Molecular Human Reproduction - Advance Access

Memoirs of an insult: sperm as a possible source of transgenerational epimutations and genetic instability

de Boer, P., Ramos, L., de Vries, M., Gochhait, S. — Fri, 06 Nov 2009 05:26:58 PST

Male transgenerational epigenetic effects have been discovered in the discipline of mouse radiation genetics, using genetic and non-genetic readouts. The mechanism to explain the origin of the transmission of epigenetic and genetic instability is still unknown. In a search for a hypothesis that could satisfy the data, we propose that regulation of chromosome structure in the germline, by the occupancy of matrix/scaffold associated regions, contains molecular memory function. The male germline is strikingly dynamic as to chromatin organization. This could explain why experience of irradiation stress leaves a persistent mark in the male germline only. To be installed, such memory requires both S-phase and chromatin reorganization during spermatogenesis and in the zygote, that likely also involves reorganization of loop domains. By this reorganization, another layer of information is added, needed to accommodate early embryonic development. Observations point at the involvement of DNA repair as inducer of transgenerational epigenetic modulation.

Nuclear structure, chromatin composition and loop domain organization are aspects of human sperm variability, that in many cases of assisted reproduction is increased due to inclusion of more incompletely differentiated/maturated sperm nuclei. Adjustment of loop domains in early embryo development can be anticipated and zygotic and cleavage stage S-phase repair activity will have to deal with potential paternal DNA lesions. Therefore, by changing male nucleus structure due to reproduction from impaired spermatogenesis, the transgenerational information content could be changed as well. We discuss aspects of male reproductive performance in the context of this hypothesis.

Genetic Association of Phase I and Phase II Detoxification Genes with Recurrent Miscarriages among North Indian Women

Parveen, F., Faridi, R.M., Das, V., Tripathi, G., Agrawal, S. — Thu, 05 Nov 2009 05:27:15 PST

Allelic variants of the detoxification genes that have impaired biotransformation functions may increase susceptibility to reproductive toxicity leading to endometriosis, recurrent miscarriage (RM) or poor pregnancy outcome. In the present study, we have investigated CYP1A1, CYP2D6, GSTT1, GSTP1 and GSTM1, which are involved in the phase I and phase II detoxification systems, in relation to their role in the etiology of unexplained recurrent miscarriages (RM). In a case control study, we have investigated 200 females with RM and 300 age and ethnically matched healthy controls with successful reproductive history from North India. The frequencies of phase I wild-type genotypes of CYP1A1 and CYP2D6 in RM cases were 0.56 and 0.60, whereas in controls these were 0.68 and 0.65 respectively (both p<0.05). The GSTM1 null-genotype frequencies were 0.66 and 0.84 among RM cases and controls respectively, the GSTT1 null- genotype frequencies were 0.52 and 0.45 (p<0.005) and the GSTP1 variant allele frequencies were 0.23 and 0.20 respectively. In conclusion, we observed significant protective effects of phase I wild-type genotypes and association of the GSTT1 null genotype with RM. Through combined analyses we have highlighted the importance of the balance of phase I/phase II detoxification systems, in the etiology of recurrent miscarriage.

CAG repeat number is not inversely associated with androgen receptor activity in vitro

Sun, 01 Nov 2009 23:52:16 PST

A negative linear association between androgen receptor (AR) function and the CAG repeat numbers is generally assumed. However, in vivo data concerning the association between CAG number and androgenic effects have been conflicting.

Since former in vitro studies mostly have been based on extreme CAG lengths and reporter-systems containing viral promoters, the objective of this study was to investigate ARs with CAG lengths within normal range (16, 22 and 28) in a reporter-assay with the human prostate specific antigen promoter as target. We also wished to elucidate whether the interpretation of the results was depending on the methods used for adjustment of transfection efficiency and protein content.

With ß-galactosidase as transfection control, 22CAG had the highest activity (set to 100%) compared to 16CAG (mean 78% [range 41- 132], p=0.005) and 28CAG (68% [26-162], p=0.006), whereas renilla-luciferase resulted in 16CAG behaving similar to 22CAG (104% [56- 165], p=0.7) and 28CAG having lower activity (59% [33-101], p=0.004). In these experiments, also the empty vector displayed considerable background activity.

