Phosphodiesterase 3 inhibitors selectively block the spontaneous resumption of meiosis by macaque oocytes in vitro.

BACKGROUND: The purpose of this study was to determine whether phosphodiesterase (PDE) 3 inhibitors selectively prevent the resumption of meiosis in primates. METHODS: Immature oocytes (intact germinal vesicles) obtained from large pre-ovulatory follicles following ovarian stimulation in rhesus macaques were incubated with or without various doses of the PDE3 inhibitors, Cilostamide, Milrinone or ORG 9935, or a selective PDE4 inhibitor, Rolipram. Oocytes were observed for germinal vesicle breakdown (GVBD) as an indicator of resumption of meiosis. RESULTS: At 24 h, 72 of 121 (60%) control oocytes progressed to GVBD compared with 9/34 (27%, P < 0.01), 4/36 (11.1%, P < 0.01) and 0/28 (0%, P < 0.01) oocytes incubated with ORG 9935 at 0.1, 0.5 and 1.0 micromol/l respectively. Similar results were achieved at 24 h with 1.0 micromol/l Cilostamide (2/24 oocytes, 8%, P < 0.01) and 100 micromol/l Milrinone (2/32, 6%, P < 0.01). In contrast, no significant difference in GVBD was noted between control oocytes and those incubated with up to 100 micromol/l Rolipram for 24 h (43/58, 74%) or 48 h (44/58, 76%). CONCLUSIONS: These experiments establish the specificity and dose-dependent ability of PDE3, but not PDE4, inhibitors to block resumption of meiosis in macaque oocytes in vitro. Thus, PDE3 inhibitors have potential use as contraceptives in primates.

Male contraception: views to the 21st century, Bethesda, MD, USA, 9-10 September 1999.

For men who still wish to father children, the contraceptive options currently available are withdrawal and the condom. Although significant progress has been made on hormonal and vaccine-related approaches to male contraception, a marketed product is, at best, several years away. Therefore, the National Institute of Child Health and Human Development convened a workshop to discuss novel strategies for development of male contraceptives that focused on the testis and epididymis. Participants recognized that exploration of these new approaches will necessitate considerable investment of funds and research efforts.

Expression of follistatin and inhibin/activin subunit genes in porcine follicles.

Expression of the follistatin (FS) and inhibin/activin (I/A) alpha, beta(A), and beta(B) subunit genes in porcine ovarian follicles was evaluated by reverse transcriptase polymerase chain reaction and/or RNase protection procedures to establish changes during the final stages of follicular development. For the I/A alpha and beta(A) subunits, expression increased (p < 0.05) as follicles progressed to the mid-stage of the follicular phase. The beta(B) subunit was expressed in lower concentrations, and all three I/A subunits showed a marked reduction (p < 0.01) in expression by the late stage of follicular development. In contrast to this pattern, FS gene expression decreased (p < 0.05) as follicles developed from the early (low estradiol) to the mid stage (high estradiol) and continued to decline in advanced follicles (after estrus). The predominant mRNA encoded for FS-315, and the ratio of mRNA for FS-315 to mRNA for FS-288 did not differ significantly during the three stages. Within an animal, concentration of FS mRNAs was related more to stage of the follicular phase than to follicular size. Follicular fluid concentration of FS changed in a manner similar to that observed for expression of its gene. We conclude that expression of the FS gene and translation of its mRNA decrease as follicles approach ovulatory status.

Circulating levels of follistatin from puberty to menopause.

