Effects of gonadotropin treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of oocytes in prepubertal gilts

This study was undertaken to compare the effects of FSH-pituitary (FSH-P), eCG, and a combination of gonadotropins containing 400 IU eCG and 200 IU hCG (PG 600) on the growth of large follicles, oocyte quality and in vitro fertilization (IVF) rate of in vitro matured (IVM) oocytes in prepubertal gilts. The ovaries were removed via midventral laparotomy 48 h (Experiment 1) or 72 h (Experiment 2) after the first injection. In Experiment 1, 30 gilts received 1 of 5 treatments: 1) saline (3 ml i.m., once, n = 6); 2) FSH-P8 (8 mg i.m., twice, with a 24-h interval, n = 6); 3) FSH-P16 (16 mg i.m., twice, with a 24-h interval, n = 6; 4) eCG (1000 IU i.m., once, n = 6); or 5) PG 600 (5 ml i.m., once, n = 6). Compared with saline, treatment with PG 600 or eCG induced significant (P < 0.05) growth of large follicles (> or = 6 mm). In Experiment 2, 16 gilts received 1 of 5 treatments: 1) saline (n = 4); 2) FSH-P8 (n = 4); 3) FSH-P16 (n = 4); 4) eCG (n = 4), or 5) PG 600 (n = 4). The same injection protocol as in Experiment 1 was used. Compared with treatment with FSH-P8 or FSH-P16, eCG increased (P<0.05) the number of large follicles. The proportion of good oocytes was increased (P<0.05) with FSH-P8 or FSH-P16 compared with treatment with eCG or PG 600. Moreover, oocytes from eCG-treated gilts had a greater (P<0.05) rate of male and female pronuclei than FSH-P or saline-treated gilts. In conclusion, treatment with FSH-P resulted in a higher proportion of oocytes with multilayer cumulus cells, whereas treatment with eCG resulted in higher pronuclear rates following in vitro fertilization in prepubertal gilts.     

In vitro development of embryos from superovulated gilts treated with the progesterone agonist, altrenogest (Regu-Mate) or the prostaglandin analogue, cloprostenol (Planate)

This study was conducted to compare the in vitro development of embryos from superovulated postpubertal gilts synchronized with progesterone agonist altrenogest (REG, Regu-Mate) and those from superovulated prepubertal gilts synchronized with prostaglandin analogue cloprostenol (PLA, Planate). Ten postpubertal gilts that had exhibited estrus at least once were fed 20 mg/d of REG from Day 0 (the first day of treatment, may have been any day of the estrous cycle) to Day 17. The gilts received intramuscularly (im) 1500 IU of equine chorionic gonadotropin (eCG) on the afternoon of Day 17, followed by 1000 IU of human chorionic gonadotropin (hCG) 84 h later. Eight prepubertal gilts received intramuscularly one dose of a combination of 400 IU of eCG and 200 IU of hCG (PG 600) on Day 0 (the first day of treatment), followed by 750 IU of hCG on Day 3. From Day 16 to Day 19, the prepubertal gilts received 350 mg/d of PLA, followed by 1500 IU of eCG on the afternoon of Day 19, then 1000 IU of hCG 84 h later. Gilts were checked for estrus with an intact boar. At estrus, all gilts were artificially inseminated and/or mated twice at 12-h intervals. Then 50 to 54 h after the hCG injection, a mid-ventral laparotomy was performed on each gilt. Corpora albicans (CA) and corpora hemorrhagica (CH) were counted, and oviducts were flushed in situ. The embryos recovered (1- to 2-cell) were cultured in modified Whitten's medium at 38.5 degrees C under an atmosphere of 5% CO2 in air for 144 h. The number of CA per gilt did not differ between the postpubertal and prepubertal gilts (11.9 vs 7.9, respectively; P > 0.05). However, the number of CH per gilt (27.5 vs 18.1, P = 0.05) and the number of embryos per gilt (26.2 vs 15.3, P < 0.05) were higher in postpubertal gilts than in prepubertal gilts. Furthermore, after 144 h of in vitro culture, the percentage of embryos cleaving to the >-16-cell (morula + blastocysts) or > or =32-cell (blastocysts) was greater (P < 0.05) in prepubertal gilts than in postpubertal gilts (85.2 vs 68.5, 55.7 vs 44.2, respectively). The total numbers of embryos examined were 122 and 260 in prepubertal and postpubertal gilts, respectively. These results show that postpubertal gilts treated with REG produced a higher number of embryos. However, better embryo development was noted with zygotes from prepubertal gilts primed with exogenous gonadotrophin, followed by synchronization with prostaglandin before induction of superovulation and insemination.     

