The goal of sperm is to fertilize the oocyte. To achieve that purpose, it must acquire motility in the epididymis and hyperactivated motility in the female reproductive tract. Motility is only achieved when the sperm presents a fully functional flagellum, is capable of producing energy to fuel the movement, and suffers epididymal maturation and capacitation. Since sperm is a transcriptionally silent cell, motility depends on the activation and/or inhibitions of key signaling pathways. This review describes and discusses the main signaling pathways involved in primary and hyperactivated motility, as well as the bioenergetic mechanisms necessary to produce energy to fuel sperm motility. Although the complete human spermmotility process is far from being fully known, we believe that in the upcoming decades extensive progress will be made. Understanding the signaling pathways behind sperm motility can help pinpoint the cause of male infertility and uncover targets for male contraception.

Summary Sentence

Sperm motility depends on energy availability, intact flagellum and the crosstalk of several signaling pathways that lead to an increase of tyrosine phosphorylation of key proteins.

Major clinical challenges for obstetricians and neonatologists result from early cervix remodeling and preterm birth. Complications related to cervix remodeling or delivery account for significant morbidity in newborns and peripartum mothers. Understanding morphology and structure of the cervix in pregnant women is limited mostly to the period soon before and after birth. However, evidence in rodent models supports a working hypothesis that a convergence of factors promotes a physiological inflammatory process that degrades the extracellular collagen matrix and enhances biomechanical distensibility of the cervix well before the uterus develops the contractile capabilities for labor. Contributing factors to this remodeling process include innervation, mechanical stretch, hypoxia, and proinflammatory mediators. Importantly, the softening and shift to ripening occurs while progesterone is near peak concentrations in circulation across species. Since progesterone is required to maintain pregnancy, the premise of this review is that loss of responsiveness to progesterone constitutes a common final mechanism for remodeling the mammalian cervix in preparation for birth at term. Various inputs are suggested to promote signaling between stromal cells and resident macrophages to drive proinflammatory processes that advance the soft cervix into ripening. With infection, pathophysiological processes may prematurely drive components of this remodeling mechanism and lead to preterm birth. Identification of critical molecules and pathways from studies in various rodent models hold promise for novel endpoints to assess risk and provide innovative approaches to treat preterm birth or promote the progress of ripening at term.

Summary Sentence

The mechanism for cervix remodeling in preparation for birth is proposed to involve a convergence of inputs and complex orchestration of inflammatory processes that direct resident macrophage activities to drive extracellular matrix degradation.

Endometrial receptivity is crucial for implantation and establishment of a normal pregnancy. The shift from proliferative to receptive endometrium is still far from being understood. In this paper, we comprehensively present the transcriptome of the human endometrium by comparing endometrial biopsies from proliferative phase with consecutive biopsies 7–9 days after ovulation. The results show a clear difference in expression between the two time points using both total and small RNA sequencing. A total of 3,297 messenger RNAs (mRNAs), 516 long noncoding RNAs (lncRNAs), and 102 small noncoding RNAs were identified as statistically differentially expressed between the two time points. We show a thorough description of the change in mRNA between the two time points and display lncRNAs, small nucleolar RNAs, and small nuclear RNAs not previously reported in the healthy human endometrium. In conclusion, this paper reports in detail the shift in RNA expression from the proliferative to receptive endometrium.

Summary Sentence

Messenger RNA, sncRNA, and lncRNA show a clear difference in expression between proliferative phase and 7–9 days after ovulation, thoroughly described together with lncRNA, snoRNA, and snRNA not previously reported in healthy human endometrium.

Absolute uterine factor infertility, or the absence of a functional uterus, has a prevalence of 3%–5% in the general population. Despite the great strides being made in reproductive medicine, patients diagnosed with absolute uterine factor infertility remain untreatable. The only available solution has been gestational surrogacy, but recently the Brannström group presented a viable alternative by reporting the first successful live birth after uterus transplantation. Similar to other transplantations, this approach has inherent limitations such as the paucity of donor organs and the need for long-term immunosuppression. Whole organ de- and recellularization, a novel tissue engineering approach within the field of regenerative medicine, could eventually provide another solution. Several groups have described animal models in which they have performed decellularization of whole uteri, while maintaining the extracellular matrix to enable recellularization attempts. Our work offers a new perspective; in decellularizing the porcine uterus, this constitutes the first pilot study using large whole reproductive organs. We demonstrated the preservation of a reusable/functional extracellular matrix while maintaining its vascular network. Furthermore, we report the first use of human side population stem cells in the successful recellularization of small acellular disk scaffolds procured from the decellularized organs. To conclude, this research opens new avenues in whole uterus bioengineering, opening the way towards the transplantation of functional bioengineered uteri into humans.

