Regulation of phosphoenolpyruvate carboxykinase and tyrosine transaminase in hepatoma cell cultures. 3. Comparative studies in H35, HTC, MH1C1 and RLC cells.

The ability of N(6), O(2)'-dibutyryl cyclic AMP (DBcAMP) to regulate a number of metabolic events in four lines of cultured rat hepatomas has been examined. Although dexamethasone induces tyrosine transaminase in all four lines, DBcAMP induces this enzyme normally only in H35 cells. A slight increase in transaminase activity was seen with MH(1)C(1) cells and HTC cells, but no effect was detectable in RLC cells. In contrast, phosphoenolpyruvate carboxykinase activity is increased by both agents in H35 and MH(1)C(1) cells, but neither had any effect in HTC or RLC cells. DBcAMP caused a rapid inhibition of the growth rate and DNA synthesis and an increase in protein content in both H35 and MH(1)C(1) cells but not in HTC or RLC cells. The effect of DBcAMP on DNA synthesis in MH(1)C(1) cells could be reversed by deoxycytidine as is also the case with H35 cells. The resistance of HTC and RLC cells to DBcAMP was not due to reduced uptake or deacylation as judged by studies with [(3)H]DBcAMP. The cyclic nucleotide appears to enter the cells by passive diffusion as the intracellular concentration approaches that in the medium within 30-60 min. Possible explanations for the differential responses observed are discussed.

DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages.

The main goal of this study was to investigate whether and at what level damage of paternal DNA influences fertilization of oocytes and early embryonic development. We hypothesized that posttesticular sperm DNA damage will only marginally affect sperm physiology due to the lack of gene expression, but that it will affect embryo development at the stage that embryo genome (including the paternal damaged DNA) expression is initiated. To test this, we artificially induced sperm DNA damage by irradiation with x- or gamma rays (doses of 0-300 Gy). Remarkably, sperm cells survived the irradiation quite well and, when compared with nonirradiated cells, sperm motility and integrity of plasma membrane, acrosome, and mitochondria were not altered by this irradiation treatment. In contrast, a highly significant logarithmic relation between irradiation dose and induced DNA damage to sperm cells was found by both terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and the acridin orange assay. Despite the DNA damage, irradiated sperm cells did not show any sign of apoptosis (nuclear fragmentation, depolarization of inner mitochondrial membranes, or phospholipid scrambling) and were normally capable of fertilizing oocytes, as there was no reduction in cleavage rates when compared with nonirradiated sperm samples up to irradiation doses of less than 10 Gy. Further embryonic development was completely blocked as the blastocyst rates at days 7 and 9 dropped from 28% (nonirradiated sperm) to less than 3% by greater than 2.5-Gy-irradiated sperm. This block in embryonic development was accompanied with the initiation of apoptosis after the second or third cleavage. Specific signs of apoptosis, such as nuclear fragmentation and aberrations in spindle formation, were observed in all embryos resulting from in vitro fertilization with irradiated sperm (irradiation doses >1.25 Gy). The results show that sperm DNA damage does not impair fertilization of the oocyte or completion of the first 2-3 cleavages, but blocks blastocyst formation by inducing apoptosis. Embryos produced by assisted reproductive techniques (ART) could have incorporated aberrant paternal DNA (frequently detected in sperm of sub/infertile males). Analogously, in the present work, we discuss the possibility of following embryo development of oocytes fertilized by ART through the blastocyst stage before embryo transfer into the uterus in order to reduce risks of reproductive failure.

Effects of recombinant human follicle stimulating hormone on follicle development and ovulation in the mare.

