Follicular Estradiol Production Research Articles

Putative role of cocaine- and amphetamine-regulated transcript (CARTPT) in dominant follicle selection in cattle

The mechanisms regulating development of a single (dominant) follicle capable of ovulation during each follicular wave in cattle and atresia of remaining follicles (dominant follicle selection) are not well understood. FSH and IGF1 are known regulators of follicle growth and granulosa cell estradiol production during follicular waves. Recent evidence indicates cocaine and amphetamine regulated transcript (CARTPT), with intraovarian expression only in single-ovulating species, is a novel regulator of follicular development. The mature CARTPT peptide (CART) is a potent negative regulator of FSH and IGF1 action on granulosa cells in vitro and can inhibit follicular estradiol production in vivo. Follicular fluid CART concentrations in healthy follicles decrease after dominant follicle selection and CARTPT mRNA is lower in healthy versus atretic follicles collected prior to and early after initiation of follicle dominance, suggestive of a regulatory role in the selection process. The inhibitory actions of CART on FSH signaling and estradiol production are dependent on the Go/i-subclass of inhibitory G proteins and linked to multiple components of the FSH signal transduction pathway resulting in reduced CYP19A1 mRNA and estradiol production. Evidence to date supports a potential important functional role for CART in regulation of dominant follicle selection and the species-specific ovulatory quota in monotocous species.

Relationship between follicular dynamics and oocyte maturation during in vitro culture as a non-invasive sign of caprine oocyte meiotic competence

The search for non-invasive signs of oocyte meiotic competence is very important for the development of in vitro follicle culture (IVFC) systems. The aims of the present study were: (1) to investigate the effect of in vitro maturation (IVM) of in vivo grown goat COCs, in group or individually, on oocyte chromatin configuration (Experiment 1), and (2) the influence of IVFC period (12 vs. 18 days) on the ability of the oocyte to resume meiosis immediately after IVFC (before in vitro maturation; IVM), or after IVM (Experiment 2). In experiment 1, in vivo grown cumulus-oocyte complexes (COCs) were submitted to IVM in groups (10 COCs/100 μL-drop) or individually (1 COC/10 μL-drop), and chromatin configuration was assessed. In experiment 2, isolated follicles were individually cultured for 12 or 18 days, and submitted to individual IVM afterwards. The following end points were evaluated: follicular growth and morphology, oocyte diameter, viability and chromatin configuration, as well as individual follicular estradiol production. Similar maturation rates were obtained between in vivo grown COCs matured individually and in groups (66.7% vs. 63.6%, respectively) (Experiment 1). Only after 18 days of IVFC, oocytes were able to grow during IVM, reaching a mean oocyte diameter of 119 μm. Also, this treatment produced the highest rate of metaphase II oocytes (46.2% out of the total number of cultured follicles). Finally, it was observed that follicles with a daily growth rate >7.1 μm/day (fast-growing) and that reached at least 600 μm in diameter, were more likely (P < 0.05) to produce oocytes capable of attaining MII. In conclusion, caprine oocytes can be individually matured in vitro, as efficiently as in groups. This result was essential to pair in vitro follicle development and in vitro oocyte maturation with specific individual follicles. Using this approach, it was possible to establish non-invasive signs for the efficiency of IVFC based on follicle daily growth rate and diameter, and oocyte diameter: follicle daily growth >7 μm, follicle diameter of at least 600 μm, and oocyte diameter ≥120 μm. In addition, 18 days seems to be the most suitable culture time for caprine early antral follicles.

Mastitis outcomes on pre-ovulatory follicle diameter, estradiol concentrations, subsequent luteal profiles and conception rate in Buffaloes

