Bone loss is associated with aging and is exacerbated by female transition tomenopause. Estrogen has long been associated with bone loss, and replacement therapy is effective at restoring bone health. In the last 10 years, pituitary and gonadal peptide hormones have been implicated in bone biology, and here we update the story on FSH and its potential role as a direct regulator of bone and adipose. If translated to humans, new approaches to ameliorate age related bone demise could be developed. Moreover, young women with hormone responsive cancers who are unable to maintain bone may have new routes to avoid debilitating bone loss.

Summary Sentence

Bone loss in women could be explained by high FSH and not loss of estradiol.

Bone loss is associated with aging and is exacerbated by female transition tomenopause. Estrogen has long been associated with bone loss, and replacement therapy is effective at restoring bone health. In the last 10 years, pituitary and gonadal peptide hormones have been implicated in bone biology, and here we update the story on FSH and its potential role as a direct regulator of bone and adipose. If translated to humans, new approaches to ameliorate age related bone demise could be developed. Moreover, young women with hormone responsive cancers who are unable to maintain bone may have new routes to avoid debilitating bone loss.

Summary Sentence

Bone loss in women could be explained by high FSH and not loss of estradiol.

Not only essential for scientific research, but also in the analysis of male fertility and for animal husbandry, sperm tracking and characterization techniques have been greatly benefiting from computational imaging. Digital image sensors, in combination with optical microscopy tools and powerful computers, have enabled the use of advanced detection and tracking algorithms that automatically map sperm trajectories and calculate various motility parameters across large data sets. Computational techniques are driving the field even further, facilitating the development of unconventional sperm imaging and tracking methods that do not rely on standard optical microscopes and objective lenses, which limit the field of view and volume of the semen sample that can be imaged. As an example, a holographic on-chip sperm imaging platform, only composed of a light-emitting diode and an opto-electronic image sensor, has emerged as a high-throughput, lowcost and portable alternative to lens-based traditional sperm imaging and tracking methods. In this approach, the sample is placed very close to the image sensor chip, which captures lensfree holograms generated by the interference of the background illumination with the light scattered from sperm cells. These holographic patterns are then digitally processed to extract both the amplitude and phase information of the spermatozoa, effectively replacing the microscope objective lens with computation. This platform has further enabled high-throughput 3D imaging of spermatozoa with submicron 3D positioning accuracy in large sample volumes, revealing various rare locomotion patterns. We believe that computational chip-scale sperm imaging and 3D tracking techniques will find numerous opportunities in both sperm related research and commercial applications.

Summary Sentence

Computational techniques are facilitating the development of unconventional sperm imaging and locomotion tracking methods that do not rely on standard opticalmicroscopes and objective lenses.

Not only essential for scientific research, but also in the analysis of male fertility and for animal husbandry, sperm tracking and characterization techniques have been greatly benefiting from computational imaging. Digital image sensors, in combination with optical microscopy tools and powerful computers, have enabled the use of advanced detection and tracking algorithms that automatically map sperm trajectories and calculate various motility parameters across large data sets. Computational techniques are driving the field even further, facilitating the development of unconventional sperm imaging and tracking methods that do not rely on standard optical microscopes and objective lenses, which limit the field of view and volume of the semen sample that can be imaged. As an example, a holographic on-chip sperm imaging platform, only composed of a light-emitting diode and an opto-electronic image sensor, has emerged as a high-throughput, lowcost and portable alternative to lens-based traditional sperm imaging and tracking methods. In this approach, the sample is placed very close to the image sensor chip, which captures lensfree holograms generated by the interference of the background illumination with the light scattered from sperm cells. These holographic patterns are then digitally processed to extract both the amplitude and phase information of the spermatozoa, effectively replacing the microscope objective lens with computation. This platform has further enabled high-throughput 3D imaging of spermatozoa with submicron 3D positioning accuracy in large sample volumes, revealing various rare locomotion patterns. We believe that computational chip-scale sperm imaging and 3D tracking techniques will find numerous opportunities in both sperm related research and commercial applications.

Summary Sentence

Computational techniques are facilitating the development of unconventional sperm imaging and locomotion tracking methods that do not rely on standard opticalmicroscopes and objective lenses.

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. Thiswork analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30–40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ∼4 × 10⁴ cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cellmass were separated. We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation ismore similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping.

