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In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a bovine pluripotent cell population, nor how to maintain these cells in vitro. The objective of this study was to determine the transcriptomic profile related to bovine pluripotency. Therefore, in vitro derived embryos were cultured in various culture media that recently have been reported capable of maintaining the naïve pluripotent state of human embryonic cells. Gene expression profiles of embryos cultured in these media were compared using microarray analysis and quantitative RT-PCR. Compared to standard culture conditions, embryo culture in 'naïve' media reduced mRNA expression levels of the key pluripotency markers NANOG and POU5F1. A relatively high percentage of genes with differential expression levels were located on the X-chromosome. In addition, reduced XIST expression was detected in embryos cultured in naïve media and female embryos contained fewer cells with H3K27me3 foci, indicating a delay in X-chromosome inactivation. Whole embryos cultured in one of the media, 5iLA, could be maintained until 23 days post fertilization. Together these data indicate that 'naïve' conditions do not lead to altered expression of known genes involved in pluripotency. Interestingly, X-chromosome inactivation and development of bovine embryos were dependent on the culture conditions.

Transforming acidic coiled-coil (TACC) proteins are key players during mitosis via stabilization of the spindle. The roles of TACCs during meiosis are however less clear. We used bovine oocytes to study the expression and function of TACC3 during meiosis. TACC3 mRNA was detected in bovine oocytes during meiosis using qRT-PCR, and while it was also expressed in cleavage stage embryos, its expression was down-regulated at the morula and blastocyst stages. Immunofluorescence was used to demonstrate that TACC3 co-localized with tubulin in the metaphase I and II spindles. However, TACC3 was not detected at anaphase or telophase of the first meiotic division. Aurora A, which is known to phosphorylate and activate TACC3 in mitotic cells, showed a similar pattern of gene expression to that of TACC3 in meiotic oocytes and preimplantation embryos. Aurora A protein was however only very transiently associated to the meiotic spindle. Pharmaceutical inhibition of Aurora A activity inhibited TACC3 phosphorylation but did not prevent TACC3 appearance in the spindle. Inhibiting Aurora A activity did however lead to abnormal meiotic spindle formation and impaired maturation of bovine oocytes. Similar results were obtained by knock-down of TACC3 expression using siRNA injection. These results suggest that TACC3 is important for stabilizing the meiotic spindle, but phosphorylation of TACC3 by Aurora A is not required for its recruitment to the meiotic spindle although phosphorylation of TACC3 by other kinases cannot be excluded.

Background: MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene targets. Accurate quantification of miRNA expression using validated internal controls should aid in the understanding of their role in epigenetic modification of genome function. To date, most studies that have examined miRNA expression levels have used the global mean expression of all expressed genes or the expression of reference mRNAs or nuclear RNAs for normalization., Results: We analyzed the suitability of a number of miRNAs as potential expression normalizers in bovine oocytes and early embryos, and porcine oocytes. The stages examined were bovine oocytes at the germinal vesicle (GV) and metaphase II stages, bovine zygotes, 2, 4 and 8 cell embryos, morulae and blastocysts, as well as porcine cumulus oocyte complexes, GV, metaphase I and II oocytes. qRT-PCR was performed to quantify expression of miR-93, miR-103, miR-26a, miR-191, miR-23b, Let-7a and U6 for bovine samples and miR-21, miR-26a, miR-93, miR-103, miR-148a, miR-182 and miR-191 for porcine oocytes. The average starting material for each sample was determined using specific standard curves for each primer set. Subsequently, geNorm and BestKeeper software were used to identify a set of stably expressed miRNAs. Stepwise removal to determine the optimum number of reference miRNAs identified miR-93 and miR-103 as the most stably expressed in bovine samples and miR-26a, miR-191 and miR-93 in porcine samples., Conclusions: The combination of miR-93 and miR-103 is optimal for normalizing miRNA expression for qPCR experiments on bovine oocytes and preimplantation embryos; the preferred combination for porcine oocytes is miR-26a, miR-191 and miR-93.

Endometrial oxytocin receptors (OXTR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are central components of the luteolytic pathway in cyclic mares, and their suppression is thought to be critical to luteal maintenance during early pregnancy. We examined the effect of pregnancy on endometrial expression of potential regulators of prostaglandin (PG) F2α secretion in mares. Expression of the nuclear progesterone receptor and oestrogen receptor ERα was high during oestrus, and depressed when progesterone was elevated; the opposite applied to the membrane progesterone receptor. PTGS2 was upregulated on Day 14 of dioestrus, but not pregnancy. Although OXTR mRNA expression was not elevated on Day 14 of dioestrus, protein abundance was; this increase in OXTR protein was absent on Day 14 of pregnancy. Intriguingly, gene and protein expression for PTGS2 and OXTR increased markedly between Days 14 and 21 of pregnancy suggesting that, although initial avoidance of luteolysis during pregnancy involves their suppression, this is a transient measure that delays rather than abolishes luteolytic pathway generation. The only oxytocin-PGF2α feedback loop component downregulated on both Days 14 and 21 of pregnancy was the PGF2α receptor we propose that downregulation of the PGF2α receptor uncouples the oxytocin-PGF2α feedback loop, thereby preventing generation of the large PGF2α pulses required for luteolysis.

