Reproductive Biology and Endocrinology - Latest Articles

Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: in vivo and ex vivo studies

Janaina Couto — 2010-02-09T00:00:00Z

Background: The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats. Methods: Twelve adult male Wistar rats were treated with rosiglitazone (5mg/kg) administered by gavage for 15 days. Twelve control animals were treated with the vehicle. The ability of rosiglitazone to directly affect the production of testosterone by Leydig cells ex vivo was evaluated using isolated Leydig cells from rosiglitazone-treated rats. Testosterone production was induced either by activators of the cAMP/PKA pathway (hCG and dbcAMP) or substrates of steroidogenesis [22(R)-hydroxy-cholesterol (22(R)-OH-C), which is a substrate for the P450scc enzyme, and pregnenolone, which is the product of the P450scc-catalyzed step]. Testosterone in plasma and in incubation medium was measured by radioimmunoassay. The StAR and P450scc expression was detected by immunocytochemistry. Results: The levels of total circulating testosterone were not altered by rosiglitazone treatment. A decrease in basal or induced testosterone production occurred in the Leydig cells of rosiglitazone-treated rats. The ultrastructural and immunocytochemical analysis of Leydig cells from rosiglitazone-treated rats revealed cells with characteristics of increased activity as well as increased StAR and P450scc expression, which are key proteins in androgen biosynthesis. However, a number of rosiglitazone-treated cells exhibited significant mitochondrial damage. Conclusion: The results revealed that the Leydig cells from rosiglitazone-treated rats showed significant reduction in testosterone production under basal, hCG/dbcAMP- or 22 (R)-OH-C/pregnenolone-induced conditions, although increased labeling of StAR and P450scc was detected in these cells by immunocytochemistry. The ultrastructural study suggested that the lower levels of testosterone produced by these cells could be due to mitochondrial damage induced by rosiglitazone.

The association between leukocytes and sperm quality is concentration dependent

Jakob Lackner — 2010-02-05T00:00:00Z

Background: To evaluate the association between leukocytes (polymorphonuclear granulocytes -PMNL) and semen parameters at different leukocyte concentrations. Methods: This was a retrospective clinical study at a university hospital andrology clinic. Semen samples from infertile men were analyzed for sperm morphology and motility according to seminal leukocytes (PMNL) concentration (category A: >0 to <0.25 x 10(6)/mL; category B: >0.25 to <0.5 x 10(6)/mL; category C: >0.5 to <0.75 x 10(6)/mL; category D: >0.75 to <1.0 x 10(6)/mL, category E: >1 x 10(6)/mL). Results: The percentage of sperm with normal morphology increased significantly from category A (14%) to category D (19%) but decreased in category E to levels (14%) similar to those in category A. Motility grades a and a+b (combined) also increased from category A (12%, 20%) to category D (18.0%, 28.5%) and decreased in category E (11%, 20.5%) to levels similar to those in category A. Sperm deformities and motility grades c and d increased progressively in all categories.SummaryLeukocytes had a positive association with normal morphology and progressive motility in semen samples at a concentration of 0-1 x 10(6)/mL. The findings suggest that the association between leukocytes (PMNL) and semen quality might be concentration dependent.

Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

Ken-Go Hayashi — 2010-02-05T00:00:00Z

Background: Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classifythem by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes. Methods: Global gene expression profiles of F1 (10.7 +/- 0.7 mm) and F2 (7.8 +/- 0.2 mm) were analyzed by hierarchical cluster analysis and expression profiles of 16 representative genes were confirmed by QPCR analysis. In addition, localization of six identified transcripts was investigated in healthy and atretic follicles using in situ hybridization. The healthy or atretic condition of examined follicles was classified by progesterone and estradiol concentrations in follicular fluid. Results: Hierarchical cluster analysis of microarray data classified the follicles into two clusters. Cluster A was composed of only F2 and was characterized by high expression of 31 genes including IGFBP5, whereas cluster B contained only F1 and predominantly expressed 45 genes including CYP19 and FSHR. QPCR analysis confirmed AMH, CYP19, FSHR, GPX3, PlGF, PLA2G1B, SCD and TRB2 were greater in F1 than F2, while CCL2, GADD45A, IGFBP5, PLAUR, SELP, SPP1, TIMP1 and TSP2 were greater in F2 than in F1. In situ hybridization showed that AMH and CYP19 were detected in granulosa cells (GC) of healthy as well as atretic follicles. PlGF was localized in GC and in the theca layer (TL) of healthy follicles. IGFBP5 was detected in both GC and TL of atretic follicles. GADD45A and TSP2 were localized in both GC and TL of atretic follicles, whereas healthy follicles expressed them only in GC. Conclusion: We demonstrated that global gene expression profiling of F1 and F2 clearly reflected a difference in their follicular status. Expression of stage-specific genes in follicles may be closely associated with their growth or atresia. Several genes identified in this study will provide intriguing candidates for the determination of follicular growth.