The impact of dose of FSH (Folltropin) containing LH (Lutropin) on follicular development, estrus and ovulation responses in prepubertal gilts

FSH is favored over chorionic gonadotropins for induction of estrus in various species, yet little data are available for its effects on follicle development and fertility for use in pigs. For Experiment 1, prepubertal gilts (n = 36) received saline, 100 mg FSH, or FSH with 0.5 mg LH. Treatments were divided into six injections given every 8 h on Days 0 and 1. Proportions of gilts developing medium follicles were increased for FSH and FSH-LH (P < 0.05) compared to saline, but follicles were not sustained and fewer hormone-treated gilts developed large follicles (P < 0.05). No gilts expressed estrus and few ovulated. Experiment 2 tested FSH preparations with greater LH content. Prepubertal gilts (n = 56) received saline, FSH-hCG (100 mg FSH with 200 IU hCG), FSH-LH5 (FSH with 5 mg LH), FSH-LH10 (FSH with 10 mg LH), or FSH-LH20 (FSH with 20 mg LH). FSH-LH was administered as previously described, while 100 IU of hCG was given at 0 h and 24 h. Hormone treated gilts showed increased (P < 0.05) medium and large follicle development, estrus (>70%), ovulation (100%), and ovulation rate (>30 CL) compared to saline. There was an increase (P < 0.05) in the proportion of hormone-treated gilts with follicular cysts at Day 5, but these did not persist to Day 22. These gilts also showed an increase in poorly formed CL (P < 0.05). FSH alone or with small amounts of LH can induce medium follicle growth but greater amounts of LH at the same time is needed to sustain medium follicles, stimulate development of large follicles and induce estrus and ovulation in prepubertal gilts.     

Effects of pregnant mare serum gonadotropin (eCG) on follicle development and granulosa-cell apoptosis in the pig

The effect of eCG on follicular development and granulosa-cell apoptosis in sexually mature and immature gilts and on granulosa-cell apoptosis in vitro were studied. The sexually mature gilts were treated with eCG on Day 11 of the estrous cycle, and effects were analyzed at different times after treatment with untreated animals at corresponding stages of the cycle as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), hematoxylin and eosin staining, and DNA ladder. The proportion of apoptotic cells in atretic follicles (39%) was significantly higher (P<0.01) than that in healthy follicles (9%). At 24h after eCG treatment in mature gilts, the total number of follicles visible on the ovarian surface (57 per ovary), the number of small (<3mm) follicles (31.5 per ovary) and the number of medium-sized (3-5mm) follicles (23 per ovary) were significantly higher (P<0.05) than those of control animals (28, 20 and 6.5 per ovary, respectively), and declined gradually thereafter to below the level of control animals. The number of large (>or=5mm) follicles began to show a marked increase at 72h after eCG (8.5 versus 2.5, P<0.05). At 24h after eCG treatment, the proportions of apoptotic cells in small (7.2%) and medium-sized follicles (7.4%) were markedly lower (P<0.01) than those in controls (21.5 and 21%, respectively) and increased gradually thereafter to approach the level in controls. The percentage of apoptotic cells in large follicles (10% at 24h post-eCG) did not change significantly. Before eCG treatment, there were markedly fewer follicles of all types on ovaries of immature gilts than of mature gilts (9 versus 25 per ovary) and the proportion of apoptotic cells in small and medium follicles was high (25 and 34%, respectively). After eCG treatment, the changes in follicle number and proportion of apoptotic cells in the immature gilts followed a similar pattern to that of the mature gilts. Equine chorion gonadotropin inhibited apoptosis of granulosa cells cultured either in vitro or in intact follicles in a dose-dependent manner. Thus, follicular atresia in the pig, as in other animals, was characterized by apoptosis of large numbers of granulosa cells, and eCG promoted follicular development by inhibition of granulosa-cell apoptosis.     

