Showing papers in "Molecular Human Reproduction in 2005"

IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch

Abstract: Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith–Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVFderived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn’s advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/ H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.

Cytoplasmic microvesicular form of Fas ligand in human early placenta: switching the tissue immune privilege hypothesis from cellular to vesicular level

Abstract: The local immune privilege of the fetus is created by the placenta. Fas ligand (FasL) expression in trophoblast has been implied as one of the mechanisms of fetal tolerance. However, the expression of membranal FasL by trophoblast has failed to explain this role of FasL. Two objections can be raised: (1) there have been contradictions considering which trophoblast cells, syncytiotrophoblast (ST) or cytotrophoblast, express FasL; (2) in vivo and in vitro studies have shown that the membranal form of FasL evokes inflammatory response and thus may promote fetal rejection. Using different assays and the FasL-specific antibody G247-4 we demonstrate beyond doubt that in vivo, (1) FasL is produced by and stored in the first trimester human ST only and (2) the human ST lacks surface membranal FasL. Instead, FasL, loaded in microvesicles, is stored in cytoplasmic granules. These results complement the recent in vitro studies of the microvesicular form of FasL secretion by cultured trophoblast cells, and suggest that placental FasL is synthesized by villous ST, stored in microvesicular form and secreted as exosomes. Secretion of the exosome-associated form of FasL may be one mechanism by which the placenta promotes a state of immune privilege. Additionally, FasL expression in Hofbauer cells is also demonstrated.

Capacitation-dependent concentration of lipid rafts in the apical ridge head area of porcine sperm cells.

Abstract: Lipid architecture of the plasma membrane plays an important role in the capacitation process of the sperm cell. During this process, an increase in membrane fluidity takes place, which coincides with a redistribution of cholesterol to the apical region of the head plasma membrane and subsequently an efflux of cholesterol. Cholesterol is also a major player in the formation of lipid rafts or microdomains in the membrane. Lipid rafts favour specific protein-protein interactions by concentrating certain proteins in these microdomains while excluding others. In this study, we investigated the organization of lipid rafts during in vitro capacitation of boar sperm cells. We report on the presence of the lipid raft-specific proteins caveolin-1 and flotillin-1 in sperm cells. Capacitation induced a change in membrane distribution of these proteins. Lipid analysis on detergent-resistant membranes (DRMs) of sperm cells indicated that capacitation induces a lipid raft concentration rather than a disintegration of lipid rafts, because the total amount of lipid in the DRM fraction remained unaltered. Using a proteomic approach, we identified several major DRM proteins, including proteins involved in capacitation-dependent processes and zona pellucida binding. Our data indicate that sperm raft reorganization may facilitate capacitation-specific signalling events and binding to the zona pellucida.

IDO expression on decidual and peripheral blood dendritic cells and monocytes/macrophages after treatment with CTLA-4 or interferon-γ increase in normal pregnancy but decrease in spontaneous abortion

Abstract: Recent data demonstrated that CD4 + CD25 + regulatory T (Treg) cells and an enzyme called indoleamine 2,3-dioxygenase (IDO) mediate maternal tolerance to the fetus. Interestingly, Treg cells express the CTLA-4 molecule on their surface, and B7 (CD80/86) ligation by CTLA-4 enhanced IDO activity of dendritic cells (DCs) and monocytes by the induction of interferon gamma (IFN-� ) production. In this study, we studied the IDO expression on peripheral blood monocytes and decidual monocytes or DCs after treatment with CTLA-4/Fc fusion protein or IFN-� using flow cytometry. IDO expressions on both peripheral blood DC and decidual DC and monocytes were up-regulated during normal pregnancy. On the other hand, both IDO expression on DC and monocytes after IFN-� treatment or CTLA-4 treatment were decreased in spontaneous abortion cases. The expression of CD86 on peripheral blood and decidual monocytes and DC in spontaneous abortion cases was lower compared with those in normal pregnancy subjects. Also, IFN-� production by decidual and peripheral blood mononuclear cells after CTLA-4/Fc treatment in spontaneous abortion cases was significantly lower than those in normal pregnancy subjects. These data suggest that CTLA-4 on Treg cells up-regulates IDO expression on decidual and peripheral blood DC and monocytes by the induction of IFN-� production.

Bisphenol-A induces cell cycle delay and alters centrosome and spindle microtubular organization in oocytes during meiosis.

