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Jaya Gamble, Joel Chick, Kelly Seltzer, Joel H Graber, Steven Gygi, Robert E Braun and Elizabeth M Snyder

The testis transcriptome is exceptionally complex. Despite its complexity, previous testis transcriptome analyses relied on a reductive method for transcript identification, thus underestimating transcriptome complexity. We describe here a more complete testis transcriptome generated by combining Tuxedo, a reductive method, and spliced-RUM, a combinatorial transcript-building approach. Forty-two percent of the expanded testis transcriptome is composed of unannotated RNAs with novel isoforms of known genes and novel genes constituting 78 and 9.8% of the newly discovered transcripts, respectively. Across tissues, novel transcripts were predominantly expressed in the testis with the exception of novel isoforms which were also highly expressed in the adult ovary. Within the testis, novel isoform expression was distributed equally across all cell types while novel genes were predominantly expressed in meiotic and post-meiotic germ cells. The majority of novel isoforms retained their protein-coding potential while most novel genes had low protein-coding potential. However, a subset of novel genes had protein-coding potentials equivalent to known protein-coding genes. Shotgun mass spectrometry of round spermatid total protein identified unique peptides from four novel genes along with seven annotated non-coding RNAs. These analyses demonstrate the testis expresses a wide range of novel transcripts that give rise to novel proteins.

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Hirofumi Nishizono, Mohamed Darwish, Takaho A Endo, Kyosuke Uno, Hiroyuki Abe and Ryohei Yasuda

Oviduct fluid is essential for the fertilization and subsequent preimplantation development. Glycine is abundant in oviduct fluid and is reported to be critical for preimplantation development of fertilized eggs in mammals. However, the mechanism by which glycine exerts its action on fertilized eggs is yet to be understood. Here we show that glycine regulates the preimplantation development of mouse fertilized eggs via glycine receptors. Among them, the alpha-4 subunit (Glra4) and the β subunit are expressed in mouse fertilized eggs, and lacking Glra4 inhibits embryonic development to the blastocyst stage, decreases the number of cells in the blastocysts and the litter size. Thus, we identify a novel function of the glycine receptor, which is considered to act mainly as a neurotransmitter receptor, as a regulator of embryonic development and our data provide new insights into the interactions between oviduct milieu and mammalian fertilized egg.

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Fang-Ju Wu, Ying-Wen Wang and Ching-Wei Luo

Bone morphogenetic proteins (BMPs) are known to play an indispensable role in preventing the precocious luteinization of granulosa cells within growing ovarian follicles. In this study, we found that the transcripts of BMP8 genes are enriched in the ovaries of humans and rodents. When analyzing transcriptomic datasets obtained from human mature granulosa cells, we further found that the BMP8 transcripts not only show the highest abundance among the searchable BMP-related ligands but also decrease significantly in women of advanced age or women with polycystic ovarian syndrome. The correlation between the BMP8 levels in granulosa cells and the decline in ovarian function in these subjects suggests that BMP8 protein may be involved in the regulation of granulosa cell function(s). Using a rat model, we demonstrated that human BMP8A protein activates the SMAD1/5/8 and the SMAD2/3 pathways simultaneously in both immature and mature granulosa cells. Furthermore, the expression of potential type I and type II receptors used by BMP8 in rat granulosa cells was characterized. We found that BMP8A treatment can significantly inhibit gonadotropin-induced progesterone production and steroidogenesis-related gene expression in granulosa cells. Pathway dissection using receptor inhibitors further revealed that such inhibitory effects occur specifically through the BMP8-activated SMAD1/5/8, but not SMAD2/3, pathway. Taken together, considering its abundance and possible functions in granulosa cells, we suggest that BMP8 may act as a novel luteinization inhibitor in growing follicles.