When adjusting for AR protein, the 22CAG genotype had the highest activity; 16CAG and 28CAG displaying 20% (10-47, p<0.0001) and 12% (5-21, p<0.0001) thereof. Similar results were obtained with adjustment for total protein

Thus, by normalising for AR-content, contrary to various control vectors, the highest AR activity was confined to the 22CAG and not 16 CAG, which may at least partly explain the discrepancy in data aiming to link physiological conditions to CAG repeat length.

Novel Pathways for Implantation and Establishment and Maintenance of Pregnancy in Mammals

Fri, 30 Oct 2009 07:26:29 PDT

Uterine receptivity to implantation varies among species, and involves changes in expression of genes that are coordinate with attachment of trophectoderm to uterine lumenal and superficial glandular epithelia, modification of phenotype of uterine stromal cells, silencing of receptors for progesterone and estrogen, suppression of genes for immune recognition, alterations in membrane permeability to enhance conceptus-maternal exchange of factors, angiogenesis and vasculogenesis, increased vascularity of the endometrium, activation of genes for transport of nutrients into the uterine lumen, and enhanced signaling for pregnancy recognition. Differential expression of genes by uterine epithelial and stromal cells in response to progesterone, glucocorticoids, prostaglandins and interferons may influence uterine receptivity to implantation in mammals. Uterine receptivity to implantation is progesterone-dependent; however, implantation is preceded by loss of expression of receptors for progesterone (PGR) so that progesterone most likely acts via PGR-positive stromal cells throughout pregnancy. Endogenous retroviruses expressed by the uterus and/or blastocyst also affect implantation and placentation in various species. Understanding the roles of the variety of hormones, growth factors and endogenous retroviral proteins in uterine receptivity for implantation is essential to enhancing reproductive health and fertility in humans and domestic animals.

The Ovary: from basic research to clinic

Mon, 26 Oct 2009 06:23:55 PDT

Oocyte peptides as paracrine tools for ovarian stimulation and oocyte maturation

Mottershead, D. G., Watson, A. J. — Wed, 21 Oct 2009 00:29:18 PDT

Recent studies report the production and isolation of a stable bioactive recombinant human bone morphogenetic protein 15 (rhBMP15) that is appropriately processed in HEK-293 cells and activates the SMAD 1/5/8 pathway in mouse granulosa cell cultures. Further, the purified rhBMP15 induces the expression of genes associated with cumulus expansion. Thanks to recent research we have a greater understanding of the importance of the dialogue that occurs between the oocyte and the granulosa cell layer with regards to regulating folliculogenesis and the acquisition of oocyte developmental competence and maturation. BMP15 is one of the critical components of these intra-follicular communication pathways. The production of recombinant human BMP15 is important for understanding the biochemistry of this specific pathway and for also fully understanding its functional contributions to mediating oocyte development. The production of a stable recombinant human BMP15 is also important for use in experiments aimed at optimizing ovarian stimulation protocols and in vitro oocyte maturation methods. This is required to improve oocyte and embryonic developmental competence and increase our ability to effectively use in vitro methods for animal production and the treatment of human infertility.

Disruption of Tsc2 in oocytes leads to overactivation of the entire pool of primordial follicles

Tue, 20 Oct 2009 08:04:39 PDT

To maintain the length of reproductive life in a woman, it is essential that most of her ovarian primordial follicles are maintained in a quiescent state to provide a continuous supply of oocytes. However, our understanding of the molecular mechanisms that control the quiescence and activation of primordial follicles is still in its infancy. In this study, we provide some genetic evidence to show that the tumor suppressor Tsc2 (tuberous sclerosis complex 2), which negatively regulates mTORC1 (mammalian target of rapamycin complex 1), functions in oocytes to maintain the dormancy of primordial follicles. In mutant mice lacking the Tsc2 gene in oocytes, the pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in oocytes. This results in depletion of follicles in early adulthood, causing premature ovarian failure (POF). Our results suggest that the Tsc1-Tsc2 complex mediated suppression of mTORC1 activity is indispensable for maintenance of the dormancy of primordial follicles, thus preserving the follicular pool, and that mTORC1 activity in oocytes promotes follicular activation. Our results also indicate that deregulation of Tsc/mTOR signaling in oocytes may cause pathological conditions of the ovary such as infertility and POF.

