Effect of calf suckling on oxytocin, prolactin, growth hormone and milk yield in crossbred Gir $\times$ Holstein cows during milking

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Effect of calf suckling on oxytocin, prolactin, growth hormone and milk yield in crossbred Gir × Holstein

cows during milking

João Negrão, Pierre-Guy Marnet

To cite this version:

João Negrão, Pierre-Guy Marnet. Effect of calf suckling on oxytocin, prolactin, growth hormone and

milk yield in crossbred Gir × Holstein cows during milking. Reproduction Nutrition Development,

EDP Sciences, 2002, 42 (4), pp.373-380. �10.1051/rnd:2002032�. �hal-00900342�

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Regulation of mammary gland remodelling and lactation

Dr. Y. Chilliard, Prof. T. Motyl

The subject matter of Session 2 was focused on three main topics:

1. genomic studies on polymorphism and expression of genes involved in regulation of mammary gland function, genes encoding milk proteins and mammary tissue enzymes;

2. apoptosis as a fundamental process responsible for mammary gland involution;

3. metabolism of lactating animals.

The following conclusions have been drawn:

1. Bovine lactogenic hormones: GH and PRL undergo mutations in 5’-flanking region, which can affect expression and secretion pattern of these hormones.

2. Relevant relationship was found between polymorphism of as1 and as2 casein genes and their expression, organization and physico-chemical properties of casein micelle, and subcellular transport and secretion of milk components.

3. Apoptosis of mammary epithelial cells is dependent on increased expression, subcellular redistribution and interaction of Bcl-2-related death promoters. Mitochondrial pathway is involved in TGF-b1-induced apoptosis of mammary epithelial cells.

4. Short-term mild hyperglycemia does not induce any insulin resistance, but it improves the insulin- stimulated glucose disposal in lactating goats.

5. New sophisticated methods for investigation of mammary gland function and reproduction were presented: laser scanning cytometry for mammary apoptosis research, septaplex PCR for “parentage control” and sexing of cattle, real-time RT-PCR to quantify stearoyl-CoA-desaturase expression, and milk somatic cells as a model to evaluate transcription of milk proteins.

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Association between sequence polymorphism in the 5’-flanking region of the bovine alpha S2 casein gene, transcription factor binding and milk composition. M. Szymanowska, T. Malewski, L. Zwierzchowski (Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, 05-552 Wólka Kosowska, Poland)

Different sites involved in the expression of bovine milk protein genes were identified in 5’-noncoding regions (promoters) of relevant genes (Schild and Geldermann, Theor. Appl.

Genet. 93 (1996) 887–893). Our computer analysis using the TESS program revealed that at least some of the polymorphic sequences colo- calised with transcription-factor-binding sites, and thus they may influence gene expression.

We analysed nuclear protein binding to variable 5’-noncoding regions of the bovine aS2 gene by mobility-shift assays (EMSA). The following synthetic oligonucleotides (probes) were used:

probe I, covering the –1113/–1087 bp region of the aS2 casein gene promoter, including two substitutions – A/C, C/T – and binding sites for TR, AP1, GCN4 and HNF3; probe II, –199/–175 bp region, with single substitution C/T and binding sites for TR, GR, AP1 and CREB; probe III, –20/+10 bp region with two mutations – A/C, C/T – and binding sites for NF, NFkB, GR and CTF. EMSA analyses showed that the proteins contained in the nuclear extracts obtained from the heifer’s and lactating cow’s mammary gland bind more efficiently to the C variant of probe II, while extracts obtained from lactating and drying cow’s bind more efficiently to the CT variant of probe I and to the AC variant of probe III. Differences in nuclear protein binding to polymorphic sequences were statistically significant (P < 0.01). EMSA com- petition experiments showed that the transcription factors AP1, CREB, TR and NFkB take part in the formation of the DNA-protein complexes.

Proteins of milk derived from cows with vari- ous aS2 gene promoter genotypes were analysed using SDS-PAGE and HPLC techniques.

Differences were shown for the separation patterns of milk proteins and casein content in the milks obtained from cows carrying specific mutations at positions –186 and –1 084 contained in probe II and I, respectively. Moreover, differences were found in the expression rates of the different variants of the aS2 casein gene using

RT-PCR analysis performed on RNA isolated from biopsies of the mammary gland or from somatic cells derived from cow’s milk. Alto- gether these results suggest that an association exists between the 5’-flanking region polymorphism of the aS2 casein gene and its expression rate which may be explained by differences in transcription factor binding to polymorphic sequences.

