Retinoic acid is dispensable for meiotic initiation but required for spermiogenesis in the mammalian testis.

Retinoic acid (RA) is the proposed mammalian 'meiosis inducing substance'. However, evidence for this role comes from studies in the fetal ovary, where germ cell differentiation and meiotic initiation are temporally inseparable. In the postnatal testis, these events are separated by more than 1 week. Exploiting this difference, we discovered that, although RA is required for spermatogonial differentiation, it is dispensable for the subsequent initiation, progression and completion of meiosis. Indeed, in the absence of RA, the meiotic transcriptome program in both differentiating spermatogonia and spermatocytes entering meiosis was largely unaffected. Instead, transcripts encoding factors required during spermiogenesis were aberrant during preleptonema, and the subsequent spermatid morphogenesis program was disrupted such that no sperm were produced. Taken together, these data reveal a RA-independent model for male meiotic initiation.

Characterization of the effects of heat stress on autophagy induction in the pig oocyte.

BACKGROUND: Heat stress (HS) occurs when body heat accumulation exceeds heat dissipation and is associated with swine seasonal infertility. HS contributes to compromised oocyte integrity and reduced embryo development. Autophagy is a potential mechanism for the oocyte to mitigate the detrimental effects of HS by recycling damaged cellular components. METHODS: To characterize the effect of HS on autophagy in oocyte maturation, we utilized an in vitro maturation (IVM) system where oocytes underwent thermal neutral (TN) conditions throughout the entire maturation period (TN/TN), HS conditions during the first half of IVM (HS/TN), or HS conditions during the second half of IVM (TN/HS). RESULTS: To determine the effect of HS on autophagy induction within the oocyte, we compared the relative abundance and localization of autophagy-related proteins. Heat stress treatment affected the abundance of two well described markers of autophagy induction: autophagy related gene 12 (ATG12) in complex with ATG5 and the cleaved form of microtubule-associated protein 1 light chain 3 beta (LC3B-II). The HS/TN IVM treatment increased the abundance of the ATG12-ATG5 complex and exacerbated the loss of LC3B-II in oocytes. The B-cell lymphoma 2 like 1 protein (BCL2L1) can inhibit autophagy or apoptosis through its interaction with either beclin1 (BECN1) or BCL2 associated X, apoptosis regulator (BAX), respectively. We detected colocalization of BCL2L1 with BAX but not BCL2L1 with BECN1, suggesting that apoptosis is inhibited under the HS/TN treatment but not autophagy. Interestingly, low doses of the autophagy inducer, rapamycin, increased oocyte maturation. CONCLUSIONS: Our results here suggest that HS increases autophagy induction in the oocyte during IVM, and that artificial induction of autophagy increases the maturation rate of oocytes during IVM. These data support autophagy as a potential mechanism activated in the oocyte during HS to recycle damaged cellular components and maintain developmental competence.

Acyl-CoA synthetase 6 enriches seminiferous tubules with the ω-3 fatty acid docosahexaenoic acid and is required for male fertility in the mouse.

Docosahexaenoic acid (DHA) is an ω-3 dietary-derived polyunsaturated fatty acid of marine origin enriched in testes and necessary for normal fertility, yet the mechanisms regulating the enrichment of DHA in the testes remain unclear. Long-chain ACSL6 (acyl-CoA synthetase isoform 6) activates fatty acids for cellular anabolic and catabolic metabolism by ligating a CoA to a fatty acid, is highly expressed in testes, and has high preference for DHA. Here, we investigated the role of ACSL6 for DHA enrichment in the testes and its requirement for male fertility. Acsl6-/- males were severely subfertile with smaller testes, reduced cauda epididymal sperm counts, germ cell loss, and disorganization of the seminiferous epithelium. Total fatty acid profiling of Acsl6-/- testes revealed reduced DHA and increased ω-6 arachidonic acid, a fatty acid profile also reflected in phospholipid composition. Strikingly, lipid imaging demonstrated spatial redistribution of phospholipids in Acsl6-/- testes. Arachidonic acid-containing phospholipids were predominantly interstitial in control testes but diffusely localized across Acsl6-/- testes. In control testes, DHA-containing phospholipids were predominantly within seminiferous tubules, which contain Sertoli cells and spermatogenic cells but relocalized to the interstitium in Acsl6-/- testes. Taken together, these data demonstrate that ACSL6 is an initial driving force for germ cell DHA enrichment and is required for normal spermatogenesis and male fertility.

Inhibition of germinal vesicle breakdown using IBMX increases microRNA-21 in the porcine oocyte.

