing to the large percentage of ovulatory cows in the GnRH-1 group (n = 6), anovulatory cows (n = 2) were removed from this treatment group for statistical analysis, as were cows with luteal tissue from the GnRH-4 (n = 2) and GnRH-C (n = 2) groups. The numbers of small (1.0-4.9 mm) and medium plus large (> or = 5 mm) follicles were not affected (P > 0.10) by treatment. However, GnRH-4 cows (n = 6) had greater (P < 0.05) concentrations of oestradiol in follicular fluid than did control (n = 8) but not GnRH-1 (n = 6) or GnRH-C (n = 6) cows. Concentrations of insulin-like growth factor I were greater (P < 0.05) in the follicular fluid of GnRH-1 cows than in all other treatment groups. Concentrations of androstenedione and progesterone in follicular fluid were not affected (P > 0.10) by treatment or follicle size. The binding activity of insulin-like growth factor binding proteins was not affected by GnRH treatment. However, the binding activity of insulin-like growth factor binding protein 2, 29-32 kDa and 22 kDa insulin-like growth factor binding proteins were greater (P < 0.05) in small versus medium plus large follicles. In Expt 2, granulosa cells were collected from nutritionally anoestrous cows to determine whether ovarian cells from anoestrous cows have the capacity to respond to insulin-like growth factor I or insulin in vitro. Both insulin-like growth factor I (20 and 200 ng ml-1) and insulin (10, 100 and 1000 ng ml-1) increased (P < 0.05) granulosa cell proliferation and progesterone production. In conclusion, pulsatile infusion of 2 micrograms GnRH (every 1 or 4 h) for 13 days into nutritionally induced anoestrous cows results in increased intrafollicular oestradiol and insulin-like growth factor I concentrations and can stimulate ovulation without markedly affecting concentrations of androstenedione or progesterone, or the binding activity of insulin-like growth factor binding proteins, in follicular fluid. In addition, granulosa cells from nutritionally induced anoestrous cows have the capacity to respond to insulin-like growth factor I and insulin in vitro, indicating that the decrease in trophic factors observed with restricted feeding does not reduce the response of the ovary to insulin-like growth factor I and insulin.     

Clinical,Morphological and Endocrinological Studies in Gilts with Delayed Puberty

Thirty-six gilts which had not shown oestrus at about 8 months of age or more were transported from the pig research station to the clinic, a journey of 12 km. The gilts were examined by laparoscopy and those which had only small follicles in the ovaries were catheterized and placed in pens, with sexually mature boars kept in adjacent pens. Oestrus detection was done twice daily and blood was sampled three times a day. After 7 days the laparoscopy was repeated and gilts which still had only small follicles in their ovaries were given 250 μg GnRH intravenously the following day. Blood samples were taken frequently before and after GnRH treatment. One week. after administration of GnRH the ovaries were inspected by laparoscopy once more. The first laparoscopic examination showed that 42 % of the gilts were sexually mature. One gilt had no uterus or ovaries. Twenty gilts had only small follicles in the ovaries and fourteen of these gilts showed ovulatory oestrus 5.5 days (4-7.5 days) after arrival. In these fourteen gilts a rise in the oestradiol-17B level (>30 pmol/1) was seen at an average time of 1.9 days and a rise in LH (preovulatory peak) was seen at an averaged 4.5 days after the start of blood sampling. Six gilts were given 250 ug GnRH. An immediate rise in LH could be seen in all the gilts (mean peak level was 6.18 μg/l) and the elevated levels had a duration of 4 hours. None of the GnRH-treated gilts responded with oestrus symptoms or increased ovarian activity.     

Feed restriction in cyclic gilts: gonadotrophin-independent effects on follicular growth

Our objective was to determine whether changes in metabolic hormones, induced by feed restriction, can alter follicle distribution in swine ovaries through effects independent of LH pulsatility. In a factorial arrangement, 24 gilts were fed a high or a low level of dietary energy (240 or 80% of maintenance requirements) and given an antagonist of GnRH or saline between days 3 and 12 of the oestrous cycle. Serial blood samples were collected on day 12 and ovaries on day 13. Antagonist treatment, that blocked LH pulsatility, decreased the number of follicles larger than 2 mm and increased the number of follicles smaller than 1 mm. The feed restriction did not alter gonadotrophin secretion, decreased the number of follicles smaller than 1 mm and increased the number of 1 - to 1.9-mm follicles. These findings indicate that feed restriction can alter the growth of small follicles independently of gonadotrophin levels.     

Evidence to support a follicle-stimulating hormone threshold theory for follicle selection in ewes chronically treated with gonadotrophin-releasing hormone agonist

The mean and peak concentrations of follicle-stimulating hormone (FSH) during the luteal phase of a normal cycle were measured in 8 Welsh Mountain ewes. Gonadotrophin secretion and follicle growth were then suppressed by the chronic administration of the GnRH agonist buserelin for 5 weeks. During the 6th week of agonist treatment, each ewe was given a continuous infusion of FSH to produce a peripheral concentration of FSH equal to either the mean or peak of the gonadotrophin measured for that individual in the cycle preceding agonist treatment. Treatment had no effect on the total number of follicles, the number of follicles less than or equal to 2.5 mm in diameter or the in-vitro production of oestradiol by the small follicles when compared with control animals. None of the animals infused with the mean luteal-phase FSH equivalent developed large follicles greater than 2.5 mm diameter which could be classified as preovulatory follicles (oestradiol greater than 1000 pg/follicle/h). All of the animals infused with the peak luteal-phase FSH equivalent developed large follicles, some of which were preovulatory. The results suggest that an individual threshold concentration exists for FSH above which the later stages of preovulatory follicular development are stimulated.     

Localization of follicle regulatory protein in the porcine ovary

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.     

Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of progesterone on follicular growth in the rabbit ovary

Studies were undertaken to measure the growth of follicles in the rabbit ovary during periods of elevated blood levels of progesterone. The progestin was increased in the blood by pregnancy or by implantation of progesterone pellets, which raised blood progesterone to near the levels measured during pregnancy. After 1, 2, 3, or 4 weeks of pregnancy or progesterone-pellet treatment, follicles of 1.0 mm external diameter or greater were dissected out of the ovaries and their external diameters were measured; then, each follicle was extracted for measurement of estradiol content. Blood levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in these animals as well. Follicles up to 2.5 mm in diameter were found in the ovaries of nonpregnant and untreated animals while 1.8 mm was the maximal size found during pregnancy or progesterone-pellet treatment. Furthermore, both in pregnant and in progesterone-treated rabbits, the follicular estradiol content and concentration were significantly suppressed compared to follicles from untreated rabbits. The progesterone pellets had no major effect on the levels of LH and FSH in the blood; the concentration of these gonadotropins in the progesterone-treated rabbits was virtually identical to levels previously measured in the blood of pregnant animals. The results of these studies indicate that progesterone exerts an inhibitory action on follicular development and steroidogenic function in the rabbit ovary. The progesterone action appears to be exerted directly on the ovary and is not indirect, by way of an inhibition of gonadotropin secretion.