Sequence polymorphism in the 5’-flanking region of the bovine prolactin gene – effect on prolactin levels and secretion patterns in cattle. M. Klauzinska, R. Grochowska, L. Zwierzchowski (Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, 05-552 Wólka Kosowska, Poland)

Due to their key role in growth promotion and mammary gland development, the genes coding for growth hormone (GH), prolactin (PRL) and their receptors (GH-R and PRL-R) seem to be promising candidate markers for selection pur- poses in animal breeding. In particular, sequence polymorphism in regulatory 5’-flanking regions (promoters) may influence gene expression and thus the physiological characteristics of an animal, thus determining its productive traits. In this study polymorphism in the 5’-regulatory region of the bovine PRL gene was analysed using the SSCP (single strand conformation polymorphism) technique. About 350 cows, heifers and bulls from different breeds – Polish Black- and-White, Polish Red and Piedmontese – were analysed. The analysis of the –1004 to –846 region of the PRL gene revealed four SSCP patterns; the PCR products derived from the A, B, and C genotypes were sequenced using an Alf- express sequencer (Pharmacia). The preliminary results of sequencing identified two nucleotide substitutions and one deletion in the analyzed fragments of different genotypes at the prolactin locus: A/C at position –996, T/C at position –928 and the deletion of the (dT-dG)₂repeat at position –877. Computer analyses of the polymorphic region in TESS and TRANSFAC programs were aimed at identifying several putative transcription factor-binding sites: IRF, BF-1, Hb, myogenin, GR, NH-3 and Ap1. The putative AP1 binding site was shown to colocalise with the C/T polymorphic site at position –928 while the (dT-dG)₂ deletion is located at GR and HNF-3 putative

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Regulation of mammary gland remodelling and lactation 497

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secretion patterns was analysed. The blood levels of prolactin were measured by RIA in animals of the different PRL genotypes, before and after single injection of TRH, PRL and GH sec- retagogues. Basic levels as well as peak ampli- tude and peak area after TRH were measured. It appears that the genotype of the PRL gene promoter had no direct influence on prolactin levels in the blood but, together with other parameters (basic level vs. the slope of prolactin increase after TRH stimulation,) it significantly (P² 0.05) modified the pattern of PRL releasing into the blood.

aS1-casein polymorphism: some clues to the existence of a particular milk secretion process in the goat species. C. Neveuâ,b, A. Riaublanc^b, G. Miranda^c, J.F. Chich^d, P. Martinê(âLaboratoire de Recherche et de Technologie Laitières, 65 rue de Saint Brieuc, 35042 Rennes Cedex, France; ^bLaboratoire d’Étude des Interactions des Molécules Alimentaires, La Géraudière, BP 71627, 44316 Nantes Cedex 3, France; ^cUnité de Biochimie et Structure des Protéines, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France;

d Unité de Virologie et Immunologie Molécu- laires, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France; ^eLaboratoire de Génétique Biochimique et de Cytogénétique, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France)

The extensive (18 alleles) and quantitative (0 to 3.6 g of as1-Cn/L/allele) polymorphism recorded in the goat at the a_s1-casein locus strongly influences the composition (protein and, unexpect- edly, lipid fractions) and the technological behaviour of milk (Grosclaude & Martin, IDF- FIL Seminar on Milk protein polymorphism, Palmerston North, New Zealand, Proceedings, 1997, pp. 241–253). Electron microscopy studies of mammary epithelial cells (MEC) have revealed a dysfunction of the intracellular transport of caseins when a_s1-casein is lacking (Chanat et al., J. Cell Sci. 112 (1999) 3399–3412). Despite a long controversy, goat milk secretion is still considered to occur through an apocrine process in opposition to the generally admitted mero- crine secretion process of cow milk (Wooding