Background: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed., Results: Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated., Conclusion: The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

The quality of an oocyte is crucial for successful generation of offspring, but few selection parameters have been identified that reliably predict oocyte developmental competence. The objective of the present study was to determine whether the developmental competence of in vivo-matured oocytes derived from superstimulated heifers could be predicted by 17β-estradiol and progesterone concentrations in follicular fluid, degree of cumulus cell expansion, and follicular diameter. Cumulus oocyte complexes were individually collected from follicles ≥8 mm 22 hours after an induced LH peak and individually fertilized and cultured in vitro. Only oocytes that originated from follicles with 17β-estradiol ≤0.25 μM and progesterone ≥0.26 μM developed into blastocysts. When a combination of these cutoff values was evaluated as a predictor of oocyte competence, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 75%, 49%, and 100%, respectively. Hormone concentrations in follicular fluid were also associated with the degree of cumulus cell expansion and only cumulus oocyte complexes with full expansion developed into blastocysts; sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 71%, 45%, and 100%, respectively, when full expansion was used as the predictive criterion for blastocyst production. Follicular diameter was not a good predictor of oocyte competence. In conclusion, concentrations of 17β-estradiol and progesterone in the preovulatory follicle and the degree of cumulus cell expansion are predictors of blastocyst production in superstimulated heifers and can be used as selection markers for oocyte developmental competency., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Metabolic conditions characterized by elevated free fatty acid concentrations in blood and follicular fluid are often associated with impaired female fertility. Especially elevated saturated fatty acid levels can be lipotoxic for several somatic cell types. The aim of this study was to determine the impact of elevated free fatty acid concentrations in follicular fluid on neutral lipids (fatty acids stored in lipid droplets) inside cumulus cells and oocytes and their developmental competence. To this end, cows were exposed to a short-term fasting period during final oocyte maturation. This resulted in elevated, but distinct, free fatty acid concentrations in blood and follicular fluid and a rise in the concentrations of in particular fatty acids with a chain length of 14-18 carbon atoms. Interestingly, elevated free fatty acid concentrations in follicular fluid resulted in a massive increase in the level of neutral lipids in cumulus cells, whereas the level of neutral lipid in oocytes was hardly affected. Furthermore, competence of oocytes to develop to the blastocyst stage after fertilization and culture of cumulus-oocyte-complexes of the experimental and control group was not different. In conclusion these data suggest that short-term elevated free fatty acid concentrations in follicular fluid do not harm oocyte developmental competence. We propose that the involvement of high levels of mobilized oleic acid in follicular fluid in combination with the induced lipid storage in cumulus cells serves to prevent harmful saturated fatty acid exposure to the oocyte.

Both Bos indicus (zebu) and Bos javanicus (banteng) contribute to the Indonesian indigenous livestock, which is supposedly of a mixed species origin, not by direct breeding but by secondary cross-breeding. Here, the analysis of mitochondrial, Y-chromosomal and microsatellite DNA showed banteng introgression of 10-16% in Indonesian zebu breeds with East-Javanese Madura and Galekan cattle having higher levels of autosomal banteng introgression (20-30%) and combine a zebu paternal lineage with a predominant (Madura) or even complete (Galekan) maternal banteng origin. Two Madura bulls carried taurine Y-chromosomal haplotypes, presumably of French Limousin origin. There was no evidence for zebu introgression in five populations of the Bali cattle, a domestic form of the banteng., (© 2012 Blackwell Verlag GmbH.)

SARCOSIN, also named Krp1, has been identified as a protein exclusively expressed in striated muscle tissue. Here we report on the role of SARCOSIN in skeletal muscle development and differentiation. We demonstrate, by means of whole-mount in situ hybridization, that Sarcosin mRNA is expressed in the myotome part of the mature somites in mouse embryos from embryonic day 9.5 onwards. Sarcosin is not expressed in the developing heart at these embryonic stages, and in adult tissues the mRNA expression levels are five times lower in the heart than in skeletal muscle. SARCOSIN protein partially co-localizes with the M-band protein myomesin and between and below laterally fusing myofibrils in adult skeletal muscle tissue. RNA interference mediated knock-down of SARCOSIN in the C2C12 myoblast cell line appeared to be stimulatory in the early phase of differentiation, but inhibitory at a later phase of differentiation.

A muscle progenitor cell population, other than muscle satellite cells, can be isolated and purified from porcine muscle tissue. We show the presence of at least two types of stem cells in porcine muscle: those that express α6 integrin and those that lack expression of this integrin type. By flow cytometry, we could select for myogenic stem cell populations expressing the neural cell adhesion molecule in the presence and absence of α6 integrin. The expression of α6 integrin showed an advantage in the formation of myotubes, possibly by an improved cell fusion capacity. This notion was strengthened by qRT-PCR analysis showing sustained PAX7, MYF5 and DESMIN expression and a strong myogenic differentiation capacity of this stem cell population. Selective inhibition of α6 integrin function, both by blocking antibodies and RNA interference, showed the importance of α6 integrin in myogenic differentiation of muscle stem cells. It is concluded that α6 integrin expression can be used as biomarker to select for highly myogenic cell populations in muscle tissue., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This study investigated the role of the leptin system in human oocyte maturation and its prognostic value for IVF outcome. The protein concentrations of leptin and soluble leptin receptor in follicular fluid were determined and the free leptin index (FLI) were established. Additionally, mRNA expression levels of different leptin receptor (ObR) isoforms and of PTX3 and HAS2 in cumulus cells were quantified, mutually compared and analysed relative to FLI, body mass index, age and number of retrieved oocytes. Expression of all target genes was detected in the cumulus cells, with relatively low concentrations of ObR-Long. Strong mutual correlations were found between mRNA expression levels of leptin receptor isoforms (P < 0.001) and also between the short isoforms of the leptin receptor and PTX3 (P < 0.001). Although the mean values of the pregnant and non-pregnant groups did not differ significantly for any of the variables, the chance that treatment resulted in ongoing pregnancy was higher with leptin 0.5 ng/mg protein compared with concentrations >0.5 ng/mg protein (P < 0.05). It is concluded that the leptin system appears to play a role in the IVF protocol, whereby signal transduction in cumulus cells occurs predominantly via the short isoforms of ObR., (2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Background: Two bovine species contribute to the Indonesian livestock, zebu (Bos indicus) and banteng (Bos javanicus), respectively. Although male hybrid offspring of these species is not fertile, Indonesian cattle breeds are supposed to be of mixed species origin. However, this has not been documented and is so far only supported by preliminary molecular analysis., Methods and Findings: Analysis of mitochondrial, Y-chromosomal and microsatellite DNA showed a banteng introgression of 10-16% in Indonesian zebu breeds. East-Javanese Madura and Galekan cattle have higher levels of autosomal banteng introgression (20-30%) and combine a zebu paternal lineage with a predominant (Madura) or even complete (Galekan) maternal banteng origin. Two Madura bulls carried taurine Y-chromosomal haplotypes, presumably of French Limousin origin. In contrast, we did not find evidence for zebu introgression in five populations of the Bali cattle, a domestic form of the banteng., Conclusions: Because of their unique species composition Indonesian cattle represent a valuable genetic resource, which potentially may also be exploited in other tropical regions.

