Benjamin J. Hale

Small RNA regulation of reproductive function

Post‐transcriptional gene regulation is one mechanism that occurs “above the genome,” allowing the cells of an organism to have dramatically different phenotypes and functions. Non‐coding ribonucleic acid (ncRNA) molecules regulate transcript and protein abundance above the level of transcription, and appear to play substantial roles in regulation of reproductive tissues. Three primary classes of small ncRNA are microRNA (miRNA), endogenous small interfering RNA (endo‐siRNA), and PIWI‐interacting RNA (piRNA). These RNA classes have similarities and clear distinctions between their biogenesis and in the interacting protein machinery that facilitate their effects on cellular phenotype. Characterization of the expression and importance of the critical components for the biogenesis of each class in different tissues is continuously contributing a better understanding of each of these RNA classes in different reproductive cell types. Here, we discuss the expression and potential roles of miRNA, endo‐siRNA, and piRNA in reproduction from germ‐cell development to pregnancy establishment and placental function. Additionally, the potential contribution of RNA binding proteins, long ncRNAs, and the more recently discovered circular RNAs (circRNAs) in relation to small RNA function is discussed. Mol. Reprod. Dev. 81: 148–159, 2014.

MicroRNA-21 and PDCD4 expression during in vitro oocyte maturation in pigs

BackgroundMicroRNA (miRNA) are small non-coding RNA molecules critical for regulating cellular function, and are abundant in the maturing oocyte and developing embryo. MiRNA-21 (MIR21) has been shown to elicit posttranscriptional gene regulation in several tissues associated with rapid cell proliferation in addition to demonstrating anti-apoptotic features through interactions with PDCD4 mRNA and other targets. In many tissues, MIR21 interacts and suppresses PDCD4 due to the strong complementation between MIR21 and the PDCD4 3′UTR.MethodsThe objective of this project was to examine the relationship between MIR21 and PDCD4 expression in porcine oocytes during in vitro maturation and assess the impact of MIR21 inhibition during oocyte maturation on early embryo development. Additionally, we evaluated the effect of gonadotropins in maturation media and the presence of cumulus cells to determine their ability to contribute to MIR21 abundance in the oocyte during maturation.ResultsDuring in vitro maturation, expression of MIR21 increased approximately 6-fold in the oocyte and 25-fold in the cumulus cell. Temporally associated with this was the reduction of PDCD4 protein abundance in MII arrested oocytes compared with GV stage oocytes, although PDCD4 mRNA was not significantly different during this transition. Neither the presence of cumulus cells nor gonadotropins during in vitro maturation affected MIR21 abundance in those oocytes achieving MII arrest. However, inhibition of MIR21 activity during in vitro maturation using antisense MIR21 suppressed embryo development to the 4–8 cell stage following parthenogenetic activation.ConclusionsMIR21 is differentially expressed in the oocyte during meiotic maturation in the pig and inhibition of MIR21 during this process alters PDCD4 protein abundance suggesting posttranscriptional regulatory events involving MIR21 during oocyte maturation may impact subsequent embryonic development in the pig.

Physiological consequences of heat stress in pigs

Heat stress negatively influences the global pork industry and undermines genetic, nutritional, management and pharmaceutical advances in management, feed and reproductive efficiency. Specifically, heat stress-induced economic losses result from poor sow performance, reduced and inconsistent growth, decreased carcass quality, mortality, morbidity, and processing issues caused by less rigid adipose tissue (also known as flimsy fat). When environmental conditions exceed the pig’s thermal neutral zone, nutrients are diverted from product synthesis (meat, fetus, milk) to body temperature maintenance thereby compromising efficiency. Unfortunately, genetic selection for both increased litter size and leaner phenotypes decreases pigs’ tolerance to heat, as enhanced fetal development and protein accretion results in increased basal heat production. Additionally, research has demonstrated that in utero heat stress negatively and permanently alters post-natal body temperature and body composition and both variables represent an underappreciated consequence of heat stress. Advances in management (i.e. cooling systems) have partially alleviated the negative impacts of heat stress, but productivity continues to decline during the warm summer months. The detrimental effects of heat stress on animal welfare and production will likely become more of an issue in regions most affected by continued predictions for climate change, with some models forecasting extreme summer conditions in key animal-producing areas of the globe. Therefore, heat stress is likely one of the primary factors limiting profitable animal protein production and will certainly continue to compromise food security (especially in emerging countries) and regionalise pork production in developed countries. Thus, there is an urgent need to have a better understanding of how heat stress reduces animal productivity. Defining the biology of how heat stress jeopardises animal performance is critical in developing approaches (genetic, managerial, nutritional and pharmaceutical) to ameliorate current production issues and improve animal wellbeing and performance.