way of secretion for goat milk, possibly associated with a low synthesis-rate of as1-Cn variants. Traces of an additional cytoplasmic contribution to milk proteins due to the putative apocrine process were investigated by comparing, by two-dimensional electrophoresis (2-DE), the protein profiles of whey prepared from high as1-Cn level (A/A) and null type (O1/O1) pooled milks. Since the ER is involved in the formation of the milk fat globule (MFG) via triglycerides and phospholipid biosynthesis, the potential effects of its dysfunction associated with low- level a_s1-Cn variants were analysed by comparing the structure of MFG in individual milks from goats of the following genotypes: A/A, F/F and O1/O1, associated with high, low and null as1-Cn contents, respectively. Particule size, lipids and phospholipid composition and MFG membrane (MFGM) protein profiles were analysed. Additional proteins were actually found in 01/01 whey. These proteins are currently under characterisation, in order to determine whether they indicate an apocrine secretion process. The effect of as1-Cn polymorphism on the fat fraction of milk was confirmed for total lipid (TL) variability (AA > FF > OO). However, no significant difference was found neither in phospholipid (PL) content and composition nor in protein profile of MFGM, mainly because of the great individual variability observed. Interestingly, significant effects on the proportion of PL in TL and on the MFG size were shown, a_s1-Cn defi- ciency being associated with higher proportions of PL in TL and smaller MFG. Since PL mainly originates in membrane materials, two interpre- tations can be put forward. First, the fat dispersion in milk is truly finer thus suggesting an internal perturbation of either lipid biosynthesis or the intracellular MFG growth mechanism. Second, these results reflect a higher content of free membrane or cell fragments in milk, possibly due to an apocrine contribution. Whatever the proposal finally retained, both are the signature of a modified secretion process associated with defective a_s1-Cn variants. The different paths explored here all seem to converge towards a global effect of the as1-Cn polymorphism on the physiology of the MEC and a deep involvement of this protein in the cellular mechanisms underlying the secretion of milk components.

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Kinetic study of growth hormone effects upon the gene expression in goat milk epithelial cells. M. Boutinaud, H. Jammes, J. Djiane (INRA, Unité Biologie cellulaire et moléculaire, Domaine de Vilvert, Bâtiment Jacques Poly, 78352 Jouy-en-Josas Cedex, France)

The secretory activity of the mammary gland during lactation is a complex process regulated by the interaction of many hormonal and paracrine influences, with particular emphasis given to the galactopoietic effects of the Growth Hormone (GH). To aid in our understanding of these influences it is useful to know which genes are affected and how they are affected, which to this point in time has been studied by repeated mammary biopsies; a less than desirable approach.

The fact that mammary epithelial cells are natu- rally shed into the milk as part of the lactogenic process is unique to the process of lactation. From these cells, RNA can be isolated and may provide a dynamic, non-invasive means of monitoring gene activity within the mammary gland. Thus, our objectives were to determine the relative expression of 3 genes of interest in goats treated with GH or untreated, as assessed from milk somatic cell RNA studied over time in experiment 1, and in experiment 2 from RNA isolated from milk somatic cells and congruent mammary gland tissue RNA. In experiment 1, 4 goats beginning their 16th wk of lactation were divided into two groups and subjected to a switch-back design with treatments of GH (as described sub- sequently) or no treatment being given during the first 6 days of each wk over a three wk inter- val. Thus, during the ewes 16th, 17th, and 18th wk of lactation one group followed successively Control-GH-Control treatments, and the other group followed successively GH-Control-GH treatments. GH treated goats received 5 mg of recombinant bovine GH (GH; Somidobove, Elanco, Indianapolis, USA,) subcutaneously once daily for 6 successive days after the morning milking. Milk yield increased significantly in response to GH (P < 0.05). Expression of the 3 major milk protein genes of interest (aS1 casein, kappa casein, and alactalbumin genes) increased 3 days after initiating GH treatment, suggesting a coordinated up-regulation by GH. In a second experiment, 6 goats in late lactation received daily treatments of recombinant bovine GH (5 mg.d^–1) for 3 weeks, or no treatment. One gland from each goat was milked thrice daily (right half udder) throughout the study, and the

other gland once daily (left half udder). Milk cells were collected twice a week from each half udder, quantified and used for total RNA prepa- ration. These serial RNA preparations allowed a kinetic study of mammary gene expression throughout GH treatment. At the end of the study, 3 control and 3 GH treated goats were killed and mammary tissue was collected and subjected to analysis by the methods of least squares ANOVA. The model used to analyse the data accounted for the effects associated with the animal, milking frequency, GH treatment and the interaction between milking frequency and GH treatment. A stimulatory effect of milking frequency was observed on milk yield and mam- mary gland weight (P < 0.01). In the once-milked half udder, no difference in milk yield existed between the control and GH-treated goats. How- ever, the weight of the once-milked half udder was greater in the GH than in the control goats (P < 0.05). After 3 weeks of treatment, the degree of mammary apoptosis, as determined by the tunnel effect, was similar in once-milked half udders from the control and GH-treated goats.

