Traditionally, research in the field of trace element biology and human and animal health has largely depended on epidemiological methods to demonstrate involvement in biological processes. These studies were typically followed by trace element supplementation trials or attempts at identification of the biochemical pathways involved. With the discovery of biological molecules that contain the trace elements, such as matrix metalloproteinases containing zinc (Zn), cytochrome P450 enzymes containing iron (Fe), and selenoproteins containing selenium (Se), much of the current research focuses on these molecules, and, hence, only indirectly on trace elements themselves. This review focuses largely on two synchrotron-based x-ray techniques: X-ray absorption spectroscopy and x-ray fluorescence imaging that can be used to identify the in situ speciation and distribution of trace elements in tissues, using our recent studies of bovine ovaries, where the distribution of Fe, Se, Zn, and bromine were determined. It also discusses the value of other techniques, such as inductively coupled plasma mass spectrometry, used to garner information about the concentrations and elemental state of the trace elements. These applications to measure trace elemental distributions in bovine ovaries at high resolutions provide new insights into possible roles for trace elements in the ovary.

The nucleolus is a dynamic nuclear compartment that is mostly involved in ribosome subunit biogenesis; however, it may also play a role in many other biological processes, such as stress response and the cell cycle. Mainly using electron microscopy, several studies have tried to decipher how active nucleoli are set up during early development in mice. In this study, we analyzed nucleologenesis during mouse early embryonic development using 3D-immunofluorescent detection of UBF and Nopp140, two proteins associated with different nucleolar compartments. UBF is a transcription factor that helps maintain the euchromatic state of ribosomal genes; Nopp140 is a phosphoprotein that has been implicated in pre-rRNA processing. First, using detailed image analyses and the in situ proximity ligation assay technique, we demonstrate that UBF and Nopp140 dynamic redistribution between the two-cell and blastocyst stages (time of implantation) is correlated with morphological and structural modifications that occur in embryonic nucleolar compartments. Our results also support the hypothesis that nucleoli develop at the periphery of nucleolar precursor bodies. Finally, we show that the RNA polymerase I inhibitor CX-5461: 1) disrupts transcriptional activity, 2) alters preimplantation development, and 3) leads to a complete reorganization of UBF and Nopp140 distribution. Altogether, our results underscore that highly dynamic changes are occurring in the nucleoli of embryos and confirm a close link between ribosomal gene transcription and nucleologenesis during the early stages of development.

The objectives of this study were to characterize changes in transcriptome of preimplantation conceptuses at the onset of elongation and associated changes in uterine histotroph composition and endometrial physiology. Lactating dairy cows (n = 160) had their ovulation synchronized by artificial insemination (study Day 0). On Day 15, uteri were flushed and endometrium tissue collected. Recovered conceptuses were classified based on morphology/length as ovoid (1–4 mm), tubular (5–19 mm), and filamentous (20–60 mm). Total RNA (n = 48) was subjected to transcriptome analysis. The uterine fluid (n = 30) was evaluated by mass spectrophotometry. Transcriptome analyses revealed drastic changes in the transition from ovoid to tubular and from tubular to filamentous. Differentially expressed genes were associated with cellular movement, cell-to-cell signaling, cellular assembly and organization, lipid metabolism, small molecule biochemistry, and molecular transport. Specific changes included reorganization of cytoskeleton and cell migration, arginine metabolism, growth factors signaling, and lipid metabolism. Functional analysis revealed fatty acids and peroxisome proliferator activated receptor gamma as upstream regulators of transcriptome changes. Expression of PPARG increased 17-fold during the onset of elongation and was highly correlated with genes involved in lipid metabolism. The histotroph was rich in amino acids, lipids, saccharides, and other intermediate metabolites, and important changes in composition occurred in the presence of a conceptus. Pregnancy had a major impact on the concentrations of important lipids in the uterine fluid and expression of genes in the endometrium. Collectively, conceptus elongation involves remarkable changes in transcriptome, composition of the histotroph, and endometrial physiology, which help elucidate important events in uterine and conceptus biology at the onset of elongation.

