All-trans retinoic acid exposure increases connexin 43 expression in cumulus cells and improves embryo development in bovine oocytes.

In developing follicles, cellular coupling within cumulus-oocyte complexes (COCs) creates a functional syncytium allowing for the passage of small molecules. In many species, intercellular coupling between granulosa cells results from the expression of connexin 43 (CX43 or Gja1) and the formation of gap junctional plaques. Previously, our lab has shown that oocytes with a higher developmental potential had higher CX43 expression in their cumulus cells compared with developmentally incompetent oocytes. All-trans retinoic acid (ATRA) has been shown to increase CX43 expression in several different cell types. In this study we investigated the effect of ATRA treatment, during maturation, on CX43 expression and localization in cumulus cells and the developmental competence of bovine oocytes. COCs and granulosa cells exposed to ATRA during maturation had significantly higher CX43 expression and increased gap junctional coupling, respectively. In addition, there was a significant increase in the maturation, cleavage, and blastocyst rates in ATRA treated COCs. Data from these studies suggest that not only can CX43 be used as a biomarker for oocyte health, it can also potentially be manipulated using ATRA to increase the number of oocytes achieving developmental competence.

Connexin 43 coupling in bovine cumulus cells, during the follicular growth phase, and its relationship to in vitro embryo outcomes.

Gap junctional coupling between cumulus cells is required for oocytes to reach developmental competence. Multiple connexins, which form these gap junctions, have been found within the ovarian follicles of several species including bovine. The aim of this study was to determine the role of connexin 43 (CX43) and its relationship to embryo development, after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) were obtained from abattoir sourced, mixed breed, bovine ovaries. COCs were isolated from follicles ranging from 2 to 5 mm in size, representing the preselected follicle pool. Immediately after isolation, two cumulus cell biopsies were collected and stored for analysis pending determination of developmental outcomes. Using in vitro procedures, COCs were individually matured, fertilized, and cultured to the blastocyst stage. Biopsies were grouped as originating from COCs that arrested at the two-cell stage (low developmental competence [LDC]) or having developed to the late morula/blastocyst stage (high developmental competence [HDC]), after IVF and embryo culture. The expression level of CX43 was found to be significantly higher in cumulus cells from COCs that had an HDC when compared with those that had an LDC. Moreover, the gap junctional intercellular coupling rate was significantly higher in cumulus from COCs deemed to have an HDC. Significantly higher expression of the cumulus health markers luteinizing hormone receptor and cytochrome p450 19A1 was found in the cumulus originating from oocytes with HDC, suggesting that this system may provide a mechanism for noninvasively testing for oocyte health in preselected bovine follicles.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography-high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Incorporation of a fixed-time artificial insemination protocol results in improved reproductive management and genetics of the beef herd. However, a subset of animals exposed to such protocols will not display estrus prior to insemination. Behavioral estrus is indicative of the preovulatory follicle's physiological maturity and is essential for both the production of an oocyte with optimal developmental competence and preparation of the maternal environment for pregnancy establishment. Animals that do not display estrus prior to insemination and are induced to ovulate a physiologically less advanced follicle have reduced oocyte developmental competence that leads to reduced embryo cleavage rates, embryo quality, and pregnancy rates. This study investigated the impacts of reduced follicle maturity at the initiation of ovulation on the energy production capacity of the oocyte as well as follicular fluid metabolic composition. Results from this study demonstrated that follicle maturity, indicated by increased serum estradiol concentration at the initiation of ovulation, resulted in increased ATP within the oocyte as well as an increased level of metabolites involved in glucose metabolism in the follicular fluid. Increased energy production ability in the oocytes from more mature follicles could contribute to the increased cleavage rates and embryo quality seen in previous studies.

Preovulatory follicular fluid and serum metabolome profiles in lactating beef cows with thin, moderate, and obese body condition.

Extremes in body condition reduce fertility and overall productivity in beef cattle herds, due in part to altered systemic metabolic conditions that influence the intrafollicular and uterine environment. Follicular fluid and serum metabolome profiles are influenced by body composition in women and dairy cattle; however, such information is lacking in beef cattle. We hypothesized that body condition score (BCS)-related alterations in the metabolome of preovulatory follicular fluid and serum may influence oocyte maturation while impacting the oviductal or uterine environment. Therefore, we performed a study with the objective to determine the relationship between BCS and the metabolome of follicular fluid and serum in lactating beef cattle. We synchronized the development of a preovulatory follicle in 130 cows of varying BCS. We collected blood and performed transvaginal follicle aspirations to collect follicular fluid from the preovulatory follicle ~18 h after gonadotropin-releasing hormone administration to stimulate the preovulatory gonadotropin surge. We then selected follicular fluid and serum samples from cows with BCS 4 (Thin; n = 14), BCS 6 (Moderate; n = 18), or BCS >8 (Obese; n = 14) for ultra-high performance liquid chromatography-high resolution mass spectrometry. We identified differences in the follicular fluid or serum of thin, moderate, and obese animals based on multiple linear regression. MetaboAnalyst 5.0 was used for enrichment analysis of significant metabolites. We identified 38 metabolites in follicular fluid and 49 metabolites in serum. There were no significant differences in follicular fluid metabolite content among BCS classifications. There were 5, 22, and 1 serum metabolites differentially abundant between thin-obese, moderate-thin, and moderate-obese classifications, respectively (false discovery rate [FDR] < 0.10). These metabolites were enriched in multiple processes including "arginine biosynthesis," "arginine/proline metabolism," and "D-glutamine/D-glutamate metabolism" (FDR < 0.04). Pathways enriched with serum metabolites associated with BCS indicate potentially increased reactive oxygen species (ROS) in serum of thin cows. ROS crossing the blood follicular barrier may negatively impact the oocyte during oocyte maturation and contribute to the reduced pregnancy rates observed in thin beef cows.

