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J. Kotwica, D. Schams, H. H. D. Meyer and Th. Mittermeier

Summary. In Exp. I oxytocin (60 μg/100 kg/day) was infused into the jugular vein of 3 heifers on Days 14–22, 15–18 and 16–19 of the oestrous cycle respectively. In Exp. II 5 heifers were infused with 12 μg oxytocin/100 kg/day from Day 15 of the oestrous cycle until clear signs of oestrus. Blood samples were taken from the contralateral jugular vein at 2-h intervals from the start of the infusion. The oestrous cycle before and after treatment served as the controls for each animal. Blood samples were taken less frequently during the control cycles. In Exp. III 3 heifers were infused with 12 pg oxytocin/100 kg/day for 50 h before expected oestrus and slaughtered 30–40 min after the end of infusion for determination of oxytocin receptor amounts in the endometrium. Three other heifers slaughtered at the same days of the cycle served as controls.

Peripheral concentrations of oxytocin during infusion ranged between 155 and 641 pg/ml in Exp. I and 18 and 25 pg/ml in Exp. II. In 4 out of 8 heifers of Exps I and II, one high pulse of 15-keto-13,14-dihydro-prostaglandin F-2α (PGFM) appeared soon after the start of oxytocin infusion followed by some irregular pulses. The first PGFM pulse was accompanied by a transient (10–14 h) decrease of blood progesterone concentration. High regular pulses of PGFM in all heifers examined were measured between Days 17 and 19 during spontaneous luteolysis. No change in length of the oestrous cycle or secretion patterns of progesterone, PGFM and LH was observed. The number of oxytocin receptors in endometrium was not affected by oxytocin infusion around the time of oestrus. These results suggest that luteolytic events were not significantly influenced by a constant infusion of oxytocin.

Keywords: oxytocin; infusion; luteolysis; oestrous cycle length

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S. Blottner, O. Hingst and H. H. D. Meyer

Quantitative changes in testes of roe deer were studied during the annual cycle. Testicular spermatozoa were counted and proportions of different cell types were estimated using DNA flow cytometry. A proliferation-specific antigen of somatic cells was evaluated by an immunoradiometric assay. Apoptosis was examined by cell death detection ELISA, and testosterone concentrations were measured with an enzymeimmunoassay. The testis mass of adults reached a maximum during the rut from mid-July to mid-August. Gonadal size corresponded to numbers of testicular spermatozoa g−1 testis. In the rutting period, epididymal spermatozoa were of the highest morphological and functional competence. The proportions of haploid (1c), diploid (2c) and tetraploid (4c) cells changed over time with the maximum of 1c cells during the breeding period. Meiotic division (1c:4c ratio) increased sharply immediately before rut, while mitosis (% cells in G2–M phase) was already high during spring. Proliferation and apoptosis revealed an opposite pattern during the annual cycle; the most intensive apoptosis occurred during the time of testis involution. Testosterone production showed a biphasic pattern. It dropped rapidly from the highest value in August to very low concentrations thereafter. Yearlings were characterized by smaller peaks of testicular growth and sperm production. Fawns started testicular growth and meiosis in winter. In conclusion, the production of spermatozoa in roe deer is intensified by enlargement of gonads as well as enhanced efficiency of spermatogenesis during the rut. Interrupted proliferation and stimulated apoptosis promote testis involution after the rut, and testosterone seems to play a role in the regulation of both processes.

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F. Göritz, K. Jewgenow and H. H. D. Meyer

Identification of epidermal growth factor (EGF) and its distribution in the ovary were examined with an immunohistochemical technique using a polyclonal rabbit antibody against mouse EGF. A combination of HPLC and enzymeimmunoassay was elaborated to quantify EGF in different compartments of the feline ovary. In addition, EGF receptors were localized in ovarian cryostat sections with a new ligand–histochemical technique using biotinylated EGF for labelling. Epidermal growth factor was present in theca interna cells, in specific aggregations of interstitial gland cells located next to tertiary follicles, in smaller, single cells of the ovarian cortex, and in the corpus luteum. The strongest EGF-positive reaction was found in vacuolized cells of the interstitium and in theca interna cells of large tertiary follicles rich in cytoplasm. The strictly cellular localization of the EGF-antibody reaction suggests the synthesis of EGF in these cells. Specific binding sites of EGF were present on granulosa cells of secondary and tertiary follicles and on interstitial gland cells. The EGF-binding capacity of granulosa cells of the cumulus oophorus was greater than that of the mural granulosa cells. Granulosa cells of atretic follicles showed a lower or no affinity for staining. In conclusion, we suggest that EGF plays an important role in ovarian folliculogenesis in cats.

