BOR Papers in Press

[Gamete Biology] Reduction of Mouse Egg Surface Integrin Alpha9 Subunit (ITGA9) Reduces the Egg's Ability to Support Sperm-Egg Binding and Fusion

Vjugina, U., Zhu, X., Oh, E., Bracero, N. J., Evans, J. P. — 2009-01-07

The involvement of egg integrins in mammalian sperm-egg interactions has been controversial, with data from integrin inhibitor studies contrasting evidence from knockouts showing that specific integrin subunits are not essential for fertility. An alpha₄/alpha₉ (ITGA4/ITGA9) integrin subfamily member has been implicated in fertilization but not extensively examined, so we tested the hypotheses that: (1) an ITGA4/ITGA9 integrin participates in sperm-egg interactions; (2) short-term acute knockdown by RNA interference of integrin subunits would result in a fertilization phenotype, differing from chronic depletion via knockout; (3) detection of a fertilization phenotype is sensitive to in vitro fertilization (IVF) assay conditions. We show that mouse and human eggs express the alpha₉ integrin subunit (ITGA9). RNAi-mediated knockdown resulted in reduced levels of Itga9 mRNA and surface protein in mouse eggs. RNAi attempts to knockdown ITGA9's likely beta partner, beta₁ (ITGB1), resulted in reduced Itgb1 mRNA but no reduction in ITGB1 surface protein. Therefore, studies using a function-blocking anti-ITGB1 antibody tested the hypothesis that ITGB1 participates in gamete interactions. Analyses of sperm-egg interactions with Itga9-knockdown eggs and anti-ITGB1 antibody-treated eggs in IVF assays using specific sperm:egg ratios revealed: (1) a reduction but not complete loss of sperm-egg binding and fusion; (2) the reduction of sperm-egg binding and fusion was not detected in inseminations with high sperm:egg ratios. These data demonstrate that ITGA9 and ITGB1 participate in sperm-egg interactions, although clearly are not the only molecules involved. This also shows that careful design of IVF parameters allows detection of deficiencies in gamete interactions.

[Gamete Biology] CDC14B Acts Through FZR1 (CDH1) to Prevent Meiotic Maturation of Mouse Oocytes

Schindler, K., Schultz, R. M. — 2009-01-07

Meiotic maturation in oocytes is a prolonged process that is unique because of cell cycle arrests at prophase of meiosis I (MI) and at metaphase of meiosis II (MII). Fluctuations in cyclin-dependent kinase 1 (CDK1/CDC2A) activity govern meiotic progression, yet little is known about how these fluctuations are achieved. CDC14 is a highly conserved, dual-specificity phosphatase that counteracts the function of proteins phosphorylated by CDK. Mammals contain two CDC14 homologs, CDC14A and CDC14B. We report that CDC14B localizes with the meiotic spindle in mouse oocytes and, unlike somatic cells, it does not localize in the nucleolus. Oocytes that over-express CDC14B are significantly delayed in resuming meiosis and fail to progress to MII, whereas oocytes depleted for CDC14B spontaneously resume meiosis under conditions that normally inhibit meiotic resumption. Depletion of FZR1 (CDH1), a regulatory subunit of the anaphase-promoting complex/cyclosome (APC/C) that targets cyclin B1 (CCNB1) for ubiquitin-mediated proteolysis, partially restores normal timing of meiotic resumption in oocytes with excess CDC14B. These studies also reveal that experimentally altering CDC14B levels generates eggs with abnormal spindles and chromosome alignment perturbations. Our data indicate that CDC14B is a negative regulator of meiotic resumption and may regulate MI in mouse oocytes.

[Female Reproductive Tract] Chlamydia Infection Causes Loss of Pacemaker Cells and Inhibits Oocyte Transport in the Mouse Oviduct

2009-01-07

Chlamydia trachomatis is a common sexually-transmitted bacterial infection that results in healthcare costs in the US exceeding US$2 billion/year. Chlamydia infections cause damage to the oviducts resulting in ectopic pregnancy and tubal factor infertility (TFI), but the reasons for defective oviduct function are poorly understood. We have investigated the role of oviduct contractions in egg transport and found that underlying electrical pacemaker activity is responsible for oviduct motility and egg transport. Specialized pacemaker cells, referred to as oviduct interstitial cells of Cajal (ICC-OVI) are responsible for pacemaker activity. ICC-OVI, labeled with antibodies to KIT protein, form a dense network associated with the smooth muscle cells along the entire length of the oviduct. Selective removal of ICC-OVI with KIT neutralizing antibody resulted in loss of electrical rhythmicity and loss of propulsive contractions of the oviduct. We tested whether infection might adversely affect the ICC-OVI. Mice, infected with Chlamydia muridarum displayed dilation of oviducts, pyosalpinx and loss of spontaneous contractile activity. Morphological inspection showed disruption of ICC-OVI networks, and electrophysiological recordings showed loss of intrinsic pacemaker activity without change in basal smooth muscle membrane potential. Chlamydia infection was also associated with upregulation of NOS2 (iNOS) and PTGS2 (COX II) in leucocytes. Loss of ICC-OVI and pacemaker activity causes oviduct pseudo-obstruction and loss of propulsive contractions for oocytes. This accompanied by retention of oviduct secretions may contribute to the development of tubal factor infertility.

[Gamete Biology] Comprehensive Analysis of Reproductive ADAMs: Relationship of ADAM4 and ADAM6 with an ADAM Complex Required for Fertilization in Mice

2009-01-07

A Disintegrin And Metalloprotease (ADAM) family members expressed in male reproductive tissues are divided phylogenetically into three major groups. In this study, we analyzed six ADAMs in one of the groups (ADAMs 4, 6, 24, 26, 29 and 30) of which function is largely unknown. Our results showed that most of the ADAMs undergo unique processing during sperm maturation and they are located at the surface of sperm head. We found that the levels of ADAM4 and ADAM6 are dramatically reduced in Adam2 and Adam3 knockout sperm defective in various fertilization processes. We observed premature processing of ADAM4 in the Adam3 null mice. Further, we obtained a result showing complex formation of ADAM6 with ADAM2 and ADAM3 in testis. Taken together, the results of our study disclose involvement of ADAM4 and ADAM6 in a reproductive ADAM system that functions in fertilization.

[Minireview] Gene Birth, Death, and Divergence: The Different Scenarios of Reproduction-Related Gene Evolution

Tian, X., Pascal, G., Fouchecourt, S., Pontarotti, P., Monget, P. — 2009-01-07

Reproductive genes are known to evolve more rapidly than genes expressed in other organs. In this paper, we present an overview and bring some new data on the evolution study of reproduction-related genes by integrating phylogeny with gene genomic localization. We focus on the gene evolutionary processes of gene birth, death and divergence. We show that phylogenetic gene birth is confirmed by gene location in genomes, which definitively localized the "place of birth" of new genes (such as Obox and KHDC1/DPPA5/ECAT1/OOEP gene families). By finding their "place of death" in genomes, it also demonstrates that ZP genes, TGM4 and OVGP1 have been lost, respectively, in certain species during vertebrate evolution. Moreover, in the case of gene divergence, comparison of gene locations across different genomes establishes orthologous relationships that are weakly supported by the phylogenetic tree. Specifically, genomic localization demonstrates that the fish and bird mtnr1c (Mel1C) receptor is orthologous to mammalian GPR50, and that ungulate genomes contain new seminal vesicle specific BSP genes that are not present in other species. Overall, the phylogenomic approach to gene evolution presented in this paper offers more insight into gene function, such as species-specific duplications for speciation, changes in gene expression due to gene divergence and functional loss by gene death.

[Mechanisms of Hormone Action] Androgens Upregulate cyp19a1b (Aromatase B) Gene Expression in the Brain of Zebrafish (Danio rerio) Through Estrogen Receptors

2009-01-07

The brain of teleosts is known for its strong aromatase expression, exhibiting unique features compared to other vertebrates. Among these features is the high sensitivity of aromatase B (the product of cyp19a1b) to estrogens. This effect involves the binding of estrogen receptors on an estrogen-responsive element (ERE) of the cyp19a1b promoter. Given the presence of potential androgen-responsive elements (ARE) on this promoter, in vivo and in vitro effects of androgens were studied. The brain of teleosts is known for its strong aromatase expression, exhibiting unique features compared to other vertebrates. Among these features is the high sensitivity of aromatase B (the product of cyp19a1b) to estrogens. This effect involves the binding of estrogen receptors on an estrogen-responsive element (ERE) of the cyp19a1b promoter. Given the presence of potential androgen-responsive elements (ARE) on this promoter, in vivo and in vitro effects of androgens were studied. Using immunohistochemistry and quantitative PCR on zebrafish embryos, we found that cyp19a1b is up-regulated by testosterone, an aromatizable androgen, and by 5alpha-dihydrotestosterone, a non-aromatizable androgen, suggesting a potential androgenic regulation of cyp19a1b through androgen receptors. To assess a putative direct regulation of the cyp19a1b gene by androgen receptors (AR), we transfected U-251MG cells with zebrafish ar together with a luciferase reporter gene driven by 3000 bp of the proximal cyp19a1b promoter, containing the ERE and potential ARE. Interestingly, although zebrafish ar activated luciferase reporter genes controlled by ARE, they failed to induce the cyp19a1b-luciferase construct. This data suggests that the androgenic regulation of cyp19a1b does not involve AR. We further showed that regulation of the cyp19a1b gene by testosterone is in fact due to aromatization, whilst the effect of 5alpha-dihydrotestosterone (DHT) involves conversion into 5alpha-androstane-3beta, 17beta-diol (betadiol), a metabolite of DHT with known estrogenic activity. The blockage of the androgen regulation of cyp19a1b expression using anti-estrogens further confirmed the involvement of estrogen receptors in mediating these effects.

