Variant Progesterone Receptor mRNAs Are Expressed in Human Endometrium During all Phases of the Menstrual Cycle

OBJECTIVE: The endogenous opioid peptides (EOPs) are important regulators of GnRH pulse frequency. Recent evidence in sheep indicates dynorphin EOP neurons play a major role in the control of GnRH pulse frequency through direct synaptic input to GnRH cells (Goodman et al., Endocrinol., 2003). Dynorphin input to GnRH neurons has not been examined in humans. The goal of this study was to determine if dynorphin terminals directly contact GnRH neurons in the human brain. DESIGN: A descriptive study using dual-label immunocytochemistry to examine close contacts between dynorphin fibers and GnRH cell bodies at a light microscopic level. MATERIALS AND METHODS: Blocks of fixed human preoptic and hypothalamic postmortem tissue were obtained from the New York Brain Bank at Columbia University. The subjects were otherwise healthy premenopausal women. Tissue blocks were infiltrated in sucrose and sectioned coronally on a freezing microtome. Tissue sections were subjected to an antigen retrieval technique involving heating in citrate buffer. Sections were stained using a dual immunoperoxidase procedure with polyclonal antibodies against dynorphin A 1-17 (Pennisula Laboratories) and GnRH (LR-5, gift of Dr. R. Benoit). A total of 27 GnRH cell bodies and dendrites in the preoptic area were examined under brightfield microscopy. Close contacts were defined as direct appositions between dynorphin-positive varicosities and GnRH cell bodies observed in the same plane of focus under 40X magnification. The number of close contacts, and whether they were in contact with GnRH somas or dendrites, were recorded for each cell. In addition, the lengths of dendritic and somal cell surfaces analyzed were recorded. The mean number of contacts per length of somal and dendritic cell surface was statistically compared by two-tailed t-test. RESULTS: We found that 23 out of 27 GnRH cell bodies and dendrites (85.2%) received close contacts from dynorphin-containing varicosities. Multiple close contacts were present 68% of the time; the mean number of contacts per GnRH neuron was 2.47 ⫾ 0.319 (mean ⫾ SEM). Although the total number of contacts observed onto GnRH dendrites was greater than that observed onto somas (p⬍0.002), the average number of contacts per unit length of dendrite (2.19 contacts/100 ␮m) was not different from that onto somas (2.06 contacts/100 ␮m). In addition, there were no obvious regional differences within the preoptic area in the number of close contacts or their location on somas vs. dendrites. CONCLUSIONS: This study revealed the existence of close contacts between dynorphin-positive fibers with GnRH neurons in the human hypothalamus. This provides supportive evidence for the role of dynorphin as a regulator of GnRH pulse frequency in the human female reproductive system. Future studies using confocal and/or electron microscopic analyses will be necessary to confirm that such contacts represent bona fide synaptic contacts. Supported by: NIH T32 HD40135-03 to S.K.D. and NIH R01 HD039916 to M.N.L.

Monday, October 17, 2005 5:15 p.m. O-21 Variant Progesterone Receptor mRNAs Are Expressed in Human Endometrium During all Phases of the Menstrual Cycle. P. B. Marshburn, J. Zhang, Z. Bahrani-Mostafavi, B. S. Hurst, M. L. Matthews, J. Wagstaff. Carolinas Medical Ctr, Charlotte, NC. OBJECTIVE: Recently, variant progesterone receptor (PR) mRNAs with whole and partial exon deletions have been described in human endometrium. These PR variant mRNAs could encode PR-like proteins with the potential to impair progesterone action in the endometrium. For example, the PR variant with deletion of exon 6 (del-6 PR) has been shown to inhibit PR-mediated gene transcription in breast cells. Such PR variants could play a role during normal endometrial development or in abnormalities of endometrial differentiation such as endometrial causes of infertility, implantation failure, and/or endometrial neoplasia. We report the relative expression profile of PR variants with whole or partial exon deletions compared to wild-type (native) PR mRNA expression during normal proliferative and secretory endometrial development. DESIGN: Prospective study; academic community-based hospital. MATERIALS AND METHODS: Patients were 18-40 years and undergoing hysterectomy for benign gynecologic causes. Eighty-one normal endometrial specimens (46 proliferative, 35 secretory) were analyzed, and