The objectives of this study was to investigate the outcome of mastitis, in its clinical or subclinical forms, on the mean diameter of pre-ovulatory follicle (POF), plasma estradiol concentration on the day of estrus, subsequent luteal profile and subsequent conception rate in buffaloes. Sixty dairy buffalo (Bubalus bubalus) conducted in this study were divided into three groups {healthy (H), n=20; subclinical mastitis (SCM), n=18; and clinical mastitis (CM), n=22}. Ultrasonography of ovaries revealed that mean diameter of POF was larger (P<0.05) in H buffalo (14.35mm) compared to SCM (12.40mm) and CM (10.25mm). Also, plasma estradiol concentration on the day of estrus was higher (P<0.05) in H buffalo compared to SCM and CM counterparts; 34.95 vs. 32.87 and 27.50pg/ml, respectively. Besides, positive correlation was observed between the POF diameter with plasma estradiol concentration in H, SCM and CM buffaloes (r=0.64, 0.74, 0.72 respectively, P<0.05). Moreover, positive correlations (P<0.01) were found on days 9, 12, 16, and 21 post-ovulation between POF diameter and luteal profile. Thus, the conception rate in H buffalo was higher (P<0.05) compared with SCM and CM counterparts; 55% vs. 38.89 and 18.18%, respectively. In conclusion, mastitis in its clinical or subclinical forms disrupts the functioning of the pre-ovulatory follicle on the day of estrus, associated with low follicular estradiol production, resulting in suppression to subsequent luteal profile leading to substantial decrease in pregnancy consequence of buffaloes.

177 DOWN-REGULATION OF P450 AROMATASE AND GLUCOSE TRANSPORTER 4 mRNAs EXPRESSION IN SHEEP OVARIAN FOLLICLES AFTER ULTRASHORT NUTRITIONAL FLUSHING

Nutritional supplementation before breeding (flushing) has become a common practice and is a reliable method to increase lambing and twining rates in sheep. We have shown that ultrashort flushing (USF: 1 day long) acutely increases ovulation rate without affecting the body condition or animal weight. A short nutritional stimulus would necessarily be acting either directly at the ovarian level or through hormones and factors other than gonadotropins. The possible mechanisms of action of USF on ovulation rate is the deregulation of the feedback loop between the ovaries and gonadotropin secretion, where nutrition causes a direct inhibition of follicular oestradiol production leading to compensatory secretion of FSH that stimulates folliculogenesis, or direct stimulation of follicular development through hormones and factors other than gonadotropins. We hypothesised that USF enhances the number of follicles selected for ovulation due to either a decrease in the expression of P450 aromatase or alternatively by advancing the maturation of the ovarian follicles, which would be reflected in an increase in LH receptor (LHr) and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression, an increase in glucose transporter 4 (GLUT4) mRNA expression, or both. To test these hypotheses, the oestrus cycle of 30 ewes was synchronized with progestin intravaginal sponges and prostaglandins. Animals were given the USF on the day of progestin withdrawal. The ovaries were removed surgically before treatment (time 0; n = 6), or at 12, 24, and 48 h after being treated with either glycerol or water (n = 4 per treatment by time category). The ovarian follicles were dissected out and counted. Follicles larger than 3 mm in diameter were pooled and the mRNA extracted for specific P450aromatase, 3β-HSD, LHr, and glucose transporter 4 determinations. USF increased the number of follicles larger than 3 mm at 48 h after treatment (P &lt; 0.05). No effect was observed in the number of small follicles. In the two largest follicles, aromatase and GLUT4 mRNA abundance decreased (P &lt; 0.01) 12 h after treatment. There was no effect of treatment on mRNA for LH receptor or 3β-HSD. These results demonstrate a reduction in aromatase expression in potentially ovulatory follicles 12 h after flushing. The decrease in aromatase may favour a transient increase in FSH concentrations 12 h after flushing that would allow the stimulation and selection of supplementary ovulatory follicles.

Pioglitazone counteracts the tumor necrosis factor-α inhibition of follicle-stimulating hormone-induced follicular development and estradiol production in an in vitro mouse preantral follicle culture system