Abstract: Bisphenol-A (BPA) is a widely used environmental estrogen-like chemical that has a weak estrogenic activity. This study aimed to test the potential inhibitory effects of BPA on meiotic cell cycle progression, centrosomes and spindle integrity in mouse cumulus-oocyte complexes (COCs). They were exposed to BPA (10-30 microM; 2.3-6.8 ppm) during meiosis-I and the formation of metaphase-II (M-II) spindle. Exposure to BPA during meiosis-I caused a dose-dependent retardation/inhibition of cell cycle progression; 74 and 61% of cells reached metaphase-I (M-I) in the presence of 10 and 30 microM BPA, respectively, (81% in controls, P<0.001). A more striking delay was noted when oocytes were exposed to BPA during the formation of M-II spindle, i.e. 61 and 41% of cells (94% in controls, P<0.001) reached M-II while the remaining cells remained at M-I. Depending on dose, both (i) loosening and elongation of meiotic spindles and (ii) compaction and dispersion of pericentriolar material (PCM) were noted in all samples, all of which resulted in a series of spindle abnormalities. Interestingly, no chromosome was detected in the first polar body after the 10 and 30 microM BPA treatments. When the cells were freed from BPA exposure at 10 and 30 microM, 70 and 61%, of the cells succeeded in reaching M-II (93% in controls, P<0.001), respectively. In conclusion, one mode of action of BPA is a moderately severe yet reversible delay in the meiotic cell cycle, possibly by a mechanism that degrades centrosomal proteins and thus perturbs the spindle microtubule organization and chromosome segregation.

The Y chromosome gr/gr subdeletion is associated with male infertility

Abstract: Men with Y chromosome (Yq) AZFc deletions lack all copies of the DAZ gene and have severe spermatogenic failure. A recently described gr/gr subdeletion of AZFc removes two of four copies of DAZ. To better understand the relative frequencies of AZFc and gr/gr deletions and their associated phenotypes, we analysed two large groups of infertile men. A total of 788 men from the Monash Male Infertility (MMI) database with a range of fertility disorders showed similar overall prevalences of AZFc (2.5%) and gr/gr deletions (3.4%). There was no association of gr/gr deletions with sperm density. In 234 control men of known or presumed fertility, only one gr/gr deletion was found. In a further 599 consecutive men presenting for assisted reproductive technologies, we detected 13 (2.2%) AZFc deletions and 28 (4.7%) gr/gr deletions. All AZFc deletions were seen with sperm densities <5 million/ml but again the gr/gr deletion occurred with similar frequency across all sperm density categories. These data show that gr/gr deletions are significantly associated with infertility in the Australian population (P = 0.0015) but not exclusively with reduced sperm density suggesting a complex interaction with other factors important for male fertility. Vertical transmission of gr/gr deletions from father to son by ICSI was demonstrated in four cases. Analysis of 130 ICSI-conceived sons revealed no de novo gr/gr deletions indicating that ICSI is not a risk factor. The data suggest that testing for gr/gr deletions should be considered in the routine genetic assessment of men with idiopathic infertility.

Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes

Abstract: Previous studies analysing the incidences of mitochondrial DNA (mtDNA) deletions and mtDNA content in unfertilized oocytes in relation to donors' age have been controversial. The objective of the study was to compare these two parameters in unfertilized oocytes and relate them to the donors' age. Fifty-two women donated 155 unfertilized metaphase II (MII) oocytes. The incidence of 4977 bp deletion was 34.6%, and the mtDNA copy number was 598 350 +/- 265 862. Women >or=35 years of age had a significantly higher incidence of 4977 bp deletion, lower mtDNA copy number, higher FSH level and poorer ovarian response when compared with younger women. The mtDNA copy number was negatively correlated with the donor's age. The higher incidence of mtDNA deletion and lower mtDNA copy number in older women suggested that these two parameters may reflect ovarian ageing.

Progesterone-dependent release of transforming growth factor-beta1 from epithelial cells enhances the endometrial decidualization by turning on the Smad signalling in stromal cells

Abstract: Endometrial decidualization results from the differentiation of stromal cells in an ovarian steroid-sensitive manner. Human endometrial tissues obtained from fertile women at various stages of the menstrual cycle were subjected to immunohistochemistry to localize the components of the transforming growth factor-beta (TGF-beta) system. TGF-beta receptor-I and -II expression was higher in stromal cells than in epithelial cells during the secretory phase while no such variation was observed during the proliferative phase. The expression of phosphorylated Smad3 (pSmad2/3), an activated form of a component of the TGF-beta signalling pathway, and translocation of pSmad2/3 from the cytoplasm to the nucleus were more pronounced in secretory endometrium. In coculture of human endometrial epithelial with stromal cells, each isolated from the proliferative endometrium, administration of progesterone stimulated decidualization as well as TGF-beta signalling activation in stromal cells. Progesterone also significantly elevated the concentration of TGF-beta1 in the coculture medium. Careful manipulation of the coculture, i.e. selective addition and omission of the cellular components, showed that this progesterone-induced increase in secretion of TGF-beta1 come mainly from epithelial cells. Moreover, administration of TGF-beta1 (10 ng/ml) directly to cultured stromal cells enhanced the expression of prolactin as well as pSamd2/3 even without progesterone. Taken together, our present data support the notion that progesterone induces stromal decidualization indirectly, i.e. by enhancing the expression and secretion of TGF-beta1 from epithelial cells. The secreted, epithelial-derived TGF-beta1 then acts on adjacent stromal cells, at least in part, to turn on Smad signalling that could lead to stromal decidualization.