Physiological mechanisms through which heat stress compromises reproduction in pigs

Seasonal variations in environmental temperatures impose added stress on domestic species bred for economically important production traits. These heat‐mediated stressors vary on a seasonal, daily, or spatial scale, and negatively impact behavior and reduce feed intake and growth rate, which inevitably lead to reduced herd productivity. The seasonal infertility observed in domestic swine is primarily characterized by depressed reproductive performance, which manifests as delayed puberty onset, reduced farrowing rates, and extended weaning‐to‐estrus intervals. Understanding the effects of heat stress at the organismal, cellular, and molecular level is a prerequisite to identifying mitigation strategies that should reduce the economic burden of compromised reproduction. In this review, we discuss the effect of heat stress on an animals ability to maintain homeostasis in multiple systems via several hypothalamic‐pituitary‐end organ axes. Additionally, we discuss our understanding of epigenetic programming and how hyperthermia experienced in utero influences industry‐relevant postnatal phenotypes. Further, we highlight the recent recognized mechanisms by which distant tissues and organs may molecularly communicate via extracellular vesicles, a potentially novel mechanism contributing to the heat‐stress response.

Heat stress induces autophagy in pig ovaries during follicular development

Abstract Hyperthermia or heat stress (HS) occurs when heat dissipation mechanisms are overwhelmed by external and internal heat production. Hyperthermia negatively affects reproduction and potentially compromises oocyte integrity and reduces developmental competence of ensuing embryos. Autophagy is the process by which cells recycle energy through the reutilization of cellular components and is activated by a variety of stressors. Study objectives were to characterize autophagyrelated proteins in the ovary following cyclical HS during the follicular phase. Twelve gilts were synchronized and subjected to cyclical HS (n = 6) or thermal neutral (n = 6) conditions for 5 days during the follicular phase. Ovarian protein abundance of Beclin 1 and microtubule associated protein light chain 3 beta II were each elevated as a result of HS (P = 0.001 and 0.003, respectively). The abundance of the autophagy related (ATG)12–ATG5 complex was decreased as a result of HS (P = 0.002). Regulation of autophagy and apoptosis occurs in tight coordination, and B-cell lymphoma (BCL)2 and BCL2L1 are involved in regulating both processes. BCL2L1 protein abundance, as detected via immunofluorescence, was increased in both the oocyte (∼1.6-fold; P < 0.01) and granulosa cells of primary follicles (∼1.4-fold P < 0.05) of HS ovaries. These results suggest that ovarian autophagy induction occurs in response to HS during the follicular phase, and that HS increases anti-apoptotic signaling in oocytes and early follicles. These data contribute to the biological understanding of how HS acts as an environmental stress to affect follicular development and negatively impact reproduction. Summary Sentence Heat stress induces autophagy in the pig ovary during the follicular stage, and autophagy is a potential mechanism by which the ovary mitigates cellular stress.

Characterizing the acute heat stress response in gilts: I. Thermoregulatory and production variables

