The matrisome contributes to the increased rigidity of the bovine ovarian cortex and provides a source of new bioengineering tools to investigate ovarian biology

The gonadotoxic effects of some cancers significantly increase the risk of developing infertility and cessation of ovary hormones (premature ovarian insufficiency, POI). Fertility preservation in the form of ovarian tissue cryopreservation (OTC) is offered to pediatric and adolescent cancer patients who cannot undergo oocyte retrieval and egg cryopreservation. The cryopreserved ovarian tissue can be transplanted back and has been found to restore fertility in 20 - 40% of transplants and restore hormone function for an average of 3 to 5 years. However, some individuals have primary or metastatic disease within their ovarian tissue and would not be able to transplant it back in its native form. Therefore, there is a need for additional methods for hormone and fertility restoration that would support a safe transplant with increased successful livebirths and long-term hormone restoration. To support this goal, we sought to understand the contribution of the ovarian microenvironment to its physical and biochemical properties to inform bioprosthetic ovary scaffolds that would support isolated follicles. Using atomic force microscopy (AFM), we determined that the bovine ovarian cortex was significantly more rigid than the medulla. To determine if this difference in rigidity was maintained in isolated matrisome proteins from bovine ovarian compartments, we cast, and 3D printed hydrogels created from decellularized bovine ovarian cortex and medulla slices. The cast gels and 3D printed bioprosthetic ovary scaffolds from the cortex was still significantly more rigid than the medulla biomaterials. To expand our bioengineering toolbox that will aide in the investigation of how biochemical and physical cues may affect folliculogenesis, we sought to confirm the concentration of matrisome proteins in bovine ovarian compartments. The matrisome proteins, COL1, FN, EMILIN1 and AGRN were more abundant in the bovine ovarian cortex than the medulla. Whereas VTN was more abundant in the medulla than the cortex and COL4 was present in similar amounts within both compartments. Finally, we removed proteins of interest, EMILIN1 and AGRN, from decellularized bovine ovarian cortex materials and confirmed that this specifically depleted these proteins without affecting the rigidity of cast or 3D printed hydrogels. Taken together our results indicate the existence of a rigidity gradient in the bovine ovary, that this rigidity gradient is maintained in resulting engineered materials strongly implicating a role for matrisome proteins in contributing to the physical properties of the bovine ovary. By establishing additional engineering tools, we will continue to explore mechanisms behind matrisome-follicle interactions.

Hyaluronan and Collagen Are Prominent Extracellular Matrix Components in Bovine and Porcine Ovaries

The extracellular matrix (ECM) is a major component of the ovarian stroma. Collagen and hyaluronan (HA) are critical ovarian stromal ECM molecules that undergo age-dependent changes in the mouse and human. How these matrix components are regulated and organized in other mammalian species with reproductive characteristics similar to women such as cows and pigs, has not been systematically investigated. Therefore, we performed histological, molecular, and biochemical analyses to characterize collagen and HA in these animals. Bovine ovaries had more collagen than porcine ovaries when assessed biochemically, and this was associated with species-specific differences in collagen gene transcripts: Col3a1 was predominant in cow ovaries while Col1a1 was predominant in pig ovaries. We also observed more HA in the porcine vs. bovine ovary. HA was distributed across three molecular weight ranges (<100 kDa, 100–300 kDa, and >300 kDa) in ovarian tissue and follicular fluid, with tissue having more >300 kDa HA than the other two ranges. Transcripts for HA synthesis and degradation enzymes, Has3 and Hyal2, respectively, were predominant in cow ovaries, whereas Has2, Kiaa1199, and Tmem2 tended to be predominant in pig ovaries. Together, our findings have implications for the composition, organization, and regulation of the ovarian ECM in large mammalian species, including humans.

84 Characterization of agouti-signalling protein expression within the bovine ovary and early embryo

Factors present in the oocyte and surrounding follicular cells aid in the attainment of oocyte competence. Agouti-signalling protein (ASIP) is a known regulator of melanocyte function through binding to melanocortin receptors including MC1R and MC4R. Additionally, ASIP has been classified as an adipokine due to a link with insulin resistance and obesity in humans. In mice, expression is limited to hair follicles where ASIP regulates hair pigmentation. In cattle, however, ASIP mRNA has been detected in a variety of tissues, including adipose, skin, heart, testis, and the ovary. Despite ovarian expression, the role of ASIP in reproduction remains undetermined. Bovine ASIP is a secreted protein consisting of 133 amino acids. The aim of this experiment was to provide a detailed description of the ASIP expression profile within the bovine ovary and during early embryonic development. Reverse transcription PCR (RT-PCR) was conducted to analyse ASIP, MC1R, and MC4R mRNA expression. Samples examined included fetal ovaries from gestational day 90 to 250, adult ovary, fetal testis, adult testis, and 12 somatic tissues including adrenal, cerebral cortex, gut, heart, intestine, kidney, liver, lung, muscle, pituitary, stomach, and thymus. Amplification of ribosomal protein L19 (RPL19) served as a positive control for all samples. Expression of ASIP was detected in the fetal testis, 9 somatic tissues, and the fetal and adult ovary. In the fetal ovary, ASIP was detected as early as 90 days of gestation and continued throughout gestation. Expression of the ASIP receptors, MC1R and MC4R, were detected exclusively in the fetal ovary. To further characterise ASIP expression, quantitative real-time PCR (RT-qPCR) was utilised to examine samples including germinal vesicle and MII oocytes (pool of 10 oocytes), invitro-produced embryos ranging from the 2-cell to blastocyst stages (pool of 10 embryos), and cumulus and granulosa cells collected from a pool from 5 cumulus–oocyte complexes (COCs) and follicles, respectively. Theca cells from a single follicle were analysed. Samples with cycle threshold values below 35 were considered to express the gene of interest. Of the follicular cells examined, ASIP expression was present in theca, granulosa, and cumulus cells. ASIP expression was detected in both GV and MII oocytes. Early embryonic expression of ASIP was detected in the 2-cell embryo and continued to the blastocyst stage of development. In conclusion, ASIP is present in the bovine adult and fetal ovary, follicular cells including cumulus, granulosa, and theca cells, GV and MII oocytes, and invitro-produced embryos from the 2-cell to blastocyst stages. Future research will focus on identifying the function of ovarian and early embryonic ASIP in cattle.

