Involvement of theca cells and steroids in the regulation of granulosa cell apoptosis in rabbit preovulatory follicles

Follicular atresia is characterized by a rapid loss of granulosa cells and, to a lesser extent, theca cells, via apoptosis. The aim of this study was to investigate the possible involvement of theca cell secretions in the regulation of apoptosis of rabbit granulosa cells. The annexin-V binding method based on externalization of phosphatidylserine to the outer layer of plasma membrane during apoptosis was used to detect apoptotic granulosa cells in flow cytometry. Regulation of apoptosis of granulosa cells was studied in three different culture systems: (i) isolated cultured granulosa cells, (ii) granulosa cells obtained from cultured preovulatory follicles and (iii) granulosa cells co-cultured with theca cells. The results of this study indicate that: (i) the rate of apoptosis of granulosa cells was significantly reduced when granulosa cells were co-cultured with theca cells or obtained from cultured preovulatory follicles in comparison with isolated cultured granulosa cells; (ii) FSH exerts its anti-apoptotic effect only on granulosa cells issued from cultured preovulatory follicles; (iii) ovarian steroids do not affect the percentage of isolated apoptotic granulosa cells; and (iv) the occurrence of an apoptotic process in rabbit theca cells could be upregulated in vitro by hCG and an analogue of the gonadotrophin second messenger cAMP. The results of this study indicate that in rabbits (i) steroids were ineffective in vitro in protecting isolated granulosa cells against apoptosis in comparison with observations in vivo in rats, and (ii) the presence of theca cells was efficient to reduce granulosa cell apoptosis but not sufficient to allow the anti-apoptotic effect of gonadotrophins observed in cultured follicles.     

Incidence of apoptosis in granulosa cells from immature human follicles

The aim of this study was to investigate the incidence of apoptosis in granulosa cells from immature human follicles undergoing in vitro maturation (IVM) and to compare the incidence of apoptotic granulosa cells (i) between FSH-primed and unprimed normal ovaries and (ii) between polycystic and normal ovaries. Furthermore, the incidence of apoptosis was related to maturation and subsequent fertilization and cleavage of the oocytes from the corresponding ovary. Seventy women undergoing 70 IVM cycles were included. Group 1 consisted of patients with normal ovaries (n = 52) and group 2 consisted of patients with polycystic ovaries (n = 18). Patients in group 1 were subdivided into two groups according to priming with FSH before aspiration. In group 1a (n = 27 cycles) oocytes were obtained in unstimulated cycles. In group 1b (n = 25 cycles) oocytes were obtained after priming with recombinant FSH for 3 days initiated on day 3 after spontaneous menstruation. In group 2 all patients were primed with recombinant FSH for 3 days before aspiration. Aspiration was performed transvaginally and cumulus-enclosed oocytes were matured for 28-30 h before fertilization. Granulosa cells were collected from follicular aspirates. An APOPTAG detection kit was used to stain the granulosa cells and to detect apoptosis. The incidence of apoptosis in granulosa cells was decreased in follicles from FSH-primed normal ovaries compared with follicles from unprimed normal ovaries and FSH-primed polycystic ovaries. No difference was found between granulosa cells from FSH-primed polycystic ovaries and granulosa cells from unstimulated normal ovaries. No differences in maturation rate, fertilization rate, cleavage rate and implantation rate were observed when oocytes from a polycystic ovary were compared with oocytes from an unstimulated normal ovary. In unstimulated cycles, the ovaries were grouped according to the presence of a dominant follicle. The incidence of apoptosis was significantly higher in granulosa cells from an ovary without a dominant follicle compared with granulosa cells from an ovary with a dominant follicle. The rates of maturation, fertilization and cleavage did not differ between the two groups.     

