Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids. Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization

Immunohistochemical localization of the bone morphogenetic protein receptors in the porcine ovary

The bone morphogenetic protein (BMP) family is emerging as playing a crucial role in regulating normal follicle growth and determining ovulation rate. BMPs exert their effects via BMP receptors (BMPR-IA, -IB and -II). However, there is a paucity of information relating to the expression of the BMPRs within the ovary of large polyovular species such as the pig. Furthermore, there is a lack of information on the expression of BMPRs by fetal ovaries of any species. The purpose of this study was to investigate temporal and spatial expression of the BMPRs in the porcine ovary, at different developmental stages. Immunohistochemistry for BMPR-IA, BMPR-IB and BMPR-II was performed using sections from paraffin wax-embedded ovaries, obtained from fetal (n = 15), prepubertal (n = 3) and cycling postpubertal (n = 4) pigs. Results confirmed the presence of all three receptors in the fetal egg nests and in the granulosa cell layer of follicles ranging from primordial to late antral stages. Immunostaining was also observed in oocytes, theca layer, corpus luteum and ovarian surface epithelium. The expression of BMPRs by fetal ovaries may be related to follicle formation, whereas expression in pre- and post-pubertal animals indicates BMPs are involved in regulating porcine ovarian follicle growth

Immunocytochemical localization of angiotensin II receptor subtypes 1 and 2 in the porcine fetal, prepubertal and postpubertal ovary

There is considerable evidence for a mammalian ovarian renin–angiotensin system, which may influence ovulation, angiogenesis and steroidogenesis via the autocrine and/or paracrine actions of the biologically active product of the cascade, angiotensin II (AngII). There are two characterized AngII receptors – type 1, AT(1) and type 2, AT(2). We report the localization of these receptor subtypes within porcine fetal, prepubertal and postpubertal ovaries. Positive staining for AT(1) and AT(2) receptors was observed in egg nests in all fetal ovaries studied, as well as in a defined two-cell layer at the ovarian periphery. In prepubertal tissue, positive AT(1) and AT(2) staining was localized to granulosa cells adjacent to the basement membrane of pre-antral and antral follicles, with no staining in the thecal layer. There was immunostaining for both receptors in prepubertal oocytes and zona pellucida. In postpubertal tissue, positive AT(1) and AT(2) immunostaining was localized to areas of putative neovascularization, the zona pellucida and the oocyte. Further AT(1) staining was located to the postpubertal antral follicle granulosa cells. The results indicate that there are higher densities of AT(1) receptors than AT(2) receptors in the porcine fetal, prepubertal and postpubertal ovary, and this has profound implications for the role of AngII in ovarian development

Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B.

Multi-subunit tethering complexes control membrane fusion events in eukaryotic cells. Class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) are two such complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named "CHEVI"), perhaps analogous in configuration to CORVET and HOPS. We utilized the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting that these proteins have distinct sub-cellular localizations. Consistent with previous reports, we observed that VPS33A was co-localized with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although VPS33A clearly co-localized with several subunits of CORVET and HOPS in this assay, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localize with VPS33B. Instead, we identified that VPS33B interacts directly with CCDC22, a member of the CCC complex. CCDC22 does not co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates, suggesting that CCDC22 interacts transiently with VPS33B/VIPAR rather than forming a stable complex with these proteins in cells. We also observed that the protein complex containing VPS33B and VIPAR is considerably smaller than CORVET/HOPS, suggesting that the CHEVI complex comprises just VPS33B and VIPAR.This work was supported by a Sir Henry Dale Fellowship, jointly funded by the Royal Society and the Wellcome Trust, to SCG [098406/Z/12/Z and 098406/Z/12/B] and an Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge Joint Research Grant to SCG. GGH was supported by a Parkinson Canada Basic Research Fellowship. ACG is a Tier 1 Canada Research Chair and supported by a CIHR Foundation grant (FDN 143301)

Nutritional effects on oocyte and embryo development in mammals: implications for reproductive efficiency and environmental sustainability

The environment in which a breeding female lives prior to conception and during the early stages of her pregnancy has striking effects on oocytes developing in the ovarian follicle and on early embryos in the reproductive tract. Of the various environmental factors known to affect oocyte and embryo development, altered nutrition during this critical period has been particularly well studied. Alterations in the quantity of food consumed or the composition of the diet imposed solely during the pre-mating period affect oocyte maturity, blastocyst yield, prenatal survival and the number of offspring born alive. Importantly, nutrition at this time also affects the quality of embryos and resultant offspring, with increasing evidence from a variety of species showing that peri-conception nutrition can alter behaviour, cardiovascular function and reproductive function throughout post-natal life. In livestock species, it is important to devise nutritional strategies that improve reproductive efficiency and the quality of offspring but that do not add to the environmental footprint of the production system and which recognize likely changes in feedstuff availability arising from predicted changes in climate

Landscape-Scale Mining and Water Management in a Hyper-Arid Catchment: The Cuajone Mine, Moquegua, Southern Peru

Peer reviewed: TrueAcknowledgements: The authors acknowledge the valuable support of A. Quispe and W. Zeballos at UNAM; Himantha Cooray for his assistance in developing the Mathematica program for data analysis; John Forrest for his contribution in arranging fieldwork; and Julia Porturas for her administrative support.Publication status: PublishedThe expansion of copper mining on the hyper-arid pacific slope of Southern Peru has precipitated growing concern for scarce water resources in the region. Located in the headwaters of the Torata river, in the department of Moquegua, the Cuajone mine, owned by Southern Copper, provides a unique opportunity in a little-studied region to examine the relative impact of the landscape-scale mining on water resources in the region. Principal component and cluster analyses of the water chemistry data from 16 sites, collected over three seasons during 2017 and 2018, show distinct statistical groupings indicating that, above the settlement of Torata, water geochemistry is a function of chemical weathering processes acting upon underlying geological units, and confirming that the Cuajone mine does not significantly affect water quality in the Torata river. Impact mitigation strategies that firstly divert channel flow around the mine and secondly divert mine waste to the Toquepala river and tailings dam at Quebrada Honda remove the direct effects on the water quality in the Torata river for the foreseeable future. In the study area, our results further suggest that water quality has been more significantly impacted by urban effluents and agricultural runoff than the Cuajone mine. The increase in total dissolved solids in the waters of the lower catchment reflects the cumulative addition of dissolved ions through chemical weathering of the underlying geological units, supplemented by rapid recharge of surface waters contaminated by residues associated with agricultural and urban runoff through the porous alluvial aquifer. Concentrations in some of the major ions exceeded internationally recommended maxima for agricultural use, especially in the coastal region. Occasionally, arsenic and manganese contamination also reached unsafe levels for domestic consumption. In the lower catchment, below the Cuajone mine, data and multivariate analyses point to urban effluents and agricultural runoff rather than weathering of exposed rock units, natural or otherwise, as the main cause of contamination