Plasma follicle stimulating hormone,ovulation rate and fertility in Merino ewes treated with bovine follicular fluid

Charcoal-treated bovine follicular fluid (bFF) given as four 5-ml subcutaneous injections to 13 Merino-Border Leicester ewes around the time of natural luteolysis suppressed (P<0.01) plasma levels of follicle stimulating hormone (FSH) [from 1.08 ± 0.05 to 0.41 ± 0.03, mean ± s.e.m. of log_e (ng+ 1) /mlplasma]. This was followed (P < 0.01) by hypersecretion or a rebound of FSH (to 1.46 ± 0.11) lasting 32 h in 10 of the treated ewes, and then by a further fall (to 0.73 ± 0.03, P < 0.05) before the surge (1.21 ± 0.07, P < 0.05) associated with the preovulatory surge of luteinizing hormone (LH).Plasma FSH at 56–72 h before the LH surge (i.e., at the time of the FSH rebound) was correlated with the subsequent ovulation rate (n=13, r= + 0.73, P < 0.01). Fewer ewes treated with four injections of 2 or 5 ml of bFF than control ewes (injected with bovine plasma) became pregnant (28 of 41 vs. 38 of 41, χ² = 4.05, P < 0.05), although plasma progesterone was similar at Day 11 in treated and control ewes. It is concluded that plasma FSH during such a rebound influences the subsequent ovulation rate in sheep.     

Effect of a dopamine D-1 agonist in rats treated chronically with zuclopenthixol

We have previously demonstrated long-lasting increases in vacuous chewing movements (VCM) and tongue protrusions in rats treated discontinuously (DISC), but not continuously (CONT), with neuroleptics. To test whether this increase in mouth movements could be a result of exaggerated activity at the D-1 site, 34 rats were divided into three groups receiving the neuroleptic zuclopenthixol (ZU) DISC or CONT for 15 weeks, or no treatment. After withdrawal DISC treated animals showed an increase in oral activity compared to CONT treated. Two weeks after termination of medication the animals were tested with the D-1 agonist SK&F 38393. The increases in VCM after SK&F 38393 did not differ among the groups, but in contrast to control (CTRL) rats, treated rats showed a significant increase in tongue protrusions. There were no significant differences in the densities of D-1 and D-2 receptors in the striatum between the groups. The increase in tongue protrusions after SK&F 38393 in neuroleptic treated animals implies behavioural D-1 receptor supersensitivity. No significant differences in the rise in tongue protrusions and VCM after SK&F 38393 were seen between DISC and CONT treated animals. Our results thus do not indicate that increased D-1 receptor responsiveness is significant for the rise in spontaneous oral activity found after neuroleptic withdrawal.     

Suppression of follicle wave emergence in cyclic ewes by supraphysiologic concentrations of estradiol-17beta and induction with a physiologic dose of exogenous ovine follicle-stimulating hormone

Follicle waves are preceded by follicle-stimulating hormone (FSH) peaks in ewes. The purpose of the present study was to see whether estradiol implant treatment would block FSH peaks to create a model in which the effect of the timing and mode of FSH peaks could be studied by ovine FSH (oFSH) injection. In Experiment 1, 10 ewes received estradiol-17beta implants on Day 4 after ovulation (Day 0, day of ovulation); five ewes received large implants, and five ewes received small implants. Five control ewes received empty implants. In Experiment 2, 12 ewes received large implants on Day 4. On Day 9, six ewes received oFSH twice, 8 h apart (0.5 microg/kg; s.c.). Implants were left in place for 10 days in both experiments. In both studies, ovarian ultrasonography and blood sampling was done daily. In Experiment 1, estradiol concentrations were significantly higher in ewes with large implants (10.4 +/- 0.7 pg/ml) compared with controls (3.9 +/- 0.7 pg/ml) and ewes with small implants (5.4 +/- 0.7 pg/ml; P < 0.001). A significant reduction was found in mean FSH peak concentration (31%; P < 0.05) and FSH peak amplitude (45%; P < 0.05) in ewes with large implants compared with controls. Mean and basal FSH concentrations were unaffected by the large implants. The large implants halted follicle-wave emergence between Day 0 and 8 after implant insertion. The small follicle pool (2-3 mm in diameter) was unaffected by the large implants. When oFSH was injected into ewes with large implants, a follicle wave emerged 1.5 +/- 0.5 days after injection; however, in ewes given saline alone, a follicle wave emerged 4.8 +/- 0.8 days after injection (P < 0.01). We concluded that truncation of FSH peaks by estradiol implants prevented follicle-wave emergence, but injection of physiologic concentrations of oFSH reinitiated follicle-wave emergence.     