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H. Sauerwein, A. Miyamoto, J. Günther, H. H. D. Meyer and D. Schams

Summary. The effect of insulin-like growth factors (IGFs) and insulin on the release of progesterone and oxytocin from bovine corpus luteum was investigated at early (days 5–7), mid- (days 8–12) and late (days 15–18) luteal phases of the oestrous cycle in an in vitro microdialysis system. The expression of specific receptors was evaluated in bovine corpora lutea of the respective luteal stages.

A 30 min infusion of IGF-1, IGF-2 (1·3, 13 and 130 nmol l−1) or insulin (13, 130 and 1300 nmol l−1) caused a stimulation of the release of progesterone (P < 0·05). IGF-1 was most effective in releasing progesterone. Oxytocin release from corpora lutea was stimulated by insulin at all doses tested (13-1300 nmol l−1), whereas the IGFs were only effective at the highest dose (130 nmol l−1) applied. The high doses of IGFs (130 nmol l−1) and insulin (1300 nmol l−1) stimulated the release of progesterone and oxytocin throughout the luteal phase (P < 0·05). For all three peptides, greatest stimulation was seen during the late luteal phase (days 15-18 of the oestrous cycle) with the peak of progesterone release directly related to peptide infusion (P < 0·05). In addition, IGF-1 stimulated total release of progesterone (units in 4 h) after the beginning of the stimulation during this phase (P < 0·05). IGF-1 caused a gradual increase of progesterone even beyond the time of peptide perfusion, whereas IGF-2 and insulin stimulated progesterone release only during the peptide perfusion.

Distinct receptors for IGF-1 and IGF-2 were present in corpora lutea membrane preparations at all stages investigated. Specific binding for insulin was also seen in all stages of the cycle without any cycle-dependent changes in the amount of binding. The displacement of labelled insulin by unlabelled IGF-1 and IGF-2 did not show the rank of order that has been described as typical for insulin receptors (i.e. insulin > IGF-1 > IGF-2), but comparable binding affinities were observed for the three unlabelled ligands. Specific binding of IGF-2 was markedly higher than that of IGF-1 or insulin throughout the cycle (1·9- and 4·9-fold higher compared with IGF-1 and insulin, respectively). Receptor specificity did not change during luteal development. Binding affinity and capacity of IGF-1 receptor was constant throughout the oestrous cycle. Specific IGF-2 binding increased and showed a positive co-operativity towards the end of the cycle. Specific binding of insulin was not significantly different in the three luteal stages examined.

Keywords: corpus luteum; IGFs; insulin; progesterone; oxytocin; cow

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H Kliem, H Welter, W D Kraetzl, M Steffl, H H D Meyer, D Schams and B Berisha

The corpus luteum (CL) offers the opportunity to study high proliferative processes during its development and degradation processes during its regression. We examined the mRNA expression of matrix metalloproteases (MMP)-1, MMP-2, MMP-9, MMP-14, MMP-19, tissue inhibitor of MMP (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), uPA-receptor (uPAR), PA-inhibitors (PAI)-1, PAI-2 in follicles 20 h after GnRH application, CLs during days 1–2, 3–4, 5–7 and 8–12 of the oestrous cycle as well as after induced luteolysis. Cows in the mid-luteal phase were injected with Cloprostenol and the CLs were collected at 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2α injection. Real-time RT-PCR determined mRNA expressions. Expression from 20 h after GnRH to day 12: MMP-1, MMP-2, MMP-14 and tPA showed a clear expression, but no regulation. TIMP-1 and uPAR mRNA increased when compared with the follicular phase. TIMP-2, MMP-9, MMP-19 and uPA increased from the follicular phase to days 8–12. PAI-1 and PAI-2 expression increased from days 1–7 and decreased to days 8–12. Induced luteolysis: MMP-1, MMP-2, MMP-9, MMP-14, MMP-19 and TIMP-1 all increased at different time points and intensities, whereas TIMP-2 was constantly decreased from 24 to 64 h. The plasminogen activator system and their inhibitors were up-regulated from 2 to 64 h, tPA was already increased after 0.5 h. Immunohistochemistry for MMP-1, MMP-2, MMP-14: an increased staining for MMP-1 and MMP-14 was seen in large luteal cells beginning 24 h after PGF2α application. MMP-2 showed a strong increase in staining in endothelial cells at 48 h.

