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1.  Rhox13 Is Translated in Premeiotic Germ Cells in Male and Female Mice and Is Regulated by NANOS2 in the Male1 
Biology of Reproduction  2011;86(4):127.
Male and female germ cells enter meiosis in response to an extrinsic cue by retinoic acid (RA), but the pathways downstream of RA signaling that regulate early gametogenesis remain uncertain. We identified a novel reproductive homeobox gene, Rhox13, transcribed in the prenatal ovary and testis beginning on Embryonic Day (E) 13.5. Translation of RHOX13 also begins in female germ cells on E13.5 but is suppressed in male germ cells until Postnatal Day 3. Translation of RHOX13 coincides with initiation of RA signaling in both male and female gonads in vivo but occurs precociously in neonatal testes exposed to RA in vitro or in fetal male germ cells when NANOS2 is absent in vivo. Conversely, RHOX13 translation in female germ cells is suppressed in the presence of ectopically induced NANOS2. These results strongly suggest that RHOX13 expression is regulated at a posttranscriptional step by direct interaction of NANOS2 with Rhox13 mRNA to suppress translation.
NANOS2 and retinoic acid regulate the temporal expression of RHOX13 protein in gonocytes.
PMCID: PMC3338663  PMID: 22190708
meiosis; NANOS2; retinoic acid; Rhox13; testis; translation
2.  Developmental Exposure to Xenoestrogens at Low Doses Alters Femur Length and Tensile Strength in Adult Mice1 
Biology of Reproduction  2011;86(3):69.
Developmental exposure to high doses of the synthetic xenoestrogen diethylstilbestrol (DES) has been reported to alter femur length and strength in adult mice. However, it is not known if developmental exposure to low, environmentally relevant doses of xenoestrogens alters adult bone geometry and strength. In this study we investigated the effects of developmental exposure to low doses of DES, bisphenol A (BPA), or ethinyl estradiol (EE2) on bone geometry and torsional strength. C57BL/6 mice were exposed to DES, 0.1 μg/kg/day, BPA, 10 μg/kg/day, EE2, 0.01, 0.1, or 1.0 μg/kg/day, or vehicle from Gestation Day 11 to Postnatal Day 12 via a mini-osmotic pump in the dam. Developmental Xenoestrogen exposure altered femoral geometry and strength, assessed in adulthood by micro-computed tomography and torsional strength analysis, respectively. Low-dose EE2, DES, or BPA increased adult femur length. Exposure to the highest dose of EE2 did not alter femur length, resulting in a nonmonotonic dose response. Exposure to EE2 and DES but not BPA decreased tensile strength. The combined effect of increased femur length and decreased tensile strength resulted in a trend toward decreased torsional ultimate strength and energy to failure. Taken together, these results suggest that exposure to developmental exposure to environmentally relevant levels of xenoestrogens may negatively impact bone length and strength in adulthood.
Developmental exposure to low, environmentally relevant doses of three xenoestrogens, bisphenol A, ethinyl estradiol, and diethylstilbestrol, has a negative impact on femoral length and strength in adult mice.
PMCID: PMC3316267  PMID: 22088916
bone; developmental origins of health and disease; endocrine disruptors; environmental contaminants and toxicants; estradiol; estradiol receptor
3.  Rethinking In Vitro Embryo Culture: New Developments in Culture Platforms and Potential to Improve Assisted Reproductive Technologies1 
Biology of Reproduction  2011;86(3):62.
The preponderance of research toward improving embryo development in vitro has focused on manipulation of the chemical soluble environment, including altering basic salt composition, energy substrate concentration, amino acid makeup, and the effect of various growth factors or addition or subtraction of other supplements. In contrast, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development within the laboratory. The goal of this review is not to reevaluate the soluble composition of past and current embryo culture media, but rather to consider how other controlled and precise factors such as time, space, mechanical interactions, gradient diffusions, cell movement, and surface interactions might influence embryo development. Novel culture platforms are being developed as a result of interdisciplinary collaborations between biologists and biomedical, material, chemical, and mechanical engineers. These approaches are looking beyond the soluble media composition and examining issues such as media volume and embryo spacing. Furthermore, methods that permit precise and regulated dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being developed and tested. While several factors remain to be investigated to optimize the efficiency of embryo production, manipulation of the embryo culture microenvironment through novel devices and platforms may offer a pathway toward improving embryo development within the laboratory of the future.
New designs of platforms on which embryos are cultured permit use of dynamic fluid conditions, offering a benefit over traditional static culture techniques.
PMCID: PMC3316262  PMID: 21998170
blastocyst; dynamic culture; embryo culture; microfluidics; surface coating; vibration
4.  Brief Exposure to Progesterone During a Critical Neonatal Window Prevents Uterine Gland Formation in Mice1 
Biology of Reproduction  2011;86(3):63.