[Gamete Biology] Dynamic Nuclear Organization of Constitutive Heterochromatin During Fetal Male Germ Cell Development in Mice

Yoshioka, H., McCarrey, J. R., Yamazaki, Y. — 2009-01-07

In mice, male germ cells enter mitotic arrest beginning at 13.5 days postcoitum (dpc) and remain suspended in the G₀/G₁ cell cycle stage until after birth. During this period, male germ cells undergo extensive epigenetic reprogramming, which is essential for their subsequent function as male gametes. A global reorganization and spatial clustering of constitutive heterochromatin has been implicated in epigenetic plasticity during cellular differentiation. Here we have studied the dynamics of heterochromatin in fetal (12.5-19.5 dpc) and neonatal (4 days postpartum [dpp]) male germ cells. We monitored constitutive heterochromatin-specific markers, and observed changes in the association of histone H3 trimethylation of lysine 9 (H3K9me3), binding of heterochromatin protein 1 (HP1A), and patterns of 4',6-diamino-2-phenylindole (DAPI) staining in pericentric regions of chromosomes, along with a coincident loss of chromocenters in fetal prospermatogonia during mitotic arrest. We also observed a transient loss of H3K9me3 associated with major and minor satellite repeat sequences, plus inactivation of histone methyltransferases (Suv39h1 and Suv39h2) and transient activation of histone demethylase (Jmjd2b) in these same cells. These epigenetic changes were correlated with relocation of centromeric regions toward the nuclear periphery in prospermatogonia during mitotic arrest. Taken together, these results show that constitutive heterochromatin undergoes dramatic reorganization during prespermatogenesis. We suggest that these dynamic changes in heterochromatin contribute to normal epigenetic reprogramming of the paternal genome in fetal prospermatogonia suspended in the G₀/G₁ stage, and that this also represents an epigenomic state that is particularly amenable to reprogramming.

[Embryo] Metabolic and Mitochondrial Dysfunction in Early Mouse Embryos Following Maternal Dietary Protein Intervention

Mitchell, M., Schulz, S. L., Armstrong, D. T., Lane, M. — 2009-01-07

Dietary supply of nutrients, periconception and during pregnancy, influence the growth and development of the fetus and offspring, and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease (DOHaD) hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. This study examined embryo development following periconception exposure of females to a high protein diet (HPD) or a low protein diet (LPD) relative to a medium control diet (MPD), and hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and the LPD reduced the number of ICM cells in the blastocyst stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from the HPD had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and potentially the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo and the nature of the perturbation differs between HPD and LPD exposure.

[Embryo] The Unfolded Protein Response Contributes to Preimplantation Mouse Embryo Death in the DDK Syndrome

Hao, L., Vassena, R., Wu, G., Han, Z., Cheng, Y., Latham, K. E., Sapienza, C. — 2009-01-07

DDK syndrome is the polar-lethal embryonic death that occurs at the morula-blastocyst transition when female mice of the DDK strain are mated with males from many other inbred strains (so-called "alien" males). Embryonic death is caused by incompatibility between a DDK oocyte factor and an alien male gene, both of which map to the Om locus on mouse chromosome 11. We have compared global transcription patterns of DDK X DDK embryos (high viability) and DDK X C57BL/6 embryos (low viability) at the morula stage, approximately 24 h before any morphological manifestations of DDK syndrome are observed. Of the transcripts that are differentially more abundant in the DDK X C57BL/6 embryos, we noted that many are the products of genes induced by the "unfolded protein response." We confirmed that a number of genes in this pathway are up-regulated in the DDK X C57BL/6 embryos by quantitative RT-PCR. Immunostaining of the endoplasmic reticulum (ER) marker BIP/GRP78 (immunoglobin-binding protein/glucose-regulated protein of 78 kDa), official symbol HSPA5, heat shock protein 5. revealed an accompanying abnormal HSPA5 accumulation and ER structure in the DDK X C57BL/6 embryos. Immunostaining for HERPUD1 (homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1) and ATF4 (activating transcription factor 4) also revealed accumulation of these stress-response products. Our results indicate that the unfolded protein response is induced in embryos destined to die from DDK syndrome and that the embryonic death observed is associated with inability to resolve the associated ER stress.

[Ovary] Survival Role of Locally Produced Acetylcholine in the Bovine Corpus Luteum

Al-zi'abi, M. O., Bowolaksono, A., Okuda, K. — 2009-01-07

The present study was conducted to explore the source of acetylcholine (ACH) in the corpus luteum (CL) and to test our hypothesis of anti-apoptotic role of ACH in the bovine CL, and further to investigate whether nerve growth factor (NGF), insulin like growth factor 1 (IGF1) and transforming growth factor beta 1 (TGFB1) influence the expression of choline acetyltransferase (CHAT), the biosynthetic enzyme of ACH, in cultured bovine luteal cells. Protein expression and immunolocalization of CHAT were carried out at different stages throughout the luteal phase and in cultured luteal and endothelial cells. ACH was measured in luteal tissue at the different luteal stages and in luteal cells cultured for 8 h and 24 h. Cell viability and TUNEL assays were performed on cultured mid luteal cells treated with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG) in the presence of ACH and its muscarinic (atropine) and nicotinic (mecamylamine) receptor antagonists. The CL was devoid of cholinergic nerve fibers. CHAT immunostaining was evident in luteal, endothelial and stromal cells in luteal tissue sections and in cultured luteal and endothelial cells. CHAT protein was expressed throughout the cycle without any significant changes. ACH concentration in luteal tissue was not changed during the luteal stages, but increased over time and with increased cell numbers in luteal cell cultures. ACH increased cell viability and prevented cell death induced by TNF/IFNG. Atropine significantly attenuated ACH action whereas mecamylamine had no effect. TNF/IFNG treatment downregulated CHAT expression while NGF, IGF1 and TGFB1 upregulated CHAT expression in cultured luteal cells. The overall findings strongly suggest a non-neural source and anti-apoptotic role of ACH in the bovine CL. Locally produced ACH appears to be regulated by NGF, IGF1 and TGFB1.

[Neuroendocrinology] Developmental Programming: Contribution of Prenatal Androgen and Estrogen to Estradiol Feedback Systems and Periovulatory Hormonal Dynamics in Sheep

Veiga-Lopez, A., Astapova, O. I., Aizenberg, E. F., Lee, J. S., Padmanabhan, V. — 2009-01-02

Prenatal testosterone excess leads to neuroendocrine and periovulatory disruptions in the offspring culminating in progressive loss of cyclicity. It is unknown whether the mediary of these disruptions is androgen or estrogen, because testosterone can be aromatized to estrogen. Taking a reproductive lifespan approach of studying control, prenatal testosterone and dihydrotestosterone-treated offspring, this study tested the hypothesis that disruptions in estradiol negative but not positive feedback effects are programmed by androgenic actions of testosterone and that these disruptions in turn will have an impact on the periovulatory hormonal dynamics. The approach was to test estradiol negative and positive feedback responses of all 3 groups of ovary-intact females during prepubertal age and then compare the periovulatory dynamics of LH, FSH, estradiol and progesterone during the first breeding season. The findings show that estradiol negative but not estradiol positive feedback disruptions in prenatal testosterone-treated females are programmed by androgenic actions of prenatal testosterone excess and that follicular phase estradiol and gonadotropins surge disruptions during reproductive life are consistent with estrogenic programming. Additional studies carried out testing estradiol positive feedback response over time found progressive deterioration of E₂ positive feedback in prenatal testosterone-treated sheep until the time of puberty. Together, these findings provide insight into the mechanisms by which prenatal testosterone disrupts the reproductive axis. The findings may be of translational relevance, since daughters of mothers with hyperandrogenism are at risk of increased exposure to androgens.

[Behavior] Juvenile Rank Can Predict Male-Typical Adult Mating Behavior in Female Sheep Treated Prenatally with Testosterone

Roberts, E. K., Flak, J. N., Ye, W., Padmanabhan, V., Lee, T. M. — 2009-01-02

Previous research with female sheep indicates that exposure to excess testosterone for 60 days (from gestational day 30-90 of the 147 day gestation) leads to virilized genitalia, severe neuroendocrine deficits, as well as masculinization and defeminization of sexual behavior (T60 females). In contrast, 30 days of testosterone exposure (days 60-90 of gestation) produce animals with female-typical genitalia, less severe neuroendocrine alterations, and variable gender patterns of sexual behavior (T30 females). Variation in adult sexual behavior of male ungulates is influenced by early social experience, but this has never been tested in females. Here we investigate the influence of rank in the dominance hierarchy on the expression of adult sexual behavior in females. Specifically, we hypothesized that juvenile rank would predict the amount of male- and female-typical mating behavior exhibited by adult female sheep. This hypothesis was tested in two treatment groups and their controls (Group 1: T60 females, Group 2: T30 females). Dominance hierarchies were determined by observing competition over resources. Both groups of prenatal testosterone-treated females were higher ranking than Controls (T60: P = 0.05; T30 P < 0.01). During the breeding season, both T60 and T30 females exhibited more male-typical mating behavior than did Controls, however the T30 animals also exhibited female-typical behavior. For the T60 group, prenatal treatment, not juvenile rank, best predicted male-typical sex behavior (P = 0.007), while juvenile rank better predicted male mating behavior for the T30 group (P = 0.006). Rank did not predict female mating behavior in the hormone-treated or control ewes. We conclude that the effect of prenatal testosterone exposure on adult male-specific, but not female-specific, mating behavior is modulated by juvenile social experiences.

