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1.  Ovarian Membrane-Type Matrix Metalloproteinases: Induction of MMP14 and MMP16 During the Periovulatory Period in the Rat, Macaque, and Human1 
Biology of Reproduction  2014;91(2):34, 1-12.
An intrafollicular increase in proteolytic activity drives ovulatory events. Surprisingly, the periovulatory expression profile of the membrane-type matrix metalloproteinases (MT-MMPs), unique proteases anchored to the cell surface, has not been extensively examined. Expression profiles of the MT-MMPs were investigated in ovarian tissue from well-characterized rat and macaque periovulatory models and naturally cycling women across the periovulatory period. Among the six known MT-MMPs, mRNA expression of Mmp14, Mmp16, and Mmp25 was increased after human chorionic gonadotropin (hCG) administration in rats. In human granulosa cells, mRNA expression of MMP14 and MMP16 increased following hCG treatment. In contrast, mRNA levels of MMP16 and MMP25 in human theca cells were unchanged before ovulation but declined by the postovulatory stage. In macaque granulosa cells, hCG increased mRNA for MMP16 but not MMP14. Immunoblotting showed that protein levels of MMP14 and MMP16 in rats increased, similar to their mRNA expression. In macaque granulosa cells, only the active form of the MMP14 protein increased after hCG, unlike its mRNA or the proprotein. By immunohistochemistry, both MMP14 and MMP16 localized to the different ovarian cell types in rats and humans. Treatment with hCG resulted in intense immunoreactivity of MMP14 and MMP16 proteins in the granulosa and theca cells. The present study shows that MMP14 and MMP16 are increased by hCG administration in the ovulating follicle, demonstrating that these MMPs are conserved among rats, macaques, and humans. These findings suggest that MT-MMPs could have an important role in promoting ovulation and remodeling of the ovulated follicle into the corpus luteum.
PMCID: PMC4435413  PMID: 24920038
extracellular matrix; granulosa cells; matrix metalloproteinase; ovary; ovulation; theca cell
2.  Expression of Progesterone Receptor Membrane Component-2 Within the Immature Rat Ovary and Its Role in Regulating Mitosis and Apoptosis of Spontaneously Immortalized Granulosa Cells1 
Biology of Reproduction  2014;91(2):36, 1-11.
Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular 3H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development.
PMCID: PMC4435414  PMID: 24990806
apoptosis; granulosa cells; mitosis; PGRMC1; PGRMC2; progesterone
3.  Murine Follicular Development Requires Oocyte DICER, but Not DROSHA1 
Biology of Reproduction  2014;91(2):39, 1-8.
Both DICER and DROSHA are RNase III enzymes involved in the biogenesis of small noncoding RNAs. DROSHA cleaves the stem-loop portion of the primary miRNAs and produces precursor miRNAs in the nucleus, whereas DICER processes double-stranded RNA precursors into mature miRNAs and endogenous small interference RNAs in the cytoplasm. Selective inactivation of Dicer in growing oocytes of primary follicles leads to female infertility due to oocyte spindle defects. However, it remains unknown if oocyte Dicer expression in the fetal ovary is required for proper follicular development in the postnatal ovary. Moreover, the role of Drosha in folliculogenesis has never been investigated. Here, we report that conditional knockout of Dicer in prophase I oocytes of the fetal ovary led to compromised folliculogenesis, premature ovarian failure, and female infertility in the adult ovary, whereas selective inactivation of Drosha in oocytes of either the fetal or the developing ovary had no effects on normal folliculogenesis and female fertility in adulthood. Our data indicate that oocyte DICER expression in the fetal ovary is required, and oocyte DROSHA is dispensable, for postnatal follicular development and female fertility in adulthood.
PMCID: PMC4435415  PMID: 24990804
fertility; meiosis; oocyte; oogenesis; ovary; ovulation; posttranscriptional regulation; premature ovarian failure; primordial germ cells; small RNA; spindle
4.  Cryptorchidism in the Orl Rat Is Associated with Muscle Patterning Defects in the Fetal Gubernaculum and Altered Hormonal Signaling1 
Biology of Reproduction  2014;91(2):41, 1-14.
Cryptorchidism, or undescended testis, is a common male genital anomaly of unclear etiology. Hormonal stimulation of the developing fetal gubernaculum by testicular androgens and insulin-like 3 (INSL3) is required for testicular descent. In studies of the orl fetal rat, one of several reported strains with inherited cryptorchidism, we studied hormone levels, gene expression in intact and hormone-stimulated gubernaculum, and imaging of the developing cremaster muscle facilitated by a tissue clearing protocol to further characterize development of the orl gubernaculum. Abnormal localization of the inverted gubernaculum was visible soon after birth. In the orl fetus, testicular testosterone, gubernacular androgen-responsive transcript levels, and muscle-specific gene expression were reduced. However, the in vitro transcriptional response of the orl gubernaculum to androgen was largely comparable to wild type (wt). In contrast, increases in serum INSL3, gubernacular INSL3-responsive transcript levels, expression of the INSL3 receptor, Rxfp2, and the response of the orl gubernaculum to INSL3 in vitro all suggest enhanced activation of INSL3/RXFP2 signaling in the orl rat. However, DNA sequence analysis did not identify functional variants in orl Insl3. Finally, combined analysis of the present and previous studies of the orl transcriptome confirmed altered expression of muscle and cellular motility genes, and whole mount imaging revealed aberrant muscle pattern formation in the orl fetal gubernaculum. The nature and prevalence of developmental muscle defects in the orl gubernaculum are consistent with the cryptorchid phenotype in this strain. These data suggest impaired androgen and enhanced INSL3 signaling in the orl fetus accompanied by defective cremaster muscle development.
