Cyproterone acetate reduced antler growth in surgically castrated fallow deer.

We studied the role of androgens in antler growth. In particular, we investigated whether the onset of antler regrowth is triggered by a short-term pulse of testosterone and if low levels of androgens are required for antler growth. The study was conducted on 12 surgically castrated fallow deer bucks (Dama dama) aged approximately 27 months. Six animals (CA group) were given the antiandrogen, cyproterone acetate (CA, 1000 mg/treatment); the others were given vehicle solution only (control). Before each CA treatment, blood was sampled and analysed for testosterone, androstenedione, IGF-1, cortisol, FSH, and LH. CA treatment and blood sampling were performed 2 days before castration, on the day of castration and afterwards at 2-day intervals until day 22. Subsequently, CA treatment and blood sampling continued at weekly intervals until day 270. All animals cast their antlers, followed by antler regrowth in all control bucks, but in only four of the six CA-treated castrates. Plasma testosterone concentrations were low in all animals (between 0.01 and 0.20 ng/ml), but were significantly (P<0001) greater in the controls. In both groups, a temporary increase in testosterone values was recorded around the time of antler regrowth, the peak being significantly (P<0.01) higher in the controls. Androstenedione showed a similar pattern as testosterone. Plasma IGF-1 concentrations increased sharply during the antler growth spurt and did not differ significantly between the two groups throughout the study period. Cortisol concentrations were greater in controls than in the CA group. However, no link with the antler cycle was apparent. FSH and LH concentrations were higher in the controls for most of the study. Antlers produced by the control bucks were significantly larger than those in the CA group (P<0.03). For antler length, testosterone, androstenedione and IGF-1, areas under the curve (AUC) were calculated over the period of antler growth. For the pooled deer (n=12) significant correlations existed between AUCs of antler length and testosterone, but not for antler length and IGF-1. Also, a trend for a positive correlation between AUCs of antler length and androstenedione was noted. It is concluded that a plasma androgen concentration at least above a minimal threshold level is a necessary prerequisite for normal antler regrowth in fallow deer, and that this androgen effect is not mediated via circulating IGF-1. The biological role of low levels of androgens may be to sensitize antler cells to the stimulating effect of IGF.

Genome reactivation in developing early pig embryos: an ultrastructural and autoradiographic analysis.

The onset of RNA synthesis in developing early pig embryos from 1-cell to 8-cell and morula stages was studied using high-resolution autoradiography of (5-3H)uridine incorporation. No transcriptional activity was detected in nuclei of 1- and 2-cell stage embryos with this technique. In these embryos nucleolus-like bodies (NLB) consist of sharply delineated, round, electron dense fibrillar masses. In the 4-cell stage embryos, the first uridine-3H incorporation in the nucleoplasm was detected and localized mainly near the regions of condensed chromatin. The first signs of reticulation and chromatin association were observed at the periphery of NLBs. In the next cell cycle (5- to 8-cell embryos) uridine-3H labelling was detected in the nucleoplasm and nucleoli. In these embryos, nucleoli consist of a central dense fibrillar mass without any transcriptional activity and fibrillo-granular cortex over which label was localized. The degree of functional restructure of nucleoli was variable within one blastomere or among different blastomeres, some nucleoli being more reticulated and showing more transcriptional activity than others. Fully developed nucleoli were present in early morulae. Electron dense unidentified structures described here as small dense round-shaped bodies (RDB) often surrounded by blocks of large chromatin granules were observed in intact 2-cell and alpha-amanitin treated 4-cell stage embryos. These structures did not show any transcriptional activity.

Localization of estrogen receptors in rhesus monkey ovary.

We have previously demonstrated that the exogenous administration of estradiol-17ß (E2) to rhesus monkeys induces atresia of the dominant preovulatory follicle (DF); and that this effect is mediated centrally, via the inhibition of follicle-stimulating hormone, and is also exerted directly at the level of the ovarian granulosa cell. We wished to investigate whether the local effect of E2 is transduced through interaction with the nuclear receptor for estrogen, particularly in light of certain evidence that suggests a general lack of estrogen receptor (E-R) in the rhesus monkey ovary, except in the germinal epithelium. In the present study, we evaluated the presence of E-R by both autoradiographic and immunocytochemical techniques. Frozen sections of ovaries from rhesus females were incubated in experiment 1 with either 3H-E2 or 125I-E2, in the presence or absence of excess, non-radioactive ligand or analogues diethylstilbestrol (DES) or the receptor antagonist 4-OH-tamoxifen (TAM). 3H-E2 binding was most intense over functional corpora lutea, and was reduced to background with excess DES; label was also evident over antral follicles, Image analysis showed specific binding of 125I-E2 by ovaries. In experiment 2, cryostat sections were processed for immunocytochemical staining using the per-oxidase-anti-peroxidase (PAP) method and the H222 monoclonal antibody to the E-R. Intense, specific label was observed over nuclei of germinal epithelium, but, additionally, for the first time, to granulosa cells of antral follicles and other compartments of the ovary. In this paper, we report the first evidence for estrogen binding to rhesus monkey ovary; tins binding is specific, apparently receptor mediated, and corroborated independently by autoradiographic and immunocytochemical means. We herein provide substantial support for estrogen's dramatic effects being exerted directly at the level of the monkey ovary. © 1993 Wiley-Liss, Inc.

