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1.  [No title available] 
PMCID: PMC4076403  PMID: 24337315
2.  [No title available] 
PMCID: PMC4076404  PMID: 24337314
3.  [No title available] 
PMCID: PMC4076405  PMID: 24352558
4.  [No title available] 
PMCID: PMC4076406  PMID: 24389876
5.  [No title available] 
PMCID: PMC4076407  PMID: 24429216
6.  [No title available] 
PMCID: PMC4076408  PMID: 24451983
7.  [No title available] 
PMCID: PMC4076409  PMID: 24352559
8.  [No title available] 
PMCID: PMC4076410  PMID: 24389875
9.  Nondividing, Postpubertal Rat Sertoli Cells Resumed Proliferation after Transplantation1 
Biology of Reproduction  2013;90(1):13.
ABSTRACT
Conventionally, it was believed that Sertoli cells (SC) stopped proliferating at puberty and became terminally differentiated quiescent cells. However, recent studies have challenged that dogma. In this study, we transplanted nondividing SC isolated from 23- to 27-day-old postpubertal rats transduced with a recombinant adenoviral vector (containing furin-modified human proinsulin cDNA) into diabetic severe combined immunodeficiency mice. Immunostaining the grafts for cell proliferation markers, proliferating cell nuclear antigen (PCNA) and MKI67, revealed that transplanted SC within the grafts were proliferating. Possible causes for resumption of proliferation of SC could be viral transduction, cell isolation and culture, higher abdominal temperature at the transplant site, and/or transplantation. To test for these possible causes, double- immunofluorescence staining was performed for GATA4 (SC marker) and MKI67. None of the SC were positive for MKI67 in tissue collected during SC isolation and culture or at higher temperature. However, nontransduced SC stained positive for MKI67 after transplantation into rats, suggesting viral transduction was not a key factor for induction of SC proliferation. Interestingly, resumption in proliferative ability of nondividing SC was temporary, as SC stopped proliferating within 14 days of transplantation and did not proliferate thereafter. Quantification of 5-bromo-2′-deoxyuridine-labeled SC demonstrated that 7%–9% of the total transplanted SC were proliferating in the grafts. These data indicate for the first time that nondividing SC resumed proliferation after transplantation and further validate previous findings that SC are not terminally differentiated. Hence, transplantation of SC could provide a useful model with which to study the regulation of SC proliferation in vivo.
After transplantation into rodents, postpubertal nondividing rat Sertoli cells reinitiate their proliferation; thus, transplantation can be used as a model to study Sertoli cell proliferation in vivo.
doi:10.1095/biolreprod.113.110197
PMCID: PMC4076399  PMID: 24285718
proliferation; Sertoli cells; terminally differentiated; transplantation
10.  SPACA7 Is a Novel Male Germ Cell-Specific Protein Localized to the Sperm Acrosome That Is Involved in Fertilization in Mice1 
Biology of Reproduction  2013;90(1):16.
ABSTRACT
Sperm acrosome associated 7 (SPACA7) is a novel protein of unknown function with no homology to any known protein. Spaca7 transcripts are detected only in testis and predict a 158-residue mature polypeptide with one potential N-glycosylation site and no cysteines. Orthologs are present in various species, including mice and humans. We developed a polyclonal antibody to mouse SPACA7 to study its expression and function. Western blotting and immunofluorescence microscopy detected SPACA7 only in testis, and it was detected in testis starting at Postnatal Day 21 and into adulthood. Immunofluorescence staining of testicular germ cells detected weak SPACA7 expression as early as zygotene spermatocytes. Higher expression was observed in round spermatids, where SPACA7 was localized to a perinuclear spot adjacent to the Golgi and to the acrosome of elongating spermatids and spermatozoa. Immunogold electron microscopy demonstrated that SPACA7 is localized within the proacrosomal granule of round spermatids and the acrosome of spermatozoa. Finally, we showed that SPACA7 was retained within the acrosome of epididymal sperm and was released upon the acrosome reaction. To assess if SPACA7 was involved in fertilization, in vitro fertilization assays in the presence of anti-SPACA7 IgG were performed. Anti-SPACA7 inhibited fertilization of cumulus-intact eggs and prominently delayed cumulus dispersal. However, anti-SPACA7 did not inhibit fertilization of cumulus-free eggs. Our findings indicate that release of SPACA7 from the acrosome accelerates cumulus dispersal and facilitates fertilization via unknown mechanisms. This study is the first to document the expression of endogenous SPACA7 and a function for this novel acrosomal protein.