Demethylation of LHR in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

Zhu, J.-Q., Zhu, L., Liang, X.-W., Xing, F.-Q., Schatten, H., Sun, Q.-Y. — Wed, 14 Oct 2009 07:27:16 PDT

The cause of polycystic ovary syndrome (PCOS), a complex endocrine disorder, is unknown, but its familial aggregation implies underlying genetic influences. Hyperandrogenemia is regarded as a major endocrine character of the PCOS. In this study, we employed bisulfite sequencing and bisulfite restriction analysis to investigate the DNA methylation status of LHR, AR, FSHR and H19 in dehydroepiandrosterone (DHEA)-induced mouse PCOS model. The results showed that methylation of LHR was lost in ovary from induced PCOS mouse. However, AR, FSHR and H19 had similar methylation pattern in DHEA-treated group and control groups. These data provide evidence for close linkage between DNA demethylation of LHR and PCOS.

Paracrine Support of Ovarian Stimulation

Hillier, S. G. — Tue, 13 Oct 2009 00:40:07 PDT

Assisted Reproductive Technology has evolved on the back of blunderbuss ovarian stimulation regimes designed to maximize the number of oocytes recoverable for treatment purposes. However, oocyte ‘quality’ is finely programmed by local paracrine and autocrine signalling events during folliculogenesis and can be adversely affected by inappropriate gonadotrophic stimulation. This brief review traces the full follicular lifespan – from initiation to ovulation – to identify gonadotrophin-responsive checkpoints likely to impact oocyte quality. It is argued that these might be targeted during controlled ovarian stimulation therapy to (1) increase responsiveness to FSH through follicular priming with LH or hCG, (2) improve follicular synchrony and oocyte quality through conditioning with FSH, and (3) promote ‘gold standard’ preovulatory maturation through follicular coasting with LH or hCG. It is concluded that while no one-size-fits-all approach to ovarian stimulation, treatment regimes based on paracrine principles and tailored to personal needs will always be more likely to achieve the desired outcomes.

Nucleolar transplantation in oocytes and zygotes: challenges for further research

Fulka, H., Fulka, J. — Fri, 09 Oct 2009 00:32:27 PDT

In germinal vesicles (GVs) of immature mammalian oocytes, including humans, as well as in pronuclei in one-cell stage embryos, prominent nuclear organelles – nucleoli, can be easily detected even under a relatively low magnification. In humans, it has been clearly documented that their number, position and distribution in pronuclei can be used as an indicator of embryonic developmental potential. In the light of some recent experiments showing the feasibility of nucleolar manipulation we discuss here if these new approaches can be used to rescue those embryos with abnormal pronuclear nucleolar patterns.

Control of Ovulation in Mice by Progesterone Receptor-Regulated Gene Networks

Kim, J., Bagchi, I. C., Bagchi, M. K. — Thu, 08 Oct 2009 07:32:57 PDT

The mid-cycle surge of luteinizing hormone (LH) induces ovulation, a process during which a fertilizable oocyte is released from a mature ovarian follicle. Although ovulation is a physiologically-well-characterized event, the underlying molecular pathways remain poorly understood. Progesterone receptor (PGR), which mediates the biological effects of the steroid hormone progesterone, has emerged as a key regulator of ovulation in mice. The development of a progesterone-receptor-null (Pgr-null) mouse model confirmed a critical role of this hormone in ovulation because in these mutant mice mature preovulatory follicles fail to release the oocytes (Lydon et al., 1995). This animal model has thus presented a unique opportunity to study the molecular pathways underlying ovulation. Gene-expression profiling experiments by several groups, using the ovaries of Pgr-null mice, revealed novel gene networks, which act downstream of PGR to control ovulation. These genes encode diverse molecules such as proteases, transcription factors, cell-adhesion molecules, modulators of vascular activities, and regulators of inflammation. Functional analyses using gene-knockout mouse models have confirmed that some of these factors play critical roles during ovulation. The knowledge gained from these studies has helped us to understand better the molecular mechanisms that facilitate the release of oocytes from preovulatory follicles. Further analysis of the role of molecular regulators of ovulation will help identify useful molecular targets that would allow the development of improved contraceptives and new therapeutics for anovulatory infertility.