Regulation of follicle-stimulating hormone-receptor messenger RNA in hen granulosa cells relative to follicle selection

Both the viability of hen prehierarchal follicles and subsequent differentiation associated with the selection of a single follicle per day into the preovulatory hierarchy depend on circulating FSH and the expression of FSH receptor (FSH-R) in granulosa cells. The present study addresses mechanisms that mediate both basal expression plus selective up-regulation of FSH-R mRNA in granulosa cells from prehierarchal follicles. Results demonstrate that FSH-R mRNA is both expressed and functional in granulosa cells collected from growing prehierarchal follicles as small as those of 1-2 mm in diameter, as indicated by rapid induction of steroidogenic acute regulatory (StAR) protein expression by FSH in vitro. Real-time polymerase chain reaction determined that relative FSH-R expression within the granulosa layer from individual prehierarchal follicles of 6-8 mm in diameter was similar among the 8-13 follicles within this cohort, with the notable exception that the granulosa layer from a single follicle (presumably the selected follicle) showed elevated expression. Levels of FSH-R mRNA expression were enhanced by both recombinant human (rh) transforming growth factor (TGF) beta1 and, to a lesser extent, rh-activin A after 20 h of culture. This stimulatory effect was effectively blocked by mitogen-activated protein (MAP) kinase signaling induced by TGF alpha treatment. Finally, inhibition of MAP kinase signaling, using the selective inhibitor U0126, promoted FSH-R expression and further enhanced TGF beta1-induced FSH-R expression in vitro. Collectively, results suggest that premature granulosa cell differentiation normally is suppressed by tonic MAP kinase signaling. At the time of follicle selection, a release from inhibitory MAP kinase signaling is proposed to occur, which enables the full potentiation of FSH-R expression mediated by intrafollicular factors.     

Circulating concentrations of ovarian steroids and follicle-stimulating hormone (FSH) in ewes with 3 or 4 waves of antral follicle emergence per estrous cycle

The mechanism governing the number of follicle-stimulating hormone (FSH) peaks and emerging follicular waves in ruminants remains unknown. The main purpose of the present study was to examine the relationships between progesterone (P(4)) levels, circulating concentrations of FSH and antral follicular development throughout the interovulatory interval in sheep. We retrospectively analyzed and compared daily serum concentrations of (P4), FSH and estradiol (E2) obtained in cyclic (November-December) Western White Face ewes (Columbia×Rambouillet) that had 3 (n=10) or 4 (n=19) follicular waves per estrous cycle. Follicular growth was monitored in all animals by daily transrectal ultrasonography. Mean P(4) concentrations were greater (p<0.05) in sheep with 4 waves per cycle compared to their counterparts with 3 waves of follicular growth. The ewes with 3 waves exceeded (p<0.05) animals with 4 follicular waves in mean serum FSH concentrations on days 0-2, 6, 7, 9-11, 14 and 15 post-ovulation. Animals with 4 follicular waves exceeded (p<0.05) the ewes with 3 waves in mean serum E(2)> concentrations on days - 1, 2 and 10 of the estrous cycle studied (day 0=ovulation). The present results are supportive of the notion that luteal P(4) is an important endocrine signal, which controls the periodicity of FSH peaks and the number of emerging follicular waves in cyclic ewes.     

Serum hormone profiles and return to estrus in early-postpartum spring-lambing ewes treated with somatostatin