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dated as early, mid, and late proliferative and secretory phases. Native PR and PR variant mRNA levels were determined using reverse transcription (RT) - polymerase chain reaction (PCR) assay. RESULTS: Native PR, and splice variants of PR with deletions of exon 4 (del-4 PR), exon 6 (del-6 PR), exons 4 and 6 (del-4&6 PR), partial of exon 4 (del-p4 PR), and partial of exon 6 (del-p6 PR) mRNAs were detected in all endometrial specimens throughout the menstrual cycle. Semi-quantitative analysis showed higher levels of native PR mRNA in the early and mid proliferative endometrial phases than in late proliferative and secretory endometrium (p⬍0.05). The relative expression of all PR variants compared to native PR mRNA was proportionately constant throughout all stages of endometrial development. The ratio of the variant PR with native PR mRNA expression indicated that the relative expression of del-4&6 PR (1%) was significantly lower than the ratios of the del-4 PR (2%) or del-p6 PR (2%) (p⬍02). Southern blot analysis indicated a 967 base pair band that revealed a novel del-partial 4 PR variant. CONCLUSIONS: We present the largest and most systematic study of the expression of mRNA for native PR and PR variants in the human endometrium throughout the menstrual cycle. We found no evidence of differential co-expression of the PR variants assessed compared with native PR during normal endometrial development. Future investigation is required to assess the potential biological significance of the novel del-p4 PR variant. Studies are underway to determine whether a different profile of PR variant expression is detected in the endometrium of patients with infertility, recurrent pregnancy loss, or endometrial adenocarcinoma. Supported by: Health Services Foundation of the Carolinas Medical Center, Charlotte, NC

Monday, October 17, 2005 5:30 p.m. O-22 Hyperglycemia Stimulates Activation of Nuclear Factor ␬B (NF␬B) in Mononuclear Cells (MNC) of Women With Polycystic Ovary Syndrome (PCOS). F. Gonzalez, J. P. Kirwan, J. Minium, H. Gaiser, N. S. Rote. Case Western Reserve University School of Medicine at MetroHealth Medical Center, Cleveland, OH. OBJECTIVE: Activatation of the proinflammatory transcription factor NF␬B culminates in the transcription of tumor necrosis factor-alpha (TNF␣), a mediator of insulin resistance. We have previously shown that in PCOS, hyperglycemia stimulates increased TNF␣ release from MNC. Thus, we examined the effect of glucose challenge on NF␬B activation in MNC of women with PCOS compared to those of ovulatory controls. DESIGN: Prospective controlled study MATERIALS AND METHODS: Eight women with PCOS (4 lean, 4 obese) between ages 18-40, diagnosed on the basis of oligo- or amenorrhea and hyperandrogenemia, and 8 ovulatory controls (4 lean, 4 obese) of similar age were selected for study after excluding diabetes, inflammatory illnesses or other endocrinopathies. A 3-hour glucose tolerance test was performed within 5-8 days of menses. Nuclear-bound activated NF␬B was quantified by an electrophoretic mobility shift assay in MNC isolated from blood samples drawn fasting (pre) and 2 hours after (post) glucose ingestion. Insulin sensitivity was derived by ISOGTT. RESULTS: There were no significant differences in age and body mass index between the women with PCOS and control subjects. Lean women with PCOS exhibited a significantly lower ISOGTT compared to lean controls (5.0⫾0.4 vs. 8.4⫾1.7, p⬍0.04). As expected, a significantly (p⬍0.008) lower ISOGTT was also evident in obese women with PCOS (2.9⫾0.1) and obese controls (3⫾.91.0) compared to lean controls. The mean percent change in MNC-derived activated NF␬B (% ⌬ NF␬B) between the pre- and post-glucose challenge samples of women with PCOS increased significantly compared to that of controls which slightly decreased (91⫾27% vs. -11⫾3%, p⬍0.005). When subjects were grouped by body weight, the mean % ⌬ NF␬B of lean and obese women with PCOS (118⫾50%, 63⫾18%) increased significantly (p⬍0.04) compared to that of lean controls which decreased (-32⫾15%). The mean % ⌬ NF␬B was also significantly greater (p⬍0.03) in obese women with PCOS compared to obese controls (12⫾8%). Finally, % ⌬ NF␬B correlated with BMI in control subjects (r⫽0.81; p⬍0.02) but not in women with PCOS. CONCLUSIONS: These preliminary data indicate that hyperglycemia activates NF␬B in MNC of women with PCOS independent of obesity. We

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