Toxicants and human sperm chromatin integrity

Delbes, G., Hales, B. F., Robaire, B. — Wed, 07 Oct 2009 00:36:56 PDT

The integrity of the paternal genome is essential as the spermatozoon can bring genetic damage into the oocyte at fertilization and contribute to the development of abnormal pregnancy outcome. During the past two decades, many assays have been developed to measure sperm DNA strand breaks, chromatin structure and compaction, and assess the proteins associated with the DNA, as well as epigenetic modifications. Using these assays, it has been shown that exposure to physical agents or to chemicals, including therapeutic drugs and environmental toxicants, can affect the integrity of sperm chromatin, inducing structural, genetic and/or epigenetic abnormalities. The mechanisms by which such damage is triggered are still largely unresolved and the susceptibility of each individual will depend on their genetic background, lifestyle and exposure to various insults. Depending on the nature of the chemicals, they may directly target the DNA, induce an oxidative stress, or modify epigenetic elements. The significance of measuring sperm chromatin integrity comes from the fact that this endpoint correlates well with low IVF and ICSI outcomes, and idiopathic infertility. Nevertheless, it is hard to establish a direct link between paternal sperm chromatin integrity and the health of the future generations. Thus, it seems essential to undertake studies that will resolve the impact of chemical and environmental factors on chromatin structure and epigenetic components of human spermatozoa and to elucidate what sperm nuclear endpoints are predictors of the quality of progeny outcome.

Prokineticin (PROK1) modulates interleukin (IL)-11 expression via prokineticin receptor (PROKR1) and the calcineurin/NFAT signalling pathway

Sat, 03 Oct 2009 03:09:30 PDT

Prokineticin-1 (PROK1) is a multifunctional secreted protein which signals via the G-protein coupled receptor (GPCR), PROKR1. Previous data from our laboratory using a human genome survey microarray showed that PROK1-PROKR1 signalling regulates numerous genes important for establishment of early pregnancy, including the cytokine IL-11. Here we have shown that PROK1-PROKR1 induces the expression of IL-11 in PROKR1 Ishikawa cells and first trimester decidua via the calcium-calcineurin signalling pathway in a guanine nucleotide-binding protein (G_q/11), extracellular signal-regulated kinases (ERK), Ca²⁺ and calcineurin-nuclear factor of activated T cells (NFAT) dependent manner. Conversely, treatment of human decidua with a lentiviral miRNA to abolish endogenous PROK1 expression results in a significant reduction in IL-11 expression and secretion. Importantly, we have also shown a regulatory role for the regulator of calcineurin 1 isoform 4 (RCAN1-4). Overexpression of RCAN1-4 in PROKR1 Ishikawa cells using an adenovirus leads to a reduction in PROK1 induced IL-11 indicating that RCAN1-4 is a negative regulator in the calcineurin-mediated signalling to IL-11. Finally, we have shown the potential for both autocrine and paracrine signalling in the human endometrium by co-localizing IL-11, IL-11R and PROKR1 within the stromal and glandular epithelial cells of non pregnant endometrium and first trimester decidua. Overall we have identified and characterised the signalling components of a novel PROK1-PROKR1 signalling pathway regulating IL-11.

Genomic assessment of follicular marker genes as pregnancy predictors for human IVF

Hamel, M., Dufort, I., Robert, C., Leveille, M.-C., Leader, A., Sirard, M.-A. — Thu, 24 Sep 2009 04:10:24 PDT