GH treatment did not affect Casein kappa and prolactin receptor mRNA levels but significantly (P < 0.05) increased STAT5 gene expression suggesting a regulation of mammary cell respon- siveness to GH. In conclusion, milk somatic cell RNA may constitute a useful source of RNA to assess hormonal effects on mammary gene expression and particularly in the dynamic study of the direct effect of GH on mammary gland metabolism.

Characterisation of the caprine Stearoyl-CoA Desaturase gene and its mammary transcript.

Development of a method of mRNA quantifi- cation by real time RT-PCR. L. Bernardâ, C. Lerouxâ, Y. Chilliardâ, P. Martin^b(âUnité de Recherche sur les Herbivores, INRA, Theix, 63122 Saint-Genès-Champanelle, France; ^bLab- oratoire de Génétique Biochimique et Cytogéné- tique, INRA, 78352 Jouy-en-Josas, France)

Some unsaturated fatty acids found in milk and beneficial for human nutrition, such as oleic (cis9 C18:1), palmitoleic (cis9 C16:1) and the major part of conjugated linoleic acids (cis9,trans11 C18:2), are synthesised in the mammary gland by

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palmitic (C16:0), and vaccenic (trans11 C18:1) acids, respectively (Griinari J.M. et al., J. Nutr.

130 (2000) 2285–2291). In order to study the regulation of SCD gene expression, we charac- terised the caprine SCD gene and its mRNA, then, developed molecular tools to quantify SCD transcript by real-time RT-PCR. Northern Blot- ting was performed using Poly(A)⁺RNA isolated from mammary tissues of lactating Alpine goats. RT-PCR products were sequenced on both strands. Amplified DNA fragments obtained from genomic DNA were sequenced. Real-time RT- PCR was realised using a primer pair and Taq- Man probe, to quantify SCD and Cyclophilin (a housekeeping gene used as the internal standard) transcripts on an ABI model 7 700 SDS (PE Applied Biosystems). A 5.1-kb long SCD transcript was identified by northern-blot analysis of poly(A+) RNA. The complete sequencing of the SCD cDNA (5 123 bp) was realised (acces- sion number AF 325 499): it contained an open reading frame of 1080 nt coding for 359 amino acids and followed by an unusually long (3.8 kb) 3’-UTR sequence, deriving from a single exon as reported in human (Zhang et al., Biochem. J. 340 (1999) 255–264) and rodent (Mihara J., Biochem.

108 (1990) 1022–1029; Kaestner K.H. et al., J. Biol. Chem. 264 (1989) 14755–14761) species.

When compared, the coding region of the caprine SCD mRNA shares 98, 95, 90 and 86% identity with its ovine, bovine, porcine and human coun- terparts, respectively. The complete structural organisation of the relevant gene deduced from PCR analysis, was determined: the transcription unit spans a 15-kb region and consists in 6 exons which are 223 (E1), 283 (E2), 131 (E3), 206 (E4), 233 (E5), and 4047 (E6) bp long, and 5 introns which are 0.6 (I1), 3.7 (I2), 1.5 (I3), 1.8 (I4) and 2.7 kb (I5) long. Characterisation of the cDNA sequence allowed us to propose a system to quantify SCD mRNA by real-time RT-PCR. A 179 bp length fragment located in the 3’UTR was amplified using both a specific primer pair and a TaqMan probe. This system was designed for studying the regulation of the caprine SCD gene in the mammary gland and adipose tissue. The first results showed that the SCD gene was highly expressed in the lactating mammary gland and subcutaneous adipose tissue while low expression was observed in the perire- nal adipose tissue suggesting a tissue specific regulation of its expression. In conclusion, we

the caprine SCD gene.