OBJECTIVE: To determine the changes in circulating levels of follistatin, a binding protein for activin and inhibin, through the reproductive life cycle in women. DESIGN: An open, prospective descriptive study. SETTING: An academic endocrine research unit. PATIENTS: Prepubertal (n = 10), midpubertal (n = 7), and postpubertal (n = 25) (early adolescent) girls, normal cycling adult women (n = 8), postmenopausal women (n = 17), and men (n = 13) were studied. INTERVENTIONS: Normal cycling women were given Nal-Glu GnRH antagonist for 3 days in the follicular phase of the cycle. MAIN OUTCOME MEASURE: Serum concentrations of follistatin determined in a heterologous RIA. RESULTS: Mean follistatin levels did not change during puberty but were higher in adult and postmenopausal women. Levels of immunoreactive follistatin in men were lower than levels found in normal cycling women and postmenopausal women. Daily immunoreactive follistatin levels during the menstrual cycle remained constant and did not change significantly after ovarian suppression with GnRH antagonist. CONCLUSION: Because dynamic changes of serum immunoreactive follistatin do not occur during ovarian activation (puberty), suppression, and age-related ovarian failure, the increase in immunoreactive follistatin levels in adult and postmenopausal women may implicate sources of follistatin other than the ovary.

Follistatin concentrations in follicular fluid of normal and polycystic ovaries.

Follistatin (FS) is an activin/inhibin binding protein which is believed to act in an autocrine/paracrine manner to regulate growth and differentiation. Although FS has been identified in human follicular fluid, it remains unclear how its concentration changes during selection and atresia, and what the concentrations of FS are in follicles of women with polycystic ovary syndrome (PCOS). Towards this goal, we have measured by radioimmunoassay the concentrations of FS in follicular fluid obtained from dominant and atretic cohort follicles of normal cycling women, preovulatory follicles of in-vitro fertilization (IVF) patients, and small Graafian follicles of patients with PCOS. In all cases, the follicular fluid concentration of FS was much higher (approximately 100-fold) than that reported in serum. The FS concentrations (ng/ml) were 203 +/- 42 (normal dominant), 185 +/- 17 (atretic cohort), 185 +/- 5 (IVF), and 250 +/- 14 (PCOS). There was no statistical difference between these mean values of FS. Further, there were no significant correlations between the follicular fluid concentrations of FS and the concentrations of oestradiol, progesterone, or androstenedione. These results indicate that human Graafian follicles, regardless of whether they are healthy or atretic, normal or PCOS, contain high steady-state concentrations of FS in the micro-environment. Collectively, these data fit with the hypothesis that major increases and decreases in the concentration of FS in the micro-environment may not play a key role in the mechanisms of selection, atresia, and PCOS in women. The possibility of regulation of intrinsic activin and inhibin activity through FS binding is discussed.

Local production and action of follistatin in human placenta.

The aim of the present study was to investigate the possible production, localization, and action of follistatin in human placenta, fetal membranes (amnion, chorion), and maternal decidua. Four different experimental approaches were used: 1) Southern blot analysis following reverse polymerase chain reaction to identify follistatin messenger RNA (mRNA) in tissue homogenates; 2) immunohistochemistry to localize immunoreactive (ir-) follistatin in the various intrauterine tissues; 3) measurement by RIA of ir-follistatin levels in culture medium of placental cells; and 4) possible action of follistatin on human CG (hCG) and progesterone release from cultured placental cells. Placental and decidual cells collected during first trimester or at term gestation express follistatin mRNA; fetal membranes (amnion, chorion) at term also express follistatin mRNA. Immunoreactive follistatin is localized in syncytial cells of placental villi at term as well as in large decidual cells, in amnion epithelium, and in chorionic cells. The placental secretion of follistatin has been confirmed by the evidence of measurable levels of ir-follistatin in the medium of cultured placental cells at term; the release is time dependent and is not modified by the addition of forskolin or progesterone. The addition of increasing doses of recombinant human follistatin does not significantly influence the release of hCG or progesterone from cultured placental cells, whereas the activin A-induced hCG and progesterone release are completely reversed. The present data showed that 1) human placenta, fetal membranes, and decidua express follistatin mRNA; 2) ir-follistatin is localized and released from placental cells at term; and 3) follistatin has a functional role in the local control system regulating placental hormone production.

Differential control of activin, inhibin and follistatin proteins in cultured rat granulosa cells.