Summary Sentence

A novel decellularization protocol using SDS and Triton X-100 successfully converts large pig uteri into acellular bioscaffolds with potential for subsequent recellularization.

Adenomyosis is a nonneoplastic condition characterized by the benign invasion of ectopic endometrium into the myometrium. Macrophages play significant roles in epithelial-mesenchymal transition (EMT) and adenomyosis. An EMT associated with adenomyosis has been extensively studied. This study investigated the process by which the interaction of macrophages with endometrial cells induces EMT in Ishikawa cells and epithelial cells of adenomyosis. Specimens were collected after hysterectomy or resection of adenomyosis lesions from women with adenomyosis and curettage from women without adenomyosis or endometriosis. Immunohistochemistry and immunofluorescent staining demonstrated that CD68-positive macrophages aggregated in adenomyosis lesions, along with the increased protein expressions of n-cadherin, vimentin, and S100A4. By contrast, the protein expressions of e-cadherin and CK7 were decreased. After the primary endometrium cells were cocultured with THP-1-derived macrophages, the protein expression levels of n-cadherin, vimentin, and S100A4 of endometrium cells were increased, whereas the protein expression levels of e-cadherin and CK7 were decreased. The proportion of alternatively activated (M2) macrophages derived from THP-1 macrophages was also increased. The M2 macrophages elicited a bidirectional effect on Ishikawa cells by inducing EMT-like or mesenchymal-epithelial transition-like processes. The apoptotic rate of the Ishikawa cells cocultured with macrophageswas increased, whereas their cell proliferation rate was decreased. Transmission electron microscopy indicated that the number of intercellular junctions of the cocultured Ishikawa cells was reduced. Microarray-based gene expression analysis revealed that transforming growth factor-β1/Smad3 and interleukin-6/JAK2/STAT3 signaling pathways were upregulated. Therefore, macrophages can induce EMT-like processes in adenomyosis and undergo polarization to M2.

Summary Sentence

Macrophages can induce EMT-like processes in adenomyosis and undergo polarization to M2.

Cystic endometrial hyperplasia (CEH)—pyometra syndrome is one of the most common diseases of noncastrated female dogs. However, determination of etiological mechanisms and differential diagnosis of CEH—pyometra syndrome are undefined. The aim of this study is to compare immunohistochemically the expression of cyclooxygenase-2 (COX-2) inflammatory mediator, Ki-67 antigen proliferation marker, vascular endothelial growth factor (VEGF-A) angiogenesis mediator and its FLT-1 and KDR receptors, and correlate with Doppler velocimetry of uterine artery and endometrial vascularization in bitches with CEH—pyometra syndrome. Bitches were allocated into CEH-mucometra Group (n = 13), Pyometra Group (n = 11), and Control Group (n = 8). Pyometra Group presented cytoplasmatic staining intensity for COX-2, VEGF-A, and FLT-1 and KDR receptors in luminal epithelium cells significantly higher compared to CEH-mucometra and Control groups. For the glandular epithelium, Pyometra Group had higher immunostaining score for VEGF-A and its receptors (FLT-1 and KDR). Hemodynamic indexes showed negative correlation with VEGF-A and its receptors as well as with COX-2. On the other hand, uterine vascularization score showed positive correlation in relation to immunostaining of COX-2, VEGF-A, and receptors in the endometrium luminal epithelium. In conclusion, uterus of bitches with CEH—pyometra syndrome show inflammatory process characterized by COX-2 expression, resulting in greater expression of proliferative Ki-67 marker as tissue response against the infectious agent. Furthermore, the increased VEGF-A expression and its receptors in CEH—pyometra reflect the increased blood flow and lower vascular resistance. Therefore, canine pyometra is characterized by an inflammatory, proliferative, and vascular disorder.

Summary Sentence

Canine CEH—pyometra syndrome is characterized by an inflammatory, proliferative and vascular disorder, with higher uterine blood flow and vascularization.

Regulation of myometrial functions during pregnancy has been considered the result of the integration of endocrine and mechanical signals. Nevertheless, uterine regeneration is poorly understood, and the cellular source within the gravid uterus is largely unexplored.