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age and is characterized by chronic anovulation. Insulin resistance may be a key component of the pathogenesis of this disorder. Pioglitazone is a thiazolidinedione derivative that acts by improving insulin resistance via the peroxisome proliferator-activated receptor-γ (PPAR-γ) pathway. Reportedly, pioglitazone improves the anovulation status in patients with PCOS. In the present study, we examined whether pioglitazone directly affects ovarian follicular development and steroidogenesis using in vitro mouse preantral follicle culture system.MethodsAn isolated individual in vitro mouse preantral follicle culture was used to test the effects of pioglitazone on the follicle development and steroidogenesis. Tumor necrosis factor-α (TNF-α), which plays a role in insulin resistance, has been reported to inhibit the follicle stimulating hormone (FSH)-induced follicular development and steroidogenesis in an in vitro mouse preantral follicle culture system. Therefore, we examined whether pioglitazone counteracts these effects by TNF-α. We assessed the follicle diameter and follicle survival and antral-like cavity formation rates, the 17β-estradiol (E2) levels in the culture medium, and the ovulation rate using the in vitro preantral follicle culture.ResultsPioglitazone treatment counteracted the inhibition of TNF-α in FSH-induced follicle development in a dose-dependent manner. Pioglitazone, at a concentration of 5 μM, which was the minimum effective concentration, significantly counteracted the inhibition of TNF-α in FSH-induced follicle survival (29 versus 56%, P < 0.05), antral-like cavity formation (29 versus 48%, P < 0.05), E2 concentration in the culture medium (mean ± SEM = 21 ± 1 versus mean ± SEM = 27 ± 1 pg/mL, P < 0.05), and human chorionic gonadotropin-induced ovulation rate (9 versus 28%, P < 0.05).ConclusionsPioglitazone counteracted the inhibition by TNF-α on FSH-induced follicle development and steroidogenesis in the in vitro mouse preantral follicle culture. The results suggest that pioglitazone may directly affect the follicular development and steroidogenesis.

Fever in women may interfere with follicular development during controlled ovarian stimulation

Purpose: This study aims to evaluate the effects of fever on follicular development in women undergoing controlled ovarian stimulation during in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles.Materials and methods: This was a retrospective observational self-controlled study at a tertiary-care fertility centre. Six gonadotropin stimulation cycles characterised by poor ovarian response in which women reported the occurrence of a febrile illness, were considered for evaluation. Fever-exposed cycles were compared to the next stimulation cycle in the same women. Primary outcome measures were final number of pre-ovulatory follicles (≥ 16 mm) and final peak serum estradiol levels (pg/mL). Other outcome measures were final number of medium-sized follicles (12–15 mm), final mean estradiol serum level per follicle ≥ 12 mm (pg/mL), total days of stimulation and total gonadotropin ampoules utilised.Results: Fever-exposed cycles were associated with significantly lower number of pre-ovulatory follicles (0.7 ± 0.8), significantly higher number of medium-size follicles (21.0 ± 4.5), and significantly reduced serum estradiol per follicle ≥12 mm (50.7 ± 11.7 pg/mL). They also required a significantly longer duration of ovarian stimulation (15.7 ± 3.3 days) and a significantly increased number of gonadotropin ampoules (47.2 ± 10.9). Four women had polycystic ovary syndrome and one hypothalamic hypogonadism.Conclusion: This preliminary report suggests a possible negative effect of fever on follicular development and ovarian estradiol production in some women undergoing controlled ovarian stimulation.

Putative role of cocaine- and amphetamine-regulated transcript (&lt;I&gt;CARTPT&lt;/I&gt;) in dominant follicle selection in cattle

The mechanisms regulating development of a single (dominant) follicle capable of ovulation during each follicular wave in cattle and atresia of remaining follicles (dominant follicle selection) are not well understood. FSH and IGF1 are known regulators of follicle growth and granulosa cell estradiol production during follicular waves. Recent evidence indicates cocaine and amphetamine regulated transcript (CARTPT), with intraovarian expression only in single-ovulating species, is a novel regulator of follicular development. The mature CARTPT peptide (CART) is a potent negative regulator of FSH and IGF1 action on granulosa cells in vitro and can inhibit follicular estradiol production in vivo. Follicular fluid CART concentrations in healthy follicles decrease after dominant follicle selection and CARTPT mRNA is lower in healthy versus atretic follicles collected prior to and early after initiation of follicle dominance, suggestive of a regulatory role in the selection process. The inhibitory actions of CART on FSH signaling and estradiol production are dependent on the G(o/i)-subclass of inhibitory G proteins and linked to multiple components of the FSH signal transduction pathway resulting in reduced CYP19A1 mRNA and estradiol production. Evidence to date supports a potential important functional role for CART in regulation of dominant follicle selection and the species-specific ovulatory quota in monotocous species.

Role of Cocaine- and Amphetamine-Regulated Transcript (CART) in Regulation of Antral Follicle Development in Cattle: Demonstration of Inhibitory Effects of the CART Peptide on Follicular Estradiol Production and Granulosa Cell Aromatase mRNA In Vivo and Potential FSH Regulation of CART Receptor Binding.