Uterine gland development (adenogenesis) in mice begins on Postnatal Day (PND) 5 and is completed in adulthood. Adenogenesis depends on estrogen receptor 1, and progesterone (P4) inhibits mitogenic effects of estrogen on uterine epithelium. This progestin-induced effect has been used to inhibit uterine gland development; progestin treatment of ewes for 8 wk from birth has produced infertile adults lacking uterine glands. The goals of the present study were to determine if a window of susceptibility to P4-mediated inhibition of uterine gland development exists in mice and whether early P4 treatment abolishes adenogenesis and fertility. Mice were injected daily with P4 (40 μg/g) or vehicle during various postnatal windows. Adenogenesis, cell proliferation, and expression of key morphoregulatory transcripts and proteins were examined in uteri at PNDs 10 and 20. Additionally, adenogenesis was assessed in isolated uterine epithelium. Treatment during PNDs 3–9, 5–9, or 3–7 abolished adenogenesis at PND 10, whereas treatments during PNDs 3–5 and 7–9 did not. Critically, mice treated during PNDs 3–9 lacked glands in adulthood, indicating that adenogenesis did not resume after this treatment. However, glands were present by PND 20 and later following treatment during PNDs 5–9 or 3–7, whereas treatment during PNDs 10–16 produced partial inhibition of adenogenesis at PND 20 and later. Epithelial proliferation at PND 10 was low following P4 treatment (PNDs 3–9) but exceeded that in controls at PND 20, indicating a rebound of epithelial proliferation following treatment. Messenger RNA for Wnt, Fzd, and Hox genes was altered by neonatal P4 treatment. All groups cycled during adulthood. Mice treated with P4 during PNDs 3–9, but not during other developmental windows, showed minimal fertility in adulthood. In summary, brief P4 treatment (7 days) during a critical neonatal window (PNDs 3–9) transiently inhibited epithelial proliferation but totally and permanently blocked adenogenesis and adult fertility. This resulted in permanent loss of uterine glands and, essentially, total infertility during adulthood. The narrow window for inhibition of adenogenesis identified here may have implications for development of this methodology as a contraceptive strategy for animals.
Exposure of neonatal mice to progesterone during a narrowly circumscribed, early postnatal window prevents uterine gland development.
PMCID: PMC3316263  PMID: 22133692
adenogenesis; ESR1; fertility; uterus
5.  Metastasis-Associated Protein 3 (MTA3) Regulates G2/M Progression in Proliferating Mouse Granulosa Cells1 
Biology of Reproduction  2011;86(3):64.
Metastasis-associated protein 3 (MTA3) is a constituent of the Mi-2/nucleosome remodeling and deacetylase (NuRD) protein complex that regulates gene expression by altering chromatin structure and can facilitate cohesin loading onto DNA. The biological function of MTA3 within the NuRD complex is unknown. Herein, we show that MTA3 was expressed highly in granulosa cell nuclei of all ovarian follicle stages and at lower levels in corpora lutea. We tested the hypothesis that MTA3-NuRD complex function is required for granulosa cell proliferation. In the ovary, MTA3 interacted with NuRD proteins CHD4 and HDAC1 and the core cohesin complex protein RAD21. In cultured mouse primary granulosa cells, depletion of endogenous MTA3 using RNA interference slowed cell proliferation; this effect was rescued by coexpression of exogenous MTA3. Slowing of cell proliferation correlated with a significant decrease in cyclin B1 and cyclin B2 expression. Granulosa cell populations lacking MTA3 contained a significantly higher percentage of cells in G2/M phase and a lower percentage in S phase compared with control cells. Furthermore, MTA3 depletion slowed entry into M phase as indicated by reduced phosphorylation of histone H3 at serine 10. These findings provide the first evidence to date that MTA3 interacts with NuRD and cohesin complex proteins in the ovary in vivo and regulates G2/M progression in proliferating granulosa cells.
Knockdown of metastasis-associated protein 3 (MTA3), a chromatin remodeling complex component, reduces cyclin B expression and slows proliferation of granulosa cells.
PMCID: PMC3316264  PMID: 22075476
cell cycle; chromatin; cohesin; granulosa cells; NuRD complex; ovary
6.  Probiotics Can Induce Follicle Maturational Competence: The Danio rerio Case1 
Biology of Reproduction  2011;86(3):65.
In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the maturation phase.
The probiotic Lactobacillus rhamnosus IMC501 enhances the ability to acquire maturational competence of zebrafish follicles.
PMCID: PMC3316265  PMID: 22088919
DNA array; fish reproduction; FPA FT-IR imaging; follicle maturation; gamete biology; gene expression; nutrition; oocyte maturation; PCR-DGGE; real-time PCR; zebrafish
7.  Protein Restriction to Pregnant Rats Increases the Plasma Levels of Angiotensin II and Expression of Angiotensin II Receptors in Uterine Arteries1 
Biology of Reproduction  2011;86(3):68.
