Marion Spitschak

Research resource: preovulatory LH surge effects on follicular theca and granulosa transcriptomes.

The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the preovulatory LH surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal cell (TC) and granulosa cell (GC) type-specific biologic functions and signaling pathways, large dominant bovine follicles were collected before and 21 hours after an exogenous GnRH-induced LH surge. Antral GCs (aGCs; aspirated by follicular puncture) and membrane-associated GCs (mGCs; scraped from the follicular wall) were compared with TC expression profiles determined by mRNA microarrays. Of the approximately 11 000 total genes expressed in the periovulatory follicle, only 2% of thecal vs 25% of the granulosa genes changed in response to the LH surge. The majority of the 203 LH-regulated thecal genes were also LH regulated in GCs, leaving a total of 57 genes as LH-regulated TC-specific genes. Of the 57 thecal-specific LH-regulated genes, 74% were down-regulated including CYP17A1 and NR5A1, whereas most other genes are being identified for the first time within theca. Many of the newly identified up-regulated thecal genes (eg, PTX3, RND3, PPP4R4) were also up-regulated in granulosa. Minimal expression differences were observed between aGCs and mGCs; however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) dominated these differences. We also identified large numbers of unknown LH-regulated GC genes and discuss their putative roles in ovarian function. This Research Resource provides an easy-to-access global evaluation of LH regulation in TCs and GCs that implicates numerous molecular pathways heretofore unknown within the follicle.

Down-regulation of genes encoding steroidogenic enzymes and hormone receptors in late preovulatory follicles of the cow coincides with an accumulation of intrafollicular steroids.

The transformation of the dominant follicle into a functional corpus luteum is accompanied by a profound molecular and morphological reorganization of somatic cell layers. Several studies have focused on gene expression during early processes of follicular differentiation as it relates to recruitment and selection of dominant follicles. However, little information exists on changes of gene expression profiles in late preovulatory follicles. This lack of information is addressed here to elucidate molecular mechanisms behind the LH-induced transition from the large dominant estrogen-active to the preovulatory follicle, an intermediate stage toward full luteinization. Transcripts encoding key molecules for the biosynthesis of steroid hormones and prostaglandins, as well as receptors for gonadotropic and growth hormones (Star, Cyp11a1, Hsd3b, Cyp17, Cyp19, Ptgs2, Fshr, Lhr, and Ghr), were quantified by real-time polymerase chain reaction (PCR) in the granulosa and theca of large dominant and late preovulatory follicles. The steroid hormones progesterone (P4) and estradiol-17beta (E2) were monitored to distinguish estrogen-active and estrogen-inactive follicles. We found that (1) independent of the follicular stage, the gene expression profile was very different in granulosa and theca; (2) the abundance of several key transcripts was lower in estrogen-inactive, compared with estrogen-active, dominant follicles; (3) in the granulosa of late preovulatory follicles, transcripts encoding steroidogenic enzymes and hormone receptors were largely down-regulated, whereas (4) progesterone and E2 were found at high concentrations in the follicular fluid. Collectively, our data show that late preovulatory follicles have a transient and unique gene expression profile and are clearly different from both the preceding and subsequent (follicular and luteal, respectively) stages.

DNA Methylation Is Not Involved in Preovulatory Down-Regulation of CYP11A1, HSD3B1, and CYP19A1 in Bovine Follicles but May Have a Role in Permanent Silencing of CYP19A1 in Large Granulosa Lutein Cells

Abstract The luteinizing hormone-induced morphological and physiological reorganization of the bovine follicle is preceded by a profound and well-orchestrated modulation of gene expression. In the present study, the cell type-specific methylation profiles of CYP11A1, HSD3B1, and CYP19A1, genes that encode key enzymes of steroid hormone biosynthesis, were analyzed to elucidate whether epigenetic parameters such as DNA methylation might be involved in gene regulation during luteinization. Transcript abundance and DNA methylation levels were determined in granulosa and theca of large dominant and late preovulatory follicles and in large granulosa lutein cells isolated from corpora lutea cyclica and graviditatis. Levels of the steroid hormones progesterone and estradiol-17beta were monitored to assess the physiological status of individual follicles. From our results, we conclude that (1) individual, even closely neighboring, CpG dinucleotides can show very different methylation levels; (2) proximal (<300 base pair [bp] from the respective transcription start sites) but not distal CpGs show cell type-specific methylation levels; (3) higher methylation levels suggestively preclude high levels of gene expression; (4) DNA methylation is not involved in the transient (HSD3B1 and CYP11A1) respectively permanent (CYP19A1) down-regulation of gene expression in late preovulatory follicles; and (5) DNA methylation may have a role in the permanent shutdown of promoter 2-directed CYP19A1 expression in large (granulosa derived) lutein cells.

