Transformative learning for a sustainable and healthy future through ecosystem approaches to health: insights from 15 years of co-designed ecohealth teaching and learning experiences.

This paper presents insights from the work of the Canadian Community of Practice in Ecosystem Approaches to Health (CoPEH-Canada) and 15 years (2008-2022) of land-based, transdisciplinary, learner-centred, transformative learning and training. We have oriented our learning approaches to Head, Hands, and Heart, which symbolise cognitive, psychomotor, and affective learning, respectively. Psychomotor and affective learning are necessary to grapple with and enact far-reaching structural changes (eg, decolonisation) needed to rekindle healthier, reciprocal relationships with nature and each other. We acknowledge that these approaches have been long understood by Indigenous colleagues and communities. We have developed a suite of teaching techniques and resources through an iterative and evolving pedagogy based on participatory approaches and operating reciprocal, research-pedagogical cycles; integrated different approaches and ways of knowing into our pedagogy; and built a networked Community of Practice for continued learning. Planetary health has become a dominant framing for health-ecosystem interactions. This Viewpoint underscores the depth of existing scholarship, collaboration, and pedagogical expertise in ecohealth teaching and learning that can inform planetary health education approaches.

Differential expression of lysosome-associated protein transmembrane-4 beta (LAPTM4B) in granulosa cells of ovarian follicles and in other bovine tissues.

BACKGROUND: LAPTM4B is a member of the lysosome-associated transmembrane protein superfamily that is differentially expressed in normal human tissues and upregulated in various types of carcinomas. These proteins are thought to be involved in the regulation of cell proliferation and survival. The objective of this study was to investigate the expression of bovine LAPTM4B during ovarian follicular development and in various bovine tissues. METHODS AND RESULTS: Northern blot analysis revealed a 1.8 kb transcript, with highly variable steady state levels among tissues. RT-PCR analysis showed that LAPTM4B mRNA transcripts were low in granulosa cells of small antral follicles, increased in large dominant follicles, and decreased in ovulatory follicles following injection of human chorionic gonadotropin (hCG; P < 0.003). Ovulatory follicles collected at various times after hCG injection revealed a significant reduction of LAPTM4B mRNA starting at 18 h post-hCG (P < 0.029). Immunoblotting analysis using antibodies generated against bovine LAPTM4B recognized proteins of 26.3 and 31.5 kDa in granulosa cells of developing follicles and corpus luteum. Further analyses of affinity-purified His-tag LAPTM4B overexpressed in HEK cells showed that the 31.5 kDa protein represented the ubiquinated isoform of the 26.3 kDa native protein. The 26.3 kDa protein was differentially expressed showing highest amounts in dominant follicles and lowest amounts in ovulatory follicles 24 h post-hCG. Immunohistochemical analyses of LAPTM4B showed marked heterogeneity of labeling signal among tissues, with LAPTM4B mainly localized to perinuclear vesicles, in keeping with its putative lysosomal membrane localization. CONCLUSION: This study reports for the first time that bovine LAPTM4B in granulosa cells is present in both unubiquinated and ubiquinated forms, and is differentially expressed in developing ovarian follicles, suggesting a possible role in terminal follicular growth.

Transforming growth factor-beta1 inhibits luteinization and promotes apoptosis in bovine granulosa cells.

We have previously shown that TGFB1 inhibits estradiol (E(2)) and progesterone (P(4)) biosynthesis in FSH-stimulated bovine granulosa cells by selective inhibition of steroidogenic enzymes. The objective of this study was to assess the effects of TGFB1 on E(2) and P(4) steroidogenesis in bovine granulosa cells cultured in the absence of FSH and to measure the effects of TGFB1 on cell proliferation and apoptosis in the presence and absence of FSH. Bovine granulosa cells from 2 to 5 mm follicles were cultured in serum-free medium for 2-6 days. In the absence of FSH, the secretion of P(4) increased with time in culture (P<0.05). Addition of TGFB1 for 6 days decreased P(4) secretion and mRNA levels of the P(4) synthesis-associated genes STAR, CYP11A1, HSD3B1, and GSTA (P<0.05). In the absence of FSH, the secretion of E(2) decreased and addition of TGFB1 for 6 days partially reversed this decline and stimulated E(2) biosynthesis, CYP19A1 and HSD17B1 mRNA levels and CYP19A1 activity (P<0.05). Conversely, TGFB1 did not affect HSD17B7 expression and HSD17B-reducing activity. TGFB1 decreased the proportion of cells in the G0/G1 and S+G2/M phases in FSH-stimulated and unstimulated granulosa cells (P<0.05). Furthermore, in the presence or absence of FSH, TGFB1 increased the proportion of cells in apoptosis measured by propidium iodide staining and flow cytometry and confirmed by increased levels of cleaved caspase-3 (P<0.05). Our results therefore indicate that TGFB1 inhibits luteinization in cultured bovine granulosa cells while maintaining an estrogenic phenotype, and this effect was associated with increased apoptosis.

Role of transforming growth factor-beta1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells.

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle.

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.

Follicular fluid concentration of transforming growth factor-beta1 is negatively correlated with estradiol and follicle size at the early stage of development of the first-wave cohort of bovine ovarian follicles.

The objectives of this study were to characterize the relationship between estradiol and transforming growth factor-beta1(TGF-beta1) concentrations in follicular fluid of growing bovine ovarian follicles, and to examine the effect of TGF-beta1 on FSH-stimulated estradiol secretion in cultured bovine granulosa cells. Follicular fluid was collected from individual follicles >5 mm in diameter by ultrasound-guided transvaginal puncture (n = 12 heifers). Follicles were sampled at four different stages of development of the first post-ovulatory wave during selection of the single dominant follicle. Estradiol, progesterone and total TGF-beta1 were measured in follicular fluid of the three or four largest follicles sampled when the largest follicle (F1) had reached either 6.5, 7.5, 8.5 or 9.5+/-0.5 mm stage of development. There was a significant negative relationship between follicular fluid TGF-beta1 and estradiol concentrations (R2 = 0.44; p < 0.002), and between TGF-beta1 concentrations and follicle diameter (R2 = 0.23; p < 0.01) in cohort follicles at the 6.5 mm stage, but not at any later stage of development of the follicle wave. There was no correlation between progesterone and TGF-beta1 concentrations at any stage. To assess the causal relationship between TGF-beta1 and estradiol, granulosa cells from follicles measuring 2-5 mm at dissection were placed in serum-free culture. TGF-beta1 caused a dose-dependent decrease in FSH-stimulated estradiol secretion (P < 0.001). These findings suggest that TGF-beta1 has an inhibitory effect on estradiol secretion in FSH-stimulated follicles and that a reduction in TGF-beta1 inhibition may be part of the mechanism of selection of a single dominant follicle.