The only gonadotrophin preparation shown to stimulate commercially useful multiple ovulation in mares is equine pituitary extract (EPE); even then, the low and inconsistent ovulatory response has been ascribed to the variable, but high, LH content. This study investigated the effects of an LH-free FSH preparation, recombinant human follicle stimulating hormone (rhFSH), on follicle development, ovulation and embryo production in mares. Five mares were treated twice-daily with 450 i.u. rhFSH starting on day 6 after ovulation, coincident with PGF(2alpha) analogue administration; five control mares were treated similarly but with saline instead of rhFSH. The response was monitored by daily scanning of the mares' ovaries and assay of systemic oestradiol-17beta and progesterone concentrations. When the dominant follicle(s) exceeded 35 mm, ovulation was induced with human chorionic gonadotrophin; embryos were recovered on day 7 after ovulation. After an untreated oestrous cycle to 'wash-out' the rhFSH, the groups were crossed-over and treated twice-daily with 900 i.u. rhFSH, or saline. At the onset of treatment, the largest follicle was <25 mm in all mares, and mares destined for rhFSH treatment had at least as many 10-25 mm follicles as controls. However, neither dose of rhFSH altered the number of days before the dominant follicle(s) reached 35 mm, the number of follicles of any size class (10-25, 25-35, >3 mm) at ovulation induction, the pre- or post-ovulatory oestradiol-17beta or progesterone concentrations, the number of ovulations or the embryo yield. It is concluded that rhFSH, at the doses used, is insufficient to stimulate multiple follicle development in mares.

Nuclear and cytoplasmic maturation of sow oocytes are not synchronized by specific meiotic inhibition with roscovitine during in vitro maturation.

The effect of roscovitine exposure prior to IVM was studied on cumulus expansion, on changes of cumulus-oocyte contacts and on nuclear and cytoplasmic maturation of sow oocytes. It was hypothesized that delayed nuclear maturation and prolonged contact with cumulus cells allows prolonged cytoplasmic differentiation and therefore improves oocyte developmental potential. Cumulus-oocyte complexes (COCs) were exposed for 22 h or 44 h to 0, 25 or 50 microM of roscovitine and subsequently cultured for 22, 29 or 44 h without roscovitine. COCs were examined for cumulus expansion and oocytes for nuclear status and dynamics of transzonal microfilaments. Oocyte developmental potential was assessed by blastocyst formation after IVF. Fifty muM of roscovitine inhibited cumulus expansion for the first 22 h of culture, and maintained oocytes in meiotic arrest for 44 h. Roscovitine treatment during 22 h prior to culture for 44 h without roscovitine did not increase embryo development, but oocytes cultured for 66 h without roscovitine had reduced blastocyst formation. Oocytes cultured for 29 h after roscovitine exposure showed reduced blastocyst rates compared with their counterparts cultured for 44 h. Roscovitine treatment during 44 h prior to culture for 22 h or 44 h without roscovitine reduced embryo development. Transzonal microfilaments were reduced after culture with roscovitine, and disappeared during culture without roscovitine. It is concluded that prolonged contact with cumulus cells does not improve oocyte developmental potential. Furthermore, it is suggested that nuclear and cytoplasmic maturation in vitro cannot be seen as two independent processes.

Expression of bone morphogenetic protein2 (BMP2), BMP4 and BMP receptors in the bovine ovary but absence of effects of BMP2 and BMP4 during IVM on bovine oocyte nuclear maturation and subsequent embryo development.

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.

Differential cardiovascular and neuroendocrine effects of epinine and dopamine in conscious pigs before and after adrenoceptor blockade.

1. The effects of epinine or dopamine (both 1-10 micrograms kg-1 min-1) on systemic haemodynamics and plasma concentrations of catecholamines and prolactin were studied in conscious pigs before and after combined non-selective alpha- and beta-adrenoceptor blockade. 2. The plasma concentrations of the two compounds did not differ from each other over the entire dose-range. 3. Epinine increased aortic blood flow (AoBF, 24 +/- 6%), which was due to an increase in heart rate (HR) for doses less than 10 micrograms kg-1 min-1. At 10 micrograms kg-1 min-1, HR decreased slightly (10 +/- 3%, as compared to the value obtained at 5 micrograms kg-1 min-1) and stroke volume increased up to 15% (P < 0.05). Mean arterial pressure (MAP, 99 +/- 3 mmHg at baseline) decreased dose-dependently (14 +/- 2%, P < 0.05) up to the infusion rate of 5 micrograms kg-1 min-1, but increased by 4.0 +/- 1.8 mmHg during infusion of 10 micrograms kg-1 min-1. Systemic vascular resistance (SVR) decreased up to 23 +/- 3% for doses less than 10 micrograms kg-1 min-1, but did not change further during infusion of the highest dose. LVdP/dtmax increased during the two highest infusion rates up to 22 +/- 6% (P < 0.05). After the infusion was stopped there was an abrupt increase in HR (18 +/- 4%, P < 0.05) and a further decrease in SVR before all parameters returned to baseline.4. Dopamine caused increases in AoBF (27 +/- 3%) similar to epinine, the only difference being that HR continued to increase (32 +/- 5%) and MAP (13 +/- 3%) and SVR continued to decrease (31 +/- 3%) over the entire dose-range. The increase in LVdP/dt,,,, at the highest dose (48 +/- 4%, P <0.05) was more pronounced than with epinine.5. Adrenoceptor blockade inhibited all epinine-induced changes, but did not affect the dopamineinduced changes in AoBF, SVR and MAP, but attenuated the increases in HR and LVdP/dtmax.6. Noradrenaline (NA) and adrenaline (Ad) concentrations did not change during infusion of epinine or dopamine, but NA increased by 50% within 2.5 min after stopping the infusion of epinine. After adrenoceptor blockade NA and Ad concentrations did not change during infusion of dopamine, which contrasted with a decrease of 55 +/- 5% (P<0.05) in NA during infusion of epinine.7. Prolactin concentrations decreased gradually from 480 +/- 40 pg ml-' to 270 +/- 50 pg ml1' (P<0.05) during infusion of epinine, but did not change significantly during dopamine infusion.8. The differential effects of epinine and dopamine on MAP, SVR, plasma NA (before and after adrenoceptor blockade) and prolactin, leads us to conclude that in conscious pigs, epinine is a more potent a, P2 and D2-receptor agonist, but a weaker D,-receptor agonist than dopamine.

