Expression of galanin in human placenta.

The neuropeptide galanin was originally implicated in the regulation of feeding behaviour. Today, galanin is implicated in several physiological functions including reproduction and feeding. Many hypothalamic neurohormones of the hypothalamo--pituitary axis (HPA) are also expressed in the placenta where the specialized topological compartments of the HPA are missing and where paracrine and autocrine regulatory mechanisms consequently prevail. Since galanin influences gonadotrophin-releasing hormone secretion in the HPA, we argued that a similar regulatory role for galanin might exist in human placenta. Since the presence of galanin in human placenta had not been previously reported, we analysed galanin expression in the human placenta by immunohistochemistry and quantitative polymerase chain reaction (PCR) throughout gestation. We found that the peptide hormone localizes to the syncytio- and cytotrophoblast layers; its RNA could be detected. By quantitative PCR we observed that throughout gestation, there is a loss of galanin mRNA which parallels the fall in signal intensity from immunohistochemical detection of the galanin oligopeptide. Furthermore, we detected secretion of galanin from isolated trophoblastic cells. We conclude that galanin may be an important and novel regulator of placental function.

Endogenous regulation of the GnRH receptor by GnRH in the human placenta.

Gonadotrophin releasing hormone (GnRH) plays an important regulatory role in the function and growth of human placenta, but its placental expression sites and co-localization with GnRH receptor (GnRH-R) are not well known. GnRH and GnRH-R expression has been found in both placenta and cultured trophoblasts; however, cultured trophoblastic cells very quickly lose GnRH-R message and subsequently receptor protein. Speculating that endogenously released GnRH induced this down-regulation, we determined GnRH-R in cultures of trophoblastic cells in the presence of a neutralizing anti-GnRH antibody. Cells incubated with this antibody showed a strong signal for the GnRH-R, while those cultured without did not (as analysed by immunofluorescence, reverse transcription-polymerase chain reaction, protein 'dot blot' and Western blotting). Furthermore, addition of the GnRH agonist buserelin led to a reduction of the receptor protein. We have therefore shown that GnRH released from trophoblastic cells down-regulates GnRH-R in these trophoblastic cells. Having previously shown that trophoblast layers were synchronously positive for GnRH and GnRH-R, these new findings support the hypothesis of an ultrashort feedback regulation of trophoblasts by GnRH involving autocrine regulation of GnRH-R.

Reproductive outcome of 32 patients with primary or secondary infertility and uterine pathology.

We did a retrospective study on 102 patients who had a laparoscopy and a hysteroscopy during investigations for primary or secondary infertility. 32 of the 102 patients had uterine pathology. Seven of them had septate uteri, eight had uterine synechiae, another six had uterine fibroids, four had a bicornuated uterus, while the remaining had either a combination of all or other uterine anomalies. After surgical treatment of these conditions ten women conceived and five pregnancies including one twin pregnancy resulted in term deliveries.

Analysis of ultrashort feedback regulation in human placenta: synthesis and secretion of GnRH by human trophoblastic cells.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) presumably controls placental growth and functions by autocrine/paracrine mechanisms, and is therefore an important part of the neuroendocrine network in human placenta. AIM: Our earlier work had indicated that GnRH was expressed in human placenta; in extension to these findings, we wanted to analyse synthesis and release of GnRH by trophoblastic cells. GnRH-associated peptide, co-linearly synthesised with GnRH, was used as indicator of actual peptide synthesis. METHOD: First, we immunised rabbits with lipopeptides containing partial sequences of GnRH-associated peptide (GAP) and developed antibodies for immunohistochemical staining. Second, we set up a competitive enzyme immunoassay to measure GnRH: Non-biotinylated GnRH, GnRH analogues or trophoblastic cell culture supernatants were used to inhibit binding of biotinylated des-pGlu1-GnRH to a monoclonal anti-GnRH antibody. RESULTS: a) Placental sections stained positive for GAP in the layers of trophoblastic cells. b) GnRH could be detect by a competitive EIA in supernatants of placental cultures in concentrations between 200 and 5 nM. CONCLUSIONS: GnRH is synthesised and released by trophoblastic cells.