Short-term mild hyperglycemia enhances insulin-stimulated glucose disposal in lactating goats. S. Lemosquetâ, E. Debras^b, M. Balage^b, J.F. Hocquette^c, H. Rulquinâ, J. Grizard^b(âUnité Mixte de Recherches sur la Production du Lait, INRA, 35590 Saint-Gilles, France; ^bUnité d’Étude du Métabolisme Azoté, INRA, Centre de Clermont-Ferrand-Theix, 63122 Saint-Genès- Champanelle, France; ^cUnité de Recherches sur les Herbivores, INRA, Centre de Clermont- Ferrand-Theix, 63122 Saint-Genès-Champanelle, France)

This work was designed to study the effect of a 3-day mild hyperglycemia (5.3 vs. 3.3 mM in control period) on the regulation of glucose metabolism in lactating goats. Glucose was intra- venously infused at variable rates, simultane- ously with a constant potassium-amino acid infusion. During hyperglycemia, the diet plus substrate infusion allowed to maintain the net energy supply whereas protein supply was decreased (–20.8%). The glucose transporters GLUT-4 were measured in skeletal muscle (Longissimus thoracis) before and after hyper- glycemia. In addition, the acute effect of medium and high-insulin doses on glucose turnover was measured in vivo during euglycemic and hyperglycemic hyperinsulinemic clamps under potassium and amino acid replacement. The medium and acute-insulin rates were slightly higher during euglycemic clamps (0.07 nmol.min^–1and 1.06 nmol.min^–1) than during hyperglycemic clamps (0.027 nmol.min^–1and 1.00 nmol.min^–1) to obtain similar plasma insulin concentrations (0.22 ± 0.09 nM at medium-insulin dose and 1.17 ± 0.25 nM at acute-insulin dose) with both types of clamps. Hyperglycemia reduced the endogenous glucose appearance rate (–0.59 nmol.min^–1) but increased glucose disposal (+0.98 nmol.min^–1). It decreased the total membrane-associated GLUT-4 protein in skeletal muscle. In contrast, during hyperglycemic clamp, it enhanced the insulin-stimulated glucose disposal (+0.28 nmol.min^–1at medium- insulin dose and +0.69 nmol.min^–1at acute- insulin dose). In addition, it enhanced the acute insulin-stimulated glucose disposal during

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euglycemic clamps (+0.36 nmol.min^–1). Both the level and the duration (3 days) of hyperglycemia contributed to this effect. Milk yield did not increase in response to hyperglycemia, suggesting that in the present conditions, non- insulin dependent glucose utilisation by the mammary gland was not a limiting factor for milk synthesis. Milk protein yield was also not affected by hyperglycemia. However, during hyperglycemia, intravenous amino-acid infusion only partially blunted the decrease of blood free amino acids because of the decrease in dietary protein intake. In these conditions, the decrease in the supply of some essential amino acids may have prevented the positive effect of an increase in glucose supply on milk yield and protein yield which has been observed in other experiments.

We conclude that short-term mild hyperglycemia does not induce any insulin resistance in lactating goats. Rather, it improves the insulin-stimulated glucose disposal as in non-lactating rodents or humans. Because our experiment is the only experiment performed with amino acid replacement (even partially), further studies are needed to analyse the possible effect of amino acid avail- ability on this improvement.

Laser Scanning Cytometer (LSC) as a method of apoptosis research in mammary epithelial cells. M.M. Godlewski, O. Kolek, T. Motyl (Department of Physiology, Biochemistry, Phar- macology and Toxicology, Warsaw Agricultural University, Nowoursynowska 166, 02-787 War- saw, Poland)

During apoptosis, the cell expresses a series of characteristic features that distinguish this kind of cell death from necrosis. In early phases of apoptosis proapoptotic proteins (Bax or Bid) are activated, then aggregate and translocate within the cell compartments. Bax insertion in the outer mitochondrial membrane facilitates the reflux of cytochrome c and other apoptosis inducing factors. As a consequence, caspase is activated and PARP (poly (ADP-ribose) polymerase) is cleaved. Phosphatidyl-serine is expressed on the cell surface, and cell membranes start leaking.

Shortly after, nuclear changes occur: chromatin condensation, pyknosis, nuclear fragmentation, and finally the formation of apoptotic bodies.