Using transgenic mice with spermatozoa expressing enhanced green fluorescent protein in their acrosome and red fluorescent protein in their midpiece mitochondria, we followed the behavior of spermatozoa within the female genital tract after natural mating. When examined 15 min after coitus, many spermatozoa were around the opening of the uterotubal junction. Spermatozoa that entered the uterotubal junction were seemingly not moving, yet they steadily migrated toward the isthmus at a speed only time-lapse video recording could demonstrate. Many spermatozoa reaching the lower isthmus were motile. The site where spermatozoa attached and detached from the isthmus epithelium shifted from the lower to the upper segment of the isthmus with time. Virtually all the live spermatozoa within the lower isthmus were acrosome intact, whereas many of the actively motile spermatozoa in the upper isthmus were acrosome reacted. As far as we could observe, all the spermatozoa we found within the lumen of the ampulla and the cumulus oophorus were acrosome reacted. Even though we saw only a very few spermatozoa within the ampulla during fertilization, all were associated with, or were already within, oocytes, indicating that mouse fertilization in vivo is extremely efficient.

Progressive fibrosis is recalcitrant to conventional therapy and commonly complicates chronic diseases and surgical healing. We evaluate here a novel mechanism that regulates scar-tissue collagen (COL1A1/Col1a1) expression and characterizes its translational relevance as a targeted therapy for fibrosis in an endometriosis disease model. Endometriosis is caused by displacement and implantation of uterine endometrium onto abdominal organs and spreads with progressive scarring. Transcription factor KLF11 is specifically diminished in endometriosis lesions. Loss of KLF11-mediated repression of COL1A1/Col1a1 expression resulted in increased fibrosis. To determine the biological significance of COL1A1/Col1a1 expression on fibrosis, we modulated its expression. In human endometrial-stromal fibroblasts, KLF11 recruited SIN3A/HDAC (histone deacetylase), resulting in COL1A1-promoter deacetylation and repression. This role of KLF11 was pharmacologically replicated by a histone acetyl transferase inhibitor (garcinol). In contrast, opposite effects were obtained with a HDAC inhibitor (suberoyl anilide hydroxamic acid), confirming regulatory specificity for these reciprocally active epigenetic mechanisms. Fibrosis was concordantly reversed in Klf11^−/− animals by histone acetyl transferase inhibitor and in wild-type animals by HDAC inhibitor treatments. Aberrant lesional COL1A1 regulation is significant because fibrosis depended on lesion rather than host genotype. This is the first report demonstrating feasibility for targeted pharmacological reversal of fibrosis, an intractable phenotype of diverse chronic diseases.

To accomplish fertilization in the oviductal ampulla, ejaculated sperm are required to migrate through the female reproductive tract. However, this fundamental process largely remains unknown. In this study, we focused on the role of oviductal smooth muscle (myosalpinx) contractions in the sperm migration. Administration of prifinium bromide, padrin, to mice effectively suppressed myosalpinx contractions, resulting in a decreased rate of fertilization in a dose-dependent manner, and an abrogation of high-speed back-and-forth/shuttling flows of oviductal fluids around the isthmus. Regardless of padrin administration, no shuttling flows were found near the ampulla. In the isthmus, sperm formed a tight assemblage that was synchronized with the shuttling flows. The sperm assemblage was gradually loosened and then completely abolished near the ampulla. No sperm assemblage was formed in the isthmus when padrin was administrated. These results suggest that myosalpinx contractions play important roles in the formation of sperm assemblage in the isthmus, and in the transport of the assemblage to the middle region of the oviduct. It is also suggested that the motility of sperm is essential for the migration of sperm from the middle oviductal region to the ampulla.

Women with endometriosis can suffer from decreased fecundity or complete infertility via abnormal oocyte function or impaired placental-uterine interactions required for normal pregnancy establishment and maintenance. Although AT-rich interactive domain 1A (SWI-like) (ARID1A) is a putative tumor suppressor in human endometrial cancers and endometriosis-associated ovarian cancers, little is known about its role in normal uterine function. To study the potential function of ARID1A in the female reproductive tract, we generated mice with a conditional knockout of Arid1a using anti-Müllerian hormone receptor 2-Cre. Female Arid1a conditional knockout mice exhibited a progressive decrease in number of pups per litter, with a precipitous decline after the second litter. We observed no tumors in virgin mice, although one knockout mouse developed a uterine tumor after pregnancy. Unstimulated virgin female knockout mice showed normal oviductal, ovarian, and uterine histology. Uteri of Arid1a knockout mice showed a normal decidualization response and appropriate responses to estradiol and progesterone stimulation. In vitro studies using primary cultures of human endometrial stromal fibroblasts revealed that small interfering RNA knockdown of ARID1A did not affect decidualization in vitro. Timed pregnancy studies revealed the significant resorption of embryos at Embryonic Day 16.5 in knockout mice in the third pregnancy. In addition to evidence of implantation site hemorrhage, pregnant Arid1a knockout mice showed abnormal placental morphology. These results suggest that Arid1a supports successful pregnancy through its role in placental function.