Alterations in anterior pituitary function of dogs with pituitary-dependent hyperadrenocorticism.

For the purpose of obtaining an integral picture of anterior pituitary function in canine pituitary-dependent hyperadrenocorticism (PDH), 47 dogs with PDH and eight control dogs received combined administration of four hypophysiotropic hormones (CRH, GHRH, GnRH and TRH) and measurements were made of ACTH, cortisol, GH, LH, PRL and TSH. Basal plasma levels in 47 dogs with PDH were higher for ACTH, cortisol and PRL, lower for GH, and not different for LH (n = 25 noncastrated dogs) and TSH compared with controls (n = 8). In dogs with PDH the responses to combined hypophysiotropic stimulation, measured as increment and area under the curve (AUC), were not different for ACTH, lower for GH and TSH (increments and AUC) and higher for cortisol (increments), LH (AUC, n = 25 noncastrated dogs) and PRL (increments and AUC) than in controls. We conclude that pituitary function is altered in several respects in dogs with PDH. 1) In spite of persisting hypercortisolemia and the neoplastic transformation of the corticotropic cells, these cells usually remain responsive to combined hypophysiotropic stimulation. 2) Basal plasma GH concentrations and GH responsiveness in the combined stimulation test are decreased, probably as a result of the glucocorticoid-induced increase in somatostatin tone. 3) Plasma PRL concentrations and the PRL response to stimulation are increased, probably as a result of cosecretion with ACTH by the transformed corticotropic cells. 4) Despite the well known effect of glucocorticoids of decreasing circulating concentrations of gonadal steroids and thyroxine, the basal plasma concentrations of LH and TSH remain unchanged and there is a tendency to hyperresponsiveness to stimulation for LH and hyporesponsiveness for TSH. The most likely explanation for these changes is a dual effect of glucocorticoids: a direct effect on the gonads and thyroids and/or the transport and metabolism of their secretory products, and an influence on the sensitivity of the feedback control at the hypothalamic-pituitary level.

Residual pituitary function after transsphenoidal hypophysectomy in dogs with pituitary-dependent hyperadrenocorticism.