[Testis] The Molecular Signature of Spermatogonial Stem/Progenitor Cells in the 6-Day-Old Mouse Testis

2008-12-23

To characterize the molecular phenotype of spermatogonial stem cells (SSCs), we examined genes that are differentially expressed in the stem/progenitor spermatogonia compared to non-stem spermatogonia. We isolated type A spermatogonia (stem and non-stem type A) from 6-day-old mice using sedimentation velocity at unit gravity and further selected the stem/progenitor cell subpopulation by magnetic activated cell sorting with an antibody to GDNF-receptor-alpha-1 (GFRA1). It has been previously shown that GFRA1 is expressed in SSCs and is required for their stemness. The purity of the isolated cells was approximately 95% to 99% as indicated by immunocytochemistry using anti-GFRA1. Comparison of GFRA1 positive and GFRA1 negative spermatogonia by microarray analysis revealed 99 known genes and 12 uncharacterized transcripts that are over-expressed in the former cell population with a >2 fold change. Interestingly, the highest level of over-expression was observed for Csf1r, encoding the receptor for macrophage colony stimulating factor (M-CSF, official symbol CSF1), which has a well-established role in the regulation of myeloid progenitor cells. Analysis of our microarray data with a bioinformatics software program (Ingenuity Systems) revealed the potential role of various signaling pathways in stem/progenitor spermatogonia and suggested a common pathway for GFRA1 and CSF1R, which may lead to their proliferation. Further investigation to test this hypothesis has shown that CSF1 promotes cell proliferation in primary cultures of the isolated type A spermatogonia and in the spermatogonial-derived stem cell line C18-4. Semi-quantitative RT-PCR and immunohistochemistry confirmed the above microarray data. Collectively, this study provides novel molecular signatures for stem/progenitor spermatogonia and demonstrates a role for CSF1/CSF1R signaling in regulating their proliferation.

[Female Reproductive Tract] Prostaglandin F2Alpha Stimulates 11Beta-Hydroxysteroid Dehydrogenase 1 Enzyme Bioactivity and Protein Expression in Bovine Endometrial Stromal Cells

Lee, H.-Y., Acosta, T. J., Skarzynski, D. J., Okuda, K. — 2008-12-23

11Beta-hydroxysteroid dehydrogenase (HSD11B) enzymes play important roles in regulating cortisol availability in target tissues. We previously demonstrated that HSD11B1 is expressed and active in bovine endometrium and that cortisol suppresses prostaglandin (PG) F2alpha and PGE2 production in cultured bovine endometrial stromal cells. The present study was conducted to examine whether locally synthesized PGF2alpha and/or PGE2 regulates the enzymatic bioactivity and/or the expression of HSD11B1 in bovine endometrium. The conversion rate of cortisone to cortisol in cultured endometrial stromal cells was significantly stimulated by PGF2alpha (1 and 10 µM). PGF2alpha but not PGE2 dose-dependently increased the net conversion of cortisone to cortisol in stromal cells after 4 h of treatment. In addition, the bioactivity of HSD11B1 was significantly inhibited by indomethacin (10 µM). The inhibitory effect of indomethacin on HSD11B1 bioactivity was abolished by PGF2alpha (1 µM) but not PGE2. Although PGF2alpha (1 µM) did not affect the expression of HSD11B1 mRNA in cultured stromal cells, it significantly stimulated the protein expression of HSD11B1. Cycloheximide (CHX), a general translational inhibitor, abolished the stimulatory effects of PGF2alpha on HSD11B1 protein expression in endometrial stromal cells, indicating that PGF2alpha increases HSD11B1 protein expression by stimulating a post-transcriptional process rather than a transcriptional mechanism. These results demonstrate that PGF2alpha but not PGE2 increases HSD11B1 bioactivity and protein expression by stimulating a post-transcriptional mechanism in stromal cells, and suggest that cortisol plays a physiologically relevant role in preventing excessive uterine PG production in the non-pregnant bovine endometrium.

[Pregnancy] Alpha-Lipoic Acid Inhibits Tumor Necrosis Factor-Induced Remodeling and Weakening of Human Fetal Membranes

2008-12-23

Untimely rupture of the fetal membranes (FM) is a major precipitant of preterm birth. Although the mechanism of FM weakening leading to rupture is not completely understood, pro-inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 beta (IL1B), have been shown to weaken FM concomitant with the induction of reactive-oxygen species, collagen remodeling, and prostaglandin release. We hypothesized that alpha-lipoic acid, a dietary antioxidant, may block the effect of inflammatory mediators and thereby inhibit FM weakening. Full thickness FM fragments were incubated with control media or TNF, with or without alpha-lipoic acid pre-treatment. FM rupture strength, as well as release of matrix metalloproteinase 9 (MMP9) and Prostaglandin E₂ (PGE2) from the full thickness FM fragments was determined. The two constituent cell populations in amnion, the mechanically strongest FM component, were similarly examined. Amnion epithelial and mesenchymal cells were treated with TNF or IL1B, with or without alpha-lipoic acid pre-treatment. MMP9 and PGE2 were analyzed by ELISA, Western Blot and Zymography. TNF decreased FM rupture strength 50% while increasing MMP9 and PGE2 release. Lipoic acid inhibited these TNF-induced effects. Lipoic acid pre-treatment also inhibited TNF and IL1B-induced increases in MMP9 protein, activity and release in amnion epithelial cells, and PGE2 increases in both amnion epithelial and mesenchymal cells. In summary, lipoic acid pre-treatment inhibited TNF-induced weakening of FM and cytokine induced MMP9 and PGE2 in both intact FM and amnion cells. We speculate that dietary supplementation with alpha-lipoic acid might prove clinically useful in prevention of preterm premature rupture of fetal membranes.

[Testis] Mitogen-Activated Protein Kinase Signaling in Male Germ Cell Apoptosis in the Rat

2008-12-23

Programmed germ cell death is critical for functional spermatogenesis. Increased germ cell apoptosis can be triggered by various regulatory stimuli, including testicular hyperthermia or deprivation of gonadotropins and intratesticular testosterone (T). We have previously shown the involvement of the mitogen-activated protein kinase (MAPK) 14 in apoptotic signaling of male germ cells across species after hormone deprivation. This study investigates the role of MAPK 14 in germ cell apoptosis in rats triggered by testicular hyperthermia. The contributions of the MAPK1/3 and the MAPK8 to male germ cell death were also examined after this intervention. We show that: 1) testicular hyperthermia results in induction of both MAPK1/3 and MAPK14 but not MAPK8; 2) inhibition of MAPK1/3 has no effect on the incidence of heat-induced germ cell apoptosis, suggesting that MAPK1/3 signaling may be dispensable for heat-induced male germ cell apoptosis; and 3) activation of MAPK14 and BCL2 phosphorylation are critical for heat-induced male germ cell apoptosis in rats. Thus, unlike hormone deprivation model, heat stress through activation of the MAPK14 signaling promotes germ cell apoptosis by provoking BCL2 phosphorylation, leading to its inactivation and the subsequent activation of the mitochondria-dependent death pathway. These novel findings point to a critical role of MAPK14 in stage-and cell-specific activation of male germ cell apoptosis triggered by hormone deprivation or heat stress.

[Toxicology] Etoposide Induces TRP53-Dependent Apoptosis and TRP53-Independent Cell-Cycle Arrest in Trophoblasts of the Developing Mouse Placenta

2008-12-23

Abnormal regulation of placental apoptosis and proliferation has been implicated in placental disorders. Recently, several DNA-damaging agents were reported to induce excessive apoptosis and reduce cell proliferation in the placenta; however, the molecular pathways of these toxic effects on the placenta are unclear. The aim of the present study was to determine the involvement of TRP53, a tumor suppressor that mediates cellular responses to DNA damage, in the induction of apoptosis and cell cycle arrest in the developing placenta. For this purpose, we treated pregnant mice on Day 12 of gestation with 10 mg/kg of etoposide and 5 Gy gamma-ray irradiation, potent inducers of DNA damage. We found an increase in the number of trophoblastic apoptosis 8 and 24 h after etoposide injection and 6 and 24 h after irradiation in the placental labyrinth zone. The number of mitoses and DNA synthesis in trophoblasts decreased after treatment. The accumulation and phosphorylation of TRP53 protein were detected 8 and 6 h after etoposide injection and irradiation, respectively. In Trp53-deficient placentas, the induction of etoposide-induced trophoblastic apoptosis is abrogated while the reduction of proliferation occurred similarly, as in wild-type placentas. CDC2A, a regulator of G2/M progression, was inactivated by phosphorylation after etoposide injection and irradiation, suggesting that the cell cycle was arrested at the G2/M border by treatment. Our present study demonstrated that etoposide injection induced TRP53-dependent apoptosis and TRP53-independent cell cycle arrest in labyrinthine trophoblasts, providing insights into the molecular pathway of placental disorders.