Two lineage segregation events in mammalian development form the trophectoderm, primitive endoderm, and pluripotent primitive ectoderm. In mouse embryos, Oct4, Cdx2, Nanog, and Gata6 govern these events, but it is unknown whether this is conserved between mammals. Here, the expression patterns of these genes and their products were determined in porcine oocytes and embryos and in bovine embryos. CDX2 and GATA6 expression in porcine and bovine blastocysts resembled that of mouse, indicating conserved functions. However, NANOG expression was undetectable in porcine oocytes and embryos. Some inner cell mass cells in bovine blastocysts expressed NANOG protein. OCT4 protein was undetectable in porcine morulae, but present in both the trophectoderm and the inner cell mass of blastocysts, suggesting that downregulation of OCT4 in the trophectoderm does not precede trophectoderm formation. Combined, the results indicate differences in lineage segregation between mammals., ((c) 2008 Wiley-Liss, Inc.)

In this study, the mechanisms of supposed leptin action on oocyte maturation were examined. Expression of leptin mRNA, as determined with RT-PCR, was present in oocytes but not in cumulus cells. The long isoform of the leptin receptor (ObR-L) was expressed exclusively in cumulus cells after 7 and 23 hr of maturation. In oocytes the expression of the short receptor isoform (ObR-S) and all the receptor isoforms combined (ObR-T) did not change during maturation, as determined by quantitative RT-PCR, but in cumulus cells there was a significant increase in ObR-S transcripts after 7 hr of maturation. To determine if leptin plays a role in resumption of meiosis, oocytes meiotically arrested by the connection of the cumulus to a piece of granulosa layer were exposed to leptin. After 23 hr of culture, the proportion of oocytes that had resumed meiosis did not differ from the control. Exposure of COCs to leptin (1,000 ng/ml) resulted after 17 hr of maturation in a smaller proportion of oocytes that was still in metaphase-I stage (M-I) compared to the control group. Fertilization of oocytes after maturation in the presence of leptin resulted in a larger proportion of embryos that had developed to the 8-cell stage on Day 5 compared to the control group and in more blastocysts on Day 8 of culture. It is concluded that leptin enhances meiotic maturation of bovine oocytes, and that this effect is cumulus cell-mediated., (Copyright 2007 Wiley-Liss, Inc.)

Progesterone and oestrogen play essential roles in the maintenance of pregnancy in eutherian mammals and are thought to exert their effects on the developing conceptus indirectly, via the endometrium. In some species, early embryos have themselves been shown to express steroid receptors, thereby suggesting that reproductive steroids may also influence embryonic development directly. The aim of this study was to determine whether early intrauterine equine conceptuses express either the classical intracellular progesterone (PR) and oestrogen receptors (ERalpha and ERbeta) or the more recently characterised membrane-bound progesterone receptors (PGRMC1 and mPR). Horse conceptuses recovered on days 7, 10 and 14 after ovulation (n=8 at each stage) were examined for steroid receptor mRNA expression using quantitative rtPCR. Where commercial antibodies were available (PR, ERbeta), receptor localisation was examined immunohistochemically in day 10, 12, 14, 15 and 16 conceptuses (n=2 at each stage). mRNA for PR, PGRMC1 and mPR was detected at all stages examined, but while PGRMC1 and mPR expression increased during the day 7-14 period, PR expression decreased. ERalpha mRNA was not detected at any stage examined, whereas ERbeta mRNA was detected in all day 14, some day 10 and no day 7 conceptuses. Immunoreactive ERbeta receptors were localised to the trophectoderm of day 14-16 conceptuses; PR were not detected immunohistochemically in conceptus tissue. In summary, this study demonstrates that equine conceptuses express mRNA and, in the case of ERbeta, protein for steroid hormone receptors during the period encompassing rapid conceptus growth, differentiation and maternal pregnancy recognition.

Background: In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction., Results: In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated., Conclusion: Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.

Nitric oxide (NO) plays a key role in the processes leading to cervical softening prior to labor. Inducible nitric oxide synthase (iNOS) contributes most to the increased production of NO during labor, as demonstrated in the rat cervix, or at term pregnancy in women. Changes in expression of iNOS during late gestation have not yet been studied longitudinally in any species, because repeatedly taking biopsies could not be performed. iNOS mRNA (n = 6) and protein expression (n = 3) in serial cervical biopsies of pregnant pluriparous cows taken around days 225, 250, and 275 of pregnancy and within 1.5 hr after calving (d225, d250, d275 and parturition biopsies, respectively) were measured using quantitative RT-PCR and Western blotting. iNOS mRNA expression decreased from the d225 biopsy onwards, differences being significant between the d250 and d275 (P < 0.05) and between the d275 and parturition biopsies (P < 0.05). iNOS protein expression decreased from d225 to d250 onwards. Immunohistochemical analysis of biopsies showed, besides positive staining in endothelium and epithelium, which remained unchanged at different time points, that iNOS expressing cells in the connective tissue cells of early biopsies were predominantly spindle shaped (mostly smooth muscle cells and some fibroblasts). In the parturition biopsies, iNOS reactivity was mainly found in mononuclear leucocytes. These results lead us to suggest that iNOS from spindle shaped cells is involved in prepartum cervical ripening, while iNOS in mononuclear inflammatory cells may be important for local tissue repair mechanisms during postpartum cervical involution., ((c) 2006 Wiley-Liss, Inc.)