Embryo selection efficiency in human IVF procedure is still suboptimal as shown by low pregnancy rates with single embryo transfer (SET). Bidirectional communication between the oocyte and follicular cells (FC) is essential to achieve developmental competence of the oocyte. Differences in the gene expression profile of FCs from follicles leading to pregnancy could provide useful markers of oocyte developmental competence. FCs were recovered by individual follicle puncture. FC expression levels of potential markers were assessed by Q-PCR with an intra-patient and an inter-patient analysis approach. Using gene expression, a predictive model of ongoing pregnancy was investigated. Using intra-patient analysis, 4 candidate genes, phosphoglycerate kinase 1 (PGK1), regulator of G-protein signalling 2 (RGS2), regulator of G-protein signalling 3 (RGS3) and cell division cycle 42 (CDC42) showed a difference between FCs from follicles leading to a pregnancy or developmental failure. The best predictors for ongoing pregnancy were PGK1 and RGS2. Additionally, inter-patient analysis revealed differences in FC expression for PGK1 and CDC42 between follicles leading to a transferred embryo with positive pregnancy results and those with negative results. Both inter-patient and intra-patient approaches must be taken into consideration to delineate gene expression variations in the context of follicular competence. A predictor model using biomarkers could improve the efficiency of predicting developmental competence of oocytes. These new approaches provide useful tools in the context of embryo selection and in the improvement of pregnancy rates with SET.

Genomic changes detected by array CGH in human embryos with developmental defects

Wed, 23 Sep 2009 23:29:21 PDT

Developmental abnormalities of human embryos can be visualized in utero using embryoscopy. Our previous embryoscopic and genetic evaluations detected developmental abnormalities in the majority of both euploid (74%) and aneuploid or polyploid (90%) miscarriages. Since we found the pattern of morphological changes to be similar in euploid and noneuploid embryos, we proposed that lethal submicroscopic changes, not detected by standard chromosome testing, may be responsible for miscarriage of euploid embryos. Whole genome oligo and bacterial artificial chromosome (BAC) array comparative genome hybridization ( CGH) was used to screen for submicroscopic chromosomal changes (DNA copy number variants or CNVs) in 17 euploid embryonic miscarriages, with a range of developmental abnormalities documented by embryoscopy. The CNV breakpoints were refined using a custom array (Agilent) with a high resolution coverage of the CNVs. Six unique CNVs, previously not reported, were identified in 5 of the 17 embryos (29% of all cases or 50% of cases studied with higher resolution arrays). All 6 unique CNVs were less than 250kb in size. Based on parental array CGH analysis, a de novo origin of a CNV was determined for one embryo (at 13q32.1) and suspected for another (at 10p15.3). Three CNVs, at Xq28, 1q25.3 and 7p14.3, were inherited and a CNV at 17p13.1 was of unknown origin. The genes contained within these unique CNVs will be discussed, with specific reference to rearrangements of syntaxin and tryptophan-aspartic acid (WD) repeat genes. Our report describes for the first time, de novo and inherited unique CNVs in euploid human embryos with specific developmental defects.

Immature rat seminiferous tubules reconstructed in vitro express markers of Sertoli cell maturation after xenografting into nude mouse hosts

Gassei, K., Ehmcke, J., Wood, M. A., Walker, W. H., Schlatt, S. — Mon, 21 Sep 2009 06:01:35 PDT

Sertoli cells undergo a maturation process during postnatal testicular development that leads to the adult - type Sertoli cell, which is required for spermatogenesis. Understanding Sertoli cell maturation is therefore necessary to gain insight into the underlying causes of impaired spermatogenesis and male infertility. The present study characterized the cellular and molecular differentiation of Sertoli cells in a xenograft model of mammalian testicular development. Immature rat Sertoli cells were cultured in a three - dimensional culture system to allow the formation of cord - like structures. The in vitro Sertoli cell cultures were then grafted into nude mice. Sertoli cell proliferation, morphological differentiation and mRNA expression of Sertoli cell maturation markers were evaluated in xenografts. Sertoli cell proliferation significantly decreased between one week and four weeks (6.7 %±0.9 vs. 1.2 %±0.1, p<0.001), and was maintained at low levels thereafter. Sertoli cell cord - like structures significantly decreased between one week and four weeks (59.6 % vs. 21 %, p<0.05), whereas Sertoli cell tubules were more frequently observed after four weeks (13.3 % vs. 73.1 %, p<0.05). Furthermore, expression of androgen binding protein, transferrin, and follicle stimulating hormone receptor, markers for mature Sertoli cells, was detected after one week of grafting and increased significantly thereafter. We conclude from these results that rat Sertoli cells continue maturation after xenografting to the physiological environment of a host. This model of in vitro tubule formation will be helpful in future investigations addressing testicular maturation in the mammalian testis.