[Embryo] Identification of Differentially Expressed Genes Between Cloned and Zygote-Developing Zebrafish (Danio rerio) Embryos at the Dome Stage Using Suppression Subtractive Hybridization

Luo, D., Hu, W., Chen, S., Xiao, Y., Sun, Y., Zhu, Z. — 2008-12-17

Comparative analyses of differentially expressed genes between somatic cell nuclear transfer (SCNT) embryos and zygote-developing (ZD) embryos are important for understanding the molecular mechanism underlying the reprogramming processes. Herein, we employed the suppression subtractive hybridization (SSH) approach and from more than 2,900 clones identified 96 differentially expressed genes between the SCNT and ZD embryos at the dome stage in zebrafish. We have reported the first database of differentially expressed genes in zebrafish SCNT embryos. Collectively, our findings demonstrate that zebrafish SCNT embryos undergo significant reprogramming processes during the dome stage. However, the majority of differentially expressed genes are down-regulated in SCNT embryos, indicating failure of reprogramming. Based on EnsEMBL description and Gene Ontology (GO) annotation, the problems of reprogramming at the dome stage may occur during nuclear remodeling, translation initiation, and regulation of the cell cycle. The importance of regulation from recipient oocytes in cloning should not be underestimated in zebrafish.

[Ovary] Developmental Programming: Differential Effects of Prenatal Testosterone and Dihydrotestosterone on Follicular Recruitment, Depletion of Follicular Reserve, and Ovarian Morphology in Sheep

Smith, P., Steckler, T. L., Veiga-Lopez, A., Padmanabhan, V. — 2008-12-17

Prenatal testosterone excess programs an array of adult reproductive disorders including LH excess, functional hyperandrogenism, neuroendocrine defects, polycystic ovarian morphology, and corpus luteum dysfunction culminating in early reproductive failure. Polycystic ovarian morphology originates from enhanced follicular recruitment and follicular persistence. We tested if prenatal testosterone treatment by its androgenic actions enhances follicular recruitment, causes early depletion of follicular reserve and disrupts the ovarian architecture. Pregnant sheep were given twice weekly injections of testosterone or dihydrotestosterone (DHT), a non-aromatizable androgen, from Days 30 to 90 of gestation. Ovaries were obtained from Day 90 and Day 140 fetuses and from 10-month old females during a synchronized follicular phase (n=5-9 per treatment). Stereological techniques were used to quantify changes in ovarian follicle/germ cell populations. Results revealed no differences in numbers of oocytes and follicles between the 3 groups on Fetal Day 90. Greater numbers of early growing follicles were found in prenatal testosterone and DHT-treated fetuses on Day 140. Increased numbers of growing follicles and reduced numbers of primordial follicles were found in 10-month old prenatal testosterone but not DHT-treated females. Antral follicles of prenatal testosterone- not DHT-treated females manifested several abnormalities, which included the appearance of haemorrhagic and luteinized follicles and abnormal early antrum formation. Both treatment groups showed morphological differences in the rete ovarii. These findings suggest that increased follicular recruitment and morphologic changes in the rete ovarii of prenatal testosterone-treated females are facilitatd by androgenic programming but postpubertal follicular growth, antral follicular disruptions and follicular depletion largely via estrogenic programming.

[Minireview] Germ Cell Research: A Personal Perspective

Yanagimachi, R. — 2008-12-17

My interest in germ cells began when I first witnessed sea urchin fertilization and embryo development during a laboratory class at the Hokkaido University (Japan) almost 60 years ago. Weismann's concept of germ cells that I learned during my undergraduate years became the driving force of my entire research career. During the early years, my associates and I used mainly the golden hamster and guinea pig as model animals because their spermatozoa had large acrosomes and we could readily follow changes in the acrosomes without killing or staining spermatozoa. We later used the mouse as our model organism because we wanted to produce live offspring with known genetic backgrounds. A summary of the findings we made during those years include: (a) first in vitro sperm capacitation, (b) discovery of sperm hyperactivation, (c) demonstration of the importance of Ca2+ in sperm acrosome reaction, hyperactivation, sperm-egg fusion, and egg activation, (d) development of the sperm's fusion-competence during the acrosome reaction, (e) characterization of sperm-oviduct relationships before and during fertilization, (f) the use of zona-free hamster eggs to examine fertilizing ability and chromosomes of human spermatozoa, (g) the development and use of intracytoplasmic sperm injection, (h) the use of pre-spermatozoal cells for the production of offspring, (i) sperm preservation by freeze-drying and freezing whole animal bodies, and (j) mouse cloning by somatic cell nuclear transfer. My current interests and my visions for the future include: (1) mass production of mature eggs and spermatozoa in vitro, (2) permanent sperm preservation at ambient temperature, (3) development of safer and more efficient assisted fertilization (reproduction) technologies, (4) development of safe and efficient methods of cloning, (5) production of artificial organs such as an artificial uterus, (6) development of safe and effective male contraceptives, and (7) prevention of cancer through germ cell research.

[Male Reproductive Tract] The Novel Epididymis-Specific Beta-Galactosidase-Like Gene Glb1l4 Is Essential in Epididymal Development and Sperm Maturation in Rats

Zhen, W., Li, P., He, B., Guo, J., Zhang, Y.-L. — 2008-12-17

We describe a novel epididymis-specific cDNA named Glb1l4, which was isolated from rat epididymis by differential display of mRNAs. Glb1l4 cDNA contains 2607 nucleotides and encodes a 637 amino acid protein with 50% similarity to mouse beta-galactosidase. The gene is located on chromosome 8q13, spanning 21 exons. Northern blot analysis reveals that Glb1l4 is specifically expressed in the caput region of epididymis and up-regulated by androgen. A specific polyclonal antiserum against the N-terminal peptide of GLB1L4 has been produced. Western blot and immunohistochemistry assay reveal that GLB1L4 is specifically expressed in principal cells of the caput epididymis. Interestingly, its expression peaks at postnatal day 45 in mRNA level and at Postnatal Day 60 in protein level while the epididymis column cells undergoing differentiation. Moreover, within this very period, this secretory protein is confined inside the cell with a change of sub-cellular distribution pattern which implies its important roles in the cell differentiation process. Only after the epididymal epithelium differentiation is completed and the spermatozoa enter the epididymal lumen, the GLB1L4 is secreted into the luminal fluid and binds on the sperm head. Our results suggest that GLB1L4 may play various roles in the principal cell differentiation and sperm maturation.

[Reproductive Technology] Effects of Ooplasm Manipulation on DNA Methylation and Growth of Progeny in Mice

2008-12-10

New techniques to boost male and female fertility are being pioneered at a rapid pace in fertility clinics to increase the efficiency of assisted reproduction methods in couples where natural conception has not been achieved. This study investigates the possible epigenetic effects of ooplasm manipulation methods on post-natal growth and development using a mouse genetic model, with particular emphasis on the possible effects of inter-genotype manipulations. We performed inter-strain and control intra-strain maternal pronuclear transfers, MII spindle transfers, and ooplasm transfer between C57BL/6 and DBA/2 mice and found no major, long-term growth defects or epigenetic abnormalities in either males or females associated with inter-genotype transfers. Ooplasm transfer itself was associated with reduced viability, and additional subtle effects of ooplasm strain of origin were observed. Both inter-strain and intra-strain ooplasm transfer were associated with subtle, transient effects on growth early in life. We also performed inter- and intra-strain germinal vesicle transfers (GVT). Inter-strain GVT-females, but not males, had significantly lower body weights at birth and thereafter compared to the intra-strain GVT and non-GVT controls. No GVT-associated changes were observed in DNA methylation of the Mup1, Rasgrf1, H19, Snrpn, or Peg3 genes, nor any difference in expression of the imprinted Rasgrf1, Igf2r, or Mest genes. These results indicate that some ooplasm manipulation procedures may exert subtle effects on growth early in life, whilst inter-genotype germinal vesicle transfer can result in significant growth deficiencies after birth.

[Testis] Functional Analysis of Male Mouse Haploid Germ Cells of Various Differentiation Stages: Early and Late Round Spermatids Are Functionally Equivalent in Producing Progeny

Ohta, H., Sakaide, Y., Wakayama, T. — 2008-12-10

Spermiogenesis is a complex process consisting of three main phases: the round, elongating, and elongated spermatid phases. Although the germ cells acquire a haploid set of paternal chromosome after meiosis, how functional these male haploid germ cells are as male gametes at various differentiation stages has remained unclear. We selectively injected specific steps of haploid male germ cells into oocytes and assessed the function of the zygotes. Applying the transillumination technique using acrosin-GFP transgenic mice, we succeeded in selecting four types of haploid male germ cells for microinsemination: early round spermatids (steps 2-3), late round spermatids (steps 7-8), elongating spermatids (steps 9-10), and elongated spermatids (step 16). The microinsemination technique revealed that the early and late round spermatids had similar developmental abilities in producing progeny, indicating that the nuclear status of newly generated round spermatids was similar to that of late round spermatids. An increased birthrate of progeny was first observed in steps 9-10 of elongating spermatids, but the frequency was slightly lower than that of the elongated spermatids. These results indicated that the transition from steps 7-8 of round spermatids to steps 9-10 of elongating spermatids is a key step in changing the nuclear status of male gametes in producing progeny.