After parturition, 10 mature spring-lambing fine-wool ewes producing twins were allotted to one of two treatments. Five ewes received sterile saline (i.v.) twice daily on Days 12 to 15 post partum (PP) while 5 ewes were treated similarly except each injection contained 500 mug somatostatin (SRIF). Jugular blood samples were collected at 15-min intervals for 1 h before to 3 h after morning treatment on Days 12 and 15 PP. Animals were observed twice daily for signs of estrus using vasectomized rams beginning on Day 31 PP and continuing until ewes returned to estrus. Interval from parturition to estrus (mean +/- SEM) was similar (P > 0.40) in ewes receiving SRIF (119 +/- 6.2 d) and in control ewes (113 +/- 6.2 d). Ewes receiving 500 mug SRIF had lower (P < 0.10) serum insulin during the first 45 min after treatment on Day 12 PP; however, on Day 15 PP, serum insulin did not differ (P > 0.40) between treatment groups. Serum growth hormone (GH) did not differ (P > 0.40) between treatment groups 1 h before treatment on Day 12 PP; however, ewes treated with SRIF had lower (P < 0.05) GH levels before treatment on Day 15 PP than control ewes (4.4 and 9.9 +/- 1.5 ng/ml, respectively). After administration of SRIF, serum GH was higher (P < 0.05) in SRIF-treated ewes than in controls (8.2 and 5.3 +/- 2.7 ng/ml, respectively) on Day 12 PP but no differences (P > 0.80) were noted between treatment groups on Day 15 PP. These data indicate that 500 mug SRIF given twice daily from Days 12 to 15 PP neither lowered serum GH nor influenced return to estrus in lactating fine-wool ewes.     

Effects of medroxyprogesterone acetate (MAP) on ovarian antral follicle development, gonadotrophin secretion and response to ovulation induction with gonadotrophin-releasing hormone (GnRH) in seasonally anoestrous ewes

When ovulation is induced with gonadotrophin-releasing hormone (GnRH) in anoestrous ewes, a proportion of animals fail to form normal (full-lifespan) corpora lutea (CL). Progesterone treatment before GnRH prevents luteal inadequacy. It remains uncertain whether a similar effect, achieved with medroxyprogesterone acetate (MAP) from intravaginal sponges, is mediated by influences on growing ovarian follicles and/or secretion of gonadotrophic hormones, before and after GnRH treatment. Two experiments were performed, on 13 and 11 anoestrous Western white-faced ewes, respectively. Seven and six ewes, respectively, received MAP-containing sponges (60 mg) for 14 days; the remaining ewes served as untreated controls. To test the effect of timing of GnRH administration after pre-treatment with MAP-releasing sponges, GnRH injections (250 ng every 2h for 24h followed by a bolus injection of 125 microg of GnRH i.v.) were given either immediately (Experiment 1) or 24h after sponge removal in the treated ewes (Experiment 2). Ovarian follicular dynamics (follicles reaching >or=5mm in size) and development of luteal structures were monitored using transrectal ultrasonography. In Experiment 1, the mean ovulation rate (0.7+/-0.3 and 1.0+/-0.4) and proportion of ovulating ewes (57 and 67%, respectively) did not vary (P>0.05) between MAP-treated and control ewes. Normal (full-lifespan) CL were detected in 29% of treated and 67% of control ewes (P>0.05). In Experiment 2, the mean ovulation rate (2.3+/-0.2 and 1.2+/-0.6; P<0.05) and percentage of ewes with normal (full-lifespan) CL (100 and 40%, respectively; P<0.10) were greater in the treated compared to control ewes. In Experiment 1, the mean peak concentration of the GnRH-induced LH surge was lower (P<0.05) in MAP-treated than in control ewes. There were no significant differences between MAP-treated and control ewes in the characteristics of follicular waves, mean daily serum FSH concentrations, and secretory parameters of LH/FSH, based on intensive blood sampling conducted 1 day before sponging and 1 day before sponge removal. It is concluded that treatment with MAP has no effect on the tonic secretion of LH/FSH or follicular wave development in anoestrous ewes. However, the GnRH-stimulated LH discharge was attenuated in the ewes that received MAP-impregnated sponges for 14 days and were treated with GnRH immediately after sponge withdrawal. Ovulatory response and CL formation were increased when GnRH was administered 24 h after sponge removal.     