Function of Sperm Chromatin Structural Elements in Fertilization and Development

Ward, W. S. — Fri, 11 Sep 2009 00:34:13 PDT

Understanding how DNA is packaged in the mammalian sperm cell has important implications for human infertility as well as for the cell biology. Recent advances in the study of mammalian sperm chromatin structure and function have altered our perception of this highly condensed, inert chromatin. Sperm DNA is packaged very tightly to protect the DNA during the transit that occurs before fertilization. However, this condensation cannot sacrifice chromosomal elements that are essential for the embryo to access the correct sequences of the paternal genome for proper initiation of the embryonic developmental program. The primary levels of sperm chromatin structure can be divided into three main categories; the large majority of DNA is packaged by protamines, a smaller amount (2-15%) retains histone bound chromatin, and the DNA is attached to the nuclear matrix at roughly 50 kb intervals. Current data suggest that the latter two structural elements are transferred to the paternal pronucleus after fertilization where they have important functional roles. The nuclear matrix organization is essential for DNA replication, and the histone bound chromatin identifies genes that are important for embryonic development. These data support the emerging view of the sperm genome as providing, in addition to the paternal DNA sequence, a structural framework that includes molecular regulatory factors that are required for proper embryonic development.

Elevated levels of oxidised low-density lipoprotein and of catalase activity in follicular fluid of obese women

Thu, 03 Sep 2009 04:18:17 PDT

The intrafollicular levels of oxidised low-density lipoprotein (oxLDL) and of enzyme antioxidants might contribute to reproductive disorders in obese and infertile women. Relevant data are missing. Eighty-four patients were grouped according to obese versus non-obese status and whether they had polycystic ovary syndrome (PCOS). The concentrations of oxLDL and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) in serum and follicular fluid (FF) were measured. Obese women with and without PCOS had significantly greater amounts of oxLDL in FF as compared to non-obese women. The level of oxLDL in FF was 1000-times lower than in serum. Obese women with and without PCOS had significantly higher catalase activity in FF as compared to non-obese women. No differences were found for SOD activity in FF. The GPx and GR acitivities were up-regulated in obese patients without and with PCOS, yet not in respect to each serum and FF sample. We conclude that elevated levels of oxLDL in the FF of obese women are associated with higher catalase activity; both parameters are independent of PCOS. The levels of oxLDL and catalase activity appear to indicate different degrees of oxidative stress.

Aromatase over expression transgenic murine models for aromatase inhibitor studies

Li, X. — Wed, 02 Sep 2009 03:57:47 PDT

In recent years the emerging importance of estrogen signaling in males in addition to its major role in the female reproductive system became highlighted. Aromatase is the key enzyme for synthesis of estrogens from androgens and is responsible for controlling the androgen/estrogen ratio. Inhibition of aromatase gene expression can be achieved in different ways, and is important for the treatment of several estrogen-dependent diseases, such as breast cancer in females or gynecomastia/breast cancer in males, or for non-tumorigenic conditions like precocious puberty and the induction of ovulation. The inhibition of aromatase could also serve as a tool for studying the role of estrogens during development or adulthood. Using transgenic models, we are able to analyze in more detail the involvement of aromatase in the molecular mechanisms underlying the essential balance of the androgen/estrogen ratio. In this review we focus on male phenotype characterization in the aromatase overexpressing transgenic murine models and how these models can further serve as a tool for aromatase inhibitor research.

Effects of highly purified urinary FSH and human menopausal FSH on Uterine Myoelectrical Dynamics

Hascalik, S., Celik, O., Tagluk, M.E., Yildirim, A., Aydin, N.E. — Mon, 31 Aug 2009 00:58:53 PDT

The aim of the study was to investigate the effects of urinary FSH compounds on the electrical activity of myometrium using signal-processing techniques. Thirty animals were involved in the experiment. After two successive normal estrous cycles, 15 of these animals were put into three equal subgroups. Group 1 was the control; animals were given solvent. Groups 2 and 3 were treated with Urofollitropin and Menotropin, respectively. The other 15 animals were ovariectomized and subjected to the same protocol. Their uterine myoelectrical signals were recorded over a period of at least three minutes at a sampling frequency of 500 Hz, and analyzed through software assisted signal processing. The results show the power and some characteristic spectral components of myoelectrical signal were differentially reduced with the administration of highly purified urinary FSH and human menopausal FSH but significant differences were not detected between their histology. In conclusion, uterine myoelectrical signals change with administration of urinary FSH preparations. Human menopausal FSH and more precisely highly purified FSH suppress the spectral components and modify the power of the myoelectrical signals which provides uterine quiescence.