In this study, we isolated and quantified the myometrial stem cells (MSC) population from pregnant female Eker rat uteri, by using Stro1/CD44 surface markers. We demonstrated that prior parity significantly increased the percentage of Stro1⁺/CD44⁺ MSC because of injured tissue response. Interestingly, we established that Stro1⁺/CD44⁺ MSC respond efficiently to physiological cues when they were treated in vitro under different dose-dependent pregnant rat serum.

Previous studies reveal strong regulatory links between O₂ availability and stem cell function. Based on these premises, cell proliferation assays showed that isolated Stro1⁺/CD44⁺ MSC possess a higher proliferative rate under hypoxic versus normoxic conditions. We also detected a total of 37 upregulated and 44 downregulated hypoxia-related genes, which were differentially expressed in Stro1⁺/CD44⁺ MSC, providing an alternative approach to infer into complexmolecular mechanisms such as energy metabolism, inflammatory response, uterine expansion, and/or remodeling.

Since these cells preferentially grow under low oxygen conditions, we propose that the increase of the rat uterus during pregnancy involves myometrial oxygen consumption, thereby enhancing MSC proliferation. Moreover, pregnancy-induced mechanical stretching results in hypoxic conditions, ultimately creating an environment that promotes stem cell proliferation and further uterine enlargement, which is essential for a successful pregnancy. In summary, all of these data support that rat Stro1⁺/CD44⁺ MSC contribute to uterine enlargement during pregnancy.

Summary Sentence

The human and murine uterus exhibits a large expansion in size and remodeling over the course of pregnancy. However, at this stage, little is known about the uterine regeneration at cellular level. Due to limited access to sequentially-timed human pregnant myometrial tissues, we proceeded to address these pivotal questions by using a rat animal model. In this study, we identified and characterized myometrial stem cells (MSC) from pregnant female Eker rat uteri, by using specific surface markers. We also demonstrated their contribution to uterine enlargement during pregnancy under hypoxic conditions. Nevertheless, further studies are needed to elucidate the exact role of myometrial stem cells in healthy pregnancy and also in pathological conditions such as preterm labor.

Although the positive effects of vaginal estrogens and the selective estrogen receptor modulator, ospemifene (OS), on the vaginal epithelium are well recognized, less is known regarding the effects of these therapies on the lower urinary tract or vaginal muscularis. Clinical evidence suggests that vaginally administered estrogenmay improve overactive bladder-related symptoms. The objective of this study was to compare the effects of OS, vaginal conjugated equine estrogens (CEE), or both on the vaginal wall and lower urinary tract in a rat model of menopause. Contractile force of the bladder neck, dome, and external urethral sphincter at optimal field stimulation did not differ significantly among treatment groups. Pharmacologic responses to atropine, carbachol, and potassium chloride were similar among groups. Vaginal epithelial thickness and differentiation were differentially regulated by CEE or OS. Ospemifene altered epithelial differentiation pathways in vaginal epithelium in a unique way, and these effects were additive with local CEE. Unless contraindicated, the beneficial effects of vaginal CEE on the vaginal wall outweigh those of OS.

Summary Sentence

Although ospemifene has no effect on the lower urinary tract, it alters vaginal epithelium in a unique way that is additive with local estrogen.

Spermmotility notably depends on the structural integrity of the flagellum and the regulation ofmicrotubule dynamics. Although researchers have started to use “omics” techniques to characterize the human sperm's molecular landscape, the constituents responsible for the assembly, organization, and dynamics of the flagellum microtubule have yet to be fully defined. In this study, we defined a core set of 116 gene products associated with the human sperm microtubulome (including products potentially involved in abnormal ciliary phenotypes and male infertility disorders). To this end, we designed and applied an integrated genomics workflow and combined relevant proteomics, transcriptomics, and interactomics datasets to reconstruct a dynamic interactome map. By further integrating phenotypic information, we defined a disease-interaction network; this enabled us to highlight a number of novel factors potentially associated with altered sperm motility and male fertility. Lastly, we experimentally validated the expression pattern of two candidate genes (CUL3 and DCDC2C) that had never previously been associated with male germline differentiation. Our analysis suggested that CUL3 and DCDC2C's products have important roles in the sperm flagellum. Taken as a whole, our results demonstrate that an integrated genomics strategy can highlight relevant molecular factors in specific sperm components. This approach could be easily extended by including other “omics” data (from asthenozoospermic men, for example) and identifying other critical proteins from the human sperm microtubulome.