Identifying traits associated with susceptibility or tolerance to heat stress (HS) is a prerequisite for developing strategies to improve efficient pork production during the summer months. Study objectives were to determine the relationship between the thermoregulatory and production responses to acute HS in pigs. Prepubertal gilts (n = 235; 77.9 ± 1.2 kg BW) were exposed to a thermoneutral (TN) period (P1, 24 h; 21.9 ± 0.5 °C, 62 ± 13% RH; fed ad libitum) followed immediately by a subsequent acute HS period (P2, 24 h; 29.7 ± 1.3 °C, 49 ± 8% RH; fed ad libitum). Rectal temperature (TR), skin temperature (TS), and respiration rate (RR) were monitored and BW and feed intake (FI) were determined. All pigs had increased TR, TS, and RR (0.80 °C, 5.65 °C, and 61.2 bpm, respectively; P < 0.01) and decreased FI and BW (29% and 1.10 kg, respectively; P < 0.01) during P2 compared to P1. Interestingly, body temperature indices did not explain variation in FI during P2 (R2 ≤ 0.02). Further, the percent change in BW during P2 was only marginally explained by each body temperature index (R2 ≤ 0.06) or percent change in FI (R2 = 0.14). During HS, TR was strongly correlated with P1 TR (r = 0.72, P < 0.01), indicating a pigs body temperature during TN conditions predicts the severity of hyperthermia during HS. Additionally, the change in TR (ΔTR, HS TR - TN TR) was larger in pigs retrospectively classified as susceptible (SUS) as compared to tolerant (TOL) pigs (1.05 vs. 0.51 °C, respectively; P < 0.01). In summary, thermoregulatory responses and production variables during acute HS are only marginally related. Further, changes in BW and FI were unexpectedly poorly correlated during acute HS (r = 0.34; P < 0.01). Collectively, suboptimal growth is largely independent on the thermoregulatory response and hypophagia during acute HS. Consequently, incorporating solely body temperature indices into a genetic index is likely insufficient for substantial progress in selecting HS tolerant pigs.

Heat stress causes dysfunctional autophagy in oxidative skeletal muscle

We have previously established that 24 h of environmental hyperthermia causes oxidative stress and have implicated mitochondria as likely contributors to this process. Given this, we hypothesized that heat stress would lead to increased autophagy/mitophagy and a reduction in mitochondrial content. To address this hypothesis pigs were housed in thermoneutral (TN; 20°C) or heat stress (35°C) conditions for 1‐ (HS1) or 3‐ (HS3) days and the red and white portions of the semitendinosus collected. We did not detect differences in glycolytic muscle. Counter to our hypothesis, upstream activation of autophagy was largely similar between groups as were markers of autophagosome nucleation and elongation. LC3A/B‐I increased 1.6‐fold in HS1 and HS3 compared to TN (P < 0.05), LC3A/B‐II was increased 4.1‐fold in HS1 and 4.8‐fold in HS3 relative to TN, (P < 0.05) and the LC3A/B‐II/I ratio was increased 3‐fold in HS1 and HS3 compared to TN suggesting an accumulation of autophagosomes. p62 was dramatically increased in HS1 and HS3 compared to TN. Heat stress decreased mitophagy markers PINK1 7.0‐fold in HS1 (P < 0.05) and numerically by 2.4‐fold in HS3 compared to TN and BNIP3L/NIX by 2.5‐fold (P < 0.05) in HS1 and HS3. Markers of mitochondrial content were largely increased without activation of PGC‐1α signaling. In total, these data suggest heat‐stress‐mediated suppression of activation of autophagy and autophagosomal degradation, which may enable the persistence of damaged mitochondria in muscle cells and promote a dysfunctional intracellular environment.

Detection of miRNA in Mammalian Oocytes and Embryos

MicroRNAs (miRNAs) play a crucial role in the regulation of many post-transcriptional processes in reproductive cells. Regulation of maternal mRNA translation and activation of zygotic mRNA are essential to successful embryonic development. Moreover, the precise development of embryonic cell and/or tissue lineages requires temporal and spatial control of gene expression, mRNA abundance, and translation into proteins, which is in part regulated via miRNA. Here, we describe some key protocols that can be utilized to detect and quantify miRNA in in vitro produced oocytes and embryos.

Regulation of Oogenesis by Noncoding RNAs

Non-coding RNAs (ncRNA) play important regulatory roles in female gamete development, steroidogenesis and female fertility. Among the various types of ncRNA, the role of microRNA influencing mRNA expression and translation during the process of oogenesis is becoming clearer. During fetal development, miRNA are involved in primordial germ cell migration and establishment of sex specific gonads. In the pre- and post-pubertal animal, under the influence of gonadotropins, specific miRNA influence follicle and oocyte growth, oocyte atresia, steroid hormone production and oocyte competence. The importance of post-translational gene regulation by miRNA in oocyte maturation and early stages post-fertilization has been demonstrated via numerous in vitro and in vivo studies across species. Defects in miRNA expression in the ovary at different stages of development can lead to pathologies such as premature ovarian failure, polycystic ovarian syndrome, infertility and ovarian cancer, emphasizing the significance of the regulatory role played by miRNA in oogenesis. Understanding oogenesis and the role of miRNA in this process can help us improve female reproductive potential.