Effect of prior FSH treatment on the estrus and ovulation responses to eCG in prepubertal gilts

The objective of this study was to determine the effect of pre-treatment of prepubertal gilts with FSH on the estrus and ovulatory responses to eCG injection at two ages. A total of 149 prepubertal Hypor gilts were selected at 150 days (n=76) or 180 days (n=73) of age and assigned to injection of 400 IU eCG plus 200 IU hCG (PG600), 600IU eCG alone (Folligon), pre-treatment with 72 mg FSH (Folltropin) administered as 6 x 12 mg injections at 12 h intervals with 600 IU Folligon 12h after last FSH injection, or non-injected controls. To facilitate detection of estrus, gilts were exposed to a mature boar for 15 min daily for 7 days. To determine ovulatory responses, blood samples were obtained on the day of injection and 10 days later and assayed for progesterone content. Following treatment at 150 days, one control gilt (5.3%) was deemed estrus but ovulation did not occur. Compared to treatment with Folligon alone, PG600 injection tended (P=0.1) to increase the estrus response (52.6% compared with. 26.3%) and increased (P<0.01) the ovulatory response (89.5% compared with. 47.4%). The estrous response in gilts pretreated with Folltropin was intermediate (42.1%) but the ovulatory response (47.4%) was the same as for Folligon alone. Following treatment at 180 days, two control gilts (10.5%) were deemed estrus and ovulation did occur in these gilts. There was no difference between hormone-treated groups for estrus or ovulatory responses, although the ovulatory response of PG600-treated gilts tended (P=0.1) to be greater than for the Folligon-treated group (89.5% compared with 66.7%), with Folltropin-pretreated gilts being intermediate (76.5%). These data demonstrate that the estrus and ovulatory responses of gilts were greater for PG600 than for Folligon and that while responses to PG600 were not affected by gilt age, for the combined Folligon groups, estrous response (P<0.02) and ovulatory response (P<0.05) improved with increased gilt age.     

Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function

The overall objective was to evaluate the use of porcine luteinizing hormone (pLH) for synchronization of ovulation in cyclic gilts and its effect on reproductive function. In an initial study, four littermate pairs of cyclic gilts were given altrenogest (15 mg/d for 14 d). Gilts received 500 microg cloprostenol (Day 15), 600 IU equine chorionic gonadotropin (eCG) (Day 16) and either 5mg pLH or saline (Control) 80 h after eCG. Blood samples were collected every 4h, from 8h before pLH/saline treatment to the end of estrus. Following estrus detection, transcutaneous real-time ultrasonography and AI, all gilts were slaughtered 6d after the estimated time of ovulation. Peak plasma pLH concentrations (during the LH surge), as well as the amplitude of the LH surge, were greater in pLH-treated gilts than in the control (P=0.01). However, there were no significant differences between treatments in the timing and duration of estrus, or the timing of ovulation within the estrous period. In a second study, 45 cyclic gilts received altrenogest for 14-18d, 600 IU eCG (24h after last altrenogest), and 5mg pLH, 750 IU human chorionic gonadotropin (hCG), or saline, 80 h after eCG. For gilts given pLH or hCG, the diameter of the largest follicle before the onset of ovulation (mean+/-S.E.M.; 8.1+/-0.2 and 8.1+/-0.2mm, respectively) was smaller than in control gilts (8.6+/-0.2mm, P=0.05). The pLH and hCG groups ovulated sooner after treatment compared to the saline-treated group (43.2+/-2.5, 47.6+/-2.5 and 59.5+/-2.5h, respectively; P<0.01), with the most synchronous ovulation (P<0.01) in pLH-treated gilts. Embryo quality (total cell counts and embryo diameter) was not significantly different among groups. In conclusion, pLH reliably synchronized ovulation in cyclic gilts without significantly affecting embryo quality.     

