The objective of this study was to detect and characterize a secreted oviduct-specific glycoprotein (OGP) in the rhesus macaque (Macaca mulatta) and to compare the characteristics of this OGP to those previously characterized in baboons and women. Oviducts were obtained from untreated ovariectomized rhesus and from ovariectomized rhesus either treated with estradiol (E2) for 14 days or treated sequentially with E2 for 14 days and then with E2 plus progesterone (P4) for an additional 14 days. Segments of oviducts were either fixed for morphological analysis, cultured for OGP synthesis and release, or frozen for RNA analysis. The proteins present in the culture media were separated by one-dimensional SDS-PAGE, and OGP was detected on Western blots using polyclonal antibodies generated against the reduced form of baboon OGP or a 17-amino acid segment of the baboon core protein. Cross-reacting antigens were present in the 120-kDa region, identical to what was observed for baboon and human OGP. Indirect immunogold localization of OGP on thin sections demonstrated specific clustering of gold particles over the apical secretory granules of the secretory cells of the oviductal epithelium. A cDNA was generated using RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE), and sequenced. The total transcript was 2237 nucleotides in length plus a poly(A) tail. The largest open reading frame was 624 amino acids, which would produce a protein of 69.3 kDa. The nucleotide sequence was more than 95% identical to the nucleotide sequences of baboon and human OGP. Northern blots revealed a single message at 2.4 kilobases (kb) in oviduct samples obtained from E2-treated rhesus. This message was absent in oviducts obtained from untreated ovariectomized and from sequential E2 plus P4-treated rhesus macaques. In summary, the rhesus oviduct synthesizes and secretes an OGP in the presence of E2 that is immunologically and structurally similar to the baboon and human OGP. The presence of a highly homologous glycoprotein in several primates suggests a similar function for OGP in the reproductive process.
We have found that microwave (MW) stabilization greatly improves detection of the estrogen receptor (ER) in frozen sections of rhesus monkey oviduct by immunocytochemistry (ICC). Fresh samples of fimbriae were MW-irradiated, frozen, and then cryosectioned. The frozen sections were also MW-treated and then fixed in a paraformaldehyde-based fixative before ICC processing. A parallel set of samples from each monkey were frozen, sectioned and processed for ICC without any MW treatment. MW stabilization clearly increased immunostaining intensity with either of two ER-specific monoclonal antibodies, namely, H222 and 1D5. The greatest increase was noted in tissues collected from spayed or progesterone-treated animals. An antibody dilution series indicated that MW stabilization increased the sensitivity approximately 20- to 40-fold. In addition, we incubated spayed macaque fimbriae at 4 C in the presence of 10 nM [3H]Moxestrol and then either froze the tissues immediately (non-MW) or treated them with MW. Slide-mounted cryosections of non-MW and MW-treated tissue were then incubated with either a Tris-EDTA buffer (low salt) or the same buffer containing 4 M KCl (high salt). The quantity of [3H]Moxestrol-occupied ER extracted from the frozen sections by each buffer was determined by a sucrose gradient shift assay. The low salt buffer extracted significantly more radiolabeled ER from non-MW sections than from MW-treated sections (P < 0.01), whereas the high salt buffer extracted equal amounts of ER from both the MW-treated and non-MW sections. MW-irradiation enhanced ICC detectability of ER in frozen sections by greatly reducing the amount of ER extracted during the various washes used during normal ICC processing.
Experiments were conducted to examine the effect of a cyclopropenoid fatty acid (CPFA) on progesterone (P4) production by the ovine corpus luteum (CL) during the estrous cycle. Ewes in Exp 1 and 2 were laparotomized on day 2 of the estrous cycle, and animals with CL in both ovaries were subjected to unilateral ovariectomy. Ewes with CL in one ovary only were not ovariectomized. During surgery, ewes were injected with a mixture of fatty acids (sterculic acid, 39%; palmitic, 29%; linoleic, 12%; malvalic acid, 9%; oleic, 8%; stearic, 3%) containing 500 micrograms sterculic acid (SA; Exp 1), 750 micrograms SA, or 750 micrograms oleic acid (Exp 2) via the artery supplying the ovary bearing the CL. Control ewes were similarly injected with vehicle only (0.1-0.2 ml dimethylsulfoxide; Exp 1 and 2, respectively). Sera from blood samples collected at 15-min intervals for 1 h after injection or once daily on alternate days of the cycle after surgery were analyzed for LH and P4, respectively. In Exp 3, slices of CL removed from five ewes on day 10 of the cycle were incubated for 90 min in medium containing 100 ng/ml SA or vehicle (10 microliters dimethylsulfoxide). Slices were then reincubated for 90 min in medium containing 10 ng/ml oLH or saline (10 microliters). Tissue and medium were analyzed for P4. Injection of 500 micrograms SA suppressed serum levels of P4 (P less than 0.01), but did not alter mean cycle length. Injection of 750 micrograms SA reduced serum concentrations of P4 and shortened estrous cycle duration (P less than 0.005). Oleic acid (750 micrograms) or as much as 1.9 mg of a mixture of fatty acids devoid of CPFA had no effect on cycle length or serum levels of P4, suggesting that altered luteal function was due to the type and not the quantity of fatty acid injected. Treatments had no effect on serum concentrations of LH. Preincubation with SA interfered with the ability of luteal slices to synthesize P4 when subsequently incubated alone or with ovine LH (P less than 0.01). It is concluded that SA acts on the CL to impair steroidogenesis and ultimately cause luteal regression.