Endometriotic cells are resistant to interferon-γ-induced cell growth inhibition and apoptosis: a possible mechanism involved in the pathogenesis of endometriosis

Abstract: In order to evaluate the involvement of cell proliferation and apoptosis in the pathogenesis of endometriosis, we investigated the effects of interferon-gamma (IFN-gamma) on cell growth inhibition and apoptosis of cultured ovarian endometriotic cyst stromal cells (ECSC), eutopic endometrial stromal cells with endometriosis (ESCwE) and normal endometrial stromal cells (NESC) by modified methylthiazoletetrazolium assay, 5-bromo-2'-deoxyuridine incorporation assay and internucleosomal DNA fragmentation assay. The expression of apoptosis-related molecules and IFN-gamma receptor 1 was also examined in ECSC, ESCwE and NESC using western blot analysis. IFN-gamma significantly inhibited cell proliferation and DNA synthesis of ESCwE and NESC, and induced apoptosis of these cells. In contrast, IFN-gamma did not show apparent effects on the viable cell number, DNA synthesis, or apoptosis of ECSC. An up-regulated expression of Bcl-2 and Bcl-X(L) proteins was observed in ECSC in comparison with ESCwE and NESC, whereas the levels of Bax, Bad, Fas and Fas ligand proteins in ECSC were similar to those in ESCwE and NESC. IFN-gamma receptor 1 expression was detected in ECSC, ESCwE and NESC. Enhanced expression of anti-apoptotic molecules in the ectopic endometrial cells may contribute to the development of endometriosis by conferring resistance to cytokine-induced apoptosis and increasing the chance that these cells will survive and implant outside the uterus. Further investigations on the regulation of cell proliferation in both the endometriotic and the normal endometrium may be important for the elucidation of the pathogenesis of endometriosis.

Identification of transcripts by macroarrays, RT–PCR and in situ hybridization in human ejaculate spermatozoa

Abstract: Round spermatids contain high levels of extremely varied mRNAs that are synthesized either throughout early spermatogenesis or during spermiogenesis from the haploid genome. Concomitantly, with major changes in the chromatin organization, arrest of transcription occurs at midspermiogenesis. However, previous investigations using RT ‐PCR have revealed the persistence of numerous and different transcripts in ejaculated spermatozoa. In the present study, a step-by-step analysis by means of macroarray hybridization, RT ‐PCR and in situ hybridization was performed to identify more accurately the different mRNA species found in the human ejaculated spermatozoa. The data showed an extended pattern of various transcripts encoding a diverse range of proteins involved in signal transduction and cell proliferation. For the first time, they demonstrated that mRNAs coding for the transcription factors NFkB, HOX2A, ICSBP, protein kinase JNK2, growth factor HBEGF and receptors RXRb and ErbB3 accumulate within the sperm nucleus. The origin and fate of the sperm transcripts remain subject to discussion.

Identification and localization of prostaglandin E2 receptors in upper and lower segment human myometrium during pregnancy

Abstract: Prostaglandin E2 (PGE2) plays a key role in the maintenance of human pregnancy and labour onset. PGE2 can elicit diverse actions within the uterus depending on the PGE2 receptors (EP1 ,E P 2 ,E P 3 and EP4) expressed. By signalling through different intracellular pathways the EP receptors may inhibit or promote smooth muscle contractility. Nine different EP3 receptor splice variants have been identified with divergent signalling pathways. RT ‐PCR and immunohistochemistry were utilized to identify and localize EP receptor isoforms within the upper segment (US) and lower segment (LS) myometrium. EP1 was significantly increased in the LS myometrium with term labour. EP3 (and EP3 splice variants EP3I(1b) ,E P 3II ,E P 3III and EP3IV) was downregulated in pregnancy (US and/or LS) with a further decrease at term labour in the LS. Overall, expression of EP2 was significantly higher in the LS while EP3 was significantly higher in the US. No significant EP4 changes were observed. Consistent with the RT ‐PCR results, immunohistochemistry confirmed the presence and, interestingly, showed nuclear localization of EP receptors in the myometrium with higher EP1 expression and lower expression of EP3. The differential regulation of EP receptors within the myometrium indicates that they may play a role in controlling the onset and maintenance of human labour.