Laser scanning cytometry (LSC) is the method of

choice for studying cells that require attachment to the surface for optimal growth and development, including mammary epithelial cells. It gives the possibility to track apoptosis in each and every of the above mentioned steps. Detec- tors for two different fluorescence channels and for transmitted light (forward scatter) offer the possibility for simultaneous analysis of the local- isation and aggregation of FITC-labelled proteins vs. DNA content and chromatin condensation (7-aminoactinomycin D or propidium iodide staining). Exposition of phosphatidylserine on the surface of the plasma membrane and DNA strand breaks, as features of apoptosis, are detected with annexin V-FITC and BrdUTP, respectively. The possibility of cell compart- mentalisation on the nucleus and cytoplasmic areas (Nf vs. Cf) is unique for LSC. This allows the measurement of FITC-labelled protein relocation within the cell compartments. Further- more, the aggregation of proteins can be measured, based on the FITC Maximal Pixel, a parameter referring to the highest value of fluorescence. Another unique feature of LSC is its property to store the cell positions on a slide along with the measurement data. This allows the relocation of a single cell with interesting features and the correlation with cell morphology and picture taking. Of course one can always restain the sample and return to (relocate) cells for further measurements.

Expression of apoptosis-related proteins in the involuting mammary gland of the sow. P. Wareskiâ, T. Motylâ, B. Gajkowska^b, U. Wojewódzka^b, A. Rekiel^c, T. Ploszajâ (âDepartment of Physiology, Biochemistry, Phar- macology and Toxicology, Warsaw Agricultural University, Nowoursynowska 166, 02-787 War- saw, Poland; ^bMedical Research Centre, Polish Academy of Sciences, Warsaw, Poland; ^cDepart- ment of Pig Breeding and Production, The Ani- mal Husbandry Faculty, Warsaw Agricultural University, Brwinow, Poland)

Apoptosis is responsible for cell loss during mammary involution after natural weaning in rodents and during drying off in ruminants. In the present study we investigated: (A) the number and morphology of apoptotic cells in low-, moderate-, and high-involuted mammary glands

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forming the growth factor-b1 (TGF-b1) cytokine as a local inductor, TGF-b-receptor, Bax and Bcl-2 proteins as a promoter and inhibitor, respectively and cysteine protease CPP-32 (Caspase 3) as an executor of apoptosis), (C) the subcellular distribution of proapoptotic protein Bax. (A) The percentage of apoptotic cells (simultaneous TUNEL and Hoechst 33342 staining) was evaluated at levels 2.7 (± SEM) 0.7, 3.4 (±) 0.7 and 3.8 (±) 1.1% for low-, moderate-, and high-involuted glands, respectively. The ultrastructural analysis (electron microscopy) showed characteristic morphological features of apoptosis such as margination and condensation of chromatin, pyknosis and fragmentation of the nucleus as well as apoptotic body formation.

Moreover, phagocytosis of apoptotic cells by intraepithelial macrophages was also observed (B/C) The immunohistochemical study (light microscope, MicroImage System) demonstrated a significant increase in the integrated optical density (IOD) of the lobuloalveolar mammary tissue labelled with anti-Bax antibody from low, through moderate-, to high-involuted glands.

Western blot analysis of the mammary gland homogenates confirmed these observations. The immunoelectron microscopy revealed that Bax was localised in the cytosol, in the nuclear envelope pores, on the mitochondrial and endoplasmic reticulum membranes and in the heterochromatin of the mammary epithelial cells. A relatively high Bax/Bcl-2 ratio (Bax IOD/Bcl-2 IOD was calculated to be about 2.3, 2.6 and 5.6 for low-, moderate-, and high-involuted glands, respectively) indicating the increasing “susceptibility”

of mammary epithelial cells to apoptosis in the course of involution. The highest Bax/Bcl-2 ratio in the high-involuted glands coincided with the relatively strongest tissue expression of TGF-b1, TGF-b-receptor and CPP-32. The results of the present study suggest the involvement of the Bax/Bcl-2 checkpoint in the regulation of apoptosis of mammary epithelial cells in the involuting mammary gland of the sow. More- over, the important role of TGF-b1 and CPP-32 in the sow mammary gland after weaning (as the inductor and executor of programmed cell death, respectively) is also discussed.