Pituitary function was assessed before and after transsphenoidal hypophysectomy in 39 dogs with pituitary-dependent hyperadrenocorticism (PDH). Anterior pituitary function was investigated using combined administration of four hypophysiotropic releasing hormones (corticotropin-releasing hormone (CRH), GHRH, GnRH, and TRH) with measurements of ACTH, cortisol, GH, LH, prolactin (PRL), and TSH Pars intermedia function was assessed by measurements of basal plasma alpha-MSH concentrations and adrenocortical function by baseline urinary corticoid/creatinine ratios. At eight weeks after hypophysectomy basal plasma ACTH, cortisol, GH, LH, PRL, and TSH concentrations were significantly lower than before surgery. In seven dogs with elevated alpha-MSH concentrations, the values returned to the normal level after surgery. In the combined anterior pituitary function test there were no plasma GH, LH, PRL, and TSH responses to stimulation, whereas plasma ACTH and cortisol responses were small but significant. Remission of hyperadrenocorticism was obtained in 35 dogs and recurrences occurred in 3 of these within 16 months postoperatively. At 8 weeks after hypophysectomy, these 3 dogs were not discernible, with respect to residual pituitary and adrenocortical function, from the 32 dogs with persisting remission. Urinary corticoid/creatinine ratios in the latter group of dogs did not increase during 22 months after hypophysectomy. In contrast to the presurgical findings, at 8 weeks after hypophysectomy there were significant positive correlations between baseline urinary corticoid/creatinine ratios and basal levels and responses for ACTH, indicating return to normal function of the pituitary-adrenocortical axis. It is concluded that among the adenohypophyseal cells present in the sella turcica after hypophysectomy, the corticotropes have a distinct behavior. Much more so than the other cell types, the unaffected corticotropes tend to remain functional, or a repressed reserve fraction of corticotropes may become functional. This may be due to the removal of the hypothalamic influence of a postulated corticotropin-release inhibiting factor or a diminished inhibitory influence of a postulated paracrine factor. The corticotropes may maintain normocorticism, but may also lead to mild recurrence after relatively long periods of remission.

Inhibin, gonadotrophins and sex steroids in dogs with Sertoli cell tumours.

Inhibin bioactivity and mRNA for inhibin subunits were measured in four dog Sertoli cell tumours and in the testes of five normal control dogs. The tumours contained increased levels of inhibin (P less than 0.05) and mRNA for the alpha and beta B subunits when compared with controls, whereas the mRNA for the beta A subunit was not detected in tumours or control testes. The inhibin bioactivity was associated with a 32 kDa molecule in both Sertoli cell tumours and normal dog testes; no higher molecular weight forms were found after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Peripheral levels of immunoassayable inhibin in dogs with Sertoli cell tumours were higher than those in the controls (P = 0.01), indicating that it might be possible to use this parameter as a marker for Sertoli cell tumours. Other testicular tumours, however, might also secrete immunoactive inhibin. The increased inhibin concentrations are likely to be the cause of the suppressed peripheral levels of FSH (P less than 0.02). However, peripheral levels of LH (P less than 0.02) and testosterone (P less than 0.01) were also suppressed in the dogs with Sertoli cell tumours, whereas the concentrations of oestradiol in the peripheral plasma of both groups did not differ. Finally, i.v. injection of the LHRH agonist buserelin caused a significant increase in LH and testosterone in the control dogs, but not in the dogs with Sertoli cell tumours. It was concluded that secretory products from the Sertoli cell tumours suppressed pituitary gonadotrophin secretion. It is unlikely that testosterone or oestradiol play a role in this respect. FSH may be suppressed by the high levels of inhibin in tumour-bearing dogs, but it remains unclear whether inhibin or another Sertoli cell product is responsible for the unresponsiveness of the pituitary gland to LHRH and the suppression of LH.

Effects of morphine and naloxone on plasma levels of LH, FSH, prolactin and growth hormone in the immature male pig.

The effects of acute i.v. administration of gonadotrophin-releasing hormone (GnRH; 0.1 micrograms/kg), morphine (3 mg/kg) and/or naloxone (0.5 mg/kg) on LH and FSH secretion was evaluated in young male pigs (approximately 6 weeks old) with venous brachiocephalic cannulae. The effects of morphine and/or naloxone treatments on prolactin and GH were also evaluated. The influence of morphine on hypophysial hormone secretion was also examined 2 days after castration. Animals treated with morphine and/or naloxone were compared with saline-injected control animals. Injection of GnRH induced 400 and 50% increases in LH and FSH respectively. Morphine and/or naloxone did not influence LH secretion in intact or castrated animals. Morphine suppressed (P less than 0.01) FSH levels 40-60 min after injection whereas naloxone had no effect. Castration eliminated morphine-induced suppression of FSH. Injection of morphine followed by naloxone resulted in acutely raised (P less than 0.05) FSH concentrations. Morphine induced a threefold increase (P less than 0.01) in prolactin within 30 min of injection and naloxone inhibited the effect of morphine. Levels of GH were increased (P less than 0.01) 20 min after morphine treatment and this increase was delayed when naloxone was given immediately after morphine. Naloxone alone did not affect prolactin or GH secretion. Castration caused increases in LH (P less than 0.05) and FSH (P less than 0.01), did not influence prolactin or GH, and reduced plasma testosterone to undetectable (less than 1.0 nmol/l) levels. These results suggest that in young male pigs the hypothalamic-hypophysial axis is responsive to GnRH and gonadal negative feedback.(ABSTRACT TRUNCATED AT 250 WORDS)

15-Hydroxy-prostaglandin dehydrogenase activity in uterine tissues of late gestational and parturient cows.