Role of Cocaine- and Amphetamine-Regulated Transcript (CART) in Regulation of Antral Follicle Development in Cattle: Demonstration of Inhibitory Effects of the CART Peptide on Follicular Estradiol Production and Granulosa Cell Aromatase mRNA In Vivo and Potential FSH Regulation of CART Receptor Binding.

Effect of GnRH down-regulation on cumulus cell viability and apoptosis as measured by fluorescence-activated cell sorting

To determine whether gonadotropin releasing hormone (GnRH)-agonist or -antagonist induces higher percentages of cumulus cell apoptosis and if the use of either is detrimental to ART outcomes. Women in a private facility under treatment for IVF had their cumulus cells isolated and analyzed by flow cytometry. Viable, apoptotic, and dead cumulus cell rates related to ovarian stimulation by GnRH-agonist or -antagonist were measured and compared with fertilization and implantation rates. Treatment with GnRH-agonist produced a greater number of follicles than treatment with GnRH-antagonist. No differences in implantation and pregnancy rates were found. While cumulus cell (CC) apoptosis was positively correlated with estradiol on the day of hCG administration, no significant difference in the percentage of apoptotic cells between treatments was detectable. Additionally, implantation rate and the average follicular estradiol production on the day of hCG administration were no different between treatments. GnRH-agonist or -antagonist treatment protocols induce similar levels of apoptosis in CCs and are not detrimental to ART outcomes.

A review of the effects of supplementary nutrition in the ewe on the concentrations of reproductive and metabolic hormones and the mechanisms that regulate folliculogenesis and ovulation rate

This paper discusses the phenomenon of nutritional flushing in ewes whereby increased nutrition stimulates folliculogenesis and ovulation rate. In addition the paper reviews recent findings on the effects of increased levels of nutrition on the blood concentrations of reproductive and metabolic hormones in the ewe and some of the intraovarian changes that take place in response to nutritional stimulation. Finally, in the paper, we propose a model of the physiological mechanism for the nutritional stimulation of folliculogenesis and we review how closely the model fits recent published and unpublished evidence examining the mechanism of flushing. Nutritional stimulation alters the blood concentrations of some metabolic hormones. By using short-term models of nutritional flushing, we have shown that as the blood concentrations of insulin and leptin increase that of growth hormone decreases while that of IGF-I appears unaffected by the nutritional flushing. Nutritional flushing also alters the blood concentrations of some reproductive hormones. Again, using the same model, we have shown that there is a transient increase in FSH and a decrease in oestradiol concentrations in the blood. The changes in oestradiol are particularly evident in the follicular phase of the oestrous cycle. In the ovary, the effect of nutrition is to stimulate folliculogenesis. These changes are associated with intra-follicular alterations in the insulin-glucose, IGF and leptin metabolic systems. The stimulation of these intra-follicular systems leads to a suppression in follicular oestradiol production. The consequence of these direct actions on the follicle is a reduced negative feedback to the hypothalamic-pituitary system and increased FSH secretion that leads to a stimulation of folliculogenesis.

Infantile 4- tert-octylphenol exposure transiently inhibits rat ovarian steroidogenesis and steroidogenic acute regulatory protein (StAR) expression

Phenolic compounds, such as 4- tert-octylphenol (OP), have been shown to interfere with rat ovarian steroidogenesis. However, little is known about steroidogenic effects of infantile OP exposure on immature ovary. The aim of the present study was to investigate the effects of infantile OP exposure on plasma FSH, LH, estradiol, and progesterone levels in 14-day-old female rats. The effect on ovarian steroidogenic acute regulatory protein (StAR) and FSH receptor (FSHr) expression was analyzed by Western blotting. Ex vivo analysis was carried out for follicular estradiol, progesterone, testosterone, and cAMP production. Sprague–Dawley rats were given OP (0, 10, 50, or 100 mg/kg) subcutaneously on postnatal days 6, 8, 10, and 12. On postnatal day 14, plasma FSH was decreased and progesterone increased significantly at a dose of 100 mg OP/kg. In addition, the highest OP dose advanced the time of vaginal opening in puberty. OP had no effect on infantile LH and estradiol levels or ovarian FSHr content. Ovarian StAR protein content and ex vivo hormone and cAMP production were decreased at all OP doses compared to controls. However, hormone levels recovered independent on FSH and even increased above the control level during a prolonged culture. On postnatal day 35, no statistically significant differences were seen between control and OP-exposed animals in plasma FSH, LH, estradiol, and progesterone levels, or in ovarian StAR protein content. The results indicate that the effect of OP on the infantile ovary is reversible, while more permanent effects in the hypothalamus and pituitary, as described earlier, are involved in the reduction of circulating FSH levels and premature vaginal opening.

Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle

Nutrition is a major factor affecting cow reproductive efficiency. Long-term moderate or chronic dietary restriction results in a gradual reduction in dominant follicle (DF) growth rate, maximum diameter and persistence. Animals become anoestrus when they lose on average 22–24% of their initial body weight. There is evidence of significant animal-to-animal variation in the interval from the imposition of dietary restriction to onset of anoestrus and from the recommencement of re-alimentation to resumption of ovulation. In contrast, acute dietary restriction to 40% of maintenance requirements rapidly reduces dominant follicle growth rate and maximum diameter and induces anoestrus in a high proportion (60%) of heifers within 13–15 days of dietary restriction. In lactating dairy and beef cows negative energy balance or reduced dietary intake in the early post-partum period, while not affecting the population of small-to-medium size follicles, adversely affects the size and ovulatory fate of the dominant follicle. Re-alimentation of nutritionally induced anoestrous heifers results in an initial gradual increase in dominant follicle growth rate and maximum diameter, followed by a more accelerated increase in dominant follicle growth rate and maximum diameter as the time of resumption of ovulation approaches. Increased dominant follicle growth rate and maximum diameter are associated with increased peripheral concentrations of IGF-I, pulsatile LH and oestradiol. Direct nutritional effects on ovarian function appear to operate through hepatic rather than follicular regulation of IGF-I, and on systemic concentrations of IGF-I BPs and insulin; cumulatively reducing follicular responsiveness to LH and ultimately shutting down follicular oestradiol production. Indirect nutritional effects are apparently mediated through altering the GnRH pulse generator and in-turn selectively reducing pulsatile LH secretion without any apparent adverse effect on FSH secretory patterns. Endogenous opioid peptides, NPY and glucose appear to play a role in the nutritional regulation of GnRH release and in turn pulsatile LH secretion.

Decline in circulating estradiol during the periovulatory period is correlated with decreases in estradiol and androgen, and in messenger RNA for p450 aromatase and p450 17alpha-hydroxylase, in bovine preovulatory follicles.

The preovulatory surge of gonadotropins induces meiotic maturation of the oocyte, the follicular/luteal phase shift in hormone production, and ovulation. This complex and rapid series of developmental changes is difficult to study in large mammals, such as primates and ruminants, because variability in the length of individual reproductive cycles makes it virtually impossible to predict the time of the LH surge. We have validated an experimental model for inducing the LH surge and ovulation in cattle and used it to study the sequence of changes in hormone secretion and some of the mechanisms of these changes. Luteolysis and a follicular phase were induced by injection of prostaglandin F(2alpha); injection of a GnRH analogue 36 h later induced an LH surge and ovulation. The LH surge peaked 2 h after GnRH and ovulation followed 22-31 h after the surge, consistent with the periovulatory interval in natural cycles. The ensuing luteal phase was normal, both in length and in concentrations of circulating progesterone. In experiment I, the uteroovarian effluent was collected, via cannulation of the vena cava, at frequent intervals relative to GnRH injection. Circulating estradiol declined progressively after GnRH, reaching a nadir by 8-10 h before ovulation, whereas concentrations of androstenedione and testosterone remained constant. In experiment II, preovulatory follicles were obtained at 0, 3.5, 6, 12, 18, or 24 h after GNRH: Concentrations of androgens and estradiol were measured in follicular fluid and medium from cultures of follicle wall (theca + granulosa cells); steady-state levels of mRNA for 17alpha-hydroxylase (17alphaOH) and P450 aromatase were measured in follicular tissue. Shortly after the LH surge (3.5 h post-GnRH) there was an acute increase in the capacity of follicular tissue to secrete androstenedione, but not estradiol, in vitro. Thereafter, both androgens and estradiol declined, both in follicular fluid and in medium collected from cultures of follicle wall. Levels of mRNA for 17alphaOH and aromatase in follicle wall decreased significantly by 6 h after GnRH, suggesting that declining levels of these enzymes underlie the decreases in steroid production by follicular cells. These results show that in cattle the preovulatory decrease in follicular estradiol production is mediated by redundant mechanisms, because androgen production and the capacity of granulosa cells to convert androgens to estradiol decline coordinately, in concert with decreases in mRNA for 17alphaOH and P450 aromatase.