SPACA7, a novel male germ cell-specific acrosomal protein, is released upon the acrosome reaction and facilitates cumulus dispersal and fertilization.
doi:10.1095/biolreprod.113.111831
PMCID: PMC4076400  PMID: 24307706
acrosome; cumulus cells; fertilization; sperm; testis
11.  Inhibitory Effect of Progesterone on Cervical Tissue Formation in a Three-Dimensional Culture System with Human Cervical Fibroblasts1 
Biology of Reproduction  2013;90(1):18.
ABSTRACT
Progesterone supplementation is recommended to prevent preterm birth in women with a short cervix, but the mechanism is unclear. We hypothesize that progesterone acts by altering the composition of the cervical extracellular matrix (ECM). We tested this hypothesis using human cervical fibroblasts in both two-dimensional (2D) and three-dimensional (3D) cultures. For 2D culture, cells were seeded in 6-well plates and cultured with media supplemented with estradiol (10−8 M), progesterone (10−7 or 10−6 M), and vehicle. For 3D culture, the cells were cultured on a porous silk protein scaffold system. Progesterone and estrogen receptors were documented by immunohistochemistry and Western blot analysis. In both 2D and 3D cultures, decreased collagen synthesis was seen with increased progesterone concentration. Three-dimensional cultures could be maintained significantly longer than 2D cultures, and the morphology of 3D cultures appeared similar to native cervical tissue. Thus, further studies were performed in 3D culture. To determine the effect of progesterone concentration, the 3D scaffolds were cultured with estradiol (10−8 M) and five conditions: vehicle; 10−9, 10−8, or 10−7 M progesterone; or 10−7 M progesterone plus 10−6 M mifepristone. The highest progesterone concentration correlated with the least amount of collagen synthesis. Collagen synthesis progressively increased as progesterone concentration decreased. This effect was partially antagonized by mifepristone, suggesting the mechanism is mediated by the progesterone receptor. This hormonally responsive 3D culture system supports the hypothesis that progesterone has a direct effect on remodeling cervical ECM during pregnancy. The 3D culture system could be useful for studying the mechanism of progesterone effects on the cervix.
Using a hormonally responsive, three-dimensional culture system and human cervical fibroblasts, estradiol increased formation of cervical-like tissue; this effect was opposed by progesterone.
doi:10.1095/biolreprod.113.112540
PMCID: PMC4076401  PMID: 24285720
cervix; human; pregnancy; preterm birth; progesterone
12.  The Forkhead Transcription Factor, FOXP3: A Critical Role in Male Fertility in Mice1 
Biology of Reproduction  2013;90(1):4.
ABSTRACT
Fertility is dependent on the hypothalamic-pituitary-gonadal axis. Each component of this axis is essential for normal reproductive function. Mice with a mutation in the forkhead transcription factor gene, Foxp3, exhibit autoimmunity and infertility. We have previously shown that Foxp3 mutant mice have significantly reduced expression of pituitary gonadotropins. To address the role of Foxp3 in gonadal function, we examined the gonadal phenotype of these mice. Foxp3 mutant mice have significantly reduced seminal vesicle and testis weights compared with Foxp3+/Y littermates. Spermatogenesis in Foxp3 mutant males is arrested prior to spermatid elongation. Activation of luteinizing hormone signaling in Foxp3 mutant mice by treatment with human chorionic gonadotropin significantly increases seminal vesicle and testis weights as well as testicular testosterone content and seminiferous tubule diameter. Interestingly, human chorionic gonadotropin treatments rescue spermatogenesis in Foxp3 mutant males, suggesting that their gonadal phenotype is due primarily to a loss of pituitary gonadotropin stimulation rather than an intrinsic gonadal defect.