The distribution of progesterone receptor immunoreactivity and mRNA in the preoptic area and hypothalamus of the ewe: upregulation of progesterone receptor mRNA in the mediobasal hypothalamus by oestrogen.

The distribution of progesterone receptors (PR) was mapped in the hypothalamus of the ewe using immunocytochemistry. These results were confirmed using in situ hybridization with a sheep-specific 35S-labelled riboprobe. In addition, the effect of oestrogen on the level of PR mRNA in the hypothalamus was examined in ovariectomized (OVX) ewes following treatment with an oestrogen implant or without treatment. PR immunoreactive (-ir) cells were readily detected in OVX animals. Labelled cells were observed in four main hypothalamic regions: the preoptic area (POA), including the organum vasculosum of the lamina terminalis, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and the arcuate nucleus (ARC) (including the region ventral to the mamillary recess). In addition, lightly stained PR-ir cells were observed in the supraoptic nucleus and a few PR-ir cells were also found in the diagonal band of Broca. No PR-ir cells were found in the brainstem. PR mRNA-containing cells were found in the same hypothalamic regions as the PR-ir cells. Image analysis of emulsion-dipped slides following in situ hybridization indicated that oestrogen treatment increased (P<0.01) the mean number of silver grains/cell and the density of labelled cells in the VMN and ARC but had no effect on the level of PR mRNA expression in the POA or PeN. The distribution of PR-containing cells in the hypothalamus is similar to that described in other species and all cells were located in nuclei that contain large populations of oestrogen receptor-containing cells. These include regions implicated in the regulation of reproductive neuroendocrine function, and reproductive behaviour. Oestrogen and progesterone synergize to inhibit GnRH secretion and the present results suggest that these functions may involve cells of the VMN and ARC, with oestrogen acting to upregulate PR.

Subjective evaluation of the therapeutic value of laparoscopic adhesiolysis: a retrospective analysis.

BACKGROUND: Adhesions are believed to be one of the principal causes of chronic pelvic pain. Although there may be some discrepancy between the degree of adhesions and the severity of the symptoms, surgical adhesiolysis is still considered to be useful for the relief of pain. METHODS: A total of 187 patients who underwent laparoscopic adhesiolysis at the Medical University of Ulm, Germany, within a 2-year period were asked to rank their discomfort on a visual pain scale before surgery and up to 1 1/2 years postoperatively. RESULTS: In this retrospective study, we found that nearly one-third of patients suffered from functional irritations that were either ameliorated or completely relieved by laparoscopic adhesiolysis. When other causes of chronic pain (such as endometriosis) are excluded, the results show that most patients benefited from laparoscopic adhesiolysis. CONCLUSIONS: It appears that laparoscopic adhesiolysis is an effective therapeutic measure to relieve chronic pelvic pain. Therefore, adhesiolysis should be performed in all patients with chronic or intermittent pain, and a complete lysis of adhesions should be planned. However, since pelvic pain may have organic or functional causes other than adhesions, complete adhesiolysis in patients with persistent pelvic pain may be of only limited importance.

Expression and functional analysis of endothelial nitric oxide synthase (eNOS) in human placenta.