Extremes in body condition affect fertility and pregnancy outcomes in beef cows. Much research has been done in women and dairy cows to evaluate body condition's effect on oocyte and embryo quality, pregnancy rates, and pregnancy outcomes. However, little work of this type has been done in beef cows and most studies do not focus on the preovulatory time period. The preovulatory time period is an essential time for the oocyte, as final stages of prematuration and the completion of oocyte maturation take place in the peri-ovulatory follicle. The follicular fluid provides the microenvironment for oocyte maturation and exchanges substances with maternal circulation at the blood follicular barrier. Alterations in maternal circulation due to extremes in body condition may pass into the follicular fluid and affect the oocyte during the preovulatory time period. Such conditions may contribute to the reduced fertility seen in beef cows with extreme body condition.

Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes.

To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method.

The Herbicide Atrazine Potentiates Angiotensin II-Induced Aldosterone Synthesis and Release From Adrenal Cells.

Atrazine is one of the most commonly used pre-emergence and early post-emergence herbicides in the world. We have shown previously that atrazine does not directly stimulate the pituitary or adrenal to trigger hormone release but acts centrally to activate a stress-like activation of the hypothalamic-pituitary-adrenal axis. In doing so, atrazine treatment has been shown to cause adrenal morphology changes characteristic of repeated stress. In this study, adrenals from atrazine treated and stressed animals were directly compared after 4 days of atrazine treatment or restraint stress. Both atrazine and stressed animals displayed reduced adrenocortical zona glomerulosa thickness and aldosterone synthase (CYP11B2) expression, indicative of repeated adrenal stimulation by adrenocorticotropic hormone. To determine if reduced CYP11B2 expression resulted in attenuated aldosterone synthesis, stressed and atrazine treated animals were challenged with angiotensin II (Ang II). As predicted, stressed animals produced less aldosterone compared to control animals when stimulated. However, atrazine treated animals had higher circulating aldosterone concentrations compared to both stressed and control groups. Ang II-induced aldosterone release was also potentiated in atrazine pretreated human adrenocortical carcinoma cells (H295R). Atrazine pretreated did not alter the expression of the rate limiting steroidogenic StAR protein or angiotensin II receptor 1. Atrazine treated animals also presented with higher basal blood pressure than vehicle treated control animals suggesting sustained elevations in circulating aldosterone levels. Our results demonstrate that treatment with the widely used herbicide, atrazine, directly increases stimulated production of aldosterone in adrenocortical cells independent of expression changes to rate limiting steroidogenic enzymes.

Correlation between Pre-Ovulatory Follicle Diameter and Follicular Fluid Metabolome Profiles in Lactating Beef Cows.

Induced ovulation of small pre-ovulatory follicles reduced pregnancy rates, embryo survival, day seven embryo quality, and successful embryo cleavage in beef cows undergoing fixed-time artificial insemination. RNA-sequencing of oocytes and associated cumulus cells collected from pre-ovulatory follicles 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the pre-ovulatory gonadotropin surge suggested reduced capacity for glucose metabolism in cumulus cells of follicles ≤11.7 mm. We hypothesized that the follicular fluid metabolome influences metabolic capacity of the cumulus-oocyte complex and contributes to reduced embryo cleavage and quality grade observed following induced ovulation of small follicles. Therefore, we performed a study to determine the correlation between pre-ovulatory follicle diameter and follicular fluid metabolome profiles in lactating beef cows (Angus, n = 130). We synchronized the development of a pre-ovulatory follicle and collected the follicular contents approximately 20 h after GnRH administration. We then performed ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) metabolomic studies on 43 follicular fluid samples and identified 38 metabolites within pre-ovulatory follicles of increasing size. We detected 18 metabolites with a significant, positive correlation to follicle diameter. Individual and pathway enrichment analysis of significantly correlated metabolites suggest that altered glucose and amino acid metabolism likely contribute to reduced developmental competence of oocytes when small pre-ovulatory follicles undergo induced ovulation.