The forkhead transcription factor FOXP3 is essential for normal pituitary gonadotropin expression and, consequently, spermatogenesis in male mice.
doi:10.1095/biolreprod.113.112375
PMCID: PMC4076402  PMID: 24258212
fertility; forkhead; FOXP3; gonadotropin; pituitary; spermatogenesis; transcription factor
13.  Regulation of Baboon Fetal Ovarian Development by Placental Estrogen: Onset of Puberty Is Delayed in Offspring Deprived of Estrogen In Utero1 
Biology of Reproduction  2013;89(6):132.
ABSTRACT
Using the baboon as a model for studies of human reproductive biology, we previously showed that placental estrogen regulates fetal ovarian follicle development. In this study, offspring of baboons untreated or treated in utero with the aromatase inhibitor letrozole (estradiol reduced >95%) or letrozole and estradiol were reared to adulthood to determine whether estrogen programming of the fetal ovary impacted puberty and reproduction in adulthood. All offspring exhibited normal growth and blood pressure/chemistries. Puberty onset in untreated baboons (43.2 ± 1.4 mo) was delayed (P < 0.01) in animals of letrozole-treated mothers (49.0 ± 1.2 mo) and normal in offspring of mothers treated with letrozole and estradiol (42.7 ± 0.8 mo). During the first 2 yr postmenarche, menstrual cycles in estrogen-suppressed animals (43.2 ± 1.3 days) were longer (P < 0.05) than in untreated baboons (38.3 ± 0.5 days) or those treated with letrozole and estrogen (39.6 ± 0.8 days). Moreover, in estrogen-suppressed offspring, serum levels of estradiol were lower and follicle-stimulating hormone greater (P < 0.05) in the follicular and luteal phases, and the elevation in luteal-phase progesterone extended (P < 0.02). Thus, puberty onset was delayed and menstrual cycles prolonged and associated with altered serum hormone levels in baboon offspring that developed in an intrauterine environment in which estradiol levels were suppressed. Because puberty and follicle development, as shown previously, were normal in baboons treated in utero with letrozole and estradiol, we propose that fetal ovarian development and timely onset of puberty in the primate is programmed by fetal exposure to placental estrogen.
Fetal ovarian development and timely onset of puberty in the primate is programmed by fetal exposure to estrogen.
doi:10.1095/biolreprod.112.107318
PMCID: PMC4076353  PMID: 24132960
estradiol; ovary; pregnancy; primate; puberty
14.  Enhanced Cellular Responses and Distinct Gene Profiles in Human Fetoplacental Artery Endothelial Cells under Chronic Low Oxygen1  
Biology of Reproduction  2013;89(6):133.
ABSTRACT
Fetoplacental endothelial cells are exposed to oxygen levels ranging from 2% to 8% in vivo. However, little is known regarding endothelial function within this range of oxygen because most laboratories use ambient air (21% O2) as a standard culture condition (SCN). We asked whether human umbilical artery endothelial cells (HUAECs) that were steadily exposed to the physiological chronic normoxia (PCN, 3% O2) for ∼20–25 days differed in their proliferative and migratory responses to FGF2 and VEGFA as well as in their global gene expression compared with those in the SCN. We observed that PCN enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. In oxygen reversal experiments (i.e., when PCN cells were exposed to SCN for 24 h and vice versa), we found that preexposure to 21% O2 decreased the migratory ability, but not the proliferative ability, of the PCN-HUAECs in response to FGF2 and VEGFA. These PCN-enhanced cellular responses were associated with increased protein levels of HIF1A and NOS3, but not FGFR1, VEGFR1, and VEGFR2. Microarray analysis demonstrated that PCN up-regulated 74 genes and down-regulated 86, 14 of which were directly regulated by hypoxia-inducible factors as evaluated using in silico analysis. Gene function analysis further indicated that the PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from our functional assays. Given that PCN significantly alters cellular responses to FGF2 and VEGFA as well as transcription in HUAECs, it is likely that we may need to reexamine the current cellular and molecular mechanisms controlling fetoplacental endothelial functions, which were largely derived from endothelial models established under ambient O2.