Effects of epidermal growth factor on early embryonic development after in vitro fertilization of oocytes collected from ewes treated with follicle stimulating hormone

Epidermal growth factor (EGF) has been shown to enhance the in vitro rate of blastocyst formation in several species. Follicular development was induced in ewes (n=15) by twice daily administration of FSH-P on Days 13 and 14 of the estrous cycle. Cumulus oocyte complexes (COCs) were collected from all visible follicles (n=25+/-2.4/ewe) on Day 15. COCs from each ewe were cultured separately for 24h in maturation medium (containing 10% serum, LH, FSH and estradiol) with (8.2+/-0.9 per ewe) or without (7.8+/-0.8 per ewe) EGF (10 ng/ml). Oocytes were then denuded by hyaluronidase treatment, and healthy oocytes were cultured in the presence of frozen-thawed semen in synthetic oviductal fluid (SOF) medium containing 2% sheep serum. After 18-20 h, zygotes were transferred to SOF medium without glucose and cultured for about 36 h until they reached the 4-8 cell stage. Embryos were transferred to SOF medium with glucose for further development. Medium was changed every other day until blastocyst formation on Day 8 of culture (Day 1=day of fertilization). The rate of embryonic development was evaluated throughout the culture period. After maturation, cumulus cells were more expanded in the presence than in the absence of EGF. The rates of fertilization (overall 75.7+/-3.9%) and morula formation (overall 40.6+/-7.1%) were similar (P>0.05) for COCs cultured with or without EGF. However, EGF increased (P<0.01) the number of blastocysts (1.4+/-0.1 versus 0.6+/-0.2 per ewe) and tended to increase (P<0.1) the rate of blastocyst formation (21.0+/-6.6% versus 13.4+/-4.3% per ewe). These data demonstrate that EGF increases blastocyst formation in FSH-treated ewes. Therefore, EGF is recommended as a supplement to maturation medium to enhance embryonic development in vitro in FSH-treated sheep.     

Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.     

Ovarian follicular development and oocyte quality in anestrous ewes treated with melatonin, a controlled internal drug release (CIDR) device and follicle stimulating hormone

The objective of the current study was to determine the effects of hormonal treatments on ovarian follicular development and oocyte quality in anestrous ewes. Multiparous crossbred (RambouilletxTarghee) ewes were given melatonin implants (MEL) and/or controlled internal drug release (CIDR) devices in conjunction with follicle stimulating hormone (FSH) during anestrus (March-May). In Experiment 1, ewes (n=25) were assigned randomly to four groups (n=4-7/group) in a 2x2 factorial arrangement [+/-MEL and +/-CIDR], resulting in Control (no treatment), CIDR, MEL, and MEL/CIDR groups, respectively. Ewes received an implant containing 18 mg of melatonin (Melovine) on Day 42 and/or a CIDR from Days 7 to 2 (Day 0: oocyte collection). In Experiment 2, ewes (n=12) were assigned randomly to two groups (n=6/group; 1CIDR or 2CIDR) and received the same type of melatonin implant on Day 60. All ewes received a CIDR device from Days -22 to -17 and 2CIDR ewes received an additional CIDR device from Days -10 to -2. In both experiments, ewes were given FSH im twice daily (morning and evening) on Days -2 and -1 (Day -2: 5 units/injection; Day -1: 4 units/injection). On the morning of Day 0, ovaries were removed, follicles>or=1 mm were counted, and oocytes were collected. Thereafter oocytes were matured and fertilized in vitro. In Experiment 1, the number of visible follicles and the rates of oocyte recovery and in vitro maturation were similar (P>0.10) for Control, CIDR, MEL and MEL/CIDR (overall 29.7+/-2.9%, 89.9+/-7.1% and 95.0+/-2.0%, respectively). The rates of in vitro fertilization (IVF) were lower (P<0.01) for CIDR and MEL/CIDR than for Control and MEL groups (10.3% and 10.1% versus 20.0% and 18.5%, respectively). In Experiment 2, the number of visible follicles, and the rates of oocyte recovery and in vitro maturation were similar (P>0.10) for 1CIDR and 2CIDR groups (overall 27.3+/-3.2%, 92.1+/-2.7% and 90.2+/-1.9%, respectively). However, the rates of IVF were lower (P<0.01) for 2CIDR than 1CIDR group (30.2% versus 58.0%, respectively). In summary, when treatment with P4 commenced only 2 d before oocyte collection, rates of IVF were reduced in both experiments. Therefore, progestin treatment protocols used in ovine IVF programs should be carefully designed to minimize adverse effects on fertilization rates. In addition, melatonin treatment did not affect follicular development and oocyte quality for anestrous ewes.     