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K. Jewgenow, L. M. Penfold, H. H. D. Meyer and D. E. Wildt

About 1500 preantral follicles can be recovered from a single cat ovary by mechanical dissection. This is a potentially rich source of genetic material if ova could be preserved and grown in vitro, especially from rare or endangered species that die abruptly or are ovariectomized for medical reasons. The aims of this study were to examine cryoprotectant toxicity and then the potential of successfully cryopreserving preantral cat follicles. In the initial toxicity trial, isolated cat follicles (40–90 μm) were exposed to dimethylsulfoxide, glycerol, 1,2-propandiol or ethylene glycol at 0°C for 15 min. Follicle viability was assessed by supravital staining using a combination of Trypan blue and Hoechst 33258 at 0 h, and after 18 h and 1 week of culture. Percentages of follicles with intact oocytes and granulosa cells were similar (P >0.05) among control (no cryoprotectant), dimethylsulfoxide, 1,2-propandiol and ethylene glycol treatments at all time points, but were reduced (P <0.05) after glycerol exposure. On the basis of this finding, dimethylsulfoxide and 1,2-propandiol were used to cryopreserve intact follicles, and post-thaw viability was assessed by supravital staining and 5-bromo-2′-deoxyuridine uptake into oocytes and granulosa cells during culture. Of control (noncryopreserved) follicles, 31.4% ± 2.9%, 18.8% ± 1.9% and 16.2% ± 1.6% were intact after 0 h, 18 h and 1 week of culture, respectively. Uptake of 5-bromo-2′-deoxyuridine occurred in approximately 20% of follicles at all time points. On the basis of the presence of both a healthy oocyte and granulosa cells, cryopreservation in dimethylsulfoxide or 1,2-propandiol allowed approximately 19% of follicles to survive. Approximately 10% demonstrated clear evidence of cell activity that was sustainable for 1 week. In conclusion, the cat ovary contains a population of preantral follicles that are not adversely affected by short-term exposure to most conventional cryoprotectants. Furthermore, there is a subpopulation of these follicles capable of surviving cryopreservation, remaining structurally intact and physiologically active after thawing.

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S E Ulbrich, K Schulke, A E Groebner, H D Reichenbach, C Angioni, G Geisslinger and H H D Meyer

Prostaglandins (PGs) are important regulators of reproductive processes including early embryonic development. We analyzed the most relevant PG in bovine uteri at different preimplantation pregnancy stages when compared with non-pregnant controls. Additionally, endometrium and trophoblast tissues were examined regarding specific enzymes and receptors involved in PG generation and function. Simmental heifers were artificially inseminated or received seminal plasma only. At days 12, 15, or 18, post-estrus uteri were flushed for PG determination by liquid chromatography–tandem mass spectrometry. Endometrium and trophoblast tissues were sampled for RNA extraction and quantitative real-time PCR analysis. At all days and points of time examined, the concentration of 6-keto PGF (stable metabolite of PGI2) was predominant followed by PGF>PGE2>PGD2≈TXB2 (stable metabolite of TXA2). At days 15 and 18, PG increased from overall low levels at day 12, with a much more pronounced increase during pregnancy. The PGF/PGE2 ratio was not influenced by status. The highest PG concentration was measured at day 15 with 6-keto PGF (6.4 ng/ml) followed by PGF (1.1 ng/ml) and PGE2 (0.3 ng/ml). Minor changes in endometrial PG biosynthesis enzymes occurred due to pregnancy. Trophoblasts revealed high transcript abundance of general and specific PG synthases contributing to uterine PG. As PGI2 and PGF receptors were abundantly expressed by the trophoblast, abundant amounts of PGI2 and PGF in the uterine lumen point towards an essential role of PG for the developing embryo. High amounts of PG other than PGE2 in the preimplantation uterus may be essential rather than detrimental for successful reproduction.

