Induction Of Follicular Development Research Articles

The presence of VEGF and Notch2 during preantral-antral follicular transition in infantile rats: Anatomical evidence and its implications

Folliculogenesis is a process that depends on angiogenesis, in which VEGF and Notch signaling pathway members are involved. Although this pathway is present in preantral and antral follicular structures during the second stage of folliculogenesis, this association has not been described. Therefore, this study aimed to identify VEGF and Notch2 in ovary structures of infantile rats after induction of follicular development with a gonadotropin stimulus. In order to explore this possibility we analyzed rat ovary morphology from days 10–25 after birth; subsequently, the transition from preantral follicle to an antral stage was analyzed by the induction of follicular development with equine chorionic gonadotropin (eCG) and VEGF and Notch were identified in the rat ovary by fluorescence. The histological analysis revealed that the ovary of a 10-day-old rat has the highest percentage of preantral follicles and based on this a 10IU eCG dose promoted an increase in the number of antral follicles, as well as a decrease in the number of preantral follicles, related to which there was an increase in ovary weight and size. In addition, a higher concentration of circulating estradiol was observed, proliferation of granulosa cells in both follicle groups was stimulated, and the accumulation of VEGF in granulosa and theca cells and in the antral follicle oocyte was increased (p<0.05), whereas the presence of Notch2 was limited to mural granulosa cells, in granulosa cells that formed the cumulus oophorus and in the oocyte of both groups of follicles. The multiple correspondence analysis allowed us to support an association between VEGF and Notch2 during the transition from preantral to antral follicles in the ovary of an infantile rat.

197 EXPRESSION OF MELATONIN-RELATED GENES IN RAT CUMULUS–OOCYTE COMPLEXES

Melatonin is a hormone usually associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function. There is evidence that melatonin acts directly on the regulation of ovary function. The mRNA expression of melatonin membrane receptors genes was detected in mammalian and non-mammalian ovaries. In spite of melatonin receptors and Asmt (limiting enzyme in melatonin biosynthesis) genes being present in cattle cumulus–oocyte complexes (COC), no information regarding rat COC has been reported. The aim of this study was to investigate the expression of melatonin receptor (Mt2) and melatonin synthesis enzyme (Asmt) genes at different meiotic cell cycle stages in COC from 27-day-old female rats. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. To obtain germinal vesicle (GV) immature COC from ovarian follicles, rats were treated with 20 IU equine chorionic gonadotropin (eCG) for induction of follicular development and killed by euthanasia 48 h later. To obtain COC at MII oocyte stage from oviducts, rats were injected with 20 IU of eCG and 48 h later with 20 IU of human chorionic gonadotropin (hCG), and then killed after 14 to 16 h. The oocytes in COC were separated from their cumulus cells by repeated pipetting through a narrow-bore pipette in culture medium. Oocytes and cumulus cells total RNA were isolated using a Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). Pools of 80 COC per cDNA sample were used. Quantitative real-time PCR (qRT-PCR) was performed (7500 Real-Time PCR System; Applied Biosystems Inc., Foster City, CA, USA) with 25-µL reactions containing 2 µL of cDNA (10 ng µL–1), SYBR green (Power SYBR Green; Applied Biosystems Inc.), and 400 nM specific intron-spanning primers. A set of 10-fold serial dilutions of each internal standard (102 to 106 copies/2 µL) was used to generate a standard curve. Transcript numbers were determined by the system software and normalized using the geometric mean calculated from the reference genes: Actb and Rpl37a. All the results were plotted as the mean ± SEM, and 4 replicates were performed. Unpaired t-test was used to evaluate the cell type (oocyte v. cumulus cells) differences. The qRT-PCR analyses revealed the presence of transcripts of the Mt2 gene only in oocytes from immature (GV) and mature (MII) COC. At both maturational stages, the copy numbers for Asmt in oocytes were significantly higher than in cumulus cells. The results confirm the presence of the Asmt gene in rat COC and suggest the possible involvement of these cells on melatonin biosynthesis. The presence of Mt2 transcript in immature and mature oocytes also suggests the potentially important role of melatonin in regulating the rat meiotic cellular cycle. Research supported by FAPESP.