PMCID: PMC4435416  PMID: 24966393
androgen receptor; cryptorchidism; gubernaculum; insulin-like 3; male reproductive tract
5.  Placental Growth Factor Influences Maternal Cardiovascular Adaptation to Pregnancy in Mice 
Biology of reproduction  2014;92(2):44.
In healthy human pregnancies, placental growth factor (PGF) concentrations rise in maternal plasma during early gestation, peak over weeks 26–30, then decline. Since PGF in non-gravid subjects participates in protection against and recovery from cardiac pathologies, we asked if PGF contributes to pregnancy-induced maternal cardiovascular adaptations. Cardiovascular function and structure were evaluated in virgin, pregnant and postpartum C56BL/6-Pgf−/− (Pgf−/−) and C57BL/6-Pgf+/+ (B6) mice using plethysmography, ultrasound, qPCR and cardiac and renal histology. Pgf−/− females had higher systolic blood pressure in early and late pregnancy but an extended, abnormal midpregnancy interval of depressed systolic pressure. Pgf−/− cardiac output was lower than gestation day (gd)-matched B6 after mid-pregnancy. While Pgf−/− left ventricular mass was greater than B6, only B6 showed the expected gestational gain in left ventricular mass. Expression of vasoactive genes in the left ventricle differed at gd8 with elevated Nos expression in Pgf−/− but not at gd14. By gd16, Pgf−/− kidneys were hypertrophic and had glomerular pathology. This study documents for the first time that PGF is associated with the systemic maternal cardiovascular adaptations to pregnancy.
PMCID: PMC4490891  PMID: 25537372 CAMSID: cams4691
Cardiac remodeling; Cardiovascular risk; Fetal growth; Placenta; Ultrasound
6.  Edaravone Mitigates Hexavalent Chromium-Induced Oxidative Stress and Depletion of Antioxidant Enzymes while Estrogen Restores Antioxidant Enzymes in the Rat Ovary in F1 Offspring1 
Biology of Reproduction  2014;91(1):12, 1-12.
Environmental contamination of drinking water with chromium (Cr) has been increasing in more than 30 cities in the United States. Previous studies from our group have shown that Cr affects reproductive functions in female Sprague Dawley rats. Although it is impossible to completely remove Cr from the drinking water, it is imperative to develop effective intervention strategies to inhibit Cr-induced deleterious health effects. Edaravone (EDA), a potential inhibitor of free radicals, has been clinically used to treat cancer and cardiac ischemia. This study evaluated the efficacy of EDA against Cr-induced ovarian toxicity. Results showed that maternal exposure to CrVI in rats increased follicular atresia, decreased steroidogenesis, and delayed puberty in F1 offspring. CrVI increased oxidative stress and decreased antioxidant (AOX) enzyme levels in the ovary. CrVI increased follicle atresia by increased expression of cleaved caspase 3, and decreased expression of Bcl2 and Bcl2l1 in the ovary. EDA mitigated or inhibited the effects of CrVI on follicle atresia, pubertal onset, steroid hormone levels, and AOX enzyme activity, as well as the expression of Bcl2 and Bcl2l1 in the ovary. In a second study, CrVI treatment was withdrawn, and F1 rats were injected with estradiol (E2) (10 μg in PBS/ethanol per 100 g body weight) for a period of 2 wk to evaluate whether E2 treatment will restore Cr-induced depletion of AOX enzymes. E2 restored CrVI-induced depletion of glutathione peroxidase 1, catalase, thioredoxin 2, and peroxiredoxin 3 in the ovary. This is the first study to demonstrate the protective effects of EDA against any toxicant in the ovary.
PMCID: PMC4434959  PMID: 24804965
chromium; edaravone; ovary; oxidative stress
7.  Age- and Species-Dependent Infiltration of Macrophages into the Testis of Rats and Mice Exposed to Mono-(2-Ethylhexyl) Phthalate (MEHP)1 
Biology of Reproduction  2014;91(1):18, 1-11.