15-Hydroxy-prostaglandin dehydrogenase activity (PGDH) was measured in endometrium, allanto-chorion, allanto-amnion and the placenta from six cows during late gestation and six parturient cows during caesarean section. Under saturated substrate conditions PGDH activity was lower than 138 pmol g wet tissue-1 min-1 in all uterine tissues in both groups. Human placental reference samples showed a PGDH activity of 16,800 +/- 1200 pmol per g wet weight min-1 (n = 4). Under subsaturational conditions enzyme activity was demonstrated in endometrium and both fetal membranes. These results indicate that PGF2 alpha catabolism in bovine uterine tissue on day 262 and during parturition, contributes little to changing plasma PGFM concentrations.

Role of growth hormone and growth hormone receptor in oocyte maturation.

Near the completion of growth, mammalian oocytes acquire the competence to resume and complete meiosis. In vivo the preovulatory LH surge triggers the resumption of meiosis in the oocyte contained in preovulatory follicles. When immature oocytes and the surrounding cumulus cells are released from their follicular environment, resumption of meiosis is induced spontaneously. Culture of bovine cumulus oocyte complexes (COCs) obtained from antral follicles results in blastocyst formation following in vitro maturation, in vitro fertilisation and in vitro embryo culture. Addition of growth hormone (GH) to the maturation medium accelerates nuclear maturation of cumulus enclosed bovine oocytes, induces cumulus expansion and promotes early embryonic development following in vitro fertilisation. The effect of GH is exerted through the cumulus cells and not mediated by IGF-I. Cumulus cells and the oocyte express mRNA for GH receptor. Using specific inhibitors it has been shown that the effect of GH on oocyte maturation and cumulus expansion is mediated by the cyclic AMP signal transduction pathway. Within COCs both cumulus cells and oocyte show GH immunoreactivity while expression of GH mRNA is only found in the oocyte. These observations point to a paracrine and/or autocrine action of GH in oocyte maturation.

Acrosome reaction in dog sperm is induced by a membrane-localized progesterone receptor.

The aim of this study was to investigate whether the dog sperm acrosome reaction can be induced by progesterone and whether the action of progesterone is mediated by binding of progesterone to a receptor on the sperm plasma. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Ten mM progesterone increased the acrosome reaction in viable spermatozoa over time from 3 +/- 1% at 0 hours to 69 +/- 8% at 6 hours (six dogs). In freshly ejaculated sperm from six dogs, P-BSA-FITC staining was observed in 13 +/- 1% of the viable, acrosome-intact cells, as characterized by bright fluorescence over the entire apical region. The proportion of P-BSA-FITC-stained, viable, acrosome-intact cells increased to 84 +/- 11% following 7 hours incubation in a low-calcium medium. In contrast, the majority (72 +/- 3%) of fresh epididymal sperm already demonstrated bright P-BSA-FITC staining. Apparently, epididymal spermatozoa already possess the progesterone receptor. The receptor is masked at ejaculation and subsequently gradually exposed.

Use of peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome reaction in stallion spermatozoa.

Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated with matching hemizonae for 1 minute (onset binding) followed by a 60-minute incubation (1-hour binding) of the sperm-hemizona complexes in sperm-free medium to assess the acrosomal status of the bound spermatozoa. For acrosome assessment, spermatozoa and washed sperm-hemizona complexes were air dried onto microscope slides, fixed, permeabilized in ethanol, stained with FITC-PNA, and counterstained with the DNA dye ethidium homodimer. Both zona-bound and non-bound spermatozoa showed similar staining patterns. Acrosome-intact spermatozoa displayed intensively green fluorescence over the acrosomal cap, whereas reacting spermatozoa showed a patchy disrupted image of fluorescence. Sperm cells that completed the acrosome reaction were principally stained on the equatorial segment or not stained at all. During prolonged incubation and during the calcium ionophore treatment, the proportion of spermatozoa with an acrosomal modification (reacting) and a complete breakdown of the acrosome (reacted) increased noticeably. Significant induction of the acrosome reaction was observed within 60 minutes of sperm-zona contact (P < 0.001). In conclusion, a rapid and reliable assessment of the acrosomal status and the incidence of the acrosome reaction of stallion spermatozoa at the zona surface were demonstrated in this study.