[Ovary] Necessity of Sequential Pulses of Prostaglandin F2alpha for Complete Physiologic Luteolysis in Cattle

Ginther, O. J., Araujo, R. R., Palhao, M. P., Rodrigues, B. L., Beg, M. A. — 2008-12-10

The luteolytic effects of exogenous prostaglandin F_2alpha (PGF) that did and did not simulate natural PGFM pulses were studied during mid-diestrus in 42 Holstein heifers. Plasma concentrations of PGF were assessed by assay of 13,14-dihydro-15-keto-PGF (PGFM). In experiment 1, a single intrauterine injection of 4.0 mg of PGF into the uterine horn ipsilateral to the corpus luteum resulted in a precipitous progesterone decline, whereas sequential injections of 0.25 or 1.0 mg every 12 h resulted in a stepwise decrease (P < 0.05) following each injection. A progesterone increase occurred during the first 5 min before the luteolytic decrease but only for the 4.0-mg dose. From the results of experiment 2, a 2-h intrauterine infusion of a total of 0.5 mg of PGF was judged to best simulate a natural PGFM pulse. In experiment 3, simulation of sequential pulses at 12-h intervals resulted in a continuous precipitous decrease in progesterone to < 1 ng/ml by the beginning of the fourth simulated pulse. In contrast, a single simulated pulse resulted in a 6-h progesterone decrease to a constant concentration for 3 d after treatment, followed by a return to control concentrations. The interval between the pretreatment and posttreatment ovulations was shorter (P < 0.05) in the group with sequential simulated pulses (14 ± 1 d) than in the group with a single pulse (21 ± 1 d). Results indicated that excessive PGF doses may stimulate nonphysiologic progesterone responses and supported the hypothesis that sequential PGF pulses are required to stimulate natural luteolysis in cattle.

[Ovary] B-Vitamin and Homocysteine Status Determines Ovarian Response to Gonadotropin Treatment in Sheep

Kanakkaparambil, R., Singh, R., Li, D., Webb, R., Sinclair, K. D. — 2008-12-10

Maternal B-vitamin status and homocysteinemia can affect fertility and pregnancy establishment, although direct effects on ovarian follicle and oocyte development are not known. We report on the effects of restricting the supply of vitamin B₁₂ and methionine from the diet of mature female sheep on ovarian folliculogenesis following FSH stimulation. The study was split into three batches and involved 76 animals. Surprisingly, the number of growing, estrogen-active antral follicles following FSH treatment was enhanced (P = 0.005) following this dietary intervention. This increase occurred even in the presence of modest live-weight loss (batch 1 only), and depressed plasma insulin concentrations, suggesting a break-down in the regulation of follicular responsiveness to FSH. This dietary intervention also increased plasma homocysteine concentrations. Physiological concentrations of homocysteine increased granulosa cell proliferation (P < 0.001), estradiol production (P = 0.05) and FSHR transcript expression (P = 0.017) during culture. Transcript levels for growth differentiation factor 9 and bone morphogenetic protein 15 in oocytes from treated ewes were increased (P < 0.05) in the first two batches. Furthermore, regression of BMP receptor 2 (BMPR2) transcript expression and diet on follicle number revealed a significant interaction (P = 0.01); BMPR2 transcript expression was associated with follicle number only in vitamin B₁₂/methionine restricted animals. As FSHR transcript expression was also positively (P = 0.007) related to follicle number, so the effects of diet may have arisen through enhanced FSH and BMP signaling. Although this remains to be confirmed the data support an intra-ovarian impact of vitamin B₁₂/methionine deficient diets.

[Testis] RAB13 Participates in Ectoplasmic Specialization Dynamics in the Rat Testis

Mruk, D. D., Lau, A. S.N. — 2008-12-10

During spermatogenesis, leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) to gain entry into the adluminal compartment for further development. At the same time, these, as well as other, germ cell types in the epithelium must retain their close association with Sertoli cells via specialized cell junctions. In this study, we demonstrate that RAB13 -- a GTPase known to participate in tight junction function in other epithelia -- also participates in the dynamics of the ectoplasmic specialization, a testis-specific type of anchoring junction. By immunohistochemistry microscopy, RAB13 localized to the ectoplasmic specialization. Moreover, RAB13 was found to associate with vinculin (VCL) and espin (ESPN), two putative ectoplasmic specialization actin (ACT)-binding proteins, by co-immunoprecipitation and immunofluorescence microscopy experiments. To address the role of RAB13 in ectoplasmic specialization dynamics, an in vivo model was utilized in which administration of Adjudin induced the disassembly of Sertoli-germ cell anchoring junctions. Following administration of this drug, the RAB13 level decreased steadily when the loss in testicular weight was taken into account. Similarly, the association of RAB13 with VCL decreased, but was not completely lost, during Adjudin-mediated ectoplasmic specialization restructuring. Taken collectively, these results suggest that RAB13 functions in ectoplasmic specialization dynamics in the testis.

[Gamete Biology] The Presence and Function of Dopamine Type 2 Receptors in Boar Sperm: A Possible Role for Dopamine in Viability, Capacitation, and Modulation of Sperm Motility

2008-12-10

Several reports have shown that dopamine and other catecholamines are present in oviduct luminal fluid. We have recently reported that dopamine type 2 receptors (DRD2) are present in a wide range of mammalian sperm, suggesting a role for dopaminergic signaling in events such as fertilization, capacitation and sperm motility. In the present study, we used Western blot analysis to show that boar sperm express DRD2 and that their activation with dopamine (100 nM) has a positive impact on cell viability, which can be correlated with AKT/PKB-phosphorylation. Bromocriptine (100 nM) and dopamine (100 nM and 10 µM) increased tyrosine phosphorylation during the capacitation period. Immunofluorescence analysis indicated that DRD2 localization is dynamic and depends on the capacitation stage, colocalizing with tyrosine phosphorylated proteins in the acrosome and midpiece region of capacitated boar sperm. This association was confirmed by coimmunoprecipitation analysis. We also showed that bromocriptine (100 nM) and low-concentration dopamine (100 nM and 10 µM) increased both total and progressive motility of sperm. However, high- concentrations of dopamine (1 mM) decreased both tyrosine phosphorylation and motility in in vitro sperm capacitation assays. This can be explained by the presence of dopamine transporters (DAT, official symbol SLC6A3) in sperm, as demonstrated by Western blot analysis and immunocytochemistry. Taken together, our results support the idea that dopamine could play a fundamental role during sperm capacitation and motility in situ, in the female upper reproductive tract.

[Testis] Levonorgestrel Enhances Spermatogenesis Suppression by Testosterone with Greater Alteration in Testicular Gene Expression in Men

2008-12-10

Prior studies have demonstrated that combined treatment of testosterone (T) with a progestin induces a more rapid and greater suppression of spermatogenesis than T treatment alone. We hypothesized that the suppressive effects of the combination of T undecanoate (TU) injections plus oral Levonorgestrel (LNG) on spermatogenesis may be mediated through a greater perturbation of testicular gene expression than TU alone. To test this hypothesis, we performed open testicular biopsy on 12 different adult healthy subjects: 1) 4 normal men as control; 2) 4 men, 2 weeks after TU treatment; and 3) 4 men, 2 weeks after TU+LNG administration. RNA isolated from biopsies was used for DNA microarray using the Affymetrix Human Genome U133 Plus 2.0 oligonucleotide microarrays. Gene expression with ≥2-fold changes (P < 0.05) compared with control was analyzed using the NIH DAVID 2008 resource. TU treatment altered the gene expression in 109 transcripts while TU+LNG altered the gene expression in 207 transcripts as compared to control. Both TU and TU+LNG administration suppressed gene expression of insulin-like 3; cytochrome P450, family 17, subfamily A1 in Leydig cells; and inhibin alpha in Sertoli cells; and increased proapoptotic transcripts BCL2-like 14, insulin-like growth factor binding protein 3, and decreased X-linked inhibitor of apoptosis protein. In comparison with TU treatment alone, TU+LNG treatment up-regulated insulin-like 6, relaxin 1 and down-regulated RNA binding protein transcripts. We conclude that TU+LNG administration induces more changes in testicular gene expression than TU alone. This exploratory study provided a novel and valuable database to study the mechanisms of action of hormonal regulation of spermatogenesis in men and identified testicular specific molecules that may serve as potential targets for male contraceptive development.