Godlewski, T. Motyl (Department of Physiology, Biochemistry, Pharmacology and Toxicology, Warsaw Agricultural University, Nowoursyn- owska 166, 02-787 Warsaw, Poland)

Transforming growth factor-b₁(TGF-b₁) is an antiproliferative factor for mammary epithelial cells (MEC). Acting in an auto/paracrine manner, it is considered as an important local regulator of mammary tissue involution. TGF-b1inhibits proliferation, differentiation and milk protein synthesis in MEC and increases extracellular matrix formation. TGF-b1is also an apoptogenic agent for MEC, however in contrast to its well- described antiproliferative effect, the mechanism of TGF-b1-induced apoptosis is still obscure.

Several studies indicate that Bcl-2-related proteins are involved in the regulation of TGF-b₁- induced programmed cell death. The increase in the expression of TGF-b1and its receptor is associated with an increase in Bax (the promoter of apoptosis) and CPP-32 (the executor of apoptosis) in MEC. Experiments on HC11 mouse mammary epithelial cells indicate that translocation of Bax from the cytosol to organellar membranes (mitochondria, Golgi apparatus, endoplasmic reticulum) and through nuclear envelope pores to the nucleus belong to early events in the cellular response to TGF-b1. The redistribution of Bax within the cell is dependent on its activation via cleavage at the N-terminal epitope and exposure of the BH3 domain. Activation and subcellular redistribution of Bax precedes the increase in p53 expression. Our studies, using MEC, suggest the involvement of the mitochondrial pathway during TGF-b₁-induced apoptosis in HC11 cells, involving the activation and redistribution of Bax to the mitochondrial membranes, release of cytochrome c from the mitochondrial inter- membrane space, and increase in CPP-32 (caspase 3) concentration in the cytoplasm and nucleus leading to the degradation of the nuclear PARP [poly (ADN-ribose) polimerase]. It has been shown that lactogenic hormones like prolactin and IGF-I abolish TGF-b₁-induced apoptosis. The protective effect of prolactin and IGF-I against TGF-b1-induced programmed cell death may occur not only at the level of the Bax/Bcl-2 rheostat but also through the inhibition of TGF-b1expression.

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Septaplex PCR for “parentage control” and sexing of cattle. J. Lubieniecka, G. Grzybowski, K. Lubieniecki (Institute of Genetics and Ani- mal Breeding, Polish Academy of Science, Jastrzebiec, 05-552 Wólka Kosowska, Poland)

The methods enabling the verification of infor- mation included in pedigree documentation are crucial for the genetic improvement of livestock animals. The need for a new method of individual identification has been intensified by the widespread use of new biotechnology in animal reproduction. This reproduction biotechnology allows a quick genetic improvement but at the same time makes it easier for mistakes in pedigree registration to appear. In cattle, pedigree control has been performed on a routine basis in most countries relying on typing reagents that have been standardised through regular com- parison tests under the auspices of the Interna- tional Society for Animal Genetics (ISAG).

Although personal identification based on blood groups and protein polymorphism is still valid, at the 1996 ISAG Conference in Tours, France, it was decided that the introduction of DNA polymorphism in pedigree control of cattle and horses should be given a priority. An international com- parison test is taking place, organised by ISAG, which will select the most appropriate markers and establish internationally standardised DNA

testing methods for cattle. The fact that the microsatellites found so far in the cattle genome consist of dinucleotide repeats makes this task very difficult. This estimate is often confounded by the presence of “shadow” (“stutter”) bands, i.e.

artificial PCR products derived from the genomic poly (TG) tract by the deletion or insertion of one or more motifs. Multiplexing PCR reactions is a more efficient way to determine the individual’s genotype at many microsatellite loci. It significantly reduces the cost and time spent on analysis. The aim of this study was to create a multiplexed PCR reaction which would include several microsatellite loci as well as an amelo- genin (AMG) locus. The AMG codes for an extracellular matrix protein which is important in mammalian tooth bud development, specifically in the formation of tooth enamel. It was found to have genes on both the X and Y chromosomes.

Due to a deletion in the AMG gene located on the Y chromosome, the genes produce slightly different migration patterns, a fortuitous event that allows forensic scientists to determine the sex of an individual. Optimal conditions for a PCR reaction consisting of 6 microsatellite loci (CSRM60, INRA005, HEL1, HEL5 and BM1818) and an AMG locus were obtained in all of the cases. Detection signals were intense and no unspecific products were detected.

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