234 EXPRESSION OF NUCLEAR AND MEMBRANE MELATONIN RECEPTORS GENES AND THE CLOCK GENES IN RAT OOCYTES: PRELIMINARY RESULTS

There is evidence that melatonin acts directly on the regulation of ovary function. This action is probably attributed in part to melatonin receptors, which are known to be present in granulosa and cumulus cells (Kang J-T et al. 2009 J. Pineal Res. 46, 22–28). Melatonin is also known to be associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function (Arendt J 1998 Rev. Reprod. 3, 13–22). Circadian rhythms and clock genes appear to be involved in reproductive processes (Dolatshad H et al. 2009 Repro. Fertil. Dev. 21, 1–9). However, the presence of melatonin receptor genes and the clock genes has not been so widely studied or has never been reported to exist in mammalian oocytes.The aim of this study was to investigate the presence of nuclear (Rorα) and membrane (Mt1 and Mt2) melatonin receptors genes and the clock genes (Clock, Bmal1, Cry1, Cry21, Per1, Per2) in rat oocytes by reverse RT-PCR. Twenty-seven-day-old Wistar female rats were treated with 20 UI of pregnant mares serum gonadotropin for induction of follicular development and slaughtered 48 h later. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. The ovaries were removed and placed in TCM-199 supplemented with 100 UI mL-1 penicillin, 100 μg mL-1 streptomycin, and 0.1% polyvinyl alcohol (H-199 medium). Germinal vesicle-intact oocytes, isolated from the ovarian follicles, were denuded from cumulus cells by vortexing for 5 to 8 min. The denuded oocytes were incubated for 5 min in H-199 medium with 0.1% pronase for removal of the zona pellucida. Pools of 80 oocytes per cDNA sample were used.As an internal control for the sample integrity, additional primers for RPL37a were included in each PCR reaction. All the 3 control PCR replicates showed a repeatable amplification. Polymerase chain reaction amplifications of cDNA yielded Rorα, Clock, Bmal1, and Cry1 products in 2 of 3 replicates. No expression of the MT1 and MT2 mRNA was observed. The preliminary results suggest the presence of a nuclear melatonin receptor gene and some clock genes in rat oocytes. However, additional studies are necessary to confirm this hypothesis. This research was supported by FAPESP.

Identification and Characterization of an Ovary-Selective Isoform of Epoxide Hydrolase1

A novel ovary-selective gene was identified by suppression subtractive hybridization (SSH) that is expressed only during the mouse periovulatory phase of a stimulated estrous cycle. Analysis of the protein encoded by the full-length cDNA revealed that the majority of it, with the exception of the first 44 amino acids, matched soluble epoxide hydrolase (Ephx2, referred to as Ephx2A). By comparing the cDNA sequence of this newly identified variant of soluble epoxide hydrolase (referred to as Ephx2B) with the mouse genome database, an exon was identified that corresponds to its unique 5' cDNA sequence. Through the use of an Ephx2A-specific probe, Northern blot analysis revealed that this mRNA was also expressed in the ovary, with the highest level of expression occurring during the luteal phase of a stimulated estrous cycle. In situ hybridization revealed that Ephx2B mRNA expression was restricted to granulosa cells of preovulatory follicles. Ephx2A mRNA expression, however, was detectable in follicles at different stages of development, as well as in the corpus luteum. Total ovarian epoxide hydrolase activity increased following the induction of follicular development, and remained elevated through the periovulatory and postovulatory stages of a stimulated estrous cycle. The change in enzyme activity paralleled the combined mRNA expression profiles for both Ephx2A and Ephx2B, thus supporting a role for epoxide metabolism in ovarian function.

Developing ovarian follicles inhibit the endotoxin-induced glomerular inflammatory reaction in pseudopregnant rats.

We tested the hypothesis that developing ovarian follicles produce factors inhibiting the endotoxin induced inflammatory response. Pseudopregnant rats were treated with FSH to induced follicular development (FSH-rats). For control we used untreated pseudopregnant rats (PSP-rats) and rats in the follicular phase of the ovarian cycle (C-rats). All rats were infused with either saline or endotoxin. Three days after the infusion rats were sacrificed and kidney specimens were snapfrozen. Cryostat kidney sections were stained for the presence of monocytes, granulocytes, CD11a- and CD11b-positive cells and for ICAM-1 expression. The results show that induction of follicular development in pseudopregnant rats inhibited glomerular infiltration of monocytes and CD11b(+) cells, while it did not affect the other parameters, i.e. glomerular granulocyte number, CD11a(+) cells and glomerular ICAM-1 expression. Developing ovarian follicles produce factors inhibiting monocyte responses to endotoxin.