Summary Sentence

An in vitro expanded trophectoderm biopsy is representative of embryo trophectoderm in terms of methylation, so is a suitable proxy for bovine embryo epigenotyping.

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. Thiswork analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30–40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ∼⃒4 × 10⁴ cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cellmass were separated. We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation ismore similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping.

Summary Sentence

An in vitro expanded trophectoderm biopsy is representative of embryo trophectoderm in terms of methylation, so is a suitable proxy for bovine embryo epigenotyping.

In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any antiluteolytic endocrine signal as found in other species that modulate uterine function during the critical period of pregnancy establishment. Nevertheless, in the dog an embryo-maternal communication must occur in order to prevent rejection of embryos. Based on this hypothesis, we performed microarray analysis of canine uterine samples collected during pre-attachment phase (days 10-12) and in corresponding non-pregnant controls, in order to elucidate the embryo attachment signal. An additional goal was to identify differences in uterine responses to pre-attachment embryos between dogs and other mammalian species exhibiting different reproductive patterns with regard to luteolysis, implantation, and preparation for placentation. Therefore, the canine microarray data were compared with gene sets from pigs, cattle, horses, and humans. We found 412 genes differentially regulated between the two experimental groups. The functional terms most strongly enriched in response to pre-attachment embryos related to extracellular matrix function and remodeling, and to immune and inflammatory responses. Several candidate genes were validated by semi-quantitative PCR. When compared with other species, best matches were found with human and equine counterparts. Especially for the pig, the majority of overlapping genes showed opposite expression patterns. Interestingly, 1926 genes did not pair with any of the other gene sets. Using a microarray approach, we report the uterine changes in the dog driven by the presence of embryos and compare these results with datasets from other mammalian species, finding common-, contrary-, and exclusively canine-regulated genes.

Summary Sentence

Pre-implantation embryos invoke functional changes in the canine uterus related to ongoing structural remodeling and immunological modulation; comparisons with different mammals reveal similarities and differences in maternal pregnancy recognition.

The first sign of mammalian germ cell sexual differentiation is the initiation of meiosis in females and of mitotic arrest in males. In the mouse, retinoic acid induces ovarian Stra8 expression and entry of germ cells into meiosis. In developing mouse testes, cytochrome P450 family 26, subfamily b, polypeptide 1 (CYP26B1) produced by the Sertoli cells degrades retinoic acid, preventing Stimulated by Retinoic Acid Gene 8 (Stra8), expression and inhibiting meiosis. However, in developing humans, there is no evidence that CYP26B1 acts a meiosis-inhibiting factor. We therefore examined aspects of the retinoic acid/STRA8/CYP26B1 pathway during gonadal development in the tammar wallaby, a marsupial, to understand whether retinoic acid stimulation of STRA8 and CYP26B1 degradation of retinoic acid was conserved between widely divergent mammals. In tammar ovaries, as in human ovaries and unlike the pattern in mice, CYP26B1 expression was not downregulated before the onset of meiosis. Exposure of pre-meiotic tammar ovaries to exogenous retinoic acid in vitro upregulated STRA8 expression compared to controls. We conclude that retinoic acid and STRA8 are conserved factors that control the initiation of meiosis amongst mammals but the role of CYP26B1 as a meiosis-inhibiting factor may be specific to rodents. The identity of the marsupial meiosis-inhibiting factor remains unknown.

Summary Sentence

This comparative study using a marsupial species suggests that the roles of retinoic acid and STRA8 but not CYP26B1 in controlling the initiation of meiosis are conserved between different mammals.

The P2X7 is an adenosine triphosphate (ATP)-gated ion channel involved in several facets of immune activation and neuronal function through its importance in interleukin (IL)-1β secretion. We hypothesized that blockade of P2X7 would prevent perinatal brain injury associated with exposure to intrauterine (IU) inflammation. Dams received 45 mg/kg of Brilliant Blue G (BBG), a specific P2X7 receptor (P2X7R) antagonist, on gestation day 17 (E17) prior to administration of lipopolysaccharide (LPS) or phosphate-buffered saline (PBS). Furthermore, we utilized embryo transfer experiments to delineate whether the P2X7 was the key mediator of IU inflammation-associated brain injury on maternal or fetal sides. In these experiments, P2X7^–/– dams were embryo-transferred wild type embryos and wild type dams were embryo-transferred P2X7^–/– embryos. In the mousemodel of intrauterine inflammation, pharmacologic blockade of P2X7R reduced preterm birth rate, improved offspring performance on neuromotor tests as well as the dendritic arborization and density of cortical neurons. Embryo transfer experiments demonstrated the importance of maternal P2X7R in IU inflammation-mediated effects on offspring. Both genetic and pharmacologic blockade of IL-1β signaling, by targeting maternal P2X7R, ameliorated perinatal brain injury following exposure to IU inflammation. Specific targeting of maternal P2X7R may provide a clinically useful tool to prevent both preterm birth and prematurity-associated perinatal brain injury, and further studies are urgently needed.