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Sakshi Chauhan, Subhashree Parida, E Prakash, G Srinivasan, Vivek Srivastava, Manjit Panigrahi, Thakur Uttam Singh and Santosh K Mishra

The aim of the present study was to reveal the effect of hyperlipidemia on β2- and β3-adrenergic signaling in late pregnant rat uterus. Hyperlipidemia was induced in female Wistar rats by feeding a high-fat high-cholesterol diet for 8 weeks before and after mating upto the 21st day of gestation. The effect of hyperlipidemia on β-adrenergic signaling was studied with the help of tension experiments, real-time PCR and cAMP ELISA in 21-day pregnant rat uterus. In tension experiments, hyperlipidemia neither altered the spontaneous contractility nor the oxytocin-induced contractions. However, it decreased the −logEC50 values of β2-adrenoceptor agonist, salbutamol and β3-adrenoceptor agonist, BRL37344. It also decreased the efficacy of adenylyl cyclase activator, forskolin. Further, there was a significant decrease in salbutamol and BRL37344-stimulated cAMP content in uterine tissues. However, there was no alteration in mRNA expressions of β2-adrenoceptor (Adrb2), β3-adrenoceptor (Adrb3) and Gs protein (Gnas) though there was a significant increase in the mRNA expression of Gi protein (Gnai). In conclusion, reduced cAMP content after beta-adrenergic receptor stimulation, which correlates with an increase in Gnai mRNA, may explain the mechanism of the impairment of uterine β-adrenergic signaling in hyperlipidemic pregnant rats. The clinical implication of the present study may relate to reduced myometrial relaxant response to β-adrenergic agonists in high fat-induced uterine dysfunction.

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Zijing Zhang, Florencia Schlamp, Lu Huang, Haley Clark and Lynae Maria Brayboy

The fertility of women declines sharply after age 35 and is essentially lost upon menopause at age 51. The ovary plays an important part in aging-associated changes in women’s physiology since it is an essential component of both the reproductive and endocrine systems. Several previous studies in mice have shown that the ovarian tissue goes through drastic changes over the course of aging and exhibits signs of aging-associated chronic inflammation (inflammaging), which may contribute to the marked decline of oocyte quality in aged individuals. To further examine aging-associated gene expression changes in the ovary and to characterize the development of inflammaging, we performed detailed transcriptomic analysis of whole ovaries from mice of six different age groups over the mouse reproductive lifespan and identified more than 5000 genes with significant expression change over the course of aging. Intriguingly, we found aging-associated changes in the expression of several markers that indicate alterations in the composition of ovarian macrophages, which are known to be central players of inflammaging. Using flow cytometry, we analyzed and compared macrophage populations and polarization in young and old ovaries and found a significant increase in monocyte recruitment and macrophage alternative activation (M2) in the old ovaries compare to those in young. Our results are consistent with previous findings of aging-associated increase of fibrosis in the ovarian stromal extracellular matrix, and they provide new clues about the development of inflammaging in the mammalian ovary.

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Jelmer R Prins, Lotte M Marissen, Sicco A Scherjon, Annemieke Hoek and Astrid E P Cantineau

Follicular fluid (FF) surrounds the granulosa cell–oocyte complex and is one of the mediating factors in the communication between the cells within the follicle. Literature reveals that human FF and its components are key factors to the success of natural fertilization. Among other substances, FF consists of multiple cytokines and immune cells, including interleukin 6 (IL6), IL12, sHLA-G, macrophages, NK cells and lymphocytes. Together, these cells and cytokines might influence the oocyte–granulosa–cell complex. Altered balances of immune content might be involved in changes on folliculogenesis, oocyte maturation, oocyte quality and ovulation. Furthermore, these altered balances are possibly involved in infertility associated with immune-mediated diseases such as endometriosis. The aim of this narrative review is to elaborate on the function and contents of FF and its immunological profile in patients with endometriosis. A comprehensive literature search was performed for the published literature on FF (immune) contents, FF function and FF content alterations in endometriosis patients. In FF of patients with endometriosis, elevated levels of macrophages and several cytokines have been reported. The role of specific immune cells in FF and a clarification of the biological mechanism in healthy women and endometriosis patients remain largely unknown. Future studies in this field will give us more insight in the role of FF immune cells and the effect of altered balances in patients with endometriosis.

