Variation in the ovarian and plasma progesterone and estradiol levels of the domestic hen during a pause in laying

Little is known about the physiological events occurring in the chicken ovary during a pause in laying, therefore the aim of the present study was to examine changes in sex steroid concentration in the follicle wall and blood plasma during cessation of egg laying. The experiment was performed on laying Isa Brown hens. Control hens were fed ad libitum whereas the experimental ones were subjected to a pause in laying by complete food deprivation for 5 days and water deprivation on 3 day followed by feeding every second day up to 9 day and then ad libitum. Blood samples were taken from the wing vein each day. The hens were decapitated on day 3, 6, 9, and 16. The ovary was isolated and the following follicles were dissected: white (1-2; 2-4; 4-6; 6-8 mm) and yellow preovulatory ones (F1-F3). Progesterone and estradiol were measured in the follicle wall and blood plasma by RIA methods. The hens stopped egg laying on day 4 and began egg restoration on day 14 of the experiment. Cessation of egg laying was preceded by a decrease in estradiol and progesterone levels in the ovary as well as in the blood plasma. The plasma level of these steroids began to increase 7 days before the start of egg restoration. Autopsy of the ovary showed that the atrophy of the chicken yellow preovulatory follicles during the pause in laying was accompanied by a significant increase in the total number of white follicles.     

Immunohistochemical localization of aromatase during the development and atresia of ovarian follicles in prepubertal horses

Ovarian steroidogenesis from the neonatal to pubertal period in horses is poorly understood. This study was designed to immunolocalize cytochrome P450 aromatase in the ovarian follicles of slaughtered fillies ages approximately (I) 6-9 mo (<10MF); (II) 1 y (1YF); and (III) 1.5 y (1.5YF). The ovaries of adult mares were used as controls. In each age group, immunoreactivity for P450arom was observed in the mural granulosa of nonatretic follicles >5 mm in diameter. Staining intensity was dependent on the size and morphology of the follicle. In nonatretic follicles 5-10 mm in diameter, the reaction was weak and heterogeneous, while most intense staining was observed in preovulatory follicles. In follicles (diameter <20 mm) in the groups <10MF and 1YF, the reaction was less intense than in adult mare follicles of similar size. In each age group, several follicles with early or advanced signs of atresia exhibited a heterogeneous staining pattern, which subsequently disappeared in late atretic follicles. No immunoreactivity was detected in the theca interna, preantral follicle, or stroma cells. Our observations reveal that the mural granulosa of viable follicles in fillies about 6-18 mo old contains aromatase, indicating that the ovary is capable of estrogen synthesis. Immunoreactivity for P450arom was dependent on follicle size and disappeared in atretic follicles.     

Progesterone injection and egg production in turkey hens

An arrest in laying associated with either a polyovarian follicle (POF) or a polycystic ovarian follicle (PCOF) syndrome has been reported in turkey hens photostimulated at an early age with a constant-light photoperiod. Hens expressing the POF or PCOF syndrome had stopped laying for several weeks, but the ovary contained an increased number of mature-size and larger follicles (POF hens), which were cystic (PCOF) in some of the hens. Hens with the POF or PCOF syndrome had plasma progesterone (P(4)) concentrations that were relatively high and without surges. We hypothesized that high plasma P(4) concentrations may block ovulatory surges of LH but not the growth or maintenance of hierarchical follicles leading to development of the POF or PCOF syndrome in turkey hens. In the first six studies, hens were photostimulated with either a 14L:10D or a 24L:0D photoperiod and, after laying for 1-38 wk, were then injected daily for up to 14 days with P(4) (up to 1.50 mg kg(-1) day(-1)) and necropsied. At all ages, the oviposition rate was reduced at a P(4) dosage of 0.17 mg kg(-1) day(-1). With dosages of 0.33 mg kg(-1) day(-1) or greater, however, ovipositions stopped in most hens within approximately 2 days. For hens laying for less than 15 wk, oviductal weight and number of hierarchical follicles of P(4)-injected hens were not different from control vehicle-injected hens, but the numbers of mature, cystic, and atretic follicles were increased. For hens laying for 38 wk, when treated with P(4), oviductal weight and number of hierarchical follicles decreased, but number of atretic follicles increased. No effect of photoperiod was found on egg production, oviductal weight, or follicle number, and none of the hens developed POF or PCOF syndrome in these experiments. Two additional experiments were conducted with hens early in the reproductive period that had been photostimulated with 14L:10D or 24L:0D and injected with P(4) (0.33 mg kg(-1) day(-1)) for 10 or 12 days but not necropsied until 3 wk after the last injection. Most of the hens photostimulated with the 24L:0D photoperiod and injected with P(4), and a few of the hens photostimulated with the 14L:10D photoperiod and injected with P(4), had developed the PCOF syndrome when necropsied. The hens with the PCOF syndrome had high levels of P(4) when necropsied. From these studies, we concluded that the PCOF syndrome can be induced early in the reproduction period by photostimulating turkey hens with a 24L: 0D photoperiod, injecting them for 10 to 12 days with P(4) at a dosage of 0.33 mg kg(-1) day(-1), and then waiting 3 wk for the PCOF syndrome to develop.     