[Gamete Biology] Immunogold Evidence Suggests That Endoplasmic Reticulum Is the Site of Protamine-Type Protein Synthesis and Participates in Translocation of These Proteins into the Nucleus During Chara vulgaris Spermiogenesis

Poplonska, K., Kwiatkowska, M., Wojtczak, A., Polit, J. — 2008-12-10

During spermiogenesis of an alga Chara vulgaris, which in many aspects resembles that of animals, histones are replaced by protamine-type proteins. Our earlier immunocytochemical studies showed that this replacement started during the short stage V of spermiogenesis when electronograms revealed an extensive system of cisternae and vesicles of endoplasmic reticulum (ER). The present studies revealed at stage V intensive incorporation of labeled ³H-arginine and ³H-lysine quickly translocating into a nucleus visualised with pulse-chase autoradiography of semi-thin sections. The immunogold technique with the use of the antibodies to protamine-type proteins isolated from C. tomentosa show that both ER cisternae and vesicles are labeled with gold grains which are absent from the spermatids not treated with the antibodies, thus ER is probably the site of the protamine-type proteins synthesis. These proteins are then translocated to a nucleus through ER channels connected with the nuclear envelope which is suggested by gold labeling of an inner membrane of the nuclear envelope adjacent to condensed chromatin. The above results correspond with those of other authors which prove that in animals protamines bind with lamin B receptors localized in the inner membrane of the nuclear envelope. A hypothesis has been put forward that during Chara spermiogenesis the inner membrane of the nuclear envelope invaginates into a nucleus together with protamine-type proteins which become separated from the membrane and penetrate into chromatin.

[Mechanisms of Hormone Action] Functional AR Signaling Is Evident in an In Vitro Mouse Follicle Culture Bioassay That Encompasses Most Stages of Folliculogenesis

Lenie, S., Smitz, J. — 2008-12-10

Androgens serve distinct physiological functions within the ovary. The biological action of androgens is primarily exerted through transcriptional regulation by the nuclear androgen receptor (AR), but the molecular cascades governed by AR remain largely unknown. At present, there is eminent concern that environmental man-made chemicals with (anti-)androgenic properties among other are capable of modulating hormonal responses thereby interfering with normal physiological processes, critical to fertility. In the present study, we aimed to further characterize a standardized and reproducible follicle culture system in terms of AR expression during in vitro folliculogenesis in order to be able to use it as a bioassay to study effects of (anti-)androgens on follicular and oocyte growth, steroid secretion profile and oocyte meiotic maturation capacity. Immunohistochemical analysis revealed cytoplasmic AR protein was translocated to the nucleus of both granulosa and theca cells in response to endogenous androgen production in theca cells during preantral follicular development. During the antral phase in vitro, AR was differentially expressed in mural and cumulus cells, implying an oocyte-mediated regulation. Treatment of follicles with either hydroxyflutamide or bicalutamide, two model anti-androgenic compounds, resulted in a reduced follicular growth during the preantral phase, an altered steroidogenic environment, and an arrest in oocyte meiotic maturation in response to hCG. In conclusion, AR expression in the culture model corresponded well to what is described in vivo and this system revealed several ovarian functions being targeted by AR antagonists that can be further investigated by more in-depth molecular techniques.

[Neuroendocrinology] Cortisol Interferes with the Estradiol-Induced Surge of Luteinizing Hormone in the Ewe

2008-12-03

Two experiments were conducted to test the hypothesis that cortisol interferes with the positive feedback action of estradiol that induces the LH surge. Ovariectomized sheep were treated sequentially with progesterone and estradiol to create artificial estrous cycles. Cortisol or vehicle (saline) was infused from 2 h before the estradiol stimulus through the time of the anticipated LH surge in the artificial follicular phase of two successive cycles. The plasma cortisol increment produced by infusion was ~1.5 times greater than maximal concentrations seen during infusion of endotoxin, which is a model of immune / inflammatory stress. In Experiment 1, half of the ewes received vehicle in the first cycle and cortisol in the second; the others were treated in reverse order. All ewes responded with an LH surge. Cortisol delayed the LH surge and reduced its amplitude, but both effects were observed only in the second cycle. Experiment 2 was modified to provide better control for a cycle effect. Four treatment sequences were tested (cycle 1-cycle 2): vehicle-vehicle, cortisol-cortisol, vehicle-cortisol, cortisol-vehicle. Again, cortisol delayed but did not block the LH surge, and this delay occurred in both cycles. Thus, an elevation in plasma cortisol can interfere with the positive feedback action of estradiol by delaying and attenuating the LH surge.

[Pregnancy] Select Nutrients in the Ovine Uterine Lumen. III. Cationic Amino Acid Transporters in the Ovine Uterus and Periimplantation Conceptuses

Gao, H., Wu, G., Spencer, T. E., Johnson, G. A., Bazer, F. W. — 2008-11-26

Arginine is an essential amino acid for conceptus (embryo/fetus and trophoblast/placenta) growth and development; however, the mechanisms for arginine transport into the uterine lumen and uptake by conceptuses are largely unknown. In this study, expression of System y⁺ (SLC7A1, SLC7A2, and SLC7A3) cationic amino acid transporters in uteri of cyclic and pregnant ewes and conceptuses were studied and effects of pregnancy, progesterone (P4) and interferon tau (IFNT) on their expression were investigated. SLC7A1 mRNA was most abundant in endometrial luminal (LE) and superficial glandular (sGE) epithelia on Day 16 of the estrous cycle and Days 16 to 20 of pregnancy, whereas SLC7A2 mRNA was most abundant in LE and mid- to deep glandular (GE) epithelia on Days 14 to 20 of gestation. Expression of both SLC7A1 and SLC7A2 was enhanced in pregnant ewes in a cell-specific manner, but abundance of SLC7A3 was not affected by either day of the estrous cycle or pregnancy status. SLC7A1, SLC7A2, and SLC7A3 mRNAs were expressed in both trophectoderm and endoderm of conceptuses. In ovariectomized ewes, short-term treatment of ewes with P4 and IFNT did not affect endometrial SLC7A1 mRNA, while long-term treatment with P4 stimulated SLC7A1 in both LE and GE, and IFNT tended to increase SLC7A1 abundance in LE. SLC7A2 mRNA abundance increased 4.1-fold in response to short-term P4 treatment and an additional 1.7-fold by IFNT primarily in endometrial LE/sGE, and these effects were ablated by a progesterone receptor antagonist. These results indicate that coordinate changes in SLC7A1, SLC7A2 and SLC7A3 expression in uterine endometria and conceptuses are likely important in transport of arginine that is critical to conceptus growth, development and survival.

[Reproductive Technology] Production of Piglets after Cryopreservation of Embryos Using a Centrifugation-Based Method for Delipation Without Micromanipulation

2008-11-26

It is still difficult to successfully cryopreserve in vitro produced (IVP) swine embryos, as they are sensitive to chilling due to the abundance of intracellular lipids. Mechanical delipation through micromanipulation is successful, but this increases the potential of pathogen transmission because of the damage inflicted upon the zona pellucida during micromanipulation, and is labor intensive. Reported here is a method to remove the lipid of IVP porcine embryos without significantly compromising the zona pellucida by trypsin treating the embryos or exposing the embryo to a high osmolality solution to enlarge the perivitelline space so that the lipid could be polarized and separated completely after subsequent centrifugation without micromanipulation. The procedures work both for nuclear transfer derived embryos as well as in vitro fertilized embryos. Both methods provide a high throughput process that leaves the zona pellucida intact (or relatively intact as for the trypsin treatment) to aid in preventing disease transmission. It is also demonstrated that this procedure results in viable piglets, a claim not able to be made by many previous reports. Although the efficiencies of cryopreservation have not been dramatically improved, these procedures allow a single person to process very large numbers of embryos without the necessity of manipulating each individual embryo on a micromanipulator. Such high throughput processing overcomes the lack of high efficiency, i.e. the system can be overloaded with embryos for transfer to surrogates.

[Pregnancy] Effect of Leptin on Mouse Trophoblast Giant Cells

Schulz, L. C., Widmaier, E. P., Qiu, J., Roberts, R. M. — 2008-11-26

Leptin plays a role in both energy homeostasis and reproduction, and is required in early pregnancy. It stimulates metalloproteinase activity in cultured human trophoblast and invasiveness of cultured mouse trophoblast. Our goal has been to examine mechanisms that underpin the ability of leptin to promote trophoblast invasiveness in primary cultures of mouse trophoblast. Leptin stimulated the phosphorylation of MEK (MAP2K1) but not STAT3 in the cultures, increased the concentration of the SOCS3 (suppressor of cytokine signaling) protein, and upregulated metalloproteinase activity. Microarray analysis revealed that leptin stimulated select genes with roles in cell motility, including Stmn, a gene linked to invasiveness in other cell types. There was also an increase in activity of several genes associated with MAPK and RhoGTPase signaling. In addition, leptin muted expression of genes correlated with terminal differentiation of trophoblast giant cells, including ones associated with TGFbeta signaling pathway and endoreduplication of DNA and up-regulated, selected prolactin-related family members. Feulgen staining of leptin treated cells revealed a loss of cells with low ploidy. The data suggest that leptin accelerates disappearance of non-giant cells while inhibiting terminal differentiation of committed giant cells, possibly by maintaining cells in an intermediate stage of differentiation.