Summary Sentence

Brilliant Blue G, a specific P2X7 channel blocker, prevents preterm birth and perinatal brain injury associated with exposure to intrauterine inflammation suggesting a role for IL-1β in both regulation of preterm birth and neuronal damage.

Feeding-related metabolic factors exert regulatory influences on the hypothalamic–pituitary–gonadal axis. Glucagon-like peptide-1 (GLP-1) is an anorexigenic hormone synthesized from the ileum in response to food intake. The purpose of this study was to examine the direct effect of GLP-1 on hypothalamic kisspeptin and gonadotropin-releasing hormone (GnRH) expression using the rat clonal hypothalamic cell line rHypoE-8. GLP-1 significantly increased Kiss-1 mRNA expression in rHypoE-8 cells up to 1.94 ± 0.22-fold. This effect of GLP-1 on Kiss-1 gene expression was also observed in GT1–7 GnRH-producing neurons and in primary cultures of fetal rat brain. GLP-1 increased cAMP-mediated signaling, as determined by cAMP response element activity assays, but failed to activate extracellular signal-regulated kinase pathways. Another anorexigenic factor, leptin, similarly increased Kiss-1 mRNA levels up to 1.34 ± 0.08-fold in rHypoE-8 cells. However, combined treatment with GLP-1 and leptin failed to potentiate their individual effects on Kiss-1 mRNA expression. Gnrh mRNA expression was not significantly increased by GLP-1 stimulation in rHypoE-8, but kisspeptin significantly stimulated the expression of Gnrh mRNA in these cells. Our current observations suggest that the anorexigenic peptide GLP-1 directly regulates Kiss-1 mRNA expression in these hypothalamic cell lines and in neuronal cells of fetal rat brain and affects the expression of Gnrh mRNA.

Summary Sentence

Anorexigenic peptide GLP-1 directly regulates Kiss-1 mRNA expression in thypothalamic cell lines and affects the expression of Gnrh mRNA.

Feeding-related metabolic factors exert regulatory influences on the hypothalamic–pituitary–gonadal axis. Glucagon-like peptide-1 (GLP-1) is an anorexigenic hormone synthesized from the ileum in response to food intake. The purpose of this study was to examine the direct effect of GLP-1 on hypothalamic kisspeptin and gonadotropin-releasing hormone (GnRH) expression using the rat clonal hypothalamic cell line rHypoE-8. GLP-1 significantly increased Kiss-1 mRNA expression in rHypoE-8 cells up to 1.94 ± 0.22-fold. This effect of GLP-1 on Kiss-1 gene expression was also observed in GT1–7 GnRH-producing neurons and in primary cultures of fetal rat brain. GLP-1 increased cAMP-mediated signaling, as determined by cAMP response element activity assays, but failed to activate extracellular signal-regulated kinase pathways. Another anorexigenic factor, leptin, similarly increased Kiss-1 mRNA levels up to 1.34 ± 0.08-fold in rHypoE-8 cells. However, combined treatment with GLP-1 and leptin failed to potentiate their individual effects on Kiss-1 mRNA expression. Gnrh mRNA expression was not significantly increased by GLP-1 stimulation in rHypoE-8, but kisspeptin significantly stimulated the expression of Gnrh mRNA in these cells. Our current observations suggest that the anorexigenic peptide GLP-1 directly regulates Kiss-1 mRNA expression in these hypothalamic cell lines and in neuronal cells of fetal rat brain and affects the expression of Gnrh mRNA.

Summary Sentence

Anorexigenic peptide GLP-1 directly regulates Kiss-1 mRNA expression in thypothalamic cell lines and affects the expression of Gnrh mRNA.