Vascular endothelial growth factor production in growing pig antral follicles

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4-5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.     

Increased ovarian follicular angiogenesis and dynamic changes of follicular vascular plexuses induced by equine chorionic gonadotropin in the gilt

Follicular angiogenesis and capillary degeneration are crucial ovarian processes in folliculogenesis. The present study was conducted to assess the changes in population of follicular vascular plexuses with different capillary status in prepubertal gilts 72 h after equine chorionic gonadotropin (eCG) (1,250 IU) treatment, using combined vascular corrosion casting and scanning electron microscopy. Follicular fluid concentrations of estradiol, progesterone and vascular endothelial growth factor (VEGF) were determined to confirm the follicular status. Based on the proliferative or degenerative characteristics of their capillaries, follicles were classified into three categories: active angiogenesis, low angiogenesis and degeneration. Irrespective of exogenous gonadotropin treatment in vivo, small follicular vascular plexuses (<4 mm in diameter) exhibited all three conditions in casted ovaries, while medium (4–5 mm) and large (>5 mm) plexuses showed only active angiogenesis or degeneration. eCG treatment significantly increased the population of large, but decreased that of small follicular plexuses. Most large follicular vascular plexuses showed active angiogenesis with higher follicular fluid estradiol:progesterone ratios and VEGF concentration. eCG also increased the percentage of medium follicular plexuses with active angiogenesis. The populations of small follicular plexuses with active angiogenesis were higher in controls, but decreased after eCG treatment. However, treatment of gilts with the gonadotropin increased the percentage of small plexuses (<1.0 mm) with low angiogenesis and those (1–3.9 mm) with extensive capillary degeneration. These findings are consistent with the hypothesis that angiogenesis is involved in selection and growth of small follicles in gilts under the regulation of gonadotropin.This work was supported by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences and Research for the Future Program, the Japan Society for the Promotion of Science (JSPS-RFTF97L00904) and the Canadian Institutes of Health Research (MOP-15691). J.Y.J. is a recipient of a CIHR-STIRRHS Postdoctoral Fellowship. A preliminary report of this work was presented at the 49th Annual Meeting of the Canadian Fertility and Andrology Society, 5–8 November 2003, Victoria, British Columbia, Canada     

Fixed-time artificial insemination protocols on brazilian locally adapted breed gilts on ovulatory response and embryo production

The aim of this study was to use estrus synchronization protocols to favor fixed-time artificial insemination and consequently fixed-time embryo collection, and increase embryo production using eCG, in gits. In a cross over design, nine Piau breed gilts were subjected to 18 days of oral progesterone; P4 group did not receive any further; GnRH group received 25µg of GnRH 104 hours after the final application of P4; and eCG+GnRH group received 1000IU of eCG 24 hours after the final P4 in addition to GnRH for subsequent embryo collection, that was performed six days after first AI, by laparotomy. Artificial insemination was performed after 12 and 24 hours of estrus in P4 group, and 128 and 144 hours in GnRH and eCG+GnRH groups. The number of CL (8.6±3.9; 8.3±2.1; 26.7±15.0) and anovulatory follicles (4.3±3.7; 3.9±3.9; 17.2±9.5) was higher in the eCG+GnRH gilts (P<0.05). However, the use of 1000 IU of eCG reduced (P<0.05) the number of total structures (5.2±3.6; 5.1±3.1; 1.7±2.7), viable embryos (5.0±3.5; 4.8±3.3; 0.4±0.7), freezable embryos (3.6±3.4; 3.3±3.8; 0.1±0.3) and recovery rate (63.7±38.9; 58.6±24.7; 5.38±9.5). P4 and GnRH protocols were effective in the production and recovery of embryos. However, the use of 1000 IU of eCG, 24 hours after P4, was not effective in promoting the production of embryos, although the animals had superovulated.     

Structure-function relationships during preovulatory development of porcine follicles following equine chorionic gonadotropin stimulation.