Whether gestational protein restriction affects the renin-angiotensin system (RAS) in uterine artery remains unknown. In this study, we hypothesized that gestational protein restriction alters the expression of RAS components in uterine artery. In study one, time-scheduled pregnant Sprague Dawley rats were fed a normal or low-protein (LP) diet from Day 3 of pregnancy until they were killed at Days 19 and 22. The uterine arteries were collected and used for gene expression of Ace, Ace2, Agtr1a, Agtr1b, Agtr2, Esr1, and Esr2 by quantitative real-time PCR and/or Western blotting. LP increased plasma levels of angiotensin II in pregnant rats. In the uterine artery, the expressions of Agtr1a, Agtr1b, and Esr1 were increased by LP at Days 19 and 22 of pregnancy, whereas the abundance of AGTR1 and AGTR2 was increased by LP at Day 19 of pregnancy. The expression of Ace2 was not detectable in rat uterine artery. In study two, virgin female rats were ovariectomized and implanted with either 17beta-estradiol (E2), progesterone (P4), both E2 and P4, or placebo pellets until they were killed 7 days later. In rat uterine artery, E2 and P4 reduced the expression of Agtr1a, and E2 increased the expression of Agtr1b and Agtr2, but neither E2 nor P4 regulated the expression of Ace. These results indicate that gestational protein restriction induces an increase in Agtr1 expression in uterine artery, and thus may exacerbate the vasoconstriction to elevated angiotensin II present in maternal circulation, and that female sex hormones also play a role in this process.
Gestational protein restriction increases plasma levels of angiotensin II and AGTR1 and AGTR2 protein levels in late pregnancy; estrogen plays a role in regulating the expression of angiotensin II receptors in rat uterine artery.
PMCID: PMC3316266  PMID: 22088913
angiotensin I converting enzyme; angiotensin II/angiotensin II receptor; developmental origins of health and disease; gestational protein restriction; nutrition; pregnancy; rat; uterine artery
8.  Two miRNA Clusters, Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3), Are Involved in the Regulation of Spermatogonial Differentiation in Mice1 
Biology of Reproduction  2011;86(3):72.
Increasing evidence indicates that microRNAs (miRNAs) may be critical players in spermatogenesis. The miRNA expression profiles of THY1+-enriched undifferentiated spermatogonia were characterized, and members of Mir-17-92 (Mirc1) and its paralog Mir-106b-25 (Mirc3) clusters are significantly downregulated during retinoic acid-induced spermatogonial differentiation, both in vitro and in vivo. The repression of microRNA clusters Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3) by retinoic acid in turn potentially upregulates the expression of Bim, Kit, Socs3, and Stat3. The male germ cell-specific Mir-17-92 (Mirc1) knockout mice exhibit small testes, a lower number of epididymal sperm, and mild defect in spermatogenesis. Absence of Mir-17-92 (Mirc1) in male germ cells dramatically increases expression of Mir-106b-25 (Mirc3) cluster miRNAs in the germ cells. These results suggest that Mir-17-92 (Mirc1) cluster and Mir-106b-25 (Mirc3) cluster miRNAs possibly functionally cooperate in regulating spermatogonial development.
Down-regulation by retinoic acid of Mir-17-92 (Mirc1) cluster and its paralog Mir-106b-25 (Mirc3) cluster miRNAs contributes to retinoic acid-induced spermatogonial differentiation.
PMCID: PMC3316268  PMID: 22116806
microRNA; Mir-106b-25 cluster; Mir-17-92 cluster; retinoic acid; spermatogonial differentiation
9.  Ovarian Expression and Regulation of the Stromelysins During the Periovulatory Period in the Human and the Rat1 
Biology of Reproduction  2011;86(3):78.
The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.
Expression of the metalloproteinase Mmp10 mRNA is stimulated by hCG prior to follicular rupture in both the human and the rat ovary, indicating involvement in ovulation and subsequent luteinization.
PMCID: PMC3316269  PMID: 22116802
extracellular matrix; granulosa cells; matrix metalloproteinase; ovulation; ovulatory cycle; proteinases; theca cells
10.  The Long and Short of the Prolactin Receptor: The Corpus Luteum Needs Them Both!1 
Biology of Reproduction  2011;86(3):85.
In this issue of Biology of Reproduction, Le et al. examine the role of the long form of the prolactin receptor in ovarian function by developing mice expressing only this isoform of the receptor either ubiquitously or specifically in the corpus luteum.
PMCID: PMC3316270  PMID: 22190702
11.  Generation of Mice Expressing Only the Long Form of the Prolactin Receptor Reveals That Both Isoforms of the Receptor Are Required for Normal Ovarian Function1 
Biology of Reproduction  2011;86(3):86.
Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.
While follicular development and ovulation are unaffected by the sole expression of the long form of the prolactin receptor, luteinization, vascularization, and maintenance of corpus luteum function are impaired.
PMCID: PMC3316271  PMID: 22190699
corpus luteum; female infertility; ovary; prolactin; prolactin receptor; vascularization
12.  Liver X Receptor Modulation of Gene Expression Leading to Proluteolytic Effects in Primate Luteal Cells1 
Biology of Reproduction  2011;86(3):89.