Intrauterine growth restriction (IUGR) is caused by dysregulation of placental metabolism. Paternally inherited IUGR mutations in the fetus influence maternal physiology via the placenta. However, it is not known whether the maternal placenta also affects the extent of IUGR in such fetuses. In cattle and other ruminants, maternal–fetal communication occurs primarily at the placentomes. We previously identified a 3′ deletion in the noncoding MER1 repeat containing imprinted transcript 1 (MIMT1) gene that, when inherited from the sire, causes IUGR and late abortion in Ayshire cattle with variable levels of severity. Here, we compared the transcriptome and genomic imprinting in fetal and maternal placentome components of wild-type and MIMT1^Del/WT fetuses before IUGR became apparent, to identify key early events. Transcriptome analysis revealed fewer differentially expressed genes in maternal than fetal MIMT1^Del/WT placentome. AST1, within the PEG3 domain, was the only gene consistently reduced in IUGR in both fetal and maternal samples. Several genes showed an imprinting pattern associated with IUGR, of which only secernin 3 (SCRN3) and paternally expressed 3 (PEG3) were differentially imprinted in both placentome components. Loss of strictly monoallelic, allele-specific expression (∼80:20) of PEG3 in the maternal MIMT1^Del/WT placenta could be associated with incomplete penetrance of MIMT1^Del. Our data show that dysregulation of the PEG3 domain is involved in IUGR, but also reveal that maternal placental tissues may affect the penetrance of the paternally inherited IUGR mutation.

Summary Sentence

Aberrant expression of imprinted genes specifically in maternal placental tissues affects the penetrance of a paternally inherited IUGR mutation in cattle.

Sepsis is the leading cause of direct maternal mortality, but there are no data directly comparing the response to sepsis in pregnant and nonpregnant (NP) individuals. This study uses a mouse model of sepsis to test the hypothesis that the cardiovascular response to sepsis is more marked during pregnancy. Female CD1 mice had radiotelemetry probes implanted and were time mated. NP and day 16 pregnant CD-1 mice received intraperitoneal lipopolysaccharide (LPS; 10 μg, serotype 0111: B4). In a separate study, tissue and serum (for RNA, protein and flow cytometry studies), aorta and uterine vessels (for wire myography) were collected after LPS or vehicle control administration. Administration of LPS resulted in a greater fall in blood pressure in pregnant mice compared to NP mice. This occurred with similar changes in the circulating levels of cytokines, vasoactive factors, and circulating leukocytes, but with a greater monocyte and lesser neutrophil margination in the lungs of pregnant mice. Baseline markers of cardiac dysfunction and apoptosis as well as cytokine expression were higher in pregnant mice, but the response to LPS was similar in both groups as was the ex vivo assessment of vascular function. In pregnant mice, nonfatal sepsis is associated with a more marked hypotensive response but not a greater immune response. We conclude that endotoxemia induces a more marked hypotensive response in pregnant compared to NP mice. These changes were not associated with a more marked systemic inflammatory response in pregnant mice, although monocyte lung margination was greater. The more marked hypotensive response to LPS may explain the greater vulnerability to some infections exhibited by pregnant women.

Summary Sentence

In pregnant mice, nonfatal sepsis is associated with a more marked hypotensive response but not a greater immune response.

Mass spectrometry (MS) approaches were used herein to identify metabolites and proteins in uterine flushings (UF) that may contribute to nourishing the conceptus. Ovine uteri collected on Day 12 of the estrous cycle (n = 5 ewes exposed to vasectomized ram) or Days 12 (n = 4), 14 (n = 5), or 16 (n = 5) of pregnancy (bred with fertile ram) were flushed using buffered saline. Metabolites were extracted using 80% methanol and profiled using ultraperformance liquid chromatography (LC) tandem mass spectrometry. The proteome was examined by digestion with trypsin, followed by the analysis of peptides with LC-MS/MS. Metabolite profiling detected 8510 molecular features of which 9 were detected only in UF from Day 14–16 pregnant ewes that function in fatty acid transport (carnitines), hormone synthesis (androstenedione like), and availability of nutrients (valine). Proteome analysis detected 783 proteins present by Days 14–16 of pregnancy in UF, 7 of which are as follows: annexin (ANX) A1, A2, and A5; calcium-binding protein (S100A11); profilin 1; trophoblast kunitz domain protein 1 (TKDP); and interferon tau (IFNT). These proteins function in endocytosis, exocytosis, calcium signaling, and inhibition of prostaglandins (annexins and S100A11); protecting against maternal proteases (TKDP); remodeling cytoskeleton (profilin 1); and altering uterine release of prostaglandin F2 alpha as well as inducing IFNT-stimulated genes in the endometrium and the corpus luteum (IFNT). Identifying metabolites and proteins produced by the uterus and conceptus advances our understanding of embryo/maternal signaling and provides insights into possible the causes of reproductive failure.