Prolactin and insulin levels in lactating sows in relation to nursing frequency.

It has been established that sows up- or down-regulate their milk production as the frequency of nursings is changed. The amount of udder massage by piglets might also influence milk production. To investigate whether these effects are associated with changes in prolactin or insulin levels, we enforced five sows each to nurse either every 35 min (MIN35) or every 70 min (MIN70) over a 26- to 28-hr period. Milk production was measured during the first 24 hr of this period. During the last three to four nursings, blood was collected every 5 min. Plasma prolactin levels increased after milk ejection (P < 0.05), whereas insulin levels increased only briefly in MIN70 sows. Sows nursing every 35 min had lower basal (P < 0.001) and maximal (P < 0.05) concentrations of insulin than MIN70 sows. There were no differences between the two groups in prolactin levels. Nursings with a postejection udder massage longer than 90 s tended to induce a higher increase in prolactin (P < 0.1) than nursings with a shorter massage. When the effects of imposed nursing frequency were removed, there was an across-sows positive residual correlation between average prolactin levels (P < 0.05) and the duration of post-ejection udder massage during the preceding 24 hr. We conclude that when milk production of a sow is changed by altering the nursing frequency within natural limits, the necessary alteration in catabolic state of energy metabolism may be associated with altered insulin levels. The duration of udder massage in a single nursing might have only a slight immediate impact on prolactin levels, but may influence prolactin levels more substantially if increased for a period of 24 hr.

Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine.

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3H-thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24h resulted in proliferation arrest in vitro. Treatment with 0.5mM mimosine significantly (P<0.05) inhibited 3H-thymidine incorporation after 24h of culture (4.6% +/- 0.1) and after 24h of culture in serum deprived medium (41.3% +/- 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.

Activation of in vitro matured pig oocytes using activators of inositol triphosphate or ryanodine receptors.

In our study, we observed the activation of in vitro matured pig oocytes and their subsequent parthenogenetic cleavage after stimulation of ryanodine receptors (RyR) using ryanodine (Ry), caffeine or cyclic adenosine diphosphate ribose (cADPri) or after stimulation of inositol triphosphate receptors (IP(3)R) using D-myo-inositol 1,4,5-triphosphate (IP(3)). Heparin, a potent blocker of IP(3)R, prevented the activation of porcine oocytes using IP(3), but blockers of RyR (ruthenium red or procaine) prevented activation after stimulation by RyR and stimulation by IP(3)R using IP(3). The drugs were injected into oocytes matured to the stage of metaphase II and activation was determined by assessment of pronuclear formation. The activity of H1 kinase was determined and our results demonstrated a significant drop in H1 activity in the activated oocytes. The cleavage of parthenogenetic embryos progresses to more advanced stages after stimulation by IP(3)R than after stimulation by RyR. Our results could indicate that, in pig oocytes, the calcium released from IP(3)-sensitive stores triggers the calcium release from ryanodine-sensitive intracellular stores, which is necessary for oocyte activation. The calmodulin inhibitors ophiobolin A and W7 reduce the activation of oocytes induced by stimulation of RyR or IP(3)R.

Ultrastructural and morphometric analysis of cytoplasmic structures in preimplantation pig embryos.

Preimplantation pig embryos (1-, 2-, 3/4-, 16-cell stage, blastocysts) were analysed morphologically and/or morphometrically by means of electron microscopy and computer-assisted image analysis. Qualitative and quantitative changes in localization, organization, number, and area of cytoplasmic inclusions were analysed on thin sections. The structure and number of mitochondria, number of endoplasmic reticulum profiles were recorded in several individual developmental stages. Three types of cytoplasmic inclusions were described (vesicles with yolk globules, vesicles with homogeneous light inclusions, lipid droplets), each having a different topographic distribution. Quantitatively, a gradual increase in the number of endoplasmic reticulum profiles and mitochondria occurred between 4-cell stage blastomeres and blastocyst trophectoderm cells. The significant differences recorded in number, morphology and distribution of organelles in different embryonic developmental stages are discussed in relation to physiological phenomena known to occur during cleavage and compaction.

Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production.

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.

Characterization of the periovulatory period in superovulated heifers.

Heifers (n=31) were superovulated with an FSH-P/cloprostenol regimen, and at 12 and 24 hours after the onset of estrus they were inseminated. Blood sampling for LH analyses and ultrasound scanning of the ovaries were performed at 4-hours intervals. The scanning, at which the first and last ovulations were recorded, was performed at 22.7 +/- 1.5 (mean +/- SD) and 31.0 +/- 1.5 hours after the LH peak, respectively. An average of 7.8 +/- 1.0 ovulations was monitored when the first ovulations were detected, while 2.8 +/- 0.7 ovulations occurred later. At 16 hours after detection of the first ovulations the oviducts were flushed and 5.6 +/- 0.5 fertilized and 2.3 +/- 0.3 unfertilized ova were isolated per animal. The fertilized ova displayed spherical pronuclei of synchronous development, and polyspermic penetration was not seen. At 24 hours after detection of the first ovulations the content of the remaining 3.3 +/- 0.5 nonovulatory follicles > 8 mm per animal was aspirated. Expanded cumulus investment was found in 69.4% of the oocytes, while 22.4% had abstricted the first polar body.

[Transfer of cattle embryos in the induction of twinning]. / Prenos embryí hovädzieho dobytka pri riadenej produkcii dvojiciek.

The superovulation of donors, heifers and cows, was induced by means of gonadotropin PMSG (Antex Leo) in combination with the synthetic prostaglandin analogue--PGF2 alpha (Estrumate, ICI) applied 48 hours later. The response of donors to this treatment was variable. The average number of ovulating follicles from one heifer was fourteen, and the number of embryos to be used was four. In cows the average number of ovulating follicles amounted to six, and on the average three embryos from one donor could be used. Seventy-four embryos were transferred to thirty-seven recipients, into each horn of uterus one embryo. Twenty-six (70%) cows were pregnant, out of this number seventeen (65%) recipients gave birth to twins. In some cases the isolated embryos were kept in the oviduct of female rabbit for 72 hours. It was proved that this deposition did not exert any negative influence on further development of embryos.

Absence of maternal cell contamination in mesenchymal stromal cell cultures derived from equine umbilical cord tissue.

This study aimed to determine whether maternal cell contamination exists in cells derived from equine umbilical cord tissue, a perspective material for cell-based therapies in veterinary medicine. Potential maternal cell contamination was analyzed at DNA level via a set of 16 microsatellite markers in cells originating from the cord tissue of 22 foals. In these cells no maternal cell contamination was detected at a sensitivity level of 0.01%. Our results suggest that equine umbilical cord tissue-derived cells are entirely of fetal origin.

Preweaning housing effects on behavior and physiological measures in pigs during the suckling and fattening periods.

The effect of the preweaning housing system on the stress response of pigs before weaning and during fattening was studied in 33 litters of domestic pigs. Three preweaning housing systems were compared: barren crate (standard farrowing crate without straw), enriched crate (20% larger crate, with straw), and as a control, a farrowing pen (pen, 60% larger than the barren crate, with straw). At 25 d of age, pigs were tested with an isolation test and 1 d later with a human approach test (HumanT). Pigs were weaned at 28 d of age. At 3 and 6 mo of age, pigs were tested with an isolation-human approach test. The latency and frequency of squeal calls and locomotor activity were analyzed for all 3 tests, whereas physical contact with the human was also analyzed for the HumanT and isolation-human approach test. At 6 mo of age, the pigs were transported to a slaughterhouse. One day before transport, immediately after transport, and 1 h after transport, saliva samples were taken for cortisol analysis. The pH of the LM was also measured 45 min after slaughter. Preweaning housing system affected (P < 0.05) the probability of squeal vocalizations, the latency of locomotion, and the duration of locomotion during the HumanT. Pigs from the enriched pens vocalized less, had a longer latency to move, and performed less overall locomotion than pigs from the barren crates. Preweaning housing system did not affect behavior of fattening pigs. Cortisol concentrations before and after transport were not affected by preweaning housing system. An interaction of cortisol concentrations and housing systems was observed between the control sample and the sample taken immediately after transport in pigs from the barren crates (P < 0.05) compared with pigs from the enriched housing systems. Meat from pigs reared in the barren crate tended to have lower pH (P < 0.10) and that of pigs reared in enriched crates had lower pH (P < 0.05) than meat of pigs reared in enriched pens. No differences were observed between pigs from barren or enriched crates. Our results suggest that enrichment of the preweaning environment through enlarged space, provision of straw, and free movement for the sow had a positive effect on the coping behavior of pigs before weaning and prevented an increase in salivary cortisol concentration immediately after transport and a decrease in meat pH 45 min postmortem at the age of 6 mo. Minimal enrichment of the commercial farrowing crate did not affect behavior and physiological measures in pigs before and after weaning.