Concentrations of steroids and expression of messenger RNA for steroidogenic enzymes and gonadotropin receptors in bovine ovarian follicles of first and second waves and changes in second wave follicles after pulsatile LH infusion

The objectives were to compare expression of mRNA for cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase Delta(4), Delta(5) isomerase (3beta-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or 4 days using a computer-controlled syringe pump (n=5-6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25 microg/h; W2LH). During infusion, blood samples were collected at 12min intervals for 8h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or 4 after emergence of follicular waves. Follicles were frozen and stored at -80 degrees C. Follicular fluid (FF, 50 microl) was collected for determination of progesterone (P4), oestradiol-17beta (E2) and androstenedione (A4) concentrations. Frozen sections (14 microm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P450scc, P450c17, P450arom, 3beta-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E2 and A4 in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E2 and A4 concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P450c17 and 3beta-HSD. In conclusion, follicular fluid concentrations of E2 and A4 were lower in W2S than in W1S and E2 and A4 concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3beta-HSD.     

Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

The effects of age and sex steroids on the macrophage population in the ovary of the chicken, Gallus domesticus

The role of macrophages in the function of the hen ovary has not yet been described, although these cells may be an important regulator of ovarian function in mammals. The aim of this study was to determine the changes in the frequency of macrophages during ageing and follicular atresia, and the effects of sex steroids on the macrophage population in the hen ovary. Cryostat sections of ovarian tissues of immature, young laying and old laying hens and those of immature hens treated with or without diethylstilboestrol (DES) or progesterone were immunostained for macrophage cells using mouse anti-chicken macrophage monoclonal antibody. Macrophages were observed under a light microscope and counted using a computer assisted image analyser. The frequency of macrophages in both the stroma and theca of primary follicles was significantly greater in young laying hens than in immature and old laying hens and these cells were more frequent in old laying hens than in immature hens (P < 0.01). Macrophages were more frequent in atretic follicles than in normal follicles (P < 0.01). The number of macrophages in both the stroma and theca of primary follicles of DES-treated birds was significantly greater than in those of progesterone-treated and control birds (P < 0.01). Progesterone had no significant effect on the population of macrophages. These results suggest that macrophages in the ovary increase in association with sexual maturation of birds and atresia of follicles and decrease during ageing. Oestrogen may be one of the factors that affect the population of macrophages in the hen ovary.     

Effect of growth hormone on steroid content,proliferation and apoptosis in the chicken ovary during sexual maturation

The present study was undertaken to examine in vivo the effect of growth hormone (GH) on progesterone and estradiol levels and on cell proliferation and apoptosis in the chicken ovary during sexual maturation. Hy-Line chickens (10 weeks old) were injected three times a week with 200 μg recombinant chicken GH (cGH) per kilogram body weight until sexual maturity. GH treatment significantly increased ovarian weight at 16 weeks of age, i.e., ∼1 week before onset of egg laying. The progesterone content in the ovary just before and at the time of sexual maturity and the estradiol content before onset of egg laying were also elevated after cGH injections. The highest number of proliferating (positive for proliferating cell nuclear antigen) and apoptotic (positive for terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labeling) cells was found in the ovarian stroma and white follicles (>1-4 mm diameter), whereas the lowest number of these cells was detected in yellow (>8-30 mm) follicles. Administration of cGH significantly stimulated cell proliferation and inhibited cell apoptosis in the ovarian stroma and small ovarian follicles. The number of ovarian follicles and the weight of the ovary prior to the first oviposition were also higher in cGH-injected hens. Thus, prior to and after the onset of egg laying, GH participates in the growth, maturation and hormonal activity of ovarian follicles in the chicken, via the regulation of steroidogenesis, proliferation and apoptosis processes.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Correlation between messenger RNA expression of cytochrome P450 aromatase and its enzyme activity during oocyte development in the red seabream (Pagrus major)