Follistatin, activin and inhibin proteins are produced by granulosa cells, but the mechanisms controlling their production remain unclear. Here, we examined how the protein kinase A (PKA) and protein kinase C (PKC) pathways act and interact to regulate production of these proteins. Granulosa cells from immature rats were cultured with activators of the PKA pathway (100 ng/ml FSH, 10 microM forskolin) and/or activators of the PKC pathway (100 nM GnRH agonist, 100nM 2-0-tetradecanoyl-phorbol-13-acetate, TPA). Conditioned media were assayed for inhibin and activin by ligand blotting using recombinant human 125I-follistatin and for follistatin by double ligand blotting using cold activin plus 125I-follistatin. FSH and forskolin stimulated inhibin but not activin production. In contrast, GnRH and TPA stimulated activin, and to a lesser degree, inhibin production; significantly, this is the first demonstration of activin dimer production by granulosa cells. Activators of the PKA pathway antagonized the actions of PKC effectors and vice versa. All agents increased follistatin protein production, and the PKA and PKC activators interacted to generate further increases in follistatin production. These results show that the FSH-PKA signalling pathway favors formation of alpha beta inhibin dimers while the GnRH-PKC pathway favors formation of beta-subunit activin dimers. Both pathways act to increase follistatin protein production.

Increased follistatin (activin-binding protein) gene expression in rat anterior pituitary tissue after ovariectomy may be mediated by pituitary activin.

For lack of evidence to the contrary, it is now believed that the FSH-suppressing actions of follistatin are due to its ability to bind endogenous pituitary activin. Recent data have demonstrated a role for pituitary activin-B in mediating FSH hypersecretion after ovariectomy (OVX) and during the secondary FSH surge on estrus. Therefore, given that follistatin is produced within anterior pituitary tissue, and considering the potentially important function of follistatin to modulate activin bioactivity, we sought to gain insights into the regulation of follistatin gene expression in the anterior pituitary gland of adult female rats. At the termination of all in vivo investigations, rats were killed, trunk blood was collected for determination of serum LH and FSH levels by RIA, and pituitary tissue was collected, pooled (two or three glands per pool), and processed for determination of follistatin messenger RNA (mRNA) levels by a solution-hybridization RNase protection assay. In the first experiment, pituitary follistatin mRNA levels were significantly (P < 0.01) increased 3 weeks after OVX. Treatment of long-term ovariectomized rats with a Nal-Glu LHRH antagonist restored serum LH levels to precastration levels and suppressed serum FSH concentrations by 70%, but follistatin message levels were not altered. In contrast, treatment of castrated rats with recombinant human follistatin-288 selectively suppressed serum FSH levels (50%) and completely abolished OVX-induced increases in follistatin mRNA levels. Subsequent experiments revealed that OVX-induced increases in follistatin gene expression could be observed in pituitary tissue grafted underneath the kidney capsule of hypophysectomized rats. Furthermore, follistatin mRNA levels were significantly (P < 0.05) higher in pituitary glands taken from estrous rats during the secondary FSH surge (0200 h) than in glands obtained from rats on proestrous morning when serum FSH levels were basal. Because increased steady state follistatin mRNA levels in the latter two instances were associated with selective FSH hypersecretion, and such hypersecretion was previously shown to be dependent to a significant degree on pituitary activin, we next tested the hypothesis that increased pituitary follistatin gene expression is mediated by activin. Using cultures of dispersed pituitary cells, addition of recombinant human activin-A for 72 h increased follistatin mRNA levels 3-fold while enhancing only FSH secretion. Collectively, the present results demonstrate a coupling of follistatin gene expression in the anterior pituitary gland with changes in pituitary FSH secretion under conditions where LH secretion is unaltered. Viewed in the context of previous work, the data also suggest that changes in follistatin mRNA levels may be linked to activin signaling.

Regulation of pulsatile gonadotropin secretion by estrogen, inhibin, and follistatin (activin-binding protein) in ovariectomized rats.