Effects of adrenocorticotrophic hormone on the expression of matrix metalloproteinases and their inhibitors in the bovine ovary

Cattle undergo numerous environmental and management stressors that reduce fertility and affect ovulation. The extracellular matrix of the follicle wall can be altered by matrix metalloproteinases (MMPs), the activities of which are regulated by interleukins and tissue-specific inhibitors of metalloproteinases (TIMPs), especially during ovulation. The aims of the present study were to: (1) evaluate changes in the hormone milieu, the localisation and activity of MMP2 and MMP9 and the localisation of MMP14, TIMP1 and TIMP2 in response to adrenocorticotrophic hormone (ACTH) during the preovulatory period in cows; and (2) determine the direct effects of ACTH on the mRNA expression of MMP2 and MMP9 in the cultured follicle wall of bovine ovaries obtained from an abattoir. 100IU ACTH was administered during pro-oestrus every 12h until ovariectomy, which was performed before ovulation. Cortisol concentrations in the plasma and follicular fluid (FF) of preovulatory follicles were higher in ACTH-treated than control cows. Progesterone presented subluteal concentrations in plasma of ACTH-treated cows (P&lt;0.05). MMP2 immunostaining and activity in ovaries were higher in ACTH-treated than control cows (P&lt;0.05), whereas MMP9 immunostaining was similar between the two groups. However, unlike in control cows, MMP9 activity was absent in the FF of ACTH-treated cows. These results suggest that the administration of ACTH during the preovulatory period in cows could cause changes that culminate in modifications in the content and activation of MMPs and TIMPs in the ovary, which could interfere with the ovulation process.

105 P75 Neuronal Cells and Fibers in the Bovine Ovary

Neurotrophins are molecules involved in the development and survival of neurons and its cellular projections. Results of recent studies have implicated the local role of the high affinity neurotropin receptor, trkA, in bovine ovarian follicle selection and early luteogenesis (Carrasco et al. 2016 Reprod. Biol. Endocrinol. 14, 47), but innervation and neuropeptide control remains an unexplored aspect of ovarian function. P75 is the low-affinity receptor for all neurotrophins and is expressed in ovarian tissue. The objective of this study was to explore the distribution of P75 neurons and fibres within the ovary and to examine the relationship of these components with follicular development. The ovaries of cows (n = 5) were collected at the time of slaughter, 36 h after induced luteolysis (i.e. proestrus). The ovaries were fixed in 4% paraformaldehyde for 48 h, and samples from the ovarian hilus, medulla, and cortex (3 blocks per ovary) were cryo-sectioned (20–50 µm). Tissue sections were incubated for 48 h with a rabbit antibody against rat P75 or a mouse monoclonal antibody against neurofilament. Immunodetection was visualised by an amplification procedure with horseradish peroxidase using nickel DAB as a chromogen. Sections were counterstained with nuclear fast red for follicle identification. Immunoreactive cell bodies were counted in 10 to 20 fields (40×) per section, and data were expressed based on ovarian areas (cortex, medulla, or hilus) as an average count per 40× field per animal. Data among ovarian regions were compared by ANOVA; differences were considered significant when P < 0.05. Antral follicles ≤5 mm displayed strong immunoreactivity in the theca layer, without reaction in the granulosa cells. In contrast, preovulatory follicles were devoid of P75 immuno-reactivity in the theca layer. Oval P75 immunoreactive neuron-like cells were present in all ovarian areas studied. The neuronal nature of the P75 immunoreactive cells was confirmed by the presence of a similar pattern when adjacent sections were stained for neurofilaments, a protein characteristic of neurons. In the stroma of the ovarian cortex and medulla, neurons were present individually (scattered) rather than grouped; however, a dense network of neurons and fibres was detected immediately beneath the ovarian surface epithelium. No differences between the cortex, medulla, and hilus were found in the mean number of immunoreactive cells (10.6 ± 2.8, 14.4 ± 3.6 and 13.9 ± 2.0 cells/40× field, respectively). Immunoreactive neuron-like cells and fibres were in close proximity to blood vessels in the ovarian medulla. Corpora lutea were devoid of P75 immunoreactivity. In conclusion, results document the existence of a neuronal network in the bovine ovary, displaying an association with follicles at different stages of development. The abundance of neuronal components (i.e. neuron cell bodies and axons) in the ovarian stromal and surface epithelium implies a role of innervation (either extrinsic or intrinsic) in the control of ovarian follicular development and function. Research was supported by the Natural Sciences and Engineering Research Council of Canada.

Proliferative activity of oocytes in multi-oocyte follicles of bovine ovary

We characterized the proliferative activity of multi-oocyte follicles with anti-nuclear antigen of proliferating cells (PCNA). Ovaries (n = 12) from heifers were processed for histology. From 789 multi-oocytes follicles observed, only 11 were considered appropriated for immunostaining, since they presented all nuclei of the oocytes clearly visible. All multi-oocyte follicles were positive for PCNA, but some oocytes showed no proliferative activity. We conclude that oocytes in multi-oocyte follicles seem to be in different stages of the cell cycle.