In teleosts, estradiol-17beta (E2) is an important hormone responsible for oocyte development. To elucidate the molecular mechanisms underlying E2 biosynthesis, we characterized the structure of red seabream (Pagrus major) cytochrome P450 aromatase (P450(arom)) that is directly involved in E2 biosynthesis and found changes in mRNA levels of P450(arom) during oocyte development induced by implantation of gonadotropin-releasing hormone analogue. A cDNA clone encoding P450(arom) is 1779 base pairs in length and encodes a protein of 519 amino acids in length, with a calculated molecular weight of 58.9 kDa. Northern blot analysis showed that P450(arom) mRNA levels increased gradually from Day 8, when oocytes reached the secondary yolk globule stage, and were maintained at high levels at the day of spawning (Day 15). The P450(arom) mRNA levels increased in association with an increase of the gonadosomatic index (gonad weight/body weight x 100%), serum E2, and P450(arom) enzyme activity (in vitro conversion of testosterone to E2 in the ovarian fragments). Furthermore, an increase in mRNA levels of the LHbeta, but not FSHbeta, correlated with increased P450(arom) mRNA levels during the course of ovarian development. In addition, the levels of P450(arom) mRNA increased in isolated ovarian follicles during the course of vitellogenic oocyte growth and became undetectable in follicles at the migratory nucleus and the mature stages. These findings, together with those of the previous studies, suggest that LH, not FSH, may regulate E2 biosynthesis via increased levels of P450(arom) mRNA during oocyte development of red seabream.     

The influence of prepubertal partial sinistral ovariectomy on the subsequent reproductive function of domestic hens (Gallus domesticus )

Each of 20 White Leghorn hens of 13 to 14 weeks were subjected to partial sinistral ovariectomy and sham-operations. In half of the hens from each group, the percentage of egg production and clutch size were noted until 50 weeks of age. The growing pattern of normal ovarian follicles was also recorded at 26 weeks of age in a rest half ofthe hens in the two groups. The percentage of egg production and the mean and variance of clutch size did not differ significantly (P / 0.05) between the partially ovariectomized and sham-operated groups. The growing yellow follicles (>8 mm) in the rapidly developing phase in these two groups did not vary, although the smaller follicles (4 to 8 mm in diameter) remained significantly (P / 0.01) more in the shamoperated control group than in the partially ovariectomized group. This observation indicates that smaller follicles (4 to 8 mm) developed in the larger (>8 mm) follicles more efficiently in partially ovariectomized hens than in the sham-operated (control) hens. In a second experiment, one group of hens had all the yellow follicles (>8 mm) removed, while a second group of hens was left untreated. On the 3rd and 6th day post treatment, the hens were examined for the presence of ovarian follicles. No significant (P / 0.05) difference in the growing pattern of subsequent follicles (2 to 4 or 4 to 8 mm) was detected due to treatment. These data demonstrate that the mechanisms that regulate follicular growth and atresia are adjust to maintain normal ovulation following partial ovariectomy.     

Onset of steroidogenic enzyme gene expression during ovarian follicular development in sheep