Summary Sentence

Although researchers have started to use “omics” techniques to characterize the human sperm's molecular landscape, the constituents responsible for the assembly, organization, and dynamics of the flagellum microtubule have yet to be fully defined.

Oxidative stress is a major determinant of mammalian sperm function stimulating lipid peroxidation cascades that culminate in the generation of potentially cytotoxic aldehydes. The aim of this study was to assess the impact of such aldehydes on the functionality of stallion spermatozoa. The impact of exposure to exogenous acrolein (ACR) and 4-hydroxynonenal (4HNE) was manifested in a highly significant dose- and time-dependent increase in mitochondrial reactive oxygen species (ROS), total cellular ROS, a decrease in sperm motility, and a time-dependent increase in lipid peroxidation. Notably, low doses of ACR and 4HNE also caused a significant decrease in zona binding. In contrast, exogenous malondialdehyde, a commonly used marker of oxidative stress, had little impact on the various sperm parameters assessed. In accounting for the negative physiological impact of ACR and 4HNE, it was noted that both aldehydes readily adducted to sperm proteins located predominantly within the head, proximal centriole, and tail. The detoxifying activity of mitochondrial aldehyde dehydrogenase 2 appeared responsible for a lack of adduction in the midpiece; however, this activity was overwhelmed by 24 h of electrophilic aldehyde exposure. Sequencing of the dominant proteins targeted for ACR and 4HNE covalent modification identified heat shock protein 90 alpha (cytosolic) class A member 1 and arylsulfatase A, respectively. These collective findings may prove useful in the identification of diagnostic biomarkers of stallion fertility and resolving the mechanistic basis of sperm dysfunction in this species.

Summary Sentence

ALDH2 activity is exacerbated following electrophilic aldehyde exposure, and HSP90AA1 and ARSA are vulnerable sites for adduction.

Male differentiation of primordial germ cells (PGCs) is initiated by the inhibition of entry into meiosis and exposure to male-inducing factor(s), which are regulated by somatic elements of the developing gonad. Fibroblast growth factor 9 (FGF9) produced by pre-Sertoli cells is essential for male gonadal differentiation and also contributes to survival and male differentiation of XY PGCs. However, it is not clear how FGF9 regulates PGC fate. Using a PGC culture system, we identified dose-dependent, fate-determining functions of FGF9 in XY PGCs. Treatment with low levels of FGF9 (0.2 ng/ml) increased expression of male-specific Dnmt3L and Nanos2 in XY PGCs. Conversely, treatment with high levels of FGF9 (25 ng/ml) suppressed male-specific gene expression and stimulated proliferation of XY PGCs. Western blotting showed that low FGF9 treatment enhanced p38 MAPK (mitogen-activated protein kinase) phosphorylation in the same cells. In contrast, high FGF9 treatment significantly stimulated the ERK (extracellular signal-regulated kinase)1/2 signaling pathway in XY PGCs. We investigated the relationship between the ERK1/2 signaling pathway stimulated by high FGF9 and regulation of PGC proliferation. An ERK1/2 inhibitor (U0126) suppressed the PGC proliferation that would otherwise be stimulated by high FGF9 treatment, and increased Nanos2 expression in XY PGCs. Conversely, a p38 MAPK inhibitor (SB202190) significantly suppressed Nanos2 expression that would otherwise be stimulated by low FGF9 in XY PGCs. Taken together, our results suggest that stage-specific expression of FGF9 in XY gonads regulates the balance between proliferation and differentiation of XY PGCs in a dose-dependent manner.

Summary Sentence

FGF9 directly regulates different fates of XY PGCs in a dose-dependent manner: low FGF9 treatment promotes p38 signaling pathway to induce PGC male differentiation, whereas high FGF9 treatment enhances ERK1/2 signaling pathway to proliferate PGCs.