Porcine follicular maturation begins by recruitment from a continually proliferating pool of small antral follicles; those receiving the appropriate stimulus differentiate rapidly through a series of structural and functional changes. Such ovarian activity can be induced in prepubertal gilts with a single injection of equine chorionic gonadotropin (eCG). Average follicular diameter in eCG treated females increased from approximately 2 mm before stimulation to 3.5 mm by 24 hr after injection, with subsequent growth to ovulatory size (8 or 9 mm) by 96 hr. Both theca and granulosa layers increased in thickness and complexity, and a prominent capillary bed evolved immediately outside the basement membrane separating the two layers. Cytoplasmic organelles associated with increased metabolic activity and steroidogenesis proliferated within the first 24 hr. Progressive changes included increasing amounts of lipid and rough and smooth endoplasmic reticulum, with the latter occurring in vesicular or lamellar forms and as lipid-associated whorls. Bizarre mitochondrial forms also appeared, often associated with lipids. The amount and proportion of rough and smooth endoplasmic reticulum shifted dramatically as follicles matured. By 24 hr, rough endoplasmic reticulum in thecal cells increased from 4.2 to 7% of cell volume, while the amount in granulosa cells increased from less than 3.5% to more than 10%; the quantity remained relatively constant in the theca but declined to prestimulation values in the granulosa layer. Rough endoplasmic reticulum predominated over smooth in the first 24 hr following stimulation but the proportions were then reversed, so that more than 10% of both layers was composed of smooth endoplasmic reticulum by the time ovulation was imminent. Some follicles had or were in the process of ovulating by 96 hr. Their walls were collapsed into prominent folds with the two cell types beginning to mix. Slight undulations and some regions of discontinuity were observed in basement membranes of large unovulated follicles at this time. In specimens collected at 96 hr poststimulation and processed for retention of lipid, lipid-like material was noticeable in the extracellular matrix surrounding cells that contained organelle configurations suggestive of steroidogenesis.     

Inhibin production by macaque granulosa cells from pre- and periovulatory follicles: regulation by gonadotropins and prostaglandin E2.

Although inhibin (IN) is secreted by granulosa cells (GC) of preovulatory follicles, the major source of immunoreactive IN circulating during the primate ovarian cycle is the corpus luteum. The aims of this study were (1) to investigate culture conditions for optimal IN production by luteinized GC (LGC) from rhesus monkeys and (2) to compare IN and progesterone (P) production by nonluteinized GC (NGC) and LGC in response to putative agonists. Animals were treated for up to 9 days with human menopausal gonadotropins to promote the development of multiple preovulatory follicles. GC were obtained from large follicles before (NGC) or 27 h after (LGC) an ovulatory injection of hCG. For Aim 1, cells were cultured in Hams F-10 medium +/- hCG (100 ng/ml) with or without the addition of insulin/transferrin/selenium, 10% fetal bovine serum, or 10% Serum-Plus (JRH Biosciences, Lenexa, KS). Medium was changed on Days 1, 2, 4, 6, and 8, and IN and P concentrations were determined by RIA. Basal (unstimulated) IN production by LGC was enhanced and maintained for 6-8 days in the presence of serum, but rapidly declined in the absence of serum. In contrast, basal P secretion declined regardless of exposure to serum. Human CG consistently increased (p less than 0.05) IN production only in the presence of serum but stimulated (p less than 0.05) P production under all conditions. For Aim 2, cells were cultured for 4 days in Ham's F-10 medium + 10% macaque serum +/- hCG (100 ng/ml), hFSH (100 ng/ml), prostaglandin E2(PGE2; 14 microns), or dibutyryl(db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin production by dispersed granulosa and theca interna cells from porcine preovulatory follicles

Prepubertal gilts were treated with 750 IU pregnant mares' serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa (GC) and theca interna (TIC) cells were prepared by microdissection and enzymatic digestion from follicles obtained 36, 72 and 108 h after PMSG treatment and incubated for up to 6 h in a chemically defined medium in the presence or absence of arachidonic acid, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and indomethacin. Production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. Both GC and TIC had the capacity to produce prostaglandins, with production by each cell type increasing markedly with follicular maturation. PGE was the major prostaglandin produced by both cellular compartments. Only PGE production by GC was consistently enhanced by addition of arachidonic acid to the incubation medium. Neither cell type was responsive to FSH and LH in vitro. Indomethacin inhibited the production of PGE and PGF by both cell types. These results provide convincing evidence for an intrafollicular source of prostaglandins and indicate that both cellular compartments contribute significantly to the increased production of prostaglandins associated with follicular rupture.     