We have investigated the expression and localization of endothelium-derived nitric oxide synthase (eNOS) and the effect of eNOS on placental human chorionic gonadotrophin (HCG) release. eNOS mRNA was found to be expressed in all tissues, with its expression significantly (P<0.05) increased across gestation. Compared to normal term gestation, placentae from term pregnancies with fetal retardation, or maternal diabetes, but not with maternal hypertension, displayed significantly more (P<0.05) eNOS mRNA. By immunocytochemistry, we found staining for eNOS in both the cyto- and syncytiotrophoblasts of first trimester and a loss of cytotrophoblast eNOS staining in term placentae, while syncytiotrophoblasts at term were strongly eNOS positive. Additional staining was found in endothelium surrounding the vascular tree. HCG was found to colocalize with eNOS in trophoblasts, but not in endothelia. When placental explants were perifused, exposure to the NOS substrate, the NO donor, I-arginine and trinitroglycerol evoked a prompt, albeit transient, increase of HCG release. The NOS inhibitor delayed, but did not block arginine-induced HCG release. Thus, eNOS is expressed in the human placenta at increasing levels during gestation with further increases during some pathological conditions. A role for NO in the acute endocrine modulation of the placenta is suggested by the colocalization of eNOS with HCG in human trophoblasts and the prompt secretion of HCG in response to agents which increase NO concentrations.

Effects of chronic opioid antagonism on gonadotrophin and ovarian sex steroid secretion during the luteal phase.

OBJECTIVES: Since short-term opioid antagonism increases LH pulsatility during the luteal phase in women, we postulated that prolonged opioid antagonism may also accelerate the LH secretory episodes at this time. If so, the functional and temporal links between secretory episodes of pituitary LH and oestradiol (E2) and progesterone (P) release from the mature human corpus luteum may be disrupted. STUDY DESIGN: Prolonged opioid blockade with the oral antagonist naltrexone (100 mg daily) was effected in eight women during the entire luteal phase of their cycles. Following documented ovulation in both placebo (control) and naltrexone cycles, blood samples were obtained daily and frequently (every 10 minutes for 10 h) on days 6-8 after ovulation. MEASUREMENTS: In all blood samples, LH, E2 and P were determined by IRMA. RESULTS: Compared to control cycles, the temporal organization and the endocrine characteristics of the luteal phase remained virtually unchanged during chronic opioid blockade. Periodic fluctuations were detected (by cluster analysis) in LH, E2 and P data series established by frequent sample collections in both the control and naltrexone cycles. LH secretory profiles were remarkably similar during control and naltrexone cycles, and the E2 and P secretory episodes tended to be coupled to LH pulses during both cycles. As determined by time-series analysis, the cross-correlations between the LH/E2 and LH/P data series remained unaltered by opioid blockade. CONCLUSIONS: Chronic opioid antagonism with naltrexone did not disrupt the temporal organization or endocrine characteristics of the luteal phase. In particular, prolonged opioid blockade did not change LH secretory patterns. The functional and temporal links between LH inputs and sex steroid release from the mature corpus luteum remained unaffected by prolonged opioid antagonism. In contrast to the effects of short-term opioid blockade on LH pulsatile release during the luteal phase, the effects of chronic opioid antagonism on LH release may be transient and may not persist throughout the entire luteal phase, suggesting desensitization of the opiate receptors.

Detection of gonadotrophin releasing hormone and its receptor mRNA in human placental trophoblasts using in-situ reverse transcription-polymerase chain reaction.

Neuropeptides such as gonadotrophin releasing hormone (GnRH) are presumed to play an important role in the regulation of the function and growth of human placenta. Knowledge about the placental site of GnRH expression and the eventual co-localization of its peptide with the GnRH receptor (GnRH-R) is crucial for a better understanding of possible autocrine/paracrine mechanisms. We therefore investigated these questions by use of in-situ reverse transcription-polymerase chain reaction (RT-PCR) alone or in combination with immunocytochemistry in human first and third trimester placentae. Paraffin-embedded placental sections (7 microm thick), or single trophoblasts in monolayer cultures for up to 3 days, were treated with proteinase K. Following RT with GnRH or GnRH-R specific oligoprimers, PCR was performed employing primers with exon-exon overlaps to exclude non-specific DNA amplification. Detection of the amplicons was accomplished by nested PCR which was performed with digoxigenin-labelled dUTP and nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP) for substrate visualization. The GnRH peptide was detected using a sandwich-antibody assay. GnRH and GnRH-R gene expression was found in all first and third trimester placentae, with abundant signals for the GnRH and GnRH-R message both in the cyto- and syncytiotrophoblasts. Single trophoblasts of different gestational ages in culture also displayed GnRH expression in individual cytotrophoblasts and in syncytiotrophoblast-like fusionates. Additional immunostaining revealed GnRH peptide to be co-localized with GnRH-R message in trophoblast layers. Since messages for GnRH and GnRH-R were found in virtually all trophoblasts, we infer that GnRH and GnRH-R are co-expressed in identical cells. These data strongly suggest that the trophoblasts are the source of GnRH, and that there is autocrine/ paracrine regulation by GnRH in human placenta.