The expressions of genes involved in cholesterol efflux increase, whereas those involved in extracellular cholesterol uptake decrease, during spontaneous functional regression of the primate corpus luteum (CL). This may result from liver x receptor (LXR) alpha (official symbol NR1H3) and/or beta (official symbol NR1H2) control of luteal gene transcription, because these nuclear receptor superfamily members are key regulators of cellular cholesterol homeostasis. Therefore, studies were conducted to assess endogenous LXR ligands in the primate CL through the luteal phase, and to determine the effect of synthetic or natural LXR ligands on cholesterol efflux and uptake in functional primate luteal cells. Using high-performance liquid chromatography tandem mass spectrometry, three LXR ligands were identified and quantified in the rhesus macaque CL, including 22R-hydroxycholesterol (22ROH), 27-hydroxycholesterol (27OH), and desmosterol. Levels of 22ROH paralleled serum progesterone concentrations, whereas mean levels of 27OH tended to be higher following the loss of progesterone synthesis. Desmosterol was present throughout the luteal phase. Functional macaque luteal cells treated with the synthetic LXR agonist T0901317 or physiologically relevant concentrations of the endogenous luteal ligands 22ROH, 27OH, and desmosterol had increased expression of various known LXR target genes and greater cholesterol efflux. Additionally, T0901317 reduced low-density lipoprotein receptor protein and extracellular low-density lipoprotein uptake, whereas 27OH decreased low-density lipoprotein receptor protein, most likely via a posttranslational mechanism. Collectively, these data support the hypothesis that LXR activation causes increased cholesterol efflux and decreased extracellular cholesterol uptake. In theory, these effects could deplete the primate CL of cholesterol needed for steroidogenesis, ultimately contributing to functional regression.
Liver x receptor activation in primate luteal cells increases cholesterol efflux and reduces extracellular cholesterol uptake from low-density lipoprotein, contributing to luteal regression by limiting cholesterol availability.
PMCID: PMC3316272  PMID: 22156476
corpus luteum; functional regression; liver x receptor; luteolysis; reverse cholesterol transport; rhesus macaque
13.  Placental TonEBP/NFAT5 Osmolyte Regulation in an Ovine Model of Intrauterine Growth Restriction1 
Biology of Reproduction  2011;86(3):94.
TonEBP/NFAT5 (the tonicity-responsive enhancer binding protein/nuclear factor of activated T cells) modulates cellular response to osmotic changes by accumulating inositol and sorbitol inside the cells. Our objective was to assess placental osmolytes, TonEBP/NFAT5 RNA and protein expression, and signaling molecules across gestation between control and intrauterine growth restriction (IUGR) ovine pregnancies. Pregnant sheep were placed in hyperthermic conditions to induce IUGR. Placental tissues were collected at 55, 95, and 130 days gestational age (dGA) to measure inositol, sorbitol, TonEBP/NFAT5 (NFAT5), sodium-dependent myo-inositol transporter (SMIT; official symbol SLC5A3), aldose reductase (AR), and NADPH (official symbol DE-CR1). Placental weight was reduced in IUGR compared to controls at 95 and 130 dGA. Osmolyte concentrations were similar between control and IUGR placentas, but both groups demonstrated a significant decrease in inositol concentration and an increase in sorbitol concentration with advancing gestation. Cytosolic NFAT5 protein decreased significantly from 55 to 95 dGA in both groups, and nuclear NFAT5 protein increased only at 130 dGA in the IUGR group, but no differences were seen between groups for either cytosolic or nuclear NFAT5 protein concentrations. DE-CR1 concentrations were similar between groups and increased significantly with advancing gestational age. AR was lowest at 55dGA, and SLC5A3 increased with advancing gestational age. We conclude that both placental osmolytes inositol and sorbitol (and their corresponding proteins SLC5A3 and AR) change with gestational age and are regulated, at least in part, by NFAT5 and DE-CR1 (NADPH). The inverse relationship between each osmolyte across gestation (e.g., inositol higher in early gestation and sorbitol higher in late gestation) may reflect nutritional needs that change across gestation.
The tonicity-responsive enhancer binding protein/nuclear factor of activated T cells (TonEBP/NFAT5) maintains normal placental osmotic homeostasis, but is altered in growth-restricted pregnancies.
PMCID: PMC3316273  PMID: 22190709
hyperthermia; IUGR; osmoregulation; placenta; TonEBP/NFAT5
14.  Dexrazoxane Ameliorates Doxorubicin-Induced Injury in Mouse Ovarian Cells1 
Biology of Reproduction  2011;86(3):96.
