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This study examined caprine follicular development in different concentrations of alginate matrix to determine the optimal conditions for culture. Caprine preantral follicles were cultured in a two-dimensional system (control) or a three-dimensional encapsulated system in 0.25%, 0.5%, or 1% alginate (ALG 0.25, ALG 0.5, and ALG 1, respectively). A higher percentage of morphologically normal follicles developed in ALG 0.5 and ALG 1 than in ALG 0.25 or the control (P < 0.05). The rate of antrum formation, however, was higher in ALG 0.25 than in ALG 0.5 and ALG 1 conditions (P < 0.05), but similar to the control. Follicles cultured in ALG 0.25 had higher growth rates and meiotic resumption than those cultured in ALG 0.5, ALG 1, or the control (P < 0.05). Moreover, follicles cultured in ALG 0.25 had higher levels of estradiol and progesterone than those cultured in ALG 0.5, ALG 1, or the control, as well as higher levels of CYP19A1 and HSD3B mRNA. In conclusion, a three-dimensional system that uses ALG 0.25 fosters the in vitro development of caprine preantral follicles and increases the rate of meiotic resumption., (© 2014 Wiley Periodicals, Inc.)

Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3β-hydroxysteroid dehydrogenase, 3β-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3β-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays.

Immunolocalization of 3β-hydroxysteroid dehydrogense (3β-HSD), cytochrome P450c17 (P450c17) and androgen receptor (AR) were investigated in rat cumuli oophori (COCs) of late pre-ovulatory follicles and in post-ovulatory COCs bearing fertilized oocytes. A gradient of intensity of 3β-HSD immunolabelling was observed in the granulosa layer of pre-ovulatory follicles, with almost negative immunolabelling in COCs and with the strongest immunoreaction in the mural granulosa cells. Post-ovulatory COCs showed strong 3β-HSD immunolabelling in the peripheral regions and weak labelling near the oocyte, suggestive of responsiveness of cumulus cells to an anti-luteinizing effect exerted by the fertilized oocyte. In pre-ovulatory follicles, a weak P450c17 immunopositivity was limited to expanded cumulus granulosa cells and the positive labelling persisted in post-ovulatory COCs. P450c17 immunopositivity was also found in ampullary epithelial cells. A strong AR immunopositivity was confined mainly to the COCs in pre-ovulatory follicles and a similar immunoreaction was present in the granulosa cells of ovulated COCs. Simultaneous AR and cytochrome P450c17 immunolabelling in the pre- and post-ovulatory COCs is suggestive of an intra- and paracrine androgen regulation of the cumulus granulosa cell function., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

The expression patterns of steroidogenic enzymes in ovarian antral follicles at various stages of growth in a follicular wave have not been reported for sheep. Ovaries were collected from ewes (n=4-5 per group) when the largest follicle(s) of the first wave of the cycle, as determined by ultrasonography, reached (i) 3 mm, (ii) 4 mm, (iii) > or =5 mm in diameter or when there was a single (iv) preovulatory follicle in the last wave of the cycle, 12h after estrus detection. The expression pattern of steroidogenic enzymes was quantified using immunohistochemistry and grey-scale densitometry. The expression of CYP19 in the granulosa and 3beta-HSD and CYP17 in the theca increased (P<0.01) progressively from 3 to > or =5 mm follicles in the first wave of the cycle and was lower (P<0.01) in the preovulatory follicle compared to > or =5 mm follicles. However, the expression of 3beta-HSD in the granulosa increased (P<0.05) from 3 to > or =5 mm follicles and was maintained (P<0.05) at a high level in the preovulatory follicles. The amount of CYP19 in the granulosa of the growing follicles correlated positively (r=0.5; P<0.03) with the concurrent serum estradiol concentrations. We concluded that the expression pattern of steroidogenic enzymes in theca and granulosa of follicles growing in each wave in the ewe, paralleled with serum estradiol concentrations, with the exception that concentrations of 3beta-HSD in granulosa increased continuously from follicles 3mm in diameter to the preovulatory follicle.

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.

Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.