Endoplasmic reticulum protein 29 (ERp29), a protein related to sperm maturation is involved in sperm-oocyte fusion in mouse

Xiaoqian Ying — 2010-02-04T00:00:00Z

Background: Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process. Methods: Laser scanning confocal microscopy (LSCM) and Western blot analysis were employed to probe for ERp29 protein in BALB/c mouse epididymal and acrosome-reacted spermatozoa. We prepared rabbit polyclonal antibodies against mouse recombinant ERp29 (rERp29) to characterize: 1) fertilization rate (FR); 2) fertilization index (FI); 3) sperm motility and 4) acrosome reaction (AR). Results: Confocal microscopy indicated that ERp29 was partially localized at the sperm head of the epididymal caput as well as over the whole head and part of the principal piece of the tail region from the epididymal cauda. However, when the acrosome reacted, ERp29 remained in the equatorial and post-acrosomal regions of the sperm head, which is the initial site of sperm-oocyte membrane fusion. Such localization changes were confirmed based on the results of Western blot analysis. Furthermore, the antibodies against mouse rERp29 inhibited the spermatozoa from penetrating into the zona pellucida (ZP)-free oocytes. The functional blocking antibodies reduced both mouse sperm-oocyte FR and FI at concentrations of 100 and 200 micro g/ml compared with pre-immunized rabbit IgG or with anti-mouse recombinant bactericidal/permeability-increasing protein (BPI, a sperm surface protein unrelated to sperm-oocyte fusion) antibodies (100 micro g/ml), but they had no effect on sperm motility and AR. Conclusion: This study demonstrates that ERp29 on mouse spermatozoa membrane changes during epididymal transit and AR. Accordingly, in mice this protein may be one of the important factors involved in sperm fertilization by facilitating sperm-oocyte membrane fusion.

Selection against spermatozoa with fragmented DNA after postovulatory mating depends on the type of damage

Juan Hourcade — 2010-01-31T00:00:00Z

Background: Before ovulation, sperm-oviduct interaction mechanisms may act as checkpoint for the selection of fertilizing spermatozoa in mammals. Postovulatory mating does not allow the sperm to attach to the oviduct, and spermatozoa may only undergo some selection processes during the transport through the female reproductive tract and/or during the zona pellucida (ZP) binding/penetration. Methods: We have induced DNA damage in spermatozoa by two treatments, (a) a scrotal heat treatment (42 degrees C, 30 min) and (b) irradiation with 137Cs gamma-rays (4 Gy, 1.25 Gy/min). The effects of the treatments were analyzed 21-25 days post heat stress or gamma-radiation. Postovulatory females mated either with treated or control males were sacrificed at Day 14 of pregnancy, and numbers of fetuses and resorptions were recorded. Results: Both treatments decreased significantly implantation rates however, the proportion of fetuses/resorptions was only reduced in those females mated to males exposed to radiation, indicating a selection favoring fertilization of sperm with unfragmented DNA on the heat treatment group. To determine if DNA integrity is one of the keys of spermatozoa selection after postovulatory mating, we analyzed sperm DNA fragmentation by COMET assay in: a) sperm recovered from mouse epididymides; b) sperm recovered from three different regions of female uterine horns after mating; and c) sperm attached to the ZP after in vitro fertilization (IVF). Similar results were found for control and both treatments, COMET values decreased significantly during the transit from the uterine section close to the uterotubal junction to the oviduct, and in the spermatozoa attached to ZP. However, fertilization by IVF and intracytoplasmatic sperm injection (ICSI) showed that during sperm ZP-penetration, a stringent selection against fragmented-DNA sperm is carried out when the damage was induced by heat stress, but not when DNA fragmentation was induced by radiation. Conclusion: Our results indicate that in postovulatory mating there is a preliminary general selection mechanism against spermatozoa with low motility and fragmented-DNA during the transport through the female reproductive tract and in the ZP binding, but the ability of the ZP to prevent fertilization by fragmented-DNA spermatozoa is achieved during sperm-ZP penetration, and depends on the source of damage.

Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes

Sonali Vora — 2010-01-25T00:00:00Z

Background: The objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB) subunit, in full-thickness fetal membranes (FM) and myometrium in the absence or presence of term or preterm labor. Methods: Paired full-thickness FM and myometrial samples were collected from women in the following cohorts: preterm no labor (PNL, N=22), spontaneous preterm labor (PTL, N=21), term no labor (TNL, N=23), and spontaneous term labor (STL, N=21). NF-kappaB p65 localization was assessed by immunohistochemistry, and DNA binding activity was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based method. Results: Nuclear p65 labeling was rare in amnion and chorion, irrespective of clinical context. In decidua, nuclear p65 labeling was greater in the STL group relative to the TNL cohort, but there were no differences among the TNL, PTL, and PNL cohorts. In myometrium, diffuse p65 nuclear labeling was significantly associated with both term and preterm labor. There were no significant differences in ELISA-based p65 binding activity in amnion, choriodecidual, and myometrial specimens in the absence or presence of term labor. However, parallel experiments using cultured term fetal membranes demonstrated high levels of p65-like binding even the absence of cytokine stimulation, suggesting that this assay may be of limited value when applied to tissue specimens. Conclusions: These results suggest that the decidua is an important site of NF-kappaB regulation in fetal membranes, and that mechanisms other than cytoplasmic sequestration may limit NF-kappaB activation prior to term.

Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida

Beena Bhandari — 2010-01-23T00:00:00Z

Background: Human egg is enveloped by a glycoproteinaceous matrix, zona pellucida (ZP), responsible for binding of the human spermatozoa to the egg and induction of acrosomal exocytosis in the spermatozoon bound to ZP. In the present manuscript, attempts have been made to delineate the downstream signalling components employed by human ZP to induce acrosome reaction. Methods: Heat-solubilized human ZP (SIZP) was used to study the induction of acrosome reaction in capacitated human spermatozoa using tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA) in absence or presence of various pharmacological inhibitors. In addition, intracellular calcium ([Ca2+]i) levels in sperm using Fluo-3 acetoxymethyl ester as fluorescent probe were also estimated in response to SIZP. Results: SIZP induces acrosomal exocytosis in capacitated human sperm in a dose dependent manner accompanied by an increase in [Ca2+]i. Human SIZP mediated induction of acrosome reaction depends on extracellular Ca2+ and involves activation of Gi protein-coupled receptor, tyrosine kinase, protein kinases A & C and phosphoinositide 3 (PI3)- kinase. In addition, T-type voltage operated calcium channels and GABA-A receptor associated chloride (Cl-) channels play an important role in SIZP mediated induction of acrosome reaction. Conclusions: Results described in the present study provide a comprehensive account of the various downstream signalling components associated with human ZP mediated acrosome reaction.

Identification of calcium-binding proteins associated with the human sperm plasma membrane

Soren Naaby-Hansen — 2010-01-15T00:00:00Z

Background: The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane. Methods: Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface. Results: Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalysed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm. Conclusion: The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.

GnRH I and II up-regulate MMP-26 expression through the JNK pathway in human cytotrophoblasts

Jing Liu — 2010-01-15T00:00:00Z

Background: Matrix metalloproteinase-26 (MMP-26), one of the main mediators of extracellular matrix (ECM) degradation, has been shown to exist in trophoblasts of human placenta and to play a role in trophoblast cell invasion. However, little is known about the regulation of MMP-26 expression in human trophoblasts. Recently, gonadotropin-releasing hormone I (GnRH I) and GnRH II have been shown to regulate the expression of MMP-2, MMP-9/tissue inhibitor of metalloproteinases 1 (TIMP-1), and urokinase plasminogen activator (uPA)/plasminogen activator inhibitor (PAI) in human trophoblasts, suggesting that these two hormones may work as paracrine and/or autocrine regulators in modulating the activities of various protease systems at the feto-maternal interface. In this study, we determined the regulatory effects of GnRH I and GnRH II on the expression of MMP-26 in human immortalized cytotrophoblast-like cell line, B6Tert-1. Methods: Real-time PCR was used to quantify mRNA levels of MMP-26 in human trophoblast-like cell line, B6Tert-1 and primary cultured cytotrophoblasts. Western blotting was used to characterize the expression of MMP-26 and the phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) in B6Tert-1 cells after treatment with GnRH I and GnRH II. Results: We found that GnRH I increased MMP-26 expression in B6Tert-1 cells after 12 h of treatment at both the mRNA and protein level, while GnRH II increased MMP-26 expression beginning at 3 h of treatment. Treatment of GnRH I at 1 nM resulted in maximal increase of MMP-26 mRNA and protein levels, whereas GnRH II treatment at a concentration of 100 nM was required to induce maximal increase in MMP-26 expression. In addition, we demonstrated that the activation of JNK, but not ERK1/2, was required for GnRH I and II-stimulated MMP-26 production in B6Tert-1 cells and primary cytotrophoblasts. Conclusions: These novel findings indicated that GnRH I and II could up-regulate MMP-26 expression through the JNK signaling pathway in human trophoblast-like/trophoblast cells.

Neurokinin B and pre-eclampsia: a decade of discovery

Nigel Page — 2010-01-14T00:00:00Z

At the start of the last decade, we provided evidence that levels of the peptide neurokinin B were highly elevated in pre-eclampsia. We hypothesized that elevated levels of neurokinin B may be an indicator of pre-eclampsia and that treatment with certain neurokinin receptor antagonists may be useful in alleviating the symptoms. At the time of the original hypothesis many questions remained outstanding. These included - Does neurokinin B have any diagnostic value in the detection and diagnosis of pre-eclampsia? - What is the cause of the elevated levels of neurokinin B during pre-eclampsia? - What is the physiological significance of neurokinin B in the placenta? This review discusses the answers to these questions taking into account the subsequent developments of the past ten years and analyzing the plethora of discoveries that have arisen from those initial observations.