[Iatrogenic androgenization]. / Iatrogene Androgenisierung.

Virilization in postmenopausal women is suspicious for androgen-secreting adrenal or ovarian tumors; however, iatrogenic androgenization needs to be additionally considered. Here we report on a 64-year-old patient who presented clinically with progressive signs of virilization. An adrenal source of androgen excess was excluded, and the patient strictly denied the use of any androgenic medication. Thus, elevated serum levels of testosterone were suspicious of ovarian hyperandrogenism. Shortly before planned surgical exploration, the clinical finding of an extensive vulvar lichen sclerosus pointed towards a possible long-term use of testosterone-containing cremes for symptomatic relief of this disease. Apparently, the patient did not consider the mere topical application of potent agents to be a medication. This case demonstrates that besides adrenal or ovarian sources of hyperandrogenism, iatrogenic androgenization has to be considered.

Laparoscopic management of ovarian tumors.

BACKGROUND: Laparoscopy can be used with minimal operative morbidity to evaluate adnexal masses. We report our experience with the endoscopic approach to the diagnosis and treatment of ovarian tumors. In particular, we describe 11 patients who incidentally underwent laparoscopy and in whom the ovarian masses were found to be malignant. METHODS: Between September 1994 and September 1996, 292 patients with 316 ovarian tumors were treated laparoscopically in the Department of Obstetrics-Gynaecology, University of Ulm. We assessed vaginal ultrasonography, clinical assessment, the tumor marker CA 12-5, and the intraoperative low-power magnification for their value in predicting the final diagnosis in all laparoscopically treated ovarian tumors. RESULTS: From a total of 292 patients with ovarian tumors, 11 were diagnosed, intraoperatively or after final histologic examination, as having a malignant or borderline ovarian tumor. All applied pre- and intraoperative diagnostic procedures were by themselves too unreliable to exclude early stages of ovarian carcinoma exactly. CONCLUSIONS: On the basis of the present findings, we are tempted to conclude that laparoscopic surgery is justified in the management of ovarian tumors. Even with an accurate preoperative selection of suitable patients for laparoscopic surgery, the presence of an undetected ovarian carcinoma cannot be entirely excluded.

Is opioidergic activity responsible for the circadian variation observed in the gonadotrophin responsiveness of early follicular phase women?

OBJECTIVES: In women, the gonadotrophin response to gonadotrophin-releasing hormone (GnRH) displays a circadian rhythm during the early follicular phase (EFP), with GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release found to be markedly decreased at night. Since the opioidergic inhibition of gonadotrophin secretion is selectively enhanced at night, we reasoned that the circadian changes in the gonadotrophin responsiveness to GnRH might be related to a nocturnal increase of opioidergic activity. STUDY DESIGN: Eleven women with normal menstrual cycles were studied in the EFP on four different occasions in random order. Studies were conducted either during the day (0900-1300 h) or at night (2100-0100 h). During these times, GnRH (25 micrograms i.v.) was administered in conjunction with either saline (as control) or naloxone (4 mg i.v.). MEASUREMENTS: Frequent blood samples were obtained before and after GnRH stimulation for determination of basal sex steroid and gonadotrophin concentrations by immunoradiometric assays. RESULTS: While oestradiol levels were comparable (P > 0.3) at all times, progesterone concentrations were significantly (P < 0.01) higher during day than during night hours, with no difference between control and naloxone conditions. Gonadotropin responses to GnRH stimulation were not significantly different between day and night times, nor did they vary between control and naloxone conditions. CONCLUSIONS: Opioidergic blockade imposed by naloxone did not noticeably change GnRH-stimulated gonadotrophin release at any of the study times. We therefore infer that mechanisms other than a nocturnal increase of opioidergic inhibition may account for eventual circadian changes in the gonadotrophin responsiveness of early follicular phase women.