During human pregnancy, paternally inherited antigens expressed by the fetal–placental unit can elicit expansion of antigen-specific CD8+T cells. These cells can persist for years asmemory T cells, but their effects on long-term maternal health are unknown. Shared placenta/tumor-associated antigens are expressed by placenta and tumors, but areminimally expressed or absent in normal adult tissues. We hypothesized that maternal T cells elicited against these antigens can alter risk of cancers expressing the same antigen after pregnancy, and tested this inmice using chicken ovalbumin (OVA) as a surrogate shared placenta/tumor antigen. Hemizygous OVA transgenicmales were bred to wild-type C57BL/6 females (H2b haplotype) such that the fetuses inherited and expressed OVA. Maternal OVA/H2K^b-specific CD8+ T cells became detectable during gestation, and persisted in some animals for up to 24 weeks. To determine whether these cells might influence growth of OVA-expressing tumors in OVA-bred females, E.G7-OVA thymoma cells were inoculated subcutaneously in OVA-bred, wild-type bred, and virgin females, and monitored for growth. OVA-bred mice had prolonged survival as compared to virgin mice and the progression of tumors was delayed in comparison to wild-type bred and virgin females. Thus, paternally inherited OVA antigen elicited a CD8+ T cell response during pregnancy that was associated with delayed growth of OVA-expressing tumors following pregnancy. These data suggest a possible role of antigen-specific T cells in protecting parous females against tumors bearing shared placenta/tumor antigens.

Summary Sentence

Gestational exposure of shared placenta/tumor-associated antigen may induce antigen-specific T cells, which may lower cancer risk against tumors bearing same antigen.

Natural killer (NK) cells are the most prevalent leukocyte population in the uterus during early pregnancy. Natural killer cells contribute to uterine vascular (spiral artery) remodeling in preparation for the increased demand on these vessels later in pregnancy. A second wave of spiral artery modification is directed by invasive trophoblast cells. The significance of the initial wave of NK-cell-mediated vascular remodeling in species exhibiting deep trophoblast invasion such as humans and rats is not known. The purpose of this study was to generate a genetic model of NK-cell deficiency in rats, and determine the consequences of NK-cell deficiency on spiral artery remodeling and reproductive outcomes. To accomplish this task, we utilized zinc finger nucleasemediated genome editing of the rat interleukin-15 (Il15) gene. Il15 encodes a cytokine required for NK-cell lineage development. Using this strategy, a founder rat was generated containing a frameshift deletion in Il15. Uteri of females harboring a homozygous mutation at the Il15 locus contained no detectable NK cells. NK-cell deficiency did not impact fetal growth or viability. However, NK-cell deficiency caused major structural changes to the placenta, including expansion of the junctional zone and robust, early-onset activation of invasive trophoblast-guided spiral artery remodeling. In summary, we successfully generated an NK-cell-deficient rat and showed, using thismodel, that NK cells dampen the extent of trophoblast invasion and delay trophoblast-directed spiral artery remodeling. This study furthers our understanding of the role of NK cells on uterine vascular remodeling, trophoblast invasion, and placental development.

Summary Sentence

Natural killer cells delay trophoblast invasion and trophoblast-directed spiral artery remodeling in rats.

The mammalian epididymis is an exceptionally long ductal system tasked with the provision of one of the most complex intraluminal fluids found in any exocrine gland. This specialized milieu is continuously modified by the combined secretory and absorptive of the surrounding epithelium and thus finely tuned for its essential roles in promoting sperm maturation and storage. While considerable effort has been focused on defining the composition of the epididymal fluid, relatively less is known about the intracellular trafficking machinery that regulates this luminal environment. Here, we characterize the ontogeny of expression of a master regulator of this machinery, the dynamin family of mechanoenzymes. Our data show that canonical dynamin isoforms were abundantly expressed in the juvenile mouse epididymis. However, in peripubertal and adult animals dynamin takes on a heterogeneous pattern of expression such that the different isoforms displayed both cell- and segment-specific localization. Thus, dynamin 1 and 3 were predominately localized in the distal epididymal segments (corpus and cauda), where they were found within clear and principal cells, respectively. In contrast, dynamin 2 was expressed throughout the epididymis, but localized to the Golgi apparatus of the principal cells in the proximal (caput) segment and the luminal border of these cells in more distal segments. These dynamin isoforms are therefore ideally positioned to play complementary, nonredundant roles in the regulation of the epididymal milieu. In support of this hypothesis, selective inhibition of dynamin altered the profile of proteins secreted from an immortalized caput epididymal cell line.

Summary Sentence

The dynamin family of mechanoenzymes is differentially expressed in the mouse epididymal epithelium and selectively regulates protein secretion.