PIWI proteins are members of the larger Argonaute family and bind to specific 24–32 nucleotide RNAs called PIWI-interacting RNAs (piRNAs). PIWI-interacting RNAs direct PIWI-mediated suppression of retrotransposon expression in the male germline in humans and mice, but their roles in bovine reproduction and embryogenesis are unknown. Although the majority of research in mammals has focused on the functions of PIWI proteins during spermatogenesis, this family of proteins and their associated piRNAs have recently been identified in early embryos. The goals of this study were to characterize the expression of PIWIL1 in bovine testis, oocytes, and early embryos. A full-length PIWIL1 transcript and protein was found in the testis, specifically in the germs cells of mature seminiferous tubules. RNA-immunoprecipitation demonstrated the presence of putative piRNAs with a mean length of 30 nucleotides bound to PIWIL1 in testes. 3′-Rapid amplification of cDNA ends analysis of PIWIL1 transcripts in testes and oocytes revealed two shorter isoforms in addition to the full-length transcript that was only present in testes. Truncated PIWIL1 isoforms in oocytes and testes were confirmed through amplification of their unique intronic fragments. Expression profiling of PIWIL1 through early embryogenesis demonstrated peak mRNA expression at the 2-cell stage with decreasing levels through to the blastocyst. PIWIL1-YFP fusion plasmids were produced for each isoform and expressed in HEK 293 cells, demonstrating nuclear exclusion and size-specific banding of the different isoforms. These data represent the first comprehensive characterization of PIWIL1 in bovine, revealing functional similarities with PIWIL1 in other species and suggest tissue-specific expression of several isoforms.

Spermatozoa must penetrate the outer investments of the oocyte, the cumulus oophorus and the zona pellucida (ZP), in order for fertilization to occur. This may require exposure of enzymes on the sperm's inner acrosomal membrane (IAM), one of which is matrix metalloproteinase (MMP) 2, to factors in oviductal fluid. Plasminogen is present in oviductal fluid and activates MMP2 in somatic tissues. The objectives of this study were: 1) to examine possible interactions between plasminogen and IAM-bound plasminogen activator receptor (SAMP14) and -MMP2, 2) to demonstrate plasminogen's presence in the extracellular environment at the site of fertilization, and 3) to provide evidence that plasminogen plays a role in fertilization. Zymographs of sonicated bull and rat sperm extracts incubated with plasmin and/or plasminogen (plasmin/ogen) showed acceleration of initiation of MMP2 activity in concentrations as low as 1 μg/ml. Immunohistochemical and immunofluorescence analysis of plasmin/ogen revealed its presence in the cytoplasm of mouse ovarian and oviductal oocytes, oviductal epithelium, around the ZP, and amongst the cumulus cells. We modified the standard in vitro fertilization (IVF) approach to more closely mimic natural fertilization by reducing sperm concentration during insemination by ∼100× and also comparing cumulus-intact and denuded oocytes. In mice, addition of plasminogen in IVF medium significantly improved fertilization, while MMP2 antibody significantly inhibited sperm penetration in these conditions. IVF improvement by plasminogen was blocked by SAMP14 antibody. Furthermore, MMP2 antibody inhibition was coincident with a failure by spermatozoa to disperse the cumulus oophorus. We provide evidence that plasminogen on its own and through an MMP2-related mechanism improves the ability of oocytes to be fertilized, and demonstrate its effect in sperm penetration of oocyte investments.