Reproductive endocrine responses to photoperiod and exogenous gonadotropins in the pallas' cat (Otocolobus manul)

AbstractFecal samples were collected for 14–26 months from three male and six female Pallas' cats (Otocolobus manul) to examine gonadal steroidogenic activity in response to changes in photoperiod and treatment with exogenous gonadotropins. Females exhibited a seasonal anestrus from May–December, excreting consistently low concentrations of fecal estrogens (overall mean, 50.2±8.5 ng/g). During the breeding season (January–April), baseline fecal estrogen concentrations were higher, averaging 128.4±18.9 ng/g, with peak concentrations ranging from 455.8–909.6 ng/g. Interpeak intervals in estrogen excretion ranged between 7 and 21 days, with an average estrous cycle length of 14.3±1.7 days. Two females became pregnant after natural mating, with overall luteal progestogen concentrations averaging ∼40 μg/g throughout gestation. Fecal estrogens increased in mid‐gestation, peaking just before birth. Induction of follicular development with eCG (100–300 IU, i.m.) resulted in an increase in fecal estrogens (peak range, 263.1–1198.1 ng/g), followed by a postovulatory increase in fecal progestogens (overall mean, 41.1±11.9 μg/g) after hCG (75–150 IU, i.m.). Despite apparently normal ovarian responses, none of the females conceived after artificial insemination (AI). The gonadotropin‐induced nonpregnant luteal phase lasted 49.8±5.3 days (range, 30–60 days), whereas gestation lasted ∼70 days. In the male Pallas' cat, fecal androgens were elevated from November–April (overall mean, 352.3±30.3 ng/g) compared with nadir concentrations during the rest of the year (82.1±3.3 ng/g). Entrainment of seasonality to photoperiod was demonstrated by stimulation of gonadal steroidogenic activity in cats exposed to increasing artificial light during natural (nonbreeding season) and artificially induced short‐day photoperiods. In summary, reproduction in Pallas' cats is highly seasonal and photoperiod‐dependent. Females exhibit elevated baseline and peak fecal estrogen concentrations for 3–4 months during late winter/early spring. Testicular steroidogenic activity precedes the rise in female estrogen excretion by about 2 months, presumably to ensure maximal sperm production during the breeding season. Zoo Biol 21:347–364, 2002. Published 2002 Wiley‐Liss, Inc.

Suppression by developing ovarian follicles of the low-dose endotoxin-induced glomerular inflammatory reaction in the pregnant rat

Objective: In the current study the role of developing ovarian follicles in the control of the endotoxin-induced pregnancy-specific inflammatory reaction was evaluated. Study Design: Follicular development was induced in pregnant rats (n = 20) by means of daily intraperitoneal injections of follicle-stimulating hormone from day 11 of pregnancy until the end of the experiment. Control pregnant rats (n = 20) received daily sodium chloride injections. All pregnant rats were infused for 1 hour with either 2 mL endotoxin solution (1.0 μg/kg body weight) or 2 mL sodium chloride solution on day 14 and killed 4 hours or 3 days later. At death, the left kidneys were snap-frozen and immunohistologically stained for the presence of polymorphonuclear leukocytes and monocytes. Results: The results show that in control pregnant rats endotoxin significantly increased glomerular polymorphonuclear leukocyte and monocyte numbers at both 4 hours and 3 days after endotoxin infusion. Induction of follicular development did not affect glomerular polymorphonuclear leukocyte number after endotoxin infusion but significantly decreased the number of monocytes in the glomeruli at both 4 hours and 3 days after endotoxin infusion. Conclusion: We conclude that follicles stimulated with follicle-stimulating hormone produce a follicular factor or factors that are able to prevent the endotoxin-induced influx of monocytes into the glomeruli of pregnant rats. It is suggested that these factors play a role in the control of inflammatory processes associated with reproduction, including the disease of pregnancy, preeclampsia. (Am J Obstet Gynecol 2000;183:89-93.)