Nitric-oxide-dependent activation of pig oocytes: the role of the cGMP-signalling pathway.

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.

Effects of cell cycle dependent kinases inhibitor on nuclear and cytoplasmic maturation of porcine oocytes.

The aims of this study were to assess the effectiveness of roscovitine, a potent inhibitor of cell cyclin kinases, to prevent meiotic resumption in porcine oocytes, and to test the subsequent fertilisability and developmental competence of these oocytes. Roscovitine blocked porcine oocytes at the GV stage during 22-44 hr of culture. This effect was dose-dependent, and a concentration of 25 microM was sufficient to prevent meiotic resumption in 92+/-5% of the oocytes after 22 hr in the presence of EGF and FSH. Cumulus expansion was also inhibited under these conditions. The histone H1 kinase activity in oocytes was inhibited in a dose-dependent way, and maintained at a basal level with 25 microM of roscovitine. Synthesis of proteins of 29, 47 and 79 kDa, normally synthesized during maturation, was inhibited too. All these effects were fully reversible. However, the kinetics of maturation were accelerated after roscovitine removal, and the acceleration was more pronounced after 44 hr of inhibition than after 22 hr. Fertilization of oocytes blocked for 22 hr before a 44 hr maturation was decreased compared to control, but was not different from that of oocytes matured for 66 hr. The developmental competence was decreased for the oocytes cultured for 66 hr, including or not an inhibition period, but it was less reduced for oocytes maintained under inhibition for 22 hr. Roscovitine may thus protect oocytes against the aging mechanisms responsible for developmental competence loss, but not against loss of fertilisability. In conclusion, roscovitine provides a useful tool to study the morphological and biochemical basis of porcine oocyte terminal differentiation.

High developmental competence of cattle oocytes maintained at the germinal vesicle stage for 24 hours in culture by specific inhibition of MPF kinase activity.

Roscovitine, a potent inhibitor of M-phase Promoting Factor (MPF) kinase activity, was used to maintain cattle oocytes at the germinal vesicle stage for a 24-hr culture period. A concentration of 25 microM of roscovitine was sufficient to reach the maximum level of meiotic resumption inhibition with 83 +/- 6% of the oocytes remaining at the germinal vesicle stage after the 24 hr of culture. The histone H1 kinase activity was maintained at a basal level after culture under roscovitine inhibition at any of the concentrations tested (12.5, 25, 50, and 100 microM). This inhibitory effect of roscovitine was fully reversible since 89 +/- 4% of the oocytes cultured for 24 hr in the presence of 25 microM of roscovitine reached the metaphase II stage after a further culture of 24 hr in permissive medium (TCM199 supplemented with 10 ng/ml EGF). The cleavage rate as well as the development to the blastocyst stage was not different for oocytes cultured for 24 hr under roscovitine (25 microM) inhibition and then matured for 24 hr in the presence of EGF as compared to oocytes not submitted to prematuration culture (82 +/- 8% cleavage and 41 +/- 4% blastocysts at 8 days post insemination for control oocytes compared to 90 +/- 7% and 36 +/- 7% respectively for roscovitine-treated oocytes). Roscovitine meiotic inhibition was also effective in the presence of EGF, and the final developmental potential as well as the kinetics of blastocyst formation were not affected after such prematuration treatment. The EGF induced cumulus expansion was also inhibited by roscovitine. These results indicate for the first time the feasibility of culturing cattle oocytes under meiotic inhibition without decreasing their resulting developmental potential.

Estrogen receptor protein and mRNA expression in the ovary of sheep.