Doxorubicin (DXR) is a frontline chemotherapy agent implicated in unintended ovarian failure in female cancer survivors. The fertility preservation techniques currently available for cancer patients are often time and cost prohibitive and do not necessarily preserve endocrine function. There are no drug-based ovary protection therapies clinically available. This study provides the first investigation using dexrazoxane (Dexra) to limit DXR insult in ovarian tissue. In KK-15 granulosa cells, a 3-h DXR treatment increased double-strand (ds) DNA breaks 40%–50%, as quantified by the neutral comet assay, and dose-dependent cytotoxicity. Dexra exhibited low toxicity in KK-15 cells, inducing no DNA damage and less than 20% cell loss. Cotreating KK-15 cells with Dexra prevented acute DXR-induced dsDNA damage. Similarly, Dexra attenuated the DXR-induced 40%–65% increase in dsDNA breaks in primary murine granulosa cells and cells from in vitro cultured murine ovaries. DXR can cause DNA damage either through a topoisomerase II-mediated pathway, based on DXR intercalation into DNA, or through oxidative stress. Cotreating KK-15 cells with 2 μM Dexra was sufficient to prevent DXR-induced, but not H2O2-induced, DNA damage. These data indicated the protective effects are likely due to Dexra's inhibition of topoisomerase II catalytic activity. This putative protective agent attenuated downstream cellular responses to DXR, preventing H2AFX activation in KK-15 cells and increasing viability as demonstrated by increasing the DXR lethal dose in KK-15 cells 5- to 8-fold (LD20) and primary murine granulosa cells 1.5- to 2-fold (LD50). These data demonstrate Dexra protects ovarian cells from DXR insult and suggest that it is a promising tool to limit DXR ovarian toxicity in vivo.
Dexrazoxane protects ovarian cells from DXR-induced DNA damage, subsequent H2AX activation, and cell death by topoII inhibition, providing a novel tool for protection of the ovary from chemotherapy insult.
PMCID: PMC3316274  PMID: 22190700
doxorubicin; female infertility; granulosa cells; ovary; premature ovarian failure
15.  Roles of Reactive Oxygen Species and Antioxidants in Ovarian Toxicity1 
Biology of Reproduction  2011;86(2):27.
Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.
Oxidative stress can have significant impacts on follicular atresia and oocyte quality.
PMCID: PMC3290661  PMID: 22034525
follicle; oocyte; ovary; oxidative stress; toxicology
16.  A-MYB (MYBL1) Stimulates Murine Testis-Specific Ldhc Expression via the cAMP-Responsive Element (CRE) Site1 
Biology of Reproduction  2011;86(2):30.
Generally, knowledge of the mechanism regulating gene expression in primary spermatocytes is incomplete. We have used the lactate dehydrogenase gene (Ldhc) as a model to explore these mechanisms during spermatogenesis. Its 100-bp core promoter contained two essential elements common to many genes, a GC box and a CRE site. Here we report results that support a model in which transcription factor MYBL1 acts as a coactivator directing tissue-specific expression via the CRE cis element. We hypothesize that this is a common mechanism involving activation of multiple genes in the primary spermatocyte. MYBL1 is expressed predominantly as a tissue-specific transcription factor in spermatocytes and breast epithelial cells. Our finding that LDHC expression is lost in 21-day testes of MYBL1 mutant mice supports our hypothesis. In the GC1-spg germ cell line exogenous MYBL1 induces activity 4- to 8-fold, although extracts from these cells do not show MYBL1 binding activity for the Myb consensus sequences in the Ldhc promoter by EMSA. Rather, MYBL1 stimulates expression from a synthetic promoter containing only CRE elements, suggesting MYBL1 activates the promoter by interacting with protein that binds to a CRE element. Mutation of three Myb sites does not affect Ldhc promoter activity significantly (P > 0.05). CREB-binding protein (CBP) is a coactivator that interacts with CRE-binding protein CREB. We show that the transactivation domain (TAD) in MYBL1 interacts with the KIX domain in CBP, and the TAD domain and DNA binding domain in MYBL1 each interact with the CREB N-terminal domain. MYBL1 also stimulated expression from testis-specific genes Pgk2 (phosphoglycerate kinase 2) and Pdha2 (pyruvate dehydrogenase alpha 2) promoters, each of which contains CRE promoter elements and is expressed in primary spermatocytes. We propose that MYBL1 directs germ cell-specific activation via the CRE site of certain genes that are activated specifically in the primary spermatocyte, although other, more indirect effects of MYBL1 remain a possible explanation for our results.
MYBL1 is a coactivator that increases Ldhc expression in meiotic stages of spermatogenesis.
PMCID: PMC3290662  PMID: 21998171
gene expression; gene regulation; sperm; spermatocyte; spermatogenesis
17.  Maternal Protein Restriction Reduces Expression of Angiotensin I-Converting Enzyme 2 in Rat Placental Labyrinth Zone in Late Pregnancy1 
Biology of Reproduction  2011;86(2):31.