Interaction of bovine granulosa and theca cells in a novel serum-free co-culture system

The objective of this study was to develop a defined culture system in which bovine follicular and granulosa cells are grown in close contact with each other and with the extracellular matrix (ECM) component laminin. Granulosa and theca cells from follicles 4-6 mm in diameter were cultured on either side of laminin-coated BioCoat cell culture inserts in a serum-free medium containing 10 ng insulin ml(-1) at plating densities of 10(5) and 3 x 10(5) cells per membrane side. The cells adopted a clumped arrangement, maintained steroidogenic activity for at least 7 days and demonstrated paracrine communication by increased steroidogenesis and enhanced cell survival compared with cells in mono-culture. Co-cultured theca cells secreted significantly more androstenedione compared with cells in mono-culture. Granulosa cell viability was doubled by co-culture with theca cells. Co-cultures at both cell plating densities were responsive to treatment with physiological combinations of either FSH, LH and LR3 insulin-like growth factor I (IGF-I) (treatment A) or FSH, LR3 IGF-I and androstenedione (treatment B). Significantly more androstenedione was secreted in the presence of treatment A compared with controls. In contrast, oestradiol secretion was increased only by treatment B. Progesterone secretion was unaffected by treatment and did not increase during culture. Co-cultures at the higher plating density demonstrated higher theca cell survival and better maintenance of the follicular cell phenotype. In conclusion, this novel co-culture system provides a unique model for the study of paracrine communication between ovarian somatic cells and cell-ECM interactions during follicle growth.     

Analysis of atresia in equine follicles using histology,fresh granulosa cell morphology and detection of DNA fragmentation

Follicular atresia has been examined previously by various biochemical and histological methods. The aim of this study was to compare, for the first time, detection of granulosa cell apoptosis by biochemical DNA analysis and microscopic examination of fresh granulosa cell morphology with the established method of detecting atresia by histology in equine follicles. DNA extracted from granulosa cells was examined by staining with ethidium bromide and end-labelling with [(32)P]dideoxy-ATP, which labels the free 3'-end of DNA fragments. In 25 of 26 follicles (96%) there was agreement between end-labelling and staining of DNA with ethidium bromide (P < 0.001). Granulosa cell apoptosis was distinguished more easily in the end-labelled samples than by staining with ethidium bromide. Histological atresia and apoptosis as detected by biochemical DNA analysis were significantly correlated (P < 0.02) with 20 of 22 follicles (91%) receiving corresponding classifications with the two methods. No follicles with granulosa cell apoptosis as detected by biochemical DNA analysis were histologically viable, but some of the histologically early atretic follicles did not display DNA laddering. Stereomicroscopic evaluation of morphology of the fresh granulosa cells was significantly correlated (P < 0.001) with the histological findings, with 29 of 33 follicles (88%) receiving corresponding classifications. There was a potential error in determining follicle health by biochemical DNA analysis only, as both histologically early and late atretic follicles in some cases did not show DNA laddering. Thus, if relying solely on biochemical detection of apoptosis, severely atretic follicles could wrongly be classified as healthy follicles.     

Differential effects of activin A on basal and gonadotrophin-induced secretion of inhibin A and progesterone by granulosa cells from preovulatory (F1-F3) chicken follicles

Previous work has shown that activin A is expressed selectively within the theca rather than the granulosa layer of preovulatory chicken follicles. In the present study, this finding was verified and the potential paracrine actions of activin A on basal and gonadotrophin-induced secretion of inhibin A, inhibin B and progesterone by granulosa cells from the three largest preovulatory follicles (F1-F3) were investigated. Treatment with activin A (0, 0.25, 2.5 and 25 ng ml(-1)) alone increased inhibin A secretion markedly in a follicle- and time-dependent manner, with the greatest response (up to 15-fold increase; P < 0.0001) in F1 follicles after 3 days of treatment. In contrast, activin A alone had no effect on progesterone output at any time. Cells from F3 follicles were more responsive to FSH than were F1 cells in terms of both inhibin A (P < 0.02) and progesterone (P < 0.01) secretion. Furthermore, activin A greatly enhanced FSH-induced secretion of both inhibin A (up to tenfold; P < 0.0001) and progesterone (up to sixfold; P < 0.0001). In terms of LH-induced inhibin A and progesterone secretion, cells from F1, F2 and F3 follicles showed similar responses. Co-treatment with activin A enhanced LH-induced secretion of inhibin A markedly (up to ninefold; P < 0.0001) but had only a marginal effect on LH-induced progesterone secretion (up to twofold; P < 0.001). The presence of activin receptor subtypes IA, IB, IIA and IIB in cultured granulosa cells from F1, F2 and F3 follicles was demonstrated using immunocytochemistry. These findings support the hypothesis that activin A secreted by the theca layers of avian preovulatory follicles exerts a local paracrine action on granulosa cells to modulate 'basal' inhibin A secretion and to upregulate gonadotrophin-induced secretion of both inhibin A and progesterone. However, the extent to which this local role of activin A contributes to the generation of the preovulatory LH-progesterone surge remains to be established.     