The mechanism by which noninfectious testicular inflammation results in infertility is poorly understood. Here the infiltration of CD11b+ immunoreactive testicular interstitial cells (neutrophil, macrophages, dendritic cells) in immature (Postnatal Day [PND] 21, 28, and 35) and adult (PND 56) Fischer rats is described at 12, 24, and 48 h after an oral dose of 1 g/kg mono-(2-ethylhexyl) phthalate (MEHP), a well-described Sertoli cell toxicant. Increases of CD11b+ cells are evident 12 h after MEHP exposure in PND 21 and 28 rats. In PND 28 rats, CD11b+ cells remained significantly elevated at 48 h, while in PND 21 rats, it returned to control levels by 24 h. The peak number of CD11b+ cells in PND 35 rat testis is delayed until 24 h, but remains significantly elevated at 48 h. In PND 56 rats, no increase in CD11b+ cells occurs after MEHP exposure. In PND 21, 28, and 35 rats, a significant increase in monocyte chemoattractant protein-1 (MCP-1) by peritubular myoid cells occurs 12 h after MEHP. Interestingly, MEHP treatment of C57BL/6J mice did not incite an infiltration of CD11b+ cells at either PND 21 or 28. The peak level of germ cell apoptosis observed 24 h after MEHP exposure in young rats is not seen in mice at any age or in PND 56 rats. Taken together, these findings implicate MCP-1 released by peritubular myoid cells in provoking the migration of CD11b+ cells into the immature rat testis early after MEHP exposure and point to a role for CD11b+ cells in triggering germ cell apoptosis in an age- and species-dependent manner.
PMCID: PMC4434960  PMID: 24876407
interstitial cells; macrophage; myoid cells; reproductive immunology; toxicology
8.  Engineered Nanomaterials: An Emerging Class of Novel Endocrine Disruptors1 
Biology of Reproduction  2014;91(1):20, 1-8.
Over the past decade, engineered nanomaterials (ENMs) have garnered great attention for their potentially beneficial applications in medicine, industry, and consumer products due to their advantageous physicochemical properties and inherent size. However, studies have shown that these sophisticated molecules can initiate toxicity at the subcellular, cellular, and/or tissue/organ level in diverse experimental models. Investigators have also demonstrated that, upon exposure to ENMs, the physicochemical properties that are exploited for public benefit may mediate adverse endocrine-disrupting effects on several endpoints of mammalian reproductive physiology (e.g., steroidogenesis, spermatogenesis, pregnancy). Elucidating these complex interactions within reproductive cells and tissues will significantly advance our understanding of ENMs as an emerging class of novel endocrine disruptors and reproductive toxicants. Herein we reviewed the recent developments in reproductive nanotoxicology and identified the gaps in our knowledge that may serve as future research directions to foster continued advancement in this evolving field of study.
PMCID: PMC4434961  PMID: 24899576
endocrine disruptors; engineered nanomaterials; nanotoxicology; reproduction; steroidogenesis
9.  Simultaneous Gene Deletion of Gata4 and Gata6 Leads to Early Disruption of Follicular Development and Germ Cell Loss in the Murine Ovary1 
Biology of Reproduction  2014;91(1):24, 1-10.
Granulosa cell formation and subsequent follicular assembly are important for ovarian development and function. Two members of the GATA family of transcription factors, GATA4 and GATA6, are expressed in ovarian somatic cells early in development, and their importance in adult ovarian function has been recently highlighted. In this study, we demonstrated that the embryonic loss of Gata4 and Gata6 expression within the ovary results in a strong down-regulation of genes involved in the ovarian developmental pathway (Fst and Irx3) as well as diminished expression of the pregranulosa and granulosa cell markers SPRR2 and FOXL2, respectively. Postnatal ovaries deficient in both Gata genes show impaired somatic cell proliferation and arrested follicular development at the primordial stage, where oocytes are either enclosed by one layer of squamous granulosa cells or remain in germ cell nests/clusters. Furthermore, germ cell nests and primordial follicles are predominantly localized to the central region of the Sf1Cre; Gata4flox/flox Gata6flox/flox ovaries, where the boundary between the medulla and cortex is almost nonexistent. Lastly, most of the oocytes are lost early in development in conditional double mutant ovaries, which confirms the importance of normally differentiated granulosa cells as supporting cells for oocyte survival. Thus, both GATA4 and GATA6 proteins are fundamental regulators of granulosa cell differentiation and proliferation, and consequently of proper follicular assembly during normal ovarian development and function.
PMCID: PMC4434962  PMID: 24899573
differentiation; granulosa cells; ovarian development
10.  Gestational Exposure to Elevated Testosterone Levels Induces Hypertension via Heightened Vascular Angiotensin II Type 1 Receptor Signaling in Rats1 
Biology of Reproduction  2014;91(1):6, 1-7.
