Several studies performed in rats have demonstrated the in vivo or in vitro antigonadal effects of GnRH analogs. In previous studies, we have demonstrated that GnRH-a treatment produces a failure in the steroidogenic luteal capability and an increase in the apoptotic process in the ovary, eliciting consequently an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins. These observations led us to study the in vivo effect of GnRH analogs on follicular development, atresia, and programmed cell death. The results presented in this work, assessed by the determination of ovarian weight and the number of follicles at different stages, confirm the inhibitory effect of the GnRH-agonist, LA, on ovarian development previously observed by our laboratory. Particularly interesting is that coinjection of the GnRH-antagonist, Ant, reverts the inhibitory effect of LA, suggesting that LA action is mainly mediated by an ovarian GnRH receptor. Of interest, the injection of Ant alone increased the number of preovulatory follicles, reflecting the augmentation also observed in ovarian weight, suggesting that this compound improves follicular development.
Prepubertal rats were superovulated with eCG (control group, C) and treated with LA (LA group; 1 ^g rat day during 48 h), or Ant (Ant group; 10 ^g rat day during 48 h), or both (LA + Ant group). When the GnRH-agonist, LA, was injected alone, the ovarian weight decreased by 0.5-fold, whereas injection of GnRH-Ant increased ovarian weight by 0.4-fold compared with controls. Coinjection of both GnRH substances did not show differences from that of controls (Table 1).
Histological ovarian slides were stained with hematox-ylin-eosin to determine the number of different follicle stages and the percentage of apoptotic cells (Fig. 1). Table 1 shows that injection of LA significantly increased the number of PFs and atretic follicles (ATFs).
Hormones and Reagents
The GnRH-a leuprolide acetate (Lupron), was a donation from Abbott Laboratories (Buenos Aires, Argentina). The original ampoule (2.8 mg/5 ml) was dissolved in saline solution. SYNTEX S.A (Buenos Aires) generously provided eCG (Novormon). HEPES, SDS, Antide [W-Ac-D-Nal1, D-pCl-Phe2, D-Pal3, Ser4, Nic-Lys5, D-Nic-Lys6, Leu7, Ipr-Lys8, Pro9, D-Ala10NH2; Nal-Lys antagonist] was purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco modified Eagle medium (DMEM, 4.5 g glucose/L), Ham F-12 nutrient mixture (F12), fungizone (250 |xg/ml), and gentamicine (10 mg/ml), were from Gibco Laboratories (Grand Island, NY). Polyclonal primary antibodies for BAX (N-20), cytochrome C (H-104), FAS (FL-335), and FASL (Q-20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit antibody against bovine cytochrome P450scc was a generous gift from Dr. Anita H. Payne (Stanford, CA). Anti-rabbit secondary antibody conjugated with horseradish peroxidase was purchased from Sigma.
In the mammalian ovary, only a small fraction of oocytes ovulate during reproductive life, whereas the majority of ovarian follicles undergo atresia by a hormonally regulated apoptotic mechanism.
Gonadotropin-releasing hormone (GnRH) and its analogs have been widely used to prevent the spontaneous LH surge in assisted reproduction techniques. Suppression of gonadotropin secretion can be achieved with either GnRH-agonist (GnRH-a) or GnRH-antagonist (GnRH-ant). Chronic administration of GnRH-a leads to pituitary desensitization and inhibition of gonadotropin and sex steroid levels by reducing the number of GnRH receptors on the cell membrane. Conversely, GnRH-ant achieves suppression of gonadotropin secretion by the competitive blockade of the GnRH receptors. canadian healthcare mall
Aside from the effects of GnRH analogs on the pituitary-gonadal axis, studies have shown that GnRH and its analogs have extrapituitary effects, particularly on the ovaries of rats and humans. However, there is some controversy about the physiological relevance of an endogenous GnRH-like molecule during folliculogenesis.
The ovarian surface epithelium (OSE) is a modified peritoneal mesothelium. It is composed of a single-layered sheet of flat to cuboidal cells that rest on a basal lamina and express microvillus toward the peritoneal cavity. The OSE is distinguished from extraovarian mesothelium by its capacity of steroid synthesis and expression of keratin and mucin. Epithelial-derived ovarian tumors are commonly believed to originate from OSE-lining inclusion cysts located in the ovarian stroma just beneath the surface. These cyst cells express E-cadherin and hence are more epithelial-like than the E-cadherin-negative OSE. It has also been shown that SV40-immortalized OSE expressing stably transfected E-cadherin have an increased tumor-igenic capacity, resembling ovarian adenocarcinoma compared with nontransfected OSE. Therefore, unlike carcinomas in most other organs in which loss of E-cadherin are common, the normal ovarian tumor precursor cells, OSE, seem to transdifferentiate and acquire characteristics of Mullerian duct-derived epithelium when present in the ovarian stroma. The mechanism by which this occurs is unknown, largely due to the fact that very little information is available on the normal biology of OSE and its relationship to the development of ovarian cancer. In the present study, we show that the normal human OSE in situ expresses tight junction (TJ) proteins, i.e., ZO-1, occludin and claudin-1. Moreover, normal OSE cells in primary and secondary cultures form a confluent monolayer that establishes a transepithelial barrier typical of low-resistance epithelium.
Tight Junction Proteins in Human Ovarian Biopsies
By immunofluorescence microscopic analysis, we could determine that the main localization of the TJ-associated protein ZO-1 and the integral membrane proteins occludin and claudin-1 was at the cell borders of normal OSE (Fig. 1, A through C). The underlying stroma were without staining, indicated by stars. TJ protein immunofluorescent staining was the same in sections from both pre- and postmenopausal women. The histological appearance of the ovarian surface and the underlying stroma is shown with hematoxylin and eosin staining (Fig. 1D). In the negative control, where primary antibody was replaced by nonfat milk, no staining was seen (Fig. 1E).
Characterization of Normal Ovarian Surface Epithelium in Culture
To further investigate TJ in human, OSE cells, isolated by the brush technique from ovaries in pre- and postmenopausal women (n = 32), were cultured and used for im-munohistochemistry, immunoblotting, and measurement of TER in Transwell experiments (Table 1). Primary cultured OSE cells typically formed a monolayer with a characteristic cobblestone-like appearance, and this growth pattern was maintained during the first three passages in approximately 45% of the patient samples (Fig. 2A).
OSE cells and tissue biopsies from normal ovaries were obtained from women operated on for benign nonovarian diseases at the Gynecology Unit, Sahlgrenska University Hospital, Goteborg, Sweden. OSE cells for culture were obtained from a total of 32 women. Normal ovarian tissue biopsies were collected from a total of nine women. A description of age at time for operation, reproductive status, and parity are found in Table 1. The study was approved by the Ethical Committee of Goteborg University and the patients had given their informed consent.
Harvest of Cells and Culture Conditions
To obtain OSE, cytobrushes (Cytobrush Plus; Medscand AB, Malmo, Sweden) were used as the first step after entering the peritoneal cavity at laparotomy or laparoscopy. The brushes were simultaneously rotated and moved 2-3 times over the ovarian surface, exerting only a slight pressure to minimize the risk of damaging or disrupting the underlying basal membrane and tunica albuginea. The brushes were withdrawn and immediately placed in culture medium, taken to the laboratory, and gently rubbed against each other to release the cells.