[Testis] TNF Alpha-Mediated Disruption of Spermatogenesis in Response to Sertoli Cell Injury in Rodents Is Partially Regulated by MMP2

Yao, P.-L., Lin, Y.-C., Richburg, J. H. — 2008-11-26

Mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury in peri-pubertal rodents results in the stimulation of germ cell apoptosis through an interaction of FAS/FASL between these two cell types. During this peri-pubertal period, an early spike in the incidence of germ cell apoptosis occurs during the first wave of spermatogenesis and is essential for the development of functional spermatogenesis in adults. Our previous observations revealed that soluble TNF alpha (TNFA) released by germ cells after MEHP exposure consequently resulted in a robust induction of FASL by Sertoli cells. Metalloproteinases (MPs) are essential for processing the TNFA precursor to its soluble form and its ability to bind to TNFRSF1A. The activity of MPs is regulated by the tissue inhibitors of metalloproteinases (TIMPs) family. Here we report that TIMP2 is predominately expressed in Sertoli cells and that protein levels decrease in a time-dependent manner after MEHP exposure. The secretion of matrix metalloproteinase 2 (MMP2) in primary rat Sertoli cell-germ cell co-cultures is induced after MEHP exposure, and its activity increases in a time-dependent manner. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP2 and significantly reduces MEHP-enhanced soluble TNFA production in primary co-cultures. In vivo challenges with SB-3CT decrease soluble TNFA and reduce MEHP-induced testicular germ cell apoptosis. In primary co-cultures, MEHP exposure causes an increase in soluble TNFA (9.46-fold), while the addition of recombinant MMP2 protein results in a 5.4-fold increase in soluble TNFA, suggesting that MEHP-induced MMP2 is, in part, responsible for the activation of TNFA in the testis. Taken together, our observations indicate the distinct role of specific MPs in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis.

[Reproductive Technology] Generation of Live Rats Produced by In Vitro Fertilization Using Cryopreserved Spermatozoa

Seita, Y., Sugio, S., Ito, J., Kashiwazaki, N. — 2008-11-26

In rats, the success of in vitro fertilization (IVF) was reported 40 years ago. Although it has been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported to date. Here we report establishment of a successful IVF system using frozen/thawed rat spermatozoa. Our data showed intracellular cAMP and free cholesterol levels in frozen/thawed rat sperm were maintained low, suppressing capacitation-associated tyrosine phosphorylation. The treatment of methyl-beta-cyclodextrin improved removal of free cholesterol from the membrane in frozen/thawed sperm but not induction of capacitation-associated tyrosine phosphorylation in the sperm. A phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), treatment dramatically increased cAMP and tyrosine phosphorylation levels in frozen/thawed rat sperm. When the IBMX-treated frozen/thawed sperm was used for IVF, the proportions of pronuclear and blastocyst formation were significantly higher than those of frozen/thawed sperm treated without IBMX (P < 0.05). The embryos were developed to term at a high success rate equivalent to the rate obtained with IVF using fresh sperm. Thus, we established for the first time a successful IVF system in rats using cryopreserved spermatozoa.

[Embryo] Poor Embryo Development in Mouse Oocytes Aged In Vitro Is Associated with Impaired Calcium Homeostasis

Takahashi, T., Igarashi, H., Kawagoe, J., Amita, M., Hara, S., Kurachi, H. — 2008-11-26

We examined whether the impairment of intracellular Ca²⁺ homeostasis is related to poor embryo development in in vitro-aged oocytes. We found that the in vitro aging of mouse oocytes affected the patterns of Ca²⁺ oscillations at fertilization: these changes in Ca²⁺ oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca²⁺ store was decreased in in vitro-aged oocytes. A decrease in the Ca²⁺ stores induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca²⁺-ATPase inhibitor, resulted in a lower fertilization rate and poorer embryo development. The frequency of Ca²⁺ oscillations at fertilization was significantly increased whereas their amplitude was lowered in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca²⁺ homeostasis, like the decrease in the ER Ca²⁺ store, caused an alteration in Ca²⁺ oscillations and the poor embryo development in vitro-aged oocytes. Because embryo fragmentation is closely related to apoptosis, we examined the expression of BAX, a proapototic protein, and BCL2, an anti-apoptotic protein, in in vitro-aged oocytes. Although BCL2 was strongly expressed in the oocytes without in vitro-aging, the expression of BCL2 was significantly reduced in the oocytes of the other treatments, such as those in in vitro-aging, and those that were pretreated with H₂O₂ or thapsigargin. Taken together, the alteration in Ca²⁺ oscillations and the decrease in BCL2 expression in in vitro-aged oocytes might lead to poor embryo development.

[Testis] Photoperiod-Modulated Testis Maturation in Atlantic Cod (Gadus morhua, L.)

2008-11-26

Precocious male puberty is a significant problem in Atlantic cod aquaculture. While photoperiod manipulation can inhibit testis growth, a detailed analysis of effects on spermatogenesis is missing. Starting July 1, prepubertal fish were exposed to different photoperiod regimes in indoor tanks for 17 months. Testis histology, germ cell dynamics (proliferation; apoptosis), and plasma androgen levels were analyzed. In the natural light (NL) group, testis growth started in September and was completed in February 2005, when a two months spawning period started. In the constant light (LL) group, none or much less spermatogenic cysts were recruited into spermatogenesis and apoptotic germ cell loss was high. A change of photoperiod from NL to LL at winter solstice (Dec. 21) resulted in a premature (2 months) completion of the reproductive cycle, while changing from LL to NL at winter solstice triggered a faster than normal testis development. Plasma testosterone levels increased in the NL group from spermatogonial proliferation toward meiosis while those of 11-ketotestosterone rose towards spermiogenesis and spermiation. Plasma androgen levels did not rise under LL conditions. Comparing fish with developing testes from all groups indicated that low androgen levels were associated with a high incidence of spermatogonial apoptosis; we also found that ar mRNA expression was most prominent in Sertoli cells contacting growing spermatogonial clones. Our data show that an inhibitory photoperiod (LL) reduced or blocked differentiation of spermatogonia, increased apoptosis, in particular among proliferating spermatogonia, and was associated with reduced androgen levels, a situation possibly reflecting insufficient gonadotropic stimulation.

[Female Reproductive Tract] Reduced Fecundity in Female Rats with Surgically Induced Endometriosis and in Their Daughters: A Potential Role for Tissue Inhibitors of Metalloproteinase 1

2008-11-19

The cause of reduced fecundity in women with endometriosis is unknown. Expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) by both ectopic and eutopic endometrium reportedly play a role in the pathogenesis of endometriosis. We hypothesize that anomalous endometriotic TIMP protein synthesis, secretion, and localization also cause reproductive pathologies resulting in reduced fecundity. An established rat model for endometriosis (Endo) compared to non-endometriotic controls (Sham) was used to investigate reduced fecundity in endometriosis. Comparing Endo and Sham rats, Endo rats had altered ovarian dynamics including fewer ovarian follicles and corpora lutea (CL) with luteinized unruptured follicles. Further, in vivo anomalies in post-ovulatory oocyte structure and preimplantation embryo development including misaligned chromosomes, nuclear and cytoplasmic fragmentation and delayed or arrested cleavage as well as spontaneous abortions were found only in Endo rats. A causative role for TIMP1 in these phenomena is supported by our findings that Endo rats have more TIMP1 in their peritoneal fluid as detected by ELISA and more TIMP1 immunolocalization in the theca of antral follicles as measured by computer-assisted morphometric analysis. These data suggest that in endometriosis, accumulation of TIMP1 disrupts the normal MMP/TIMP enzymatic milieu in the peritoneal cavity and negatively impacts ovarian dynamics, oocyte quality and preimplantation embryo development, thereby decreasing fecundity. Most intriguingly, daughters from Endo rats which had no experimental interventions exhibited these same reproductive abnormalities. We predict that developmental exposure to endometriosis leads to permanent epigenetic changes in subsequent generations.

[Testis] Effects of Different Temperatures on Testis Structure and Function, with Emphasis on Somatic Cells, in Sexually Mature Nile Tilapias (Oreochromis niloticus)

de Alvarenga, E. R., de Franca, L. R. — 2008-11-19

The Nile tilapia (Oreochromis niloticus) is one of the economically most important freshwater fish nowadays, representing also an excellent model for studies under laboratory conditions. Temperature is considered a very important modulator of reproductive activity in fish, despite that there are very few studies specifically addressing the effects of this key factor on the morphological and functional aspects of the teleosts testes. Therefore, our main objectives in the present study were to analyze the effects of different temperatures (20, 25, 30 and 35°C) on testicular somatic and germ cells, in sexually mature Nile tilapias. In comparison with fish kept at others temperatures, tilapias mantained at 20°C presented higher (P < 0.05) Sertoli and Leydig cells proliferation, volume density and frequency of most type B spermatogonia, as well as germ cells apoptosis. Conversely, tubular fluid secretion was lower (P < 0.05), in the same animals. Although not significant, the type A spermatogonia proliferation followed the pattern established for Sertoli and Leydig cells mitotic activity, suggesting that they preferentially would proliferate at lower temperatures. Based on most results found in our study, and considering that tilapias are non-seasonal breeders, we suggest a model for temperature action on tilapia testes where lower temperature (20°C) would favor the type A spermatogonial renewal, Sertoli and Leydig cells proliferation and germ cells apoptosis, whereas higher temperatures (30-35°C) would trigger rapid germ cell differentiation. Thus, tilapias could potentially be utilized in studies involving hormones/factors related to Sertoli and Leydig cells proliferation and spermatogonial self-renewal and/or differentiation.