Mammalian spermatozoa acquire fertilizing potential as they undergo a series of changes during epididymal transit. One major facet of such is the alterations in the sperm glycome. Modifications of the sialic acid content within glycan moieties are known to regulate epitope presentation and cellular adhesion and signaling, all of which may be critical for sperm to successfully reach and fertilize the egg. To date, there is paucity of information regarding the sialic acid changes that occur on spermatozoa during epididymal transit. Therefore, the aim of this study was to identify N-linked sialylated glycoproteins in rat epididymal sperm and investigate whether they are regulated during epididymal transit. Sialylated glycopeptides from caput, corpus, and cauda spermatozoa were enriched using titanium dioxide beads. Bound N-linked glycopeptides were released by enzymatic deglycosylation using PNGase F and then analyzed by liquid chromatography tandem-mass spectrometry. A total of 92 unique N-linked sialylated glycopeptides were identified from 65 different proteins. These included members of the disintegrin and metalloproteinase domain-containing protein family (ADAM), Basigin, and Testis-expressed protein 101 (TEX101). Remarkably, label-free quantification showed that more than half of these peptides (48/92) were regulated during epididymal transit. Of interest, the protein TEX101 exhibited PNGase F-resistant deglycosylation under the conditions used in this study. The results from this study showed that changes in the N-linked sialoglycoprotein profile is a major hallmark of sperm maturation in rats.

Quiescent sperm survive in cauda epididymis for long periods of time under extreme crowding conditions and with a very limited energy substrate, while after ejaculation, motile sperm live for a much shorter period with an unlimited energy resource and without crowding. Thus, the energy metabolism in relation to the energy requirement of the two may be quite different. A simple physiological technique was evolved to collect viable quiescent sperm from rat cauda epididymis to compare its energy metabolism with motile sperm. Quiescent sperm exhibited 40%–60% higher activities of mitochondrial electron transport chain complexes I–IV and ATP synthase in comparison to motile sperm and accumulated Ca² in the midpiece mitochondria to enhance oxidative phosphorylation (OxPhos). In contrast, motile sperm displayed up to 75% higher activities of key glycolytic enzymes and secreted more than two times the lactate than quiescent sperm. Quiescent sperm phosphorylated AMPK and MAPK-p38, while motile sperm phosphorylated AKT and MAPK/ERK. Glycolytic inhibitor iodoacetamide prevented motility activation of quiescent rat sperm and inhibited conception in rabbits more effectively than OxPhos uncoupler 2,4-dinitrophenol. Apparently, quiescent sperm employ the most energy efficient OxPhos to survive for extended periods of time under extreme conditions of nutrition and crowding. However, on motility initiation, sperm switch predominantly to glycolysis to cater to their high- and quick-energy requirement of much shorter periods. This study also presents a proof of concept for targeting sperm energy metabolism for contraception.

The establishment of the primordial follicle pool is crucial for fertility in mammalian females, and the interruption of overall micro-RNA production by Dicer1 conditional knockout in the female reproductive system results in infertility. However, there are few reports about the functions of individual micro-RNA in regulating primordial follicle assembly. The present study aimed to investigate the function of miR-125b, which is conserved and preferentially expressed in mammalian ovary during primordial follicle assembly. Detection of miR-125b in the developing mouse ovaries by real-time PCR and in situ hybridization showed that it was highly expressed perinatally and specifically located in the ovarian somatic cells. MiR-125b overexpression blocked the process of primordial follicle assembly in cultured newborn mouse ovaries, while its knockdown promoted this process. Further studies showed that miR-125b regulated the activin/Smad2 signaling in neonatal mouse ovary by directly targeting the 3′-untranslated region of activin receptor type 2a (Acvr2a). Overexpression of miR-125b in neonatal mouse ovary suppressed the Acvr2a protein level, attenuating activin/Smad2 signaling, while knockdown of miR-125b showed the opposite effects. In addition, recombinant human activin A (rh-ActA) down-regulated miR-125b in the neonatal mouse ovary. Overexpression of miR-125b attenuated the promoting effects of rh-ActA on primordial follicle assembly. Taken together, these data suggest that miR-125b blocks the process of primordial follicle assembly, and miR-125b may play this role by regulating the expression of Acvr2a in the activin/Smad2 signaling pathway.