Expression and functional activity of phosphodiesterase type 5 in human and rabbit vas deferens.

Abstract: The molecular mechanisms underlying the regulation of vas deferens (VD) motility and semen emission are still poorly understood. We now report evidence on VD expression of phosphodiesterase type 5 (PDE5), which regulates nitric oxide (NO)-induced relaxation and cGMP breakdown in smooth muscle cells. In human VD, the PDE5 abundance was relatively high (>3 x 10(6) molecules/microg total RNA), although 10-fold lower than in corpora cavernosa (CC). Also cGMP metabolising activity was higher in CC than in VD. However, both tissues share the same sensitivity to a broad panel of cGMP-related PDE inhibitors: sildenafil, tadalafil, dipyridamole, zaprinast, vinpocetine, EHNA and cilostamide. Based on the rank order of potency of these PDE inhibitors, we found that the cGMP metabolizing activity in human VD mostly corresponds to PDE5. PDE5 was immunolocalized in all the muscular layers of human and rabbit VD and was found to be negatively involved in regulating NO-induced relaxation. In addition, by using a rabbit model of hypogonadotropic hypogonadism, we found that PDE5 gene expression and activity are androgen-dependent in VD, as previously demonstrated in CC. In fact, the sensitivity to a NO-donor (NCX4040), its enhancement by PDE5 inhibitors and the PDE5-related cGMP breakdown were all affected by androgen manipulation. Our results provide a hypothesis explaining the beneficial effects of PDE inhibitors in patients with rapid ejaculation.

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Abstract: Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.

Estrogen-induced changes in IGF-I, Myb family and MAP kinase pathway genes in human uterine leiomyoma and normal uterine smooth muscle cell lines.

Abstract: Many studies have implicated numerous hormones, growth factors, cytokines and other signal transduction molecules in the pathogenesis of uterine leiomyoma. Estrogen and estrogen-related genes are thought to play a key role in the growth of uterine leiomyomas, but the molecular mechanisms are unclear. In an attempt to investigate various pathways that might be involved in estrogen-regulated uterine leiomyoma growth as well as to identify any novel effector genes, microarray studies comparing estrogen-treated uterine leiomyoma cells (UtLM) and normal myometrial cells to untreated cells were performed. Several genes were differentially expressed in estrogen treated UtLM cells, including insulin-like growth factor-I (IGF-I) and others potentially involved in the IGF-I signalling pathway, specifically genes for A-myb, a transcription factor which promotes cell cycle progression and for MKP-1, a dual specificity phosphatase that dephosphorylates mitogen-activated protein kinase. IGF-I and A-myb were up-regulated in estrogen-treated cells while MKP-1 was down-regulated. Two other cell cycle promoting genes, c-fos and myc, were also down-regulated in estrogen treated UtLM cells. These genes are typically up-regulated in response to estrogen in some cells, notably breast epithelial cells, yet consistently have lower expression levels in uterine leiomyoma tissue when compared to autologous myometrium. Our results demonstrate some novel genes that may play a role in the growth of uterine leiomyoma, strengthen the case for involvement of the IGF-I pathway in the response of UtLM to estrogen and corroborate evidence that uterine smooth muscle cells respond to estrogen with a different gene expression pattern than that seen in epithelial cells.

Bufalin induces apoptosis and the G0/G1 cell cycle arrest of endometriotic stromal cells: a promising agent for the treatment of endometriosis.

Abstract: Most of the current medical treatments for endometriosis aim to down-regulate the estrogen activity. However, a high recurrence rate after medical treatments has been the most significant problem. Bufalin is a major digoxin-like immunoreactive component isolated from the skin and parotid venom glands of toad and is considered an apoptosis-inducing agent. To apply bufalin to the medical treatment of endometriosis, we investigated the effects of this agent on the cell proliferation and apoptosis of cultured ovarian endometriotic cyst stromal cells (ECSC) by a modified methylthiazoletetrazolium (MTT) assay, a 5-bromo-2´-deoxyuridine (BrdU) incorporation assay and internucleosomal DNA fragmentation assays. The effect of bufalin on the cell cycle of ECSC was also determined by flow cytometry. The expression of apoptosis- and cell cycle-related molecules was also examined in ECSC using Western blot analysis. Bufalin significantly inhibited the cell proliferation and DNA synthesis of ECSC and induced apoptosis and the G0/G1 phase cell cycle arrest of these cells. The down-regulation of the cyclin A, Bcl-2, and Bcl-XL expression with the simultaneous up-regulation of the p21 and Bax expression, and caspase-9 activation was observed in ECSC after bufalin treatment. It is suggested that bufalin induces apoptosis of ECSC by simultaneously suppressing anti-apoptotic proteins and inducing pro-apoptotic proteins. Caspase-9-mediated cascade is involved in this mechanism. Therefore, bufalin could be used as a therapeutic agent for the treatment of endometriosis.