Relatively little information is available on the local factors that regulate folliculogenesis in goats. To examine the possibility that the Kit ligand (KL) system is expressed throughout the folliculogenesis, we studied the presence and distribution of KL and its receptor, c-Kit, in goat ovaries. Ovaries of goats were collected and either fixed in paraformaldehyde for immunohistochemical localization of KL and c-Kit proteins, or used for the isolation of follicles, luteal cells, surface epithelium and medullary samples to study mRNA expression for KL and c-Kit, using the reverse transcriptase polymerase chain reaction (RT-PCR). KL protein and mRNA were found in follicles at all stages of development, i.e. primordial, primary, secondary, small and large antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue. Antral follicles expressed both KL-1 and KL-2 mRNAs, while earlier staged follicles expressed KL-1 transcript only. KL protein was demonstrated in granulosa cells from the primordial follicle onward. Its mRNA could be detected in granulosa cells isolated from antral follicles and occasionally in their theca cells. c-Kit mRNA was expressed in all antral follicular compartments and at all stages of follicular development. c-Kit protein was predominantly found in oocytes from the primordial follicle stage onwards, in theca cells of antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue, particularly in the wall of blood vessels, which may indicate these cells as the main sites of action of KL. It is concluded that the KL/c-Kit system, in goat ovaries, is widespread and that it may be involved in the regulation of various local processes, including folliculogenesis and luteal activity.

Zearalenone (ZEA) is a mycoestrogen found in diverse food and feed materials, particularly in corn and small grains. Following ingestion, the parent zearalenone is converted predominantly into alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) by hepatic hydroxy steroid dehydrogenases (HSD). The present study demonstrated by standard RT-PCR the expression of 3alpha- and 3beta-HSD also in porcine cumulus oocyte complexes (COCs) and granulosa cells isolated form cumulus oocyte complexes. Analysis of the rate of bioconversion of zearalenone (ZEA) by the cultured granulose cells showed the extra-hepatic production of both hydroxy metabolites of ZEA with alpha-ZOL being the dominating metabolites as previously observed in incubations with liver microsomes. The endogenous steroids 5alpha-dihydrotestosterone (5alpha-DHT), and progesterone (PGTN), both known substrates for 3alpha-HSD inhibited the conversion of ZEA into alpha-ZOL. In the presence of pregnelonone (PGN), a major substrate for 3beta-HSD only a slight inhibitory effect on the apparent beta-ZOL formation could be observed. In conclusion, these data indicate that both 3alpha- and 3beta-HSDs are expressed in porcine COCs and GCs, whereas the biotransformation experiments confirm the involvement of these enzymes in the extra-hepatic biotransformation of ZEA.

Bone morphogenetic proteins (BMPs) have been implicated in the regulation of ovarian follicular development and are promising candidates to apply in IVM and IVF protocols. We investigated the expression of BMP2, BMP4 and BMP receptors in bovine ovaries and the effects of BMP2 and BMP4 during oocyte maturation on bovine IVM. Reverse transcription polymerase chain reaction studies with antral follicles showed the expression of BMPR-IA, BMPR-IB, ActR-IA, ActR-IIB, BMPR-II and BMP4 mRNA in all follicular compartments, while BMP2 mRNA was generally restricted to theca and cumulus tissue. Immunohistochemistry demonstrated the presence of BMPR-II in oocytes and granulosa cells of preantral follicles but only in oocytes of antral follicles. The immunostaining of BMP2 and BMP4 was limited to theca interna and approximately 25% of oocytes of antral follicles. Exogenously added BMP2 or BMP4 to IVM medium did not affect oocyte nuclear maturation, cumulus cell expansion, nor blastocyst formation following IVF. It is concluded that a BMP-signaling system, consisting of BMP2, BMP4, type II and I receptors, is present in bovine antral follicles and that this system plays a role in development and functioning of these follicles rather than in final oocyte maturation and cumulus expansion.

The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. In goats, relatively little information is available on the local factors that regulate this process. We studied the presence and distribution of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and BMP receptors types 2 (BMPR2), 1A (BMPR1A), and 1B (BMPR1B) in goat ovaries to find evidence for their possible roles in folliculogenesis. Ovaries of cyclic goats were collected and fixed in paraformaldehyde for immunohistochemical localization of GDF9 and BMP15 proteins or used to collect follicles and luteal tissue to study the mRNA expression of GDF9, BMP15, and BMP receptors using reverse transcriptase polymerase chain reaction (RT-PCR). GDF9 and BMP15 proteins were found in oocytes of all types of follicles and granulosa cells of primary, secondary, and antral but not primordial follicles. The mRNAs for GDF9, BMP15, BMPR2, BMPR1A, and BMPR1B were detected in primordial, primary, and secondary follicles as well as in oocyte and granulosa cells of antral follicles. Transcripts for BMPR2, BMPR1A, BMPR1B, and GDF9, and GDF9 protein were furthermore found in corpora lutea. It is concluded that the mRNAs and proteins of GDF9 and BMP15 and the mRNAs of BMP receptors are expressed in goat ovarian follicles at all stages of their development, and that they form a complex intrafollicular regulatory system during folliculogenesis. Expression of all BMP receptor mRNAs and GDF9 mRNA and protein in luteal tissue additionally points to a role of GDF9 in corpus luteum function., ((c) 2004 Wiley-Liss, Inc.)

We studied the protein and mRNA expression of activin-A, follistatin and activin receptors in goat ovaries to find evidence of their possible role in ovarian activity, particularly in the various stages of follicle development. Ovaries of cyclic goats were collected and then either fixed in paraformaldehyde for immunohistochemical localisation of activin-A, follistatin, activin receptors IIA/B (ActR-IIA/B) and IA (ActR-IA) proteins or used to obtain samples to demonstrate mRNA expression of activin-A (betaA subunit), follistatin, ActR-IIA, -IIB, -IA and -IB, using RT-PCR. For this latter goal, primordial, primary and secondary follicles were isolated mechanically, washed to remove the stromal cells and then used for RT-PCR. In addition, oocytes, cumulus, mural granulosa and theca cells from small (<3 mm) and large (3-6 mm) antral follicles, luteal cells and surface epithelium were collected to study mRNA expression. Activin-A and follistatin proteins were found in oocytes of all follicle classes, granulosa cells from the primary follicle stage onwards, theca cells of antral follicles, corpora lutea and ovarian surface epithelium. In antral follicles, these proteins were detected both in cumulus and mural granulosa cells. ActR-IIA/B protein was found at the same follicular sites, and also in granulosa cells of primordial follicles onward. The localisation of ActR-IA corresponded with that of ActR-IIA/B, but the former protein was absent in the theca of large antral follicles. The mRNAs for activin-A (betaA subunit), follistatin, and ActR-IIA, -IIB, -IA and -IB were detected at all follicular and cellular types studied, except that ActR-IIB was not found in follicles that had not developed an antrum yet. In conclusion, in goat ovaries, transcripts of activin-A (betaA subunit), its receptors and its binding protein follistatin are expressed and their proteins formed at all follicular stages and in corpora lutea. These findings indicate a role of activin-A in the local regulatory system during the entire follicular development and during luteal activity.

Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling pathway was studied in both oocytes and cumulus cells by polymerase chain reaction after reverse transcription, immunoblotting, and indirect immunofluorescence. Furthermore, the Fas signaling pathway was activated in cumulus oocyte complexes with an agonistic anti-Fas antibody during in vitro maturation in the presence or absence of FSH. Viability and incidence of apoptosis in cumulus cells were evaluated by assessing membrane integrity and nuclear morphology. Oocyte nuclear maturation was also analyzed, as well as cleavage rates, blastocyst formation rates, and blastocyst quality, following in vitro fertilization. Fas mRNA and protein were expressed both in oocytes and cumulus cells. FasL protein was found in cumulus cells but could not be detected in oocytes, despite its mRNA expression. Both activation of the Fas pathway and presence of FSH during in vitro maturation increased the incidence of apoptosis in cumulus cells, affecting predominantly the middle and peripheral regions of the cumulus. The observed increase, however, had no effect on the developmental competence of the oocytes.

In various cell types, there is increasing evidence for nongenomic steroid effects, i.e., effects that are not mediated via the classical steroid receptors. However, little is known about the involvement of the nongenomic pathway of estradiol (E2) on mammalian oocyte in vitro maturation (IVM). The aim of this study was to investigate whether the effects of E2 on bovine oocyte IVM are mediated via a plasma membrane receptor (nongenomic). First, we investigated the expression of estradiol (classical) receptor alpha (ERalpha) and beta (ERbeta) mRNA in oocytes and cumulus cells (CC). We also studied the effects of different exposure times to E2 (before and after germinal vesicle breakdown, GVBD) on nuclear maturation. To study the possible involvement of the putative estradiol plasma membrane receptor on the IVM of oocytes, we used E2 conjugated with bovine serum albumin (E2-BSA), which cannot cross the plasma membranes. Our results demonstrate that oocytes expressed ERbeta mRNA, while CC expressed both ERalpha and ERbeta mRNA. Exposure to E2 during the first 8 h of culture (before GVBD) induced a block at the metaphase I stage (MI). However, the presence of E2 after GVBD induced an increase of oocytes with nuclear aberrations. Meiotic spindle organization was severely affected by E2 during IVM and multipolar spindle was the most frequently observed aberration. Exposure of oocytes to E2-BSA did not affect nuclear maturation, blastocyst formation rate, nor embryo quality. Our results suggest that the detrimental effects of E2 on in vitro nuclear maturation of bovine oocyte are not exerted via a plasma membrane receptor.

GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced blastocysts have lower cell numbers than in vivo blastocysts and exhibit higher incidences of apoptosis. Therefore we investigated the effects of 100 ng GH/ml NCSU23 medium during in vitro culture of presumptive in vitro fertilized sow zygotes on embryo development and blastocyst quality (defined by diameter, cell number, apoptosis and survival after non-surgical transfer). In vivo produced blastocysts were analysed concurrently as a reference value. GHR was expressed in embryos from the 2-cell to blastocyst stages. GH had no effect on blastocyst development or cell numbers, but increased the mean blastocyst diameter. The incidence of apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), was decreased by GH, but when non-TUNEL-labelled apoptotic fragmented nuclei were included, no difference was seen. GH appeared to slow down the progression of apoptosis though. In vivo produced blastocysts presented no apoptotic nuclei, and contained higher cell numbers and larger diameters. Pregnancy rates on day 11 were similar for all groups, but survival was poorer for in vitro than in vivo produced blastocysts. In this study GH appeared to be beneficial only from the blastocyst stage, but the presence of GHR from early cleavage stages nevertheless indicates a role for GH throughout porcine embryo development and deserves further investigation.

It has been suggested that preculturing immature oocytes in a manner that maintains them in meiotic arrest may improve cytoplasmic maturation and, thereby, the eventual developmental competence of oocytes matured in vitro. This study examined the ability of follicular cells to maintain meiotic arrest in equine oocytes. Cumulus-oocyte complexes (COCs) recovered from dead mares were cultured for 38 h in M199 either attached to, or together with, different follicle wall components, as follows: (1) attached to the follicle wall, (2) cocultured with separated follicle wall, (3) attached to membrana granulosa (COCG), (4) COCGs cocultured with sheets of theca cells, (5) COCGs cultured in theca-cell conditioned medium, and (6) control COCs without any follicle wall components. When oocytes were cultured attached to their follicle wall, 79% remained in the GV stage throughout the 38 h incubation. However, when oocytes were cocultured with separate pieces of follicle wall, meiosis resumed and a similar proportion of oocytes progressed to metaphase II (79%) as under control conditions (84%). Only 16% of oocytes cultured while still attached to the membrana granulosa (COCGs) maintained the GV stage, whereas when COCGs were cocultured with theca cells or in theca-cell conditioned medium, significantly more oocytes remained in the GV stage (64 and 52%, respectively), indicating that theca cells secrete a meiosis-inhibiting factor. The effect of FSH on the meiosis-inhibiting activity of follicular cells was investigated by culturing COCs attached to the follicle wall and COCGs in the presence or absence of theca cells in medium containing FSH. Addition of 0.05 iu recombinant human FSH ml(-1) to the culture medium did not affect nuclear maturation and failed to overcome the suppressive effect exerted by the follicle wall or by theca cells, despite the fact that mRNA for the FSH receptor was found using RT-PCR in both cumulus and granulosa cells. These results demonstrate that the maintenance of meiotic arrest in equine oocytes during culture can be promoted by theca cells, which appear to act via a secreted inhibitory factor that cannot be suppressed or counteracted by FSH.