Both the systemic and the uteroplacental renin-angiotensin system (RAS) display dramatic changes during pregnancy. However, whether gestational protein insufficiency affects the expressions of RAS in the placenta remains unknown. In this study, we hypothesized that the expression of Ace2 in the placental labyrinth was reduced by maternal protein restriction. Pregnant Sprague-Dawley rats were fed a normal diet or a low-protein diet (LP) from Day 1 of pregnancy until they were killed at Day 14 or Day 18. The labyrinth zone (LZ) of the placenta was then dissected and snap frozen for expression analysis by quantitative real-time PCR of Ace, Ace2, Agtr1a, Agtr1b, and Agtr2. Formalin-fixed placentas were used for immunohistochemical analysis on ACE and ACE2 proteins. The findings include 1) the expression of Ace2 in rat LZ was reduced by maternal protein restriction in late pregnancy; 2) ACE protein was mainly present in syncytiotrophoblasts, whereas ACE2 protein was found predominantly in fetal mesenchymal tissue and fetal capillaries; 3) Agtr1a was predominant in the rat LZ, and its mRNA levels, but not protein levels, were reduced by LP; 4) expressions of Ace, Ace2, and Agtr1a in the rat LZ and their response to LP occurred in a gender-dependent manner. These results may indicate that a reduced expression of Ace2 and perhaps an associated reduction in angiotensin (1–7) production in the placenta by maternal protein restriction may be responsible for fetal growth restriction and associated programming of adulthood hypertension.
Maternal protein restriction reduces expression of Ace2 and alters expression of Ace and Agtr1 in the rat labyrinth in a gender-specific manner, which may be responsible for the placental programming on adulthood hypertension.
PMCID: PMC3290663  PMID: 22011389
angiotensin I-converting enzyme; labyrinth zone; maternal protein restriction; placenta; pregnancy; rodents; (guinea pigs; mice; rats; voles)
18.  Expression of Structural Proteins in Human Female and Male Genital Epithelia and Implications for Sexually Transmitted Infections1 
Biology of Reproduction  2011;86(2):32.
Men and women differ in their susceptibility to sexually transmittable infections (STIs) such as human immunodeficiency virus (HIV). However, a paucity of published information regarding the tissue structure of the human genital tract has limited our understanding of these gender differences. We collected cervical, vaginal, and penile tissues from human adult donors. Tissues were prepared with hematoxylin and eosin stains or immunofluorescence labeling of epithelial cell proteins and were analyzed for structural characteristics. Rhesus macaque genital tissues were evaluated to assess the use of this model for HIV/simian immunodeficiency virus transmission events. We found the stratified squamous epithelia of the male and female genital tract shared many similarities and important distinctions. Expression of E-cadherins, desmogleins 1/2, and involucrin was seen in all squamous epithelia, though expression patterns were heterogeneous. Filaggrin and a true cornified layer were markedly absent in female tissues but were clearly seen in all male epithelia. Desmogleins 1/2 were more consistent in the outermost strata of female squamous genital epithelia. Macaque tissues were similar to their respective human tissues. These initial observations highlight how male and female genital epithelia resemble and differ from one another. Further information regarding tissue structural characteristics will help to understand how STIs traverse these barriers to cause infection. This knowledge will be essential in future HIV pathogenesis, transmission, and prevention studies.
The expression of filaggrin, involucrin, desmogleins 1/2, and E-cadherin in male and female genital epithelia are characterized and compared using high-definition epifluorescence microscopy.
PMCID: PMC3290664  PMID: 21976595
barrier function; cervix; developmental origins of health and disease; filaggrin; intercellular junctions; keratin; male reproductive tract; vaginal epithelium
19.  Initiating Meiosis: The Case for Retinoic Acid1 
Biology of Reproduction  2011;86(2):35.
The requirement for vitamin A in reproduction and development was first determined from studies of nutritional deficiencies. Subsequent research has shown that embryonic development and both male and female reproduction are modulated by retinoic acid (RA), the active form of vitamin A. Because RA is active in multiple developmental systems, its synthesis, transport, and degradation are tightly regulated in different tissues. A growing body of evidence implicates RA as a requirement for the initiation of meiosis in both male and female mammals, resulting in a mechanistic model involving the interplay of RA, RA synthesis enzymes, RA receptors, and degradative cytochrome P450 enzymes in this system. Recently, that model has been challenged, prompting a review of the established paradigm. While it remains possible that additional molecules may be involved in regulating entry into meiosis, the weight of evidence supporting a key role for RA is incontrovertible.
This review summarizes the evidence that supports a role for retinoic acid in meiotic initiation in both sexes.
PMCID: PMC3290665  PMID: 22075477
germ cells; meiosis; ovary; retinoic acid; retinoids; testis
20.  Transforming Growth Factor Beta 1 Induces Tight Junction Disruptions and Loss of Transepithelial Resistance Across Porcine Vas Deferens Epithelial Cells1 
Biology of Reproduction  2011;86(2):36.