Chronic low oxygen enhances human endothelial cell proliferation and migration and alters gene expression.
doi:10.1095/biolreprod.113.110551
PMCID: PMC4076354  PMID: 24152727
angiogenesis; artery endothelial cells; growth factors; physiological chronic low oxygen; transcriptome
15.  Estrogen Alters Remodeling of the Vaginal Wall after Surgical Injury in Guinea Pigs1 
Biology of Reproduction  2013;89(6):138.
ABSTRACT
Loss of pelvic organ support (i.e., pelvic organ prolapse) is common in menopausal women. Surgical reconstruction of pelvic organ prolapse is plagued with high failure rates. The objective of this study was to determine the effects of estrogen on biomechanical properties, lysyl oxidase (LOX), collagen content, and histomorphology of the vagina with or without surgical injury. Nulliparous ovariectomized guinea pigs were treated systemically with either 50 μg/kg/day estradiol (E2,) or vehicle. After 2 wk, vaginal surgery was performed, and animals were treated with either beta-aminopropionitrile (BAPN, an irreversible LOX inhibitor), or vehicle to determine the role of LOX in recovery of the vaginal wall from injury with or without E2. Estradiol resulted in (i) significant growth, increased smooth muscle, and increased thickness of the vagina, (ii) increased distensibility without compromise of maximal force at failure, and (iii) increased total and cross-linked collagen. In the absence of E2, BAPN resulted in decreased collagen and vaginal wall strength in the area of the injury. In contrast, in E2-treated animals, increased distensibility, maximal forces, and total collagen were maintained despite BAPN. Interestingly, LOX mRNA was induced dramatically (9.5-fold) in the injured vagina with or without E2 at 4 days. By 21 days, however, LOX levels declined to near baseline in E2-deprived animals. LOX mRNA levels remained strikingly elevated (12-fold) at 21 days in the estrogenized vagina. The results suggest that prolonged E2 induced increases in LOX, and collagen cross-links may act to sustain a matrix environment that optimizes long-term surgical wound healing in the vagina.
Estradiol not only increases baseline vaginal distensibility, vaginal epithelium and muscularis growth, and collagen content, but also prolonged increases in lysyl oxidase and collagen cross-links in the vaginal muscularis after injury.
doi:10.1095/biolreprod.113.112367
PMCID: PMC4076355  PMID: 24174572
collagen; estradiol/estradiol receptor; lysyl oxidase; matrix remodeling; menopause; pelvic organ prolapse; vagina
16.  Transcriptome Analysis of the Dihydrotestosterone-Exposed Fetal Rat Gubernaculum Identifies Common Androgen and Insulin-Like 3 Targets1 
Biology of Reproduction  2013;89(6):143.
ABSTRACT
Androgens and insulin-like 3 (INSL3) are required for development of the fetal gubernaculum and testicular descent. Previous studies suggested that the INSL3-exposed fetal gubernacular transcriptome is enriched for genes involved in neural pathways. In the present study, we profiled the transcriptome of fetal gubernaculum explants exposed to dihydrotestosterone (DHT) and compared this response to that with INSL3. We exposed fetal (Embryonic Day 17) rat gubernacula to DHT for 24 h (10 and 30 nM) or 6 h (1 and 10 nM) in organ culture and analyzed gene expression relative to that of vehicle-treated controls using Affymetrix arrays. Results were annotated using functional, pathway, and promoter analyses and independently validated for selected transcripts using quantitative RT-PCR (qRT-PCR). Transcripts were differentially expressed after 24 h but not 6 h. Most highly overrepresented functional categories included those related to gene expression, skeletal and muscular development and function, and Wnt signaling. Promoter response elements enriched in the DHT-specific transcriptome included consensus sequences for c-ETS1, ELK1, CREB, CRE-BP1/c-June, NRF2, and USF. We observed that 55% of DHT probe sets were also differentially expressed after INSL3 exposure and that the direction of change was the same in 96%. The qRT-PCR results confirmed that DHT increased expression of the INSL3-responsive genes Crlf1 and Chrdl2 but reduced expression of Wnt4. We also validated reduced Tgfb2 and Cxcl12 and increased Slit3 expression following DHT exposure. These data suggest a robust overlap in the DHT- and INSL3-regulated transcriptome that may be mediated in part by CREB signaling and a common Wnt pathway response for both hormones in the fetal gubernaculum.