The long-term effects of chronic hypothyroidism on ovarian follicular development in adulthood are not well known. Using a rat model of chronic diet-induced hypothyroidism initiated in the fetal period, we investigated the effects of prolonged reduced plasma thyroid hormone concentrations on the ovarian follicular reserve and ovulation rate in prepubertal (12-day-old) and adult (64-day-old and 120-day-old) rats. Besides, antioxidant gene expression, mitochondrial density and the occurrence of oxidative stress were analyzed. Our results show that continuous hypothyroidism results in lower preantral and antral follicle numbers in adulthood, accompanied by a higher percentage of atretic follicles, when compared to euthyroid age-matched controls. Not surprisingly, ovulation rate was lower in the hypothyroid rats. At the age of 120 days, the mRNA and protein content of superoxide dismutase 1 (SOD1) were significantly increased while catalase (CAT) mRNA and protein content was significantly decreased, suggesting a disturbed antioxidant defense capacity of ovarian cells in the hypothyroid animals. This was supported by a significant reduction in the expression of peroxiredoxin 3 (Prdx3), thioredoxin reductase 1 (Txnrd1), and uncoupling protein 2 (Ucp2) and a downward trend in glutathione peroxidase 3 (Gpx3) and glutathione S-transferase mu 2 (Gstm2) expression. These changes in gene expression were likely responsible for the increased immunostaining of the oxidative stress marker 4-hydroxynonenal. Together these results suggest that chronic hypothyroidism initiated in the fetal/neonatal period results in a decreased ovulation rate associated with a disturbance of the antioxidant defense system in the ovary.

Maternal obesity predisposes offspring to metabolic and reproductive dysfunction. We have shown previously that female rat offspring born to mothers fed a high-fat (HF) diet throughout pregnancy and lactation enter puberty early and display aberrant reproductive cyclicity. The mechanisms driving this reproductive phenotype are currently unknown thus we investigated whether changes in ovarian function were involved. Wistar rats were mated and randomized to: dams fed a control diet (CON) or dams fed a HF diet from conception until the end of lactation (HF). Ovaries were collected from fetuses at Embryonic Day (E) 20, and neonatal ovaries at Day 4 (P4), prepubertal ovaries at P27 and adult ovaries at P120. In a subset of offspring, the effects of a HF diet fed postweaning were evaluated. The present study shows that fetuses of mothers fed a HF diet had significantly fewer oocytes at E20, and in neonates, have reduced AMH signaling that may facilitate an increased number of assembled primordial follicles. Both prepubertally and in adulthood, ovaries show increased follicular atresia. As adults, offspring have reduced FSH responsiveness, low expression levels of estrogen receptor alpha (Eralpha), the oocyte-secreted factor, Gdf9, oocyte-specific RNA binding protein, Dazl, and high expression levels of the granulosa-cell derived factor, AMH, in antral follicles. Together, these data suggest that ovarian compromise in offspring born to HF-fed mothers may arise from changes already observable in the fetus and neonate and in the long term, associated with increased follicular atresia through adulthood.

Advanced reproductive age is unequivocally associated with increased aneuploidy in human oocytes, which contributes to infertility, miscarriages, and birth defects. The frequency of meiotic chromosome segregation errors in oocytes derived from reproductively aged mice appears to be similar to that observed in humans, but a limitation of this important model system is our inability to accurately identify chromosome-specific aneuploidy. Here we report the validation and application of a new low-pass whole-genome sequencing approach to comprehensively screen chromosome aneuploidy in individual mouse oocytes and blastocysts. First, we validated this approach by using single mouse embryonic fibroblasts engineered to have stable trisomy 16. We further validated this method by identifying reciprocal chromosome segregation errors in the products of meiosis I (gamete and polar body) in oocytes from reproductively aged mice. Finally, we applied this technology to investigate the incidence of aneuploidy in blastocysts derived from in vitro- and in vivo-matured oocytes in both young and reproductively aged mice. Using this next generation sequencing approach, we quantitatively assessed meiotic and mitotic segregation errors at the single chromosome level, distinguished between errors due to premature separation of sister chromatids and classical nondisjunction of homologous chromosomes, and quantified mitochondrial DNA (mtDNA) segregation in individual cells. This whole-genome sequencing technique, therefore, greatly improves the utility of the mouse model system for the study of aneuploidy and is a powerful quantitative tool with which to examine the molecular underpinnings of mammalian gamete and early embryo chromosome segregation in the context of reproductive aging and beyond.

Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxP system has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carrying cre with a transgenic female carrying the hsc-LRLG gene; cre was driven by rainbow trout vasa regulatory regions and the hsc-LRLG gene was made up of the rainbow trout heat-shock-cognate71 promoter, the DsRed gene flanked by two loxP sites, and the Egfp gene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of the loxP-flanked sequence and activation of Egfp occurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency of loxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two different cre-transgenic males frequently excised the loxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxP system in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.

Although time-lapse analysis of early embryo cleavage parameters (morphokinetics) predicts blastocyst development, it has not been definitively linked to establishing pregnancy and live birth. For example, a direct comparison of the developmental potential of embryos with optimal kinetic parameters compared to suboptimal kinetics has not been performed with human embryos. To ascertain whether such a linkage exists, we developed a mouse model of morphokinetic analysis of early embryo cleavage using time-lapse microscopy to predict blastocyst formation and tested whether cleavage parameters predict pregnancy outcome by transferring morphokinetically optimal and suboptimal embryos into a single host. Using classification and regression trees, we established that the timing of the second and third mitotic divisions (division from two to three and three to four cells, respectively) predicts blastocyst development in the mouse. Using this prediction model, we found that the incidence of sustained implantation at mid-gestation was significantly higher for the optimal compared to suboptimal embryos. In addition, the incidence of resorption among implanted embryos was significantly higher in the suboptimal compared to the optimal group. Transcript profiling of optimal and suboptimal embryos revealed minimal differences between the two groups, suggesting that time-lapse imaging of early embryo cleavage events provides additional information regarding developmental competence apart from gene expression.

The success of semen cryopreservation depends on sperm membrane integrity and function after thawing. Cholesterol-loaded cyclodextrin (CLC) is used for in vitro incorporation of cholesterol to protect cells against cold temperatures. We hypothesized that CLC treatment also enhances sperm cholesterol content to increase tolerance to osmotic shock and cryoresistance, thereby improving fertility. We confirmed the fact that treatment of goat semen with 3 mg/ml CLC increases sperm cholesterol content using both the Liebermann-Burchard approach and filipin III labeling of membrane cholesterol. Sperm were then treated with or without CLC and cryopreserved. After thawing, sperm cholesterol dramatically fell, even in the presence of CLC, which explains the mechanism of cryocapacitation. CLC treatment, however, maintained a normal prefreeze cholesterol level in sperm after cryopreservation. Furthermore, fresh sperm treated with CLC and subjected to either cold shock or incubated in hypo-, iso-, and hyperosmotic media, designed to mimic stresses associated with freezing/thawing, displayed increased temperature and osmotic tolerance. CLC treatment also improved sperm viability, motility, and acrosome integrity after thawing. Furthermore, CLC treatment did not affect the sperm's ability to undergo in vitro capacitation according to chlortetracycline fluorescence and protein tyrosine phosphorylation. A pilot field trial demonstrated that artificial insemination with sperm that underwent increased cholesterol levels following CLC treatment yielded higher fertility (P < 0.1) and proliferation (P < 0.05) rates in vivo than untreated semen from the same ejaculate samples. These observations suggest that CLC treatment could be used to improve cryoprotection during the freezing and thawing of goat sperm.

Thousands of long noncoding RNAs (lncRNAs) have been identified in mouse, rat, and human testes, some of which play important roles in testis development and spermatogenesis. However, systematic analysis of lncRNAs expressed in postnatal pig testes has not been reported. Thus, in this study, we present the expression and characterization of lncRNAs in immature (30-day-old [D30]) and mature (180-day-old [D180]) pig testes. A total of 90 440 168 (85.75%) and 97 001 700 (95.35%) 150-base-pair paired-end clean reads were generated in D30 and D180 cDNA libraries, respectively, using the Illumina HiSeq 4000 platform; 36 727 transcripts were assembled in those two libraries, 777 lncRNA transcripts from 752 lncRNA gene loci were identified using the highly stringent pipeline, and 101 of those lncRNA transcripts were significantly differentially expressed. Those lncRNAs shared some characteristics with other mammals, including fewer exons, shorter length and exon length, and lower expression level compared with those of protein-coding genes; 402 protein-coding genes (<10 kb) were found as nearest neighbors of 294 out of 752 lncRNA genes, and gene ontology enrichment showed that they were enriched in transcription- and development-related processes. Fifteen differentially expressed and 10 novel lncRNAs were randomly selected and validated by quantitative polymerase chain reaction (PCR) and reverse transcriptase PCR. In addition, one of the 10 novel lncRNAs was further confirmed using RACE clone technology. This study provides a catalog of porcine testes lncRNAs for further understanding their regulation roles in pig testis development and spermatogenesis.