BACKGROUND: Germinal vesicle breakdown (GVBD) occurs during oocyte meiotic maturation, a period when transcriptional processes are virtually inactive. Thus, the maturing oocyte is reliant on processes such as post-transcriptional gene regulation (PTGR) to regulate the mRNA and protein repertoire. MicroRNA (miRNA) are a class of functional small RNA that target mRNA to affect their abundance and translational efficiency. Of particular importance is miRNA-21 (MIR21) due to its role in regulating programmed cell death 4 (PDCD4). The objective of this study was to characterize the abundance and regulation of MIR21 in relation to GVBD. METHODS: Oocytes were collected from aspirated porcine tertiary follicles. Relative abundance of mature MIR21 was quantified at 0, 8, 16, 24, 32, and 42 h of in vitro (IVM) with or without treatment with 3-isobutyl-1-methylxanthine (IBMX). RESULTS: IBMX increased abundance of MIR21 at 24 h approximately 30-fold compared to control oocytes (P < 0.05), and the induced increase in MIR21 abundance at 24 h was concomitant with premature depletion of PDCD4 protein abundance. To characterize the effect of artificially increasing MIR21 on oocyte competence without inhibiting GVBD, a MIR21 mimic, scrambled microRNA negative control, or nuclease free water was micro-injected into denuded oocytes at 21 h of IVM. The maturation rate of oocytes injected with synthetic MIR21 (63.0 ± 7.5%) was higher than oocytes injected with negative controls (P < 0.05). CONCLUSIONS: Inhibition of nuclear meiotic maturation via IBMX significantly increased MIR21 and decreased its target, PDCD4. Injection of a MIR21 mimic increased oocyte maturation rate. Our results indicate MIR21 is active and important during meiotic maturation of the oocyte.

Heat stress induces autophagy in pig ovaries during follicular development.

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 autophagy-related 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.

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 animal's 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.

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

BACKGROUND: MicroRNA (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. METHODS: The 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. RESULTS: During 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. CONCLUSIONS: MIR21 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.

Small RNAs: Their Possible Roles in Reproductive Failure.

Posttranscriptional gene regulation is a regulatory mechanism which occurs "above the genome" and confers different phenotypes and functions within a cell. Transcript and protein abundance above the level of transcription can be regulated via noncoding ribonucleic acid (ncRNA) molecules, which potentially play substantial roles in the regulation of reproductive function. MicroRNA (miRNA), endogenous small interfering RNA (endo-siRNA), and PIWI-interacting RNA (piRNA) are three primary classes of small ncRNA. Similarities and distinctions between their biogenesis and in the interacting protein machinery that facilitate their function distinguish these three classes. Characterization of the expression and importance of the critical components for the biogenesis of each class in different tissues contributes a clearer understanding of their contributions in specific reproductive tissues and their ability to influence fertility in both males and females. This chapter discusses the expression and potential roles of miRNA, endo-siRNA, and piRNA in the regulation of reproductive function. Additionally, this chapter elaborates on investigations aimed to address and characterize specific mechanisms through which miRNA may influence infertility and the use of miRNA as biomarkers associated with several reproductive calamities such as defective spermatogenesis in males, polycystic ovarian failure, endometriosis and obesity, and chemical-induced subfertility.

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.

Pyruvate modulation of redox potential controls mouse sperm motility.

Sperm gain fertilization competence in the female reproductive tract through a series of biochemical changes and a requisite switch from linear progressive to hyperactive motility. Despite being essential for fertilization, regulation of sperm energy transduction is poorly understood. This knowledge gap confounds interpretation of interspecies variation and limits progress in optimizing sperm selection for assisted reproduction. Here, we developed a model of mouse sperm bioenergetics using metabolic phenotyping data, quantitative microscopy, and spectral flow cytometry. The results define a mechanism of motility regulation by microenvironmental pyruvate. Rather than being consumed as a mitochondrial fuel source, pyruvate stimulates hyperactivation by repressing lactate oxidation and activating glycolysis in the flagellum through provision of nicotinamide adenine dinucleotide (NAD)+. These findings provide evidence that the transitions in motility requisite for sperm competence are governed by changes in the metabolic microenvironment, highlighting the unexplored potential of using catabolite combination to optimize sperm selection for fertilization.

Identification of measures predictive of age of puberty onset in gilts.