Regulation of adiponectin and its receptors in rat ovary by human chorionic gonadotrophin treatment and potential involvement of adiponectin in granulosa cell steroidogenesis

In mammals, adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various tissues. However, the cellular expression and the role of adiponectin system have never been investigated in rat ovary. Here, we report the presence of adiponectin, AdipoR1 and AdipoR2 in rat ovaries, and we have investigated its role in granulosa cells. Using RT-PCR and western blot, we show that the mRNAs and proteins for adiponectin, AdipoR1 and AdipoR2 are found in the ovaries. Immunohistochemistry localized adiponectin, AdipoR1 and AdipoR2 in theca-interstitial T-I cells, corpus luteum, oocyte and less abundantly in granulosa cells. In the KGN human granulosa cell line, adiponectin mRNA and protein were undetectable; AdipoR2 was weakly expressed, whereas AdipoR1 was clearly present. Human chorionic gonadotrophin (hCG) injection (48 h) after pregnant mare serum gonadotrophin (PMSG) injection (24 h) in immature rats increased the level of adiponectin (protein) by about threefold (P < 0.05) and those of AdipoR1 by threefold (mRNA, P < 0.05) and 1.5-fold (protein, P < 0.05) in ovary, whereas the mRNA and protein levels of AdipoR2 were unchanged. Interestingly, hCG injection (48 h) after the PMSG treatment (24 h) decreased plasma adiponectin levels and increased insulin plasma levels. In vitro in primary rat granulosa cells, human adiponectin recombinant (5 microg/ml) in the presence or absence of follicle-stimulating hormone (10(-8) M, 48 h) had no effect on the steroidogenesis. However, it increased progesterone secretion (P < 0.05) by about twofold and oestradiol production (P < 0.05) by about 1.6-fold in response to insulin-like growth factor-I (IGF-I) (10(-8) M). Furthermore, it improved IGF-I-induced IGF-I receptor-beta subunit tyrosine phosphorylation and ERK1/2 phosphorylation. In basal state, human adiponectin recombinant also increased rapidly but transiently the ERK1/2, p38 and Akt phosphorylations, whereas it increased more lately the adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation. Thus, AdipoR1 and AdipoR2 are regulated by hCG treatment in rat ovary and adiponectin enhances IGF-I-induced steroidogenesis in granulosa cells.     

Dose-response effects of equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) on early embryonic development and viable pregnancy rate in rats

The present study examined the dose-response effects of eCG treatment alone and in combination with various doses of hCG on early embryonic development in vivo and viable pregnancy rate in rats. Mated female Wistar rats were treated with eCG alone (0, 10, 20 or 40 iu), or with 20 iu eCG in combination with various doses of hCG (10, 20, 40 or 80 iu) administered 48 h later. The animals were killed on days 2, 3, 4, 5 or 14 of pregnancy and the numbers of embryos and fetuses recovered were scored. All rats treated with 0 or 10 iu eCG were pregnant. The pregnancy rate was reduced from 62.5% on day 2 to 25% on day 14 and from 31% on day 2 to 10% on day 14 in the groups treated with 20 and 40 iu eCG, respectively. The reduction in pregnancy rate induced by 20 iu eCG was negated by the increasing doses of hCG used. A 100% pregnancy rate was noted on days 2 and 3 in the groups treated with doses of hCG between 10 and 80 iu and from day 2 to day 4 in the groups treated with doses of hCG between 20 and 80 iu. However, a higher viable pregnancy rate was observed only in the group treated with 10 iu hCG compared with the group treated with 20 iu eCG and 0 iu hCG. These results imply that hyperstimulation of rats with high doses of eCG compromises pregnancy rate and markedly reduces litter size and that the addition of hCG is required for complete ovulation, which results in higher embryo yield and a delay in early embryo demise.     

Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation,granulosa cell proliferation,steroid hormone and PGE2 release in cultured rat ovarian follicles

Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.     