The impact of sleep on gonadotropin secretion.

Comparable to the period of pubertal transition, sleep also exerts profound effects on episodic gonadotropin secretion in adult women. During the early follicular phase of the menstrual cycle, a sleep-induced slowing of luteinizing hormone (LH) secretion occurs concurrently with a rise in LH pulse amplitude. A selective increase in opioidergic, but not in dopaminergic or serotoninergic activity may account for this decline in LH pulsatility. In addition, sleep-reversal studies have confirmed that the presence of sleep is essential for the expression of this neuroendocrine function. Since pituitary gonadotropin responsiveness to gonadotropin-releasing hormone (GnRH) is virtually unchanged during sleep, the reasons for the enhanced LH pulse amplitude remain unresolved. This sleep-associated increase in opioidergic activity may be restricted to a hypothalamic site, since opiate blockade does not modify the gonadotropin response to GnRH stimulation. In addition, circadian variability is shown in terms of gonadotropin secretion in regularly cycling women; this may again represent sleep-associated effects on gonadotropin release. Although the physiological importance of sleep-associated neuroendocrine phenomena remains basically unexplained, the observed changes in LH secretory profiles during sleep in adult women suggest close functional links between the endocrine secretion and the rest-activity cycle of the brain.

A comparative randomized trial on the impact of two low-dose oral contraceptives on ovarian activity, cervical permeability, and endometrial receptivity.

In a double-blind randomized study, the suppression of ovarian activity and anti-conceptive effects on the cervix and endometrium were assessed during administration of two low-dose monophasic oral contraceptives (20 micrograms ethinyl estradiol [EE], 500 micrograms norethisterone--Eve 20 [Grünenthal, Aachen, Germany]; 20 micrograms EE, 150 micrograms desogestrel --Lovelle [Organon, Munich, Germany]). One hundred eighteen healthy women (ages: 18-35 years) were studied in 10 investigation centers during medication with either Eve 20 (n = 59) or Lovelle (n = 59). During three treatment cycles, ovarian activity was evaluated by sonographic determination of follicle-like structures (FLS) and by simultaneous assessment of serum endocrine profiles (gonadotropins LH and FSH, ovarian steroids estradiol [E2] and progesterone [P]). While on either treatment, no ovarian activity (as judged by no FLS and/or reduced sex steroid levels) was found in 90.8% (Eve 20) and 97.2% (Lovelle) of all investigated cycles. Follicular activity or cyst formation were detected in 18 of 173 cycles (Eve 20) and in 5 of 175 cycles (Lovelle), respectively. Gonadotropin levels were suppressed (LH < 6 IU/L, FSH < 8 IU/L) in most treatment cycles (Eve 20 76.6% vs. Lovelle: 84.8%). Serum E2 concentrations exceeding 0.1 nmol/L indicated residual follicular activity in 19.3% (Eve 20) versus 12.2% (Lovelle) of all cycles. An estimated by serum P levels over 5 nmol/L, ovulation had presumably occurred in 4.1% (Eve 20) versus 2.9% (Lovelle) of treatment cycles. However, when the sonographical and endocrinological data were combined, no ovulation was documented in any pill cycle. The quality and quantity of the cervical mucus was found to be minimal in the majority of women. Moreover, the endometrial layer was determined to be low by ultrasound during most pill cycles, indicating equally strong suppressive effects on endometrial receptivity by the two contraceptives. These observations suggest that ovarian activity is suppressed in the majority of cycles during use of low-dose contraceptives. This effect may mainly be medicated by pronounced suppression of serum gonadotropin levels. Strong anti-conceptive effects of these formulations on both cervical permeability and endometrial receptivity are additional factors ensuring the contraceptive efficacy of these formulations.