Summary Sentence

Discovery of metabolites and proteins in uterine flushings contributes to understanding how the conceptus communicates with the endometrium during early pregnancy in sheep.

Mass spectrometry (MS) approaches were used herein to identify metabolites and proteins in uterine flushings (UF) that may contribute to nourishing the conceptus. Ovine uteri collected on Day 12 of the estrous cycle (n = 5 ewes exposed to vasectomized ram) or Days 12 (n = 4), 14 (n = 5), or 16 (n = 5) of pregnancy (bred with fertile ram) were flushed using buffered saline. Metabolites were extracted using 80% methanol and profiled using ultraperformance liquid chromatography (LC) tandem mass spectrometry. The proteome was examined by digestion with trypsin, followed by the analysis of peptides with LC-MS/MS. Metabolite profiling detected 8510 molecular features of which 9 were detected only in UF from Day 14–16 pregnant ewes that function in fatty acid transport (carnitines), hormone synthesis (androstenedione like), and availability of nutrients (valine). Proteome analysis detected 783 proteins present by Days 14–16 of pregnancy in UF, 7 of which are as follows: annexin (ANX) A1, A2, and A5; calcium-binding protein (S100A11); profilin 1; trophoblast kunitz domain protein 1 (TKDP); and interferon tau (IFNT). These proteins function in endocytosis, exocytosis, calcium signaling, and inhibition of prostaglandins (annexins and S100A11); protecting against maternal proteases (TKDP); remodeling cytoskeleton (profilin 1); and altering uterine release of prostaglandin F2 alpha as well as inducing IFNT-stimulated genes in the endometrium and the corpus luteum (IFNT). Identifying metabolites and proteins produced by the uterus and conceptus advances our understanding of embryo/maternal signaling and provides insights into possible the causes of reproductive failure.

Summary Sentence

Discovery of metabolites and proteins in uterine flushings contributes to understanding how the conceptus communicates with the endometrium during early pregnancy in sheep.

Preterm birth continues to be a significant public health problem. Infection (bacterial and or viral) and inflammation, by stimulating proinflammatory cytokines, adhesion molecules, and matrix metalloproteinase 9 (MMP9), play a central role in the rupture of membranes and myometrial contractions. SMAD7 has been implicated in regulating the inflammatory response; however, no studies have been performed with regard to human labor. In this study, we determined the effect of spontaneous human labor and prolabor mediators on SMAD7 expression in myometrium and fetal membranes. Functional studies were employed to investigate the effect of siRNA knockdown of SMAD7 (siSMAD7) in regulating infection and inflammation-induced prolabor mediators. SMAD7 mRNA and protein expression were significantly higher with spontaneous term labor, compared to no labor, inmyometrium and fetalmembranes. SMAD7 expression was also significantly higher in amnion from women with preterm chorioamnionitis. The proinflammatory cytokines IL1B and TNF, the bacterial product fsl-1, and the viral dsRNA analog poly(I:C) significantly increased SMAD7 in myometrial cells and amnion cells. In myometrial cells, siSMAD7 cells significantly decreased cytokine (IL6) and chemokine (CXCL1, CXCL8, CCL2 are also known as GRO-alpha, interleukin (IL)-8 and monocyte chemotactic protein-1 (MCP-1)) production induced by IL1B, TNF, and fsl-1. There was also a decrease in the expression of adhesion molecules intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in siSMAD7 cells, and MMP9 expression. In amnion, siSMAD7 cells treated with IL1B also decreased cytokine and chemokine production, ICAM1 and MMP9 expression. In conclusion, we report a proinflammatory role for SMAD7 in human gestational tissues, with SMAD7 silencing attenuating the inflammatory response.