Two high-fertility mouse lines show differences in component fertility traits after long-term selection

UNLABELLED Two selected high-fertility mouse lines, namely FL1 and FL2, and a non-selected control (Fzt:DU), all derived from the same genetic pool, were analysed as an animal model for polytocous species to elucidate the effects of long-term selection and to identify relevant component traits that may be responsible for fertility performance. The index trait used for breeding selection was largely increased by 104% and 142% in the FL1 and FL2 lines, respectively, resulting in an average litter size of 17.3 pups and 18.7 pups per litter in the FL1 and FL2 lines, respectively, compared with a litter size of 11.0 pups per litter in the control (Fzt:DU). In addition, different component fertility traits were analysed in females of all three lines at different stages of the oestrous cycle and pregnancy. IN CONCLUSION (1) early embryonic development was accelerated in the FL1 and FL2 lines compared with control; (2) plasma progesterone levels were not correlated with fertility performance; (3) a largely increased ovulation number (i.e. number of corpora lutea) was responsible for high prolificacy in both lines; however, (4) the number of ova shed, as well as the rate of loss of ova and pre- and postimplantation conceptuses, was very different in the FL1 and FL2 lines, suggesting that different genetic components may be responsible for the high prolificacy in both high-fertility lines.

The Pre-Ovulatory Luteinizing Hormone Surge Is Followed by Down-Regulation of CYP19A1, HSD3B1, and CYP17A1 and Chromatin Condensation of the Corresponding Promoters in Bovine Follicles

The pre‐ovulatory luteinizing hormone (LH) surge induces an extensive molecular, physiological, and morphological reorganization of the bovine follicle. This study was designed to elucidate if chromatin modulation is involved in the LH‐induced gene regulation. Granulosa and theca of well‐characterized large bovine follicles were isolated before and after the LH surge. CYP19A1, HSD3B1, and CYP17A1 transcripts, which encode key enzymes of steroid hormone biosynthesis, were quantified by real‐time PCR (qPCR) and the degree of chromatin condensation was determined by DNase I protection assays. After LH, granulosa‐specific CYP19A1 and theca‐specific CYP17A1 transcripts were almost completely down‐regulated. Also, the abundance of HSD3B1 transcripts was reduced. The promoter chromatin of HSD3B1 and particularly of CYP19A1 was significantly less accessible to DNAse I in both cell types after LH, whereas the chromatin accessibility of the CYP17A1 promoter changed only in the theca. Correlation analysis revealed partly, highly significant negative correlations between transcript abundance and protection from DNase I digestion of the corresponding chromatin. The data strongly suggest that LH induces cell type‐ and gene‐specific chromatin condensation in the pre‐ovulatory bovine follicle. This epigenetic mechanism might be involved in the pre‐ovulatory down‐regulation of genes. Mol. Reprod. Dev. 77:1040–1048, 2010.

Quantitative Western ligand blotting reveals common patterns and differential features of IGFBP-fingerprints in domestic ruminant breeds and species

The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (P<0.05) in Cameroon sheep, if compared to 3 of 4 other sheep breeds, as well as in Dwarf goats versus Toggenburg and Boer goats (P<0.01). IGFBP-2 was elevated in Cameroon sheep and Boer goats, if compared to other breeds of these species (P<0.01), respectively. Holstein Friesian dairy cows had higher levels of IGFBP-4 (P<0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P<0.001). The total IGFBP amount of Toggenburg goats was reduced (P<0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds.