High rates of body weight gain during the juvenile period appear to program molecular events within the hypothalamus, leading to advancement of puberty. Methylation of DNA, an epigenetic mechanism that controls gene expression, is associated with metabolic programming events and is proposed to play a role in the pubertal process. In this study, DNA methylation was assessed in genomic DNA obtained from the arcuate nucleus (ARC) of juvenile heifers fed to gain body weight at low (0.5 kg/d; low-gain, LG, n = 4) or high (1 kg/d; high-gain, HG, n = 4) rates from 4.5 to 8.5 mo of age (earliest puberty expected at 9 mo of age in HG heifers). Using a customdesigned oligonucleotide array targeted to imprinted genes and genes associated with nutritional inputs and the control of puberty, a comparative-genomic-hybridization array was used to identify differentially methylated regions between LG and HG heifers. Differential methylation of genomic regions associated with alteredmRNA expression was observed for genes whose activity has been reported to be involved in the modulation of growth and metabolism (GHR) and puberty (HMGA2). Hence, increased rates of body weight gain during the juvenile period alter the methylation pattern of genomic DNA obtained from the ARC and these changes may be involved in programming the age at puberty in heifers.

Summary Sentence

Increased rates of body weight gain during the juvenile period alter the methylation pattern of genomic DNA obtained from the ARC and these changesmay be involved in programming the age at puberty in heifers.

Fibroblast growth factor 2 (FGF2) is amitogen that induces proliferation, differentiation, and migration of cells, as well as angiogenesis and carcinogenesis via autocrine or paracrine actions. Fibroblast growth factor 2 expression is abundant in porcine conceptuses and endometrium during the estrous cycle and peri-implantation period of pregnancy. However, its intracellular actions in uterine epithelial cells have not been reported. The results of this study indicated abundant expression of FGFR1 and FGFR2 predominantly in uterine luminal and glandular epithelia during early pregnancy and that their expression decreased with increasing parity of the sows. Treatment of porcine uterine luminal epithelial (pLE) cells with FGF2 increased proliferation and DNA replication based on increases in proliferating cell nuclear antigen (PCNA) and initiation of G1/S phase progression. In addition, FGF2 increases phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38, and P90RSK in a time-dependent manner, and increases in their expression was suppressed by Wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), U0126 (an ERK1/2 inhibitor), SP600125 (a JNK inhibitor), and SB203580 (a P38 inhibitor) based on western blot analyses. Also, the abundance of cytoplasmic p-AKT protein was decreased by Wortmannin and U0126, and p-ERK1/2 protein was reduced only by U0126. Furthermore, inhibition of each signal transduction protein reduced the ability of FGF2 to stimulate proliferation and migration of pLE cells. Collectively, these results indicate that activation of FGFR1 and FGFR2 by uterine- and endometrial-derived FGF2 stimulates PI3K/AKT and mitogen-activated protein kinase pathways for development of the porcine uterus and improvement of litter size.

Summary Sentence

Activation of FGFR1 and FGFR2 by uterine- and endometrial-derived FGF2 enhances uterine development for implantation and placentation and improvement of litter size.

Endothelial microparticle (MP) release was increased in numerous cardiovascular diseases including preeclampsia. Oxidative stress is a potent inducer of endothelial dysfunction. In this study, we aimed to investigate if vitamin D could protect endothelial cells (ECs) from MP release induced by oxidative stress. Endothelial cell (from human umbilical vein) oxidative stress was induced by cultivation of cells under lowered oxygen condition (2%O₂) for 48 h and cells cultured under standard condition (21%O₂) served as control. 1,25(OH)₂D₃ was used as bioactive vitamin D. Using annexin-V as a marker of released MP assessed by flow cytometry and cytochrome c reduction assay to measure EC superoxide generation, we found that MP release and superoxide generation were significantly increased when cells were cultured under 2%O₂, which could be significantly inhibited by 1,25(OH)₂D₃. To study the potential mechanisms of 1,25(OH)₂D₃ protective effects on ECs, EC expression of endothelial nitric oxide synthase (eNOS), p-eNOS^Ser1177, p-eNOS^Thr495, caveolin-1, extracellular signal-regulated kinase (ERK), p-ERK, Akt, p-Akt^Ser473, Rho-associated coiled-coil protein kinase 1 (ROCK1), and vitamin D receptor were determined. Microparticle expression of eNOS and caveolin-1 were also determined. We found that under lowered oxygen condition, 1,25(OH)₂D₃ could upregulate EC eNOS, p-eNOS^Ser1177, and p-Akt^Ser473 expression, but inhibit cleaved ROCK1 expression. The upregulatory and inhibitory effects induced by 1,25(OH)₂D₃ were dose dependent. Strikingly, we also found that oxidative stress-induced decrease in ratio of eNOS and caveolin-1 expression in MP could be attenuated when 1,25(OH)₂D₃ was present in culture. These results suggest that upregulation of eNOS^Ser1177 and Akt^Ser473 phosphorylation and inhibition of ROCK1 cleavage in EC and modulation of eNOS and caveolin-1 expression in MP could be plausible mechanisms of vitamin D protective effects on ECs.