Exposure of the developing fetal rat gubernaculum to dihydrotestosterone in vitro produces a transcriptome response characterized by overrepresentation of Wnt signaling pathway genes and strong overlap with the effects of INSL3.
doi:10.1095/biolreprod.113.112953
PMCID: PMC4076356  PMID: 24174575
androgens/androgen receptor; cryptorchidism; gene expression; gubernaculum; hormone action; male reproductive tract
17.  How the Kidney Is Impacted by the Perinatal Maternal Environment to Develop Hypertension1 
Biology of Reproduction  2013;89(6):144.
ABSTRACT
Environmental conditions during perinatal development such as maternal undernutrition, maternal glucocorticoids, placental insufficiency, and maternal sodium overload can program changes in renal Na+ excretion leading to hypertension. Experimental studies indicate that fetal exposure to an adverse maternal environment may reduce glomerular filtration rate by decreasing the surface area of the glomerular capillaries. Moreover, fetal responses to environmental insults during early life that contribute to the development of hypertension may include increased expression of tubular apical or basolateral membrane Na+ transporters and increased production of renal superoxide leading to enhanced Na+ reabsorption. This review will address the role of these potential renal mechanisms in the fetal programming of hypertension in experimental models induced by maternal undernutrition, fetal exposure to glucocorticoids, placental insufficiency, and maternal sodium overload in the rat.
The impact of adverse events during gestational life, on later cardio-renal health of the offspring is highlighted, implicating the importance of fetal life on long-term health.
doi:10.1095/biolreprod.113.111823
PMCID: PMC4076357  PMID: 24227755
developmental origins of health and disease; hypertension; intrauterine growth restriction (IUGR); kidney; oxidative stress
18.  Investigating the Role of Tbx4 in the Female Germline in Mice1 
Biology of Reproduction  2013;89(6):148.
ABSTRACT
Normal development of germ cells is essential for fertility and mammalian reproduction. Although abnormal development of oocytes or follicles may lead to primary ovarian insufficiency (POI), a disorder that causes infertility in 1% of women less than 40 yr of age, the genes and signaling pathways activated in POI are not as yet fully elucidated. Tbx4, a member of the T-box family of transcription factors, is expressed in embryonic germ cells and postnatal oocytes at all stages of folliculogenesis. To investigate the requirement for Tbx4 in the germline, we analyzed germ cell development in the absence of Tbx4. We show that primordial germ cells (PGCs) are reduced in Tbx4 homozygous null (Tbx4−/−) embryos at Embryonic Day (E) 10.0. Tbx4−/− embryos die by E10.5; to study later time points in vitro, a tamoxifen-inducible estrogen receptor Cre recombinase was used to delete Tbx4 conditional mutant alleles. In addition, Gdf9cre and Zp3cre, two oocyte-specific Cre recombinases, were used to delete Tbx4 from postnatal primordial and primary follicles, respectively. We show that in vitro differentiation of the gonad into morphologically distinct testes and ovaries occurs normally starting at E11.5 when Tbx4 is deleted. In Gdf9cre; Tbx4fl/− and Zp3cre; Tbx4fl/− adult females, primordial, primary, secondary, and antral follicles form, ovulation occurs, corpus luteum formation is normal, and the mice are fertile without any evidence of diminished ovarian reserve. Although postnatal deletion of Tbx4 in oocytes does not obviously impair fertility, it is possible that the reduction in PGCs observed in Tbx4 homozygous null mutant embryos could affect long-term fertility in adults.
Postnatal oocyte-specific deletion of Tbx4 does not obviously impair fertility but reduces primordial germ cell number.
doi:10.1095/biolreprod.113.107649
PMCID: PMC4076358  PMID: 24089201
fertility; oocytes; primordial germ cells; T-box; Tbx4
19.  1,25-Dihydroxyvitamin D3 Reduces Extracellular Matrix-Associated Protein Expression in Human Uterine Fibroid Cells1 
Biology of Reproduction  2013;89(6):150.