Steroidogenesis is a major function of the developing follicle. However, little is known about the stage of onset of steroid regulatory proteins during follicular development in sheep. In this study, several steroidogenic enzymes were studied by immunohistochemistry and/or in situ hybridization; cytochrome P450 side chain cleavage (P450(scc)), cytochrome P450 17alpha-hydroxylase (17alphaOH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 aromatase (P450(arom)), steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), and LH receptor (LH-R). To define the stages of follicular growth, ovarian maps were drawn from serial sections of ovine ovaries, and follicles were located and classified at specific stages of growth based on morphological criteria. In this way, the precise onset of gene expression with respect to stages of follicular growth for all these proteins could be observed. The key findings were that ovine oocytes express StAR mRNA at all stages of follicular development and that granulosa cells in follicle types 1-3 express 3beta-HSD and SF-1. Furthermore, the onset of expression in theca cells of StAR, P450(scc), 17alphaOH, 3beta-HSD, and LH-R occurred in large type 4 follicles just before antrum formation. This finding suggests that although the theca interna forms from the type 2 stage, it does not become steroidogenically active until later in development. These studies also confirm that granulosa cells of large type 5 follicles express SF-1, StAR, P450(scc), LH-R, and P450(arom) genes. These findings raise new questions regarding the roles of steroidogenic regulatory factors in early follicular development.     

Follicular growth and atresia in the ovaries of hens (Gallus domesticus) with diminished egg production rates

Diets containing 3.5, 1.0 and 0.1% calcium were fed from the age of 42 weeks to individually caged laying hens. Ovaries were examined at 46-49 and 70 weeks of age for changes in the follicular population corresponding to the lowered egg production rates of birds given calcium-deficient diets (1.0% and 0.1%) and older birds given a normal diet (3.5%). Growth rates of follicles from 3.5 mm diameter to ovulation were not changed by the level of dietary calcium in 46-49-week-old birds. The number of atretic small follicles (less than or equal to 8 mm diam.) increased in old and calcium-deprived birds, resulting in lower numbers of viable follicles in the intermediate stages of growth (3-8 mm diam.). There was also an increase in the number of small follicles (1-2 mm diam.) starting to grow in 70-week-old birds which may have partly compensated for the increased loss by atresia. Birds of all ages on all diets were able to produce large follicles up to ovulable size. The main feature of poor laying birds was a reduction in the ovulation rate due to the loss of large follicles (greater than 8 mm diam.) by atresia, an event seen rarely in the birds with good laying performance. As atresia is the normal fate of most of the small follicles, the mechanisms controlling atresia in the small follicles and the large follicles appear to be independent.     

Decreased granulosa cell luteinizing hormone sensitivity and altered thecal estradiol concentration in the aged hen, Gallus domesticus

Few studies have examined the effect of age on the ovulation cycle of the hen. Our aim was to determine if changes in the ovary account for the decrease in egg production with age. Young hens (28-38 wk of age) laying at least 20 eggs per sequence and old hens (53-63 wk of age) laying 3-6 eggs per sequence were used. We determined luteinizing hormone (LH) sensitivity of the ovary of young and old hens by measuring LH stimulable adenylyl cyclase (AC) activity of the granulosa layer. We also measured theca- and granulosa-layer weights and steroid concentrations of these layers and of the serum in young and old hens. Mean basal AC activity (pg/min/mg protein) for the largest (F1) and second largest (F2) follicles from young and old hens did not differ. A significant dose-response relationship to LH was present in all groups, and AC responsiveness to increasing doses of LH was greater in the F1 and F2 follicles of young hens than in the same follicles of old hens. The F4 and F5 follicles of young hens had a significantly greater estradiol (E2) concentration (pg/mg theca protein) compared to old hens, while the E2 concentration in the F2 follicle was greater in old hens. The theca layer of the F1 follicle of old hens weighed significantly more than that of young hens, whereas the theca layer of the F3, F4 and F5 follicles from young hens weighed more than those of old hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian steroid production in vitro during gonadal regression in the turkey. I. Changes associated with incubation behavior.

The mechanism regulating ovarian regression during incubation behavior in the domestic turkey has not been elucidated. This study was designed to determine whether ovarian steroidogenic potential is depressed during gonadal regression associated with the onset of incubation behavior. Hens were housed in floor pens equipped with trap nests that were checked 7 times per day. Hens were grouped, according to nesting frequency and egg production, into the following classifications: laying (laid an egg every day and trapped in the nest only once/day); transitional (laid an egg every day but trapped in the nest 4 or more times/day); and Day 1, Day 3, and Day 5 incubating (no egg for 2, 4, or 6 days, respectively, while trapped in the nest at least 4 times/day). Follicular atresia was evident in the largest preovulatory follicle (F1) in transitional hens, extensive in F1 through the third largest follicle (F3) in Day 1 incubating hens, and extensive in F1 through F7in Day 3 incubating hens. Levels of circulating LH, progesterone (P), androgen (A), and estradiol (E) decreased in transitional hens relative to concentrations in laying hens and remained low thereafter. In contrast, levels of prolactin were greater in Day 3 and Day 5 incubating hens than in laying, transitional, or Day 1 incubating hens. Basal production of P by F1 granulosa cells was lower from Day 1 incubating hens than from the other groups. Production of P in response to porcine-luteinizing hormone (pLH) was greater by cells from transitional and Day 1 incubating hens than from those of laying hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of exogenous LH on plasma concentrations of progesterone and oestradiol in relation to the cessation of egg laying induced by different moulting methods