Control of ovulation with a GnRH analog in gilts and sows

Methods for the control of ovulation with GnRH or the GnRH analog D-Phe6 -LHRH (GnRH-A), were evaluated in gilts and sows as the last step in development of a fixed-time Al protocol. This involved 3 field trials using 2,744 gilts (10 units) and 71,628 sows (33 units). In Trial 1, the GnRH-A (75 microg) was given subsequent to treatment with altrenogest for cycle control or eCG for the stimulation of uniform follicle development in gilts. The release of LH was followed by ovulations which commenced within 36.4 +/- 3.3 hr and were terminated at 39.0 +/- 2.8 hr after administration of GnRH-A. This degree of synchronization of ovulations enabled the use of fixed-time AI. Consequently, subsequent to pretreatment with altrenogest and eCG, in 10 production units 1,285 gilts received 50 microg GnRH-A and 1,459 gilts 500 IU hCG serving as positive controls (Trial 2); all the gilts were inseminated 24 and 42 hr after treatment. Pregnancy rate and piglet index (n of piglets per 100 first inseminations) following GnRH-A vs hCG were 78.8% and 779 vs 74.4% and 728, respectively (P < 0.05). In field trials with first litter gilts and multiparous sows (33 units holding from 250 to 6,000 sows), 1,000 IU eCG was used for estrus control after weaning and 25 microg or 50 microg GnRH-A were given 55 to 58 hours after eCG (n = 19,954 and 20,701) (Trial 3). Sows treated during the same time period with 300 microg GnRH plus 300 IU. hCG (n = 30,973) served as positive controls; all sows were inseminated 24 and 42 hours after treatment. Pregnancy rates for 50 microg GnRH-A, 25 microg GnRH-A and 300 microg GnRH plus 300 IU hCG were 83.0%, 81.7% and 80.7%, and the piglet indices 913, 899 and 880, respectively (P < 0.05). Unit size and parity had significant effects on fertility and productivity. In all studies, results with 50 microg GnRH-A were superior. In year-long studies, highest levels of fertility in response to these treatments were seen from December to May.     

Changes in and partial identification of the plasminogen activator and plasminogen activator inhibitor systems during ovarian follicular maturation in the pig

Plasminogen, plasmin, and plasminogen activator (PA) activities and PA and PA inhibitor (PAI) contents were measured in granulosa (GC) and theca interna cell extracts and follicular fluid (FF) obtained from preovulatory follicles of prepubertal gilts treated with eCG and hCG to induce follicular growth and ovulation. Plasmin activity in FF increased just before the time of expected ovulation. This increase was not attributable to changes in plasminogen levels, which remained relatively constant during preovulatory follicular development. The increase in follicular plasmin levels was associated with significant (p less than 0.01) increases in PA activity and content and decreases in PAI content in GC and FF. Western blot analysis suggested that follicular PA activity was represented principally by two forms of tissue type PA (t-PA) each with a pI of 7.8 and with molecular masses of 72,000 and 78,000 daltons, respectively. Two PA-PAI complexes of 126,000 and 130,000 daltons were observed. These complexes were partially dissociated with nucleophilic agents into two t-PA-like forms and a 52,000-dalton PAI protein with a pI of 4.8. Biochemical characteristics of the PAI protein suggest that it belongs to the same class of inhibitors as bovine and human PAI-1. These data indicate that rupture of the porcine ovarian follicle is temporally associated with a net increase in PA activity and an increase in plasmin activity. The increase in PA activity appears to be regulated by changes in PA and PAI content.     