ABSTRACT
Uterine fibroids (leiomyomas) are the most common benign tumors associated with excessive deposition of extracellular matrix (ECM)-associated proteins that increase fibroid tumorigenicity. Herein, we determined the expression levels of vitamin D receptor (VDR) protein in human uterine fibroids and compared these levels to those in adjacent normal myometrium. Using Western blot analysis, we found that more than 60% of uterine fibroids analyzed (25 of 40) expressed low levels of VDR. We also found that the biologically active 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), which functions via binding to its nuclear VDR, induced VDR in a concentration-dependent manner and reduced ECM-associated fibrotic and proteoglycans expression in immortalized human uterine fibroid cell line (HuLM). At 1–10 nM concentrations, 1,25(OH)2D3 significantly induced (P < 0.05) nuclear VDR, which was further stimulated by higher concentrations of 1,25(OH)2D3 in HuLM cells. 1,25(OH)2D3 at 10 nM also significantly reduced (P < 0.05) the protein expression of ECM-associated collagen type 1, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) in HuLM cells. We also found that 1,25(OH)2D3 reduced mRNA and protein expressions of proteoglycans such as fibromodulin, biglycan, and versican in HuLM cells. Moreover, the aberrant expression of structural smooth muscle actin fibers was reduced by 1,25(OH)2D3 treatment in a concentration-dependent manner in HuLM cells. Taken together, our results suggest that human uterine fibroids express reduced levels of VDR compared to the adjacent normal myometrium and that treatment with 1,25(OH)2D3 can potentially reduce the aberrant expression of major ECM-associated proteins in HuLM cells. Thus, 1,25(OH)2D3 might be an effective, safe, nonsurgical treatment option for human uterine fibroids.
1,25-dihydroxyvitamin D3 is an antifibrotic agent that suppresses major extracellular matrix-associated protein expression in human uterine fibroid cells and might be useful as an alternative therapeutic option for fibroid treatment.
doi:10.1095/biolreprod.113.107714
PMCID: PMC4076359  PMID: 24174578
1,25-dihydroxyvitamin D3 (1,25[OH]2D3); extracellular matrix (ECM); fibroids; vitamin D receptor (VDR)
20.  Parturition and Recruitment of Macrophages in Cervix of Mice Lacking the Prostaglandin F Receptor1 
Biology of reproduction  2007;78(3):438-444.
Parturition does not occur in transgenic mice lacking the prostaglandin F receptor (Ptgfr−/−) because luteolysis is forestalled and progesterone production persists. Ovariectomy of pregnant Ptgfr−/− mice leads to a decline in circulating progesterone and delivery of live pups. The objective of the present study was to test the hypothesis that immigration of macrophages and increased innervation of the cervix of Ptgfr−/− mice was associated with ripening and parturition. The cervix of pregnant Ptgfr−/− mice was studied on Days 15–21 after breeding; additional groups were ovariectomized on Day 19 of pregnancy, and the cervix obtained on Day 20 of pregnancy before birth or the next day at about 24 h after birth. On Days 18–19 of pregnancy, macrophage numbers and nerve fiber density increased more than 3-fold compared with findings in nonpregnant or Day 15 or 21 pregnant Ptgfr−/− mice. The magnitude and time course of these changes were comparable to those found in wild-type controls that delivered on Day 19 after breeding. Thus, the mechanism regulating macrophage immigration, innervation, and cervical remodeling in Ptgfr−/− mice with delayed parturition is similar to wild-type controls that deliver at term. By contrast, ovariectomy forestalled the decrease in cervical macrophages in Ptgfr−/− mice. By Day 21 after breeding, macrophage numbers more than double those after ovariectomy, relative to those found in pregnant Ptgfr−/− mice, whereas nerve fiber density was the same regardless of birth. Density of collagen structure in these mice directly matched macrophage traffic in the cervix. The findings indicate that the prostaglandin F2alpha receptor and progesterone withdrawal are a necessary part of the final common pathway for ripening of the cervix and the process of parturition.
doi:10.1095/biolreprod.107.063404
PMCID: PMC4237585  PMID: 18003949
cervix; cyclooxygenase; labor; leukocyte; macrophage; neuropeptides; parturition; progesterone; progesterone receptor
21.  Expression Profiling Reveals Developmentally Regulated lncRNA Repertoire in the Mouse Male Germline1 
Biology of Reproduction  2013;89(5):107.
