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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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T. G. Cooper, C. H. Yeung, R. Meyer and H. Schulze

Summary. Cells liberated by enzyme treatment of tubules dissected out of human epididymides obtained at castration for prostatic carcinoma were cultured for up to 42 days on permeable supports. Outgrowth and monolayer formation was unrelated to the age of the patient or his treatment with anti-androgens. The cells comprising the monolayer were cuboidal and shorter than those in situ but their ultrastructure was characteristic of epithelial cells. There was no evidence of smooth muscle cells or fibroblast overgrowth. The cells manifested fluid-phase and adsorptive endocytosis from both apical and baso-lateral aspects and released into the medium alkaline and acid phosphatases and N-acetylglucosaminidase.

Keywords: man, epididymis; cell culture; ultrastructure; endocytosis; secretion

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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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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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Progesterone and pregn-5-ene-3,20-dione, the proposed intermediate between pregnenolone and progesterone in the rat ovary, were able to overcome the phenobarbital (PB)-induced block of ovulation in the PMSG-primed immature rats when given 3½ hr after PB.

Pregnenolone failed to cause ovulation in any of the animals treated with phenobarbital, providing support for the hypothesis that the ovulation-inhibiting action of PB may be due in part to its inhibitory action on the step in steroidogenesis between pregnenolone and progesterone.

The principal metabolites of progesterone in the hypothalamus and uterus, 5α-pregnane-3,20-dione and 3α-hydroxy-5α-pregnan-20-one, were not able to overcome the PB-induced block of ovulation. These results suggest that the action of progesterone in overcoming the PB-induced block of ovulation is not because of its transformation to these two compounds.

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The facilitative effects of certain progestational steroids on ovulation were investigated by using 25-day-old female rats which had been treated on Day 22 with a non-ovulatory dose of PMSG (12 i.u.). Ovulation was caused by treatment with pregnenolone or progesterone on the morning of Day 24. The dose of pregnenolone required was higher than that of progesterone. Progesterone had this effect throughout the morning of Day 24, while prenenolone was only effective between 07.00 and 10.00 hours on Day 24. The two metabolites of progesterone in the hypothalamus and uterus, 5α-dihydroprogesterone and 3α-hydroxy-5α-pregnan-20-one, were also tested for their ability to cause ovulation. Although 5α-dihydroprogesterone possessed this ability, a dose three to four times that of progesterone was required to produce a comparable effect. Doses of up to 1·5 mg 3α-hydroxy-5α-pregnan-20-one were without effect. Neither 17α-hydroxypregnenolone nor 17α-hydroxyprogesterone had any effect in influencing ovulation.

Pregn-5-ene-3,20-dione had a facilitative ability comparable to that of progesterone in doses of 0·25 mg or higher. The facilitative effect of progesterone decreased when it was injected earlier on Day 24 or during the evening of Day 23; no potentiating effect was obtained at 14.00 hours on Day 23. The results obtained with pregnenolone, progesterone and related steroids support the hypothesis that the inhibition of ovulation by phenobarbital may be due in part to its interference with the conversion of pregnenolone to pregn-5-ene-3,20-dione.

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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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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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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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J. F. Quirke, H. H. Meyer, A. Lahlou-Kassi, J. P. Hanrahan, G. E. Bradfords and G. H. Stabenfeldt

Summary. Ovulation rate, in mixed-age groups of prolific and non-prolific ewe breed types, after administration of a range of doses of PMSG (0, 375, 750 and 1500 i.u.) during the follicular phase of the oestrous cycle, were compared in Ireland, Morocco and New Zealand. The ewes in Ireland and Morocco were from the Finnish Landrace and Galway, and D'Man and Timhadite breeds, respectively. In New Zealand Booroola Merino × Romney ewes which had been previously identified as heterozygous carriers (F +) of the Booroola high fecundity gene and purebred Romneys were used to represent the prolific and non-prolific genotypes respectively; in addition a group of Booroola Merino × Romney non-carriers (++) of the major gene were also included for comparison. Ovulation rate at the oestrus which preceded stimulation with PMSG was also measured in all animals.

In all 3 locations the ewes of the prolific genotype had a greater ovulation rate after PMSG stimulation than did the non-prolific controls. However, this association between prolificacy and response to PMSG was removed when ovulation rate after PMSG was transformed by dividing by the ovulation rate observed before PMSG administration. Despite the differences in the genetic basis of their high prolificacy the pattern of response to PMSG over the range of dosages used was similar in Finnish Landrace, D'Man and Booroola Merino × Romney (F +) ewes and all breeds had means of about 10 ovulations in response to 1500 i.u. PMSG. Amongst the non-prolific breeds, the Timhadite was the most responsive to PMSG although it had the lowest natural ovulation rate.

Information on fertility and litter size was available for all breeds in Ireland and New Zealand. In all breeds fertility was depressed at the highest dose of PMSG. Litter size at birth increased in all non-prolific breeds as the dose of PMSG was raised. For the prolific breeds, increasing the dose of PMSG from 750 to 1500 i.u. resulted in a substantial reduction in the number of lambs born.