Differential effects of various estradiol-17beta treatments on follicle-stimulating hormone peaks, luteinizing hormone pulses, basal gonadotropin concentrations, and antral follicle and luteal development in cyclic ewes

In a previous study, 10-day estradiol implant treatment truncated the FSH peaks that precede follicular waves in sheep, but subsequent ovine FSH (oFSH) injection reinitiated wave emergence. The present study's objectives were to examine the effects of a 20-day estradiol and progesterone treatment on FSH peaks, follicle waves, and responsiveness to oFSH injection. Also, different estradiol doses were given to see whether a model that differentially suppressed FSH peaks, LH pulses, or basal gonadotropin secretion could be produced in order to study effects of these changes on follicular dynamics. Mean estradiol concentrations were 11.8 +/- 0.4 pg/ml, FSH peaks were truncated, wave emergence was halted, and the number of small follicles (2-3 mm in diameter) was reduced (P < 0.05) in cyclic ewes given estradiol and progesterone implants (experiment 1). On Day 15 of treatment, oFSH injection failed to induce wave emergence. With three different estradiol implant sizes (experiment 2), estradiol concentrations were 5.2, 19.0, 27.5, and 34.8 (+/-4.6) pg/ml in control and treated ewes, respectively. All estradiol treatments truncated FSH peaks, except those that created the highest estradiol concentrations. Experiment 2-treated ewes had significantly reduced mean and basal FSH concentrations and LH pulse amplitude and frequency. We concluded that 20-day estradiol treatment truncated FSH peaks, blocking wave emergence, and reduced the small-follicle pool, rendering the ovary unresponsive to oFSH injection in terms of wave emergence. Varying the steroid treatment created differential FSH peak regulation compared with other gonadotropin secretory parameters. This provides a useful model for future studies of the endocrine regulation of ovine antral follicular dynamics.     

Patterns of antral follicular wave dynamics and accompanying endocrine changes in cyclic and seasonally anestrous ewes treated with exogenous ovine follicle-stimulating hormone during the inter-wave interval

In the ewe, ovarian follicular waves emerge every 4 to 5 days and are preceded by a peak in FSH secretion. It is unclear whether large antral follicle(s) in a wave suppress the growth of other smaller follicles during the inter-wave interval, as is seen in cattle. In this study, anestrous (n = 6; experiment 1) and cyclic (n = 5; experiment 2) Western white face ewes were given ovine FSH (oFSH) (0.5 microg/kg; two s.c. injections, 8 h apart) during the growth phase (based on ultrasonography) of a follicular wave (wave 1). Control ewes (n = 5 and 6, respectively) received vehicle. In oFSH-treated ewes, serum FSH concentrations reached a peak (P < 0.05) by 12 h after oFSH treatment, and this induced FSH peak did not differ (P > 0.05) from the endogenous FSH peaks. In all ewes, emergence of follicular waves 1 and 2 was seen (P > 0.05). However, in oFSH-treated ewes, an additional follicular wave emerged approximately 0.5 days after treatment: during the interwave interval of waves 1 and 2 without delaying the emergence of wave 2. The growth characteristics and serum estradiol concentrations did not differ (P > 0.05) between oFSH-induced waves and waves induced by endogenous FSH peaks. We concluded that, unlike in cattle, the largest follicle of a wave in sheep has limited direct effect on the growth of other follicles induced by exogenous oFSH. In addition, the largest follicle of a wave may possibly not influence the rhythmicity of follicular wave emergence, as it does in cattle.     

Serum bioactive and immunoreactive follicle stimulating hormone during chronic treatment with gonadotropin releasing hormone agonist in elderly men.

Chronic administration of GnRH agonists "down regulates" the pituitary and decreases LH and FSH serum levels. Changes in the bioactivity of FSH have not been adequately assessed under such treatment, for lack of a proper test. We examined serum changes under GnRH agonist treatment among 12 healthy elderly men suffering only from benign prostatic hypertrophy, for up to one year, using a modification of a granulosa cell bioassay for the determination of FSH bioactivity. While radioimmunoassay-FSH decreased, we noticed a significant increase in the bioactivity of this hormone. The clinical importance of this increase is discussed.     

Growth progression and 24-hour hormone profile in an infant treated chronically with a long-acting somatostatin derivative.

