The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.
Abstract:Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K D for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC 50 s that decrease from 15000 nM (1500 µg mL -1 ) for the 25 mol % functionalized polymers to 11 nM (1 µg mL -1 ) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.
Topical microbicide products are being developed for the prevention of sexually transmitted infections. These include vaginally-applied gels that deliver anti-HIV molecules. Gels may also provide partial barriers that slow virion diffusion from semen to vulnerable epithelium, increasing the time during which anti-HIV molecules can act. To explore the barrier function of microbicide gels, we developed a deterministic mathematical model for HIV diffusion through realistic gel distributions. We applied the model to experimental data for in vivo coating distributions of two vaginal gels in women. Time required for a threshold number of virions to reach the tissue surface was used as a metric for comparing different scenarios. Results delineated how time to threshold increased with increasing gel layer thickness and with decreasing diffusion coefficient. We note that for gel layers with average thickness > approximately 100 microm, the fractional area coated, rather than the gel layer thickness, was the primary determinant of time to threshold. For gel layers < approximately 100 microm, time to threshold was brief, regardless of fractional area coated. Application of the model to vaginal coating data showed little difference in time to threshold between the two gels tested. However, the protocol after gel application (i.e., with or without simulated coitus) had a much more significant effect. This study suggests that gel distribution in layers of thickness >100 microm and fractional area coated >0.8 is critical in determining the ability of the gel to serve as a barrier to HIV diffusion.
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