Sperm surface proteomics: from protein lists to biological function

Brewis, I. A., Gadella, B. M. — Fri, 28 Aug 2009 00:50:35 PDT

Proteomics technologies have matured significantly in recent years and proteomics driven research papers in reproductive biology and medicine are increasingly common. The key challenge is to move from lists of identified proteins to informed understanding of biological function. This review introduces the range of proteomics workflows most commonly used for protein identification before focusing on the mammalian sperm cell at fertilization as an exemplar for proteomic studies. We review the work of others on entire cells but then argue that proper subcellular fractionation and proper solubilisation strategies offers critical advantages to achieving increased biological understanding. In relation to understanding initial gamete recognition events at fertilization (capacitation, zona binding and acrosomal exocytosis) it is imperative to study the sperm surface proteome by using purified plasma membrane fractions. Whilst this task is challenging there are now strategies at our disposal to achieve comprehensive coverage of the proteins at the sperm surface. Within this context it is also important to understand the milieu of the sperm cell during transit from the testis to the oviduct as proteins (or other entities) from the genital tract epithelia and fluids may also affect the composition and organisation of proteins on the sperm surface. Finally the arguments presented for studying the cell plasma membrane proteome to understand the role of the cell surface equally apply to all cell types with important roles in reproductive function.

The primordial pool of follicles and nest breakdown in mammalian ovaries

Tingen, C., Kim, A., Woodruff, T. K. — Wed, 26 Aug 2009 08:07:23 PDT

The creation of the pool of follicles available for selection and ovulation is a multi-faceted, tightly regulated process that spans the period from embryonic development through to the first reproductive cycle of the organism. In mice, this development can occur in mere weeks, but in humans, it is sustained for years. Embryonic germ cell development involves the migration of primordial germs cells to the genital ridge, and the mitotic division of germ cell nuclei without complete cytokinesis to form a multi-nucleated syncytia, or germ cell nest. Through combined actions of germ cell apoptosis and somatic cell migration, the germ cell nuclei are packaged, with surrounding granulosa cells, into primordial follicles to form the initial follicle pool. Though often dismissed as quiescent and possibly uninteresting, this initial follicle pool is actually quite dynamic. In a very strictly controlled mechanism, a large portion of the initial primordial follicles formed is lost by atresia before cycling even begins. Remaining follicles can undergo alternate fates of continued dormancy or selection leading to follicular growth and differentiation. Together, the processes involved in the fate decisions of atresia, sustained dormancy, or activation carve out the follicle pool of puberty, the pool of available oocytes from which all future reproductive cycles of the female can choose. The formation of the initial and pubertal follicle pools can be predictably affected by exogenous treatment with hormones or molecules such as activin, demonstrating the ways the ovary controls the quality and quantity of germ cells maintained. Here, we review the biological processes involved in the formation of the initial follicle pool and the follicle pool of puberty, address the alternate models for regulating germ cell number, and outline how the ovary quality-controls the germ cells produced. This work is supported by NIH/NICHD Hormone Signals that Regulate Ovarian Differentiation, P01 HD021921; NIH/NICHD.

The forkhead transcription factor FOXL2 is expressed in somatic cells of the human ovary prior to follicle formation

Duffin, K., Bayne, R.A.L., Childs, A.J., Collins, C., Anderson, R.A. — Tue, 25 Aug 2009 08:47:39 PDT