The pharmacokinetics and pharmacodynamics of Implanon®, a single-rod etonogestrel contraceptive implant

ABSTRACTThis paper reviews pharmacokinetic and pharmacodynamic studies with Implanon®, which provides serum etonogestrel levels sufficient to inhibit ovulation within 8 h of insertion. After a peak of 813 pg/ml at 4 days, levels reach steady state (200 pg/ml) after 4–6 months and remain sufficient to prevent ovulation for 3 years. Variability is lower than with Norplant®. Etonogestrel levels are similar in most ethnic groups, but 40% higher in women weighing < 50 kg. After implant removal, etonogestrel is not detectable within 1 week. Implanon® inhibits ovulation by preventing the mid-cycle luteinizing hormone peak. Although it initially suppresses follicular development and estradiol production, ovarian activity slowly increases after 6 months, and follicle stimulating hormone and estradiol levels are almost normal. Endogenous progesterone levels remain in the subovulatory range for > 3 yean in most subjects. In ovarian ultrasound studies, ovulation occurred in < 5% of users after 30 months of use. Ovulation was observed in most women within 3–4 weeks of implant removal. The pharmacokinetics and pharmacodynamics of Implanon® indicate that it has high contraceptive efficacy, as reflected in a zero pregnancy rate over 5629 woman-years of use. Its excellent reliability, ease of use, and reversibility make Implanon® a valuable addition to current contraceptives.

Effect of administration of eCG to postpartum cows on folliculogenesis in the ovary ipsilateral to the previously gravid uterine horn and uterine involution

This study tested the hypothesis that increased oestradiol secretion by large follicles in the ovary ipsilateral to the previously gravid uterine horn has a local effect to increase the rate of uterine involution. Cows were administered an i.m. water placebo (n = 19), 250 iu equine chorionic gonadotrophin (eCG) (n = 18) or 750 iu eCG (n = 20) 14 days post partum (day 0). Transrectal ultrasonography at the time of treatment and 2, 4 and 6 days later monitored uterine horn diameter and ovarian structures. Blood samples collected contemporaneously were assayed for 15-keto-13, 14-dihydro-prostaglandin F(2alpha) and oestradiol concentration. For control cows, accumulated diameter of the largest follicle in the ovary ipsilateral to the previously gravid uterine horn, compared with the contralateral ovary, was smaller on day 0 (P < 0.05) and days 2, 4 and 6 (P < 0.001). There were no significant differences in the eCG-treated animals. There were 12, 8 and 11 cows with a plasma oestradiol concentration < 1 pg ml(-1) on day 0 in the control, 250 and 750 iu eCG treatment groups, respectively. For control cows, the peripheral oestradiol concentrations were higher on day 6 compared with days 0, 2 and 4 (P < 0.05); for cows treated with 250 iu eCG, concentrations were higher on days 4 and 6 compared with day 0 (P < 0.05); and for cows treated with 750 iu eCG, concentrations were higher on days 2 and 4 compared with day 0 (P < 0.01). Treatment with eCG, or the presence of a follicle > 8 mm in diameter in the ovary ipsilateral to the previously gravid uterine horn, did not affect the rate of uterine involution or plasma 15-keto-13,14-dihydro-prostaglandin F(2alpha) concentration. In conclusion, administration of eCG to increase follicular growth and oestradiol production overcame the inhibition of follicular growth in the ovary ipsilateral to the previously gravid uterine horn, but did not affect uterine involution.

Effect of transport on pulsatile and surge secretion of LH in ewes in the breeding season.