During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV collagen is an important component of many basal laminae. Each molecule is composed of three alpha chains; however, six different type IV collagen chains have been identified. It is not known which of these chains are present in the follicular basal lamina and whether the type IV collagen composition of the basal lamina changes during follicular development. Therefore, we immunolocalized each of the six chains in bovine ovaries using antibodies directed to the nonconserved non-collagenous (NC) domains. Additionally, dissected follicles were digested with collagenase to release the NC domains, and the NC1 domains were then detected by standard Western immunoblot methods. The follicular basal lamina of almost all primordial and preantral follicles was positive for all type IV collagen alpha chains. Colocalization of type IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen alpha1, alpha2, alpha3, alpha4, and alpha5 chains were present within the follicular basal lamina of only a proportion of antral follicles (17 of 22, 20 of 21, 15 of 18, 14 of 28, and 12 of 23, respectively), and staining was less intense than in the preantral follicles. Staining for the alpha1 and alpha2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining for alpha1 and alpha2 chains of type IV collagen was confirmed by Western immunoblots. As well as being detected in the basal lamina of approximately half of the antral follicles examined, type IV collagen alpha4 also colocalized with 3beta-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna. Type IV collagen alpha6 was detected in the basal lamina of only one of the 16 antral follicles examined. Thus, the follicular basal lamina changes in composition during follicular development, with immunostaining levels being reduced for all type IV collagen chains and immunoreactivity for type IV collagen alpha6 being lost as follicle size increases. Additionally, immunoreactivity for alpha1 and alpha2 appears in the extracellular matrix of the theca as it develops.

This experiment was conducted to determine why follicles luteinize faster in the Meishan breed than in the Large White breed of pig. Follicles were recovered during the late follicular phase from ovaries of both breeds before and after administration of hCG given to mimic the LH surge. First, the patterns of cholesterol transporters (high and low density lipoproteins: HDL and LDL) were compared. Cholesterol transporters detected in follicular fluid consisted of HDL only. Similar amounts of Apolipoprotein A-I were found in all samples. There was no obvious breed effect on minor lipoproteins found in the HDL-rich fraction, and this pattern was altered similarly by hCG in the two breeds. The LDL-rich samples of serum from both breeds contained similar amounts of protein. Second, three steroidogenic enzymes, adrenodoxin, 17 alpha-hydroxylase-lyase (P450(17) alpha) and 3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD) were detected by immunohistochemistry and quantified by image analysis on sections of the two largest follicles. Before hCG treatment, theca interna cells demonstrated immunoreactivities for adrenodoxin (strong), P450(17) alpha and 3 beta-HSD (very strong), whereas granulosa cells displayed immunoreactivities for adrenodoxin only. After hCG treatment, the localization of the enzymes was unchanged but the staining intensity of adrenodoxin on granulosa cells and 3 beta-HSD on theca cells increased (P < 0.01 and P < 0.05, respectively). Breed effects were detected for the amounts of adrenodoxin in theca cells (Meishan > Large White; P < 0.05) and of 17 alpha-hydroxylase (Large White > Meishan, P < 0.01). Breed x treatment interactions were never detected. Finally, gelatinases, plasminogen activator, plasminogen activator inhibitor, tissue inhibitors of metalloproteases (TIMP-1 and TIMP-2) were visualized by direct or reverse zymography or western blotting. Whatever the stage relative to LH administration, follicular fluid from Large White gilts contained more TIMP-1, and TIMP-2 (P < 0.02 and P < 0.01, respectively). No breed effect was detected for the amounts of gelatinases and plasminogen activator inhibitor 1. However, for these parameters, a significant breed x time interaction was obvious, as the Meishan follicles had a greater response to hCG (P < 0.01). Since proteolysis plays a key role in the bioavailability of growth factors such as insulin-like growth factor 1, fibroblast growth factor and transforming growth factor beta, which have the ability to alter gonadotrophin-induced progesterone production in pigs, the differences observed in its control in the present study may explain, at least in part, the different patterns of luteinization observed in Meishan and Large White follicles.

Ovarian macrophages may be involved in the degradation of the basal membrane after rupture of a preovulatory follicle, or in angiogenesis during follicle and corpus luteum development, or may support steroidogenesis. The present study describes easy access to macrophage-like cells in the ovary and characterizes them. Cells from bovine antral follicles with 1-4 ml of their naturally occurring fluid were plated on day zero. On day 10, the granulosa cell culture showed macrophage-like cells. They developed as non-adherent cells and also adhered to the monolayer as single cells or as hemopoietic-like clones. The macrophage-like cells contained strong activity of non-specific esterase, acid phosphatase or 3beta-hydroxy-steroid dehydrogenase (3beta-HSD). Only the steroid enzyme was absent on day 19. As detected by immunolocalization, macrophage-like cells of various sizes showed a response for the CD18 surface molecule of leukocytes, for the CD45 molecule related to hemopoietic progenitor cells, and for the CD14 molecule selectively expressed on cells of the monocyte-macrophage lineage. The macrophage-like cells exhibited a granular-like actin structure as revealed by fluorochrome-labeled phalloidin. The ultrastructure of small, intermediate or large macrophage-like cells was similar to that of monocytes or of macrophages. In situ immunolabeling of intact or regressing follicles revealed single cells positive for CD14, CD18 or CD45. In order to establish that in vitro proliferation of the macrophage-like cells had occurred, their number was assessed between days 1 to 19 of cultivation. The cultures derived from 23 follicles were classified into groups A, B, or C according to the number of non-adherent cells (<5000, <12 500, or >/=12 500 per 16-mm well) on day 12 of cultivation. The total number of macrophage-like cells (non-adherent and adherent) increased approximately fourfold in all groups between days 1 to 12. The total number decreased to day 5 levels between days 15 to 19. Thus, the macrophage-like cells probably represent a subpopulation of the macrophage family. They proliferate in co-culture with granulosa cells.