The diagnosis of nesidioblastosis was established in a 9-month-old male child with a history of recurrent convulsive seizures and hypoglycemia. After unsuccessful subtotal pancreatectomy, treatment was started with the long-acting somatostatin derivative Sandostatin (Octreotide, Sandoz) at a dosage of 25 micrograms t.i.d. spaced between carbohydrate-enriched meals. With this regime, blood glucose was maintained at the low normal range and seizures ceased. During a 30-month observation period, growth velocity and weight progression were well within the predicted limits. A 24-hour hormone profile recorded at the end of the observation period revealed the following: (1) failure to improve blood glucose with carbohydrate-enriched food due to reactive hyperinsulinemia; (2) hyperglycemic reaction after administration of Sandostatin caused by a reduction of plasma insulin; this effect was particularly marked during sleep; (3) low mean GH, decreased spiking frequency and reduced area covered by the nocturnal peaks by recognized standards, and (4) normal somatomedin C levels for age. Interpretation of growth hormone (GH) data is hindered by the lack of pertinent information from the patient's age group. Recording of normal growth progression in the case illustrated here can only be explained by the capability of a reduced GH secretory rate to maintain full biological activity as shown by the normal plasma level of somatomedin C. Indeed, recent evidence has been provided elsewhere for normal growth progression in the presence of low GH secretion, although other factors unrelated to this hormone may also be operative at this early age. Further reports concerning the treatment of non-GH-dependent conditions with somatostatin derivatives will certainly contribute to the better understanding of the mechanisms governing growth in the postnatal period.     

Appropriate cut-off value for follicle-stimulating hormone in azoospermia to predict spermatogenesis

Background  

This study was undertaken to determine the optimal cut-off value for FSH to predict the presence of spermatogenesis in patients with non-obstructive azoospermia.     

Ovarian response to injections of charcoal-extracted porcine follicular fluid and porcine follicle-stimulating hormone in gilts fed a progesterone agonist (altrenogest)

This experiment was conducted to compare the negative effects of charcoal-extracted porcine follicular fluid (pFF) and the positive effects of purified porcine follicle-stimulating hormone (pFSH) on growth of follicles and on plasma hormone concentrations. Twenty gilts were fed altrenogest for 18 days (20 mg.day-1.gilt-1) to suppress spontaneous growth of large follicles (greater than 6 mm in diameter). Gilts, assigned at random to receive pFF and pFSH administered in a 2 x 2 factorial arrangement, were injected 9 times at 8-h intervals starting 48 h before the last feeding of altrenogest and ending 8 h before slaughter (24 h after the last feeding of altrenogest). Blood was collected periodically through vena cava catheters. Treatment groups and mean number of medium follicles (3 to 6 mm in diameter)/gilt at necropsy were 1) 20 ml of charcoal-extracted porcine serum i.v. + 4 ml saline i.m., 30.8; 2) 20 ml of pFF i.v. + saline i.m., 0.2; 3) serum i.v. + 8 micrograms of pFSH (USDA-pFSH-B1)/kg BW in saline i.m., 59.0; and 4) pFF i.v. + pFSH in saline i.m., 36.2. Injections of pFF decreased (p less than 0.01) and injections of pFSH increased the number of medium follicles, and the interaction of pFF and pFSH was not significant. Plasma FSH decreased (p less than 0.01) during pFF treatment of saline-injected gilts at a rate of 0.29 ng.ml-1.h-1. During pFSH treatment, plasma FSH increased (p less than 0.05) at statistically identical rates of 0.33 and 0.32 ng.ml-1.h-1 in serum- and pFF-injected gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of a plasma membrane phosphodiesterase in differentiating granulosa cells. Reciprocal actions of follicle-stimulating hormone and a gonadotropin-releasing hormone agonist on cAMP degradation