The aim of this study was to elucidate the mechanism(s) involved in stress-induced subfertility by examining the effect of 4 h transport on surge and pulsatile LH secretion in intact ewes and ovariectomized ewes treated with steroids to induce an artificial follicular phase (model ewes). Transport caused a greater delay in the onset of the LH surge in nine intact ewes than it did in ten ovariectomized ewes (intact: 41.0 +/- 0.9 h versus 48.3 +/- 0.8 h, P < 0.02; ovariectomized model: 40.8 +/- 0.6 h versus 42.6 +/- 0.5 h, P < 0.02). Disruption of the hypothalamus-pituitary endocrine balance in intact ewes may have reduced gonadotrophin stimulation of follicular oestradiol production which had an additional effect on the LH surge mechanism. In the ovariectomized model ewes, this effect was masked by the exogenous supply of oestradiol. However, in these model ewes, there was a greater suppression of maximum LH surge concentrations (intact controls: 29 +/- 4 ng ml-1 versus intact transported 22 +/- 5 ng ml-1, P < 0.02; ovariectomized model controls: 35 +/- 7 ng ml-1 versus model transported 15 +/- 2 ng ml-1, P < 0.02). Subsequent exposure to progesterone for 12 days resulted in the resumption of a normal LH profile in the next follicular phase, indicating that acute stress leads to a temporary endocrine lesion. In four intact ewes transported in the mid-follicular phase, there was a suppression of LH pulse amplitude (0.9 +/- 0.3 versus 0.3 +/- 0.02 ng ml-1, P < 0.05) but a statistically significant effect on pulse frequency was not observed (2.0 +/- 0.4 versus 1.7 +/- 0.6 pulses per 2 h). In conclusion, activation of the hypothalamus-pituitary-adrenal axis by transport in the follicular phase of intact ewes interrupts surge secretion of LH, possibly by interference with LH pulsatility and, hence, follicular oestradiol production. This disruption of gonadotrophin secretion will have a major impact on fertility.

Microtubular dynamics in granulosa cells of periovulatory follicles and granulosa-derived (large) lutein cells of sheep: relationships to the steroidogenic folliculo-luteal shift and functional luteolysis.

Indirect immunofluorescence light microscopy was used to monitor temporal perturbations in the microtubular (tubulin) system of granulosa/lutein cells in paraffin-embedded sections of periovulatory follicles and corpora lutea of sheep. Estrogen-active granulosa cells of preovulatory follicles not yet exposed to the gonadotropin surge immunostained intensely for tubulin. Immunostaining of the microtubular matrix diminished after the onset of the surge and coincident with an abrupt fall in follicular estradiol production. A transient period of microtubular retraction was characterized by low-level steroid hormone output. Microtubules reappeared with the approach of ovulation and increase in follicular progesterone biosynthesis (luteinization). Treatment of animals during the preovulatory period with colchicine, a drug that binds specifically with tubulin and interferes with microtubular assembly, obstructed the follicular shift toward progesterone. Microtubular dynamics (polymerization<->depolymerization) evidently underpin fundamental mechanisms of follicular steroidogenesis. Finally, corpora lutea were isolated from ewes on Day 10 of the estrous cycle before (0 h) and after administration of prostaglandin (PG) F2 alpha. There was a small augmentation in luteal concentrations of progesterone at 2 h, followed by a sharp decrease from 4 to 16 h. Luteal weights were reduced (structural regression) at 24 h. Sections of large (PG-sensitive) steroidogenic cells of control corpora lutea typically displayed a radiating microtubular network. After administration of PGF2 alpha, tubular matrices of large cells were scant; mitochondrial clustering was evident in transmission electron micrographs. Affixed disassembly of the cytoskeleton of large luteal cells may be a heretofore unrecognized event in the biomechanics of functional luteolysis--perhaps uncoupling cholesterol translocation to mitochondrial cytochrome P450 side-chain cleavage.

Effects of epidermal growth factor, transforming growth factor α and androstenedione on follicular growth and aromatization in culture

The effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and androstenedione on follicular growth and oestradiol production were studied in cultured mouse preantral follicles. Cultured follicles in the control group showed growth and steroidogenic capacities up to the antral follicle stage at day 4 of culture. However, many of these follicles failed to resume their growth during the culture. Increasing concentrations of EGF and TGF alpha decreased the number of follicles reaching the large antral stage at day 4 of culture. All treatments with EGF, TGF alpha and androstenedione caused significant inhibitions in both follicular growth and oestradiol production, regardless of the dose used (1-20 ng/ml). Moreover, no synergistic effect between androstenedione and either of the two growth factors was observed. These results suggest that EGF, TGF alpha and/or androstenedione may play important roles in the modulation of gonadotrophin-controlled ovarian function. The effects observed in this study suggest that the aberration in the regulation of these growth factors may result in the creation and maintenance of certain ovarian disorders, such as the polycystic ovarian disease.