Summary Sentence

SMAD7 expression is increased in laboring myometrium and by mediators of labor; SMAD7 is involved in the regulation of prolabor mediators induced by inflammation.

Cation channels of sperm (CatSper) are sperm-specific calcium channels with identified roles in the regulation of sperm function in humans, mice, and horses. We sought to employ a comparative genomics approach to identify conserved CATSPER genes in the bovine genome, and profile their expression in reproductive tissue. We hypothesized that CATSPER proteins expressed in bull testicular tissue mediates sperm hyperactivation and their rheotactic response in the reproductive tract of the cow. Bioinformatic analysis identified all four known CATSPER genes (CATSPER 1–4) in the bovine genome, and profiling by quantitative real-time polymerase chain reaction identified site-specific variation in messenger ribonucleic acid (mRNA) expression for all four genes along the reproductive tract of the bull. Using a novel antibody against CATSPER 1, protein expression was confirmed and localized to the principal piece of bull sperm, in agreement with what has been reported in other species. Subsequent treatment of bull sperm with either the calcium chelator ethylene glycol tetraacetic acid;mibefradil, a specific blocker of CatSper channels in human sperm; or CATSPER1 antibody all significantly inhibited caffeine-induced hyperactivation and the rheotactic response, supporting the concept that the calcium influx occurs via CatSper channels. Taken together, the work here provides novel insights into expression and function of CatSper channels in bull testicular tissue and in the function of ejaculated sperm.

Summary Sentence

The effect of blocking calcium channels on hyperactivation and rheotactic response.

Cation channels of sperm (CatSper) are sperm-specific calcium channels with identified roles in the regulation of sperm function in humans, mice, and horses. We sought to employ a comparative genomics approach to identify conserved CATSPER genes in the bovine genome, and profile their expression in reproductive tissue. We hypothesized that CATSPER proteins expressed in bull testicular tissue mediates sperm hyperactivation and their rheotactic response in the reproductive tract of the cow. Bioinformatic analysis identified all four known CATSPER genes (CATSPER 1–4) in the bovine genome, and profiling by quantitative real-time polymerase chain reaction identified site-specific variation in messenger ribonucleic acid (mRNA) expression for all four genes along the reproductive tract of the bull. Using a novel antibody against CATSPER 1, protein expression was confirmed and localized to the principal piece of bull sperm, in agreement with what has been reported in other species. Subsequent treatment of bull sperm with either the calcium chelator ethylene glycol tetraacetic acid;mibefradil, a specific blocker of CatSper channels in human sperm; or CATSPER1 antibody all significantly inhibited caffeine-induced hyperactivation and the rheotactic response, supporting the concept that the calcium influx occurs via CatSper channels. Taken together, the work here provides novel insights into expression and function of CatSper channels in bull testicular tissue and in the function of ejaculated sperm.

Summary Sentence

The effect of blocking calcium channels on hyperactivation and rheotactic response.

Mammalian reproduction requires that males and females produce functional haploid germ cells through complex cellular differentiation processes known as spermatogenesis and oogenesis, respectively. While numerous studies have functionally characterized protein-coding genes and small noncoding RNAs (microRNAs and piRNAs) that are essential for gametogenesis, the roles of regulatory long noncoding RNAs (lncRNAs) are yet to be fully characterized. Previously, we and others have demonstrated that intergenic regions of the mammalian genome encode thousands of long noncoding RNAs, and many studies have now demonstrated their critical roles in key biological processes. Thus, we postulated that some lncRNAs may also impact mammalian spermatogenesis and fertility. In this study, we identified a dynamic expression pattern of lncRNAs during murine spermatogenesis. Importantly, we identified a subset of lncRNAs and very few mRNAs that appear to escape meiotic sex chromosome inactivation, an epigenetic process that leads to the silencing of the X- and Y-chromosomes at the pachytene stage of meiosis. Further, some of these lncRNAs and mRNAs show a strong testis expression pattern suggesting that they may play key roles in spermatogenesis. Lastly, we generated a mouse knockout of one X-linked lncRNA, Tslrn1 (testis-specific long noncoding RNA 1), and found that males carrying a Tslrn1 deletion displayed normal fertility but a significant reduction in spermatozoa. Our findings demonstrate that dysregulation of specific mammalian lncRNAs is a novel mechanism of low sperm count or infertility, thus potentially providing new biomarkers and therapeutic strategies.