Pre-eclampsia is a life-threatening pregnancy disorder whose pathogenesis remains unclear. Plasma testosterone levels are elevated in pregnant women with pre-eclampsia and polycystic ovary syndrome, who often develop gestational hypertension. We tested the hypothesis that increased gestational testosterone levels induce hypertension via heightened angiotensin II signaling. Pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate from Gestational Day 15 to 19 to induce a 2-fold increase in plasma testosterone levels, similar to levels observed in clinical conditions like pre-eclampsia. A subset of rats in these two groups was given losartan, an angiotensin II type 1 receptor antagonist by gavage during the course of testosterone exposure. Blood pressure levels were assessed through a carotid arterial catheter and endothelium-independent vascular reactivity through wire myography. Angiotensin II levels in plasma and angiotensin II type 1 receptor expression in mesenteric arteries were also examined. Blood pressure levels were significantly higher on Gestational Day 20 in testosterone-treated dams than in controls. Treatment with losartan during the course of testosterone exposure significantly attenuated testosterone-induced hypertension. Plasma angiotensin II levels were not significantly different between control and testosterone-treated rats; however, elevated testosterone levels significantly increased angiotensin II type 1 receptor protein levels in the mesenteric arteries. In testosterone-treated rats, mesenteric artery contractile responses to angiotensin II were significantly greater, whereas contractile responses to K+ depolarization and phenylephrine were unaffected. The results demonstrate that elevated testosterone during gestation induces hypertension in pregnant rats via heightened angiotensin II type 1 receptor-mediated signaling, providing a molecular mechanism linking elevated maternal testosterone levels with gestational hypertension.
PMCID: PMC4434963  PMID: 24855104
AGTR1; angiotensin; blood pressure; losartan; mesenteric arteries; pre-eclampsia; pregnancy; testosterone; vascular function
11.  Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 
Biology of Reproduction  2014;91(1):9, 1-11.
Luteinizing hormone (LH) regulation of the epidermal growth factor (EGF) network is critical for oocyte maturation and the ovulatory process. Recent studies have indicated that C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR2) play an important role in the control of meiotic arrest. Here, we investigated the involvement of the EGF network in the LH-dependent regulation of the CNP/NPR2 axis and cGMP accumulation. LH/hCG treatment causes a major decrease in both cGMP and the CNP precursor (natriuretic peptide precursor C [Nppc]) mRNA accumulation in vivo and in vitro. However, the cGMP downregulation precedes the decrease in Nppc mRNA by more than 1 h. Amphiregulin, an EGF-like factor, suppresses Nppc mRNA levels in cultured follicles to the same extent as LH, and this effect is completely prevented by the EGF receptor (EGFR) kinase inhibitor AG1478. However, the LH-dependent suppression of Nppc is insensitive to AG1478. Similarly, Nppc suppression by LH occurs in follicles from EGFR null mice. These findings document that EGFR signaling is sufficient to downregulate CNP, but is not necessary for LH action. When cGMP concentration in the follicle is measured, the short-term, but not long-term, LH effects on cGMP are prevented by AG1478, suggesting that ligand availability may be responsible for the late response. Human CG decreases the CNP-dependent cGMP synthesis in wild-type and EGFR knockdown cumulus-oocyte complexes. These findings demonstrate that redundant pathways are involved in the regulation of cGMP. EGFR-dependent events are involved in the short-term regulation of cGMP, whereas the long-term effects may involve regulation of the CNP.
PMCID: PMC4434964  PMID: 24740605
cyclic-AMP; cyclic-GMP; epidermal growth factor; granulosa cells; luteinizing hormone (LH/LH receptor); natriuretic peptide precursor type C; oocyte maturation; oocyte meiotic arrest; ovary; signal transduction; preovulatory follicle
12.  Impaired Sperm Maturation in Rnase9 Knockout Mice1 
Biology of Reproduction  2014;90(6):120, 1-10.
Ribonuclease, RNase A family, 9 (RNASE9) is a ribonuclease A superfamily member that is expressed only in the epididymis. It is a small, secreted polypeptide, it lacks ribonuclease activity, and its function(s) is unknown. However, epididymis-specific expression suggests a role in sperm maturation. We generated Rnase9−/− mice to study RNASE9 function in vivo. We confirm that RNASE9 expression is restricted to the epididymis. Within the epididymis, RNASE9 is first detected in midcaput, persists through the distal caput and corpus, and wanes in the cauda. Rnase9−/− mice are born at the expected Mendelian ratio, have normal postnatal growth and development, and have no outwardly apparent phenotype. Spermatogenesis is normal, and Rnase9-null sperm are morphologically normal. Rnase9−/− males have normal fertility in unrestricted mating trials, and fertilization rates in in vitro fertilization assays are indistinguishable from wild-type mice. Visual observations coupled with analyses of sperm velocities shortly after swim out from the corpus shows that motility of Rnase9-null sperm is significantly impaired. However, no differences between wild-type and Rnase9-null sperm are detected by computer-assisted sperm analysis 10–90 min after sperm isolation from the corpus or cauda. Assessment of capacitation-dependent signaling pathways in Rnase9-null sperm showed that, while levels of tyrosine phosphorylation of sperm proteins were normal, there was decreased phosphorylation of protein kinase A substrates upon capacitation compared to wild-type mice. In conclusion, RNASE9 is dispensable for fertility, but the absence of RNASE9 during epididymal transit results in impaired sperm maturation.
PMCID: PMC4093998  PMID: 24719258
capacitation; epididymis; male reproductive tract; sperm; sperm maturation
13.  Treatment with Bazedoxifene and Conjugated Estrogens Results in Regression of Endometriosis in a Murine Model1 
Biology of Reproduction  2014;90(6):121, 1-7.