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Dieter Schams, Vera Steinberg, Martin Steffl, Heinrich H D Meyer and Bajram Berisha

The aim of this study was to investigate the possible participation of fibroblast growth factor (FGF) family members (FGF1, FGF2 and FGF7 and their receptors) in porcine follicles (polyovulatory species) under special consideration for FGF2 during final growth. A classification of follicles was done by size and follicular fluid content of oestradiol-17β, progesterone and prostaglandin F2α. The mRNA expression of examined factors was analysed by real-time PCR. The hormone concentration was estimated by enzyme immunoassay, protein characterisation by western blotting and localisation by immunohistochemistry. Follicle tissue separated in theca interna and granulosa cells was extracted and tested for mRNA of FGF1, FGF2, FGF7 and receptors (FGFR1IIIc, FGFRIIIb and FGFR2IIIc). Additionally, the mRNA expression of FSHR, LHR and aromatase cytochrome P450 for further characterisation of follicles was analysed. Significantly, higher FGF2 protein levels were measured in stroma when compared with total follicle or corpus luteum tissue. This result was confirmed by western blot with two strong bands. Immunological localisation of FGF2 only in stroma (fibroblasts) confirms the protein measurements. The results show a clear difference for FGF2 protein expression during final growth of follicles if monovulatory (bovine) and polyovulatory (porcine) species are compared. FGF2 protein in porcine ovary may be (due to localisation and concentration in stroma) important for support of angiogenesis of more follicles (polyovulatory species) and not of a single follicle like in cows.

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Anna E Groebner, Isabel Rubio-Aliaga, Katy Schulke, Horst D Reichenbach, Hannelore Daniel, Eckhard Wolf, Heinrich H D Meyer and Susanne E Ulbrich

Amino acids (AAs) are crucial for the developing conceptus prior to implantation. To provide insights into the requirements of the bovine embryo, we determined the AA composition of the uterine fluid. At days 12, 15, and 18 post-estrus, the uteri of synchronized pregnant and non-pregnant Simmental heifers were flushed for the analysis of 41 AAs and their derivatives by liquid chromatography–tandem mass spectrometry. The ipsilateral endometrium was sampled for quantitative PCR. In addition to a pregnancy-dependent increase of the essential AAs (P<0.01), we detected elevated concentrations for most non-essential proteinogenic AAs. Histidine (His) and the expression of the His/peptide transporter solute carrier 15A3 (SLC15A3) were significantly increased at day 18 of pregnancy in vivo. In addition, SLC15A3 was predominantly stimulated by trophoblast-derived interferon-τ in stroma cells of an in vitro co-culture model of endometrial cells. Our results show an increased concentration of AAs most likely to optimally provide the elongating pre-attachment conceptus with nutrients.

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Miguel A Velazquez, Klaus-Gerd Hadeler, Doris Herrmann, Wilfried A Kues, Susanne Ulbrich, Heinrich H D Meyer, Benoît Rémy, Jean-François Beckers, Helga Sauerwein and Heiner Niemann

The present study investigated the role of IGF1 in lactating lean and non-lactating obese dairy cows by injecting 1 μg IGF1 into the ovaries prior to superovulation. This amount of IGF1 has been linked with pregnancy loss in women with the polycystic ovary syndrome (PCOS) and was associated with impaired bovine oocyte competence in vitro. Transcript abundance and protein expression of selected genes involved in apoptosis, glucose metabolism, and the IGF system were analyzed. Plasma concentrations of IGF1 and leptin, and IGF1 in uterine luminal fluid (ULF), were also measured. IGF1 treatment decreased embryo viability in lean cows to the levels observed in obese cows. Obese cows were not affected by IGF1 treatment and showed elevated levels of IGF1 (in both plasma and ULF) and leptin. Blastocysts from lean cows treated with IGF1 showed a higher abundance of SLC2A1 and IGFBP3 transcripts. IGF1 treatment reduced protein expression of tumor protein 53 in blastocysts of lean cows, whereas the opposite was observed in obese cows. IGF1 in plasma and ULF was correlated only in the control groups. Blastocyst transcript abundance of IGF1 receptor and IGFBP3 correlated positively with IGF1 concentrations in both plasma and ULF in lean cows. The detrimental microenvironment created by IGF1 injection in lean cows and the lack of effect in obese cows resemble to a certain extent the situation observed in PCOS patients, where IGF1 bioavailability is increased in normal-weight women but reduced in obese women, suggesting that this bovine model could be useful for studying IGF1 involvement in PCOS.