The present study was conducted to determine if progesterone (P4) would inhibit oxytocin-stimulated phosphoinositide hydrolysis in COS-7 cells expressing transfected ovine oxytocin receptor (OTR) with little or no nuclear P4 receptor (nPR) protein present. The relative absence of nPR in these cells was confirmed by immunocytochemistry and RT-PCR. To investigate the effects of P4 on oxytocin (OT) signaling, cells were transiently transfected with the ovine OTR. Radioreceptor assay for [ 3 H]-OT binding confirmed the presence of a high affinity binding site for OT in transfected cells, while treatment with P4 and GTPγS (which uncouples the OTR from the heterotrimeric G-protein) increased the K d for OT binding slightly. Cells were then assayed for inositol phosphate hydrolysis 48 h post-transfection. Pre-treatment of cells with P4 for 10 min significantly interfered with rapid (20 min) OT-stimulated inositol trisphosphate (IP 3 ) production. This inhibition was specific to P4, because pre-treatment of cells with promegestone (R5020), testosterone, mifepristone (RU 486), or cortisol did not decrease OT-stimulated IP 3 levels. By radioreceptor assay for PR, no measurable specific binding of R5020 was observed for either transfected or nontransfected cells. We conclude that P4 can inhibit OTR-mediated phosphoinositide hydrolysis in COS-7 cells that express little or no nPR protein. These data support a role for a nongenomic action for P4 in OTR signaling via some mechanism other than by binding to a membrane progestin receptor in an immortalized, transfected cell.
BackgroundOvarian hyperstimulation syndrome (OHSS) is a disorder associated with elevated serum VEGFA following chorionic gonadotropin (hCG) exposure in controlled ovarian stimulation (COS) cycles in women. In this study, we tested the effect of intravenous VEGFA neutralization on OHSS-like symptoms and vascular function in rhesus macaques during COS cycles.MethodsMonkeys (n = 8) were treated with 3 COS protocols and assigned randomly to groups as follows: 1) COS alone (Control, n = 5); 2) COS + VEGF mAb Avastin 19 ± 5 h before hCG (Avastin pre-hCG; n = 6); 3) COS + Avastin 3–4 days post-hCG (Avastin post-hCG; n = 4); 4) COS + Simulated Early Pregnancy (SEP n = 3); or 5) COS + SEP + Avastin (SEP + Avastin n = 3). Follicles were aspirated 36 h post-hCG, fluid was collected from one follicle for analysis of steroid and vascular hormone content. Remaining follicles were aspirated, and luteinized granulosa cells (LGCs) cultured for 24 h. Ovarian/uterine vascular flow (VF) and blood volume (BV) were analyzed by contrast enhanced ultrasound (CEUS) before hCG bolus and 6–8 days post-hCG bolus/time of peak SEP response. Ovarian permeability to albumin was analyzed by Dynamic Contrast Enhanced-MRI (DCE-MRI) post-hCG.ResultsAbdominal fluid was present in 4/5 Control, 2/6 Avastin pre-hCG, and 3/4 Avastin post-hCG females. Neutralization of VEGFA before hCG reduced ovarian VF, BV, and permeability to albumin (P < 0.05), while only ovarian VF and permeability were reduced in Avastin-post hCG group (P < 0.05). There was no effect of Avastin on ovarian vascular function during COS + SEP. VEGF levels in follicular fluid were reduced 78-fold by Avastin pre-hCG, and LGCs exposed to Avastin in vivo also released 4-fold less VEGF into culture media (P < 0.05). Culture medium of LGCs exposed to VEGFA neutralization in vivo had lower levels of P4 and ANGPT1, and an increased ratio of ANGPT2/1 (P < 0.05). Uterine VF was reduced by SEP + Avastin in the basalis/junctional zone (P < 0.05).ConclusionsAvastin treatment before hCG prevents the development of symptoms associated with ovarian hyperstimulation syndrome. In vitro data suggest neutralization of VEGFA alters expression of other vascular factors typically induced by hCG in the luteinizing follicle. Neutralization of VEGFA action alters the vascular function of the basalis zone of the uterus during simulated early pregnancy, indicating a potential effect on embryo implantation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-017-0340-5) contains supplementary material, which is available to authorized users.
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