The following study was conducted to examine the effects of estrogen and polypeptides, given either alone or in combination, on pulsatile gonadotropin secretion. One week after ovariectomy, rats received s.c. injections of oil or various doses (0.5, 5, 20 micrograms) of estradiol benzoate (EB) followed 1 day later by i.v. administration of 60 micrograms purified porcine follistatin, 10 micrograms recombinant inhibin, or the appropriate vehicle. Four hours after injection of the nonsteroids, blood was collected at 10-min intervals for 2 h, and the effects on pulsatile hormone release were assessed. Administration of EB alone dose-dependently suppressed mean and trough (lowest point between two pulses) FSH levels and all parameters of pulsatile LH release. Both follistatin and inhibin at the doses employed suppressed mean FSH levels to an equivalent extent (40%). Follistatin, but not inhibin, suppressed FSH pulse amplitude, while neither polypeptide alone influenced FSH pulse frequency or any parameter of pulsatile LH release. The effects of follistatin and EB on mean FSH levels were additive at all EB doses, whereas the effects of inhibin and EB were additive only at the middle EB dose. Follistatin in combination with the lowest EB dose significantly suppressed mean LH levels. These studies are the first to demonstrate that combined treatment with estrogen and the nonsteroids follistatin and inhibin is more efficacious in suppressing FSH release than treatment with either agent alone, thereby indicating that both steroids and nonsteroids are probably important in the physiological regulation of FSH secretion in rats. The additive effects of these compounds on FSH secretion could form the basis for exploring novel contraceptive interventions.

Passive immunoneutralization with a monoclonal antibody reveals a role for endogenous activin-B in mediating FSH hypersecretion during estrus and following ovariectomy of hypophysectomized, pituitary-grafted rats.

Recently, we reported that ovariectomy (OVX)-induced FSH hypersecretion can be elicited in hypophysectomized rats bearing renal pituitary allografts isolated from direct hypothalamic intervention. The possible role of the FSH-stimulating protein, activin-B, in eliciting this response was investigated using passive immunoneutralization with a monoclonal antibody (MAb) generated against activin-B. Other hypophysectomized/pituitary-grafted (H/G) rats serving as controls received an equivalent amount of a MAb incapable of neutralizing the biological actions of activin-B. Administration of increasing doses of the MAb prior to OVX dose-dependently suppressed serum FSH levels 12 h after OVX. Less consistent effects were observed 24 h after OVX even though an additional injection of the MAb was given 12 h after OVX in one study. Since it has been postulated that the periovulatory increase in additional injection of the MAb was given 12 h after OVX in one study. Since it has been postulated that the periovulatory increase in FSH secretion on estrus which is important for recruitment of follicles is a hypothalamic-independent phenomena, a separate experiment was performed in order to ascertain whether a local regulatory mechanism involving activin-B is operative on estrus. As in the preceding study using H/G rats, administration of the activin-B MAb on the evening of proestrus significantly attenuated serum FSH rises early on estrus. These results are consonant with the evolving concept that an important mechanism exists within the anterior pituitary proper for regulation of FSH secretion that involves the autocrine actions of activin-B.

Isolation and biochemical properties of four forms of glycosylated porcine prolactin.

Four isoforms of glycosylated prolactin (G-pPRL) were isolated from porcine pituitary glands by affinity chromatography and concanavalin A-Sepharose, based upon differences in their affinity for the lectin. Structural analysis indicated differences in the carbohydrate units of the four G-pPRLs. N-glycanase treatment cleaved the oligosaccharide from the G-pPRLs, establishing N-linked glycosylation. The binding of G-pPRLs to receptors from lactating rabbit mammary glands was only 3-8% that of nonglycosylated pPRL (NG-pPRL). The immunological crossreactivity of the G-pPRLs varied from 36 to 65% that of NG-pPRL. When tested in the pigeon crop sac bioassay, G-pPRLs were only 11-40% as active as NG-pPRL. The metabolic clearance rate of one of the G-pPRLs was slower and another faster than that of NG-pPRL. We conclude that there are several forms of G-PRL of variable immuno- and bio-potencies in the porcine pituitary, and that the current radioimmunoassay for the hormone does not measure the actual bioactivity.

Cyclic changes in follistatin messenger ribonucleic acid and its protein in the rat ovary during the estrous cycle.