Prolactin binding in benign and malignant mammary tissue of female dogs.

Prolactin receptor (PRL-R) concentrations were determined in membrane preparations of canine mammary tumours and of non-affected mammary tissues by a radioreceptor-assay using ovine prolactin (oPRL) both for 125I-labelling and for displacement. Receptor levels greater than or equal to 3 fmol/mg membrane protein were considered positive. Histologically non-affected samples of mammary tissue from 6 dogs were PRL-R positive (12-195 fmol/mg protein). These levels were positively correlated with epithelium content (based on surface area in microscopic sections; r = 0.943, P less than 0.02). In tumour samples where pre-existing mammary epithelium (PME) was present (3 non-malignant and 6 malignant tumour samples; PME content 5-10%), the cut-off limit for PRL-R positivity was increased to 50 fmol/mg protein to forestall false positives due to non-affected tissue. If no PME was present the general limit of 3 fmol/mg protein was maintained. All 18 non-malignant tumours showed PRL-R (18-162 fmol/mg protein). The PRL-R levels were positively correlated with levels of oestrogen-(ER; r = 0.735, P less than 0.002) and progesterone receptors (PgR; r = 0.556, P less than 0.02) as measured by a multi-concentration dextran-coated charcoal method. ER and PgR levels were also proportional (r = 0.660, P less than 0.01). In 6 dogs bearing primary cancers with 5-10% PME, 1 out of a total of 6 tumours was PRL-R positive. In 9 dogs bearing primary or locally recurrent cancers without PME, significant PRL-R levels were measured in 2 out of a total of 10 tumours. Three metastatic sites in 2 other dogs were PRL-R positive. In 2 dogs (1 with a PRL-R negative local recurrence) the metastatic lesions were PRL-R negative. Thus 5 dogs of a total of 18, had PRL-R positive mammary cancers (3-377 fmol/mg protein). Unlike in non-malignant lesions the ER, PgR, and PRL-R levels were not related. In mammary cancer the presence of PRL-R was less common (P less than 0.001), and the ultimate levels less high (P less than 0.001) than in non-malignant tumours. In comparative studies using pooled membrane preparations from benign mammary tissues, oPRL was far more effective than canine prolactin (cPRL) in displacing 125I-oPRL; canine growth hormone (cGH) in this respect was ineffective. It is concluded that non-malignant mammary tissue in the dog generally is PRL-R positive; only some mammary cancers retain the PRL receptors.

Anterior pituitary function in female dogs with mammary tumors: II. Prolactin.

Plasma prolactin (PRL) concentrations at rest and during dynamic tests were measured in dogs with benign or malignant proliferative mammary lesions and in age-matched healthy control dogs. Basal PRL concentrations of dogs with benign or malignant lesions were not found to be elevated in comparisons controlled for the effects of estrous cycle phase or progestin treatment. A statistically non-significant increase of the PRL response to thyrotropin-releasing hormone (TRH) was seen in separate groups of affected dogs as compared to matched controls. A tendency towards a significantly elevated response to TRH appeared in the combined (benign plus malignant disease) group of affected dogs in anestrus (P = 0.05) and in metestrus (P = 0.09). The PRL response to clonidine was very variable and did not differ between the 3 groups of dogs. The bromocriptine-induced suppression of PRL secretion did not vary significantly among the three groups. These results do not indicate an unequivocal association of mammary tumor development and altered PRL secretion. It is only warranted to conclude that in some dogs the occurrence of benign or malignant mammary lesions is associated with hyper-responsiveness of the PRL secretion to stimulation with TRH.

Assessment of a combined anterior pituitary function test in beagle dogs: rapid sequential intravenous administration of four hypothalamic releasing hormones.