Bazedoxifene (BZA), a selective estrogen receptor modulator (SERM), inhibits the action of estrogens on endometrial proliferation. Here, we evaluate the effect of a tissue-selective estrogen complex (TSEC) containing BZA and conjugated estrogens (CE) on ectopic endometrial lesions in a mouse model of endometriosis. Experimental endometriosis was created in 60 female CD-1 mice. The mice were randomly divided into 10 groups that received varying doses of either BZA (1, 2, 3, or 5 mg/kg/day), BZA (1, 2, 3, or 5 mg/kg/day) in combination with CE (3 mg/kg/day), CE treatment alone (3 mg/kg/day), or vehicle control for 8 wk. Treatment with BZA alone or the TSEC containing BZA/CE led to a decrease in endometriotic lesion size compared to controls. The mean surface area of the untreated lesions was 19.6 mm2. Treatment with BZA or BZA/CE resulted in reduced lesion size (to 8.8 and 7.8 mm2, respectively). No significant difference was found in lesion size between the BZA and BZA/CE treatment groups or between different doses of either treatment. Ovarian cyst formation was not evident in the treated groups. Treatment with the TSEC containing higher BZA dosages (3 and 5 mg/kg/day) led to significantly lower levels of estrogen receptor (Esr1) mRNA expression compared to the control treatment. No differences were observed in expression of progesterone receptor (Pgr). Immunohistochemical analysis also demonstrated a decrease in ESR protein. The combination of CE and BZA may prove to be a novel treatment option for endometriosis.
PMCID: PMC4093999  PMID: 24740602
bazedoxifene (BZA); conjugated estrogen (CE); endometriosis; hormone receptors; tissue-specific estrogen complex (TSEC)
14.  Regulation of the Proliferation and Differentiation of Leydig Stem Cells in the Adult Testis1 
Biology of Reproduction  2014;90(6):123, 1-7.
We reported previously that stem cells associated with adult rat testis seminiferous tubules are able to give rise to differentiated Leydig cells in vitro. The regulatory mechanisms by which they do so, however, are uncertain. Herein, we hypothesized that the proliferation and differentiation of Leydig cell stem cells (stem Leydig cells, SLCs) depend upon locally produced factors from the seminiferous tubules. Microarray analysis revealed that platelet-derived growth factor receptor alpha (PDGFRalpha) is up-regulated and PDGFRbeta is down-regulated with postnatal differentiation of SLCs. This suggested that their ligands, PDGF-AA and PDGF-BB, respectively, might have important roles in SLC proliferation and differentiation. To test this, we developed a unique in vitro culture system in which SLCs proliferate on the surfaces of cultured seminiferous tubules largely during Week 1 of culture and their progeny subsequently differentiate to testosterone-forming Leydig cells during Weeks 2 through 4. Using this system, seminiferous tubules from adult rat testes were cultured with PDGF-AA or PDGF-BB for up to 4 wk. Both ligands stimulated SLC proliferation during the first week of culture, with PDGF-BB significantly more potent than PDGF-AA. Furthermore, PDGF-AA had a stimulatory effect on SLC differentiation from Weeks 2 through 4 of culture. In contrast, PDGF-BB, which stimulated cell proliferation during Week 1, had a significant inhibitory effect on differentiation during Weeks 2 through 4. These findings, made possible by the development of the seminiferous tubule culture system, reveal distinct roles by locally produced PDGFs in SLC regulation.
PMCID: PMC4094000  PMID: 24740597
Leydig cells; PDGF; stem cells; testosterone
15.  Tissue-Specific Changes in Molecular Clocks During the Transition from Pregnancy to Lactation in Mice1 
Biology of Reproduction  2014;90(6):127, 1-15.
Circadian clocks regulate homeostasis and mediate responses to stressors. Lactation is one of the most energetically demanding periods of an adult female's life. Peripartum changes occur in almost every organ so the dam can support neonatal growth through milk production while homeostasis is maintained. How circadian clocks are involved in adaptation to lactation is currently unknown. The abundance and temporal pattern of core clock genes' expression were measured in suprachiasmatic nucleus, liver, and mammary from late pregnant and early lactation mice. Tissue-specific changes in molecular clocks occurred between physiological states. Amplitude and robustness of rhythms increased in suprachiasmatic nucleus and liver. Mammary rhythms of core molecular clock genes were suppressed. Attenuated rhythms appeared to be a physiological adaptation of mammary to lactation, because manipulation of timing of suckling resulting in significant differences in plasma prolactin and corticosterone had no effect on amplitude. Analysis of core clock proteins revealed that the stoichiometric relationship between positive (CLOCK) and negative (PER2) components remained 1:1 in liver but was increased to 4:1 in mammary during physiological transition. Induction of differentiation of mammary epithelial cell line HC11 with dexamethasone, insulin, and prolactin resulted in similar stoichiometric changes among positive and negative clock regulators, and prolactin induced phase shifts in HC11 Arntl expression rhythm. Data support that distinct mechanisms drive periparturient changes in mammary clock. Stoichiometric change in clock regulators occurs with gland differentiation. Suppression of mammary clock gene expression rhythms represents a physiological adaptation to suckling cues. Adaptations in mammary clock are likely needed in part to support suckling demands of neonates.