A factor, secreted by theca cells, inhibits FSH induced resumption of meiosis in bovine oocytes that are surrounded by cumulus cells which are attached to a piece of the membrana granulosa (COCGs). In order to characterize this factor, theca cell conditioned medium (CMt) was heat-treated, filtered through a 5 kD spin off filter, charcoal treated, chloroform extracted and protease treated. To investigate whether the meiosis inhibiting factor produced by theca cells was also present in follicular fluid (FF), the same treatments were done with 50% bovine follicular fluid (bFF). COCGs, originating from 2 to 8 mm follicles of bovine ovaries collected at a slaughterhouse, were cultured in groups of 15 per 600 microl medium supplemented with 0.05 IU ml FSH for 22 hr at 39 degrees C in a humidified atmosphere of 5% CO(2). After culture the oocytes were denuded, stained with orcein, and the nuclear status assessed. Heat treatment did not affect the meiosis arresting capacity of CMt since a similar proportion of the oocytes remained at the GV stage after 22 hr of culture in heat treated CMt as compared to the proportion of oocytes in the GV stage after culture in untreated CMt. Filtering through a 5 kD spin-off filter revealed that the meiosis inhibiting action was maintained in the <5 kD fraction, although there was a significant (P < 0.05) loss of inhibiting activity compared to nonfiltered CMt. No significant decrease was observed in the meiosis arresting capacity of the <5 kD fraction after charcoal or protease treatment. Extraction of the <5 kD fraction with chloroform also did not affect the theca cell produced factor. The effect of the theca cell factor on the progression of meiosis of the oocytes that resumed meiosis, as demonstrated by a very low percentage of the oocytes that matured up to the M2 stage, was not affected following any of the treatments. With regard to bFF, the results show a lower percentage of the oocytes in the GV stage after culture in 50% bFF as compared to culture in CMt, but progression of meiosis was clearly inhibited as demonstrated by a significant higher proportion of the oocytes blocked in the M1 stage after resumption of meiosis. In general, with regard to meiotic inhibition, bFF showed the same pattern as CMt following the various treatments. It is concluded that the theca cell secreted factor which inhibits the FSH-induced resumption of meiosis in COCGs is a small, stable, polar molecule which is not a peptide., (Copyright 2001 Wiley-Liss, Inc.)

In previous studies we demonstrated that bovine cumulus oocyte complexes (COCs) obtained from small and medium sized follicles express growth hormone receptor (GHR) mRNA and respond to growth hormone (GH) addition during in vitro maturation. The aim of this study was to investigate whether bovine zygotes and preimplantation embryos continue the expression of GHR gene after in vitro fertilization and during early embryo development and whether supplementation of GH during embryo culture affects embryo development. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. After in vitro fertilization the embryos were cultured: (a) on a monolayer of buffalo rat liver (BRL) cells in M199 supplemented with 10% FCS and 100 ng/ml bovine GH (NIH-GH-B18); (b) in droplets of serum-free BRL-conditioned medium supplemented with 100 ng/ml GH; (c) in droplets of synthetic oviductal fluid (SOF) supplemented with 100 ng/ml GH. Cultures without GH served as controls. Embryos were scored morphologically and the efficiency of the culture system was evaluated (a) as the percentage of cleaved embryos 4 days after IVF, (b) the percentage of blastocysts on Day 9 expressed on the basis of the number of oocytes at the onset of culture, and (c) the percentage of hatched blastocysts on Day 11 expressed on the basis of the total number of blastocysts present at Day 9. For gene expression, immature (GV) and mature (MII) oocytes (as positive control), embryos with less than 8 cells, 16-32 cells, and hatched blastocysts were prepared for reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of mRNA of GHR. Messenger RNA for GHR was found in GV and MII oocytes and in all stages of embryo development. No mRNA for GH could be detected in early and expanded blastocysts produced in SOF medium. Immunoreactive GHR was found both in trophoblastic and embryonic cells of hatched blastocysts. Addition of 100 ng/ml GH during embryo culture on a monolayer of BRL cells in M199 supplemented with 10% FCS did not affect embryo development. However, GH (100 ng/ml) supplementation during embryo culture in droplets of serum-free BRL conditioned medium significantly (P < 0.05) enhanced the proportion of > 8-cell embryos. Similarly, culture of embryos in droplets of SOF medium in the presence of GH (100 ng/ml) significantly (P < 0.05) enhanced the number of > 8-cell embryos from 53.8% in control to 70.6% in GH-treated group. Day 9 blastocyst formation in SOF medium was also significantly (P < 0.01) increased in the presence of GH (33.9%) compared to the control (20.2%). Embryos cultured in SOF without GH rarely resulted in hatched blastocysts (0.7%). However, GH supplementation remarkably enhanced the proportion of the hatched blastocysts (13%). In conclusion, expression of GHR gene in preimplantation bovine embryos, presence of the receptor, and the beneficial effect of GH on cleavage, blastocyst formation and hatchability of the embryos point to the involvement of GH in early embryonic development.

The aim of the present study was to investigate whether growth hormone (GH) has any effect on the development of cultured rat pre-antral follicles. Pre-antral follicles with a diameter between 120 microm and 160 microm were mechanically isolated from 10-day-old rat ovaries and cultured in groups for 6 days in serum-free medium without GH or with GH supplemented at concentrations of 1, 10 and 100 ng/ml, respectively. DNA content of the follicles before and after culture was measured to determine whether possible growth is due to proliferation of follicular cells. To investigate the quality of follicles cultured under different conditions, the ultrastructure of the cultured follicles was studied with transmission electron microscopy. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the expression of growth hormone receptor (GHR) in pre-antral follicles. GH, regardless of the concentration, stimulated the growth of pre-antral follicles. However, follicles cultured in medium supplemented with high-dose GH (100 ng/ml) showed a significantly lower survival rate compared with the other groups. Follicles cultured in GH-containing medium showed a better ultrastructure in comparison with those cultured in medium without GH. Remarkably, scattered cortical granules were observed in oocytes of follicles cultured in the presence of GH. With RT-PCR, the presence of the mRNA of GHR was demonstrated in pre-antral follicles. It can be concluded that GH promotes rat pre-antral follicle development in vitro and better supports the morphology of cultured pre-antral follicles. The gene expression of GHR suggests that the action of GH could be mediated by its receptors present in pre-antral follicles.