A combined anterior pituitary (CAP) function test was assessed in eight healthy male beagle dogs. The CAP test consisted of sequential 30-second intravenous administrations of four hypothalamic releasing hormones in the following order and doses: 1 microgram of corticotropin-releasing hormone (CRH)/kg, 1 microgram of growth hormone-releasing hormone (GHRH)/kg, 10 micrograms of gonadotropin-releasing hormone (GnRH)/kg, and 10 micrograms of thyrotropin-releasing hormone (TRH)/kg. Plasma samples were assayed for adrenocorticotropin, cortisol, GH, luteinizing hormone (LH), and prolactin (PRL) at multiple times for 120 min after injection. Each releasing hormone was also administered separately in the same dose to the same eight dogs in order to investigate any interactions between the releasing hormones in the combined function test. Compared with separate administration, the combined administration of these four hypothalamic releasing hormones caused no apparent inhibition or synergism with respect to the responses to CRH, GHRH, and TRH. The combined administration of these four hypothalamic releasing hormones caused a 50% attenuation in LH response compared with the LH response to single GnRH administration. The side effects of the combined test were confined to restlessness and nausea in three dogs, which disappeared within minutes after the administration of the releasing hormones. It is concluded that with the rapid sequential administration of four hypothalamic releasing hormones (CRH, GHRH, GnRH, and TRH), the adenohypophyseal responses are similar to those occurring with the single administration of these secretagogues, with the exception of the LH response, which is lower in the CAP test than after single GnRH administration.

Assessment of pituitary function after transsphenoidal hypophysectomy in beagle dogs.

Pituitary function was assessed in healthy adult beagle dogs before and after hypophysectomy. Anterior pituitary function was tested by use of the combined anterior pituitary (CAP) function test, which consisted of sequential 30-sec intravenous injections of four hypothalamic releasing hormones, in the following order and doses: 1 microgram of corticotropin-releasing hormone (CRH)/kg, 1 microgram of growth hormone-releasing hormone (GHRH)/kg, 10 micrograms of gonadotropin-releasing hormone (GnRH)/kg, and 10 micrograms of thyrotropin-releasing hormone (TRH)/kg. Plasma samples were assayed for adrenocorticotropin (ACTH), cortisol, GH, luteinizing hormone (LH), and prolactin (PRL) at multiple times for 120 min after injection. Pars intermedia function was assessed by the alpha-melanotropin (alpha-MSH) response to the intravenous injection of the dopamine antagonist haloperidol in a dosage of 0.2 mg/kg. Posterior pituitary function was assessed by the plasma vasopressin (AVP) response to the intravenous infusion of 20% saline. Basal plasma ACTH, cortisol, thyroxine, LH. PRL, and AVP concentrations were significantly lower at 10 wk after hypophysectomy than before hypophysectomy. In the CAP test and the haloperidol test, the peaks for the plasma concentrations of ACTH, cortisol, GH, LH, PRL, and alpha-MSH occurred within 45 min after injection. At 2 and 10 wk after hypophysectomy, there were no responses of plasma GH, LH, PRL, and alpha-MSH to stimulation. In four of eight hypophysectomized dogs, there were also no plasma ACTH and cortisol responses, whereas in the other four dogs, plasma ACTH and cortisol responses were significantly attenuated. The basal plasma ACTH and cortisol concentrations were significantly lower in the corticotropic nonresponders than in the responders. Plasma AVP responses were completely abolished by hypophysectomy, although water intake by the dogs was normal. Histopathological examinations at 10 wk after hypophysectomy revealed that adrenocortical atrophy was much more pronounced in the corticotropic nonresponders than in the responders. No residual pituitary tissue was found along the ventral hypothalamic diencephalon. However, in all hypophysectomized dogs that were investigated, islets of pituitary cells were found embedded in fibrous tissue in the sella turcica. A significant positive correlation was found between the number of ACTH-immunopositive cells and the ACTH increment in the CAP test at 10 wk after hypophysectomy. It is concluded that 1) stimulation of the anterior pituitary with multiple hypophysiotropic hormones, stimulation of the pars intermedia with a dopamine antagonist, and stimulation of the neurohypophysis with hypertonic saline do not cause side effects that would prohibit routine use, 2) in the routine stimulation of the anterior pituitary and the pars intermedia, blood sampling can be confined to the first 45 min, 3) the ACTH and cortisol responses to hypophysiotropic stimulation are the most sensitive indicators for residual pituitary function after hypophysectomy, 4) small islets of pituitary cells in the sella turcica, containing corticotropic cells, are the most likely source of the attenuated corticotropic response that may occur after hypophysectomy, and 5) residual AVP release from the hypothalamus after hypophysectomy is sufficient to prevent diabetes insipidus, despite the fact that the AVP response to hypertonic saline infusion is completely abolished.