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Nanae Makino, Nozomi Sato, Eriko Takayama-Watanabe and Akihiko Watanabe

Sperm intracellular Ca2+ is crucial for the induction of sperm-egg interaction, but little is known about the significance of Ca2+ maintenance prior to induction. In sperm of the newt Cynops pyrrhogaster, intracellular Ca2+ is localized to the midpiece during storage in the vas deferens, while extracellular Ca2+ is influxed in modified Steinberg’s salt solution to promote a spontaneous acrosome reaction related to the decline of sperm quality. In the present study, sperm from the vas deferens were loaded with the Ca2+ indicator Fluo8H, and changes in Ca2+ localization in modified Steinberg’s salt solution were examined. Calcium ions expanded from the cytoplasmic area of the midpiece to the entire tail in most sperm during a 1-hour incubation and localized to the principal piece in some sperm within 24 hours. Similar changes in Ca2+ localization were observed in reconstructed vas deferens solution that included ions and pH at equivalent levels to those in the vas deferens fluid. Sperm with Ca2+ localization in the entire tail or the principal piece weakened or lost responsiveness to sperm motility-initiating substances, which trigger sperm motility for fertilization, but responded to a trigger for acrosome reaction. The change in Ca2+ localization was delayed and transiently reversed by ethylene glycol tetraacetic acid or a mixture of Ca2+ channel blockers including Ni2+ and diltiazem. These results suggest that C. pyrrhogaster sperm localize intracellular Ca2+ to the midpiece through Ca2+ transport in the vas deferens to allow for responses to sperm motility-initiating substances.

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Xiaoyan Li, Jinling Zhu, Jie Tian, Dongmei Li, Xiaodong Han and Jiang Wu

Health risk of human exposure to microcystin-leucine arginine (MC-LR) has drawn more and more attention in recent years. In the present study, MC-LR inhibited miR-3473g expression of mouse granulosa cells both in vitro and in vivo. By dual-luciferase reporter assay, we confirmed miR-3473g is able to bind the 3′-UTR of StAR protein (StAR) mRNA and suppress StAR expression. Thus, downregulation of miR-3473g after MC-LR exposure led to StAR overexpression. Excessive StAR probably transported much more cholesterol into the inner membrane of the mitochondria and finally resulted in overproduction of progesterone. Our results revealed that MC-LR exposure was associated with premature luteinization of granulosa cells and may adversely affect women’s fertility.

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Wei Cui, Agnes Cheong, Yongsheng Wang, Yuran Tsuchida, Yong Liu, Kimberly D Tremblay and Jesse Mager

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

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Daniel A Dumesic, Luis R Hoyos, Gregorio D Chazenbalk, Rajanigandha Naik, Vasantha Padmanabhan and David H Abbott

Developmental origins of adult disease (DoHAD) refers to critical gestational ages during human fetal development and beyond when the endocrine metabolic status of the mother can permanently program the physiology and/or morphology of the fetus, modifying its susceptibility to disease after birth. The aim of this review is to address how DoHAD plays an important role in the phenotypic expression of polycystic ovary syndrome (PCOS), the most common endocrinopathy of women characterized by hyperandrogenism, oligo-anovulation and polycystic ovarian morphology. Clinical studies of PCOS women are integrated with findings from relevant animal models to show how intergenerational transmission of these central components of PCOS are programmed through an altered maternal endocrine–metabolic environment that adversely affects the female fetus and long-term offspring health. Prenatal testosterone treatment in monkeys and sheep have been particularly crucial in our understanding of developmental programming of PCOS because organ system differentiation in these species, as in humans, occurs during fetal life. These animal models, along with altricial rodents, produce permanent PCOS-like phenotypes variably characterized by LH hypersecretion from reduced steroid-negative feedback, hyperandrogenism, ovulatory dysfunction, increased adiposity, impaired glucose-insulin homeostasis and other metabolic abnormalities. The review concludes that DoHAD underlies the phenotypic expression of PCOS through an altered maternal endocrine–metabolic environment that can induce epigenetic modifications of fetal genetic susceptibility to PCOS after birth. It calls for improved maternal endocrine–metabolic health of PCOS women to lower their risks of pregnancy-related complications and to potentially reduce intergenerational susceptibility to PCOS and its metabolic derangements in offspring.