PMCID: PMC4094001  PMID: 24759789
circadian clock; lactation; liver; mammary; pregnancy; prolactin; SCN
16.  Adenine Nucleotide Metabolism and a Role for AMP in Modulating Flagellar Waveforms in Mouse Sperm1 
Biology of Reproduction  2014;90(6):128, 1-14.
While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase.
PMCID: PMC4094002  PMID: 24740601
ADP; AK8; AMP; ATP; adenine nucleotides; adenylate kinase; motility; sperm
17.  Increasing Maternal Body Mass Index Is Associated with Systemic Inflammation in the Mother and the Activation of Distinct Placental Inflammatory Pathways1 
Biology of Reproduction  2014;90(6):129, 1-9.
Obese pregnant women have increased levels of proinflammatory cytokines in maternal circulation and placental tissues. However, the pathways contributing to placental inflammation in obesity are largely unknown. We tested the hypothesis that maternal body mass index (BMI) was associated with elevated proinflammatory cytokines in maternal and fetal circulations and increased activation of placental inflammatory pathways. A total of 60 women of varying pre-/early pregnancy BMI, undergoing delivery by Cesarean section at term, were studied. Maternal and fetal (cord) plasma were collected for analysis of insulin, leptin, IL-1beta, IL-6, IL-8, monocyte chemoattractant protein (MCP) 1, and TNFalpha by multiplex ELISA. Activation of the inflammatory pathways in the placenta was investigated by measuring the phosphorylated and total protein expression of p38-mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK)-MAPK, signal transducer-activated transcription factor (STAT) 3, caspase-1, IL-1beta, IkappaB-alpha protein, and p65 DNA-binding activity. To determine the link between activated placental inflammatory pathways and elevated maternal cytokines, cultured primary human trophoblast (PHT) cells were treated with physiological concentrations of insulin, MCP-1, and TNFalpha, and inflammatory signaling analyzed by Western blot. Maternal BMI was positively correlated with maternal insulin, leptin, MCP-1, and TNFalpha, whereas only fetal leptin was increased with BMI. Placental phosphorylation of p38-MAPK and STAT3, and the expression of IL-1beta protein, were increased with maternal BMI; phosphorylation of p38-MAPK was also correlated with birth weight. In contrast, placental NFkappaB, JNK and caspase-1 signaling, and fetal cytokine levels were unaffected by maternal BMI. In PHT cells, p38-MAPK was activated by MCP-1 and TNFalpha, whereas STAT3 phosphorylation was increased following TNFalpha treatment. Maternal BMI is associated with elevated maternal cytokines and activation of placental p38-MAPK and STAT3 inflammatory pathways, without changes in fetal systemic inflammatory profile. Activation of p38-MAPK by MCP-1 and TNFalpha, and STAT3 by TNFalpha, suggests a link between elevated proinflammatory cytokines in maternal plasma and activation of placental inflammatory pathways. We suggest that inflammatory processes associated with elevated maternal BMI may influence fetal growth by altering placental function.
PMCID: PMC4094003  PMID: 24759787
cytokines; innate immune response; obesity
18.  Daily Exposure to Di(2-ethylhexyl) Phthalate Alters Estrous Cyclicity and Accelerates Primordial Follicle Recruitment Potentially Via Dysregulation of the Phosphatidylinositol 3-Kinase Signaling Pathway in Adult Mice1 
Biology of Reproduction  2014;90(6):136, 1-11.
Humans are exposed daily to di(2-ethylhexyl) phthalate (DEHP), a plasticizer found in many consumer, medical, and building products containing polyvinyl chloride. Large doses of DEHP disrupt normal ovarian function; however, the effects of DEHP at environmentally relevant levels, the effects of DEHP on folliculogenesis, and the mechanisms by which DEHP disrupts ovarian function are unclear. The present study tested the hypothesis that relatively low levels of DEHP disrupt estrous cyclicity as well as accelerate primordial follicle recruitment by dysregulating phosphatidylinositol 3-kinase (PI3K) signaling. Adult CD-1 mice were orally dosed with DEHP (20 μg/kg/day–750 mg/kg/day) daily for 10 and 30 days. Following dosing, the effects on estrous cyclicity were examined, and follicle numbers were histologically quantified. Further, the ovarian mRNA and protein levels of PI3K signaling factors that are associated with early folliculogenesis were quantified. The data indicate that 10- and 30-day exposure to DEHP prolonged the duration of estrus and accelerated primordial follicle recruitment. Specifically, DEHP exposure decreased the percentage of primordial follicles and increased the percentage of primary follicles counted following 10-day exposure and increased the percentage of primary follicles counted following 30-day exposure. DEHP exposure, at doses that accelerate folliculogenesis, increased the levels of 3-phosphoinositide-dependent protein kinase-1, mammalian target of rapamycin complex 1, and protein kinase B and decreased the levels of phosphatase and tensin homolog, potentially driving PI3K signaling. Collectively, relatively low levels of DEHP disrupt estrous cyclicity and accelerate primordial follicle recruitment potentially via a mechanism involving dysregulation of PI3K signaling.