Gonadotropic regulation of prostaglandin production by ovarian follicular cells of the pig

Prepubertal gilts were treated with 750 IU pregnant mare's serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa cells (GC) and theca interna cells (TC) from follicles of gilts 72 h (GC-72 and TC-72, respectively) and 108 h (GC-108 and TC-108 h, respectively) after PMSG treatment were cultured for 0, 12, 24, and 36 h in medium with or without luteinizing hormone (LH), dibutyryl cyclic adenosine 3',5'-monophosphate [Bu)2cAMP), calcium ionophore (A23187), and/or arachidonic acid (AA), and the production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. TC-72 was the principal source of PGs 72 h after PMSG. At 108 h, the production of PGE and PGF by GC was increased 10- and 30-fold, respectively, whereas corresponding increases by TC were 2-fold. LH and A23187 significantly stimulated PGE and PGF production by both GC-72 and TC-72, but only thecal PG production was stimulated by (Bu)2cAMP. LH had minimal or no effect on PG production by GC-108 and TC-108, but A23187 (GC-108, TC-108) and (Bu)2cAMP (TC-108) were stimulatory. Basal PG production by GC-72, GC-108, and TC-108 was stimulated by AA. However, production by GC and TC cultured in medium containing AA and LH, A23187, or (Bu)2cAMP was not different from that produced by AA alone. These findings suggested that GC and TC can synthesize PGs in vitro, but AA availability is rate-limiting in GC. After exposure to hCG in vivo, the capacity of both cell types to produce PGs is increased but is limited by AA availability.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH is superior to eCG for promoting ovarian response in Chinese Bamei gilts

The Bamei gilt is a Chinese native breed located in northwest China, which adapts to the extremely dry and cold environment and is distinguished for its excellent reproductive and maternal characters. To ensure sufficient numbers of embryos for transgenic and nuclear transfer research, hormonal induction of gilt estrus and superovulation may be necessary. The objective of this study was to compare the superovulation effects of equine chorionic gonadotropin (eCG, Group A) and FSH (Groups B-D) in Chinese Bamei gilts. The results show that though eCG could produce more corpora lutea (CL, 14.3) than the control (CL, 9.2), and the FSH treatments had significantly increased the number of CL compared with the eCG treatment. Within the different FSH protocols, the numbers of CL were significantly greater in Groups B (CL, 77.8) and C (CL, 66.8) than in Group D (CL, 42.7), however, ovarian cysts were observed in Groups B and C, but not in Group D. These data suggest that Group D (280 IU FSH) is a suitable protocol to facilitate the development of ovarian follicles and increase the number of useful embryos per gilt for embryos recovery. The optimal FSH protocol of superovulation in Bamei gilts appears to be: D13/100 IU, D14/80 IU, D15/60 IU, D16/40 IU plus prostaglandin (PG) 0.2mg, D17/hCG 1000 IU.     

Gonadotropin-independent mechanisms participate in ovarian responses to realimentation in feed-restricted prepubertal gilts.

Short-term feed restriction in prepubertal gilts suppresses episodic LH secretion in the absence of changes in body weight or composition. To assess non-gonadotropin-mediated effects of realimentation at the ovarian level, 52 gilts were assigned to six treatments after 7 days (Days 1-7) of maintenance feeding (approximately 30% ad libitum). Groups R12 and R9 were maintenance-fed Days 8-12 or Days 8-9, respectively; A12 and A9 were fed to appetite Days 8-12 or Days 8-9, respectively. Groups R9P and A9P were fed as groups R9 and A9 were but received 750 IU eCG at 1500 h on Day 8. Groups R12 and A12 were ovariectomized at 1500 h on Day 12, and all other groups were ovariectomized at 1500 h on Day 9. All gilts received oral progestogen (15 mg allyl trenbolone) from Day 1 to ovariectomy, to antagonize the usual increases in endogenous gonadotropins that follow realimentation. Blood samples were obtained at 10-min intervals during selected windows during the experiment. Ovarian follicles were analyzed for development and steroidogenesis, and plasma samples were analyzed by RIA to determine concentrations of LH, FSH, insulin, and insulin-like growth factor-1 (IGF-1). Allyl trenbolone abolished pulsatile LH secretion, and realimentation did not stimulate LH or FSH secretion, with the exception of FSH secretion on Day 8 in A9 gilts. Postprandial insulin concentrations on Day 9 were greater after feeding to appetite (A9, A9P, and A12) than after feed restriction (R9, R9P, and R12). Pre- and postprandial IGF-1 concentrations were higher in re-fed gilts on Day 9 (A9 and A12) and Day 12 (A12) than in feed-restricted gilts. Follicular diameter, fluid volume, and basal granulosa cell estradiol synthesis per follicle were greater in A12 gilts than in R12 gilts, although there was no difference between A9 and R9 gilts. There was no effect of realimentation on follicular fluid concentrations of estradiol or testosterone, or on androgen-driven granulosa cell estradiol synthesis. Treatment with eCG increased follicular diameter, fluid volume, basal and androgen-driven estradiol synthesis, and fluid estradiol concentrations without interaction with feeding level. In conclusion, in the absence of LH elevations, realimentation over 5 days exerts effects at the ovary, increasing follicular growth and estradiol synthesis. These effects may be mediated by insulin, IGF-1, or unmeasured growth factors and would be expected to synergize with increases in endogenous gonadotropin that follow realimentation.     