Summary Sentence

1,25(OH)₂D₃ induced upregulation of eNOS^Ser1177 and Akt^Ser473 phosphorylation and inhibition of ROCK1 cleavage could be plausible mechanisms of vitamin D protective effects to suppress MP release by ECs.

In ovine pregnancy, uterine space restriction (USR) resulting from decreased space for placental attachment caused intrauterine growth restriction and impaired nephrogenesis. The fetal kidney renin—angiotensin system (RAS) is involved in nephrogenesis, fluid balance, and iron deposition. Angiotensin II exerts its effects via multiple receptors: angiotensin II_1-8 receptor type 1 (AT₁R) and type 2 (AT₂R), and angiotensin II_1-7 Mas receptor (MASR).

Objective: To test the hypothesis that ovine USR is associated with dysregulation of the fetal renal RAS.

Methods: Multiparous pregnant ewes (n = 32), 16 with surgical bifurcated disconnection of one uterine horn to further reduce placental attachment sites, were studied. USR (n = 31) ovine fetuses were compared to nonspace restricted (NSR) singleton controls (n = 22) on gestational day (GD) 120 or GD130, term GD147. Fetal plasma was collected to evaluate plasma renin activity and iron indices. Fetal kidney AT₁R, AT₂R, and MASR proteins were assessed by Western immunoblotting and immunohistochemistry.

Results: AT₁R, AT₂R, and MASR protein expression was higher in USR at GD130 than aged-matched NSR and USR at GD120, (P < 0.05 all). AT₁R and AT₂R localization was homogenous throughout proximal and distal tubules in both USR and NSR at both gestational dates. MASR localization was punctate throughout renal cortical structures including tubules and glomeruli in both USR and NSR, shifted to intranuclear at GD130. Plasma renin activity was inversely related to plasma osmolarity (P < 0.02) and was downregulated in USR at GD130 (P < 0.05).

Conclusions: By late gestation, USR upregulated renal angiotensin receptor expression, an effect with potential functional implications.

Summary Sentence

Multifetal gestation-induced intrauterine growth restriction alters expression and localization of fetal renal angiotensin receptors and the associated relationships with fetal plasma chemistries.

Spermatogenesis is a complicated process that originates from spermatogonial stem cells (SSCs), which have self-renewal activity. Because SSCs are the only stem cells in the body that transmit genetic information to the next generation, they are an attractive target for germline modification. Although several virus vectors have been successfully used to transduce SSCs, cell toxicity or insertional mutagenesis of the transgene has limited their usage. Adeno-associated virus (AAV) is unique among virus vectors because of its target specificity and low toxicity in somatic cells, and clinical trials have shown that it has promise for gene therapy. However, there are conflicting reports on the possibility of germline integration of AAV into the genome of male germ cells, including SSCs. Here, we examined the usefulness of AAV vectors for exploring germline gene modification in SSCs. AAV1 infected cultured SSCs without apparent toxicity.Moreover, SSCs that were infected in fresh testis cells generated normal appearing spermatogenic colonies after spermatogonial transplantation. A microinsemination experiment produced offspring that underwent excision of the floxed target gene by AAV1-mediated Cre expression. Analysis of the offspring DNA showed no evidence of AAV integration, suggesting a low risk of germline integration by AAV infection. Although more extensive experiments are required to assess the risk of germline integration, our results show that AAV1 is useful for genetic manipulation of SSCs, and gene transduction by AAV will provide a useful approach to overcome potential problems associated with previous virus vector-mediated gene transduction.

Summary Sentence

A new system for spermatogonial stem cell gene transfection is demonstrated.