PMCID: PMC4435463  PMID: 24804967
di(2-ethylhexyl) phthalate; endocrine disruptors; environmental contaminants and toxicants; follicular development; folliculogenesis; ovary; phosphatidylinositol 3-kinase signaling pathway; toxicology
19.  Riding the Spermatogenic Wave: Profiling Gene Expression Within Neonatal Germ and Sertoli Cells During a Synchronized Initial Wave of Spermatogenesis in Mice1  
Biology of Reproduction  2014;90(5):108.
Continual sperm production relies on germ cells undergoing spermatogenesis asynchronously. As a result, the testis always contains a mixed population of germ cells at different stages of their differentiation process. The heterogeneous nature of the testis makes profiling gene expression within Sertoli cells or specific populations of germ cells impossible when a wild-type testis is assessed. We recently reported a unique method for synchronizing spermatogenesis without affecting fertility by manipulating RA levels within the neonatal testis. Using this protocol, combined with the RiboTag transgenic mouse line, we have mapped the Sertoli and germ cell translatome during the initial synchronized wave of spermatogenesis. Using microarray analysis, we identified 392 and 194 germ cell and Sertoli cells transcripts, respectively, that dynamically change during spermatogonial differentiation, division, and the onset of meiosis. Functional annotation clustering revealed that transcripts enriched in germ cells were mostly associated with meiosis (21 transcripts), chromatin organization (12 transcripts), and cell cycle (3 transcripts). In addition, glycoproteins (65 transcripts), cell adhesion (15 transcripts), and cell junction (13 transcripts) transcripts were overrepresented in the Sertoli cell-enriched list. These datasets represent the first transcriptional analysis of spermatogonial differentiation, division, and meiotic onset. These data suggest that several of the genes encoding meiotic proteins are expressed and are actively being translated well before germ cells enter meiosis. In addition, this study provides novel candidate genes, Asf1b and Esyt3, that may be involved in the regulation of spermatogonial chromatin reorganization, germ-Sertoli cell interactions, and/or blood-testis barrier formation.
WIN 18,446/RA treatment of neonatal mice was used to synchronize the initial wave of spermatogenesis and identify novel messages expressed within either germ or Sertoli cells as spermatogonia enter meiosis.
PMCID: PMC4076369  PMID: 24719255
gene expression; germ cells; retinoic acid; retinoids; Sertoli cells; spermatogonia; testis; translational profiling
20.  Sustained Expression of Insulin by a Genetically Engineered Sertoli Cell Line after Allotransplantation in Diabetic BALB/c Mice1 
Biology of Reproduction  2014;90(5):109.
Immune-privileged Sertoli cells (SCs) exhibit long-term survival after allotransplantation or xenotransplantation, suggesting they can be used as a vehicle for cell-based gene therapy. Previously, we demonstrated that SCs engineered to secrete insulin by using an adenoviral vector normalized blood glucose levels in diabetic mice. However, the expression of insulin was transient, and the use of immunocompromised mice did not address the question of whether SCs can stably express insulin in immunocompetent animals. Thus, the objective of the current study was to use a lentiviral vector to achieve stable expression of insulin in SCs and test the ability of these cells to survive after allotransplantation. A mouse SC line transduced with a recombinant lentiviral vector containing furin-modified human proinsulin cDNA (MSC-EhI-Zs) maintained stable insulin expression in vitro. Allotransplantation of MSC-EhI-Zs cells into diabetic BALB/c mice demonstrated 88% and 75% graft survival rates at 20 and 50 days post-transplantation, respectively. Transplanted MSC-EhI-Zs cells continued to produce insulin mRNA throughout the study (i.e., 50 days); however, insulin protein was detected only in patches of cells within the grafts. Consistent with low insulin protein detection, there was no significant change in blood glucose levels in the transplant recipients. Nevertheless, MSC-EhI-Zs cells isolated from the grafts continued to express insulin protein in culture. Collectively, this demonstrates that MSC-EhI-Zs cells stably expressed insulin and survived allotransplantation without immunosuppression. This further strengthens the use of SCs as targets for cell-based gene therapy for the treatment of numerous chronic diseases, especially those that require basal protein expression.
A mouse Sertoli cell line (MSC-1) genetically engineered using a lentiviral construct carrying furin-modified human proinsulin cDNA stably expresses insulin mRNA and protein and survives allotransplantation into diabetic BALB/c mice.
PMCID: PMC4076370  PMID: 24695630
cell-based gene therapy; immune privilege; insulin; lentivirus; mouse Sertoli cell line; Sertoli cells; testis; transplantation
21.  Morphological and Phenotypic Analyses of the Human Placenta Using Whole Mount Immunofluorescence1 
Biology of Reproduction  2014;90(5):110.