The aim of this study was to investigate whether bovine cumulus oocyte complexes (COCs) obtained from 2 to 8 mm follicles synthesize growth hormone (GH) during in vitro maturation. In addition the expression of growth hormone releasing hormone receptor (GHRH-r) in the COCs before and after in vitro maturation was investigated. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. At 0, 6, 12, and 24 hr after the onset of culture, COCs were removed and were prepared for immunohistochemical staining to detect the presence of GH. In addition, sections of ovary were stained to study the differential localization of GH in the ovary. At 0 and 24 hr COCs were removed and together with samples from granulosa cells and theca cells were prepared for reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of mRNA of GH and GHRH-r. Within COCs, cumulus cells and oocytes showed GH immunoreactivity, while expression of GH mRNA was only found in the oocyte. At the onset of culture, oocytes and cumulus cells in the majority of COCs generally showed moderate and strong staining intensity for GH, respectively. While GH staining in the cumulus cells did hardly change during 24 hr of culture, GH staining in the oocyte was absent after 24 hr of culture in 70% of COCs. Within the ovary, GH was localized in antral follicles larger than 2 mm and no staining was found in primordial, primary and secondary follicles or in the stroma. The intensity of the staining increased with the size of the follicles. Within the follicular wall the GH was persistently observed in granulosa cells, while theca cells were occasionally negative. GH mRNA in follicular compartments was only found in the oocyte and mural granulosa cells. No GHRH-r mRNA was found in the COCs nor in the granulosa or the stroma. In conclusion, the gradual increase of GH staining during follicular development and the consistent synthesis of GH in oocytes and granulosa cells, suggest a paracrine and/or autocrine action for GH in bovine follicular growth and oocyte maturation. The absence of mRNA for GHRH receptor in the COCs indicates that ovarian production of GH is not regulated by GHRH., (Copyright 1999 Wiley-Liss, Inc.)

We describe isolation and characterization of the bovine ortholog of POU5F1 (bPOU5F1) encoding octamer-binding transcription factor-4 (Oct-4). The organization of bPOU5F1 is similar to its human and murine orthologs, and it shares 90.6% and 81.7% overall identity at the protein level, respectively. Transient transfection of luciferase reporter constructs in murine P19 embryonal carcinoma cells demonstrated that bPOU5F1 has a functional promoter and contains two enhancer elements, of which one is repressed by retinoic acid. bPOU5F1 was mapped to the major histocompatibility complex on chromosome 23. bPOU5F1 mRNA was detected by nested reverse transcription-polymerase chain reaction in immature oocytes and in in vitro-produced preattachment-stage embryos. Oct-4 in oocytes and in vitro-produced preattachment-stage embryos was demonstrated by indirect immunofluorescence. Confocal laser scanning microscopy revealed Oct-4 in both the inner cell mass and trophoblast cells of the blastocyst until Day 10 of development. Immunofluorescence performed on the outgrowths formed at Day 13 postfertilization from in vitro-produced Day 8 blastocysts showed Oct-4 staining in all cells. This expression pattern suggests that bPOU5F1 acts early in bovine embryonic development but that its expression is not restricted to pluripotent cells of the blastocyst.

The effect of follicular cells and their conditioned media on the FSH-induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca-cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell-cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis-inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that progressed up to the MII stage was significantly reduced. Attachment of the COCs to the membrana granulosa enhanced this inhibiting action of CMt on the progression of meiosis. It is concluded that theca cells secrete a stable factor that inhibits the progression of FSH-mediated meiosis in oocytes of COCGs.

The aim of this study was to investigate whether bovine cumulus-oocyte complexes (COCs) synthesize activin A, inhibin, and follistatin and whether they contain activin receptor during in vitro maturation. Therefore, COCs obtained from small and medium-sized follicles were cultured in M-199 supplemented with 10% fetal calf serum (FCS) and gonadotropins for 24 hr. At 0, 6, 12, and 24 hr after the onset of culture, COCs were removed for immunohistochemical staining to detect the expression of activin A, inhibin, follistatin, and activin receptor type II proteins. At 0 and 24 hr, COCs were removed and prepared for reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA of these proteins. It appeared that cumulus cells and oocytes express activin, follistatin, and activin receptor proteins as well as their mRNA. While expression of inhibin mRNA was found exclusively in cumulus cells, the inhibin protein was present in cumulus cells and oocytes. Immunohistochemical study both in cumulus cells and in oocytes often showed a moderate and strong staining intensity for activin and follistatin, respectively. Activin staining underwent little or no change during culture except at 24 hr of maturation, where about 60% of the oocytes showed no staining. Follistatin immunoreactivity remained strong in the majority of COCs. At the onset of culture, a spotlike inhibin staining was observed in the oocyte, which increased after 12 hr and was absent at the end of culture. Activin receptor immunoreactivity in cumulus cell membranes and oolemma increased during oocyte maturation to maximum values at the end of culture in most of the COCs. It is concluded that the consistent presence of activin and the increase in activin receptor in cumulus cells and oocytes during in vitro maturation indicate a paracrine and/or autocrine action for activin on bovine oocyte maturation. This action may be modulated by inhibin and/or follistatin.