The purpose of this research was to characterize the localization of follistatin mRNA and protein in the adult rat ovary during the 4-day estrous cycle. Analysis of ovarian sections using in situ hybridization and immunohistochemistry demonstrated the presence of follistatin messenger RNA (mRNA) and its protein in granulosa and luteal cells; no follistatin (message or protein) was detected in any of the other ovarian cell types. An important observation was that the intensity of follistatin signals changed during granulosa differentiation and the estrous cycle. During folliculogenesis, the first detectable hybridization signal appeared in the granulosa cells of secondary follicles, but the signal was weak. However, when a preantral follicle reached the early tertiary stage (beginning antrum formation), the message signal was very strong, being expressed in all granulosa cells of all such follicles (300-400 microns in diameter). In atretic follicles, follistatin mRNA was localized to granulosa cells, but only during the early stages. The above hybridization pattern of follistatin mRNA in prenatral and atretic follicles appeared constant throughout the estrous cycle. Interestingly, immunohistochemistry studies showed that the follistatin protein was detected only in certain follicles, being restricted to those which were healthy. On the morning of estrus, the follistatin protein was localized to a subpopulation of early tertiary follicles, presumably the dominant follicles selected to ovulate in the next cycle. As the dominant preovulatory follicles matured through diestrus and proestrus, the follistatin mRNA and protein signals appeared more intense in the granulosa cells. After ovulation, the hybridization and immunohistochemical signals continued to be strong in the newly formed corpora lutea on estrus morning. After luteolysis on diestrus-I, neither the follistatin message nor the protein was detectable in the corpora lutea. In conclusion, these results suggest that the follistatin message is present in all the granulosa cells of every developing follicle throughout the estrous cycle, but the follistatin protein appears to be present in only the selected dominant follicles. Accordingly, the possibility that follistatin might be an important regulatory molecule for selection/atresia should be considered.

Hypersecretion of follicle-stimulating hormone (FSH) after ovariectomy of hypophysectomized, pituitary-grafted rats: implications for local regulatory control of FSH.

Previous observations have shown that a portion of the acute (less than 12 h) FSH hypersecretion after ovariectomy (OVX) is LHRH independent, thereby suggesting that mechanisms governing the acute FSH hypersecretory response to OVX may reside largely within the anterior pituitary gland. Accordingly, the present studies were conducted to determine whether acute OVX-induced FSH hypersecretion can be elicited in an animal model in which the anterior pituitary gland is isolated from diencephalic chemical signals, and if so, whether the hypersecretion could be abated by the FSH-suppressing protein, follistatin. Adult female rats hypophysectomized (H) 1 week earlier received anterior pituitary grafts (H/G) (one to three glands per rat) under the kidney capsule. In order to increase ovarian secretion of negative feedback effectors substances (i.e. estrogen, inhibin), some H/G rats were injected sc with 30 IU PMSG 4-6 days after receiving pituitary transplants, whereas other rats were given the saline vehicle. Two days later (0830 h), a blood sample was obtained via an indwelling atrial catheter inserted the previous day. H/G rats given saline or PMSG then were further subdivided and either castrated or sham castrated. Additional blood samples were obtained from the catheter, and trunk blood was collected from decapitated rats 24 h after OVX for measurement of serum estradiol and PRL levels. For comparison, H rats not receiving renal pituitary transplants were subdivided into similar experimental groups as the H/G rats. Blood samples were also obtained after sham OVX or OVX of pituitary-intact, 4-day cycling rats on diestrous day 1. Ovariectomy of PMSG-treated H rats receiving either one or three pituitary allografts resulted in a significant (P less than 0.01) increase in serum FSH levels by 12 h after OVX followed by a 2- to 3-fold increase in FSH levels by 24 h relative to either the pre-OVX FSH levels measured in this group or the FSH levels measured in PMSG-treated H/G rats 24 h after sham OVX. In contrast, OVX of saline-treated H/G rats failed to elicit FSH hypersecretion. Similarly, FSH hypersecretion was not observed after OVX of saline- or PMSG-treated H rats. Whereas serum LH levels were increased 24 h after OVX of diestrous rats, no such increases were detected 24 h after OVX of any H or H/G rats. In an additional experiment, H rats receiving two pituitary allografts were treated with PMSG and subsequently castrated. Twenty-four hours later, rats were injected iv with either 60 micrograms purified porcine follistatin or saline.(ABSTRACT TRUNCATED AT 400 WORDS)

Ovarian intrabursal administration of insulin-like growth factor-binding protein inhibits follicle rupture in gonadotropin-treated immature female rats.