Epithelial cells lining the male excurrent duct contribute to male fertility by employing a number of physiological mechanisms that generate a luminal microenvironment conducive to spermatozoa maturation and storage. Among these mechanisms, male duct epithelia establish intercellular tight junctions that constitute a barrier to paracellular diffusion of water, solutes, large molecules, and cells. Mechanisms regulating the male duct epithelial barrier remain unidentified. Transforming growth factor beta (TGFB) is a regulatory cytokine present in high concentrations in human semen. This study examined whether TGFB has any effects on epithelial function exhibited by primary cultures of porcine vas deferens epithelia. TGFB1 exposure caused a 70%–99% decrease in basal transepithelial electrical resistance (RTE, a sensitive indicator of barrier integrity), while a significant decrease in anion secretory response to forskolin was detected at the highest levels of TGFB1 exposure employed. SB431542, a selective TGFB receptor I (TGFBR1) inhibitor, prevented decreases in barrier function. Results also demonstrated that TGFB1 exposure modifies the distribution pattern of tight junction proteins occludin and claudin 7. TGFBR1 is localized at the apical border of the native porcine vas deferens epithelium. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) 11 (also known as p38-MAPK) did not alter the effect of TGFB1 on RTE significantly. These data suggest that epithelia lining the vas deferens are subject to disruptions in the physical barrier if active TGFB becomes bioavailable in the luminal fluid, which might be expected to compromise fertility.
Epithelia lining the vas deferens respond to Transforming Growth Factor Beta 1 exposure with severe losses in transepithelial resistance and tight junction organization.
PMCID: PMC3290666  PMID: 21957188
cytokines; growth factors; male epithelial barrier; male reproductive tract; vas deferens
21.  Temporal Differences in Granulosa Cell Specification in the Ovary Reflect Distinct Follicle Fates in Mice1 
Biology of Reproduction  2011;86(2):37.
The embryonic origins of ovarian granulosa cells have been a subject of debate for decades. By tamoxifen-induced lineage tracing of Foxl2-expressing cells, we show that descendants of the bipotential supporting cell precursors in the early gonad contribute granulosa cells to a specific population of follicles in the medulla of the ovary that begin to grow immediately after birth. These precursor cells arise from the proliferative ovarian surface epithelium and enter mitotic arrest prior to upregulating Foxl2. Granulosa cells that populate the cortical primordial follicles activated in adult life derive from the surface epithelium perinatally, and enter mitotic arrest at that stage. Ingression from the surface epithelium dropped to undetectable levels by Postnatal Day 7, when most surviving oocytes were individually encapsulated by granulosa cells. These findings add complexity to the standard model of sex determination in which the Sertoli and granulosa cells of the adult testis and ovary directly stem from the supporting cell precursors of the bipotential gonad.
Granulosa cells arise from mitotically arrested supporting cell precursors and the ovarian surface epithelium during fetal and perinatal mouse development.
PMCID: PMC3290667  PMID: 21976597
Foxl2; granulosa cell; mitotic arrest; ovarian surface epithelium; ovary
22.  Changes of Large Molecular Weight Hyaluronan and Versican in the Mouse Pubic Symphysis Through Pregnancy1 
Biology of Reproduction  2011;86(2):44.
During pregnancy, the mouse pubic symphysis undergoes expansion and remodeling resulting in formation of a flexible and elastic interpubic ligament allowing passage of a term fetus. In the current study, we sought to identify and characterize components of the extracellular matrix that likely play an important role in elongation and flexibility of the interpubic ligament during parturition. Mouse pubic symphyses and interpubic ligaments collected at time points during pregnancy and postpartum were utilized to evaluate collagen type, collagen content, processing and solubility, matricellular protein, and proteoglycan expression and quantitative assessment of all glycosaminoglycans. These studies revealed increased gene expression for hyaluronan synthase 1, hyaluronan synthase 2, and versican on Gestation Day 18 as well as a decline in protein expression for the versican-degrading protease a disintegrin-like and metalloprotease with thrombospondin type 1 (ADAMTS1) motif. These findings suggest that the primary mediators of increased elongation and flexibility of the interpubic ligament at term result from increased synthesis and reduced metabolism of viscoelasticity-promoting molecules such as high molecular weight hyaluronan and versican.
Increased synthesis of a matrix containing large molecular weight hyaluronan and versican increases elasticity of the mouse interpubic ligament at birth.
PMCID: PMC3290668  PMID: 22011392
collagen; extracellular matrix; hyaluronan; mouse; pregnancy; pubic symphysis
23.  Spata22, a Novel Vertebrate-Specific Gene, Is Required for Meiotic Progress in Mouse Germ Cells1 
Biology of Reproduction  2011;86(2):45.
The N-ethyl-N-nitrosourea-induced repro42 mutation, identified by a forward genetics strategy, causes both male and female infertility, with no other apparent phenotypes. Positional cloning led to the discovery of a nonsense mutation in Spata22, a hitherto uncharacterized gene conserved among bony vertebrates. Expression of both transcript and protein is restricted predominantly to germ cells of both sexes. Germ cells of repro42 mutant mice express Spata22 transcript, but not SPATA22 protein. Gametogenesis is profoundly affected by the mutation, and germ cells in repro42 mutant mice do not progress beyond early meiotic prophase, with subsequent germ cell loss in both males and females. The Spata22 gene is essential for one or more key events of early meiotic prophase, as homologous chromosomes of mutant germ cells do not achieve normal synapsis or repair meiotic DNA double-strand breaks. The repro42 mutation thus identifies a novel mammalian germ cell-specific gene required for meiotic progression.