Ovarian follicle vascularization in fasted pig

The authors have investigated in the different classes of ovarian follicles the vascular area, the blood vessel distribution, the vascular endothelial growth factor (VEGF) mRNA expression and the VEGF secretion during equine chorionic gonadotropin (eCG) induced follicle growth in prepubertal gilts fed ad libitum or fasted. Immunohistochemistry staining of Von Willebrand factor showed that fasting caused a dramatic increase in the vascular area of medium-large tertiary follicles. The increase involved the two concentric vessel networks and the area between them that, becoming crossed by several anastomosis, modified the whole vessel architecture. Both in situ hybridization and in vitro culture experiments demonstrate that granulosa cells from medium-large follicles are engaged in a copious VEGF production upon eCG stimulation both in gilts fed ad libitum or fasted. More surprisingly, the production of VEGF becomes diffuse amongst theca cells of fasted animals thus recruiting a compartment that in condition of normal feeding regimen appears nearly quiescent. In conclusion, the data presented describe a local angiogenic process that develops in the follicle wall of growing antral follicle in case of acute severe food restriction. The mechanism, essentially confined to follicles that potentially approach ovulation, appears to assume the meaning of a local compensatory mechanism that may help maintaining adequate nutrient delivery to follicles that undergo ovulation.     

Measurement and characterization of swine uterine estradiol receptors: the effects of puberty induction on estradiol receptors and corpus luteum function

Two types of cytoplasmic 17 beta-estradiol (E2) binding activity were identified and characterized in the uteri of pregnant, cycling and prepubertal, cycle-induced (400 IU pregnant mare's serum gonadotrophin (PMS) + 200 IU human chorionic gonadotrophin (hCG)) gilts. Overall, type I affinity and capacity were Kd 1.94 +/- 0.51 nM and 5.410 +/- 1.09 pmol/mg protein, respectively; type II apparent dissociation constant and capacity were Kd 21.34 +/- 6.83 nM and 62.58 +/- 15.96 pmol/mg protein, respectively. Cytoplasmic luteal E2 receptors were undetectable in all groups. Uterine E2 receptor activity was eluted from diethylaminoethyl columns by a 0.05-0.15 M KCl gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated a molecular weight of 70 400-79 000. Excluding gilts with cystic ovarian follicles (16.67%), prepubertal gilts treated with PMS + hCG versus cycling sows had lower serum progesterone on days 6 and 9-13 of the estrous cycle and lower 13,14-dihydro-15-keto prostaglandin F2 alpha levels on days 0-9 and 13-17 of the cycle. Implants, containing 200 mg estrone inserted subcutaneously on days 12-19 after PMS + hCG treatment in gilts, had no discernible effects on these parameters. These results indicate that the diminished reproductive capacity of the gilt, in which cycle activity is induced by PMS + hCG, is likely due to decreased luteal progesterone secretion. Preliminary data also suggest that the lack of E2 receptors may contribute to the low reproductive performance in gilts with cystic ovarian follicles.