The theca cells (TC) first become identifiable in preantral follicles after the granulosa cells (GC) begin to divide. It remains unknown when the TC first respond to LH and acquire the capacity to produce androgens. The signal initiating TC differentiation is also unknown since pre-theca cells do not contain LH receptors. Since the first wave of follicle development in the rat occurs postnatally, we correlated the function of dispersed ovarian cells from 4-, 5-, 6-, 7-, and 10-day-old rats with the morphological differentiation of TC. The largest follicles in ovaries from 4-day-old rats were primary follicles without associated TC. These cells were unable to produce cAMP or steroids in vitro in response to hCG. At 5 days, the first theca were associated with follicles containing 2-3 layers of GC. These cells were responsive to hCG, producing cAMP, progesterone, androstenedione, and androsterone. Responses to hCG increased progressively through 10 days of age. Cholesterol side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha)) enzymes were localized exclusively to the theca interna. Messenger RNAs for LH receptor, P450scc, 3 beta-HSD, and P450(17 alpha) were expressed prior to the time the TC become responsive to LH or morphologically differentiated. To determine the source of the signal regulating TC differentiation, dispersed cells from 4-day-old rat ovaries that were unresponsive to LH were treated with preantral follicle-conditioned medium containing thecal differentiating factor (TDF) activity. The TDF activity stimulated androgen production and expression of LH receptor, P450scc, 3 beta-HSD, and P450(17 alpha) mRNAs. These data demonstrate that a paracrine signal from the preantral follicle can initiate TC differentiation prior to expression of LH receptors. TC become responsive to LH and capable of producing androgens coincident with morphological differentiation.

Twenty nonlactating beef cows were used to determine the effects of dietary energy restriction on ovarian follicular and corpus luteum (CL) development. Cows were fed to either gain (controls) or lose (restricted; RES) body weight. Observations continued until RES cows developed a subfunctional CL (progesterone [P4] < 1.5 ng/mL on d 10 of a cycle; n = 4) or had functional CL (P4 > or = 1.5 ng/mL on d 10 of a cycle; n = 6) followed by anestrus, at which time observations were discontinued on individual controls. Estrous cycles were then standardized for all cows. For RES cows developing subfunctional CL, cycle A was the cycle before development of the subfunctional CL, and cycle B was the 11-d period during development of a subfunctional CL. For RES cows with a functional CL, cycle A was the next to last cycle before anestrus, and cycle B was the 11-d period during formation of a functional CL. Daily P4 concentrations did not differ (P > .10) between controls or RES cows developing functional CL during cycle B but were lower (P < .05) in RES cows developing subfunctional CL. Ovulatory follicles and CL were smaller (P < .05) in RES cows during cycles A and B compared with controls. Daily IGF-l concentrations were higher on d 2 through 4 of both cycles in RES cows developing functional CL compared with RES cows developing subfunctional CL (P < .05). Feeding diets limited in energy resulted in two types of CL. These differences may have been due to IGF-I concentrations.