Using immunohistochemistry and in situ hybridization, we attempted to identify the estrogen receptor (ER) protein and messenger RNA (mRNA) in sheep ovaries during the follicular phase of the estrous cycle. Monoclonal anti-ER antibodies H222 and 1D5 were used for localizing estrogen receptor on ovarian cryo-sections. Labeling for ER was found over the nuclei of surface epithelium, interstitial tissue, and granulosa cells of small as well as large ovarian follicles. In the preantral and small antral follicles, intense nuclear ER labeling was observed in mural granulosa cells and particularly in cumulus/granulosa cells surrounding the oocyte. In the large healthy looking follicles, greater diversity in labeling for ER was observed, which is characterized by mixed populations of granulosa cells expressing positive and more or less negative nuclear labeling. Such a pattern of labeling was particularly evident in follicles showing the signs of atresia. Generally, more intense nuclear staining was localized in granulosa cells proximal to basal membrane. In situ hybridization studies revealed the presence of ER mRNA in ovarian tissue. Autoradiographic visualization localized ER mRNA expression over the granulosa cells of healthy follicles of all sizes. Level of hybridization signal was comparable in mural and cumulus granulosa cells. In atretic follicles, the level of hybridization signal in granulosa cells was comparable to that of healthy follicles. A relatively weaker level of labeling was observed in granulosa cells dispersed in follicular antrum in follicles with advanced atretic lesions. Theca cells expressed a lower level of labeling than granulosa cells. Specificity of labeling for both ER protein and mRNA in ovary was proved by parallel probing the ovine uterus. Ovine ER recognition by both H222 and 1D5 antibodies was also proved by immunoblotting. These studies demonstrate the presence of the estrogen receptor and its messenger RNA in the sheep ovary and suggest an autocrine/paracrine role of estradiol and its receptor in the regulation of ovarian follicle development in sheep.

Chemically enucleated mouse oocytes: ultrastructure and kinetics of histone H1 kinase activity.

The objective of the study was to characterize the ultrastructure changes and biochemical mechanisms underlying the expulsion of the entire chromosome complement in chemically enucleated mouse oocytes. The ultrastructural studies demonstrated that the morphology of cytoplasts produced by etoposide-cycloheximide treatment were indistinguishable from intact metaphase I and II oocytes. Moreover, polar bodies formed by chemical enucleation were in almost all cases completely separated from the parent cytoplast and differed from normal polar bodies only in their chromatin content morphology and because they contained a slightly higher number of cytoplasmic organelles. The mode of polar body formation, however, in normal and chemically enucleated oocytes differs substantially: spindle involvement is important for normal polar body extrusion but plays no part in the protracted expulsion of chromosomes during chemical enucleation. After etoposide-cycloheximide treatment, histone H1 kinase activity remains high for the ensuing 6-8 h before declining gradually to basal levels 14 h after treatment. The expulsion of the polar body occurred only after the slowly declining H1 kinase activity reached basal levels. The activity of this kinase rose sharply to reach maximal levels within 4 h when the enucleated oocytes were removed from the inhibitor-supplemented medium and placed in normal medium. The findings in this paper indicate that cytoplasts produced by chemical enucleation are morphologically normal, thus suggesting that these enucleated cells are suitable for cloning studies. Although effective in mouse oocytes, we postulate that certain modifications to the enucleation technology are necessary before a reliable non-invasive protocol for ungulate oocytes will be available.

Autoradiography of 3H-uridine incorporation in the normal early blastocysts of cattle.

RNA synthesis in morphologically normal early bovine blastocysts collected 7 days after insemination was investigated using fine-structure autoradiography after 3H-uridine incorporation. The level of autoradiographic labelling is proposed as a criterion for assessing metabolic activity in the individual cells of the blastocyst. There was a detectable difference between the cells of the trophoblast and those of the inner cell mass (higher labelling), indicating a divergence of metabolic patterns according to blastomere differentiation. The occasional absence of nuclear labelling was correlated with cell degeneration. A low nuclear labelling was observed mainly in cells sequestered in the subzonal space or in the blastocoele cavity, respectively. Estimation of RNA synthesis in different blastomeres by fine structure autoradiography opens the way to a better interpretation of the relationship between morphology and metabolism during the development of early cow embryos.

[Control of terminal follicular maturation during the follicular phase in domestic mammals]. / Contrôle de la maturation terminale des follicules au cours de la phase folliculaire chez les mammifères domestiques.

Terminal follicular maturation in the ovine and the bovine species involves growth and differentiation processes in follicles between 1-2 mm diameter and the preovulatory stage. During this maturation, the follicle acquires the ability to ovulate and the oocyte becomes able to be fertilized and to develop after fertilization. Selection of ovulatory follicles results from the integration of different parameters such as the circulating levels of gonadotropins, the structure of follicular populations and the sensitivity of the hypothalamo-pituitary axis to ovarian hormones. Differences between follicles for FSH and LH responsiveness can be amplified by paracrine intrafollicular regulations. These mechanisms are probably determinant for selection of ovulatory follicles.