The effects of an insulin-like growth factor-binding protein (IGF-BP) on rat follicular function were examined by using the technique of ovarian intrabursal (IB) injection. Immature female rats were injected with 15 IU of eCG followed immediately with IB injections of 4 micrograms IGF-BP3 (right ovary) and vehicle (left ovary). Forty-eight hours later, the same animals were either killed (eCG-treated group) or injected with 1 microgram of hCG as an ovulatory stimulus. These animals were killed 24 h later (eCG/hCG-treated group). Intrabursal administration of IGF-BP3 inhibited ovulations in the eCG/hCG-treated rats by 55% when compared with the contralateral vehicle-treated ovary (p = 0.01). Examination of the ovaries exposed to IGF-BP3 revealed the presence of unruptured follicles containing a matured oocyte and a disintegrated basement membrane, in addition to normal follicles and corpora lutea. In contrast, IB injection of IGF-BP3 had no effect on ovarian weights or circulating estradiol concentrations in the eCG-treated animals, and the ovaries appeared to be morphologically normal. Ligand blotting experiments using [125I]-labeled insulin-like growth factor I revealed that granulosa cells obtained from both untreated and eCG-treated rats synthesized and secreted two IGF-BPs of Mr 35,000 and 30,000. Equine chorionic gonadotropin treatment reduced the amount of the 30,000 Mr form of IGF-BP. These data suggest that locally produced ovarian IGF-BPs may modulate follicle functions in vivo.

In vivo comparison of the follicle-stimulating hormone-suppressing activity of follistatin and inhibin in ovariectomized rats.

The present study was performed to compare and contrast the effects of two gonadal polypeptides, inhibin and follistatin, on ovariectomy-induced hypersecretion of FSH and LH. Ovariectomies were performed 1 week before study. Follistatin was purified from porcine follicular fluid, and human inhibin A was produced by recombinant DNA technology. On the day of study, a blood sample was taken from intraatrial cannulae inserted on the previous day, the materials were injected, and additional blood samples were taken at various times thereafter. Serum FSH and LH levels were determined by RIA. Both follistatin and inhibin exhibited dose-dependent suppression of circulating FSH but not LH levels, with initial decreases in FSH levels by both materials observed between 2-4 h post injection. Maximal suppression of FSH by each polypeptide occurred between 4-6 h depending on dose. Based on the dose-response relationships, it was determined that inhibin was approximately five times as potent as follistatin in suppressing FSH release. However, despite the greater biopotency of inhibin than follistatin, the duration of action of even the highest dose of inhibin (50 micrograms) was between 4-9 h, whereas the duration of FSH-suppressing activity by the two highest doses of follistatin (40 and 80 micrograms) was between 10-21 h. Data obtained from a second experiment conducted to examine the effects of inhibin and follistatin on anterior pituitary gonadotropin responses to LHRH were consistent with in vitro data showing direct pituitary effects of the gonadal polypeptides. Collectively, these results demonstrate that both purified porcine follistatin and recombinant human inhibin A profoundly suppress serum FSH levels in a dose- and time-dependent manner, with inhibin being more potent in this regard. Whereas the onset of action is similar for the two polypeptides, the duration of action of follistatin is longer than that for inhibin, suggesting, among other factors, different metabolic clearance rates or different pretranscriptional mechanisms of action of follistatin and inhibin.

Novel ovarian regulatory peptides: inhibin, activin, and follistatin.