Metabolism of curcumin and induction of mitotic catastrophe in human cancer cells

In cultured cells, curcumin (CUR) causes cell death by interfering with mitosis and leading to fragmented nuclei and disrupted microtubules, a process named mitotic catastrophe. In order to clarify the role of the known CUR metabolites hexahydro-CUR (HHC) and CUR-glucuronide (CUR-gluc) in mitotic catastrophe, the effects of CUR were studied in three human cancer cell lines with different metabolism of CUR. In Ishikawa and HepG2 cells, CUR was metabolized to HHC and small amounts of octahydro-CUR (OHC), whereas the only metabolism in HT29 cells was the formation of CUR-gluc. Despite their different metabolism, all three cell systems responded to CUR with arrest in G2/M phase and mitotic catastrophe. Fractionation of the cells showed that concentrations of CUR were higher in the ER and cytosol than in the incubation medium by a factor of up to about 150 and 8, respectively. In contrast to CUR, the metabolite HHC and the products of spontaneous degradation did not elicit any effects in Ishikawa cells. These results imply that the causative agent of mitotic catastrophe is the parent CUR molecule, whereas reductive metabolism and chemical degradation render CUR inactive.     

The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro

The human corpus luteum expresses genomic progesterone receptors (PRs) suggesting that progesterone may have an autocrine or paracrine role in luteal function. We hypothesised that the reduction in luteal PR reported in the late-luteal phase augmented progesterone withdrawal and had a role in luteolysis. We therefore tested the hypothesis that luteal rescue with human chorionic gonadotrophin (hCG) would maintain PR expression. PR was immunolocalised to different cell types in human corpora lutea (n = 35) from different stages of the luteal phase and after luteal rescue with exogenous hCG. There was no change in the staining intensity of theca-lutein cell or stromal cell PR throughout the luteal phase or after luteal rescue. In the late-luteal phase, granulosa-lutein cell PR immunostaining was reduced (P < 0.05) but the trend to reduction was also seen after luteal rescue with hCG (P = 0.055). To further investigate the effect of hCG on granulosa-lutein cell PR expression, an in vitro model system of cultured human luteinised granulosa cells was studied. Cells were cultured for 12-13 days exposed to different patterns of hCG and aminoglutethamide to manipulate progesterone secretion (P < 0.0001). Expression of PR A/B and PR B isoforms was examined by quantitative real-time RT-PCR. PR A/B mRNA was lower (P < 0.05) after 11-13 days of culture than after 7 days of culture. This reduction could not be prevented by hCG in the presence (P < 0.05) or absence (P < 0.05) of stimulated progesterone secretion. The expression of PR B mRNA showed a similar pattern (P = 0.054). Simulated early pregnancy in vivo and hCG treatment of luteinised granulosa cells in vitro did not appear to prevent the down-regulation of PR seen during luteolysis.     

Relationships of changes in B-mode echotexture and colour-Doppler signals in the wall of the preovulatory follicle to changes in systemic oestradiol concentrations and the effects of human chorionic gonadotrophin in mares

A duplex grey-scale and colour-Doppler ultrasound instrument was used to study the changes in the wall of the preovulatory follicle in mares. When the follicle reached > or =35 mm (hour 0), mares were randomized into control (n = 16) and human chorionic gonadotropin (hCG)-treated (n = 16) groups. The hCG treatment was given at hour 0. Scanning was done every 12 h until hour 36, every hour between hours 36 and 48, and every 12 h thereafter until ovulation. Blood was sampled every 12 h for oestradiol assay. During the period 0-24 h post-treatment, oestradiol concentrations decreased in the hCG group and increased in the controls (significant interaction). During the period 0-36 h post-treatment, thickness and echogenicity of the granulosa increased in the hCG group but not in the controls. During the period 36 to 12 h before ovulation, granulosa and colour-Doppler end-points increased in the control and hCG groups (hour effects), while oestradiol was decreasing in both groups. The prominence and percentage of follicle circumference with an anechoic band peripheral to the granulosa and colour-Doppler signals in the follicle wall, indicating arterial blood flow, decreased during the period 4 to 1 h before ovulation (hour effects). Results indicated that the ultrasonographic changes of the wall of the preovulatory follicle were not associated temporally with changes in oestradiol concentrations and prominence of an anechoic band, and colour-Doppler signals decreased during the few hours before ovulation. The hypothesis that the latter portion of the ovulatory LH surge has a negative effect on systemic oestradiol was supported by the immediate decrease in oestradiol concentrations when hCG was injected.     