A potential indicator of female lifetime productivity in swine is age of puberty, when a gilt achieves her first behavioral estrus. Follicular activity, as determined by tertiary follicle development, in prepubertal gilts begins during postnatal day (PND) 75 to 115. The central hypothesis of this study is that gilts demonstrating tertiary follicle development earlier in life, assessed using vulva size as a proxy, achieve puberty earlier in life compared with counterparts of a similar age and weight that lack tertiary follicle development. The objectives of this project were to identify a developmental time point when variation in ovarian development exists and to determine whether a relationship between the age prepubertal ovarian development and the age at onset of puberty exists. To accomplish this, 155 gilts of similar age (± 2 d) were weighed and vulva size measured on PND 75, 85, 95, 105, and 115. Vulva measures, including vulva width (VW), vulva length (VL), and vulva area (VA), were utilized as developmental proxies for follicular activity. At each time point, gilts (n = 10) were sacrificed and ovarian follicular activity recorded. In a subset of gilts (n = 105), estrus detection was conducted daily on PND days 126 to 200. Mean VA on PND 75, 85, 95, 105, and 115 was 596 ± 206, 683 ± 190, 864 ± 212, 1014 ± 228, and 1265 ± 252 mm2, respectively. Of the gilts demonstrating behavioral estrus, 28 were within PND 140 to 160, 36 between PND 161 to 180, 15 between PND 181 to 200, and 26 did not demonstrate estrus behavior within 200 d of age. All gilts euthanized at PND 75 lacked follicular activity as defined by having a minimum of 2 antral follicles per ovary, whereas 60%, 80%, 90%, and 100% demonstrated follicular activity on PND 85, 95, 105, and 115, respectively. Body weight at PND 75 and VW at PND 115 were correlated to age at first estrus (P < 0.05). Of the gilts whose VA was less than 1 SD from the mean on PND 95 (i.e., <652 mm2), 31% and 50% demonstrated their first behavioral estrus by PND 180 and 200, respectively. However, of gilts whose VA was within or greater than 1 SD of the mean (i.e., ≥652 mm2), 66% and 79% exhibited estrus prior to PND 180 and 200, respectively. These data support utilization of VA changes between 95 and 115 d of age as a useful tool to identify replacement gilts prior to puberty for inclusion into the sow herd.

Methods for reproductive tract scoring as a tool for improving sow productivity.

Improving sow lifetime productivity is essential for maximizing farm profitability. Study objectives were to determine the accuracy for different vulva scoring methods in a commercial production system and to assess whether gilt reproductive tract scoring [evaluated by vulva width (VW)] prior to puberty could serve as useful gilt selection criteria. To accomplish this objective, 958 prepubertal replacement gilts in a commercial system were evaluated at approximately 15 wk of age. Gilt body weight (BW) was recorded in addition to 4 different methods to evaluate VW. Methods for VW assessment included digital caliper measurement (mm), visual evaluation and scoring by trained farm personnel [Farm Score (FS)], and 2 methods using scoring tools [Vulva Score Method A and B (VSA and VSB, respectively)] specifically calibrated from the VW distribution measured on gilts from previous studies. The VSA and FS methods assigned gilts to one of 3 categories (S, M, L, and 1, 2, 3, respectively) whereas VSB classified gilts vulvas using a 5-point scoring system (1 to 5). At 15 wk of age, a low proportion of variability in vulva size (27.8 ± 0.1 mm) could be explained by BW (62.2 ± 0.2 kg; R 2 = 0.05). All 3 scoring methods were effective in categorizing gilts based upon VW, as the measured VW size within methods differed by score (P < 0.01). The proportion of gilts achieving their first parity increased with score for VSA (64.7%, 73.2%, and 84.4%; P = 0.02), VSB (66.0%, 71.7%, 79.2%, 76.4%, and 84.2%; P = 0.02), and FS (67.2%, 75.0%, and 88.8%; P = 0.03), but VSA, VSB, and FS did not influence percentage of gilts achieving their second parity (P = 0.32, 0.29, and 0.30, respectively). Litter performance of gilts scored as M or L using VSA improved with an increased total born over 2 parities compared to those scored as S (23.96 vs. 26.38 pigs; P < 0.01) as well as born alive (21.13 vs. 23.05 pigs; P < 0.05). Results were similar for VSB, where scores 2 to 5 had greater total born (23.97 vs. 26.33 pigs; P < 0.01) and born alive (21.11 vs. 23.02 pigs; P < 0.05) through 2 parities compared to gilts scored 1. Using the FS method, total born pigs tended to be increased (P = 0.06) through 2 parities for gilts having a 2 or 3 vulva score compared to those scored as a 1. Collectively, assessing VW at approximately 15 wk of age may identify sows with improved productivity through 2 parities as breeding herd females.

Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis.

Spermatogonial stem cells (SSCs) both self-renew and give rise to progenitors that initiate spermatogenic differentiation in the mammalian testis. Questions remain regarding the extent to which the SSC and progenitor states are functionally distinct. Here we provide the first multiparametric integrative analysis of mammalian germ cell epigenomes comparable with that done for >100 somatic cell types by the ENCODE Project. Differentially expressed genes distinguishing SSC- and progenitor-enriched spermatogonia showed distinct histone modification patterns, particularly for H3K27ac and H3K27me3. Motif analysis predicted transcription factors that may regulate spermatogonial subtype-specific fate, and immunohistochemistry and gene-specific chromatin immunoprecipitation analyses confirmed subtype-specific differences in target gene binding of a subset of these factors. Taken together, these results show that SSCs and progenitors display distinct epigenetic profiling consistent with these spermatogonial subtypes being differentially programmed to either self-renew and maintain regenerative capacity as SSCs or lose regenerative capacity and initiate lineage commitment as progenitors.

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.

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.