ABSTRACT
In mammals, the transcriptome of large noncoding RNAs (lncRNAs) is believed to be greater than that of messenger RNAs (mRNAs). Some lncRNAs, especially large intergenic noncoding RNAs (lincRNAs), participate in epigenetic regulation by binding chromatin-modifying protein complexes and regulating protein-coding gene expression. Given that epigenetic regulation plays a critical role in male germline development, we embarked on expression profiling of both lncRNAs and mRNAs during male germline reprogramming and postnatal development using microarray analyses. We identified thousands of lncRNAs and hundreds of lincRNAs that are either up- or downregulated at six critical time points during male germ cell development. In addition, highly regulated lncRNAs were correlated with nearby (<30 kb) mRNA gene clusters, which were also significantly up- or downregulated. Large ncRNAs can be localized to both the nucleus and cytoplasm, with nuclear lncRNAs mostly associated with key components of the chromatin-remodeling protein complexes. Our data indicate that expression of lncRNAs is dynamically regulated during male germline development and that lncRNAs may function to regulate gene expression at both transcriptional and posttranscriptional levels via genetic and epigenetic mechanisms.
Dynamic expression of large noncoding RNAs and their interactions with chromatin-remodeling proteins suggests a critical role in the epigenetic regulation of male germline development and spermatogenesis.
doi:10.1095/biolreprod.113.113308
PMCID: PMC4076377  PMID: 24048575
epigenetics; fertility; germ cell; reproduction; spermatogenesis
22.  Mouse Strain Does Not Influence the Overall Effects of Bisphenol A-Induced Toxicity in Adult Antral Follicles1 
Biology of Reproduction  2013;89(5):108.
ABSTRACT
Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) widely used in common consumer products containing polycarbonate plastics and epoxy resins. Previous studies indicate that other EDCs have species-dependent effects. Furthermore, some EDCs are known to have different effects in different strains within the same species. Little information, however, is known about whether the effects of BPA on the ovary differ by strain. Previous studies have shown that BPA inhibits follicle growth, induces atresia, and inhibits steroidogenesis and expression of steroidogenic enzymes in antral follicles from adult FVB mice. Thus, this study was designed to expand previous work by testing the hypothesis that mouse strain may differentially affect the susceptibility of adult antral follicles to BPA-induced toxicity. To test this hypothesis, antral follicles were mechanically isolated from adult FVB, CD-1, and C57BL/6 mice, individually cultured for 6–120 h and treated with either vehicle control (dimethylsulfoxide) or various concentrations of BPA (1.0 μg/ml, 10 μg/ml, or 100 μg/ml). After culture, media were subjected to measurements of hormone production via ELISA, and follicles were subjected to real-time PCR for analysis of genes known to regulate steroidogenesis, the cell cycle, and atresia. Overall, BPA inhibited follicle growth and steroidogenesis in all tested strains, but CD-1 follicles were slightly more sensitive to BPA at early time points than FVB and C57BL/6 follicles. These data suggest that CD-1, FVB, and C57BL/6 mice can all be used to investigate the effects of BPA on ovarian follicles.
Mouse strain does not influence BPA-induced inhibition of follicle growth or steroidogenesis.
doi:10.1095/biolreprod.113.111864
PMCID: PMC4076378  PMID: 24025742
bisphenol A; follicle growth; steroidogenesis; strain
23.  Impact of Vitrification on the Meiotic Spindle and Components of the Microtubule-Organizing Center in Mouse Mature Oocytes1 
Biology of Reproduction  2013;89(5):112.