PIP: The impact of two low-dose monophasic oral contraceptives (OCs) on suppression of ovarian activity, cervical permeability, and endometrial receptivity was investigated in a randomized double-blind study involving 118 healthy women 18-35 years of age recruited from 10 study centers in Germany. 59 women received Eve (20 mcg of ethinyl estradiol and 500 mcg of norethisterone) and 59 were given Lovelle (20 mcg of ethinyl estradiol and 150 mcg of desogestrel) for a total of 3 cycles. No ovarian activity, as assessed by sonographic determinations of follicle-like structures and serum endocrine profiles, was detected in 90.8% of cycles of Eve users and 97.2% of cycles in the Lovelle group. Follicular activity or cyst formation was found in 18 of 173 cycles of Eve users and 5 of 175 cycles of Lovelle users. Gonadotropin levels were suppressed (luteinizing hormone under 6 IU/L and follicle-stimulating hormone less than 8 IU/L) in 76.6% of treatment cycles in the Eve group and 84.8% of cycles in the Lovelle group. Serum estradiol concentrations exceeding 0.1 nmol/L, indicative of follicular activity, were recorded in 19.3% of cycles of Eve users and 12.2% of cycles in the Lovelle group. Although serum progesterone levels were over 5 nmol/L in 4.1% of cycles in the Eve group and 2.9% of those in the Lovelle group, consolidation of sonographic and endocrinologic data failed to document ovulation in any treatment cycles. The quantity and quality of cervical mucus was minimal in most women in both groups. Finally, the endometrial layer was determined to be low by ultrasonography during most pill cycles, confirming the OCs' equally strong suppressive effects on endometrial receptivity.

Laparoscopic extirpation of an aplastic ectopic uterus in a patient with Mayer-Rokitansky-Küster-Hauser syndrome.

The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome comprises the combined hypoplasia of the vagina and the uterus. In recent years, variable anomalies of the development of the Mullerian duct [as classified by the American Fertility Society (AFS)] have been operated on by the laparoscopic approach. We describe here a laparoscopic extirpation of an aplastic ectopic uterus in a patient found to have a hypoplastic vagina and ectopic uterus attached to the right pelvic wall. The uterus was linked to a normotopic right Fallopian tube and right ovary which were covered with endometriosis and adhesions. While the uterine vessels and the round ligament could be demonstrated on the right side, these structures were missing on the contralateral left side of the uterus. However, a normal tube and ovary were present on the left pelvic wall. Although not explicitly described in the AFS classification, this constellation most likely corresponds to the AFS classification stage Ie.

[Ovarian activity, cycle behavior and tolerance of low dosage oral contraceptives: a comparative study]. / Ovarielle Aktivität, Zyklusverhalten und Verträglichkeit bei niedrigdosierten oralen Kontrazeptiva: Eine Vergleichsstudie.