Synthetic Peptides as Therapeutic Agents: Lessons Learned From Evolutionary Ancient Peptides and Their Transit Across Blood-Brain Barriers.

Peptides play a major role in the transmission of information to and from the central nervous system. However, because of their structural complexity, the development of pharmacological peptide-based therapeutics has been challenged by the lack of understanding of endogenous peptide evolution. The teneurin C-terminal associated peptides (TCAP) possess many of the required attributes of a practical peptide therapeutic. TCAPs, associated with the teneurin transmembrane proteins that bind to the latrophilins, members of the Adhesion family of G-protein-coupled receptors (GPCR). Together, this ligand-receptor unit plays an integral role in synaptogenesis, neurological development, and maintenance, and is present in most metazoans. TCAP has structural similarity to corticotropin-releasing factor (CRF), and related peptides, such as calcitonin and the secretin-based peptides and inhibits the (CRF)-associated stress response. Latrophilins are structurally related to the secretin family of GPCRs. TCAP is a soluble peptide that crosses the blood-brain barrier and regulates glucose transport into the brain. We posit that TCAP represents a phylogenetically older peptide system that evolved before the origin of the CRF-calcitonin-secretin clade of peptides and plays a fundamental role in the regulation of cell-to-cell energy homeostasis. Moreover, it may act as a phylogenetically older peptide system that evolved as a natural antagonist to the CRF-mediated stress response. Thus, TCAP's actions on the CNS may provide new insights into the development of peptide therapeutics for the treatment of CNS disorders.

Characterization of Activation of the Hypothalamic-Pituitary-Adrenal Axis by the Herbicide Atrazine in the Female Rat.

Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.

Plasma metabolomic profiles differ at the time of artificial insemination based on pregnancy outcome, in Bos taurus beef heifers.

Infertility remains the most prevalent reason for cattle being removed from production environments. We utilized metabolomic profiling to identify metabolites in the blood plasma that may be useful in identifying infertile heifers at the time of artificial insemination (AI). Prior to AI, phenotypic parameters including body condition, weight, and reproductive organ measurements were collected. These were determined not effective at differentiating between fertile and infertile heifers. Analysis of the resulting metabolomic profiles revealed 15 metabolites at significantly different levels (T-test P ≤ 0.05), with seven metabolites having a greater than 2-fold difference (T-test P ≤ 0.05, fold change ≥2, ROC-AUC ≥ 0.80) between infertile and fertile heifers. We further characterized the utility of using the levels of these metabolites in the blood plasma to discriminate between fertile and infertile heifers. Finally, we investigated the potential role inflammation may play by comparing the expression of inflammatory cytokines in the white blood cells of infertile heifers to that of fertile heifers. We found significantly higher expression in infertile heifers of the proinflammatory markers tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), and the C-X-C motif chemokine 5 (CXCL5). Our work offers potentially valuable information regarding the diagnosis of fertility problems in heifers undergoing AI.

Kisspeptin Stimulates Growth Hormone Release by Utilizing Neuropeptide Y Pathways and Is Dependent on the Presence of Ghrelin in the Ewe.

Although kisspeptin is the primary stimulator of gonadotropin-releasing hormone secretion and therefore the hypothalamic-pituitary-gonadal axis, recent findings suggest kisspeptin can also regulate additional neuroendocrine processes including release of growth hormone (GH). Here we show that central delivery of kisspeptin causes a robust rise in plasma GH in fasted but not fed sheep. Kisspeptin-induced GH secretion was similar in animals fasted for 24 hours and those fasted for 72 hours, suggesting that the factors involved in kisspeptin-induced GH secretion are responsive to loss of food availability and not the result of severe negative energy balance. Pretreatment with the neuropeptide Y (NPY) Y1 receptor antagonist, BIBO 3304, blocked the effects of kisspeptin-induced GH release, implicating NPY as an intermediary. Kisspeptin treatment induced c-Fos in NPY and GH-releasing hormone (GHRH) cells of the arcuate nucleus. The same kisspeptin treatment resulted in a reduction in c-Fos in somatostatin (SS) cells in the periventricular nucleus. Finally, blockade of systemic ghrelin release or antagonism of the ghrelin receptor eliminated or reduced the ability of kisspeptin to induce GH release, suggesting the presence of ghrelin is required for kisspeptin-induced GH release in fasted animals. Our findings support the hypothesis that during short-term fasting, systemic ghrelin concentrations and NPY expression in the arcuate nucleus rise. This permits kisspeptin activation of NPY cells. In turn, NPY stimulates GHRH cells and inhibits SS cells, resulting in GH release. We propose a mechanism by which kisspeptin conveys reproductive and hormone status onto the somatotropic axis, resulting in alterations in GH release.