ABSTRACT
Cryopreservation of oocytes is becoming a valuable method for fertility preservation in women. However, various unphysiological alterations occur in the oocyte during the course of cryopreservation, one of which is the disappearance of the meiotic spindle. Fortunately, the meiotic spindle does regenerate after thawing the frozen oocytes, which enables completion of meiosis and further development after fertilization. Nonetheless, the mechanistic understanding of the meiotic spindle regeneration after cryopreservation is still scarce. Here, to gain insight into the mechanisms of the spindle disappearance and regeneration, we examined the status of spindle microtubules as well as the key components of the microtubule-organizing center (MTOC), specifically gamma-Tubulin, NEDD1, and Pericentrin, in mature (metaphase II) mouse oocytes at different steps of vitrification, a major cryopreservation technique. We found that the configuration of the spindle microtubules dynamically changed during the process of vitrification and that spindle regeneration was preceded by excessive microtubule polymerization, followed by reduction into the normal size and shape. Also, all three MTOC components exhibited disappearance and reappearance during the vitrification process, although Pericentrin appeared to regenerate in earlier steps compared to the other components. Furthermore, we found that the localization of the MTOC components to the spindle poles persisted even after depolymerization of spindle microtubules, suggesting that the MTOC components are impacted by vitrification independently from the integrity of the microtubules. The present study would set the stage for future investigations on the molecular mechanisms of the meiotic spindle regeneration, which may contribute to further improving protocols for oocyte cryopreservation.
The integrity of the meiotic spindle in the metaphase II oocyte, including the localization of microtubule bundles and distribution of the MTOC components, is dynamically altered during the course of the vitrification process.
doi:10.1095/biolreprod.113.108167
PMCID: PMC4076379  PMID: 24025740
assisted reproductive technology; cryopreservation; meiotic spindle; oocyte; vitrification
24.  Estrogen Responsiveness of the TFIID Subunit TAF4B in the Normal Mouse Ovary and in Ovarian Tumors1 
Biology of Reproduction  2013;89(5):116.
ABSTRACT
Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation.
The transcription factor TAF4B, which is required for murine fertility, is upregulated by estrogen in the normal mouse ovary and in ovarian tumors via nuclear estrogen receptors.
doi:10.1095/biolreprod.113.111336
PMCID: PMC4076380  PMID: 24068106
estradiol/estradiol receptor; ovarian cancer; ovary; premature ovarian failure; TAF4B
25.  Signaling in Sperm: Toward a Molecular Understanding of the Acquisition of Sperm Motility in the Mouse Epididymis1 
Biology of Reproduction  2013;89(5):127.
ABSTRACT
Sperm motility encompasses a wide range of events involving epididymal maturation and activation of biochemical pathways, most notably cyclic AMP (cAMP)-protein kinase A (PKA) activation. Following the discovery of guanine-nucleotide exchange factors (RAPGEFs), also known as exchange proteins activated by cAMP, we investigated the separate roles of PKA and RAPGEFs in sperm motility. RT-PCR showed the presence of Rapgef3, Rapgef4, and Rapgef5, as well as several known RAPGEF partner mRNAs, in spermatogenic cells. However, Rapgef3 and Rapgef4 appeared to be less abundant in condensing spermatids versus pachytene spermatocytes. Similarly, many of these proteins were detected by immunoblotting. RAPGEF5 was detected in germ cells and murine epididymal sperm. Indirect immunofluorescence localized SGK1, SGK3, AKT1 pT308, and RAPGEF5 to the acrosome, while PDPK1 was found in the postacrosomal region. SGK3 was present throughout the tail, while PDPK1 and AKT1 pT308 were in the midpiece. When motility was assessed in demembranated cauda epididymal sperm, addition of ATP and the selective ligand for RAPGEFs, 8-pCPT-2′-O-Me-cAMP, resulted in motility, but the sperm were unable to undergo hyperactivated-like motility. In contrast, when demembranated cauda epididymal sperm were incubated with ATP plus dibutyryl cAMP, sperm became motile and progressed to hyperactivated-like motility. However, no significant difference was observed when intact sperm were examined. GSK3 phosphorylation was altered in the presence of H89, a PKA inhibitor. Significantly, intact caput epididymal sperm became motile when incubated in the presence of extracellular ATP. These results provide evidence for a new pathway involved in endowing sperm with the capacity to swim.
Caput epididymal sperm become motile after exposure to extracellular ATP.
doi:10.1095/biolreprod.113.110163
PMCID: PMC4076381  PMID: 24006282
epididymis; rodents (rats, mice, guinea pigs, voles); sperm; sperm maturation; sperm motility and transport

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