Based on the extensive amount of research on inhibin and related polypeptides accomplished during the past 5 years, the inhibin concept put forth more than 50 years ago has not only become well established but also more complex than originally imagined. The closed-loop feedback mechanism of ovarian inhibin and pituitary FSH has been joined by possible "inhibin-like" actions of follistatin and FSH-stimulatory effects of activin. In addition, in vitro experiments suggest possible autocrine and paracrine functions for the gonadal polypeptide hormones. Figure 3 shows a simplistic diagram summarizing our current understanding of inhibin/activin and follistatin action along the hypothalamic-pituitary-gonadal axis. Hopefully, research in the coming years will allow us to remove the many question marks still remaining but will undoubtedly add others.

Examination of a possible mechanism by which anisomycin suppresses pulsatile luteinizing hormone release in ovariectomized rats.

An initial study was performed to ascertain the effects of anisomycin, a reversible inhibitor of protein synthesis, on pulsatile luteinizing hormone (LH) release in adult, ovarietomized (OVX) rats. For this experiment, rats OVX 3-4 weeks earlier were fitted with indwelling atrial cannulae. On the next day (approximately 13.00 h), the rats received a subcutaneous injection of either 100 mg/kg body weight (BW) anisomycin or its saline vehicle. Administration of anisomycin significantly suppressed mean plasma LH levels, mean trough values, and both LH pulse frequency (saline: 6.3 pulses/3 h vs. anisomycin: 2.7 pulses/3 h) and amplitude. To determine whether anisomycin affected anterior pituitary LH responses to LH-releasing hormone (LHRH), a second experiment was performed in which saline- and anisomycin-treated OVX rats were given an intravenous injection of 10 ng/100 g BW LHRH 1.5 h later (14.30 h). Rats then were sacrificed and the anterior pituitary and brain removed. Whereas preinjection plasma LH levels were significantly lower in anisomycin-treated rats, they were significantly higher in anisomycin-treated rats 20 min after LHRH. Consequently, mean maximal increments and percent increments were significantly higher in anisomycin-treated rats. AP LH content and content of LHRH in the medial preoptic and suprachiasmatic nuclei were not influenced by anisomycin treatment. However, median eminence (ME) LHRH concentrations in anisomycin-treated rats were almost double the LHRH levels measured in control rats. A third study was conducted to assess the effects of anisomycin on basal and potassium (K+)-stimulated LHRH release from superfused ME explants.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide-Y suppresses pulsatile secretion of luteinizing hormone in ovariectomized rats: possible site of action.

The hypothesis that neuropeptide-Y (NPY) suppresses pulsatile LH secretion in ovariectomized (OVX) rats was examined. Rats were bilaterally OVX and 6 weeks (Exp 1) or 2 weeks (Exp 2) later a stainless steel cannula was implanted in the third cerebral ventricle (3V). Seven to 10 days later, an intraatrial cannula was inserted. The next day, a blood sample was withdrawn, and each conscious unrestrained animal received a 3V injection of synthetic porcine NPY (5 or 0.5 micrograms/2 microliters saline) or vehicle in Exp 1. Blood samples were taken every 10 min for 2 h and centrifuged, and the plasma was analyzed for LH by RIA. In Exp 2, OVX rats received a 3V injection of NPY (5 micrograms/2 microliters) or vehicle. Blood samples were taken before and 60 min after injection. At 60 min, LHRH (10 ng/100 g BW) was injected iv, and blood was withdrawn 10, 20, 60, and 120 min later. NPY caused a dramatic dose-related reduction in the pulsatile release of LH compared to that in vehicle-treated rats. The 5.0-micrograms dose of NPY significantly reduced LH pulse frequency (P less than 0.05), pulse amplitude (P less than 0.01), and trough levels (P less than 0.01) compared to those in saline-injected controls. The lower dose of NPY (0.5 micrograms) significantly decreased the mean LH levels throughout the 2-h sampling period and slightly, though not significantly, the pulse frequency. Administration of LHRH increased plasma LH levels by 124% in control animals and by 1239% in NPY-injected rats. The results of these studies indicate that the suppressive effects of NPY on pulsatile LH release appear to be exerted through inhibition of pulsatile LHRH secretion from the hypothalamus.