Bovine large luteal cells show increasing de novo DNA methylation of the main ovarian CYP19A1 promoter P2

Transformation of the estrogen producing large dominant follicle into a functional progesterone producing corpus luteum involves profound and well-orchestrated changes in cell type-specific gene expression profiles, possibly involving epigenetic mechanisms of gene silencing. As an experimental paradigm to examine the involvement of de novo DNA methylation in the process of luteinization, the transcript abundance and promoter-specific DNA methylation levels of CYP19A1, which encodes the key enzyme for estrogen biosynthesis, were analyzed in enzymatically dispersed and purified large granulosa luteal cells of early- to mid-cycle bovine corpora lutea. To characterize the morphology and physiology of isolated corpora lutea, their weights and the respective plasma progesterone levels were analyzed. Transcript abundance of CYP19A1, HSD3B1, GHR, and of LHGCR was quantified by real-time PCR. Methylation levels were analyzed by bisulfite direct sequencing. The data indicated that corpora lutea weights and plasma progesterone levels significantly increased during the early luteal phase (days 3-6 of the cycle). The growth of small and large luteal cells was particularly pronounced between days 3 and 4. Large luteal cells are characterized by high HSD3B1 and GHR, but low LHCGR transcript abundance, whereas CYP19A1 expression was very low or undetectable. The DNA methylation levels of the main ovarian CYP19A1 promoter P2 significantly increased from day 5. In conclusion, the data indicated de novo DNA methylation approximately five days after the luteinizing hormone-induced down-regulation of CYP19A1 expression, suggesting that DNA methylation during the early luteal phase might play a role for permanent silencing of previously down-regulated genes.

Control of IGFBP-2 Expression by Steroids and Peptide Hormones in Vertebrates

IGFBP-2 (1) has been described as a brain tumor oncogene (2) and is widely expressed in cancers from different origins (3–8). IGFBP-2 alone cannot cause malignant transformation, yet progression of brain tumors to higher grade (9) and also has been provided as a protective element in earlier stages of multistage colon carcinogenesis (10). Therefore, it is crucial to understand the factors that determine expression patterns of IGFBP-2 under normal and malignant conditions. The present review provides a comprehensive update of known factors that have an impact on expression of IGFBP-2.

Bioanalytical validation for simultaneous quantification of non-aromatic steroids in follicular fluid from cattle via ESI-LC–MS/MS

The family of steroid hormones is quite attractive for the approach of phenotype monitoring in farm animals. Therefore, we developed a new protocol for the quantitative analysis of natural steroids in follicular fluid from dairy cows. The corresponding steroid profile, which consists of progesterone, corticosterone, hydrocortisone, testosterone, and androstenedione covering three distinct steroid classes, was determined by LC/MS. Quantification is achieved by use of steroid standards diluted in steroid-free follicular fluid as calibrators. Thus, the new protocol does not require deuterated standards. In order to correct for conditional performance of the analytical system we have used dexamethasone as an internal standard. The method was validated according to EMA guidelines. Within- and between-day variations were below 20% for most parameters assessed. All steroids assessed had lower limits of quantification in the range of 2.1 to 4.4ng/ml. We have established a simple and sensitive analytical system in order to step towards a broader and cost-efficient phenotyping analysis in follicular fluid from dairy cows.

Dissociation of somatic growth, time of sexual maturity, and life expectancy by overexpression of an RGD-deficient IGFBP-2 variant in female transgenic mice

Impaired growth is often associated with an extension of lifespan. However, the negative correlation between somatic growth and life expectancy is only true within, but not between, species. This can be observed because smaller species have, as a rule, a shorter lifespan than larger species. In insects and worms, reduced reproductive development and increased fat storage are associated with prolonged lifespan. However, in mammals the relationship between the dynamics of reproductive development, fat metabolism, growth rate, and lifespan are less clear. To address this point, female transgenic mice that were overexpressing similar levels of either intact (D‐mice) or mutant insulin‐like growth factor‐binding protein‐2 (IGFBP‐2) lacking the Arg‐Gly‐Asp (RGD) motif (E‐ mice) were investigated. Both lines of transgenic mice exhibited a similar degree of growth impairment (−9% and −10%) in comparison with wild‐type controls (C‐mice). While in D‐mice, sexual maturation was found to be delayed and life expectancy was significantly increased in comparison with C‐mice, these parameters were unaltered in E‐mice in spite of their reduced growth rate. These observations indicate that the RGD‐domain has a major influence on the pleiotropic effects of IGFBP‐2 and suggest that somatic growth and time of sexual maturity or somatic growth and life expectancy are less closely related than thought previously.