Suppression by developing ovarian follicles of the low-dose endotoxin-induced glomerular inflammatory reaction in the pregnant rat

<h2>Abstract</h2> <b>Objective:</b> In the current study the role of developing ovarian follicles in the control of the endotoxin-induced pregnancy-specific inflammatory reaction was evaluated. <b>Study Design:</b> Follicular development was induced in pregnant rats (n = 20) by means of daily intraperitoneal injections of follicle-stimulating hormone from day 11 of pregnancy until the end of the experiment. Control pregnant rats (n = 20) received daily sodium chloride injections. All pregnant rats were infused for 1 hour with either 2 mL endotoxin solution (1.0 μg/kg body weight) or 2 mL sodium chloride solution on day 14 and killed 4 hours or 3 days later. At death, the left kidneys were snap-frozen and immunohistologically stained for the presence of polymorphonuclear leukocytes and monocytes. <b>Results:</b> The results show that in control pregnant rats endotoxin significantly increased glomerular polymorphonuclear leukocyte and monocyte numbers at both 4 hours and 3 days after endotoxin infusion. Induction of follicular development did not affect glomerular polymorphonuclear leukocyte number after endotoxin infusion but significantly decreased the number of monocytes in the glomeruli at both 4 hours and 3 days after endotoxin infusion. <b>Conclusion:</b> We conclude that follicles stimulated with follicle-stimulating hormone produce a follicular factor or factors that are able to prevent the endotoxin-induced influx of monocytes into the glomeruli of pregnant rats. It is suggested that these factors play a role in the control of inflammatory processes associated with reproduction, including the disease of pregnancy, preeclampsia. (Am J Obstet Gynecol 2000;183:89-93.)

Inducible expression of cellular retinoic acid-binding protein II in rat ovary: gonadotropin regulation during luteal development.

Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular retinoic acid-binding protein and cellular retinoic acid-binding protein II [CRABP(II)]. CRABP is found in many adult tissues, whereas CRABP(II) is more restricted and is reported as abundant primarily in skin. Here we report a much greater expression of CRABP(II) in rat corpus luteum than in any other organ/tissue examined, including skin. A rat complementary DNA clone encoding CRABP(II) was isolated and the ovarian expression followed during gonadotropin induction of follicular development in the pseudopregnant rat. The pattern of rat CRABP(II) messenger RNA and protein expression correlated with the appearance of corpora lutea and the rise in progesterone production as the corpora lutea developed, and was similar to the induction of 3 beta-hydroxysteroid dehydrogenase. Immunohistochemical localization revealed that CRABP(II) appeared in luteal cells and was dramatically restricted to their cytoplasmic compartment, with no apparent presence in the nucleus. This suggests that CRABP(II) may be expressed to restrict retinoic acid from occupying nuclear retinoic acid receptors, implying that the differentiation and maintenance of the rat corpus luteum may involve in part a release of certain pathways from retinoid suppression.

Follicular development and superovulation response in cows administered multiple FSH injections early in the estrous cycle

To determine whether follicular development, superovulation and embryo production were affected by the absence or presence of a dominant follicle, cows were administered injections of FSH twice daily in the early (Days 2 to 6, estrus = Day 0) or middle stage (beginning on Day 10 or 11) of the estrous cycle. Treatment with FSH early in the cycle stimulated follicular development in 83 to 100% of all cows from 4 groups evaluated at different times after PGF2α treatment on Days 6 and 7. However, the proportion of cows with > 2 ovulations varied from 31 to 62.5%, indicating that induction of follicular development may occur in the absence of superovulation. When compared with cows treated in the middle of the cycle, no differences were observed in the proportion of cows with > 2 ovulations (31 vs 20%), ovulation rate. (26.0 ± 6.3 vs 49.6 ± 25.8), production of ova/embryos (13.3 ± 3.2 vs 14.4 ± 3.4), or the number of transferable embryos (8.0 ± 3.6 vs 5.4 ± 1.5; early vs middle, respectively). The proportion of the total number of embryos collected that were suitable for transfer was greater (P<0.01) in cows treated early in the cycle (60%) than at midcycle (37.5%). The diameter of the largest follicle observed by ultra-sound prior to initiation of FSH treatment in the early stage of the cycle (10.0 ± 2.0 mm) was smaller (P<0.05) than at midcyle (16.8 ± 1.3 mm). These results demonstrate that superinduction of follicular development is highly consistent after FSH treatment at Days 2 to 6 of the cycle and that superovulation and embryo production are not less variable than when FSH is administered during the middle of the cycle. However, superovulation in the early stage of the cycle may increase the proportion of embryos suitable for transfer.