3 beta-hydroxysteroid dehydrogenase inhibitor reduces ovarian steroid production but increases ovulation rate in the ewe: interactions with gonadotrophins and inhibin.

Two experiments were carried out during the breeding season in ewes, first to investigate the effects of oral administration of a 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) inhibitor (epostane) on the number of corpora lutea, and secondly to investigate the mechanism through which epostane acts. In the first experiment Dorset Horn ewes were treated orally with 25, 50, 100 or 200 mg epostane twice daily between days 10 and 15 of the oestrous cycle. All doses of epostane resulted in an increase in the number of corpora lutea per ewe, although the response was curvilinear, with the 25 mg dose showing the largest response and the 200 mg group the smallest response. Although there was no difference between groups in the number of ewes showing oestrus, the higher doses of epostane had a detrimental effect on fertility. In the second experiment Welsh Mountain ewes were treated twice daily with 25 mg epostane from day 10 of the oestrous cycle and the ovaries were removed for analysis during either the luteal or the follicular phases. Treatment significantly increased the number of follicles greater than 6 mm in diameter, but significantly reduced in-vitro follicular oestradiol and testosterone production. Despite a marked increase in peripheral inhibin concentrations there was no effect on in-vitro inhibin production. Epostane treatment also caused a significant reduction in peripheral FSH concentrations and an increase in mean LH concentration. The latter was due to an increase in LH pulse frequency during the luteal phase and LH pulse amplitude during the follicular phase. These results confirm that treatment of ewes with epostane orally has a significant effect on follicular steroidogenesis and causes a significant increase in the number of corpora lutea per ewe. This effect on ovulation rate is not via an increase in peripheral FSH concentration, but may be caused by a reduction in follicular steroid activity either directly on the ovary or via an alteration in the pattern of LH secretion.

Intermittent GNRH antagonist plus progestin contraception conserving tonic ovarian estrogen secretion and reducing progestin exposure

The present study was designed to evaluate the effectiveness of a once-weekly regimen of GnRH antagonist followed by a progestin as a potential new contraceptive method. On menstrual cycle days 2, 9, 16, and 23 (onset of menses = Day 1) monkeys were divided into two groups: 1) those injected sc with 0.1 mg/kg Nal-Glu GnRH antagonist in saline and those given only vehicle (control). On cycle days 15 to 26, each treated female was administered 25 μg norgestlmate/day orally. This was continued for three treatment cycles (84 days). Weekly injections of Nal-Glu GnRH antagonist effectively blocked completion of folliculogenesis, ovulation, and corpus luteum function as judged by serum LH, E 2, and P levels. Serum progesterone was undetectable (<0.1 ng/ml) during the treatment cycles. Importantly, serum estradiol levels during GnRH antagonist plus norgestimate treatments were maintained at 35 ± 7 pg/ml. Upon the cessation of norgestimate treatment on day 26 in each cycle, menses uniformly began within 2 or 3 days. Regarding recovery, apparently normal and presumably ovulatory menstrual cycles, as judged by timely estradiol elevations, mldcycle LH surges, and luteal phase progesterone patterns, were manifest immediately following termination of the final GnRH antagonist plus norgestimate treatment cycle. Endometrial biopsies removed on day 26 of control cycles, and on day 26 of the third treatment cycle revealed appropriate late secretory phase endometrium having tortuous endometrial glands and superficial stromal edema. Histological sections of ovaries removed at the end of the GnRH antagonist plus norgestimate treatment revealed multiple small and medium-sized developing and atretic follicles, having maintained serial ablation of the potentially maturing follicles. Our findings indicate that once-weekly GnRH antagonist plus norgestimate treatment is a feasible method of ovulation inhibition. The intermittent (weekly) GnRH antagonist regimen allows follicular estradiol production to continue at tonic levels. Transformation of secretory to proliferative endometrium by the 12 day/month progestin regimen attenuated the potentially adverse effects of unopposed estrogen on target tissues.