In a double-blind randomized study, the suppression of ovarian activity, the effects on the cervix and endometrium, menstrual bleeding patterns and overall tolerance were assessed during administration of two low-dose oral contraceptives (20 micrograms ethinylestradiol EE, 500 micrograms norethisterone--Eve 20, Grünenthal, Aachen; 20 micrograms EE, 150 micrograms Desogestrel--Lovelle, Organon, Munich), 118 healthy women (ages: 18 to 35 years) with comparable bioprofiles (height, weight, menstrual cycle patterns) were studied in 10 investigation centres during medication with either Eve 20 (n = 59) or Lovelle (n = 59). During 3 treatment cycles, ovarian activity was evaluated by sonographic determination of follicular size and by simultaneous assessment of serum endocrine profiles (gonadotropins LH and FSH, ovarian steroids estradiol [E2] and progesterone [P]). Treatment cycles 4 to 6 served to evaluate the patterns of menstrual bleeding and the overall subjective tolerance on each contraceptive. While on the preparations, no ovarian activity (as judged by a lack of follicular growth and suppressed sex steroid levels) was found in over 90% of all investigated cycles. Follicular activity and/or cyst formation were detected in 18 of 173 cycles (Eve 20) and in 5 of 175 cycles (Lovelle) respectively. Gonadotropin levels were suppressed (LH < 6 IU/l, FSH < 8 IU/l) in most treatment cycles (Eve 20: 76.6% vs. Lovelle: 84.8%). Serum E2 concentrations exceeding 0.1 nmol/l indicated residual follicular activity in 19.3% (Eve 20) vs. 12.2% (Lovelle) of all cycles. As estimated by serum P levels over 5 nmol/l, ovulation had presumably occurred in 4.1% (Eve 20) vs. 2.9% (Lovelle) of treatments respectively. However, when the sonographic and endocrinological data were combined, no ovulation was documented in any treatment cycle. In addition, the quality of the cervical mucus was minimal and a low endometrial thickness was found in the majority of women, indicating strong progestogen effects of both contraceptives. Menstrual irregularities (intermenstrual spotting, break-through bleeding) occurred initially on each preparation, but were mostly resolved when the pills were continued. The acceptance of each investigated drug was rated as very good or good by most subjects. These observations allow us to conclude that the rate of ovarian suppression with inhibition of follicular activity is high under low-dose oral contraceptives. The different progestogens as components of these contraceptive pills display equally good anti-conceptive effects on both the cervix and the endometrium. Furthermore, the rate of irregular menstrual bleeding is acceptable for these low-dose contraceptives. The high acceptance of each preparation suggests that such agents will have a high rate of acceptability in clinical use.

The demonstration of progesterone, but not of estrogen, receptors in the developing human placenta.

Since presence of steroid receptors in the human placenta has been the subject of dispute, we have investigated the existence of estrogen (ER) and progesterone (PR) receptors in trophoblasts across gestational age by a variety of different techniques. Fresh human placental tissue of trimesters 1 to 3 was paraffin-embedded or snap-frozen (-80 degrees C) and sliced (5 microns). Other tissue fragments from identical placentae were dispersed and incubated in monolayer cultures for up to 5 days. Immunocytochemistry (ICC) was performed for ER and PR in both trophoblast cells in culture and in whole tissue slices, using the sandwich antibody technique with subsequent horse-radish peroxidase reaction for colorization. In addition, long-term perifusion studies were conducted with explants of term placentae, using perifusion medium with estradiol (E, 2 ng/ml) and/or progesterone (P, 200 ng/ml). Perifused explants were then subjected to further ICC staining. Furthermore, RT-PCR for both ER and PR mRNA was performed for detection of the gene products in placentae of different gestational ages. Lastly, binding studies with iodine or tritium-labeled E and P were conducted on cytosol fractions. In placental sections and cultured trophoblasts, PR was clearly demonstrable in all placentae across different gestational ages. Abundant PR signal was found adjacent to the nuclei, and additionally in the dendrite-like pseudopods of syncytiotrophoblast cells. In contrast, no such staining signal was detected for the ER; this finding applied under all conditions investigated and at all gestational ages. Again, no staining for ER by ICC was detected in any tissue after perifusion with sex steroids. RT-PCR revealed no product for ER, but only for PR, in placentae across all gestational ages. Binding studies with labeled E and P showed no binding for either compound. Taken together, these observations suggest the presence of PR, but not of ER, in human placenta throughout gestation. Our failure to detect the ER does not entirely preclude the presence of this receptor in human trophoblasts, but might be attributed to a relatively low number and density of ER on these cells. Alternatively, estrogen's action on the placenta may be mediated by a different type of ER, such as by a non-classical membrane-bound receptor.