Induction of follicular development and estrus in prepubertal gilts with gonadotropins

Two experiments were conducted to examine the effect of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on follicular development in gilts. The gonadotropins were given via hourly i.v. infusions. In Experiment 1, gilts were given saline ( n = 9), a pituitary extract ( n = 6), or 7.5 ( n = 4), 15 ( n = 4), 30 ( n = 4) or 60 ( n = 5) ng LH kg −1 body weight (BW) hourly for 144 h, after which ovaries were examined by laparoscopy. The proportion of gilts given pituitary extract, or 30 or 60 ng LH kg −1 BW that had corpora lutea or follicles greater than 7 mm in diameter at the time of laparoscopic examination was significantly greater than the proportion of gilts treated with saline or lower amounts of LH. Hourly infusions of a pituitary extract and LH increased ( P < 0.05) serum LH concentrations with each infusion, but did not change FSH concentrations around each infusion, or alter basal LH, FSH or estradiol concentrations. In Experiment 2, gilts were given saline ( n = 4) or 15 ng LH kg −1BW and FSH at a rate of 1 ( n = 5), 2 ( n = 5) or 4 ( n = 5) ng kg −1 BW. Four of five gilts given 15 ng LH and 4 ng FSH kg −1 BW exhibited estrus, whereas none of the other gilts showed estrus ( P < 0.02). Administration of LH and FSH increased LH concentration within 10 min after each treatment, but did not influence concentrations of FSH after each treatment, or basal concentrations of LH, FSH or estradiol. The pattern of endocrine events observed in gilts exhibiting estrus was similar regardless of treatment or experiment and was similar to that observed previously in gilts undergoing follicular development. These results indicate that LH can stimulate follicular development, estrus and ovulation in prepubertal gilts if given hourly in sufficient quantities and that FSH acts synergistically with LH in causing follicular development when given in a pulsatile manner.

Pharmacologic induction of multiple follicular development improves the success rate of artificial insemination with husband's semen in couples with male-related or unexplained infertility

The use of artificial insemination with husband's semen (AIH) as treatment for couples with male-related or unexplained infertility is often disappointing. The aim of the present study was to evaluate whether the induction of multiple ovulation can increase the pregnancy rates in couples treated with AIH. Multiple follicular development was induced by means of clomiphene citrate (CC) plus purified follicle-stimulating hormone (FSH) in 17 couples undergoing AIH for male-related or unexplained infertility. In spite of the long duration of infertility (greater than 6 years), 10 couples (58.8%) achieved pregnancy within 6 months of treatment. This figure was significantly higher than that obtained in 120 couples who underwent AIH either during spontaneous cycles or after induction of follicular development with CC alone. These results suggest that the availability of multiple mature oocytes increases the conception rate during each cycle, as reported for in vitro fertilization (IVF) programs. It is therefore suggested that a trial of induction of multiple follicular development be performed in couples with male-related or unexplained infertility before their inclusion in protocols for invasive procedures, such as IVF or gamete intrafallopian transfer.

Induction of Follicular Development with Luteinizing Hormone - Releasing Hormone

Induction of Follicular Development with Luteinizing Hormone - Releasing Hormone

Pharmacologic induction of multiple follicular development improves the success rate of artificial insemination with husband’s semen in couples with male-related or unexplained infertility

The use of artificial insemination with husband's semen (AIH) as treatment for couples with male-related or unexplained infertility is often disappointing. The aim of the present study was to evaluate whether the induction of multiple ovulation can increase the pregnancy rates in couples treated with AIH. Multiple follicular development was induced by means of clomiphene citrate (CC) plus purified follicle-stimulating hormone (FSH) in 17 couples undergoing AIH for male-related or unexplained infertility. In spite of the long duration of infertility (greater than 6 years), 10 couples (58.8%) achieved pregnancy within 6 months of treatment. This figure was significantly higher than that obtained in 120 couples who underwent AIH either during spontaneous cycles or after induction of follicular development with CC alone. These results suggest that the availability of multiple mature oocytes increases the conception rate during each cycle, as reported for in vitro fertilization (IVF) programs. It is therefore suggested that a trial of induction of multiple follicular development be performed in couples with male-related or unexplained infertility before their inclusion in protocols for invasive procedures, such as IVF or gamete intrafallopian transfer.