Bone morphogenetic protein (BMP) signaling plays an important role in spermatogenesis and follicle development. Our previous studies have shown that repulsive guidance molecule b (RGMb, also known as Dragon) is a coreceptor that enhances BMP2 and BMP4 signaling in several cell types and that RGMb is expressed in spermatocytes and spermatids in the testis and in oocytes of the secondary follicles in the ovary. Here, we demonstrated that specific deletion of Rgmb in germ cells in the testis and ovary did not alter Smad1/5/8 phosphorylation, gonadal structures, and fertility. In addition, ovaries from postnatal global Rgmb knockout mice showed similar structures to the wild-type ovaries. Our results suggest that RGMb is not essential for normal gonadal function.

The F-box and leucine-rich repeat protein 10 (Fbxl10) gene encodes a protein that catalyzes demethylation of H3K4 and H3K36. In this study, we show the important roles of FBXL10 as a histone demethylase in sustainable sperm production using mice in which the JmjC domain of Fbxl10 was deleted (Fbxl10^{DeltaJ/DeltaJ}). In histological analysis, testis sections from 10-wk-old Fbxl10^{DeltaJ/DeltaJ} mice appeared normal. On the other hand, testes from 7-mo-old Fbxl10^{DeltaJ/DeltaJ} mice contained a greater ratio of seminiferous tubules exhibiting degeneration of spermatogenesis. Further analysis using an in vitro spermatogonia culture system, that is, germline stem cells (GSCs), revealed that Fbxl10^{DeltaJ/DeltaJ} GSCs expressed a significantly higher level of P21 and P19 mRNA, cyclin-dependent kinase inhibitors and also known as cellular senescence markers, than wild-type (WT) GSCs. Furthermore, the ratio of Fbxl10^{DeltaJ/DeltaJ} GSCs in G0/G1 phase was higher and the ratios in S and G2/M phases were lower than the corresponding ratios of WT GSCs, and the doubling speed of Fbxl10^{DeltaJ/DeltaJ} GSCs was significantly slower than that of WT GSCs. In addition to these in vitro results, an in vivo study indicated that recovery of spermatogenesis after a transient reduction in the number of testicular germ cells by busulfan treatment was significantly slower in Fbxl10^{DeltaJ/DeltaJ} mice than in WT mice. These data suggest that Fbxl10 plays important roles in long-term sustainable spermatogenesis via regulating cell cycle.

The coadministration of bleomycin, etoposide, and cisplatin (BEP) has increased the survival rate of testicular cancer patients to over 90%. Previous studies have demonstrated that BEP induces germ cell damage during the final stages of spermatogenesis, when major chromatin remodeling occurs. Chromatin remodeling permits histone-protamine exchange, resulting in sperm head chromatin compaction. This process involves different epigenetic modifications of the core histones. The objective of these studies was to investigate the effects of BEP on epigenetic modifications to histones involved in chromatin remodeling. Brown Norway rats were treated with BEP, and their testes were removed to isolate pachytene spermatocytes and round spermatids by unit gravity sedimentation. Western blot analyses were conducted on extracted proteins to detect the expression of key modified histones. In a second cohort testes were prepared for immunohistochemical analysis. The stage-specific expression of each modified histone mark in rat spermatogenesis suggests the involvement of these modifications in chromatin remodeling. BEP treatment significantly increased expression of H3K9m and decreased that of tH2B (or Hist1h2ba) in pachytene spermatocytes, suggesting that nucleosomes were not destabilized to allow for transcription of genes involved in chromatin remodeling. Moreover, BEP treatment altered the expression of H4K8ac in round and elongating spermatids, suggesting that histone eviction was compromised, leading to a looser chromatin structure in mature spermatozoa. Less-compacted sperm chromatin, with alterations to the sperm epigenome, may have an adverse effect on male fertility.