Between prepartum luteolysis and onset of expulsion.

In the cow the foetal endocrine signals that initiate the calving process result in prepartum luteolysis. Withdrawal of progesterone (P4) action is a prerequisite for a normal calving. The rather abrupt declining influence of P4 is followed by a cascade of physiological processes in the myometrium and cervix. This contribution will focus on some of these events. Like in many other species, the myometrium in cows is not completely inactivated during pregnancy. So-called contractures have been registered during the final weeks of gestation and their EMG-characteristics in cows show a low frequency (on average: 13.6 per day) and long duration (on average 12.1 min). They are not evenly spread over the day because they occur less frequently when the cows are disturbed for feeding or cleaning their stables. Contractures affect several foetal functions. In the cow these contractures disappear during a period of about 8-9h when maternal plasma P4 levels are rapidly declining before calving. There is experimental evidence that this temporary inhibition is associated with prepartal luteal regression. The cause of this inhibition is still unknown. Because nitrous oxide inhibits smooth muscle cells and evidence in laboratory animals indicates that expression of the inducible form of nitrous oxide (iNOS) is downregulated in myometrium, but upregulated in the cervix around the onset of parturition, we started to investigate the role of this enzyme in bovine tissues around calving. By means of a RT-PCR technique, we obtained a first indication that iNOS is hardly expressed in the myometrium during calving, while expression was clearly detected at day 4 after calving. Analysis of prepartum en periparturient biopsies from myometrium and cervix with quantitative PCR is still underway. In six pregnant cows, provided with uterine EMG-electrodes and with ultrasonic crystals implanted on the caudal cervical rim to measure cervical dilatation, calving was induced with an injection of prostaglandin (PG) F2alpha. While maternal plasma P4 levels had significantly declined within 8h after PG treatment, the myometrium escaped from temporary inhibition with the development of a parturient contractility pattern on average at 13.5h after injection. However, it was only at 28 h after PG treatment that the first sustained increase of the opening of the vaginal ostium of the cervix was measured.

In vitro maturation of bovine oocytes in the presence of bovine activin A does not affect the number of embryos.

This study was carried out to investigate whether bovine recombinant activin A present during the in vitro maturation (IVM) of cumulus enclosed bovine oocytes affects the proportion of embryos that develop to the blastocyst stage. In addition, the effect of the presence of activin A during maturation and during embryo culture was studied. Therefore, bovine cumulus oocyte complexes were matured at 39 degrees C in a humidified atmosphere with 5% CO2 in air for 24 h in: (1) culture medium M199 supplemented with 10% foetal calf serum (FCS), luteinising hormone (LH) and follicle-stimulating hormone (FSH) and 10 ng ml-1 activin A; (2) M199 without FCS but supplemented with LH and FSH and 10 ng ml-1 activin A; (3) M199 without FCS, LH and FSH but supplemented with 10 ng ml-1 activin A. Cultures without activin served as controls. After IVF the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of buffalo rat liver (BRL) cells. For the second part of the study, COCs were matured in vitro in M199 supplemented with LH and FSH and 10 ng ml-1 activin A, fertilized in vitro and the embryos were cultured (1) on a monolayer of BRL cells in M199 supplemented with 10% FCS and 10 ng ml-1 of activin A, and (2) in droplets of serum free BRL-conditioned medium supplemented with 10 ng ml-1 activin A. IVM in the presence of LH, FSH and 10 ng ml-1 activin A did not change the proportion of blastocysts present at Day 9 or the proportion of hatched blastocyst at Day 11. Activin present during maturation in the absence of serum and gonadotrophic hormones also did not alter the proportion of blastocysts or hatched blastocysts. In vitro culture of embryos on BRL cells or in BRL-conditioned medium in the presence of activin had no effect on embryonic development. It is concluded that IVM in the presence of bovine activin A has no effect on subsequent embryonic development.