Galanin gene expression in hypothalamic GnRH-containing neurons of the rat: a model for autocrine regulation.

Increasing experimental evidence has accumulated to suggest that galanin may be one of the essential regulators in the reproductive system of the rat. Galanin is a polypeptide which has been found in hypothalamic neurons, where it is colocalized with GnRH. GnRH-containing cells begin to express detectable levels of galanin mRNA at the onset of puberty, and this process is dependent on the presence of the gonads. This indicates that the induction of galanin expression in GnRH neurons is an important requirement for the maturation of the reproductive axis. The pattern of co-expression of galanin and GnRH in hypothalamic neurons is sexually dimorphic, with much greater galanin mRNA content in the GnRH neurons of females compared to males. The role of galanin may therefore be important in reproductive processes unique to the female, such as the LH surge and ovulation. During the estrous cycle, galanin mRNA levels are enhanced in GnRH neurons at the time of the LH surge. This suggests a tight coupling of galanin biosynthesis and release to the activity state of the GnRH neurons. Expression of the galanin gene in GnRH neurons is regulated by ovarian sex steroids: estradiol appears to support the basal expression of galanin mRNA in GnRH neurons. In addition, estradiol is capable of dramatically increasing galanin gene expression, either when administered alone or in conjunction with progesterone. Nevertheless, neuronal activation with concomitant induction of galanin gene expression requires transsynaptic signals by afferent inputs impinging on the GnRH cells. Galanin may serve as a critical autocrine regulator for the activity of GnRH cells, in that it may be co-released with GnRH and act presynaptically on the GnRH neuron, helping to shape GnRH release into distinct pulses. Collectively, these observations indicate that galanin gene expression in GnRH neurons may represent a pivotal signal for the activation of the neuroendocrine reproductive axis.

Inhibition of steroid-induced galanin mRNA expression in GnRH neurons by specific NMDA-receptor blockade.

Galanin mRNA levels in GnRH neurons increase in association with a steroid-induced LH surge in female rats. Both the steroid-induced LH surge and the concomitant increase of galanin mRNA in GnRH neurons are blocked by non-specific inhibition of central nervous system activity imposed by pentobarbital and specific central alpha-adrenergic receptor blockade. Based on these observations, we hypothesized that galanin gene expression in GnRH neurons is induced whenever GnRH neurons become activated to generate an LH surge. If this were the case, then any neurotransmitter receptor blocking agent that inhibits the LH surge by central mechanisms would likewise block the associated increase in galanin mRNA in GnRH neurons. We tested this hypothesis by examining the effects of an N-methyl-D-aspartate (NMDA) receptor antagonist on the steroid-induced LH surge and on levels of galanin mRNA in GnRH neurons. Three groups of ovariectomized rats were used: Group 1 -treated with estradiol and progesterone (E/P) and sacrificed at the peak of the LH surge; Group 2-treated the same as Group 1 except that dizocilpine (MK801, an NMDA receptor antagonist) was used to block the LH surge; and Group 3-treated the same as Group 1 except they received vehicle instead of E/P. Double-and single-label in situ hybridization followed by computerized image analysis were used to measure levels of galanin mRNA and GnRH mRNA in GnRH neurons [as grains/cell (g/c)]. E/P treatment induced a 3-fold increase in LH levels and a 5-fold increase in the galanin mRNA signal content of GnRH neurons. Treatment with MK801 completely prevented the LH surge in all animals and also blocked the steroid-induced increase in galanin mRNA in GnRH neurons. As assessed by 2 independent GnRH single-labeled assays, neither GnRH message content nor the number of identifiable GnRH neurons differed among the experimental groups. We conclude that the increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a steroid-evoked LH surge, and we infer that induction of galanin gene expression in GnRH neurons is induced as a consequence of synaptic activation of GnRH neurons.