An unbiased mutagenesis strategy identified a novel gene, Spata22, that is required for mammalian meiotic progression.
PMCID: PMC3290669  PMID: 22011390
meiosis; mutagenesis; oogenesis; Spata22; spermatogenesis
24.  TIMP1 Contributes to Ovarian Anomalies in Both an MMP-Dependent and -Independent Manner in a Rat Model1 
Biology of Reproduction  2011;86(2):47.
Ovulatory dysfunction occurs in women with endometriosis, yet the mechanisms are unknown. We have shown that endometriotic lesions synthesize and secrete tissue inhibitor of metalloproteinase (TIMP) 1 into the peritoneal cavity in humans and a rat model of endometriosis, where excess TIMP1 localizes in the ovarian theca in endometriosis and modulating peritoneal TIMP1 alters ovarian dynamics. Here, we evaluated whether mechanisms whereby excessive peritoneal fluid TIMP1 negatively impacts ovarian function are matrix metalloproteinase (MMP)-dependent and/or MMP-independent actions. Rats were treated with a mutated TIMP1 without MMP inhibitory function (Ala-TIMP1), wild-type TIMP1 (rTIMP1), or PBS. Rats treated with Ala-TIMP1 or rTIMP1 had fewer antral follicles, fewer new corpora lutea, and the presence of luteinized unruptured follicle syndrome compared with PBS rats. Ala-TIMP1 and rTIMP1 differentially caused downstream changes in gene expression and protein localization related to ovulation, as measured by whole-genome microarray with quantitative real-time PCR validation and immunohistochemistry. More vascular endothelial growth factor and FN were expressed and localized in ovaries of Ala-TIMP1-treated rats compared to rTIMP1- and PBS-treated rats inferring MMP-independent functions. Less caspase 3 localized in ovaries of rTIMP1 compared with the other two groups, and was thus dependent on MMP action. Furthermore, after coimmunoprecipitation, more CD63 was bound to TIMP1 in ovaries of rats treated with Ala-TIMP1 than in rTIMP1-treated rats, providing evidence for another MMP-independent mechanism of ovulatory dysfunction. We predict that MMP-dependent and MMP-independent events are involved in improper fortification of the follicular wall through multiple mechanisms, such as apoptosis inhibition, extracellular matrix components and angiogenesis. Collectively, excessive peritoneal TIMP1 causes changes in ovarian dynamics, both dependently and independently of MMP inhibition.
TIMP1-affected ovulation in rats is both dependent on and independent of MMP function.
PMCID: PMC3290670  PMID: 22053095
endometriosis; extracellular matrix; ovary; ovulation
25.  Cold-Shock-Domain Protein A (CSDA) Contributes Posttranscriptionally to Gonadotropin-Releasing Hormone-Regulated Expression of Egr1 and Indirectly to Lhb1 
Biology of Reproduction  2011;86(2):53.
Gonadotropin-releasing hormone (GnRH), a hypothalamic neurohormone, regulates transcription of Lhb in gonadotrophs indirectly through transient induction and accumulation of EGR1, a zinc finger transcription factor. AlphaT3 and LbetaT2 cell lines model gonadotrophs at two distinct stages of development, prenatal and postnatal expression of Lhb. Although GnRH induces EGR1 in both cell lines, the levels of the DNA-binding protein are lower and disappear more quickly in alphaT3 than in LbetaT2 cells. Herein we show that overexpression of Egr1 in alphaT3 cells rescues activity of a transfected LHB promoter-reporter, suggesting that its transcription is dependent on EGR1 crossing a critical concentration threshold. We also show that Csda, a gene that encodes an RNA-binding protein and is a member of the cold-shock-domain (CSD) family, is expressed at higher levels in LbetaT2 compared to alphaT3 cells. Transient expression studies indicate that at least one Csd element, residing in the 3′ untranslated region of Egr1 mRNA, increases activity of a chimeric pGL3 luciferase reporter vector in LbetaT2 cells. Additional experiments indicate that CSDA physically interacts with Egr1 mRNA. Furthermore, siRNA-mediated reduction of endogenous Csda mRNA attenuates GnRH regulation of a transiently transfected LHB reporter vector. Taken together, these studies suggest that CSDA contributes posttranscriptionally to GnRH-regulated expression of Egr1, thereby enabling the transcription factor to cross a critical concentration threshold necessary for maximal accumulation of Lhb mRNA in response to the neurohormone.
The RNA binding protein CSDA contributes posttranscriptionally to GnRH-regulated expression of Egr1 by interacting with the 3′ UTR of Egr1 mRNA, and this control indirectly regulates Lhb transcription.
PMCID: PMC3290671  PMID: 22053098
cold-shock-domain protein A (CSDA); Egr1; GnRH; gonadotroph

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