A close correlation in the expression patterns of Af-6 and Usp9x in Sertoli and granulosa cells of mouse testis and ovary

Usp9x, an X-linked deubiquitylating enzyme, is stage dependently expressed in the supporting cells (i.e. Sertoli cells and granulosa cells) and germ cells during mouse gametogenesis. Af-6, a cell junction protein, has been identified as a substrate of Usp9x, suggesting a possible association between Usp9x and Af-6 in spermatogenesis and oogenesis. In this study, we examined the expression pattern of Af-6 and Usp9x and their intracellular localization in testes and ovaries of mice treated with or without pregnant mare serum gonadotropin (PMSG), an FSH-like hormone. In both testes and ovaries, Af-6 expression was predominantly observed in supporting cells, as well as in steroidogenic cells, but not in any germ cells. In Sertoli cells, Af-6 was continuously expressed throughout postnatal and adult stages, where both Af-6 and Usp9x were enriched at the sites of Sertoli-Sertoli and Sertoli-spermatid junctions especially at stages XI-VI. In the granulosa cells, Af-6, as well as Usp9x, was highly expressed in primordial and primary follicles, but its expression rapidly decreased after the late-secondary follicle stage. Interestingly, in PMSG-treated mice, the expression levels of Af-6 and Usp9x were synchronously enhanced, slightly in Sertoli cells and strongly in granulosa cells of the late-secondary and Graafian follicles. Such closely correlated expression patterns between Af-6 and Usp9x clearly suggest that Af-6 may be deubiquitylated by Usp9x in both Sertoli and granulosa cells. It further suggests that the post-translational regulation of Af-6 by Usp9x may be one potential pathway to control the cell adhesion dynamics in mammalian gametogenesis.     

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed BMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K(d) 0.28 +/- 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.     

Anti-invasion and apoptosis induction of chlorella (Chlorella sorokiniana) in Hep G2 human hepatocellular carcinoma cells

The effects of 80% ethanolic extract derived from commercial granule chlorella (GPE) on cell viability, invasion capacity and apoptosis in human hepatoma cell line (Hep G2 cells) were investigated. The results demonstrated that GPE decreased cell viability, induced apoptosis and showed invasion inhibitory effects in the Hep G2 cells. GPE-triggered apoptosis was confirmed by 4′-6-diamidino-2-phenyindole (DAPI) staining and comet assay. GPE promoted an increase of reactive oxygen species (ROS) and Ca²⁺, and loss of mitochondrial membrane potential (ΔΨm) accompanied by cytochrome c release that was due to the decrease of Bcl-2 in the Hep G2 cells. GPE also induced the protein levels of apoptosis-inducing factor (AIF), increased the levels of caspase-3, -8 and -9, and stimulated the levels of fatty acid synthase (Fas) and Fas ligand (FasL) in the Hep G2 cells. Additionally GPE inhibited invasion of Hep G2 cells by down-regulation of the expression of matrix metalloproteinase (MMP)-2 and -9. Furthermore, cellular glutathione content and superoxide dismutases (SOD) activities were significantly reduced and thiobarbituric acid-reactive substances (TBARS) levels were significantly increased after GPE treatment. These results suggest that GPE can induce cytotoxicity on Hep G2 cells and inhibit the invasive capacity of malignant cells.     

Molecular characterization of bovine CSN1S2*B and extensive distribution of zebu-specific milk protein alleles in European cattle

The B allele of the bovine α_S2-casein gene (CSN1S2) was characterized at the molecular level and the distribution of zebu-specific milk protein alleles was determined in 26 cattle breeds originating from 3 continents. The CSN1S2 *B allele is characterized by a C → T transition affecting nucleotide 17 of exon 3, which leads to a change in the eighth amino acid of the mature protein, from Ser to Phe (i.e., TCC →TCC). DNA-based methods were developed to identify carriers of CSN1S2*B and the other alleles (CSN1S2 *A, C, and D) at the same locus. CSN1S2*B and other zebu-specific milk protein alleles and casein haplotypes are widely distributed in European cattle breeds, particularly those of southeastern origin. Alleles CSN1S2 *B and CSN3*H are important in searching for zebu imprints in European cattle breeds. Diversity estimates at the milk protein loci were highest in the zebus followed by southeastern European taurines. Anatolian Black had the highest number of zebu alleles among European taurines. Common, group, and intergroup haplotypes occurred in the breeds and demonstrated relationships that concurred with developmental histories, genetic makeup, and, in particular, exposed the extent of zebu influence on southeastern European cattle.