A previous study reported that the addition of bovine growth hormone (bGH) during in vitro maturation of bovine oocytes accelerates nuclear maturation and stimulates subsequent embryonic development. The aim of this study was to investigate whether bovine cumulus oocyte complexes (COCs) contain growth hormone receptor (GHR), and whether the stimulatory effect of GH on oocyte maturation is cumulus-dependent and mediated by insulin-like-growth factor (IGF-I). The expression of growth hormone receptor mRNA in mural granulosa cells, in cumulus cells, and in the oocyte was studied using reverse transcriptase polymerase chain reaction (RT-PCR). To investigate the importance of cumulus cells for GH-promoted maturation, COCs and denuded oocytes were cultured for 16 hr in M199 with or without bGH s(NIH-GH-B18). To investigate whether GH action is mediated by IGF-I, COCs were cultured in 1) 100 ng/ml bGH, 2) 100 ng/ml bGH plus anti-IGF-I, 1:100 dilution, 3) 100 ng/ml h-IGF-I, 4) 100 ng/ml h-IGF-I plus anti-IGF-I, 1:100 dilution, and 5) anti-IGF-I, 1:100 dilution. Culture was performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air, and the nuclear stage of oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining. PCR on cDNA of mural granulosa cells, cumulus cells, and oocytes revealed that mRNA for GHR was present in all cell types. Addition of GH (100 ng/ml) to the culture medium of denuded oocytes did not affect the number of metaphase II oocytes after 16 hr, while a significant (P < 0.001) increase was observed, when COCs were cultured in the presence of GH. Addition of the antibody against IGF-I to the culture medium completely suppressed the stimulatory effect of IGF-I on oocyte maturation and cumulus expansion, while stimulation by GH was not affected by the antibody. It is concluded that bovine cumulus cells, mural granulosa, and oocytes express mRNA for the GH receptor. The stimulatory effect of GH on bovine oocyte maturation is dependent on the cumulus cells and is not mediated by IGF-I.

Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage. Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 microns vs. 240 microns in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group) nor on cumulus expansion (246 microns vs. 240 microns in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles.

The effect of recombinant bovine activin A on the in vitro maturation of bovine oocytes was investigated. Culture of cumulus enclosed bovine oocytes in the presence of activin at the concentration of 100 or 500 ng/ml did not change the proportion of oocytes in which germinal vesicle breakdown had occurred at 4 and 7 h after the onset of culture. Activin had also no effect on the progression of maturation to the M II stage. The transient inhibition of germinal vesicle breakdown by 10 mM dibutyryl cyclic AMP was not affected by the addition of activin A at the onset of culture. Radiolabeling with 35S-methionine at 4 h and at 18 h after culture in the presence or absence of activin A did not show any effect of activin either on the total incorporation of radiolabel into acid precipitable material or on the protein synthesis patterns obtained after SDS-PAGE.

Six heifers were injected i.m. with 2500 i.u. PMSG followed by 15 mg prostaglandin 48 h later. Serial blood samples were collected through a catheter in the caudal vena cava every 10 min for 8 h on Day 10 (7 h after PMSG administration), during luteal regression (7 h after prostaglandin administration) and on the day thereafter. Four normally cyclic heifers served as a control group. Concentrations of progesterone, androstenedione, oestradiol, LH, FSH, and PMSG in the vena cava samples were measured and the frequency and amplitudes of episodic pulses of all hormones were estimated except for PMSG. Ovaries were collected by ovariectomy at 50 h after onset of luteal regression to determine the number of preovulatory follicles (non-atretic follicles greater than or equal to 10 mm). Stimulation of follicular growth by administration of PMSG resulted in the following effects on the secretion of steroids and endogenous gonadotrophins. (1) There were no alterations in progesterone concentration and the amplitude and frequency of episodic pulses. Mean (+/- s.e.m.) concentrations were 54.1 +/- 5.8, 19.1 +/- 3.1 and 3.4 +/- 0.9 nmol/l on Day 10 (L), during luteal regression (LR) and on the day thereafter (F) respectively. (2) There were no alterations in the episodic secretion patterns of androstenedione. Mean concentrations were 0.20 +/- 0.02, 0.15 +/- 0.02 and 0.11 +/- 0.02 nmol/l for the L, LR and F periods respectively. (3) There was an increase in oestradiol concentration from 17.1 +/- 3.0 pmol/l during the L period to 233.7 +/- 86.4 pmol/l during the F period. Pulse amplitude was enhanced compared to corresponding periods in control animals whereas pulse frequency remained the same. The oestradiol concentration was significantly correlated with the number of preovulatory follicles (r = 0.82, P less than 0.05). (4) There was a suppression of the frequency of episodic LH pulses (/8 h) during the LR (3.2 +/- 0.7) and F (4.3 +/- 0.4) periods compared to corresponding periods in control heifers (9.5 +/- 0.9 and 7.0 +/- 1.5 respectively). The preovulatory LH peak occurred earlier in 4 of 6 treated heifers. (5) There was a suppression of FSH concentrations, pulse amplitude and frequency during the LR and F (17.4 +/- 0.9 mg/l, 4.7 +/- 0.8 microgram/l and 7.5 +/- 0.4 pulses/8 h) periods compared to the corresponding F-period values (35.6 +/- 6.2 mg/l, 9.8 +/- 1.6 micrograms/l and 9.3 +/- 0.3 pulses/8 h) in control heifers.(ABSTRACT TRUNCATED AT 400 WORDS)

Preovulatory bovine follices (n = 73) were collected at different times after the onset of oestrus until shortly before ovulation, which occurred at 24 +/- 1 X 4 h after the peak concentration of LH in the peripheral blood. Non-atretic antral follicles (n = 9) of 15-19 mm were also collected from cows during the luteal phase of the oestrous cycle. Follicular fluid concentrations of dehydroepiandrosterone, androstenedione and oestrone, and of LH, FSH and prolactin were compared in 2-h periods relative to the LH plasma peak. Before the LH surge the concentrations of the steroids were much higher than in non-atretic luteal-phase follicles of similar size. From 0 to 6 h after the LH peak the steroid concentrations decreased sharply to remain low until ovulation; only that of androstenedione increased again after 14 h to remain constant. The ratio between the concentrations of androstenedione and dehydroepiandrosterone remained constant until 14 h after the LH peak; at 14 h it increased about 4-fold and remained high until ovulation. The ratio between the oestrone and androstenedione concentration increased gradually to a 10-fold higher value until at 14 h an abrupt decrease was observed. These changes indicate that after the LH peak androgen production is directly inhibited and, at a slower rate, the aromatizing activity. Androstenedione appeared to be the major aromatase substrate. Before the plasma LH peak the follicular fluid concentration of FSH was higher than in luteal-phase follicles; the concentrations of LH and prolactin were not different from those in luteal-phase follicles.(ABSTRACT TRUNCATED AT 250 WORDS)