The activity of a plasma membrane cAMP-phosphodiesterase in cultured ovarian granulosa cells was regulated by follicle-stimulating hormone (FSH) and the gonadotropin-releasing hormone (GnRH) agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). Degradation of cAMP was similar in cultures treated with FSH alone or FSH plus GnRHa when the labeled cyclic nucleotide was added from 24 to 42 h of culture. However, at 48 h and subsequent times of incubation, cAMP phosphodiesterase activity was significantly higher in cells incubated with FSH plus GnRHa. Phosphodiesterase activity was progressively increased by GnRHa concentrations between 10(-13) and 10(-10) M, and was maximally stimulated by 10(-9) M GnRHa. In comparison with control cells, FSH lowered the Vmax of cAMP catabolism by the high (1 microM cAMP substrate) and the low (50 microM) affinity phosphodiesterase, while GnRHa raised enzyme activity toward control levels. These actions of FSH and GnRHa were specific for a plasma membrane phosphodiesterase that was accessible to extracellular cAMP, since extracellular substrate was hydrolyzed, no intracellular uptake of [3H]cAMP was observed, and only a small fraction (10%) of cAMP was catabolized in the incubation medium in the absence of cells. Further, the actions of FSH and GnRHa on the membrane enzyme were the opposite of those observed when total phosphodiesterase activity was measured in cellular sonicates. Hormonal changes in phosphodiesterase activity were not due to leakage of the enzyme from damaged cells since a constant percentage of cAMP hydrolysis in the medium was observed during culture. Analysis of cAMP catabolites in granulosa cells indicated that the phosphodiesterase reaction product, 5'-AMP, was rapidly converted to adenosine by a plasma membrane 5'-nucleotidase, independent of the cellular hormonal status. These results indicate that the opposing actions of FSH and GnRHa upon granulosa cell differentiation include modulation of cAMP degradation at the plasma membrane level.     

The role of luteinizing hormone in regulating gene expression during selection of a dominant follicle in cattle

At approximately 8.5 mm in diameter, the future dominant follicle is "selected" for continued growth in cattle. In the present study, cows were treated with a gonadotropin-releasing hormone receptor antagonist, acyline, just before follicle selection (near 7.8 mm) to investigate the role of LH in changing mRNA concentrations during selection of a dominant follicle. The ovaries containing the expected dominant follicle (EDF; first largest follicle) and expected largest subordinate follicle (ESF) were removed after 12 or 24 h of treatment. Real-time PCR was used to determine mRNA concentrations. ELISA was used to measure testosterone and 17beta-estradiol (E(2)) and radioimmunoassay to measure androstenedione (A(4)) in follicular fluid. Concentrations of E(2) were greater in EDF than in ESF of untreated cows near the time of follicle selection (12 h) or at 12 h after selection (24 h). Testosterone, E(2), and A(4) were all dramatically decreased by acyline treatment at both times. In theca cells, acyline treatment reduced CYP17A1 (P450 17alpha) in EDF and STAR (steroidogenic acute regulatory protein) in both EDF and ESF but did not alter CYP11A1 (P450scc). In granulosa cells (GCs), LHCGR (luteinizing hormone [LH] receptor) was much greater in EDF than in ESF at both time of selection (739% greater) and 12 h after selection (2837% greater) and was decreased by acyline in EDF (87% decrease). The mRNA for CYP19A1 (cytochrome P450 aromatase) and PAPPA (pregnancy-associated plasma protein-A) tended to be greater in EDF than in ESF at follicle selection, and both mRNAs were much greater at 12 h after selection, with acyline significantly decreasing PAPPA mRNA after 24 h of treatment. The mRNA for FSHR (follicle-stimulating hormone receptor) was not different in EDF versus ESF and was not altered by acyline. Thus, induction of LHCGR mRNA in GCs is an early event during the follicle selection process, and surprisingly, expression of LHCGR mRNA is dependent on circulating LH. Production of follicular A(4), testosterone, and E(2) are also acutely related to LH but due to changes in expression of STAR and CYP17A1 in TC.     

Hypersecretion of follicle-stimulating hormone (FSH) on estrous afternoon in rats treated with RU486 in proestrus

Summary 1. Intact or ovariectomized (OVX) cyclic rats injected or not with RU486 (4 mg/0.2 ml oil) from proestrus onwards were bled at 0800 and 1800h on proestrus, estrus and metestrus. Additional RU486-treated rats were injected with: LHRH antagonist (LHRHa), estradiol benzoate (EB) or bovine follicular fluid (bFF) and sacrified at 1800 h in estrous afternoon. LH and FSH serum levels were determined by RIA.2. RU486-treated intact or OVX rats had decreased preovulatory surges of LH and FSH, abolished secondary secretion of FSH and hypersecretion of FSH in estrous afternoon. The latter was decreased by LHRHa and abolished by EB or bFF. In contrast, EB induced an hypersecretion of LH in RU486-treated rats at 1800h in estrus.3. It can be concluded that in the absence of the proestrous progesterone actions, the absence of the inhibitory effect of the ovary in estrus evoked a LHRH independent secretion of FSH.