Changes in the concentration of prostaglandins in preovulatory human follicles after administration of hCG.

The concentrations of prostaglandins F-2 alpha, E, D-2 and 13,14-dihydro-15-keto PGE were measured in follicular fluid collected from women undergoing routine laparoscopy following induction of follicular development with clomiphene and hCG. Laparoscopy was performed before, or at 12, 24 or 36 h after administration of hCG. Prostaglandins were measured as the methyloxime derivative by radioimmunoassay. Peaks in PGE and PGF-2 alpha concentration occurred at 12 and 36 h with a significant nadir at 24 h, whereas PGD-2 production was very low at 36 h. The concentration of PGF-2 alpha rose significantly between 0 and 36 h and was greatest in follicles yielding oocytes, suggesting a possible role for this prostaglandin in the mechanism of follicle rupture.

The effect of GnRH on induction of follicular development and ovulation in anovulatory and ovulatory mares

The effects of estradiol cypionate (ECP) and GnRH injections were tested on mares during January and February. Sixteen mares were blocked on their ovarian status and equally allotted to two groups. Group one received daily injections of 500 μg ECP (im) for 14 days followed by a 21 day period of twice daily injections of 200 μg GnRH (im). Group two received the carrier vehicle. Mean length of diestrus of ovulatory mares was 14.3 ± 1.6 days and 17.8 ± 3.5 days for treated and control groups respectively. Corresponding estrus lengths were 8.0 ± 1.4 days and 6.3 ± 2.1 days. Plasma LH levels, number of follicles < 20 mm, number of follicles > 20 mm and diameter of the largest follicle in ovulatory mares were not significantly affected by treatment with ECP or GnRH. Anovulatory mares treated with ECP and GnRH exhibited estrus more frequently (54% and 70% of the time) than sham injected controls (17% and 15% of the time). Plasma LH levels were significantly elevated (P<.05) in anovulatory mares treated with GnRH. Also more follicles < 20 mm (P<.09) were detected on the ovaries of GnRH treated mares than on those of control mares. Effects of the treatment were transient since LH levels and ovarian activity were similar in both mare groups after cessation of treatment.

Induction of Follicular Development, Maturation and Ovulation by Gonadotropin Releasing Hormone Administration to Acyclic Mares

Five acyclic mares with inactive ovaries were given three courses of gonadotropin releasing hormone (GnRH) beginning on Days 1, 11 and 21 of treatment to reproduce the gonadotropin surges responsible for causing follicular growth and ovulation during the normal estrous cycle. The first two courses consisted of 3–4 im injections of 1.0 mg GnRH, and the third course of 5 im injections of 0.4–1.4 mg GnRH, at approximately 12 h intervals. Exogenous progesterone was given on Days 5 or 6 through 16 to simulate luteal phase progesterone levels. Serum FSH, LH, progesterone and estradiol were measured daily throughout the experimental period and at frequent intervals following GnRH administration. The GnRH injections in all courses induced peak FSH concentrations of 2.5–4 times baseline by 0.1–5 h after injection, and the levels were almost back to pre-injection concentrations by 12 h. Each GnRH injection in the first two courses resulted in increases in serum LH to 1.5–3 times baseline by 0.5–1 h after injection. The third course caused a greater release of LH which was thought to be related to the increases in serum estradiol occurring at this time. Typical pre-estrous follicles were present in all mares by Day 19 of treatment. All mares were in estrus by Day 21, and each ovulated a single large follicle between Days 24 and 27 of treatment. In three mares high LH levels occurred for 5 days after the last GnRH course and these mares established corpora lutea; the FSH, LH and estradiol levels induced by GnRH were similar to those which lead to ovulation in the normal cycle. In the other two mares LH fell to pre-treatment levels by 12 h after the last GnRH injection; these mares failed to establish corpora lutes. Thus it appears that GnRH in combination with appropriate progesterone treatment in the acyclic mare treated towards the end of the nonbreeding season can consistently induce normal cyclic pituitary and ovarian activity culminating in ovulation.