Interactions between germ cells and surrounding somatic cells are central to ovarian development as well as later function. Disruption of these interactions arising from abnormalities in either cell type can lead to premature ovarian failure (POF). The forkhead transcription factor FOXL2 is a candidate POF factor, and mutations in the FOXL2 gene are associated with syndromic and non-syndromic ovarian failure. Foxl2-deficient mice display major defects in primordial follicle activation with consequent follicle loss, and earlier roles in gonadal development and sex determination have also been suggested. However, despite its importance no data presently exist on its expression in the developing human ovary. Expression of FOXL2 mRNA was demonstrated in the human fetal ovary between 8 weeks and 19 weeks gestation, thus from soon after sex determination to primordial follicle development. Expression in the ovary was higher after 14 weeks than at earlier gestation weeks and was very low in the fetal testis at all ages examined. Immunolocalisation revealed FOXL2 expression to be confined to somatic cells, both adjacent to germ cells and those located in the developing ovarian stroma. These cells are the site of action of oocyte-derived activin signalling, but in vitro treatment of human fetal ovaries with activin failed to reveal any regulation of FOXL2 transcription by this pathway. In summary, the expression of FOXL2 in somatic cells of the developing human ovary before and during follicle formation supports a conserved and continuing role for this factor in somatic/germ cell interactions from the earliest stages of human ovarian development.

Genetic and gene expression analyses of the polycystic ovary syndrome candidate gene fibrillin-3 and other fibrillin family members in human ovaries

Wed, 19 Aug 2009 01:35:23 PDT

Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884 which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-β binding proteins (LTBP) and thereby control the bioactivity of TGFβs. TGFβs stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook SNP analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n=4) and non-PCO ovarian stroma (n=9), follicles (n=3) and corpora lutea (n=3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCA-3, OVCA-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200-1000 fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.

Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15

Li, Q., Rajanahally, S., Edson, M. A., Matzuk, M. M. — Mon, 03 Aug 2009 02:08:19 PDT

Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6, and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), and activin/inhibin βA (Inhba) and βB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed, and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes.

On the possible origins of DNA damage in human spermatozoa

Aitken, R.J., De Iuliis, G.N. — Fri, 31 Jul 2009 00:37:58 PDT

DNA damage in the male germ line has been linked with a variety of adverse clinical outcomes including impaired fertility, an increased incidence of miscarriage and an enhanced risk of disease in the offspring. The origins of this DNA damage could, in principle, involve: (1) abortive apoptosis initiated post meiotically when the ability to drive this process to completion is in decline (2) unresolved strand breaks created during spermiogenesis to relieve the torsional stresses associated with chromatin remodelling and (3) oxidative stress. In this article we present a 2-step hypothesis for the origins of DNA damage in human spermatozoa that highlights the significance of oxidative stress acting on vulnerable, poorly protaminated cells generated as a result of defective spermiogenesis. We further propose that these defective cells are characterized by several hallmarks of ‘dysmaturity’ including the retention of excess residual cytoplasm, persistent nuclear histones, poor zona binding and disrupted chaperone content. The oxidative stress experienced by these cells may originate from infiltrating leukocytes or, possibly, the entry of spermatozoa into an apoptosis-like cascade characterized by the mitochondrial generation of reactive oxygen species. This oxidative stress may be exacerbated by a decline in local antioxidant protection, particularly during epididymal maturation. Finally, if oxidative stress is a major cause of sperm DNA damage then antioxidants should have an important therapeutic role to play in the clinical management of male infertility. Carefully controlled studies are now needed to critically examine this possibility.

Oogenesis and cell death in human prenatal ovaries: what are the criteria for oocyte selection?

Hartshorne, G. M., Lyrakou, S., Hamoda, H., Oloto, E., Ghafari, F. — Tue, 07 Jul 2009 08:15:10 PDT

Prenatal oogenesis produces hundreds of thousands of oocytes, most of which are discarded through apoptosis before birth. Despite this large scale selection, the survivors do not constitute a perfect population and the factors at the cellular level that result in apoptosis or survival of any individual oocyte are largely unknown. What then are the selection criteria that determine the size and quality of the ovarian reserve in women? This review focuses on new data at the cellular level, on human prenatal oogenesis, offering clues about the importance of timing of entry to meiotic prophase I by linking the stages and progress through meiotic prophase I with the presence or absence of apoptotic markers. The characteristics and responsiveness of cultured human fetal ovarian tissue at different gestational ages to growth factor supplementation and the impact of meiotic abnormalities upon apoptotic markers are discussed. Future work will require the use of a tissue culture model of prenatal oogenesis in order to investigate the fate of individual living oocytes at different stages of development.