Follicular and luteal morphology and steroidogenic function were investigated by immunohistochemistry for cytochrome P450 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) during the estrous cycle in pigs, sheep, and cows. The theca interna of all species expressed P450c17 during follicular development. In the pig, this constituted a continuous layer of cells around the follicle, but a sheath of cells lining the basement membrane appeared not to express P450c17. Neither was expression of P450c17 in ovine and bovine follicles uniform throughout the theca interna. In these two species, a beaded appearance was given by P450c17, since it was expressed in some regions but not in others. Therefore, staining for P450c17 defined functional sub-populations of cells within the theca interna of pigs, sheep, and cows. Ovulation was associated with a decrease in P450c17 in all species, but some expression persisted in theca-derived cells of developing and mature porcine CL. Expression of 3 beta-HSD in the preovulatory follicle was confined to the theca of the pig and sheep; in contrast, in the cow, it was highest in the granulosa. In general, 3 beta-HSD expression appeared to be greater in porcine than ovine or bovine follicles, the physiological relevance of which is discussed. Porcine and ovine theca continued to express 3 beta-HSD after ovulation, and granulosa-derived cells increased their 3 beta-HSD expression markedly as they luteinized in all three species. During early luteal development in pigs and sheep, theca-derived cells with high 3 beta-HSD encircled luteal lobules, but these cells appeared throughout the parenchyma of the mature CL. Luteal regression in sheep and cows was typified by the loss of many cells expressing 3 beta-HSD, whereas others, adjacent to them, appeared to be intact without loss of enzyme expression. These data further define differences in steroidogenesis during follicular and luteal development among the pig, sheep, and cow.

Pre- and postovulatory ovarian follicles of the amago salmon were investigated with special consideration given to the steroid production site. In the preovulatory specimens, the special thecal cells contained abundant mitochondria and smooth endoplasmic reticulum, as in the case of other steroid-producing cells, while the granulosa cells developed rough endoplasmic reticulum like protein-secreting cells. Remarkably ultrastructural changes occurred in the granulosa and the special thecal cells during the oocyte maturation stage; dilation of rough endoplasmic reticulum in the former cell and further development of smooth endoplasmic reticulum in the latter one. Histochemical reactions of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity were only positive in the special thecal cells of the postovulatory follicles. These results strongly suggest that the special thecal cells are the major sites of estrogen precursor synthesis in the amago salmon ovary.

The steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase and delta 4-5 isomerase (3 beta-HSD) that convert delta 5-hydroxysteroids to delta 4-ketosteroids were measured in human follicular tissue collected during the follicular phase of the menstrual cycle. This study was done in order to determine whether 3 beta-HSD enzyme activity fluctuated during the follicular phase of the menstrual cycle and, if so, whether these changes were reflected by the concentration of steroids in the follicular fluid. A microsomal fraction was prepared from each of 14 follicular-phase follicles, and 3 beta-HSD enzyme activity was estimated by the amount of androstenedione synthesized in the presence of excess substrate (dehydroepiandrosterone) and cofactor (nicotinamide adenine dinucleotide). Androstenedione, dehydroepiandrosterone, and progesterone were measured in the aspirated follicular fluid of each follicle. 3 beta-HSD enzyme activity was undetectable in the smallest (3 to 5 mm) follicles, increased in 5 to 6 mm follicles to 363.2 +/- 60 pmol of androstenedione per milligram per hour, and increased still further in 9 to 11 mm follicles to 5,000 +/- 200 pmol of androstenedione per milligram per hour. The increase in 3 beta-HSD enzyme activity was accompanied by an increase in the concentration of androstenedione in the follicular fluid as well as of progesterone in larger follicles. These data indicate that 3 beta-HSD activity increases during the follicular phase of the menstrual cycle. It is suggested that the primary product of the increased enzyme activity is androstenedione. Since androstenedione is the principal C19 steroid produced by the ovulatory follicle and serves as a substrate for estradiol production, this increase in 3 beta-HSD activity may be important for the associated changes in the late follicular phase that lead to ovulation.

Previous studies have shown that biosynthesis of progesterone, the major steroid product of hen granulosa cells, increases during follicular maturation. However, the contribution of individual granulosa cells to the total progesterone production of each follicle is not known. The objective of the present study was to determine the presence and relative activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in individual granulosa cells isolated from each of the five largest yolk-filled preovulatory follicles of laying hens. 3 beta-HSD cytochemistry in the presence or absence of pregnenolone substrate was performed on digitonin-permeabilized granulosa cells in suspension. The stained cells were fixed in a 70% ethanol solution until 1) the percentage of cells from each follicle that stained dark blue-indicating the presence of 3 beta-HSD activity-was determined by counting under light microscopy, and 2) the intensity of staining-indicating the relative amount of enzyme activity-was quantified using video image analysis. There were three findings. First, 100% of granulosa cells from each of the five largest preovulatory follicles stained positively for the presence of 3 beta-HSD activity. Second, the amount of 3 beta-HSD activity was normally distributed among granulosa cells in the population from each follicle. Third, as follicles matured from the fifth largest to the largest follicle, 3 beta-HSD activity increased steadily in individual cells, as indicated by increased staining intensities. The results indicate uniformity in the steroidogenic capacity of cells in the granulosa layer of hen preovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)