Artificially induced cessation of egg laying caused regression of the reproductive tract in hens, as well as changes in circulating concentrations of sex steroids. Hens were bled at several stages during and after artificial moult induced by fasting or overfeeding a diet low in calcium or high in zinc. Hens received a single injection of 200 i.u. of horse LH at Day 0, 7, 21, 35 and 77 (Exp. 1) or Day 0, 8, 23, 35 and 71 (Exp. 2) after start of the treatment to induce moult. Blood samples were taken before and 20, 40 and 60 min (Exp. 1) or 15, 30 and 45 min (Exp. 2) after LH injection. Hens which were fasted or given the diet high in zinc had low plasma progesterone concentrations and the response to LH was reduced or delayed. In hens fed low calcium the reduction in plasma progesterone was less pronounced and the responsiveness to LH was more or less maintained. Conversely, there was no response of oestradiol to LH in laying hens. However, oestradiol concentrations increased in moulting hens after LH injection, due to the high oestradiol secretion from the small white follicles, since all yolky follicles were atretic.     

Effects of early prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on postnatal reproduction in the laying hen (Gallus gallus)

This study demonstrates the long-term effects of very early embryonic exposure to a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0, 10 and 20 ng/egg), administered before the beginning of embryonic development, on growth and reproductive performance in laying hens. Hatchability and body weight gain from 11 weeks onwards were significantly depressed in 20 ng treated hens. All hens started laying egg at around the same age and the laying performance of TCDD-treated hens was normal. No disturbances in the age-related pattern and concentrations of oestradiol, LH or FSH in plasma could be found but mean progesterone concentrations were significantly lower in 20 ng treated hens. Moreover, follicular distribution was changed with less small white follicles and smaller yellow follicles, which probably resulted in the lower egg weight of the 20 ng treated hens. At 43 weeks of age, hens treated in ovo with TCDD showed a retained right oviduct, mostly filled with clear fluid. From these results, it seems that in ovo exposure to TCDD interferes in the right oviduct regression during embryonic development and induces some changes in follicular distribution but without impairment of reproductive performance in the adult laying hen.     

Immunolocalization of MHC-II+ cells in the ovary of immature, young laying and old laying hens Gallus domesticus

The aim of this study was to localize major histocompatibility complex class II positive (MHC-II+) cells in the hen ovary, and to determine the effects of ageing and sex steroids on their frequency. Cryostat sections of ovarian tissues of immature, young laying and old laying hens and those of immature hens treated with or without diethylstilboestrol or progesterone were prepared. Sections were immunostained for MHC class II antigens using mouse anti-chicken MHC class II monoclonal antibody and observed under a light microscope. Positive cells were counted using a computer-assisted image analyser. MHC-II+ cells were localized in the ovarian stroma and theca layer of primary follicles in all birds examined. The frequency of MHC-II+ cells in the stroma and theca of primary follicles (approximately 400-600 microns in diameter) was significantly greater in young laying hens than it was in immature and old laying hens (P < 0.01). In the stroma and the theca of primary follicles of diethylstilboestrol-treated birds, the frequency of MHC-II+ cells was significantly greater than it was in the stroma and theca of control and progesterone-treated birds (P < 0.01). Progesterone had no significant effect when compared with controls. These results indicate that both the ovarian stroma and theca of follicles in the hen ovary contain MHC-II+ cells, the frequency of MHC-II+ cells increases in association with sexual maturation and decreases thereafter during ageing, and oestrogen may be one of the factors enhancing the induction of MHC-II+ cells in the ovary.