Presence of NGF and its receptors in ovaries from human fetuses and adults.

Abstract: The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as nerve growth factor (NGF) may play a role in this process. To investigate the expression of NGF and its receptors, p75 and TrkA, in early developing follicles (mostly primordial, primary and secondary follicles), ten ovarian samples from adolescents/adults aged 13-39 and 33 ovaries from human fetuses aged 19-33 gestational weeks (GW) were obtained and immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of NGF and its two receptors. Total RNA was extracted from the frozen ovarian samples, and the expression of NGF, TrkA and p75 was investigated by RT-PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunocytochemical staining revealed the expression of NGF in granulosa cells (GC) and oocytes; TrkA was mainly in oocytes and in GC in minority of the samples; and p75 was in some of the stroma cells from fetuses aged less than 22 GW. Transcripts of NGF and TrkA were identified by RT-PCR in all samples, while those for p75 were detected only in ovarian samples from fetuses aged less than 22 GW. To elucidate if NGF is indeed involved in growth initiation of human primordial follicles, it should be added to their culture medium. The immunocytochemical detection of p75 in some of the stroma cells and transcripts in ovarian samples of fetuses less than 22 GW may suggest its role in follicular assembly.

HCG increases trophoblast migration in vitro via the insulin-like growth factor-II/mannose-6 phosphate receptor.

Abstract: We have previously shown that both HCG and insulin-like growth factor-II (IGF-II) stimulate trophoblastic invasion. Furthermore, the invasion-promoting function of IGF-II resulted from IGF-II mannose 6-phosphate receptor (IGF-II/M6PR) activation. Since HCG and IGF-II did not have an additive effect on cell migration of extravillous trophoblast (EVT) cell line, HTR-8 SVneo, we hypothesized that HCG actions are mediated via alterations in the expression and/or function of IGF-II axis. HCG treatment (50-50,000 mU/ml) of the HTR-8/SVneo cells did not alter the expression of either insulin-like growth factor-I or IGF-II mRNA or peptide synthesis, but caused (i) an increase in the (125)I-IGF-II binding to EVT cells, and (ii) an increase in the externalization rate of the IGF-II binding sites without affecting their internalization. This effect was due to the increase in the number of IGF-II binding sites in the plasma membrane without any change in the IGF-II binding affinity. Although HCG did not influence the abundance of IGF-II/M6PR mRNA or protein, anti-IGF-II/M6PR antibody decreased HCG-induced migration of EVT, supporting the hypothesis that HCG might stimulate EVT migration by increasing IGF-II binding to the plasma membrane and subsequently by increasing the IGF-II effect probably mediated via the IGF-II/M6PR.

Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation.

Abstract: A novel profilin, named profilin IV, was cloned and characterized as a testicular isoform, distinct from the previously described testis-specific profilin III. Profilin IV showed only 30% amino acid identity with the other mammalian profilins; nevertheless, database searches produced significant alignments with the conserved profilin domain. Northern blot analysis and in situ transcript hybridization suggested that profilin IV, like profilin III, is transcribed in the germ cells. However, the timing of their expression during post-natal development of rat testis and in the rat spermatogenetic cycle was distinct. In the human testis, profilin IV mRNA expression correlates with the presence of germ cells suggesting that it may be a suitable molecular diagnostic parameter to supplement conventional histopathological diagnostics in the assessment of testicular biopsies. The predicted profilin IV protein was verified employing an anti-oligopeptide antibody. Western blot analysis detected an immunorelated testicular protein of approximately 14 kDa. Immunohistochemistry revealed an intracellular protein of the rat, the mouse and the human testis accumulating asymmetrically in the cytoplasm of round and elongating spermatids with its perinuclear location coinciding with the position of the developing acrosome-acroplaxome and the manchette. Profilin IV thus may regulate testicular actin cytoskeleton dynamics and play a role in acrosome generation and spermatid nuclear shaping.

DAZL expression in human oocytes, preimplantation embryos and embryonic stem cells.