[Testis] Characterization of Two Cytoplasmic Poly(A)-binding Proteins, PABPC1 and PABPC2, in Mouse Spermatogenic Cells

Kimura, M., Ishida, K., Kashiwabara, S.-i., Baba, T. — 2008-11-19

Mouse spermatogenic cells are known to contain at least two isoforms of cytoplasmic poly(A)-binding proteins, PABPC1 and PABPC2 (PABPT). In this study, we have characterized PABPC1 and PABPC2. PABPC2 was present in pachytene spermatocytes and round spermatids, whereas elongating spermatids still included PABPC1. These two proteins are capable of binding mRNA poly(A) tails nonspecifically, and being directly associated with each other, and with several translational regulators, EIF4G1, PAIP1, PAIP2, and PIWIL1 (MIWI). Moreover, both PABPC1 and PABPC2 exhibited the ability to enhance translation of a reporter mRNA in vitro. Despite these similarities, PABPC2 was distinguished from PABPC1 by the absence in actively translating polyribosomes of testicular cells. PABPC1 was distributed in both polyribosomes and translationally inactive messenger ribonucleoprotein particles, mRNPs. Importantly, PABPC2 as well as PIWIL1 was noticeably enriched in the chromatoid body of round spermatids. These results suggest that PABPC2 may function in translational repression during spermatogenesis.

[Reproductive Technology] Heritable Imprinting Defect Caused by Epigenetic Abnormalities in Mouse Spermatogonial Stem Cells

2008-11-19

Male germ cells undergo dynamic epigenetic reprogramming during fetal development, eventually establishing spermatogonial stem cells that can convert into pluripotent stem cells. However, little is known about the developmental potential of fetal germ cells and how they mature into spermatogonial stem cells. We herein developed a culture system for fetal germ cells that proliferate for long periods of time. Male germ cells from embryos 12.5-18.5 days post-coitum could expand by glial cell line-derived neurotrophic factor, a self-renewal factor for spermatogonial stem cells. These cells did not form teratomas, but repopulated seminiferous tubules and produced spermatogenesis, exhibiting spermatogonia potential. However, the offspring from cultured cells showed growth abnormalities and were defective in genomic imprinting. The imprinting defect persisted in both the male and female germlines for at least four generations. Moreover, germ cells in the offspring showed abnormal histone modifications and DNA methylation patterns. These results indicate that fetal germ cells have a limited ability to become pluripotent cells and lose the ability to undergo epigenetic reprogramming by in vitro culture.

[Gamete Biology] Mouse TEX14 Is Required for Embryonic Germ Cell Intercellular Bridges but Not Female Fertility

Greenbaum, M. P., Iwamori, N., Agno, J. E., Matzuk, M. M. — 2008-11-19

A conserved feature of germ cell cytokinesis is the formation of stable intercellular bridges between daughter cells. These intercellular bridges are seen in diverse species from Drosophila melanogaster to Homo sapiens and have been shown to play roles in communication of large numbers of germ cells. In Tex14 knockout mice, intercellular bridges do not form during spermatogenesis, and male mice are sterile, showing an essential role for intercellular bridges in postnatal spermatogenesis in mammals. Intercellular bridges also form between dividing germ cells in both male and female embryos. However, little is known about the formation or role of the embryonic intercellular bridges in mammals. In females, embryonic intercellular bridges have been proposed to play a role in development of the presumptive oocyte. Herein, we show that TEX14 is an essential component of male and female embryonic intercellular bridges. We also demonstrate that mitotic kinesin-like protein (MKLP1, official symbol KIF23), which we have discovered is a component of intercellular bridges during spermatogenesis, is also a component of male and female embryonic intercellular bridges. Germ cell intercellular bridges are readily identified by KIF23 immunofluorescence between the gonocytes and oogonia of control mice but are absent between germ cells of Tex14 null mice. Furthermore, by electron microscopy, intercellular bridges are present in all control newborn ovaries but absent in the Tex14 knockout ovaries. Despite the absence of embryonic intercellular bridges in the Tex14 null mice, male mice initiate spermatogenesis, and female mice are fertile. Although fewer oocytes were present in Tex14 null neonatal ovaries, folliculogenesis was still active at one year of age. Thus, while TEX14 and intercellular bridges have an essential role in postnatal spermatogenesis, they are not required in the embryo.

[Gamete Biology] Expression and Functional Analyses of Circadian Genes in Mouse Oocytes and Preimplantation Embryos: Cry1 Is Involved in the Meiotic Process Independently of Circadian Clock Regulation

2008-11-19

In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study, we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in 1- to 4-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of GV oocytes and 1- to 4-cell stage embryos. Since CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and 1- to 4-cell stage embryos, CLOCK, ARNTL and CRY1 might suppress the transcription of these genes in GV oocytes and 1- to 4-cell stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3 but reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies such as meiosis.

[Immunology] Vitamin D Induces Innate Antibacterial Responses in Human Trophoblasts via an Intracrine Pathway

Liu, N., Kaplan, A. T., Low, J., Nguyen, L., Liu, G. Y., Equils, O., Hewison, M. — 2008-11-12

he active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)₂D) is a potent inducer of the antimicrobial protein cathelicidin, CAMP (LL37). In macrophages this response is dependent on intracrine synthesis of 1,25(OH)2D from precursor 25-hydroxyvitamin D (25OHD), catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). In view of the fact that trophoblastic cells also express abundant CYP27B1, we postulated a similar intracrine pathway for induction of CAMP in the placenta. Analysis of placenta explants, primary cultures of human trophoblast, and the 3A trophoblastic cell line treated with 1,25(OH)₂D (1-100 nM) revealed dose-dependent induction of CAMP similar to that observed with primary cultures of human macrophages. Also consistent with macrophages, induction of trophoblastic CAMP was enhanced via intracrine conversion of 25OHD to 1,25(OH)₂D. However, in contrast to macrophages, induction of CAMP by vitamin D in trophoblasts was not enhanced by co-stimulation with toll-like receptor ligands such as lipopolysaccharide. Despite this, exposure to vitamin D metabolites significantly enhanced antibacterial responses in trophoblastic cells: 3A cells infected with Escherichia coli (E. coli) showed decreased numbers of bacterial colony-forming units compared to vehicle-treated controls when treated with 25OHD (49.6 ± 10.9 %) or 1,25(OH)₂D (45.4 ± 9.2 %), both P < 0.001. Treatment with 25OHD (1-100 nM) or 1,25(OH)₂D (0.1-10 nM) also protected 3A cells against cell death following infection with E. coli (13.6-26.9 and 22.3-40.2% protection respectively). These observations indicate that 1,25(OH)₂D can function as an intracrine regulator of CAMP in trophoblasts, and may thus provide a novel mechanism for activation of innate immune responses in the placenta.

[Gamete Biology] ADP-Ribosylation Factor 1 Regulates Asymmetric Cell Division in Female Meiosis in the Mouse

Wang, S., Hu, J., Guo, X., Liu, J. X., Gao, S. — 2008-11-12

Mouse oocytes undergo two successive meiotic divisions to generate one large egg with two small polar bodies, which is essential for preserving the maternal resources to support embryonic development. Although previous studies have shown that some small GTPases, such as RAC, RAN and CDC42, play important roles in cortical polarization and spindle pole anchoring, no oocytes undergo cytokinesis when the mutant forms of these genes are expressed in mouse oocytes. Here we show that the ADP-ribosylation factor 1 (ARF1) plays an important role in regulating asymmetric cell division in mouse oocyte meiosis. Microinjection of mRNA of a dominant negative mutant form of Arf1 (Arf1^T31N) into fully grown germinal vesicle oocytes led to symmetric cell division in meiosis I, generating two metaphase II (MII) oocytes of equal size. Subsequently, the two MII oocytes of equal size underwent the second round of symmetric cell division to generate a 4-cell embryo (zygote) when activated parthenogenetically or via sperm injection. Furthermore, inactivation of MAPK, but not MDK (also known as MEK), has been discovered in the ARF1 mutant oocytes, and further demonstrated that ARF1, MAPK pathway plays an important role in regulating asymmetric cell division in meiosis I. Similarly, ARF1^T31N-expressing superovulated MII oocytes underwent symmetric cell division in meiosis II when activation was performed. Rotation of MII spindle for 90 degree was prohibited in ARF1^T31N expressing MII oocytes. Taken together, our results suggest that ARF1 plays an essential role in regulating asymmetric cell division in female meiosis.Microinjection of mRNA of a dominant negative mutant form of ARF1 (ARF1^T31N) into fully grown germinal vesicle oocytes led to symmetric cell division in meiosis I, generating two metaphase II (MII) oocytes of equal size. Subsequently, the two MII oocytes of equal size underwent the second round of symmetric cell division to generate a 4-cell embryo (zygote) when activated parthenogenetically or via sperm injection. Furthermore, inactivation of MAP kinase, but not MEK, has been discovered in the ARF1 mutant oocytes, and further demonstrated that ARF1, MAPK pathway plays an important role in regulating asymmetric cell division in meiosis I. Similarly, ARF1^T31N-expressing superovulated MII oocytes underwent symmetric cell division in meiosis II when activation was performed. Rotation of MII spindle for 90 degree was prohibited in ARF1^T31N expressing MII oocytes. Taken together, our results suggest that ARF1 plays an essential role in regulating asymmetric cell division in female meiosis.