Summary Sentence

Long non-coding RNAs are critical for mammalian spermatogenesis.

Mammalian reproduction requires that males and females produce functional haploid germ cells through complex cellular differentiation processes known as spermatogenesis and oogenesis, respectively. While numerous studies have functionally characterized protein-coding genes and small noncoding RNAs (microRNAs and piRNAs) that are essential for gametogenesis, the roles of regulatory long noncoding RNAs (lncRNAs) are yet to be fully characterized. Previously, we and others have demonstrated that intergenic regions of the mammalian genome encode thousands of long noncoding RNAs, and many studies have now demonstrated their critical roles in key biological processes. Thus, we postulated that some lncRNAs may also impact mammalian spermatogenesis and fertility. In this study, we identified a dynamic expression pattern of lncRNAs during murine spermatogenesis. Importantly, we identified a subset of lncRNAs and very few mRNAs that appear to escape meiotic sex chromosome inactivation, an epigenetic process that leads to the silencing of the X- and Y-chromosomes at the pachytene stage of meiosis. Further, some of these lncRNAs and mRNAs show a strong testis expression pattern suggesting that they may play key roles in spermatogenesis. Lastly, we generated a mouse knockout of one X-linked lncRNA, Tslrn1 (testis-specific long noncoding RNA 1), and found that males carrying a Tslrn1 deletion displayed normal fertility but a significant reduction in spermatozoa. Our findings demonstrate that dysregulation of specific mammalian lncRNAs is a novel mechanism of low sperm count or infertility, thus potentially providing new biomarkers and therapeutic strategies.

Summary Sentence

Long non-coding RNAs are critical for mammalian spermatogenesis.

The sex identity of fish can be easily manipulated by exogenous hormones. Treatment with 17-methyltestosterone (MT) has been widely used to induce a male fate, but the molecular and cellular processes underlying sex changes induced by MT treatments and the withdrawal of MT are not well studied. In this study, we systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels, and cellular changes during sex changes induced by MT-feeding and MT-feeding withdrawal in the protogynous orange-spotted grouper, Epinephelus coioides. Based on gonadal histology, we demonstrated that MT-feeding-induced sex reversal can be divided into early and late phases: in the early phase, male and female germ cells coexist, and MT-feeding withdrawal leads to a female fate; in the late phase, only male germ cells are observed, and MT-feeding withdrawal does not reverse the process, leading to a male fate. In both the early and late phases, cytochrome P450 family19 subfamily A member 1 (cyp19a1a) gene expression increased in response to MT-feeding withdrawal. Finally, by tracing doublesex- and Mab-3-related transcription factor 1 (dmrt1)-expressing cells, we found that gonia-like cells in the germinal epithelium might be the major germ cell sources for developing testes during sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex changes induced by exogenous hormones.

Summary Sentence

We systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels, and cellular changes during sex changes induced by MT-feeding and MT-feeding withdrawal in the protogynous orange-spotted grouper.

The sex identity of fish can be easily manipulated by exogenous hormones. Treatment with 17-methyltestosterone (MT) has been widely used to induce a male fate, but the molecular and cellular processes underlying sex changes induced by MT treatments and the withdrawal of MT are not well studied. In this study, we systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels, and cellular changes during sex changes induced by MT-feeding and MT-feeding withdrawal in the protogynous orange-spotted grouper, Epinephelus coioides. Based on gonadal histology, we demonstrated that MT-feeding-induced sex reversal can be divided into early and late phases: in the early phase, male and female germ cells coexist, and MT-feeding withdrawal leads to a female fate; in the late phase, only male germ cells are observed, and MT-feeding withdrawal does not reverse the process, leading to a male fate. In both the early and late phases, cytochrome P450 family19 subfamily A member 1 (cyp19a1a) gene expression increased in response to MT-feeding withdrawal. Finally, by tracing doublesex- and Mab-3-related transcription factor 1 (dmrt1)-expressing cells, we found that gonia-like cells in the germinal epithelium might be the major germ cell sources for developing testes during sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex changes induced by exogenous hormones.

Summary Sentence

We systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels, and cellular changes during sex changes induced by MT-feeding and MT-feeding withdrawal in the protogynous orange-spotted grouper.