Abstract: In humans, the Deleted in Azoospermia Like (DAZL) gene is believed to function in the development of primordial germ cells and in germ cell differentiation and maturation because the expression of DAZL is only found in the germ and non-germ lineage of the reproductive system and in embryonic stem (ES) cells. The present study examined the presence of DAZL transcripts in the last stages of oocyte maturation, in ES cells, and throughout the preimplantation development; the link between gametes and ES cells. The finding of DAZL transcripts in the last stages of oogenesis and during the first two cell cycles of the preimplantation development was expected, because DAZL is a germ cell marker and the transcripts present at that time are generally encoded by the maternal genome. During the third cell cycle, DAZL showed a variable expression pattern, which may point to the maternal to embryonic transition. After the third cell cycle, transcripts were again consistently detected, suggesting embryonic DAZL transcription. In blastocysts, DAZL transcripts were only detected in those of good quality and this as well in the inner cell mass (ICM) as in the trophectoderm (TE). The presence of DAZL transcripts in the ICM and in ES cells was not surprising since both can lead to the formation of germ cells, but TE cells cannot. The quality-related expression of DAZL in blastocysts, and especially its trophectodermal expression, might imply other functions for DAZL beyond germ cell development.

Post-ovulatory aging of mouse oocytes leads to decreased MAD2 transcripts and increased frequencies of premature centromere separation and anaphase

Abstract: Numerous cytological and biochemical alterations occur as mammalian oocytes age post-ovulation. Some of these changes can predispose cells to aneuploidy. The objective of this study was to test the hypothesis that the level of MAD2 spindle assembly checkpoint (SAC) transcripts decrease as mouse oocytes age post-ovulation and that this decrease was associated with chromosome missegregation. Female Institute of Cancer Research (ICR) mice were superovulated and oocytes collected at 14 h, 19 h and 24 h post-HCG for cytogenetic and quantitative real-time rapid cycle fluorescent RT-PCR analyses. Premature centromere separation (PCS) is now generally recognized as a predisposition to aneuploidy. The data showed that the frequencies of PCS-incomplete (PCS-I) did not significantly (P > 0.05) increase with time post-ovulation; whereas the proportions of oocytes displaying PCS-complete (PCS-C) and premature anaphase (PA) were significantly (P < 0.01) greater at 19 h and 24 h post-HCG, respectively. The higher frequencies of PCS-C and PA found at 19 h and 24 h coincided with decreased levels of MAD2 transcripts at these same times. Although the decline in MAD 2 transcripts with oocyte aging represents only one of many potential mechanisms responsible for aneuploidy, a compromised SAC appears to have a role in the unfavourable reproductive outcome associated with post-ovulatory aged oocytes.

Progesterone receptor expression in human decidua and fetal membranes before and after contractions: possible mechanism for functional progesterone withdrawal.

Abstract: In humans, progesterone levels are sustained before the onset of labour. Therefore, the mechanism for parturition that has been proposed for humans is ‘functional’ progesterone withdrawal. Immunohistochemical staining for the progesterone receptor (PR) was positive in the decidua with a decline after contractions began. Western blot analysis revealed a number of PR isoforms expressed in the decidua, with the PR-B form being dominant. After contractions began, all PR isoforms decreased sharply. PR-B and PR-A decreased by 85.8% 6 6.7 and 78.2% 6 7.1, respectively ( P< 0.001). Incubation of decidua with Prostaglandin F2a 1.0mg/ml decreased the expression of all forms of PR isoforms. PR-B was reduced by 64% 6 6.09 ( P< 0.01); PR-A was reduced by 77% 6 5.9 ( P< 0.05), while PR-C was reduced by 80% 6 7.24 ( P< 0.05). Progesterone (80mg/ml) increased the PR-B, PR-C the 45 and 36 kDa isoforms to 150% 6 7.89, 210% 6 12.4, 270% 6 9.7 and 216% 6 13.5, respectively ( P< 0.05). In immunohistochemical studies, the PR was not identified in the amnion or in the chorion, regardless of the presence or absence of contractions. Western blot analysis demonstrated that PR-C (60 kDa) and the 36 kDa isoforms were dominant in the amnion. After contractions began, PR-A decreased significantly by 61.9% 6 7.1 ( P< 0.001).

A heterozygous mutation in the desert hedgehog gene in patients with mixed gonadal dysgenesis.

Abstract: Aetiology of mixed gonadal dysgenesis (MGD) has not been completely elucidated. Molecular analyses have failed to demonstrate the presence of mutations in sex-determining region on Y chromosome (SRY); it has been suggested that these individuals may bear mutations in other genes involved in the testis-determining pathway. Desert hedgehog's (DHH) importance regarding male sex differentiation has been demonstrated in various studies we describe here, for the first time, two cases of MGD in which a monoallelic single base deletion in DHH is associated with the disorder. Genomic DNA was isolated from paraffin-embedded gonad tissue from 10 unrelated patients with MGD and three controls; in addition to, DNA from peripheral blood leukocytes in 100 controls. Coding sequence abnormalities in DHH were assessed by exon-specific PCR, single-stranded conformation polymorphism (SSCP) and direct sequencing. In two patients, a heterozygous 1086delG in exon 3 was found. Comparing previously described mutations in DHH to the one observed in this study, we can affirm that the phenotypic spectrum of patients with gonadal dysgenesis due to mutations in DHH is variable. This study continues to demonstrate the importance that DHH has in mammalian male sexual differentiation, providing extended evidence that DHH constitutes a key gene in gonadal differentiation.