The human placenta performs multiple essential functions required for successful pregnancy. Alterations in the placental vasculature have been implicated in severe complications of pregnancy. Despite the importance of placental vascular function during pregnancy, there are gaps in our knowledge regarding the molecular pathways that control vessel development. Furthermore, there are limited tools available to simultaneously examine the morphology, phenotype, and spatial arrangement of cells within intact placental structures. To overcome these limitations, we developed whole mount immunofluorescence (WMIF) of the human placenta. Morphological analyses using WMIF revealed that blood vessel structures were consistent with an immature, angiogenic morphology in first-trimester placentas and mature, remodeled endothelium at term. To investigate placental expression of factors that control blood vessel development, we utilized WMIF to examine gestation age-specific expression of 1) the receptors for vascular endothelial growth factor (VEGFR-1, VEGFR-2, and VEGFR-3), which are required for placental vascular development in mice, and 2) activated, tyrosine phosphorylated STAT3 (pSTAT3), a transcription factor that mediates VEGFR2 signaling. We detected high levels of VEGFR2, VEGFR3, and pSTAT3 expression in early placental blood vessels that were significantly diminished by term. VEGFR1 was expressed primarily in trophoblast and Hofbauer cells throughout gestation. Based on our collective results, we propose that VEGFR2, VEGFR3, and STAT3 play essential roles in the development of the human placental vasculature. In addition, we anticipate that WMIF will provide a powerful approach for comparing placental morphology and protein expression in normal versus pathological pregnancies and for investigating the effects of environmental factors on placental function.
Early human placental blood vessels express high levels of the pro-angiogenic receptors VEGFR2 and VEGFR3, and the activated transcription factor pSTAT3, which suggests that these molecules play a role in regulation of placental vascular development.
PMCID: PMC4076371  PMID: 24648400
angiogenesis; blood vessels; placenta; pregnancy; signal transduction; STAT3; trophoblast; VEGF
22.  Unresolved Questions Concerning Mammalian Sperm Acrosomal Exocytosis1 
Biology of Reproduction  2014;90(5):112.
In recent years, the study of mammalian acrosomal exocytosis has produced some major advances that challenge the long-held, general paradigms in the field. Principally, the idea that sperm must be acrosome-intact to bind to the zona pellucida of unfertilized eggs, based largely on in vitro fertilization studies of mouse oocytes denuded of the cumulus oophorus, has been overturned by experiments using state-of-the-art imaging of cumulus-intact oocytes and fertilization experiments where eggs were reinseminated by acrosome-reacted sperm recovered from the perivitelline space of zygotes. In light of these results, this minireview highlights a number of unresolved questions and emphasizes the fact that there is still much work to be done in this exciting field. Future experiments using recently advanced technologies should lead to a more complete and accurate understanding of the molecular mechanisms governing the fertilization process in mammals.
Unresolved questions concerning acrosomal exocytosis are highlighted; advanced technologies are anticipated to lead to a more complete understanding of the molecular mechanisms governing the fertilization process in mammals.
PMCID: PMC4076372  PMID: 24671881
acrosomal exocytosis; capacitation; fertilization; sperm
23.  Epithelial Basal Cells Are Distinct from Dendritic Cells and Macrophages in the Mouse Epididymis1 
Biology of Reproduction  2014;90(5):90.
The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system.
The basal region of the murine epididymal duct is heavily populated by basal cells, macrophages, and dendritic cells, which are morphologically and phenotypically distinct.
PMCID: PMC4076373  PMID: 24648397
basal cells; dendritic cells; epididymis; immunology; macrophages; male reproductive tract; reproductive immunology
24.  Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids1 
Biology of Reproduction  2014;90(5):93.
The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.
The production of transgenic pigs by cytoplasmic injection of piggyBac-based pmGENIE-3 plasmids into in vivo-produced one cell zygotes results in efficient transgenesis rates; at least one transgenic piglet was born every time a sow farrowed.
PMCID: PMC4076374  PMID: 24671876
pig; piggyBac; pmGENIE-3; transgenesis; transposase
25.  Toll-like Receptor 4 and MyD88 Dependent Signaling Mechanisms of the Innate Immune System are Essential for the Response to Lipopolysaccharide by Epithelial and Stromal Cells of the Bovine Endometrium 
Biology of reproduction  2012;86(2):51.
Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial lipopolysaccharide (LPS), stimulating the secretion of the pro-inflammatory cytokines interleukin (IL)-1β and IL-6, and the chemokine IL-8. As the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators was investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6 and IL8 mRNA expression, and IL-6 protein accumulation in epithelial cells; and increased IL1B and IL8 mRNA expression, and IL-6 and IL-8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IκB and the nuclear translocation of NF-κB, as well as rapid phosphorylation of MAPK3/1 and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, MYD88, using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK, or MAPK14, reduced LPS-induced IL1B, IL6 and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4 and MyD88 dependent cell signaling pathways.
PMCID: PMC4396703  PMID: 22053092
Bovine; uterus; infection; immunity; Toll-like receptors; inflammation; endometrium; innate immunity; lipopolysaccharide; TLR4; MyD88

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