Uterine luminal epithelium (LE) is essential for establishing uterine receptivity. Previous microarray analysis revealed upregulation of Atp6v0d2 in gestation day 4.5 (D4.5) LE in mice. Realtime PCR showed upregulation of uterine Atp6v0d2 starting right before embryo attachment ∼D4.0. In situ hybridization demonstrated specific uterine localization of Atp6v0d2 in LE upon embryo implantation. Atp6v0d2 encodes one subunit for vacuolar-type H⁺-ATPase (V-ATPase), which regulates acidity of intracellular organelles and extracellular environment. LysoSensor Green DND-189 detected acidic signals in LE and glandular epithelium upon embryo implantation, correlating with Atp6v0d2 upregulation in early pregnant uterus. Atp6v0d2^-/- females had significantly reduced implantation rate and marginally reduced delivery rate from first mating only, but comparable number of implantation sites and litter size compared to control and comparable fertility to control from subsequent matings, suggesting a nonessential role of Atp6v0d2 subunit in embryo implantation. Successful implantation in both control and Atp6v0d2^-/- females was associated with uterine epithelial acidification. No significant compensatory upregulation of Atp6v0d1 mRNA was detected in D4.5 Atp6v0d2^-/- uteri. To determine the role of V-ATPase instead of a single subunit in embryo implantation, a specific V-ATPase inhibitor bafilomycin A1 (2.5µg/kg) was injected via uterine fat pad on D3 18:00 h. This treatment resulted in reduced uterine epithelial acidification, delayed implantation, and reduced number of implantation sites. It also suppressed oil-induced artificial decidualization. These data demonstrate uterine epithelial acidification as a novel phenomenon during embryo implantation and V-ATPase is involved in uterine epithelial acidification and uterine preparation for embryo implantation.

Summary Sentence

Our findings that mouse uterine epithelium becomes more acidic upon embryo implantation initiation and suppression of uterine epithelial acidification adversely affects embryo implantation provide a novel direction for understanding mechanisms in establishing uterine receptivity.

Adenosine to inosine (A-to-I) RNA editing occurs in a wide range of tissues and cell types and can be catalyzed by one of the two adenosine deaminase acting on double-stranded RNA enzymes, ADAR and ADARB1. Editing can impact both coding and noncoding regions of RNA, and in higher organisms has been proposed to function in adaptive evolution. Neither the prevalence of A-to-I editing nor the role of either ADAR or ADARB1 has been examined in the context of germ cell development in mammals. Computational analysis of whole testis and cell-type specific RNA-sequencing data followed by molecular confirmation demonstrated that A-to-I RNA editing occurs in both the germ line and in somatic Sertoli cells in two targets, Cog3 and Rpa1. Expression analysis demonstrated both Adar and Adarb1 were expressed in both Sertoli cells and in a celltype dependent manner during germ cell development. Conditional ablation of Adar did not impact testicular RNA editing in either germ cells or Sertoli cells. Additionally, Adar ablation in either cell type did not have gross impacts on germ cell development or male fertility. In contrast, global Adarb1 knockout animals demonstrated a complete loss of A-to-I RNA editing in spite of normal germ cell development. Taken together, these observations demonstrate ADARB1 mediates A-to-I RNA editing in the testis and these editing events are dispensable for male fertility in an inbred mouse strain in the lab.

Summary Sentence

Testicular A to I editing occurs primarily in meiotic and postmeiotic germ cells and is catalyzed by the editing enzyme ADARB1.

The insecticide acetamiprid is used to control noxious agricultural pests. However, it can cause mammalian toxicity. We evaluated the reproductive toxicity of acetamiprid in adult male Sprague Dawley rats. Rats were given oral acetamiprid alone or with vitamin E for 35 days. Rat plasma testosterone concentration and sperm quality decreased significantly as the levels of luteinizing hormone (LH) increased after exposure. At the same time, acetamiprid increased malondialdehyde and nitric oxide (NO) levels of Leydig cells. Further analysis showed that acetamiprid reduced the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) production of Leydig cells, but the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and the activity of adenylyl cyclase were not changed. Acetamiprid exposure also significantly diminished protein levels of steroidogenic acute regulatory protein (STAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B), and cytochrome P450, family 11, subfamily a, polypeptide 1 (CYP11A1), and testicular mRNA levels, which are cAMP-dependent proteins that are essential for steroidogenesis. Electron microscopy indicated mitochondrial membrane damage in the Leydig cells of the testes of exposed rats. Vitamin E ameliorated the impairment of acetamiprid on Leydig cells. Our results indicate that acetamiprid causes oxidative stress and mitochondrial damage in Leydig cells and inhibits the synthesis of testicular ATP and cAMP. Acetamiprid disrupts subsequent testosterone biosynthesis by decreasing the rate of conversion of cholesterol to testosterone and by preventing cholesterol from entering the mitochondria within the Leydig cells. These effects caused reproductive damage to the rats.

Summary Sentence

Mitochondrial inner membrane damage may be the mechanism by which acetamiprid inhibits the testosterone secretion of testicular interstitial Leydig cells.