Macrophage migration inhibitory factor in the human epididymis and semen.

Abstract: During epididymal transit, mammalian spermatozoa acquire new proteins involved in the acquisition of motility and of male gamete fertilising ability. We have previously shown that membranous vesicles called epididymosomes are involved in the transfer of epididymal-originating proteins to spermatozoa. The cytokine macrophage migration inhibitory factor (MIF) is one of these proteins but the role played by MIF in relation to epididymal sperm maturation still remains unclear. As this protein has already been shown to bear different functions depending on its location, we investigated its distribution along the epididymis and in different compartments of human semen. Northern and Western blot analysis as well as immunohistochemical studies show that MIF is expressed all along the epididymis with a higher level of transcript in the proximal segment. MIF is associated with two types of membranous vesicles, i.e. epididymosomes and prostasomes, the latter being prostate-originating membranous vesicles present in the semen. In semen, MIF is associated with spermatozoa, prostasomes as well as the soluble fraction. The amount of MIF in the seminal fluid varies from one individual to another but does not correlate with the amount of MIF associated with ejaculated spermatozoa. There is a negative correlation between the amount of sperm-associated MIF and the percentage of motility in different semen samples. Sperm separation using discontinuous Percoll gradient centrifugation shows a higher amount of MIF associated with poorly motile spermatozoa compared to highly motile spermatozoa present in the lower Percoll fraction. These results are discussed with regards to the possible involvement of MIF in sperm motility acquisition during the epididymal transit.

Bicarbonate and bovine serum albumin reversibly 'switch' capacitation-induced events in human spermatozoa

Abstract: We have investigated the reversibility of biochemical and physiological changes that occur upon suspension of ejaculated human spermatozoa during in vitro capacitation. Cells were swum up in a simple HEPES-based saline [lacking bicarbonate and bovine serum albumin (BSA)], then resuspended either in supplemented Earle's balanced salt solution (sEBSS) (25 mM bicarbonate) with 0.3% BSA (for in vitro capacitation) or in medium-lacking bicarbonate and/or BSA. Progesterone-induced acrosome reaction (AR) developed during in vitro capacitation (6 h). A progesterone-induced [Ca2+]i signal was detectable in cells maintained in the simple HEPES-based saline, but upon transfer to sEBSS, the response increased three- to four-fold, saturating within <30 min. Serine/threonine phosphorylation saturated within minutes of resuspension, but tyrosine phosphorylation developed over 3 h. Return of cells to non-capacitating conditions caused reversal of all capacitation-dependent changes. The [Ca2+]i signal reverted to its 'uncapacitated' size within <30 min. Protein phosphorylation reversed gradually and could be reinduced (kinetics resembling the first response) upon resuspension in sEBSS. The ability of cells to undergo progesterone-induced AR fell to levels similar to those in uncapacitated cells within 1 h of resuspension in medium not supporting capacitation. Loss of protein phosphorylation occurred only in the absence of both bicarbonate and BSA, but effects on [Ca2+]i signalling and AR could be seen after removal of only one of these factors. We conclude that key events in the capacitation of human spermatozoa are both reversible and repeatable.

Up-regulation of the endocannabinoid system in the uterus of leptin knockout (ob/ob) mice and implications for fertility

Abstract: The levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) are under the negative control of leptin in the rodent hypothalamus. As leptin and endocannabinoids play opposite roles in the control of reproduction, we have investigated whether the impaired fertility typical of leptin-defective ob/ob mice is due, in part, to enhanced uterine endocannabinoid levels. We found that levels of both anandamide and 2-AG in the uterus of ob/ob mice are significantly elevated with respect to wild-type littermates, due to reduced hydrolase activity in the case of anandamide, and to reduced monoacylglycerol lipase and enhanced diacylglycerol lipase activity in the case of 2-AG. Furthermore, the process mediating endocannabinoid cellular uptake was also impaired in ob/ob mice, whereas the levels of cannabinoid and anandamide receptors were not modified. Although ineffective in wild-type mice, treatment of ob/ob mice with leptin re-established endocannabinoid levels and enzyme activities back to the values observed in wild-type littermates. Finally, treatment of ob/ob females with the CB1 receptor antagonist SR141716A did not improve their fertility, and inhibition of endocannabinoid inactivation with the endocannabinoid uptake inhibitor OMDM-1 in wild-type females did not result in impaired fertility.

Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

Abstract: LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.

Expression of the epidermal growth factor system in human endometrium during the menstrual cycle

Abstract: The epidermal growth factor (EGF) system is ubiquitous in humans and plays fundamental roles in embryogenesis, development, proliferation and differentiation. As the endometrium of fertile women is characterized by proliferation and differentiation, we hypothesize a role for the EGF system. Fourteen premenopausal women had endometrial samples removed on day 6 +/- 1 and day 6 +/- 1 and 12 +/- 1 after ovulation during one menstrual cycle. RNA was extracted and analysed by real-time PCR, and immunohistochemistry was performed to localize the components of the EGF system. Human EGF Receptor 1 (HER1) showed highest expression during the proliferative phase, HER2 and HER4 during the early and HER3 during the late secretory phase. Amphiregulin (AR) and transforming growth factor alpha (TGFalpha) expression is highest in proliferative phase. Heparin binding (HB)-EGF and betacellulin (BCL) show no variation. Epiregulin (EP) is detectable in some samples. EGF is undetectable. HER1, HER2, HER3 and HER4 were localized to the epithelium and glands HER3 and HER4 solely in the secretory phase. Amphiregulin was seen in leucocytes and stromal cells, TGFalpha and betacellulin in the epithelial lining, epiregulin in stromal cells whereas HB-EGF and EGF are undetectable. In conclusions, we observed cyclical expression of the four EGF receptors and two ligands and localized all four receptors and four ligands in endometrial biopsies. This suggests a role for the EGF system in growth of the endometrium.

α-SNAP and NSF are required in a priming step during the human sperm acrosome reaction

Abstract: The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.

Fibromodulin is expressed in leiomyoma and myometrium and regulated by gonadotropin-releasing hormone analogue therapy and TGF-β through Smad and MAPK-mediated signalling

Abstract: Microarray gene expression profiling revealed fibromodulin (FMOD) is among differentially expressed genes in leiomyoma (L) and myometrium. Using realtime PCR, western blotting and immunohistochemistry, we validated the expression of FMOD in paired leiomyoma and myometrium (N = 20) during the menstrual cycle, from women who received gonadotropin-releasing hormone analogue (GnRHa) therapy (N = 7) and in leiomyoma and myometrial (M) smooth muscle cells (SMC) due to transforming growth factor (TGF)-beta and GnRHa treatment. The results indicated that FMOD is expressed at significantly higher levels in leiomyoma as compared to myometrium from proliferative phase (two- to three-folds; P < 0.05), but not the secretory phase of the menstrual cycle, whereas GnRHa therapy reduced FMOD expression to levels detected in myometrium from proliferative phase (P = 0.05). By using western blotting and immunohistochemistry immunoreactive FMOD was detected in leiomyoma and myometrial tissue-extract and in LSMC and MSMC, connective tissue fibroblasts and arterial walls. In a time- and cell-dependent manner, TGF-beta1 (2.5 ng/ml) increased the expression of FMOD in MSMC, whereas GnRHa (0.1 microM) inhibited that in MSMC and LSMC (P < 0.05). The effect of TGF-beta and GnRHa on FMOD expression was reversed following pretreatment of LSMC and MSMC with Smad3 SiRNA and U0126 (MEK1/2 inhibitor), respectively. In summary, menstrual cycle-dependent expression of FMOD and suppression following GnRHa therapy in leiomyoma and myometrium, as well as differential regulation by TGF-beta and GnRHa in vitro suggests that FMOD, a key regulator of tissue organization, plays a critical role in leiomyoma fibrotic characteristics.

Glutathione S-transferases M1/T1 gene polymorphisms and endometriosis: a meta-analysis of genetic association studies

Abstract: In view of the controversies surrounding the glutathione S-transferases (GST) M1/T1-endometriosis association, a meta-analysis of the GSTM1/GSTT1 genetic association studies of endometriosis was performed. In this meta-analysis involving 14 GSTM1 studies with 1539 cases and 1805 controls and nine GSTT1 studies with 746 cases and 834 controls, respectively, substantial heterogeneities among studies were found. In addition, asymmetry in funnel plot was evident, which is likely to stem from publication bias, given no apparent indication of true heterogeneity. The bias appears to be prominent for GSTM1 studies, but is less so for GSTT1 studies. After correction for this bias, there is no evidence that women with GSTM1 null genotype have increased risk of developing endometriosis as compared with women with other genotypes. For GSTT1, the risk associated with the null genotype is 29% higher than other genotypes. However, even this estimate should be viewed with a large grain of salt, because the estimate could easily lose its statistical significance if there is a realistic 69-80% publication probability.